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Identification of small-molecules targeting CXCR2 function and signaling

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Identification of small-molecules targeting CXCR2 function and signaling

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IDENTIFICATION OF SMALL-MOLECULES TARGETING CXCR2 FUNCTION
AND SIGNALING

by

Helen Nen Ha

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

August 2013

Copyright 2013 Helen Nen Ha
ii

“Trust in the LORD with all your heart and lean not on your own understanding.”
~ Proverbs 3:5
iii
DEDICATION
I dedicate my dissertation to my loving parents whom I am forever indebted to for
the sacrifices they made to help me pursue my dreams.
iv
ACKNOWLEDGEMENTS
First, I thank God for granting me the strength to accomplish the work presented in
this dissertation. I thank Him for His abounding grace, mercy, and love that sustains me
each and every day. Most importantly, I thank my husband, Mathias Nakatsui, for his
unwavering love and support as I pursue my doctoral degree. look forward to a new
chapter in our lives together. I also want to thank my parents, Sang Ha and Co Soi Cun.
My mom and dad immigrated to the US in the 1980s and had to work very hard to
provide for me and my siblings.
I would like to sincerely thank my mentor, Dr. Nouri Neamati for his guidance and
support. He has offered me valuable expertise in the field of drug design and discovery
and high throughput assay development that has been indispensable to my research. I
thank him for his openness to new ideas and technologies that has given me the freedom
to explore different areas. I also appreciate how Dr. Neamati encourages a cooperative
and “family-like” environment in the lab; a workplace where we freely exchange ideas,
offer assistance to one another, and develop lifelong friendships and collaborations. I am
grateful for all the opportunities he has given me in the lab and all the life lessons he has
taught me along the way.
I would also like to thank all my colleagues and friends in the Neamati lab (Kavya
Ramkumar, Divya Pathania, Yuting Kuang, Si Li, Rasha Alsafi, Dr. Melissa Millard, Dr.
Shili Xu, Dr. Yumna Shabaik, Dr, Erik Serrao, Dr. Tino Sanchez, Dr. Roppei Yamada,
Dr. Srinivas Odde, Dr. Bikash Debnath, and Dr. Xuefei Cao). It has been such a blessing
to work with each and every one of them. I appreciate their friendship and willingness to
v
always lend a helping hand. Special thanks to Dr. Bikash Debnath and Dr. Srinivas Odde
for their collaboration on the computational studies included in this dissertation.
I would also like to thank my committee members, Dr. Roger Duncan, Dr. Curtis
Okamoto and Dr. Paul Beringer for their time, guidance, and support. I also thank Dr.
Tim Bensman and Henry Ho for their collaboration on the in vivo mice studies. I would
like to extend my thanks to Dr. Heinz Lenz, Dr. Young Hong, and Dr. Daryl Davies for
their expertise and generously providing reagents.
A special thanks to my friends (Dr. Daphne Ma, Alice Liao, Fuychun Abamonga,
Marco Abamonga, Kavya Ramkumar, Divya Pathania, Dr. Yumna Shabaik, Dr. Shili
Xu); my lovely siblings (Wie, Phu, Ping, and Peter Ha); and my gracious mother and
father-in-law (Carol and Mo Nakatsui) for their prayers, love, and support all these years.
Last, but not least, I thank my church, Evergreen Baptist Church of San Gabriel Valley,
for all their prayers and support.
The research presented in this dissertation was funded by the Department of Defense
Lung Cancer Research Program, Grant # LC090363

vi
TABLE OF CONTENTS
Dedication .......................................................................................................................... iii
Acknowledgements ............................................................................................................ iv
List of Tables ..................................................................................................................... ix
List of Figures .................................................................................................................... xi
Abstract ............................................................................................................................ xiv
Preface.............................................................................................................................. xvi
Chapter 1: Introduction ................................................................................................... 1
1.1. CXCL8 ............................................................................................................................. 3
1.1.1. Structural features of CXCL8 ....................................................................... 3
1.1.2. CXCL8 secretion .......................................................................................... 4
1.2. CXCR1 and CXCR2 ........................................................................................................ 4
1.2.1. Activation of CXCR1 and CXCR2 ............................................................... 5
1.2.2. Regulation of CXCR1 and CXCR2 .............................................................. 8
1.2.3. Structural features of CXCR1 and CXCR2 ................................................ 11
1.2.4. Differential functions of CXCR1 and CXCR2 ........................................... 14
1.2.5. CXCR2 knockout studies ............................................................................ 16
1.3. Inflammatory roles of CXCL8 in diseases ..................................................................... 17
1.3.1. Chronic lung diseases ................................................................................. 17
1.3.2. Cancer ......................................................................................................... 20
1.3.3. CXCR2: linking inflammation-mediated diseases to cancer ...................... 23
1.4. CXCR1/2 inhibition ....................................................................................................... 25
Chapter 2: Design of CXCR2 functional assays used to identify novel inhibitors ...... 39
2.1. CXCR2 binding assays .................................................................................................. 39
2.2. CXCR2 Functional Assays ............................................................................................ 41
2.2.1. β-arrestin1/2 recruitment and rearrangement .............................................. 42
2.2.2. Receptor internalization .............................................................................. 49
2.2.3. Receptor turnover........................................................................................ 55
2.2.4. Second messengers ..................................................................................... 59
2.2.5. Kinase phosphorylation .............................................................................. 63
2.3. Cellular Functions .......................................................................................................... 66
vii
2.4. Cell culture ..................................................................................................................... 72
2.5. Compounds and reagents ............................................................................................... 73
Chapter 3: Identification of Novel phenylcyclohex-1-enecarbothioamide-based
Compounds that inhibits CXCL8-mediated chemotaxis through selective regulation of β-
arrestin-2 74
3.1. Identification of CX4338 (2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-
enecarbothioamide). ................................................................................................................... 75
3.2. CX4338 inhibits receptor internalization ....................................................................... 82
3.3. CX4338 enhances CXCR2-mediated G-protein signaling ............................................ 84
3.4. CX4338 inhibits CXCL8-mediated chemotaxis ............................................................ 90
3.5. CXCR2 expression in a panel of cancer cell lines ......................................................... 92
3.6. CXCR2 knockdown with siRNA reduced cell proliferation .......................................... 95
3.7. Discussion and Conclusions .......................................................................................... 97
3.8. Materials and Methods ................................................................................................. 101
Chapter 4: IDENTIFICATION and Mechanistic Studies of Novel Pyrimidine-based
CXCR2 Inhibitors ........................................................................................................... 107
4.1. CX797 inhibits the effects of CXCL8 on cAMP signaling .......................................... 108
4.2. CX797 enhances CXCL8-mediated β-arrestin-2 recruitment ...................................... 111
4.3. CX797 inhibits CXCL8-mediated CXCR2 degradation .............................................. 113
4.4. CX797 inhibits CXCL8-mediated CXCR2 internalization.......................................... 116
4.5. CX797 enhances CXCL8-mediated ERK1/2 phosphorylation. ................................... 118
4.6. CX797 inhibits cell migration in wound healing assay ............................................... 120
4.7. CX797 induces cytostatic effects on HL60, Jurkat, and NSCLC cell lines. ................ 121
4.8. Discussions and conclusions ........................................................................................ 123
Chapter 5: Design and Discovery of Novel Compounds Based on a CXCR2 Inhibitor
Pharmacophore Model. ................................................................................................... 128
5.1. Development of a robust CXCR2 inhibitor-based pharmacophore model .................. 129
5.2. Identification of CX compounds in CXCR2 screening................................................ 136
5.3. CX compounds potently inhibit cell migration in wound healing assay ..................... 158
5.4. CX compounds inhibit cancer cell proliferation .......................................................... 159
5.5. CX compounds inhibit colony formation ..................................................................... 161
5.6. CX compounds differentially arrest cell cycle progression ......................................... 162
5.7. CX compounds induce rapid intracellula calcium release in a dose dependent manner
163
viii
5.8. CX4152 exhibit a different mechanistic profile than CX25 on CXCR2...................... 163
5.9. CX4152 induce CXCR2 internalization ...................................................................... 165
5.10. CX4152 inhibit cell proliferation and PMN cell migration ..................................... 169
5.11. Discussion and conclusions ..................................................................................... 171
5.12. Materials and Methods ............................................................................................. 175
5.12.1. Molecular modeling .................................................................................. 175
5.12.2. Data set and modeling. .............................................................................. 175
5.12.3. Pharmacophore model validation. ............................................................ 177
5.12.4. Database Search. ....................................................................................... 177
Chapter 6: Concluding Remarks and Future Perspectives ......................................... 179
Bibliography ................................................................................................................... 185
ix
LIST OF TABLES
Table 1.1 Chemokine Receptors and antagonists .............................................................. 2
Table 1.2 CXCL chemokines and CXCR1/2 and cancer ................................................. 28
Table 1.3 Different classes of CXCR2 inhibitors ............................................................ 32
Table 2.1 Antibody conditions for Western blotting ....................................................... 64
Table 3.1 IC50 values for CXCR2 and CXCR4 inhibition ............................................. 76
Table 3.2 Percent cell viability upon CX4338 treatment ................................................. 78
Table 3.3 IC
50
values for CXCR2/4 Tango inhibition for CX compounds identified in
HTS screen ............................................................................................................ 80
Table 3.4 CXCR2 transcriptional expression in a panel of cancer cell lines. .................. 93
Table 3.5 CXCL8 transcriptional expression in a panel of cancer cell lines ................... 94
Table 4.1 CX Compounds inhibits CXCL8 down-regulation of forskolin-induced
cyclicAMP .......................................................................................................... 110
Table 5.1 Results obtained from pharmacophore hypothesis generation using compounds
from training set. ................................................................................................. 132
Table 5.2 Experimental and estimated IC50 values of the training set CXCR2 inhibitors
based on Hypo-1 pharmacophore model. ........................................................... 134
Table 5.3 Hypo-1 validation using a database of previously reported CXCR2 Inhibitors
from literature ..................................................................................................... 135
Table 5.4 CXCR2 inhibition and cancer cell proliferation results of active CX
compounds selected from Hypo-1 screening ...................................................... 139
Table 5.5 CXCR2 inhibition and cancer cell proliferation results of CX compounds with
Scaffold A selected from CX25, CX86 and CX815 similarity search ............... 140
x
Table 5.6 CXCR2 inhibition and results of CX compounds with Scaffold B selected
from CX25, CX86 and CX815 similarity search ................................................ 144
Table 5.7 CXCR2 inhibition and cancer cell proliferation results of CX compounds with
Scaffold C selected from CX25, CX86 and CX815 similarity search ................ 148
Table 5.8 CXCR2 inhibition of inactive CX compounds selected from CX25, CX86 and
CX815 similarity search ..................................................................................... 152
Table 5.9 CXCR2/4 Tango and MTT IC50s for CX4152 Analogs ............................... 166
xi
LIST OF FIGURES
Figure 1.1 CXCR2 signaling cascades and receptor recycling. ......................................... 8
Figure 1.2 CXCR2 protein structure and domains........................................................... 13
Figure 1.3 CXCR1 and CXCR2 mediate neutrophil recruitment during infection. ........ 15
Figure 1.4 The multiple roles of CXCL chemokines and CXCR1/2 during tumor
development. ......................................................................................................... 23
Figure 2.1 Competitive CXCR2 binding assay schematic. ............................................. 40
Figure 2.2 Schematic of the Tango assay. ....................................................................... 43
Figure 2.3 CXCL8/SDF-1 activates CXCR2/CXCR4 in Tango assay. ........................... 45
Figure 2.4 CXCL8 induces β-arrestin-2 rearrangement. ................................................. 47
Figure 2.5 CXCL8 stimulates receptor internalization and turnover. .............................. 50
Figure 2.6 CXCL8 induces CXCR2 internalization. ....................................................... 53
Figure 2.7 CXCL8 induces CXCR2 degradation. ........................................................... 56
Figure 2.8 CXCL8 dose-dependently induce rapid and transient calcium flux in 293T-
CXCR2 cells. ........................................................................................................ 59
Figure 2.9 Schematic of CXCR2 Glosensor cyclic AMP assay. ..................................... 61
Figure 2.10 CXCL8 inhibits forskolin-induced cAMP in CXCR2 expressing cells. ...... 62
Figure 3.1 Chemical structures of CX4338 and previously reported CXCR2 antagonists
............................................................................................................................... 75
Figure 3.2 CX4338 selectively inhibit CXCR2-mediated β-arrestin coupling. ............... 77
Figure 3.3 CX4338 inhibit β-arrestin-2 rearrangement. .................................................. 79
Figure 3.4 Chemical structures of CX compounds identified from Tango screening ..... 81
Figure 3.5 CX4338 inhibits CXCL8-mediated CXCR2 internalization. ......................... 83
xii
Figure 3.6 CX4338 enhances CXCL8 induced calcium release. ..................................... 86
Figure 3.7 CX4338 enhances ERK1/2, p38, and JNK phosphorylation. ........................ 87
Figure 3.8 CX4338 does not alter CXCL8-mediated cyclic AMP signaling. ................. 88
Figure 3.9 CX4338 inhibits CXCL8-mediated cell migration......................................... 91
Figure 3.10 CXCR2 expression in a panel of cell lines. .................................................. 95
Figure 3.11 CXCR2 siRNA reduced cell proliferation in CXCR2-expressing cell lines. 96
Figure 3.12 Proposed mechanism of CX4338. .............................................................. 100
Figure 4.1 Chemical structure of CX compounds ......................................................... 108
Figure 4.2 CX compounds inhibit CXCL8-mediated cyclic AMP signaling. ............... 109
Figure 4.3 Kinetic cyclic AMP curves of (A) CX797, (B) CX143, (C) CX119, and (D)
SB265610. ........................................................................................................... 110
Figure 4.4 CX797 selectively increases CXCL8-mediated CXCR2 β-arresti-2
recruitment. ......................................................................................................... 112
Figure 4.5 CX797 and SB265610 treatment alters CXCR2 expression. ....................... 115
Figure 4.6 CX797 enhance total and surface CXCR2 expression. ................................ 117
Figure 4.7 CX797 inhibits CXCL8-mediated CXCR2 degradation in In-cell Western
assays. ................................................................................................................. 118
Figure 4.8 CX797 up-regulates CXCL8-mediated ERK1/2 phosphorylation without
greatly affecting AKT phosphorylation. ............................................................. 119
Figure 4.9 CX797 dose dependently inhibit cell migration. .......................................... 120
Figure 4.10 CX797 induces cytostatic effects on HL60, Jurkat, and NSCLC cell lines.
............................................................................................................................. 122
xiii
Figure 5.1 Chemical structures of training set used to generate Hypo-1 pharmacophore
hypothesis. .......................................................................................................... 129
Figure 5.2 Chemical structures of test set used to validate Hypo-1 pharmacophore
hypothesis. .......................................................................................................... 131
Figure 5.3 Experimental pIC50 vs. predicted pIC50 of training (•) and test set (о). ..... 133
Figure 5.4 Pharmacophore modeling of Hypo-1. .......................................................... 136
Figure 5.5 Pharmacophore mapping of CX compounds and CXCR2 inhibitors. .......... 137
Figure 5.6 Chemical structures of compound scaffold A, B, and C .............................. 138
Figure 5.7 Chemical structures of CX compounds selected for further studies. ........... 158
Figure 5.8 CX compounds inhibit cell migration. ......................................................... 159
Figure 5.9 CX compounds inhibit cancer cell proliferation. ......................................... 160
Figure 5.10 CX compounds inhibit colony formation. .................................................. 161
Figure 5.11 CX compounds differentially regulate cell cycle progression. .................. 162
Figure 5.12 CX compounds induce intracellular calcium flux. ..................................... 163
Figure 5.13 CX4152 exhibit a different mechanistic profile than CX25. ...................... 164
Figure 5.14 CX4152 induce CXCR2 internalization and turnover................................ 165
Figure 5.15 CX4152 inhibit cell proliferation and PMN cell migration........................ 170
Figure 6.1 Schematic view of CXCR2 inhibition of previously reported CXCR2
inhibitors and CX25. ........................................................................................... 181
Figure 6.2 SB265610 enhance efficacy of PDE4 inhibitors in the cyclic AMP Glosensor
assay. ................................................................................................................... 183

xiv
ABSTRACT
Chemokine receptor, CXCR2, and its ligands are an essential component of the
immune system that mediates the trafficking of neutrophils to sites of infection. It is also
important in angiogenesis by activating and coordinating the assembly of endothelial
cells. Given its roles in the immune system and angiogenesis, CXCR2 has been
implicated in a number of inflammation-mediated diseases such as chronic obstructive
pulmonary diseases (COPD) and various types of cancer. CXCR2 inhibition has shown
promising anti-inflammatory effects in the clinics as well as anti-cancer effects in several
in vivo animal models. And within the last two decades, several classes of small-
molecules targeting CXCR2 has been developed and advanced onto clinical trials.
However, current CXCR2 inhibitors are limited to four major chemical classes of
compounds and are by far scarce compared to other clinically relevant therapeutic targets.
Thus, we sought to identify additional classes of CXCR2 inhibitors using cell-based,
functional assays rather than conventional competitive ligand binding assays. We also
capitalized on the literature of previously described CXCR2 inhibitors and developed a
pharmacophore model to identify important chemical features of CXCR2 inhibitors and
to perform in silico screening of large databases of compounds.
We have identified several classes of compounds that modulate CXCR2 signaling
and cellular functions in CXCR2-expressing cells. These compounds are chemically
different from any reported CXCR2 inhibitors. Mechanistic studies of these compounds
showed that they differentially regulate CXCR2-mediated G-protein and β-arrestin1/2
signaling. For example, CX4338 inhibited CXCR2-mediated β-arrestin1/2 recruitment,
while enhancing CXCR2-mediated G-protein signaling (calcium and ERK1/2 activation).
xv
Selective inhibition of CXCR2/β-arrestin1/2 association by CX4338 was sufficient to
reduce cell migration and LPS-induced PMN migration. We also discovered CX797 in
our screening endeavors using a cell-based cyclic AMP assay. CX797 inhibited the
effects of CXCL8 on forskolin-induced cyclic AMP signaling, while enhancing
CXCR2/β-arrestin2 association and up-regulating total CXCR2 expression. CX797 also
inhibited CXCL8-mediated cell migration and cell proliferation in CXCR2-expressing
cell lines.
Lastly, using a pharmacophore model, we identified CX25 as a potent inhibitor of
CXCR2 in the Tango assay. However, these class of compounds did not show much
selectivity amongst different chemokine receptors and inhibited cancer cell proliferation
in a CXCR2-independent manner. CX4152 was also identified from the pharmacophore
screen and exhibit similar chemical features as CX25. Though CX4152 was not as active
as CX25 against CXCR2, it showed 8-fold selectivity for CXCR2 over CXCR4 (another
chemokine receptor). Mechanistic studies suggest that CX4152 inhibit CXCR2/β-
arrestin-2 association via CXCR2 down-regulation. Treatment with CX4152 reduced
surface CXCR2 expression within 10 minutes in a dose and time dependent manner.
Taken together, compounds identified from our screens have differential effects on
CXCR2 signaling and provide several novel avenues to mechanistically modulate
CXCR2 function and signaling.

xvi
PREFACE
The main goal of the research project presented in this dissertation is to develop
small-molecule CXCR2 inhibitors that are chemically different from previously reported
CXCR2 inhibitors using cell-based functional assays that has been miniaturized into high
throughput screening platforms.
Chapter 1 provide a thorough review of the current literature on CXCR2 and its
ligands. We have focused in on the various diseases that CXCR2 has been implicated in
and the different classes of CXCR2 inhibitors. Chapter 2 presents the various assays that
were used in this research project. Detail descriptions of assay development and protocol
is provided in this section. Chapter 3 describes the identification and characterization of
CX4338 using various CXCR2 assays described in Chapter 2. Neutrophil migration and
LPS-induced inflammation mice studies was performed by Dr. Tim Bensman and Henry
Ho from Dr. Paul Beringer’s lab in the USC School of Pharmacy. Chapter 4 describes
the identification and characterization of another class of compounds that are structurally
and mechanistically different those described in Chapter 3. Chapter 5 describes the
development of a CXCR2 pharmacophore model and the identification of CX25 and
CX4152. Pharmacophore modeling and in silico screening presented in this chapter was
performed by Dr. Srinivas Odde and Dr. Bikash Debnath. Dr. Tim Bensman and Henry
Ho also performed the neutrophil cell migration studies and LPS-induced inflammation
mice studies presented in this section. Lastly, we conclude with chapter 6 with closing
remarks and a discussion of future studies.
1
CHAPTER 1: INTRODUCTION
Chemokines and their cognate receptors plays an essential role in the immune system
by mediating the activation and trafficking of immune cells during innate and adaptive
responses. Chemokines are also involved in hematopoiesis and development by directing
and mobilizing precursor cells to sites of maturation. A subset of chemokine receptors
are also expressed on endothelial cells to regulate angiogenesis (Le et al., 2004).
Chemokines are small (6-14 kD) secreted proteins that have four cysteine residues that
are essential for their structural integrity. Arrangement of the four cysteine residues is
used to group chemokines into four different classes: CC, CXC, XC, and CX3C (Rollins,
1997). There are 50 chemokines and 20 chemokine receptors identified thus far with CC
and CXC being the two major classes of chemokines (Table 1.1). CXC is further
subdivided into ELR+ or ELR- denoting CXC chemokines that contain or lack the three
amino acid motif (Glu-Leu-Arg) that precedes the first cysteine residue on the N-
terminus (Hebert et al., 1991).
Given the vital roles of chemokines in the immune system and during inflammatory
responses, a number of chemokines are involved in diseases such as HIV, arthritis,
multiple sclerosis (MS), chronic obstructive pulmonary diseases (COPD), lupus, pain,
asthma, inflammatory bowel diseases (IBD), Crohn’s disease, reperfusion injury (RI),
and cystic fibrosis (CF) (Horuk et al., 1997). The drugability of chemokine receptors has
led to the development of small-molecule compounds that has advanced onto clinical
trials (Table 1.1). However, Maraviroc from Pfizer is the only FDA approved chemokine
receptor (CCR5) inhibitor for HIV thus far (Dorr et al., 2005).
2
Table 1.1 Chemokine Receptors and antagonists
Class Receptors Ligand Cell Expression Antagonists
CCR CCR1 CCL3, 5, 7, 8,
13, 15, 16, 23
Monocytes, immature
DCs, T cells, PMNs,
eosinophils, mesangial
cells, platelets
CP-481,715 (arthritis); MLN3897
(arthritis); BX471 (multiple sclerosis);
AZD-4818 (COPD)(Gladue et al., 2010)
CCR2 CCL2, 7, 8, 12,
13
Monocytes, immature
DCs, basophils, PMNs, T
cells, NK cells, endothelial
cells, fibroblasts
MLN 1202 (MS, RA, atherosclerosis);
INCB8696 (MS, lupus); CCX140 (MS);
PF-4136309 (pain); MK-0812
(rheumatoid arthritis , multiple sclerosis);
CCR3 CCL5, 7, 8, 11,
13, 14, 15, 24, 26
Eosinophils, basophils, T
cells, DCs, platelets, mast
cells
TPI ASM8 (asthma); KW-0761 (cancer);
776994 (asthma, allergic rhinitis); DPC-
168 (asthma); GW766944 (asthma)
CCR4 CCL17, 22 Immature DCs, basophils,
T cells (Th2 T-cells),
platelets
KW-0761(antibody, lymphoma)
CCR5 CCL3, 4, 5, 8,
11, 13, 14, 20
T-cells (Th1 cells),
immature DCs, monocytes,
NK cells, thymocytes
Maraviroc (approved for HIV, rheumatoid
arthritis); Vicriviroc (HIV); Aplaviroc
(HIV, exhibit toxicity); INCB9471 (HIV);
Pro 140 (HIV); CCR5mAb004 (HIV);
TBR-652 (HIV); Cenicriviroc (HIV)
CCR6 CCL20 Immature DCs, T cells, B
cells
None reported
CCR7 CCL19, 21 Naïve and memory T cells
Mature DCs, T cells, B
cells
None reported
CCR8 CCL1, 4, 16 Monocytes, B cells, T cells
(Th2 cells), thymocytes
AZ084 (preclinical)
CCR9 CCL25 T cells, thymocytes, DCs,
macrophages
CCX-282 (IBD, Crohn’s disease);
CCX8037; CCX282-B (IBD); GSK-
1605786 (Crohn’s disease)
CCR10 CCL27, 28 T cells, melanocytes,
dermal endothelia, dermal
fibroblasts, Langerhans
cells astrocytes
None reported
CXCR CXCR1 CXCL6, 8 PMNs, monocytes,
astrocytes, endothelia,
mast cells
SCH527123 (COPD); Reparixin
(reperfusion injury)
CXCR2 CXCL1, 2, 3, 5,
6, 7, 8
PMNs, monocytes,
eosinophils, endothelia,
mast cells
SCH527123 (COPD); Reparixin
(reperfusion injury); SB656933 (COPD,
cystic fibrosis); AZD5069 (neutrophil
function); GSK1325756 (pulmonary
disease)
CXCR3 CXCL9, 10, 11 T cells, B cells, NK cells,
mesangial cells, smooth
muscle cells, endothelia
T-487/AMG-487 (Psoriasis)
CXCR4 CXCL12 Hematopoietic progenitors,
T cells, immature DCs,
monocytes, B cells, PMNs,
platelets, astrocyte,
endothelia
Plerixafor (multiple myeloma, NHL);
BKT-140 (multiple myeloma); AMD 3100
(Myelokathexis); AMD11070 (HIV);
MSX-122 (cancer)
3
CXCR5 CXCL13 T cells, B cells, astrocytes None reported
CXCR6 CXCL16 Memory T cells None reported
CXCR7 CXCL12 None reported
XCR XCR1 XCL1, XCL2 T cells None reported
CX3CR CX3CR1 CX3CL1 PMNs, monocytes, NK
cells, T cells, astrocytes
None reported
Duffy CXCL1, 7, 8,
CCL1, 5
Red blood cells, endothelia None reported
D6 CCL2, 4, 5, 8,
13, 14, 15
B cells None reported
Table adapted from reviews in (Horuk et al., 1997; Le et al., 2004; Viola and Luster, 2008).
1.1. CXCL8
One of the first and most studied chemokine is CXCL8. CXCL8 is a pro-angiogenic
and pro-inflammatory chemokine that has been of interest in the last three decades. In
the late 1980s, Peveri et al. found that LPS-stimulated blood monocytes produced a
secretory protein (neutrophil activating factor, NAF) that stimulated neutrophil
exocytosis (granule release) and oxidative burst (superoxide and hydrogen peroxide
production) that appears to be mediated by cell surface receptors (Peveri et al., 1988).
NAF was the first chemokine to be purified and sequenced in 1987 that is also referred to
as interleukin-8 (CXCL8) and CXCL8 upon identification of additional chemokines
(Schroder et al., 1987; Walz et al., 1987; Yoshimura et al., 1987).
1.1.1. Structural features of CXCL8
The crystal structure of CXCL8 was first solved in 1991. It is a 72-amino acid
peptide that has three antiparallel β-strands and one α-helix made up of carboxyl residues
57-72 (Baldwin et al., 1991). The structure is stabilized by two disulfide bonds formed
by Cys7-Cys34 and Cys9-Cys50, hence the classification of CXCL8 as a CXC
chemokine. The crystal structure also revealed that CXCL8 exists as a dimer stabilized
4
by hydrogen bonds between the first β-strand. However, later studies show that CXCL8
is biologically more active as a monomer (Nasser et al., 2009; Rajarathnam et al., 1994).
Scanning mutagenesis on CXCL8, in which the first 15 amino acids of CXCL8 was
individually mutated to alanine revealed a critical N-terminus motif. Mutants E4A, L5A
and R6A were inactive in receptor activation assays and showed reduced affinity to its
receptors in competitive binding assays (Clark-Lewis et al., 1991; Hebert et al., 1991).
1.1.2. CXCL8 secretion
CXCL8 is secreted by different cell types including blood monocytes, alveolar
macrophages, endothelial cells, fibroblasts, epithelial cells, and hepatoma cells (Kwon et
al., 1994; Peveri et al., 1988; Strieter et al., 1988; Wanninger et al., 2009). CXCL8
expression is stimulated by cytokines (interleukin-1, interleukin-6, and TNFα), hypoxia,
reactive oxygen species (ROS), bacterial particles, and other environmental stresses,
mediated by transcription factors, NFκB and activator protein-1 (AP-1) (Brat et al.,
2005).
1.2. CXCR1 and CXCR2
CXCL8 is the ligand for both CXCR1 and CXCR2 receptor. The two receptors
share 76% sequence homology with each other and binds to CXCL8 with similar affinity
(K
d
~ 4 nM) (Holmes et al., 1991; Kunsch and Rosen, 1993; Murphy and Tiffany, 1991).
Most of the differences between the two receptors occur in the second extracellular loop,
C-terminal (intracellular) and N-terminal (extracellular) region (Holmes et al., 1991;
Murphy, 1994). While CXCR2 interacts with all other ELR+ chemokines (CXCL1-3,5-
5
8) with high affinity (EC
50
> 10 nM), CXCR1 only weakly binds to other ELR+
chemokines (EC
50
between 40 and 65 nM) (Ahuja and Murphy, 1996).
1.2.1. Activation of CXCR1 and CXCR2
G-protein signaling. Since its discovery in 1991, a number of studies have been
performed to characterize CXCR1 and CXCR2 receptor signaling and regulation. These
studies were mainly carried out in neutrophils, or HEK293 and RBL-2H3 cells over-
expressing CXCR1 and/or CXCR2. Upon chemokine binding, CXCR1/2 couples to
pertussis toxin-sensitive G-protein via physical interaction with the Gαi subunit to
regulate several signaling cascades that mediate neutrophil chemotaxis and activation
(Figure 1.1) (Damaj et al., 1996). Activation of CXCR1/2 induces dissociation of the
receptor with the G-protein and release of the Gβγ subunit from the Gα subunit. Release
of Gβγ subunit activates phospholipase C (PLC) and results in calcium mobilization
(from the endoplasmic reticulum to cytosol) and phosphokinase C (PKC) activation (β-2
form), which is critical for neutrophil chemotaxis (Figure 1.1 1.1) (Wu et al., 1993; Wu et
al., 2012b)
CXCL8 also induce rapid and transient phosphorylation of extracellular signal
related kinases (ERK1/2) and phosphatidylinositide 3-kinase (PI3K)/Akt in human
neutrophils (Fuhler et al., 2005; Knall et al., 1997; Knall et al., 1996; Xythalis et al.,
2002). ERK1/2 is a component of the Ras-Raf-MEK-ERK signaling cascade (Roskoski,
2012). However, the role of CXCL8-mediated ERK1/2 activation in neutrophils
migration remains unclear. Xythalis and Fuhler et al. showed PD098059 (MEK
inhibitor) inhibited CXCL8-induced neutrophil chemotaxis, while other studies showed
6
no effects of PD098059 on CXCL8-induced neutrophil chemotaxis (Fuhler et al., 2005;
Knall et al., 1997; Xythalis et al., 2002). Inhibition of PI3K with small-molecule
inhibitor, LY294002, significantly reduced CXCL8-mediated cell migration in human
neutrophils and L1.2 cells over-expressing CXCR2 independent of ERK1/2 (Knall et al.,
1997; Lane et al., 2006)
In CXCR1- and CXCR2-RBL (rat basophil leukemia) transfected cells, CXCL8
induced focal adhesion kinase (FAK) phosphorylation and re-localization. It also
induced actin and β-tubulin re-localization to promote cell spreading and motility which
is directly correlated CXCL8-induced migratory response (Cohen-Hillel et al., 2006;
Feniger-Barish et al., 2003). FAK regulates cell motility by directing processes involved
in cell spreading, attachment, and detachment (Parsons et al., 2000). Raman et al.
showed that LIM and SH3 protein 1 (LASP-1) directly associates with chemokine
receptors (CXCR1-4), and its association is critical for chemotaxis, suggesting that
LASP-1 may serve as an adaptor protein that connects chemokine receptors to
components of the cytoskeleton (Raman et al., 2010). CXCR2 also regulates other key
regulators of actin polymerization such as RacGTPases (small monomeric GTPases)
(Schraufstatter et al., 2001; Schraufstatter et al., 2003).
The uncoupling of CXCR2 to the Gαi subunit upon ligand activation inhibits the
enzyme that converts ATP to cyclic AMP, adenylyl cyclase (AC), and results in
decreased intracellular cyclic AMP concentrations and decreased downstream
phosphokinase A (PKA) activation (Damaj et al., 1996). To detect the effects of CXCL8
and CXCL1 on cyclic AMP levels, Hall et al. stimulated CXCR1/2-overexpressing CHO
7
cells with forskolin (AC activator) in the presence of CXCL8 or CXCL1. They showed
both chemokines dose dependently inhibited CXCR2-mediated forskolin-induced cyclic
AMP accumulation. Meanwhile, only CXCL8 inhibited CXCR1-mediated forskolin-
induced cyclic AMP accumulation (Hall et al., 1999).
Though CXCR1 and CXCR2 induce cell migration and granule release in
neutrophils through similar pathways, phospholipase D (PLD) activation is exclusively
mediated by CXCR1 (Baggiolini et al., 1997; L'Heureux et al., 1995; Richardson et al.,
1998b). PLD is the enzyme that converts phosphatidycholine into phosphatidic acid (PA)
and choline. PA has been shown to activate NAPDH oxidase and subsequent superoxide
anion production and oxidative burst in neutrophils (Sozzani et al., 1994). CXCR1 but
not CXCR2 in neutrophils significantly induced superoxide anion production, suggesting
that CXCR1 (and not CXCR2) is essential for CXCL8-mediated oxidative burst (Jones et
al., 1996).
8

Figure 1.1 CXCR2 signaling cascades and receptor recycling.
CXCL8 binding to CXCR2 activates several G-protein-mediated signaling cascades. Receptor activation
immediately leads to the dissociation of the Gαi subunit from the β and γ subunit, and subsequently
activates growth and stress kinases such as ERK1/2, JNK1, and p38. G-protein activation also induces
rapid intracellular mobilization released from the endoreticulum (ER) and inhibition of adenylyl cyclase
resulting in decreased cyclic AMP production. CXCR2 activation also leads to receptor phosphorylation
on the C-terminus by GRK6 and recruitment of β-arrestin1/2 and components of endocytosis such as
clathrin and dynamin to mediate receptor internalization. Internalized receptors are either recycled back to
the cell surface or routed to lysosomes for degradation.
1.2.2. Regulation of CXCR1 and CXCR2
The G-protein signaling of CXCR1/2 is tightly regulated and quickly turned off to
prevent constitutive G-protein signaling. Receptor desensitization is regulated through
several mechanisms including receptor phosphorylation/β-arrestin1/2-recruitment, AP-2
adaptor protein association, and receptor cross-desensitization.
9
Homologous desensitization (agonist-dependent). Upon ligand stimulation,
CXCR1/2 is quickly phosphorylated by G-protein-coupled receptor kinases (GRKs) and
associates with β-arrestin1/2 and AP-2 to promote dynamin- and clathrin-mediated
receptor internalization (Barlic et al., 1999; Raghuwanshi et al., 2012; Richardson et al.,
2003; Yang et al., 1999). Interestingly, CXCR2 internalization occurs at a faster rate and
at lower ligand concentrations than CXCR1, suggesting differential regulation of receptor
signaling (Prado et al., 1996; Rose et al., 2004). CXCR1 and CXCR2 are also
differentially regulated by GRKs. GRK2 mainly associates with and phosphorylates
CXCR1, while GRK6 is mediates CXCR2 phosphorylation (Raghuwanshi et al., 2012).
Receptor phosphorylation recruits β-arrestin1/2 to the receptor to terminate G-protien
signaling via two distinct mechanisms. β-arrestin1/2 association inhibits receptor and G-
protein coupling as well as recruits the endocytic machinery such as clathrin and AP-2 to
mediate receptor internalization and sequestration (Luttrell and Lefkowitz, 2002). For
CXCR2 (but not CXCR1), it appears that β-arrestin1/2 may not be absolutely necessary
for receptor internalization. Receptor internalization (though reduced) was still observed
in phosphorylation deficient CXCR2 (truncated C-terminal or GRK knockout) and β-
arrestin-2 deficient cells, suggesting that receptor internalization is also mediated through
alternative phosphorylation independent mechanisms (Barlic et al., 1999; Fan et al.,
2001a; Fan et al., 2001b; Raghuwanshi et al., 2012; Su et al., 2005; Zhao et al., 2004).
Phosphorylation or β-arrestin-2 deficiency also exhibits enhanced G-protein signaling
resulting in ROS generation that induces cell death (Su et al., 2005; Zhao et al., 2004).
Unlike β-arrestin1/2, receptor association with AP-2 does not require phosphorylation
10
and AP-2 receptor association is absolutely required for CXCR1/2 internalization (Fan et
al., 2001b). AP-2 is a critical adaptor protein that directly links membrane bound
receptors to the clathrin lattice during endocytosis (Kirchhausen et al., 1997; Robinson,
1994; Schmid, 1997).
Heterologous desensitization (agonist-independent). CXCR1/2 are also regulated
through the activation of other receptors (heterologous desensitization). CXCR1 and
CXCR2 are cross phosphorylated and desensitized (Ca
2+
mobilization, receptor
internalization) to CXCL8 by receptors for N-formylated peptides (fMLP) or complement
cleavage product (C5a) (Richardson et al., 1998a; Richardson et al., 1998b). fMLP and
C5a are strong chemoattractants for leukocytes that mediate chemotaxis and leukocyte
activation (Perez, 1984; Wittmann et al., 2002). CXCR1, but not CXCR2 also cross
phosphorylate and desensitize fMLP and C5a receptors when these receptors were co-
expressed together in RBL-2H3 cells. However, C-terminal truncated CXCR2 were able
to activate PLD and cross-phosphorylate and desensitize fMLP and C5a receptors,
suggesting that PLD activation determines the ability for CXCR1/2 to regulate other
receptors (Richardson et al., 1998b).
Receptor transactivation. In endothelial cells, CXCL8 activation of CXCR1/2
complexes with and transactivates vascular endothelial growth factor receptor 2
(VEGFR2) via receptor phosphorylation mediated by Src kinases. VEGFR2
transactivation is required for CXCL8-induced endothelial cell permeability (Petreaca et
al., 2007). CXCL8/CXCR2 also stimulate VEGFR2 activation by inducing the
transcription of VEGF in endothelial cells via the NFκB pathway (Martin et al., 2009).
11
CXCL8/CXCR2 also transactivates epithelial growth factor receptor (EGFR) via receptor
phosphorylation to mediate endothelial cell migration and tube formation (Kyriakakis et
al., 2011; Schraufstatter et al., 2003).
1.2.3. Structural features of CXCR1 and CXCR2
The difficulty of purifying membrane-bound receptors and the inherent flexibility of
GPCRs has impeded to the crystallization of chemokine receptors. However, since the
creation of GPCR Network in 2010, it has led to the structural characterization of eight
GPCR, including chemokine receptor CXCR4 (Stevens et al., 2013; Wu et al., 2010).
The GPCR Network is a collaborative effort of the scientific community at implementing
high throughput structure determination pipelines to characterize 15-25 representative
human GPCR within the next few years (Stevens et al., 2013).
N-terminus of CXCR1/2. Given the lack of structural data, a number of CXCR1 and
CXCR2 chimera, receptor truncation, and single amino acid point mutation studies have
revealed several essential structural features that are critical for receptor binding,
activation, and regulation. The N-terminus of CXCR1/2 and the first extracellular loop
of CXCR2 are critical for ligand binding and specificity (Figure 1.2) (Catusse et al.,
2003; Gayle et al., 1993; LaRosa et al., 1992; Wu et al., 1996) as well as determine the
rate of receptor internalization (Prado et al., 2007). For CXCR2, the N-terminus alone
did not determine ligand specificity, but also included other regions of the receptor that
involves the transmembrane domain 4 and extracellular loop 2 (Ahuja et al., 1996; Wu et
al., 1996). The same studies show that CXCR2 ligands bind to overlapping but distinct
sites on CXCR2 and affinity of the ligand did not necessarily correlate with potency on
12
receptor activation suggesting that sites of receptor binding and activation are distinct
(Ahuja et al., 1996). Wu et al. also showed that monoclonal antibodies raised against
human CXCR1 that binds to the first 45 residues of the receptor inhibited chemotaxis
without affecting binding, further supporting the idea that ligand binding and receptor
activation are distinct (Wu et al., 1996).
C-terminus of CXCR1/2. The C-terminus of CXCR1/2 regulate receptor
phosphorylation, internalization, G-protein coupling and association with other
cytoplasmic proteins. Truncation of the C-terminal of CXCR1/2 impaired receptor
phosphorylation, β-arrestin1/2 association, and internalization, as well as enhanced G-
protein signaling (calcium release) and reduced chemotaxis (Ben-Baruch et al., 1995; Fan
et al., 2001b; Prado et al., 1996; Richardson et al., 2003). Reduced chemotaxis without
receptor internalization suggest that G-protein signaling negatively regulates chemotaxis
and not receptor internalization. (Richardson et al., 2003). Alanine point mutations show
serines 342, 346-348 are involved in receptor desensitization and sequestration but not
receptor phosphorylation, suggesting other hydroxylated residues may be involved in
receptor phosphorylation (Mueller et al., 1997). However, this conflicts with studies
performed by Luo et al. that showed involvement of serine 346-348 in agonist-induced
phosphorylation in primary cultured cells isolated from mice (Luo et al., 2005).
The C-terminus of CXCR2 is also a binding site for several adaptor proteins that
regulate receptor desensitization and endocytosis. β-arrestin1/2 associates with CXCR1/2
upon receptor phosphorylation and mediates the recruitment of endocytic components
(clathrin and dynamin) (Barlic et al., 1999; Richardson et al., 2003; Yang et al., 1999).
13
AP-2 also binds to the LLKIL motif on CXCR2 and regulate receptor internalization and
sequestration in HEK293 cell (Fan et al., 2001b). C-terminal deletion of CXCR2 also
reduced G-protein activation (measured with GTPγS exchange), suggesting it is also
involved in G-protein coupling (Schraufstatter et al., 1998). The third intracellular loop
of CXCR2 is also involved in G-protein coupling and signaling (Yang et al., 1997).
Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, also directly associates
with CXCR2 on residues KFRHGL on the C-terminus independent of receptor
phosphorylation and mediate receptor dephosphorylation and receptor recycling (Fan et
al., 2001a). Cells stimulated with CXCL8 also phosphorylates vasodilator–stimulated
phosphoprotein (VASP) via PKC and PKA signaling and promote the association of
VASP to the C-terminus of CXCR2 (331-355). VASP association is critical for CXCR2-
mediated chemotaxis and polarization (Neel et al., 2009).

Figure 1.2 CXCR2 protein structure and domains.
The N-terminus (extracellular face) of CXCR2 is critical for ligand binding and specificity.
Transmembrane domain 4 and extracellular loop 2 is also important for ligand binding. G-protein couples
to the C-terminus (cytoplasmic face) of CXCR2 and involves intracellular loop 3. Several proteins, such as
G protein coupled receptor kinase 6 (GRK6) Vasodilator-stimulated phosphoprotein (VASP), β-arrestin1/2,
adaptor protein-2 (AP-2), protein phosphatase 2A (PP2A) also associate with the C-terminus of CXCR2 to
mediate different signaling cascades.
14
1.2.4. Differential functions of CXCR1 and CXCR2
Both receptors mediate common GPCR signaling pathways and cellular functions
such as calcium release, activation of Ras/MAPK and PI3K signaling cascade, as well as
receptor internalization and chemotaxis. However, CXCR1 but not CXCR2 was shown
to activate PLD and subsequently mediate ROS generation and oxidative burst in
neutrophils (Baggiolini et al., 1997; L'Heureux et al., 1995; Richardson et al., 1998a).
Also, it was initially thought that mice did not have the CXCR1 homologue. However, in
2007, a CXCR1 homologue having 68% identity to the human CXCR1 was identified
and shown to be activated by mouse CXCL6 and human CXCL8 (Fan et al., 2007).
Another difference observed between the two receptors is the receptor
desensitization rate. CXCR2 is internalized more rapidly and at lower ligand
concentrations than CXCR1 (Prado et al., 1996; Rose et al., 2004). It is also recycled
back to the surface at a much slower rate than CXCR1. In studies with CXCR2 mutants,
where the C-terminus is truncated and receptor internalization is impaired, CXCR2 was
able to activate PLD and mediate CXCL8-mediated superoxide anion production
(Richardson et al., 1998b). This is also corroborated with studies that inhibit the
internalization mechanism of CXCR2 (cell lines with β-arrestin1/2 deficiency or
dominant negative dynamin), which show similar increase in ROS production. This
suggests that the functional differences between CXCR1 and CXCR2 may be regulated
by the duration of the signal.
Higher ligand concentrations are required for receptor desensitization than receptor
activation (chemotaxis). And the fact that CXCR2 can interact with all ELR+
15
chemokines suggest that CXCR2 may play a more important role in chemotaxis (low
ligand concentration), whereas CXCR1 may be more important at sites of infection (high
ligand concentration) by inducing neutrophil oxidative burst and granule release essential
for microbial killing (Figure 1.3) (Chuntharapai and Kim, 1995; Hartl et al., 2007).
In endothelial cells, CXCR1 and/or CXCR2 knockdown with shRNAs show that
both receptors were critical for CXCL8-mediated endothelial cell proliferation, survival,
migration and invasion, tube formation and angiogenesis, which corroborate previous
studies performed with antibodies against CXCR1/2 and in vivo studies with CXCR2-/-
mice (Addison et al., 2000; Keane et al., 2004a). Interestingly, double knockdown of
CXCR1 and CXCR2 did not show additive effects on endothelial cells, suggesting the
knockout of either receptor is sufficient to alter CXCL8-mediated angiogenesis (Singh et
al., 2011).

Figure 1.3 CXCR1 and CXCR2 mediate neutrophil recruitment during infection.
16
In the presence of a microbial infection, macrophages at the site of infection begin to secrete CXCL8 to
attract CXCR1/2-expressing neutrophils to the site of infection. Since CXCR2 is more sensitive to low
ligand concentrations, CXCR2 is believed to play a more important role at recruiting neutrophils to the site
of infection, whereas CXCR1 mediates oxidative burst and granule release to combat the microbes at the
site of infection.

CXCR2 knockout studies
A number of CXCR2 mice knockout studies have been performed to elucidate the
roles of CXCR2. In general, most of these studies show CXCR2 knockout mice were
overall healthy but exhibit impaired wound healing and angiogenesis, and increased
susceptibility to pathogens and decreased pathogen clearance due to reduced neutrophil
recruitment (Bonnett et al., 2006; Del Rio et al., 2001; Devalaraja et al., 2000; Goncalves
and Appelberg, 2002; Milatovic et al., 2003; Nagarkar et al., 2009; Reutershan et al.,
2006; Schuh et al., 2002). CXCR2 knockout mice also exhibit neurological defects
including decreased spinal cord white matter area and reduced myelin sheath thickness
(Padovani-Claudio et al., 2006). These mice also had enlarged lymph nodes and spleen
due to increased B-cells and neutrophils, suggesting that CXCR2 plays a role in B-cell
and neutrophil expansion and development (Cacalano et al., 1994). CXCR2 is also
involved in neutrophil trafficking from the bone marrow during development (Eash et al.,
2010). Lastly, CXCR2 knockouts were less susceptible to spontaneous tumorigenesis
including melanoma, prostate and renal cancer (Cataisson et al., 2009; Jamieson et al.,
2012; Mestas et al., 2005; O'Hayer et al., 2009; Shen et al., 2006; Singh et al., 2009c).
17
1.3. Inflammatory roles of CXCL8 in diseases
Since CXCL8 is a critical component of inflammation-mediated processes, aberrant
regulation of CXCL8 and its receptors has been implicated in number of inflammatory-
mediated diseases that include cystic fibrosis (CF), chronic obstructive pulmonary
disorder (COPD), asthma, psoriasis, rheumatoid arthritis, and inflammatory bowel
diseases (IBD) (Banks et al., 2003; Bento et al., 2008; Grespan et al., 2008; Ina et al.,
1997; Izzo et al., 1992; Kulke et al., 1998; Pickens et al., 2011; Podolin et al., 2002) It is
also involved in tumorigenesis of various cancers such as lung, colon, prostate,
pancreatic, breast, ovarian, and melanoma (Table 1.2).
1.3.1. Chronic lung diseases
Chronic obstructive pulmonary disorder. COPD is a leading cause of morbidity and
mortality in developed countries characterized with progressive and irreversible airflow
obstruction caused by (1) fibrosis and narrowing of small airways and (2) destruction of
alveolar attachments (emphysema), which are heavily mediated by neutrophils and
lymphocytes (Barnes, 2000, 2004). CXCL8 contributes to the pathogenesis of COPD
through several mechanisms. First, CXCL8 and other chemokines secreted by lung
macrophages orchestrate the trafficking of neutrophils to the lungs in response to external
stimulus (cigarette smoke, air pollutants) (Barnes, 2003; Traves et al., 2004). Second,
CXCL8 also stimulates airway epithelium, causing it to contract and increase its
permeability to inflammatory cells (Reutershan et al., 2006). Third, protease secretion
from the accumulation of neutrophils and other inflammatory cells leads to sustained and
extensive tissue damage (Reutershan et al., 2006; Smit and Lukacs, 2006; Traves et al.,
18
2004). Sputum and bronchoalveolar lavage (BAL) concentrations of CXCL8 are higher
in patients with COPD than healthy volunteers, which correlates with increased
neutrophil accumulation (Aaron et al., 2001; Keatings et al., 1996; Tanino et al., 2002;
Woolhouse et al., 2002; Yamamoto et al., 1997). Additionally, neutralizing CXCL8
antibodies significantly reduced neutrophil chemotatic activity of sputum from patients
with COPD (Beeh et al., 2003) and CXCR2 inhibition with a small-molecule CXCR2
antagonist reduced neutrophillic inflammation in lungs of mice exposed to acute cigarette
smoke, suggesting that CXCL8 plays an important role in lung inflammation that
contributes to the development of COPD (Thatcher et al., 2005) .
Asthma. Asthma is characterized by episodes of reversible airflow obstruction,
bronchial constriction, and lung inflammation induced by allergens (Bousquet et al.,
2000). Neutrophils and eosinophils are increased in the lung epithelium and sputum
during severe asthma exacerbations accompanied by increased expression of ELR+
chemokines and its receptors (Kurashima et al., 1996; Lamblin et al., 1998; Nakagome et
al., 2012; Norzila et al., 2000; Qiu et al., 2007). CXCR1/2 are also expressed on airway
smooth muscle and mediate cell contraction and migration to enhance airway
responsiveness and airway remodeling (bronchoconstriction) that is observed in asthma
(Fujimura et al., 1999; Govindaraju et al., 2006). Lastly, CXCR2 deficient mice
exhibited reduced bronchial hyperresponsiveness and neutrophil recruitment induced by
ozone challenge compared to wild type mice (Johnston et al., 2005)
Cystic Fibrosis. CF is an autosomal recessive genetic disorder caused by genetic
mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that
19
leads to abnormal transport of chloride and sodium ions across epithelium of tissue
organs, such as the lungs and pancreas (Clunes and Boucher, 2007; Riordan et al., 1989).
The most effected organ of CF are the lungs, which exhibit increased mucus buildup and
subsequent cycles of lung infection and neutrophillic inflammation leading to
bronchiectasis and respiratory failure (Jacquot et al., 2008). As with other lung diseases
discussed thus far, the airways of CF patients also have elevated levels of CXCL8 and
other chemotactic cytokines (IL-1, IL-6, TNF) that coordinates the infiltration of
neutrophils (Adib-Conquy et al., 2008; Dean et al., 1993; Konstan and Berger, 1997;
Sagel et al., 2001; Tabary et al., 1998). Antibodies against CXCL8 were able to
significantly inhibit the chemotactic activity of sputum from CF patients (Richman-
Eisenstat et al., 1993). Though CXCR1 and CXCR2 expression in airway smooth
muscles from CF and non-CF patients were about the same, CXCL8 induced greater
contractions in airway smooth muscle of CF patients, which might be due to increased
myosin light chain that contribute to bronchial constriction observed in CF (Govindaraju
et al., 2008). Hartl et al. showed CXCR1, but not CXCR2 promotes bacterial killing.
However, this function is lost in the airways of CF patients. CXCR1 is cleaved by airway
proteases and the fragments of CXCR1 stimulates bronchial epithelial cells to secrete
CXCL8 via the Toll-like receptor 2 (TLR2) (Hartl et al., 2007). CF patients carrying a
particular CXCR1 and CXCR2 haplotype (CXCR1_HA and CXCR2_HA) is also
associated with decreased CXCR1, increased CXCR2 mRNA and protein expression, and
exhibit impaired antibacterial function. These haplotypes were also associated with
decreased lung function in CF patients (Kormann et al., 2012).
20
1.3.2. Cancer
Chemokines for CXCR1/2 such as CXCL1, CXCL5, CXCL7, and CXCL8 as well as
the receptors are secreted and expressed by various cancer cell types (lung, colon, breast,
ovarian, and melanoma) and stimulate cancer cell proliferation and migration in an
autocrine fashion (Acharyya et al., 2012; Erreni et al., 2009; Kim et al., 2001; Sun et al.,
2008; Tang et al., 2008; Wen et al., 2006; Wente et al., 2006; Wu et al., 2012a).
Chemokine expression is also associated with tumor grade and metastatic potential in
human tumors. For example, CXCL8 serum levels were increased in patients with
prostate cancer compared to healthy volunteers, and CXCL8 serum levels were correlated
with the stage of metastasis (Veltri et al., 1999). In pancreatic cancer, CXCL5 expression
was associated with shorter patient survival (25.5 months shorter than patients that did
not express CXCL5) and correlated with clinical stage of the cancer (Li et al., 2011b).
Table 1.2 attempts to summarize the roles and involvement of CXCR1/2 and its ligands
in major cancer.
Cancer cells are also stimulated by other sources of chemokines, mainly derived
from tumor-associated macrophages. The hypoxic and stressed tumor microenvironment
stimulate macrophages to secrete CXCL chemokines, a process mediated by NFκB (Brat
et al., 2005; Holz et al., 2010). Many cancer cells also secrete high levels of CXCR1/2
ligands without expressing the receptors, suggesting that CXCR1/2 ligands are also
involved in the tumor microenvironment in a paracrine fashion (199, 203). Indeed,
CXCR1/2 ligands play a significant role in neutrophil tumor infiltration that facilitates
cancer cell proliferation, invasion, and chemoresistance via increased levels of
21
angiogenic and growth factors produced by these tumor-associated neutrophils (Gregory
and Houghton, 2011; Houghton et al., 2010; Jamieson et al., 2012; Tazzyman et al.,
2009).
CXCR1/2 ligands also stimulate CXCR1/2 expressing endothelial cells and promote
tumor angiogenesis. In order for a tumor to progress beyond 2-3 mm
3
, it must acquire the
capacity to induce angiogenesis (blood vessel formation from a pre-existing blood vessel
network) (Folkman and Hanahan, 1991). The tumor vasculature delivers essential
nutrients and oxygen to the tumor cells that facilitate the uncontrolled growth and
invasion of tumor cells. For example, upon CXCL8 stimulation, endothelial cells begin
the angiogenic process by secreting MMPs to breakdown the ECM and begin to
proliferate and migrate to form capillaries (Li et al., 2003). The involvement of
CXCR1/2 (mainly CXCR2) in tumor angiogenesis and progression is further
demonstrated by several in vivo cancer models that showed depletion of chemokines
and/or CXCR1/2 significantly reduced tumor growth associated with decreased
microvessel density. CXCR2 knockout mice implanted with Lewis lung carcinoma
(LLC) exhibited reduced tumor growth, vascular density, and spontaneous metastases in
orthotopic tumor models compared to wild-type (Keane et al., 2004a). Similar results
were observed in prostate and pancreatic cancer, in which CXCR2 knockout mice had
smaller tumors as well as reduced tumor angiogenesis (Li et al., 2011a; Shen et al., 2006).
Small-molecule inhibition of CXCR2 have also shown promising anticancer effects
in the preclinical setting. Treatment with SCH527123 (CXCR2 antagonist developed by
Schering-Plough) reduced tumor growth and angiogenesis as well as improve sensitivity
22
to oxaliplatin treatment in colon cancer (HCT116) mice xenografts (Ning et al., 2012).
However, Varney et al. showed SCH527123 inhibited colon cancer liver metastases but
had no effects on tumor growth in mice xenografts derived from KM12L4 cell lines
(Varney et al., 2011). Both CXCR2 knockout studies using shRNA and small-molecules
against CXCR2 did not significantly affect cancer cell proliferation in vitro studies,
further supporting that CXCR2 and its ligands play an essential role in the tumor
microenvironment which is present in in vivo studies.
CXCL8 is also up-regulated in response to various anticancer agents and may
contribute to chemoresistance. For example, NFκB-mediated CXCL8 synthesis and
secretion was elevated in prostate cancer cells treated with oxaliplatin or 5-FU (Wilson et
al., 2012; Wilson et al., 2008a). Additionally, the inhibition of CXCR2 with small-
molecule antagonists or shRNA knockdown significantly enhanced sensitivity (increased
cytotoxicity) to oxalipalitin, 5-FU, ansamycin, TRAIL, doxorubicin, paclitaxel, and
lapatinib in prostate, colon, and breast cancer cells (Maxwell et al., 2007; Ning et al.,
2012; Seaton et al., 2009; Sharma et al., 2013; Singh et al., 2013; Wilson et al., 2012;
Wilson et al., 2008a; Wilson et al., 2008b). Taken together, the CXCR1/2 axis facilitates
tumor progression by stimulating tumor cells, as well as critical components of the tumor
microenvironment (Figure 1.4).
23

Figure 1.4 The multiple roles of CXCL chemokines and CXCR1/2 during tumor development.
CXCR2 and CXCL ligands promote tumor growth through several mechanisms. Secretion of CXCL8 by
tumor cells and tumor-associated macrophages stimulate cancer cell proliferation, survival, and
chemoresistance. CXCL8 secretion also mediate neutrophil recruitment to the tumor site and stimulate
neutrophils to secrete growth factors (GF) and matrix metalloproteinase (MMPs) to facilitate cancer cell
migration, invasion, and metastases. Lastly, CXCL8 stimulates CXCR2-expressing endothelial cells that
form blood vessels within the tumor and stimulate tumor angiogenesis.
1.3.3. CXCR2: linking inflammation-mediated diseases to cancer
COPD and lung cancer. The manifestations of lung cancer and COPD are
diametrically opposed. Lung cancer is characterized by uncontrolled cell proliferation,
whereas COPD is characterized by inflammation-mediated destruction of the
extracellular matrix and cell death (Hann and Rudin, 2007; Taraseviciene-Stewart et al.,
2006). Hence, treatment for these two diseases has been separately developed, targeting
different cellular pathways. However, several studies suggest that inflammation may be
an underlying mechanism that contributes to the development of both diseases. Mice
studies with activating K-ras mutation suggest COPD-like airway inflammation promote
24
the progression of lung cancer development in mice. K-ras mutations are found in 30%
of all the lung adenocarcinomas from smokers (Westra et al., 1993). Exposure of mice
with an activating K-ras mutation to aerosolized NTHi lysate (Haemophilus influenza,
commonly found in lower respiratory tract of COPD patients) resulted in
neutrophil/macrophage/CD8 T cell associated COPD-like airway inflammation. These
mice exhibited a 3.2-fold increase in lung surface tumor number (156 ± 9 versus 45 ± 7).
The authors conclude COPD-like airway inflammation promotes lung carcinogenesis in a
background of an activated K-ras allele in airway secretory cells (Moghaddam et al.,
2009). Observational studies have also found that smokers with COPD have a 1.3 to 4.9
fold increased risk of lung cancer compared to smokers without COPD (Shacter and
Weitzman, 2002; Skillrud et al., 1986; Tockman et al., 1987), which suggest that COPD
and lung cancer may be potentially linked. Studies among ex-smokers with COPD
showed concurrent, regular use of inhaled corticosteroids reduced risk of lung cancer by
50%, which suggest that reduced inflammation in COPD patients offers a protective
effect against cancer (Kiri et al., 2009).
Houghton et al. proposed a model that explores the common origins of lung cancer
and COPD. Upon cigarette smoke or pathogen exposure in the lungs, inflammatory cells
are recruited and activated, releasing serine and matrix metalloproteinases (MMPs) and
reactive oxygen species (ROS). Emphysema (a major feature of COPD) occurs when
extracellular matrix destruction and cell death exceeds reparative capacity resulting in
airspace enlargement. To compensate for the loss of alveolar cells, bronchioalveolar stem
cells (BASCs) proliferate to replace damaged cells. However, the over-compensation of
25
BASC proliferation predisposes these cells to become malignant (Houghton et al., 2008).
According to this model, both diseases arise from inflammatory recruitment of
macrophages and neutrophils to the lungs. Hence, developing therapeutics that will
reduce the inflammatory response upon pathogen exposure will diminish episodes of
emphysema as well as prevent the development of cancerous cells. Since CXCR1/2 and
its ligands play an important role in inflammatory response and have been implicated in
both lung cancer and COPD, it may be a potential common molecular pathway that links
these two diseases together.
1.4. CXCR1/2 inhibition
Given the therapeutic potential of CXCR1/2 inhibition and the drugability of these
receptors, several pharmaceutical companies have sought to develop inhibitors of
CXCR1 and CXCR2 within past two decades. Several classes of small-molecule
compounds as well as peptide-based inhibitors selectively inhibit CXCR1/2 receptors
with IC
50
s as low as sub-nanomolar (Table 1.3). In general, many of the early small-
molecule inhibitors were designed to be selective for CXCR2 over CXCR1 (with the
exception of repertaxin). CXCR2-selective inhibitors were sought after for several
reasons. First, since CXCR2 binds to all ELR+ chemokines, CXCR2 inhibition might
provide a wider therapeutic application, especially in pathologies that may predominantly
involve CXCR2-selective ligands. Second, CXCR1 and CXCR2 play a critical role in
the immune system and complete inhibition of both receptors might lead to immune
compromise. Third, it was previously thought that most preclinical disease models (mice
26
and rat) used to assess the efficacy of these compounds only expressed the CXCR2
homologue and not CXCR1.
Several small-molecule antagonists have advanced to clinical trials in various
inflammatory-mediated diseases such as asthma, COPD, and cystic fibrosis. In general,
most of these studies show that CXCR1/2 inhibition is safe and well tolerated with very
few adverse side effects. The first series of small-molecule CXCR2 antagonists were
phenol-containing diarylureas developed by GlaxoSmithKline (White et al., 1998).
Further optimizations to increase potency and reduce clearance resulted in the addition of
sulfonamide substituent adjacent to the phenol group (Jin et al., 2000). SB-656933, a
representative of this class of CXCR2 antagonists, is currently in clinical trials for cystic
fibrosis and COPD (Busch-Petersen et al., 2004). SB656933 is CXCR2 selective with
IC
50
values for CXCL8 inhibition of 5 nM (CXCR1 IC
50
>1µM) (Carpenter et al., 2004).
Recently disclosed clinical results showed that SB656933 reduced ozone-induced sputum
neutrophils by 74% when pretreated with a single dose at 150 mg in healthy patients
(Lazaar et al., 2011). SB656933 also reduced sputum neutrophils by 30% compared to
baseline in CF patients treated with 50mg of SB656933 for 28 days (Moss et al., 2012).
However, no changes in lung function was observed, suggesting that perhaps a longer
treatment duration is required or CXCR2 inhibition alone is not sufficient to enhance
lung function in CF patients.
Isosteric replacements of the urea from early CXCR2 antagonists led to phenol-
containing N, N’-diarylsquaramides. In these compounds, the urea is replaced with 3,4-
diamino-1,2-dioxocyclobutene (squaramide) (Palovich et al., 2001). A compound from
27
this class, SCH527123, is in Phase II clinical trials for COPD. SCH527123 is a very
potent, non-competitive CXCR2 antagonist (CXCR2 K
d
=49ρM; CXCR1 K
d
=3.9 nM)
(Chapman et al., 2007; Gonsiorek et al., 2007). In the clinics, SCH527123 reduced
ozone-induced sputum neutrophil in healthy patients (Holz et al., 2010). It was also able
to reduce sputum neutrophils (36.3% reduction) in asthmatic patients treated with a daily
dose of 150 mg of SCH527123 for 4 weeks. However, it had no changes in lung function
(FEV1) (Nair et al., 2012). In preclinical studies, SCH527123 exhibit anticancer effects
in colon and melanoma cancer mice models by inhibiting tumor growth and microvessel
density (Ning et al., 2012; Singh et al., 2009b).
Reparixin is another class of non-competitive CXCR1 and CXCR2 dual inhibitor
developed from molecular modeling studies with CXCR1. Reparixin is structurally
different from the earlier classes of antagonists. Mutation analysis and molecular
modeling shows Reparixin binds to a pocket in the transmembrane region of CXCR1 and
inhibit CXCL8 induced receptor signaling in intracellular compartments without altering
CXCL8 binding affinity (Bertini et al., 2004; Moriconi et al., 2007). Reparixin potently
inhibited CXCL8-induced human PMN migration (IC
50
= 1 nM) in in vitro studies
(Casilli et al., 2005). In in vivo reperfusion injury/ischemia rat models, reparixin
successfully prevented neutrophil influx and drastically reduced organ/tissue damage
(Souza et al., 2004). Phase I clinical trials show that reparixin is safe and well-tolerated
in patients (Bertini et al., 2012).

28
Table 1.2 CXCL chemokines and CXCR1/2 and cancer
Cancer
Type
Summary of findings
Lung Cancer
(NSCLC)
 IL-1β stimulates more CXC chemokines secretion in A549 (lung adenocarcinoma cell line) than in human tracheobronchial epithelium
cells via CREB and NFκB activation (Sun et al., 2008).
 LLC (lewis lung carcinoma) cells transduced with human IL-1β exhibited increased tumor growth, which was inhibited with CXCR2
antibodies (Saijo et al., 2002).
 CXCL8 stimulates epithelial cell proliferation (A549 and NCI-H292) via EGFR transactivation involving the MAPkinase pathways (Luppi
et al., 2007).
 CXCL8 stimulates H460 and MOR/P (NSCLC cell lines) cell proliferation via CXCR1 and not CXCR2 (Zhu et al., 2004).
 CXCR2-/- mice implanted with LLC primary tumors in heterotopic and orthopic models show reduced tumor growth and vascular density
as well as reduced spontaneous metastases (Keane et al., 2004b).
 Inhibition of CXCR2 with antibodies impeded the progression of premalignant alveolar lesions in mice with KRAS mutations known to
develop lung adenocarcinma (Wislez et al., 2006).
 Inhibition of CXCR2 with AZ10397767 (small-molecule antagonist) reduced neutrophil infiltration in A549 tumor spheroid and primary
tumors in mice (Tazzyman et al., 2011).
 CXCR2 antibodies inhibited SNAIL-mediated tumor burden in orthotopic and heterotopic lung cancer mice models (Yanagawa et al.,
2009).
 Depletion of CXCR2 via shRNA knockdown in a highly metastatic adenocarcinoma cell line with Kras/p53 from mice (344SQ) reduced
tumor invasion and metastasis in in vitro and in vivo orthotopic syngeneic mouse model (Saintigny et al., 2013).

Colorectal
Cancer
 CXCL8 and CXCR2 (but not CXCR1) was upregulated in colorectal tumor samples (n=8) (Erreni et al., 2009).
 CXCL8, CXCR1, and CXCR2 expression was higher in metastatic colon cancer cell lines (KM12C and KM12L4) than in Caco-2 cells.
CXCL8 also induced cell proliferation of colon cancer cells, which was attenuated with neutralizing antibodies to CXCR1/2 or CXCL8 (Li
et al., 2001).
 CXCL1 expression is higher in primary colon adenocarcinoma than in normal colon epithelium (from human samples). Inhibition of
CXCL1 with RNA silencing reduced proliferation and increased apoptosis (Wen et al., 2006).
 Primary colorectal cancer samples expressed CXCL1 and its expression is associated with tumor size and stage, metastasis, and patient
survival; colon cancer cell lines also express CXCR2, and CXCL1 stimulation increased their invasiveness (Ogata et al., 2010).
 Over-expression of CXCL8 via stable transfection in human colon cancer cells (HCT116 and Caco-2) enhanced cell proliferation,
migration, and invasion, and resistance to oxaliplatin. CXCL8 over-expressing cells also formed larger tumors with increased microvessel
density in xenograft models (Ning et al., 2011).
 Single nucleotide polymorphisms (SNPs) on CXCL8, CXCR1, and CXCR2 are associated with colon and rectal cancer risk (Bondurant et
al., 2013).
 Immunodificient mice expressing human CXCL8 on the skin had enhanced human and mouse colon cancer tumor growth, angiogenesis,
29
and metastases to the lung and liver. Conversely, CXCR2 knockout mice exhibited reduced tumor growth and angiogenesis, and increased
necrosis (Lee et al., 2012).
 SCH527123 (CXCR1/2 antagonist) inhibits human colon cancer liver metastases in a mice xenograft model; however, it had no effects on
tumor growth (Varney et al., 2011).
 SCH527123 inhibit colon cancer cell (HCT116 and Caco-2) proliferation, migration, and invasion, and increased apoptosis. SCH also
reduced tumor growth and angiogenesis as well as improve oxaliplatin treatment in mice xenograft studies (Ning et al., 2012).
Breast
Cancer
 Increased copy numbers of CXCL1/2 gene contributed to higher expression of CXCL1/2 in invasive breast tumors. CXCL1/2 participates
in a paracrine loop involving the tumor microenvironment and cancer cells to enhance chemoresistance and metastasis in breast tumors
(Acharyya et al., 2012).
 Thrombin stimulates CXCL1 expression and secretion in tumor and endothelial cells. Antibodies against CXCL1 inhibited thrombin-
induced angiogenesis (endothelial tube fromation). Depletion of CXCL1 via shRNA in 4T1 (mouse breast cancer cell line) reduced tumor
growth, angiogenesis, and metastasis (Caunt et al., 2006).
 CXCL7 and CXCR2 expression are higher in malignant (MCF10CA1a.c11) than in premalignant (MCF10AT) cells. Premalignant cells
transfected with CXCL7 showed increased invasiveness, which was attenuated with CXCL7 antibodies (Tang et al., 2008).
 Activation of the fibroblast growth factor receptor (FGFR) in epithelial breast cancer cells led to downregulation of the TGFβ/SMAD3
pathways in tumor-associated macrophages, which is associated with increased expression of CXCL chemokines. These chemokines also
stimulate breast epithelial cancer cell invasiveness which is inhibited by SB225002 (CXCR2 inhibitor) (Bohrer and Schwertfeger, 2012).
 Mesenchymal stem cells produce CXCL1 and CXCL5 and recruit mammary cancer cells, facilitating bone metastasis. This process is
inhibited with antibodies against CXCL1, CXCL5, andCXCR2 and SB265610 (CXCR2 antagonist) (Halpern et al., 2011).
 CXCR2 knockdown via shRNA in metastatic mammary tumor cell lines derived from mice (C166, 4T1) reduced cell invasion, but did not
alter cell proliferation. Implantation of these cells into orthotopic mice model showed CXCR2 knockdown exhibited reduced spontaneous
lung metastasis (40%). These cells also enhanced cytotoxicity of doxorubicin and paclitaxel in vitro and in vivo mice models (Nannuru et
al., 2011; Sharma et al., 2013).
 CXCR1 blockade with CXCR1 antibodies or reparixin (CXCR1 inhibitor) depleted breast cancer stem cells in HCC1954 and MDA-MB-
453 cell lines. Reparaxin also retard tumor growth and metastasis in xenograft studies (Ginestier et al., 2010).
 CXCL8 induced activation of EGFR/HER2 signaling pathways mediated by SRC, PI3K, and MEK in breast cancer stem cells from
metastatic and invasive human breast cancers derived from human patients. CXCL8 also enhanced colony formation of these cells.
Inhibition of CXCR1/2 with SCH563705 inhibited colony formation and improved the efficacy of lapatinib (tyrosine kinase inhibitor)
(Singh et al., 2013)
 CXCL8 levels are increased in breast cancer patients compared to healthy volunteers and its level is associated with the stage of the
disease (Benoy et al., 2004).

Prostate
Cancer
 Oxaliplatin increased NFκB activity and the transcription of CXCL8, CXCL1, and CXCR2. CXCR2 antagonist (AZ10397767) inhibited
oxaliplatin-induced NFκB activity and increased oxaliplatin-induced apoptosis in androgen-independent prostate cancer resistance to
chemotherapy (Wilson et al., 2008a).
 5-FU increased CXCL8 seceretion and CXCR1 and CXCR2 gene expression in metastatic prostate cancer (PC3) cells. AZ10397767
increased 5-FU cytotoxicity and apoptosis (Wilson et al., 2012).
30
 TRAMP (tumor adenocarcinoma of the mouse prostate)/CXCR2-/- mice were smaller than CXCR2 wild-type mice and had reduced
angiogenesis (Shen et al., 2006).
 CXCL1 and CXCL8 increased PC3 invasion and adhesion to laminin, while CXCR2 antibodies inhibited CXCL8-induced cell invasion
(Reiland et al., 1999).
 PC3 express CXCR1, CXCR2, and CXCL8. High-producing and low-producing CXCL8 clones of PC3 cells were isolated and injected
into the prostate of nude mice. Tumors with high-producing CXCL8 showed increased growth, vascularization, and lymph node
metastasis compared to low-producing CXCL8 tumors (Kim et al., 2001).
 Hypoxia induced CXCL8, CXCR1, and CXCR2 expression in PC3 cells via HIF-1 and NFκB transcriptional activity. CXCR1/2 siRNA
enhanced etoposide-induced cell death in hypoxic PC3 cells (Maxwell et al., 2007).
 CXCR2 inhibition with AZ10397767 and NFκB inhibition with BAY11-7082 enhanced ansamycin cytotoxicity in PC3 cells and not
DU145 cells (Seaton et al., 2009).
 CXCL8 upregulates cFLIP (caspase 8 inhibitor) expression and pretreatment with AZ10397767 inhibited CXCL8-induced cFLIP
expression in LnCAP and PC3 cells. It also sensitized PC3 cells to TRAIL treatment (TRAIL induce CXCL8 expression in PC3 and
LnCAP cells) (Wilson et al., 2008b).
 PTEN repression via siRNA and shRNA increased CXCL8, CXCR1 and CXCR2 expression in PCa cells. CXCL8 depletion (siRNA)
decreased cell viability in PTEN deficient cells through G1 cell cycle arrest and apoptosis (Maxwell et al., 2012).
 CXCL8 serum levels is correlated with stage of metastatic prostate cancer and increased compared to healthy volunteers (Veltri et al.,
1999).
 CXCL8 induce cyclin D1 translation via Akt and activation of translational components in PC3 and DU145 cells (MacManus et al., 2007).
 PC-3M-LN4 cells (highly metastatic) overexpress CXCL8 compared to PC-3P cells (poorly metastatic). Knockdown of PC-3M-LN4 cells
with antisense CXCL8 cDNA reduced MMP-9 expression, collagenase activity, and invasion in vitro and in vivo orthotopic model.
Conversely, upregulation of CXCL8 in PC-3P cells had the opposite effects (Inoue et al., 2000).
Ovarian
Cancer
 CXCR2 shRNA knockdown in ovarian cancer cells (T29Gro-1, T29H, and SKOV3) inhibited tumor growth and arrested cells in G0/G1
phase by regulating cell cycle modulators. CXCR2 induced apoptosis and angiogenesis. CXCR2 expression is correlated with poor
overall survival for ovarian cancer (Yang et al., 2010).
 Matrix metalloprotease-1 (MMP-1) activation of protease-activated receptor-1 (PAR1) induced CXCL8, CXCL1, and CCL1 secretion and
stimulates endothelial cell proliferation, tube formation, and migration. These activities were attenuated with CXCR1/2 inhibition with
X1/2pal-i3 (cell-penetrating pepducin that targets third intracellular loop of CXCR1/2), which also reduced tumor growth in mice and
MMP1-mediated angiogenesis (Agarwal et al., 2010).
 CXCL8 suppress TRAIL-medaited OVCAR3 apoptosis via downregulation of death receptors (Abdollahi et al., 2003).
 CXCL1 enhanced epithelial ovarian cancer cell (SKOV3 and OVCAR-3) growth and transactivates EGFR via EGF release that involves
the ERK1/2 signaling pathway (Bolitho et al., 2010).
 Ovarian cancer patients with A/A (0%) or A/T(19%) genotype for CXCL8T-251A gene polymorphism are less responsive to
cyclophosphamide and bevacizumab treatment than T/T (50%) genotype. The A/A genotype is associated with increased CXCL8
production (Schultheis et al., 2008).
 Paclitaxel induced CXCL8 promoter activation in ovarian cancer through the activation of both AP-1 and NFκB. CXCL8 inhibition with
antibodies stimulated tumor growth via recruitment of neutrophils to tumor site (Lee et al., 2000; Lee et al., 1998; Lee et al., 1996).
31
Melanoma
 Low tumorigenecity melanoma cell line (A375P) overexpressing CXCR1 or CXCR2 had enhanced in vivo tumor growth associated with
increased microvessel density and reduced apoptosis (Singh et al., 2009a).
 CXCL8 serum level was associated with patient response to dacarbazine, cisplatin, and vindesine with or without DVP/IFN-2/IL-2
chemotherapy/immunochemotherapy (Brennecke et al., 2005).
 CXCR2 (but not CXCR1) and CXCL8 correlated with melanoma tumor grade (Varney et al., 2006).
 Melanoma cell lines secrete CXCL8 and express CXCR1 and CXCR2. CXCL8 stimulation of melanoma cells enhanced cell proliferation,
migration, and invasion, which was reversed with inhibition of CXCR2 (but not CXCR1) with neutralizing antibodies (Gabellini et al.,
2009).
 CXCL8 overexpression in CXCL8 low-expressing melanoma cells (A375P) or CXCL8 knockdown (via antisense) in CXCL8 high-
expressing cells (A375SM) showed that CXCL8 regulate cell proliferation, migration, invasion, and colony formation. CXCL8
overexpression is associated with enhanced tumor growth and lung metastasis in vivo (Wu et al., 2012a).
 CXCR1 and/or CXCR2 knockdown of A375-SM cells via shRNA inhibited cell proliferation, migration, and invasion in vitro and reduced
tumor growth and mircovessel density, and increased apoptosis in nude mice compared to control cells. Similar in vitro and in vivo results
were obtained with CXCR2/CXCR2 inhibition with small molecule antagonists, SCH-479833 and SCH-527123 (Singh et al., 2009b;
Singh et al., 2010).
Pancreatic
cancer
 Capan-1cells express CXCL1, CXCL8, and CXCR. CXCL1 and CXCL8 antibodies inhibit Capan-1 growth (Lee et al., 2011).
 BxPC3 cells secrete CXCL3, CXCL5, CXCL8 (low expression in Panc-1 and MiaPaca). The supernatant from BxPC3 cells induced
neovascularization in corneal micropocket assay, which was impaired in the presence of CXCR2 antibodies. CXCL5 and CXCL8 are
expressed in pancreatic cancer tissue samples (Wente et al., 2006).
 ELR+ chemokines are higher exocrine pancreatic secretions from pancreatic cancer patients than in healthy individuals. Pancreatic cancer
cell lines (BxPC3, Colo-357, and Panc-28) also express higher ELR+ chemokines (no CXCR2 expression) than normal pancreatic ductal
epithelial cell line. Supernatants from pancreatic cancer cell line stimulated HUVEC tube formation, which was attenuated with CXCR2
antibodies. These results were replicated in pancreatic cancer orthotopic nude mice model (Matsuo et al., 2009b).
 CXCR2 knockout mice with orthotopic and heterotopic pancreatic cancer tumors had impaired mobilization of bone marrow derived
endothelial progenitor cells assocaited with reduced tumor angiogenesis and tumor growth (Li et al., 2011a).
 K-Ras4B
G12V
transformed human pancreatic duct epithelial cells show enhanced secretion of CXC chemokines and VEGF via MEK1/2
and cJun pathways. When these cells were cocultured with HUVECs, it enhanced HUVEC tube formation and invasiveness which was
inhibited with CXCR2 antagonist, SB225002, or VEGF antibody (Matsuo et al., 2009a).
 CXCL5 expression correlated with clinical stage and shorter patient survival (25.5 months shorter than patients that did not express
CXCL5) in pancreatic cancer. CXCL5 knockdown with siRNA inhibited tumor growth in pancreatic cancer xenograft in nude mice (Li et
al., 2011b).

32
Table 1.3 Different classes of CXCR2 inhibitors
CXCR2 Inhibitors Activities of CXCR2 inhibitors

(R)-Ketoprofen

Repertaxin

DF2162

DF2156A

Analogue 1

Analogue 2
 (R)-Ketoprofen 1 inhibited CXCL8-mediated PMN migration (IC
50
= 34 nM) and interacts with the TM2 and TM7 region
of CXCR1 (Allegretti et al., 2005).
 Repertaxin inhibited human PMN migration induced by CXCL8 (IC
50
= 1 nM) and CXCL1 (IC
50
= 400 nM) (Bertini et
al., 2004).
 Repertaxin inhibited CXCL8-induced migration in CXCR1-transfected (IC
50
= 1 nM) and CXCR2-transfected (IC
50
= 100
nM) cells (Bertini et al., 2004).
 Repertaxin reduced lung neutrophil recruitment and vascular permeability in LPS-induced acute lung injury model (50%
inhibition at 15mg/kg) (Zarbock et al., 2008).
 Repertaxin reduced acute inflammation and autonomic dysreflexia in a model of spinal cord injury in rats (Marsh and
Flemming, 2011).
 Repertaxin inhibited CXCL8-induced PMN adhesion to fibrinogen, CD11b upregulation, and neutrophil activation
(granule release) (Casilli et al., 2005).
 Repertaxin inhibited CXCL8-induced T-cell and NK cell migration (Casilli et al., 2005).
 Repertaxin reduced granulocyte graft infiltration and serum creatinine post syngeneic transplantation in Lewis rats
(Cugini et al., 2005).
 Repertaxin inhibited neutrophil recruitment into reperfused livers and reduced myeloperoxidase content in a rat model of
liver postischaemia reperfusion injury (Bertini et al., 2004).
 Repertaxin reduced levels of hypertension-related mediators associated with reduced blood pressure in spontaneously
hypertensive rat models (Kim et al., 2011).
 Repertaxin reduced oligodendrocyte apoptosis and neutrophil migration to site of injury post traumatic spinal cord injury
in rats (Gorio et al., 2007).
 Repertaxin inhibited CXCL8- or CINC-1-induced migration and calcium flux in human or rat neutrophils(Souza et al.,
2004).
 Repertaxin reduced neutrophil influx and vascular permeability in a model of mild and severe I/R injury in rats (Souza et
al., 2004).
 DF2162 is a noncompetitive allosteric inhibitor that is stabilized by a direct ionic bond interaction with Lys99 on CXCR1
and Asp293 on CXCR2 (Bertini et al., 2012).
 DF2162 prevented chemotaxis of rat and human neutrophils induced by chemokines acting on CXCR1/2. It is orally
bioavailable and inhibited neutrophil influx and production of inflammatory factors in an arthritis rat model (Barsante et
al., 2008).
 DF2162 inhibited chemotaxis in CXCR1 and CXCR2 over-expressing transfectants and leukocytes (Bertini et al., 2012).
 DF2162 inhibited mice sponge-induced angiogenesis by reducing leukocyte influx and vessel formation (Bertini et al.,
2012).
 DF2162 inhibited CXCL8-mediated HUVEC proliferation, migration and tube formation (Bertini et al., 2012).
33
Analogue 3

 DF2162 decreased PMN influx in a rat model of ischemia and reperfusion injury (Bertini et al., 2012).
 DF2162 reduced airway neutrophil transmigration and increased of neutrophils in lung parenchyma in a bleomycin-
induced pulmonary fibrosis mice model (Russo et al., 2009).
 DF2156A is a dual inhibitor of CXCR1 and CXCR2. It inhibited human and rat neutrophil migration in response to
CXCL1 and CXCL8 (Garau et al., 2006).
 DF2156A improved in vivo half-life compared to repertaxin (Garau et al., 2006).
 DF2156A decreased cerebral artery PMN infiltrate and improved neurological function in cerebral ischemia/reperfusion
rat model(Garau et al., 2006)
 Analogue 1-3 inhibit CXCL1- and CXCL8-mediated human PMN migration with IC50s less than 10 nM(Moriconi et al.,
2007)

SCH527123

SCH563705
SCH479833
 SCH527123 inhibit CXCL8 binding to CXCR1 (IC
50
=36 nM) and CXCR2 (IC
50
=2.6 nM) and inhibit neutrophil
chemotaxis to CXCL8 (IC
50
=16 nM) and CXCL1 (IC
50
<1 nM) (Dwyer et al., 2006).
 Optimization to improve potency of SCH527123 were performed and reported (Aki et al., 2009; Biju et al., 2009a; Liu et
al., 2009; Yu et al., 2008).
 SCH527123 bind to CXCR2 receptors of mice (K
d
=0.2 nM), rat (K
d
=0.02 nM), and monkey (K
d
=0.08 nM) (Chapman et
al., 2007).
 SCH527123 exhibit less affinity for monkey CXCR1 (5-fold decreased) and >100 fold less potent in CXCR1-mediated
chemotaxis (Chapman et al., 2007).
 SCH527123 blocked LPS induced pulmonary neutrophils (ED
50
-1.2-1.8mg/kg) in mice and rat (Chapman et al., 2007).
 SCH527123 bind to CXCR1 (K
d
=3.9 nM) and CXCR2 (K
d
=0.049 nM) in CXCR1/2-overexpressing cell lines and
inhibited CXCL1 and CXCL8-mediated neutrophil chemotaxis and myeloperoxidase release (Gonsiorek et al., 2007).
 SCH527123 reduces sputum neutrophils in patients with severe asthma but had no changes in FEV1, sputum
myeloperoxidase, CXCL8 or elastase (Nair et al., 2012).
 SCH527123 reduced ozone induced sputum neutrophil in healthy volunteers. Treatment was safe and well-tolerated (4
day, 50mg once daily dose) (Holz et al., 2010).
 SCH527123 reduced LPS-induced sputum neutrophil influx (79% inhibition) from healthy volunteers (Aul et al., 2012).
 SCH527123/SCH479833 inhibited colon cancer metastasis, reduced tumor neovascularization, and increased tumor cell
apoptosis in mice model (Varney et al., 2011).
 SCH527123/SCH479833 inhibit melanoma cell proliferation, chemotaxis, and invasivon in vitro.
SCH527123/SCH479833 reduced tumor growth associated with decreased tumor cell proliferation and microvessel
density in in vivo mice model of melanoma (Singh et al., 2009b).
 SCH563705 exhibit potent inhibitory activities against CXCL8 binding to CXCR2 (K
i
= 1 nM) and CXCR1 (K
i
= 3 nM)
(Chao et al., 2007).
 SCH563705 inhibit CXCL1 and CXCL8 induced human PMN migration (CXCR2 IC
50
=0.5 nM, CXCR1 IC
50
=37 nM)
(Chao et al., 2007).
34
 SCH563705 reduced inflammation and bone and cartilage degradation in a model of anti-collagen antibody-induced
arthritis in mice (Min et al., 2010).

N-(3-bromo-4-cyano-2-
hydroxyphenyl)-N-(2-
bromophenyl)urea

SB332235

SB225002

SB265610

SB656933

SB455821 (undisclosed structure)
SB-517785-M (undisclosed structure)

 N-(3-bromo-4-cyano-2-hydroxyphenyl)-N-(2-bromophenyl)urea is a competitive CXCR2 inhibitor. It inhibited human
PMN chemotaxis mediated by CXCL1 (IC
50
=14 nM) and CXCL8 (IC
50
=35 nM) and inhibit CXCL8-mediated
neutroprenia in rabbit models (Widdowson et al., 2004).
 SB332235 inhibit human CXCL8 binding to rabbit CXCR2 (IC
50
=40.5 nM) and CXCL8-induced calcium mobilization
(IC
50
=7.7 nM). It has less affinity for CXCR1 (IC
50
>1000 nM) and less active in CXCR1-mediated calcium mobilization
(IC
50
=2200 nM). SB332235 inhibit human CXCL8-induced chemotaxis of rabbit neutrophils (IC
50
=0.75 nM) (Podolin et
al., 2002).
 SB332235 was optimized to improve pharmacokinetics (Jin et al., 2004).
 SB332235 blocked T-cell entry when rat hippocampus was injected with amyloid β (Liu et al., 2010).
 SB225002 inhibit CXCL8 binding to CXCR2 (IC
50
=22 nM) and binding to CXCR1 with IC
50
>150 fold higher than
CXCR2. SB225002 inhibit CXCL1 and CXCL8-induced chemotaxis in rabbit and human neutrophils (White et al.,
1998).
 SB225002 reduced alveolar neutrophil and exudate macropage influx in mice infected with S. pneumoniae (Herbold et
al., 2010).
 SB225002 inhibit CXCL8 binding to CXCR2 (IC
50
=9.9 nM) and CXCL1 binding to CXCR2 (IC
50
=87.9 nM) (Catusse et
al., 2003).
 The N-terminus of CXCR2 is important for SB225002 binidng as well as other amino acids disseminated throughout
receptor and not necessarily overlapping with agonist binidng (Catusse et al., 2003).
 In a mice model of hepatic ischemia and reperfusion, post treatment with SB225002 increased hepatocyte proliferation
and regeneration, similar to CXCR2-/- mice (Kuboki et al., 2008).
 Optimization and other analogues of SB225002 were performed and reported (Bakshi et al., 2009).
 SB225002 exhibit antinociceptive effects in several mouse models of pain (spontaneous nociception) (Manjavachi et al.,
2010).
 SB225002 enhanced the activities of agonists for the DOP opiod receptor acting in an allosteric fashion (Parenty et al.,
2008).
 SB225002 reduced tumor progression in a mouse model of pancreatic cancer associated with reduced angiogenesis and
improved survival (Ijichi et al., 2011).
 SB265610 reduced superoxide accumulation and lipid peroxidation in lungs, and preserve alveolar development in
hypoxic newborn rats (exposed to 60% oxygen) (Liao et al., 2006).
 SB265610 inhibit BAL neutrophil influx and myeloperoxidase accumulation in the lungs in hypoxia-induced newborn rat
lung injury model (Auten et al., 2001).
 SB265610 inhibit rat neutrophil calcium mobilization (IC
50
=3.7 nM) and chemotaxis (IC
50
=70 nM) to CINC-1 (Auten et
al., 2001).
 SB265610 acts as allosteric, inverse agonist. SB265610 reduced maximal [125I]-CXCL8 binding without affecting its
K
d
. It also reduced agonist-induced (CXCL1 and CXCL8) CXCR2 activation and basal [35S]-GTPγS binding (Bradley
35
et al., 2009).
 SB656933 reduced LPS-induced sputum neutrophil influx (52% inhibition) from healthy volunteers (Aul et al., 2012).
 SB656933 inhibit neutrophil CD11b upregulation (IC
50
=261 nM) and shape change (associated with chemotaxis,
IC
50
=311 nM). Neutrophils collected from COPD patients (Nicholson et al., 2007).
 SB656933 inhibit CXCL1-induced CD11b expression on peripheral blood neutrophils (70% inhibition) and reduced
sputum neutrophils which is correlated with reduced myeloperoxidase concentrations in ozone-induced airway
inflammation in healthy patients. SB656933 was safe and well-tolerated at single doses as high as 1100mg (Lazaar et al.,
2011).
 Cystic fibrosis patients receiving 50mg of SB656933 for 28 days were well tolerated (common adverse event was
headaches) and had reduced sputum neutrophils (30% reduction) and elastase (26% reduction) compared to baseline.
Patients had increased blood levels of fibrinogen, C-reactive protein (CRP), and CXCL8 compared to placebo. No
changes in lung function was observed (NCT00903201)(Moss et al., 2012).
 SB455821 inhibit MIP-2-induced neutrophil migration in in vitro (IC
50
~20 nM) and in in vivo mice models (Matzer et al.,
2004).
 SB-517785-M reduced Angiotensin II-induced neutrophil and mononuclear cell recruitment, reduced arteriolar
mononuclear leukocyte adhesion, and decreased levels of MIP-1 and RANTES (Abu Nabah et al., 2007).
AZD8309 (undisclosed structure,
Pyrimidine-based)

thiazolopyrimidine

thiazolo[4,5-d]pyrimidine-2(3H)-one
 AZD8309 reduced leukocyte count (48% inhibition) post nasal LPS challenge in healthy volunteers. No adverse effects
were detected from 3 days of dosing (Virtala et al., 2012).
 Thiazolopyrimidine inhibit CXCL8 binding to CXCR2 (IC
50
=14 nM) and calcium mobilization (IC
50
=40 nM) (Baxter et
al., 2006).
 Thiazolo[4,5-d]pyrimidine-2(3H)-one inhibit CXCL8 binding to CXCR2 (IC
50
=1-60 nM). It showed improved potency
and oral bioavailability of thiazolopyrimidine (Walters et al., 2008).
 AZ10397767 increased cytotoxicity of geldanamycin and 17-AAG (HSP90 inhibitors) in PC3 but not DU145 prostate
cancer cells (Seaton et al., 2009).
 AZ10397767 reduced neutrophil infiltration into A549 (NSCLC) spheroids and A549 tumor xenograft models in mice.
Treatment did not reduce microvascular density (Tazzyman et al., 2011).
 Optimization studies to improve potency of AZ10397767 were performed and reported (Hunt et al., 2007).

36

AZ10397767

3-Arylamino-2H-1,2,4-
benzothiadiazin-5-ol 1,1-dioxides

Compound 6i

Compound 6j
 3-Arylamino-2H-1,2,4-benzothiadiazin-5-ol 1,1-dioxides inhibit CXCL8 binding to CXCR2 with IC
50
s as low as 30 nM
and CXCR1 (IC
50
=3.2µM) and inhibit FLLPR CXCR2 calcium assay (IC
50
~300-600 nM) (Wang et al., 2007).
 Compound 6i – inhibit CXCL8 binding to CXCR1 and CXCR2 with similar IC
50
’s (~20 nM) (Nie et al., 2006).
 Compound 6j – inhibit CXCL8 binding to CXCR1 (6.2µM) and CXCR2 (IC
50
=30 nM) (Nie et al., 2006).

3,4-Diamino-1,2,5-thiadiazole
 3,4-Diamino-1,2,5-thiadiazole inhibit CXCL8 binding to CXCR2 (IC
50
=13-126 nM) and CXCR1 (IC
50
=44 nM-10µM)
(Biju et al., 2008; Biju et al., 2009b).

6-amino-4-oxo-1,3-diphenyl-2-
 6-amino-4-oxo-1,3-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonyl inhibit CXCL8 induced PMN migration
(IC
50
=0.02 nM) (Cesarini et al., 2009).

37
thioxo-1,2,3,4-tetrahydropyrimidine-
5-carbonyl

Carboxylic acid bioisosteres
acylsulfonamides, acylsulfamides,
and sulfonylureas
 Carboxylic acid bioisosteres acylsulfonamides, acylsulfamides, and sulfonylureas inhibit CXCL8 binding to CXCR2
(IC
50
=50 nM) and CXCL8-induced calcium mobilization (IC
50
=5 nM) (Winters et al., 2008).
 Inhibit rabbit neutrophil chemotaxis (IC
50
=700 nM) (Winters et al., 2008).
 Inhibit hyperoxia induced neutrophil (BAL) accumulation in newborn rats (Winters et al., 2008).

 Inhibit CXCL8 binding to CXCR2 (IC
50
=28 nM) and calcium mobilization (IC
50
=48 nM), and exhibit good
bioavailability (Baxter et al., 2003).

Compound 1
Imidazolylpyrimidine
 Compound 1 binds to CXCR2 in transmembrane helices 3, 5, and 6 (de Kruijf et al., 2011).
 Compound 1 blocked CXCL8 binding to CXCR2 with K
i
=60 nM (Ho et al., 2006).
CXCL8(3–73)K11R  Blocked CXCL8 binding to human neutrophils (IC
50
=1.8pmol) and CXCL1 binding with less potency (Li and Gordon,
2001).

6-chloronicotinamide N-oxides
 6-chloronicotinamide N-oxides (Compound 4a) inhibit CXCL8-induced human neutrophil chemotaxis (IC
50
=1.3-2.3 nM)
(Cutshall et al., 2001)
 Inhibit CXCL8 binding to CXCR1 and CXCR2 with similar IC
50
’s (~1µM) (Cutshall et al., 2001).
 Well-tolerated in mice and stable to rat liver microsomes (Cutshall et al., 2001).
38

N-Pyrazolyl-N’-
alkyl/benzyl/phenylureas:
 N-Pyrazolyl-N’-alkyl/benzyl/phenylureas inhibit human neutrophil migration to CXCL8 (IC
50
=10 nM) (Bruno et al.,
2007).

CXCL8(3-72)K11R/G31P (G31P)
CXCL8 peptide
 G31P was effective at 10ng/mL in vitro. G31P inhibit both CXCR1- and CXCR2-mediated neutrophil migration and
calcium mobilization. G31P blocked neutrophil infiltration in guinea pig model of airway endotoxemia (Zhao et al.,
2009).
 G31P protect against ischemia and reperfusion injury in rats (Zhao et al., 2010).
 G31P had no agonist or chemotactic activity and antagonized the binidng of CXCR1 and CXCR2 antibodies to CXCR1/2
(Li et al., 2002).
 Other analogues and variations of this peptide was also generated (Zhao et al., 2007).
 G31P treatement prior to E.coli and LPS challenge in guinea pigs reduced pulmonary neutrophil recruitment (85%
inhibition) (Gordon et al., 2005).
39
CHAPTER 2: DESIGN OF CXCR2 FUNCTIONAL ASSAYS USED TO IDENTIFY
NOVEL INHIBITORS
In this chapter, the rationale and optimization of CXCR2 assays used to screen for
CXCR2 inhibitors and the methods of each technique used to characterize these
compounds will be presented in this chapter. All reagents and materials will also be
listed at the end of the chapter. Data obtained from these assays and methods will be
presented in the subsequent chapters.
2.1. CXCR2 binding assays
The competitive ligand binding assay is the most widely used assay to screen for
small-molecule compounds for GPCRs. This assay detects compounds that inhibit
natural ligand binding to the receptor by measuring the amount of ligand displaced from
the receptor. CXCL8 is usually labeled with a radioisotope (
I125
CXCL8) or
nonradioactive lanthanides such as europium (Eu-CXCL8) to measure natural ligand
displacement in the presence of compounds (Figure 2.1). Though this seems to be a
straightforward assay, there are several factors that may account for assay differences
across laboratories as well as functional implications of identified compounds.
40

Figure 2.1 Competitive CXCR2 binding assay schematic.
In the presence of a competitive CXCR2 inhibitor, Eu-CXCL8 binding is displaced.

Since CXCR2 is a membrane bound protein and cannot be isolated; membranes of
cells expressing CXCR2 are commonly used. However the integrity and structure of the
receptor in these membrane preparations may be compromised and may not represent
CXCR2 in intact cells. Thus, it was suggested that intact cells should be used for this
assay; however, this is also complicated with receptor activation (internalization) that
may alter receptor surface expression.
Many of previously reported CXCR2 inhibitors behave as allosteric antagonists,
rather than competitive inhibitors as previously thought. Using site-directed mutagenesis
strategies, Salchow et al. demonstrated that SB265610 and SCH527123 both bind to
CXCR2 at a common, allosteric, intracellular site, in close proximity to G-protein
coupling (Salchow et al., 2010). Using similar techniques, Catusse et al., previously
showed the N-terminus and second extracellular loop of CXCR2 is critical for ligand
binding. Most importantly, these studies showed amino acids critical for activation were
not involved in binding (Catusse et al., 2003). This offers several insights to developing
41
CXCR2 inhibitors. First, since the activation site and the binding site are distinct,
inhibitors designed to specifically target either one of these sites is sufficient to partially
or completely inhibit receptor activity. Second, conventional ligand competition assays
may not capture allosteric inhibitors that do not alter ligand binding. Thus, using
functional assay to perform initial screens may improve the rate of identification of
potential new compounds. Third, multiple sites for receptor inhibition explains the
structurally diverse, though limited, set of CXCR2 inhibitors that have been reported thus
far. This further suggests there may be many more structural classes of CXCR2
inhibitors yet to be discovered.
This shortcoming can be overcome with direct binding assays in which the
compound of interest is radiolabeled. However, direct binding assays are very costly and
time-consuming and not suitable for high-throughput screening endeavors. For our
studies purposes and given the drawbacks of conventional GPCR binding assays, we
designed several CXCR2-specific functional assays to identify and characterize novel
CXCR2 inhibitors.
2.2. CXCR2 Functional Assays
Recent technological advances have miniaturized functional assays into 96- and 384-
well plate formats, making it even more suitable for large screening projects. Many
biotechnology companies have realized the potential of functional GPCR assays and have
capitalized on it by developing several technologies to detect receptor activation.
42
2.2.1. β-arrestin1/2 recruitment and rearrangement
β-arrestin1/2 recruitment. Since the binding of CXCL8 to CXCR2 triggers
desensitization, a process mediated by the recruitment of β-arrestin1/2 to the activated
receptor (Barlic et al., 1999; Raghuwanshi et al., 2012; Richardson et al., 2003; Yang et
al., 1999), receptor activation may be detected by monitoring the interaction between β-
arrestin1/2 and CXCR2 (Figure 2.2). The association of β-arrestin1/2 and receptor can be
detected using immunofluorescence and co-immunopercipitation, however, these
methods are time consuming and not very quantitative. Several companies have
developed several technologies to measure to GPCR and β-arrestin1/2 association using
live cells that can be performed in high-throughput formats. For the identification of
CXCR2 inhibitors in our studies, we employed the Tango assay from Invitrogen.
Tango CXCR2-bla U2OS cells were genetically modified to stably over-express
CXCR2 linked to a TEV protease site and a GAL4-VP16 transcription factor (Hanson et
al., 2009). These cells also stably express a β-arrestin-2/TEV protease fusion protein and
the β-lactamase reporter gene. Upon CXCL8 binding and CXCR2 activation, the β-
arrestin-2/TEV fusion protein is recruited to the receptor and cleaves the peptide linker
that links CXCR2 to the GAL4-VP16 transcription factor. GAL4-VP16 now can enter
the nucleus and promote the transcription of the β-lactamase gene (Figure 2.2A). β-
lactamase activity is detected using a FRET-based fluorescence assay with CCF4-AM, a
β-lactamase FRET substrate. CCF4-AM is cleaved in the presence of β--lactamase. The
cleaved substrate excites at 409 nm and emit at 460 nm. In the absence of β-lactamase,
CCF4-AM will not be cleaved and excite at 409 nm and emit at 540 nm (Figure 2.2B).
43
Thus, CXCL8 activation of CXCR2 is directly correlated with the amount of cleaved β-
lactamase substrate.

Figure 2.2 Schematic of the Tango assay.
44
(A) Tango CXCR2-bla U2OS cells from Invitrogen are genetically modified to stably over-express CXCR2
linked to a TEV protease site and a GAL4-VP16 transcription factor. These cells also stably express a β-
arrestin-2/TEV protease fusion protein and the β-lactamase reporter gene. Upon CXCL8 binding and
CXCR2 activation, the β-arrestin-2/TEV protease fusion protein is recruited to the receptor and cleaves the
GAL4-VP16 transcription factor. GAL4-VP16 enters the nucleus and transcribes the β-lactamase gene. (B)
β-lactamase activity is detected using a FRET-based fluorescence assay with CCF4-AM (β-lactamase
FRET substrate). CCF4-AM is cleaved in the presence ofβ-lactamase. The cleaved substrate excites at 409
nm and emits at 460 nm. In the absence of β-lactamase, uncleaved CCF4-AM excites at 409 nm and emits
at 540 nm. Thus, CXCL8 activation of CXCR2 is directly correlated with the amount of cleaved β-
lactamase substrate.

Though the Tango technology has proven to be a robust and high throughput assay,
there are a few limitations. For example, any compound that may inhibit the readout
process of the assay (transcription of β-lactamase) and β-lactamase activity may produce
false positives. Thus, it is important to develop counter screens and secondary assays to
determine selectivity of these compounds and identify potential false positives. For our
studies, we also developed a CXCR4 Tango assay to counter screen and detect selectivity
amongst different chemokine receptors. CXCR4 is another chemokine receptor that
belongs to the same class as CXCR2 and binds to CXCL12 (also known as SDF-1α)
(Ganju et al., 1998). In Figure 2.3A, CXCR2 antagonist, SB265610 dose-dependently
reduced CXCL8-mediated β-arrestin-2/CXCR2 coupling in the Tango assay. Similarly, a
CXCR4 antagonist (AMD3100) inhibited SDF-1-mediated β-arrestin-2/CXCR4 coupling
(Figure 2.3B)
45
A. B.
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5
0
1
2
3
C X C L 8 lo g [n M ]
R a tio (4 6 0 /5 4 0 )
V e h ic le
0 .1  M
1  M
1 0  M

0 .0 0 .5 1 .0 1 .5 2 .0 2 .5
0
2
4
6
V e h ic le
0 .0 1  M
0 .1  M
1  M
S D F -1 lo g [n M ]
R a tio (4 6 0 /5 4 0 )

Figure 2.3 CXCL8/SDF-1 activates CXCR2/CXCR4 in Tango assay.
(A) CXCL8 induced β-arrestin-2 recruitment in CXCR2 Tango assay (vehicle). Pretreatment with a
CXCR2 antagonist (SB265610) at 10, 1, and 0.1 μM for 30 minutes prior to CXCL8 stimulation reduced β-
arrestin-2 recruitment, shifting the CXCL8 activity curve to the right. (B) SDF-1 induced β-arrestin-2
recruitment in CXCR4 Tango assay in a dose-dependent manner (vehicle). Pretreatment with a CXCR4
antagonist (AMD3100) at 1, 0.1, and 0.01 μM for 30 minutes prior to SDF-1 stimulation reduced β-
arrestin-2 recruitment, shifting the SDF-1 activity curve to the right.
CXCR2 and CXCR4 Tango assay protocol:
Reagents and materials
 384-well tissue culture treated black, clear bottom plates (BD Falcon, Cat #
353962)
 CXCR2-bla and CXCR4-bla U2OS Tango cells (Invitrogen, Carlsbald, CA)
 Growth media (McCoy5A supplemented with 10% dialyzed FBS (Invitrogen, Cat
# 26400-036, 0.1mM NEAA, 25 mM HEPES (pH 7.3), 1mM sodium pyruvate,
zeocin (200 μg/mL), hygromycin (50 μg/mL), and geneticin (100 μg/mL)
 Assay media (DMEM supplemented with 1% dialyzed FBS)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol)
 LiveBLAzerTM-FRET B/G Subsrate Mixture (CCF4-AM) kit, (Invitrogen, Cat#
K1095)
 CXCR2 antagonists and CX compounds (10 mM stock diluted in DMSO and
diluted in assay media)
 Envision multilabel plate reader (Perkin Elmer)

Experimental Procedures
Day 1 - CXCR2 or CXCR4-bla U2OS cells were grown in growth media until 70-80%
confluence was reached. Growth media was removed and washed with 1X PBS. Cells
46
were detached with 0.25% Trypsin-EDTA for 5 minutes at 37°C. Tryspin was
neutralized with 5 mL of growth media and cells were centrifuged at 1200 RPM for 5
minutes at room temperature. Cells were seeded (11,000 cells/well) in 384-well tissue
culture plates in 40 μL of assay media and incubated at 37°C overnight.
Day 2 – The next morning, assay media was removed and cells were pre-treated with 1X
concentrations of CXCR2 antagonists or CX compounds (36 μL total volume) for 30
minutes. Four microliter of 10X CXCL8 (10-20 nM final concentration) or SDF-1α (30
nM final concentration) was added to each well. Plates were incubated for 5 hours at
37°C . Eight microliter of β-lactamase substrate (LiveBLAzer kit) was loaded for 2
hours. β-lactamase substrate was prepared by mixing the following reagents provided in
the kit in this order:
 6 μL of Solution A (1 mM CCF4-AM dissolved in dry DMSO, stored at -20°C)
 60 μL of Solution B (stored at room temperature)
 904 μL of Solution C (loading dye, stored at room temperature)
 30 μL of Solution D (transport inhibitor, stored at -20°C, Invitrogen, Cat #
K1156)
Plates were read on the Envision microplate reader at 409 nm excitation (400/25 filter)
and 460/540 nm emissions (460/25 and 535/25 filters). Percent inhibition was calculated
using the following formulas: Ratio was calculated for each sample well: cleaved
(409/460)/uncleaved (409/540) and % Inhibition = [1-((Compound treated-unstimulated
control)/(CXCL8/SDF stimulated-unstimulated control))] x 100

47
β-arrestin1/2 rearrangement. Upon CXCL8 stimulation, cytoplasmic β-arrestin1/2 is
rearranged to co-localize with CXCR2. Using cells expressing GFP-tagged β-arrestin-2
and CXCR2, we show that cytoplasmic β-arrestin-2 is rearranged into punctate vesicles
upon CXCL8 stimulation that is inhibited by a CXCR2 antagonist, SB265610 (Figure
2.4).

Figure 2.4 CXCL8 induces β-arrestin-2 rearrangement.
SB265610 inhibited CXCL8-mediated β-arrestin-2 recruitment and rearrangement in HEK293 cells co-
expressing CXCR2 and GFP tagged β-arrestin-2. Cells were pretreated with compounds for 30 minutes
and stimulated with CXCL8 (25 nM) for additional 30 minutes. Cells were fixed and β-arrestin-2
localization was imaged on the BD Pathway.
β-arrestin-2 rearrangement assay protocol:
Reagents and materials
 384-well tissue culture treated black, clear bottom plates (BD Falcon, Cat #
353962)
48
 Growth media (DMEM supplemented with 10% FBS, 800 μg/mL geneticin, and
2 μg/mL puromycin)
 HEK293-CXCR2-β-arrestin-2-GFP (obtained from O.M. Zack Howard from the
National Cancer Institute)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in growth media
 CXCR2 antagonists (SB265610, SB225002, Tocris, Bristol, UK) and CX
compounds (10 mM stock diluted in DMSO and diluted in growth media)
 BD Pathway Bioimager (Franklin Lakes, NJ)

Experimental Procedures
Day 1 - HEK293-CXCR2-β-arrestin-2-GFP cells were cultured in growth media until 70-
80% confluence was reached. Growth media was removed and washed with 1X PBS.
Cells were detached with 0.25% Trypsin-EDTA for 5 minutes at room temperature.
Tryspin was neutralized with 5 mL of growth media and cells were centrifuged at 1200
RPM for 5 minutes at room temperature. Cells were seeded (10,000 cells/well) in 384-
well tissue culture plates in 32 μL of growth media and incubated at 37°C overnight.
Day 2 - Cells were pre-treated with four microliter of 10X CXCR2 antagonists or CX
compounds for 30 minutes at 37°C . Cells were then stimulated with four microliter of
10X CXCL8 (25 nM final concentration) for an additional 30 minutes. Cells were fixed
with 8% formaldehyde (40 μL) for 20 minutes at room temperature. Formaldehyde was
removed and wells were washed with 1X PBS and imaged on BD Pathway Bioimager
(Franklin Lakes, NJ) with the GFP filter at 20X magnification.

49
2.2.2. Receptor internalization
CXCR2 is rapidly internalized upon ligand stimulation and recycled back to the cell
surface or degraded at high ligand concentration (Baugher and Richmond, 2008). Using
immunofluorescence, we tracked the internalization of GFP-tagged CXCR2 from the cell
surface into intracellular vesicles as represented as punctate staining (Figure 2.5).
50

Figure 2.5 CXCL8 stimulates receptor internalization and turnover.
293T-CXCR2 cells over-expressing GFP-tagged CXCR2 was treated with CXCL8 (50 nM) for various
time points and fixed with formaldehyde for 30 minutes, permeabilized with 100% methanol for 10
minutes and stained with DRAQ5 for five minutes. Samples were imaged on Nikon confocal microscopy
and processed with ImageJ. GFP-tagged CXCR2 is shown in green and nuclear staining with DRAQ5 is
shown in red.
51
CXCR2 internalization immunofluorescence protocol:
Reagents and materials
 293T-CXCR2-GFP cells were stably generated by Dr. Daryl Davies (University
of Southern California, School of Pharmacy, Los Angeles, CA) using a lentiviral
system
 Growth media (DMEM supplemented with 10% FBS and puromycin (2 μg/mL)
 Glass coverslips
 24-well clear tissue culture plates
 0.01% polylysine
 1X PBS
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in growth media
 DRAQ5 (5 mM stock, Cell Signaling Cat# 4084)
 Vectashield mounting media
 Nikon confocal microscope (Melville, NY)

Experimental Procedures
Day 1 – Glass coverslips were placed in a 24-well clear tissue culture plate with sterile
tweezers. Coverslips were treated with 100 μL of polylysine at room temperature for 30-
60 minutes. Polylysine was removed and washed with 1X PBS. 293T-CXCR2-GFP
cells were cultured in growth media until 70-80% confluence was reached. Growth
media was removed and washed with 1X PBS. Cells were detached with 0.25% Trypsin-
EDTA for 5 minutes at room temperature. Tryspin was neutralized with 5 mL of growth
media and cells were centrifuged at 1200 RPM for 5 minutes at room temperature. Cells
were seeded (50-100,000 cells/well) in 24-well tissue culture plates containing the
polylysine coated glass coverslips in 450 μL of growth media and incubated at 37°C
overnight.
Day 2 – Cells were treated with 50 μL of 10X CXCL8 (50 nM final concentration) for
various time points. Growth media was removed and cells were fixed with 8%
52
formaldehyde for 30 minutes, permeabilized with 100% methanol for 10 minutes and
stained with DRAQ5 (1:1000) diluted in 1X PBS for 5 minutes. Samples were washed
with 1X PBS and mounted on to glass slides with mounting media and edges were sealed
with nail polish. Samples were imaged on Nikon confocal microscope with 100X
magnification. Stacks were processed with ImageJ.

In-cell Western assay to detect CXCR2 internalization
To measure receptor internalization in a high throughput manner, we used an in-cell
Western technique to design a high throughput (384-well) assay to monitor the surface
expression of CXCR2. This assay significantly reduced sample preparation time as well
as detect direct CXCR2 surface detection. (Other assays detect binding of a labeled
ligand, in which the amount of ligand binding is correlated with CXCR2 surface
expression). Using this assay, we show that CXCL8 dose-dependently and time-
dependently induced receptor internalization (Figure 2.6). At higher CXCL8
concentrations (>20 nM), CXCR2 is internalized within five minutes of stimulation.
53

Figure 2.6 CXCL8 induces CXCR2 internalization.
(A) Surface expression of CXCR2 treated with various concentrations of CXCL8 (0-100 nM) for various
time points (0-300 minutes). IgG wells were not stimulated with CXCL8 and was incubated with mouse
IgG instead of CXCR2 antibodies as a negative control. CXCR2 expression is detected using the IR680
channel on the Licor Odyssey. (B) CXCL8 dose-dependently induce receptor internalization. Quantitation
of A and represented as log of CXCL8 concentrations vs. percent receptor internalized. (C) CXCL8 induce
receptor internalization in a time-dependent manner. Quantitation of A and represented as time vs. percent
receptor internalized.
In-cell Western assay protocol:
Reagents and materials
 384-well tissue culture treated black, clear bottom plates (BD Falcon, Cat #
353962)
 CXCR2-bla U2OS Tango cells (Invitrogen)
 Growth media (McCoy5A supplemented with 10% dialyzed FBS (Invitrogen, Cat
# 26400-036, 0.1mM NEAA, 25 mM HEPES (pH 7.3), 1mM sodium pyruvate,
zeocin (200 μg/mL), hygromycin (50 μg/mL), and geneticin (100 μg/mL)
 Assay media (DMEM supplemented with 1% dialyzed FBS)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol)
 CXCR2 antagonists and CX compounds (10 mM stock diluted in DMSO and
diluted in assay media)
 Licor blocking buffer (Licor, Cat # 927-40000), stored at 4°C
54
 0.3% TritonX100 diluted in Licor blocking buffer
 1X PBS
 4% formaldehyde (diluted in 1X PBS)
 CXCR2 antibodies (mouse IgG
1
isotype, epitope against human CXCR2, amino
acids 1-19, Santa Cruz Biotechnology, Cat # sc-7304) diluted to 1:1000 in
blocking buffer, stored at 4°C
 Mouse IgG (Cell Signaling) diluted to 1:1000 in blocking, stored at -20°C
 IRDye 680RD secondary antibodies (goat anti-mouse IgG (H+L), Licor, Cat #
926-68070) diluted to 1:1000 in blocking buffer, stored at 4°C in the dark
 Licor Odyssey Bioimager

Experimental Procedures
Day 1 - CXCR2-bla U2OS cells were grown in growth media until 70-80% confluence
was reached. Growth media was removed and washed with 1X PBS. Cells were
detached with 0.25% Trypsin-EDTA for 5 minutes at 37°C. Tryspin was neutralized
with 5 mL of growth media and cells were centrifuged at 1200 RPM for 5 minutes at
37°C. Cells were seeded (11,000 cells/well) in 384-well tissue culture plates in 40 μL of
assay media and incubated at 37°C overnight.
Day 2 – The next morning, assay media was removed and cells were pre-treated with 1X
concentrations of CXCR2 antagonists or CX compounds (36 μL total volume) for 30
minutes. Four microliter of 10X CXCL8 was added to each well. Plates were incubated
for 5-300 minutes at 37°C . Compounds and CXCL8 was removed and cells were fixed
with 4% formaldehyde (25 μL/well) for 20-30 minutes are room temperature.
Formaldehyde was removed and wells were washed with 1X PBS and stored at 4°C.
55
Day 3 – Cells were blocked in blocking buffer (25 μL/well) for 2 hours at room
temperature and incubated with CXCR2 antibodies or mouse IgG (25 μL/well) for 2
hours at room temperature or overnight at 4°C.
Day 4 – Cells were then washed with 1X PBS six times and incubated with mouse IRDye
680RD secondary antibodies (25 μL/well) for additional 1-2 hours in the dark. Wells
were washed with 1X PBS six times. Plates were spun upside down on top of paper
towels at 1200 RPM for five minutes to remove excess liquid. Plates were allowed to
completely dry before imaging and quantification on the Licor Odyssey bioimager. To
detect total CXCR2, cells were blocked in blocking buffer with 0.3% Triton X100 for 1-2
hours. Primary and secondary antibodies were also diluted in 0.3% Triton X100 in
blocking buffer.
Percent inhibition was calculated using the following formulas: % Receptor
Internalization= [1-(CXCL8 stimulated/unstimulated)] x 100 and % Inhibition of receptor
internalization= [(Compound treated - CXCL8stimulated)/(unstimulated control –
CXCL8 stimulation)] x 100.
2.2.3. Receptor turnover
At high ligand concentration, CXCR2 is rapidly internalized and instead of being
recycled back onto the surface, the receptor is directed to lysosomes for degradation
(Baugher and Richmond, 2008). Using the same assay to detect for receptor
internalization, we were also able to adapt the assay to measure receptor turnover. Prior
to incubation with CXCR2 antibodies, cells were permeabilized with 0.3% Triton X100
56
for 1-2 hours. This step allows the antibodies to enter the cells and both surface and
cytoplasmic (vesicle bound) CXCR2 are detected. CXCL8 dose-dependently induce
sustained receptor turnover within 10 minutes of CXCL8 treatment (Figure 2.7).

Figure 2.7 CXCL8 induces CXCR2 degradation.
(A) Total expression of CXCR2 treated with various concentrations of CXCL8 (1-100 nM) for various time
points (0-300 min). Lanes 1-8 were permeabilized to detect total CXCR2 expression. Lane 9 was not
permeabilized to detect surface CXCR2 expression in unstimulated cells. (B) CXCL8 dose-dependently
induce receptor degradation. Quantitation of A and represented as log of CXCL8 concentrations vs.
percent receptor degraded. (C) CXCL8 induced receptor degradation in a time-dependent manner.
Quantitation of A and represented as time vs. percent receptor internalized.

Alternatively, total CXCR2 expression can be detected in 293T-CXCR2-GFP cells using
the flow cytometer or microscopy.
Flow cytometric assay to detect changes in CXCR2-GFP expression protocol:
57
Materials and Reagents
 293T-CXCR2-GFP cells
 Growth media (DMEM supplemented with 10% FBS and puromycin (2 μg/mL)
 96-well clear tissue culture plates
 1X PBS
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in growth media
 CXCR2 antagonists and CX compounds (10 mM stock diluted in DMSO and
diluted in assay media)
 BD LSR II flow cytometer (BD Biosciences, San Jose, CA)

Experimental Procedures
Day 1 – 293T-CXCR2-GFP cells were grown in growth media until 70-80% confluence
was reached. Growth media was removed and washed with 1X PBS. Cells were
detached with 0.25% Trypsin-EDTA for 5 minutes at 37°C. Tryspin was neutralized
with 5 mL of growth media and cells were centrifuged at 1200 RPM for 5 minutes at
room temperature. Cells were seeded at 50,000 cells per well in 96-well plates overnight.
Day 2 - Cells were treated with CX compounds and/or CXCL8 (50 nM) for 24 hours in
50 μL growth media.
Day 3 – Compounds and CXCL8 was removed and cells were collected in 1XPBS (300
μL). GFP expression was measured on the FITC channel on the BD LSR II flow
cytometer. Fold change was normalized to untreated samples and 293T background
signal.
Microscopy to detect CXCR2-GFP expression protocol:
Reagents and Materials
 293T-CXCR2-GFP cells
 Growth media (DMEM supplemented with 10% FBS and puromycin (2 μg/mL)
58
 384-well tissue culture treated black, clear bottom plates (BD Falcon, Cat #
353962)
 1X PBS
 4% formaldehyde (diluted in 1X PBS)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in growth media
 DAPI (1mg/mL in PBS), diluted to 1:5000
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in growth media
 CXCR2 antagonists and CX compounds (10 mM stock diluted in DMSO and
diluted in assay media)
 BD Pathway Bioimager (Franklin Lakes, NJ)

Experimental Procedures
Day 1 – 293T-CXCR2-GFP cells were cultured in growth media until 70-80% confluence
was reached. Growth media was removed and washed with 1X PBS. Cells were
detached with 0.25% Trypsin-EDTA for 5 minutes at room temperature. Tryspin was
neutralized with five mL of growth media and cells were centrifuged at 1200 RPM for 5
minutes at room temperature. Cells were seeded (10,000 cells/well) in 384-well tissue
culture plates in 30 μL of growth media and incubated at 37°C overnight.
Day 2 - Cells were treated with CX compounds for five hours and/or CXCL8 (25 nM) in
40 μL of growth media. Compounds and CXCL8 was removed and cells were fixed with
4% formaldehyde for 20 minutes at room temperature. Formaldehyde was removed from
wells and washed with 1X PBS. Cells were stained with DAPI for 30 minutes at room
temperature. DAPI was removed and cells were washed with 1X PBS. Wells containing
100 μL of 1X PBS were imaged on BD Pathway Bioimager with GFP and DAPI filter at
20X magnification.
59
2.2.4. Second messengers
Calcium. CXCR2 activation rapidly induces intracellular calcium mobilization,
which can be measured using chemical indicators that fluorescence upon Ca
2+
binding
such as Fura-2, Fluo-3 and Fluo-4. Cells may also be genetically modified to express a
calcium sensor to detect calcium signaling. We used the calcium dye, Fluo-4, to detect
CXCL8-mediated calcium mobilization in 293T-CXCR2-GFP cells and found that
CXCL8 (100 nM) induces rapid and transient calcium mobilization (Figure 2.8).

Figure 2.8 CXCL8 dose-dependently induce rapid and transient calcium flux in 293T-CXCR2-GFP
cells.
293T-CXCR2-GFP cells were loaded with Fluo-4NW dye and probenecid (anion transport inhibitor) for 30
minutes at 37°C and 30 minutes at room temperature. Cells were stimulated with CXCL8 at indicated
concentrations and immediately read on the Envision plate reader (490/535). Signal is normalized to
baseline signal prior to CXCL8 stimulation.
Calcium assay protocol:
Reagents and materials
 293T-CXCR2-GFP cells
 Growth media (DMEM supplemented with 10% FBS and puromycin (2 μg/mL)
 384-well tissue culture treated black, clear bottom plates (BD Falcon, Cat #
353962)
 Fluo-4 NW calcium assay kit (Invitrogen, Cat# F36206), stored at -20°C
-20000
0
20000
40000
60000
80000
100000
120000
0 50 100 150 200 250 300 350
Fluo-4 signal (normalized)
Time (seconds)
400nM 200nM 100nM 50nM Basal
60
 Assay buffer (1X HBSS, 20 mM HEPES), stored at -20°C
 Probenecid (250 mM stock diluted in assay buffer), stored at -20°C
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in growth media,
stored at -20 or -80°C
 CXCR2 antagonists and CX compounds (10 mM stock diluted in DMSO and
diluted in assay media), stored at -20°C
 Envision multilabel plate reader (Perkin Elmer)

Experimental procedures
Day 1 - 293T-CXCR2-GFP cells were seeded in 384-well black/clear bottom plates at
30,000 cells/well in growth medium overnight.
Day 2 - The next day, cells were loaded with Fluo-4 NW and probenecid. Dye mixture
included in kit was diluted with 10 mL assay buffer and 100 μL of probenecid (25 mM
final concentration). Growth media was removed from cells and 25 μL of the dye
mixture was added to each well. Cells were incubated with the dye mixture for 30
minutes at 37°C for 30 minutes and at room temperature for 30 minutes (protected from
light). To detect CXCL8-stimulated calcium flux, 5X CXCL8 (5 μL) was added to each
well and fluorescence signal detected immediately on the Envision plate reader with 490
nm excitation and 535 nm emission filters. Fluorescence signal was normalized to
baseline signal (before CXCL8 stimulation).

Cyclic AMP. CXCR2 is coupled to Gαi, which inhibits adenylyl cyclase (enzyme
that converts ATP to cyclic AMP). Thus, in the presence of CXCL8, cyclic AMP
production is significantly reduced. There are several methods to detect intracellular
cyclic AMP production in a high throughput platform including using genetically
61
modified cells that express a cyclic AMP biosensor. Using the pGloSensor-22F cAMP
plasmid that encodes a cyclic AMP sensitive firefly luciferase (Promega, cat # E2301),
293T cells over-expressing CXCR2 was stably transfected with the pGloSensor-22F
plasmid with lipofectamine 2000. The firefly luciferase is activated in the presence
cyclic AMP and release light that can be detected with a luminescence plate reader
(Figure 2.9). Decreases in basal cyclic AMP in the presence of CXCL8 are too low to
detect, therefore to enhance to signal, we used forskolin to induces cyclic AMP and
measured the effects of CXCL8 on forskolin-induced cyclic AMP. Forskolin dose-
dependently induce cyclic AMP production, which can be detected in cells expressing the
pGloSensor-22F plasmid. This effect was inhibited by CXCL8 in cells expressing
CXCR2 (Figure 2.10).

Figure 2.9 Schematic of CXCR2 Glosensor cyclic AMP assay.
CXCR2 negatively regulates adenylyl cyclase and thereby inhibits cyclic AMP production. Intracellular
cyclic AMP levels can detected in cells expressing the cyclic AMP Glosensor, which is a cyclic AMP
sensitive firefly luciferase that luminescence in the presence of cyclic AMP.
62

Figure 2.10 CXCL8 inhibits forskolin-induced cylic AMP in CXCR2 expressing cells.
(A) Forskolin dose-dependently increased cyclic AMP in cells expressing the pGloSensor-22F plasmid
(293T-CXCR2-GFP-p22F). (B) CXCL8 pretreatment (five minutes) reduced forskolin induced cyclic
AMP in CXCR2-expressing cells (293T-CXCR2-p22F) and not in cells that do not over-express CXCR2
(293T-p22F).
Glosensor cyclic AMP assay protocol:
Reagents and materials
 293T-CXCR2-GFP-p22F cells
 Growth media for 293T-CXCR2-GFP-p22F (DMEM supplemented with 10%
FBS, puromycin (2 μg/mL), and hygromycin B (200 μg/mL))
 Growth media for 293T-p22F (DMEM supplemented with 10% FBS and
hygromycin B (200 μg/mL))
 384-well tissue culture treated white plates (BD Falcon, Cat # 353988)
 Assay buffer (CO2-independent media supplemented with 10% FBS, Invitrogen,
Cat # 18045-088)
 Cyclic AMP reagent (Promega, Cat# E1290), dissolved in HEPES buffer (pH 7.5)
with a stock concentration of 1.22 μg/ μL, stored at -80°C
 1% cyclic AMP reagent (stock cyclic AMP reagent diluted 1:100 in assay buffer)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in assay media, stored
at -20 or -80°C
 CXCR2 antagonists and CX compounds (10 mM stock diluted in DMSO and
diluted in assay media), stored at -20°C
 Envision multilabel plate reader (Perkin Elmer)

Experimental procedures
63
Day 1 - 293T-CXCR2-GFP-p22F or 293T-p22F cells were seeded in assay buffer at
30,000 cells/well in white 384-well plates overnight.
Day 2 – Cells were incubated with 1% cyclic AMP reagent (32 μL) for 2 hours at room
temperature in the dark. In most assays unless otherwise specified, cells were pre-treated
with various concentrations 10X CX compounds (4 μL) for 10 minutes, then stimulated
with 10X CXCL8 (50 nM final concentration, 4 μL) for additional 10 minutes. Cells
were then stimulated with 10X forskolin (50 µM final concentration, 4 μL) until max
cyclic AMP signal is reached (10-20 minutes). Luminescence signals were detected
using the Envision microplate reader (Perkin Elmer, Waltham, MA). Percent inhibition
of CXCL8 was calculated using the max luminescence signal with the following formula:
% Inhibition of CXCL8 = [(Compound/CXCL8/forskolin – forskolin/CXCL8)/(forskolin
only – forskolin/CXCL8)] x 100.
2.2.5. Kinase phosphorylation
The activation of CXCR2 leads to rapid and transient phosphorylation of several
signaling cascades as described in Chapter 1. To further assess the functional effects of
compounds identified in our studies, we used Western blotting to detect activation of the
Akt, ERK1/2, JNK, p38, and FAK.
Western blotting protocol:
Reagents and materials
 293T-CXCR2-GFP cells
 Growth media for 293T-CXCR2-GFP-p22F (DMEM supplemented with 10%
FBS, puromycin (2 μg/mL), and hygromycin B (200 μg/mL))
 6-well clear tissue culture plates
64
 DMEM (no FBS)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol) diluted in DMEM (no FBS),
stored at -20 or -80°C
 CXCR2 antagonists and CX compounds (10 mM stock in DMSO and diluted in
DMEM, no FBS), stored at -20°C
 RIPA lysis buffer (1% NP-40, 0.1% SDS, 50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 0.5% sodium deoxycholate, and 1mM EDTA)
 10X protease inhibitor cocktail (SIGMAFAST protease inhibitor tablets dissolved
in RIPA lysis buffer, stored at 4°C)
 100X sodium orthovanadate (20 mM stock concentration), stored at -20°C
 5X SDS loading dye (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-
mercaptoethanol, 0.01% bromophenol blue)
 SDS running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS)
 Transfer buffer (25 mM Tris, 192 mM glycine, and 10% methanol)
 TBST (0.1% Tween-20, 25 mM Tris, 150 mM NaCl, 2mM KCl, pH 7.4)
 Blocking buffer (5% Milk in TBST)
 5% BSA in TBST
 HRP-linked anti-mouse or anti-rabbit secondary antibodies
 Primary antibodies
 SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific, Cat #
34076)
 Restore Western Blot Stripping buffer (Pierce, Cat # 21059)
 BCA protein estimation assay (Pierce, Rockford, IL)
 ChemiDoc system (Biorad. Hercules, CA)
Table 2.1 Antibody conditions for Western blotting
Antibodies Species Company Dilution conditions
CXCR2 (E2 Clone) Mouse Santa Cruz Biotechnology 1:1000 in 5% milk
p-ERK1/2
(Thr202/Tyr204)
Rabbit Cell Signaling 1:3000 in 5% BSA
ERK1/2 Rabbit Cell Signaling 1:3000 in 5% BSA
p-p38 Rabbit 1:1000 in 5% BSA
p38 Rabbit Cell Signaling 1:1000 in 5% BSA
p-JNK (Thr183/Tyr185) Rabbit Cell Signaling 1:500 in 5% BSA
JNK Rabbit Santa Cruz Biotechnology 1:1000 in 5% milk
p-FAK (Tyr925) 1:1000 in 5% BSA
FAK Rabbit Cell Signaling 1:5000 in 5% BSA
Actin Rabbit Santa Cruz Biotechnology 1:5000 in 5% milk
GAPDH Rabbit Cell Signaling 1:5000 in 5% BSA
Β-Tubulin Rabbit Cell Signaling 1:5000 in 5% BSA
65
Experimental procedures
Day 1 – 293T-CXCR2-GFP cells were seeded in 6-well plates at 500,000 to 1X10^6
cells/well in 2 mL of growth media. After five hours, growth media was replaced with
DMEM without FBS and serum starved overnight.
Day 2 – Media was removed from cells and replaced with 1.8 mL of 1X CX compounds
diluted in DMEM. After 30 minutes incubation with CX compounds, cells were
stimulated with 200 μL of 10X CXCL8 (10-20 nM final concentration) for five minutes
to one hour at 37°C . CX compounds and CXCL8 was immediately removed and cells
were lysed with RIPA lysis buffer solution supplemented with protease inhibitor cocktail
and sodium vanadate on ice for 30 minutes. Total protein concentrations were calculated
using the BCA assay. Protein lysates was prepared in SDS loading dye and loaded in
10% SDS page gels. Gels were transferred to a PVDF membrane. Membranes were
incubated for 1 hour in blocking buffer and incubated with primary antibodies against
respective phosphoproteins overnight at 4°C on a rocker.
Day 3 – Membranes were washed with 1X TBST three times every five minutes and
incubated with HRP-linked anti-rabbit or anti-mouse IgG antibodies for 1 hour at room
temperature. Membranes were washed with 1X TBST three times every five minutes
before detection by enhanced chemiluminescence (ECL) system on the ChemiDoc
system (Biorad. Hercules, CA). Membranes were stripped with stripping buffer for 20
minutes to re-probe for loading controls.
66
2.3. Cellular Functions
To assess the effects of CXCR2 inhibitors on cellular functions, wound healing and
transwell cell migration assays were used evaluate cell migration. MTT (3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), AlamarBlue, and colony
formation assays were used to assess cell proliferation and cytotoxicity. Propidium iodide
(PI) staining was used to analyze cell cycle perturbations and 2',7'-
dichlorodihydrofluorescein diacetate (DCF) staining was also used to measure reactive
oxygen species (ROS) production.
Wound healing assay protocol:
Reagents and materials
 CXCR2-bla U2OS cells
 96-well clear tissue culture plates
 Growth media (McCoy5A supplemented with 10% dialyzed FBS (Invitrogen, Cat
# 26400-036, 0.1mM NEAA, 25 mM HEPES (pH 7.3), 1mM sodium pyruvate,
zeocin (200 μg/mL), hygromycin (50 μg/mL), and geneticin (100 μg/mL)
 Assay media (DMEM supplemented with 1% dialyzed FBS)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol)
 CX compounds (Asinex or Enamine) or CXCR2 antagonists (SB265610 or
SB225002, Tocris, Bristol, UK)
 Geimsa stain (10X stock solution)
o Dissolve 3.8g Giemsa powder into 250 mL of methanol
o Heat solution to ~60°C
o Add 250 mL of glycerin to heated Giemsa solution
o Filter solution
o Working solution: add 10 mL of stock solution to 80 mL of distilled water
and 10 mL of methanol

Experimental Procedures

Day 1 - CXCR2-bla U2OS cells were seeded in 96-well plates (30,000 cells/well) in
DMEM supplemented with 1% FBS overnight.
67
Day 2 - A single scratch wound was made using a sterile pipette tip the next day. Wells
were washed with 1X PBS and treated with compounds at various concentrations and
recombinant CXCL8 at 100-200 nM in assayt media for 24 hours at 37°C.
Day 3 – Compounds and CXCL8 was removed and cells were fixed with 100% methanol
for 15 minutes at room temperature. Methanol was removed and cells were stained with
1X Giemsa stain for one hour. The stain was removed and wells were rinsed with
distilled water to remove excess dye. Plates were spun dried in a centrifuge and air dried
overnight. Each well was imaged on BD Pathway 435 Bioimager with transmitted light
with 4X magnification.

Cell migration assay protocol:
Reagents and materials
 CXCR2-bla U2OS cells
 24-well transwell inserts (BD, 8 μM pore size)
 Growth media (McCoy5A supplemented with 10% dialyzed FBS (Invitrogen, Cat
# 26400-036, 0.1mM NEAA, 25 mM HEPES (pH 7.3), 1mM sodium pyruvate,
zeocin (200 μg/mL), hygromycin (50 μg/mL), and geneticin (100 μg/mL)
 Assay media (DMEM supplemented with 1% dialyzed FBS)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol)
 CX compounds (Asinex or Enamine) or CXCR2 antagonists (SB265610 or
SB225002, Tocris, Bristol, UK)
 Sterile Q-tips
 Geimsa stain (10X stock solution)
o Dissolve 3.8g Giemsa powder into 250 mL of methanol
o Heat solution to ~60°C
o Add 250 mL of glycerin to heated Giemsa solution
o Filter solution
o Working solution: add 10 mL of stock solution to 80 mL of distilled water
and 10 mL of methanol

68
Experimental Procedures

Day 1 - CXCR2-bla U2OS cells were seeded on 24-well transwell inserts (BD, 8 μM
pore size, 100.000/well) in assay media overnight.
Day 2 – The next day, cells were treated with various concentrations of compounds and
CXCL8 (200 nM) for 24-48 hours at 37°C .
Days 3-4 – Cells on the top of the chamber were removed with Q-tips and rinsed with 1X
PBS. Cells on the bottom of the chamber were fixed with 100% methanol for 10 minutes
and stained with Giesma stain for 45 minutes and washed with distilled water to remove
excess dye. Dried transwells were imaged on BD Pathway with 10X magnification.

Cell viability assay protocol:
Reagents and materials
 96-well clear tissue culture plates
 384-well black, clear bottom tissue culture plates
 Growth media (Tissue culture media supplemented with 10% FBS)
 CX compounds (Asinex or Enamine) or CXCR2 antagonists (SB265610 or
SB225002, Tocris, Bristol, UK)
 MTT solution (dissolved in 1X PBS at 3 mg/mL, Amresco, Solon, OH or Sigma,
St. Louis, MO)
 DMSO
 Alamar Blue solution (AbD Serotec, Kidlington, UK)
 Microplate reader (Molecular Devices, Sunnyvale, CA).
 Envision multilabel plate reader (Perkin Elmer)

Experimental Procedures

69
Day 1 - Cell viability was assessed by MTT assay as described previously (Carmichael et
al., 1987). Briefly, cells were seeded in 96-well plates (1-8,000 cells/well) and allowed
to attach overnight. HL60 and Jurkat cells (non-adherent) were seeded in 384-well plates
(1000 cells/well) in growth media overnight.
Day 2 – The next day, cells were continuously treated with compounds for 24-72 hours in
growth media.
Day 3-5 – At the end of treatment, cells were incubated with MTT solution (at a final
concentration of 0.5 mg/mL, Amresco, Solon, OH or Sigma, St. Louis, MO) for 3-4
hours at 37 C. Cell supernatant was removed and 150 μL of DMSO (Sigma) was added.
Absorbance was read at 570 nm on a microplate reader (Molecular Devices, Sunnyvale,
CA). Non-adherent HL60 and Jurkat cells were incubated with AlamarBlue solution
(1:10 dilution, AbD Serotec, Kidlington, UK) for 4 hours and fluorescence was read at
535 nm emission and 590 nm excitation on Envision plate reader. Percentage of cell
viability was calculated using the following formula: % Cell Viability = [(OD
570
treated
cells – OD
570
blank) / (OD
570
untreated cells – OD
570
blank)] x 100

Colony formation assay protocol:
Reagents and materials
 6-well, 24-well, or 96-well clear tissue culture plates
 Growth media (Tissue culture media supplemented with 10% FBS)
 CX compounds (Asinex or Enamine) or CXCR2 antagonists (SB265610 or
SB225002, Tocris, Bristol, UK)
 2% crystal violet solution (0.2g crystal violet dissolved in 2.5 mL methanol and
7.5 mL distilled water)
 ChemiDoc system (Biorad. Hercules, CA)
70

Experimental Procedures

Day 1 – Cells were seeded at 100 cells/well in 96- 24- or 6-well tissue culture plates in
growth media overnight.
Day 2 – The next day, cells were treated with various concentrations of compounds for
24 hours. Compounds were removed and replaced with fresh media. Cells were
incubated until colonies formed (5-10 days). Next, colonies were washed with 1X PBS
and stained with a solution of crystal violet (2%) for 30 minutes. Colonies were washed
with distilled water and allowed to air dry. Colonies were imaged on a ChemiDoc system
(Biorad. Hercules, CA).

Cell cycle flow cytometry assay protocol:
Reagents and materials
 6-well clear tissue culture plates
 Growth media (Tissue culture media supplemented with 10% FBS)
 CX compounds (Asinex or Enamine) or CXCR2 antagonists (SB265610 or
SB225002, Tocris, Bristol, UK)
 Propidium Iodide solution (1 mg/mL in 1X PBS, stored at 4°C)
 RNAse A (Amresco, Solon, OH)
 BD LSRII flow cytometer
 1X PBS supplemented with 1% FBS
 100% ethanol

Experimental Procedures

71
Day 1 – Cell cycle perturbations were analyzed by propidium iodide DNA staining.
Exponentially growing cells were seeded in 6-well plates at 100-300,000 cells/well in
growth media.
Day 2 – The next day, cells were treated with various concentrations of compounds for
24-72 hours in growth media. At the end of treatment, cells were collected and washed
with 1X PBS supplemented with 1% FBS. Cells were thoroughly resuspended in 0.3 mL
of PBS and fixed in 70% ethanol overnight at 20 °C. Ethanol-fixed cells were
centrifuged at 3000 rpm for 5 min and washed twice in PBS to remove residual ethanol.
For cell cycle analysis, the pellets were resuspended in 0.5mL of PBS containing 0.02
mg/mL of propidium iodide and 0.05 mg/mL of DNase-free RNase A and incubated at
room temperature for 0.5-1 hour. Cell cycle profiles were obtained using a BD LSRII
flow cytometer. Propidium iodide was excited using a 488 nm blue argon laser. The
resulting fluorescence was measured using a PMT detector equipped with 550 nm long
pass dichroic mirror and 575/26 bandpass filter. Data were analyzed with ModFit LT
software package (Verify Software House, Inc., Topsham, ME).

DCF (2',7'-dichlorodihydrofluorescein diacetate) assay protocol:
Reagents and materials
 96-well clear tissue culture plates
 Growth media (Tissue culture media supplemented with 10% FBS)
 CX compounds (Asinex or Enamine) or CXCR2 antagonists (SB265610 or
SB225002, Tocris, Bristol, UK)
 CXCL8 (100 ng/μL in 10% BSA and 10% glycerol)
 1X PBS
 BD LSRII flow cytometer
72
 2',7'-dichlorodihydrofluorescein diacetate (dissolved in DMSO at 50 mM,
Invitrogen, Carlsbald, CA)

Experimental Procedures

Day 1 – DCF assays were used to detect reactive oxygen species generation. 293T-
CXCR2-GFP cells were seeded in 96-well plates in growth media at 100,000 cells/well
overnight.
Day 2 – The next day, cells were loaded with 25 μM of 2',7'-dichlorodihydrofluorescein
diacetate (H
2
DCFDA, Invitrogen) and CXCL8 (100 nM) and/or compounds for 30
minutes at 37°C in PBS. Cells were washed with 1X PBS and fluorescence was
measured on the flow cytometer using the FITC channel.
2.4. Cell culture
Tango CXCR2-bla and CXCR4-bla U2OS cells were purchased from Invitrogen and
grown in McCoy5A supplemented with 10% dialyzed FBS, zeocin (200 μg/mL),
hygromycin (50 μg/mL), geneticin (100 μg/mL), 1 mM sodium pyruvate, 0.1 mM
nonessential amino acids (NEAA), and 25 mM HEPES. HEK293 cells overexpressing
HA tagged CXCR2 and GFP tagged β-arrestin-2 (HEK293-HA-CXCR2-β-arrestin-
2GFP) were kindly provided by Dr. O. M. Zack Howard, NCI. These cells were grown
in DMEM supplemented with 10% FBS and geneticin (800 μg/mL) and puromycin (2
μg/mL). Cells over-expressing GFP-tagged CXCR2 (293T-CXCR2-GFP) cells were
stably generated by Dr. Daryl Davies (University of Southern California, School of
Pharmacy, Los Angeles, CA) using a lentiviral system. Cells were cultured in DMEM
73
(Invitrogen, Carlsbad, CA) supplemented with 10% FBS and puromycin (2 μg/mL). To
generate 293T-CXCR2-GFP-p22F cells, 293T-CXCR2-GFP cells were transfected with
the pGlosensor-22F cAMP plasmid (Promega, Madison, WI) using Lipofectamine 2000
(Invitrogen). 293T-CXCR2-GFP-p22F cells were cultured in DMEM supplemented with
10% FBS, puromycin (2 μg/mL), and hygromycin B (200 μg/mL). H1299, H460, A549,
293T, HCT116p53
+/+
, HCT116p53
-/-
, Jurkat, HL60, and U2OS were purchased from
ATCC (Manassas, VA) and were grown in RPMI-1640 (Invitrogen) supplemented with
10% FBS. All cells were grown at 37°C in a humidified atmosphere of 5% CO
2
. All
cell lines used were maintained in culture under 35 passages and tested regularly for
mycoplasma contamination using Plasmo Test
TM
(InvivoGen, San Diego, CA).
2.5. Compounds and reagents
Compounds were prepared in DMSO at 10 mM stock solution and stored at -20 °C.
All compounds were purchased from Asinex (Moscow, Russia) and Enamine (Kiev,
Ukraine). SB265610 and SB225002 was purchased from Tocris Bioscience (Ellisville,
MI). Human CXCL8 cDNA was inserted into the expression vector, pET32a at EcoR1
and XhoI restriction sites to generate His-tagged CXCL8. The sequence of the clone was
confirmed by DNA sequencing. SDF-1α expression vector was kindly provided by Dr.
Ghalib Alkhatib. Recombinant CXCL8 and SDF-1α was expressed in BL21-Gold (DE3)
pLysS strain of E. Coli (Stratagene, La Jolla, CA) and purified using previously reported
protocol (Altenburg et al., 2007).

74
CHAPTER 3: IDENTIFICATION OF NOVEL PHENYLCYCLOHEX-1-
ENECARBOTHIOAMIDE-BASED COMPOUNDS THAT INHIBITS CXCL8-
MEDIATED CHEMOTAXIS THROUGH SELECTIVE REGULATION OF Β-
ARRESTIN-2
We used the CXCR2 Tango assay to screen an in-house library of highly diverse
chemical compounds. CX4338 was identified from our screen and additional studies to
characterize the compound were performed. Receptor internalization and second
messenger assays were used to assess the effects of CX4338 on CXCR2-mediated
signaling. Wound healing, transwell cell migration, and LPS-induced lung inflammation
in mice were performed to determine the in vitro and in vivo efficacy of CX4338.
CX4338 inhibited CXCR2-mediated β-arrestin-2 recruitment and receptor
internalization, while enhancing CXCR2-mediated G-protein signaling. CX4338
enhanced CXCL8-mediated calcium mobilization and ERK1/2 phosphorylation and
subsequently increased ROS levels. CX4338 dose-dependently inhibited CXCL8-
induced chemotaxis in CXCR2 over-expressing cells and human neutrophils. In vivo
studies demonstrated CX4338 also significantly reduced LPS-induced bronchoalveolar
lavage neutrophils in mice.

75
3.1. Identification of CX4338 (2-(benzylamino)-4,4-dimethyl-6-oxo-N-
phenylcyclohex-1-enecarbothioamide).

Figure 3.1 Chemical structures of CX4338 and previously reported CXCR2 antagonists
(SB265610, SB468477, SCH527123, AZ10397767, and Repertaxin).
Using the CXCR2 Tango assay, we screened over 2,000 compounds from an in-
house library of highly diverse compounds belonging to the Enamine 10K commercial
database. To test for receptor selectivity, we also used the CXCR4 Tango assay to
counter screen compounds that show activity on CXCR2. From our screening endeavors,
we identified several different classes of compounds that show activity on CXCR2.
CX4338 was chosen for further mechanistic studies due to its selectivity for CXCR2
(Figure 3.1 and Table 3.1). CX4338 inhibited CXCR2-mediated β-arrestin-2 recruitment
with an IC
50
of 6.3 μM, while showing little to no inhibition of CXCR4-mediated β-
arrestin-2 recruitment (IC
50
> 100 μM, Figures 3.2A and Table 3.1). At concentrations as
high as 50 and 100 μM, CX4338 did not decrease cell proliferation in several CXCR2-
76
expressing cell lines presented in Table 3.2. These cell lines includes the Tango cell lines
used in the screening (CXCR2-bla U2OS, CXCR4-bla U2OS and parental cell line
U2OS); 293T cells over-expressing GFP-tagged CXCR2 (293T-CXCR2-GFP) and
parental cell line 293T; non-small cell lung cancer (NSCLC) cell lines (H460 and
H1299); leukemia cell lines (HL60 and Jurkat); and lung epithelial cell lines (NuLi and
CuFi).
Table 3.1 IC
50
values for CXCR2 and CXCR4 inhibition
Compound CXCR2
(IC
50
, μM)
1

CXCR4
(IC
50
, μM)
1

Selectivity
2

SB265610 0.28±0.09 >20 >71.4
CX4338 6.3±1.3 >100 >15.9
1
IC
50
= concentration (µM) required to inhibit 50% of CXCR2- or CXCR4-mediated β-arrestin-2
recruitment in the Tango assay.
2
Selectivity was calculated by dividing CXCR4 IC
50
values over CXCR2
IC
50
values. Data are presented as mean ± SD of 3 independent experiments.

Since the chemical structure of CX4338 is distinct from previously reported CXCR2
antagonists (Figure 3.1), we sought to compare CX4338 with a well-characterized
CXCR2 antagonist, SB265610. CX4338 at 10 µM had the same potency as 100 nM of
SB265610 (100-fold) on CXCL8-mediated β-arrestin-2 recruitment (Figure 3.2C).
Additionally, CX4338 and SB265610 inhibit β-arrestin-2 recruitment in an additive
manner (Figure 3.2D). SB265610 at 10 and 100 nM enhanced the inhibitory effects of
CX4338 in the CXCR2 Tango assay.
77

Figure 3.2 CX4338 selectively inhibits CXCR2-mediated β-arrestin-2 recruitment.
(A) Dose inhibition curve of CX4338 and SB265610 in the CXCR2 Tango assay. CXCR2-bla U2OS cells
were pre-treated with indicated concentrations of CX4338 or SB265610 for 30 minutes. Cells were then
stimulated with 20 nM of CXCL8 for 5 hours. Cells were loaded with a β-lactamase substrate for 2 hours
and amount of cleaved and uncleaved substrate was measured using the Envision plate reader (excitation at
409 nm and emissions at 460 and 540 nm). Data represents mean +/- SEM of 3-9 independent
experiments. (B) CX4338 and SB265610 inhibits CXCL8 dose response in the CXCR2 Tango assay.
Cells were pre-treated with assay media (control), 10 μM of CX4338, or 100 nM of SB265610 for 30
minutes prior to stimulation with CXCL8 at the indicated concentrations for 5 hours. Activation ratio was
calculated using the following formula: (cleaved β-lactamase substrate (emission at 460 nm)) / ((uncleaved
β-lactamase substrate (emission at 540 nm)). (C) CX4338 selectively inhibits CXCR2 over CXCR4.
CXCR2-bla or CXCR4-bla U2OS cells were pre-treated with CX4338 for 30 minutes prior to stimulation
with 20 nM of CXCL8 or 30 nM of SDF-1 for 5 hours. Data represents mean +/- SEM of 3-9 independent
experiments. p-values were calculated using the student t-test. ** indicates p-value > 0.001 and ***
indicates p-value > 0.0001. (D) CX4338 and SB265610 inhibits CXCR2-mediated β-arrestin-2 recruitment
in an additive manner. CXCR2-bla U2OS cells were pre-treated with assay media (CX4338 only) or
SB265610 at 10 or 100 nM. Cells were also co-treated with CX4338 at 2, 5, 10, 20, and 50 μM for 30
minutes prior to stimulation with 50 nM of CXCL8 for 5 hours. Data represents mean +/- SD of one
experiment performed in duplicates.

78
Table 3.2 Percent cell viability upon CX4338 treatment
Cell Lines % Cell Viability at
100 μM
% Cell Viability at
50 μM
CXCR2-bla U2OS
,1
86.0 ± 3.3 NT
CXCR4-bla U2OS
,1
97.8 ± 9.4 NT
293T-CXCR2
1
75 NT
293T
1
90.8 NT
H460
1
86.4 ± 11.4 NT
H1299
1
106.7 ± 5.1 NT
U2OS
1
97 NT
Jurkat
2
NT 89.1 ± 9.8
HL60
2
NT 74.6 ± 25.4
NuLi
3
102.42 ± 3.42 NT
CuFi
3
117.38 ± 7.38 NT
Cells were seeded at 1-4000 cells/well in 96-well plates in complete media one day prior to CX4338
treatment.
1
Cells were treated with CX4338 for 72 hours and cell viability was assessed with MTT assay.
2
Nonadherent cells were treated with CX4338 for 72 hours and cell viability was assessed with the
AlamarBlue assay.
3
Cells were treated with CX4338 for 24 hours and cell viability was assessed with
MTT assay.
4
NT = not tested. Data represents mean ± SD of 1-4 independent experiments.
Further, upon CXCL8 stimulation, β-arrestin-2 is rearranged into vesicles
(represented as punctate staining) in HEK293 cells over-expressing CXCR2 and GFP-
tagged β-arrestin-2 (Figure 3.3). In the presence of CX4338 (50 μM) or SB265610 (5
μM), β-arrestin-2 rearrangement is inhibited and no punctate staining was observed
(Figure 3.3). Other compounds identified from the screen with IC
50
between 0.5 μM and
50 μM are reported in Table 3.3 and Figure 3.4.
79

Figure 3.3 CX4338 inhibits β-arrestin-2 rearrangement.
HEK293-CXCR2-β-arrestin-2-GFP cells were seeded at 10,000 cells/well in a 384-well black, clear bottom
plate in 32 μL of complete media overnight. Cells were pre-treated with 50 and 20 μM of CX4338 or 5 and
1 μM of SB265610 for 30 minute prior to stimulation with 25 nM of CXCL8 for an additional 30 minutes
at 37°C . Cells were fixed with 8% formaldehyde for 20 minutes, stained with nuclei stain (DAPI), washed
with 1X PBS, and imaged with the GFP filter on the BD Pathway. DAPI is represented in blue and GFP is
represented in green. Arrows indicate punctate staining.

80
Table 3.3 CXCR2 and CXCR4 Tango IC
50
values for CX compounds
CMPD
CXCR2
(IC
50
)
1

CXCR4
(IC
50
)
1

Selectivity
2

H1299
(IC
50
)
3

% Cell Viability at 100 μM for 72hrs
239T
239T-
CXCR2
Tango
CXCR2
Tango
CXCR4
CX1142 32.0±5.5 38.1±9.2 1.2 >50 38.6 63.1 107.1 111.2
CX8875 0.6±0.01 5.5±4.2 9.4 15.9 39 30.9 63 57.6
CX2790 1.6±0.9 0.7±0.4 0.4 2.0 4.2 3.6 15.1 8
CX6766 2.8±1.7 5.6±2.1 2.0 15.9 4.2 3.7 38.7 32.9
CX8926 3.7±1.0 7.2±3.3 2.0 2.5 3.8 3.4 11.7 9.9
CX4596 4.4±2.7 24.7±9.8 5.6 25.1 NT
4
NT NT NT
CX7064 4.7±2.2 8.2±2.6 1.7 5.0 NT NT NT NT
CX0133 7.0±4.3 12.9±4.1 1.8 31.7 NT NT NT NT
CX2990 10.9±1.5 31.5±21.9 2.9 12.6 6.5 5.4 11.9 9.7
CX0518 20.1±8.7 63.1 3.1 >50 NT NT NT NT
CX1815 25.2±12.8 21.5±5.2 0.9 31.7 NT NT NT NT
CX8015 22.4 31.6 1.4 20.0 26.6 23.3 81.5 73.6
CX4071 27.7±10.4 45.2±24.2 1.6 15.9 5.9 3.3 68.6 60.2
CX9009 39.5±21.9 30.8±22.7 0.8 >50 39.7 25.9 65.2 63.5
CX2992 41.8±14.4 22.5±3.7 0.5 0.5 8.4 6.6 55.6 93.9
CX5574 0.5±0.04 3.5±2.1 6.8 2.0 3.9 3.4 12.4 7.7
CX1590 12.9±6.8 3.7±2.3 0.3 0.50 11.1 7.2 24.9 21.7
CX9294 18.0±3.8 30.1±15.3 1.7 31.62 61.1 51 129.2 83.3
CX9366 14.6±9.2 52.3±38.4 3.6 >50 102.8 91.3 91.3 85.1
CX7476 20.0 >100 >5 0.40 27.6 30.4 110 86.3
CX8000 2.1±0.4 11.5 5.5 28.9 10.05 6.35 NT NT
CX6104 4.8±6.4 45.4±35.9 9.5 13.5 3.55 3.55 NT NT
CX1424 5.0±3.2 21.3±2.6 4.3 35.5 15.85 7.95 NT NT
CX5065 6.6±3.2 7.7±5.0 1.2 25.5 39.85 33.15 NT NT
1
IC
50
= concentration (µM) required to inhibit 50% of CXCR2- or CXCR4-mediated β-arrestin-2
recruitment in the Tango assay.
2
Selectivity was calculated by dividing CXCR4 IC
50
values over CXCR2
IC
50
values.
3
H1299 cells were treated with CX compounds for 72hrs and cell proliferation inhibition was
assessed with MTT assay.
4
NT = not tested. Data represents mean ± SD of 2-3 independent experiments.
81

Figure 3.4 Chemical structures of CX compounds identified from the CXCR2 Tango.
In addition to CX4338, other classes of compounds from the Enamine database were also identified to
show activity against CXCR2. However, many of these compounds also inhibit CXCR4 and show anti-
proliferative properties.

82
3.2. CX4338 inhibits receptor internalization
Upon β-arrestin-2 recruitment during CXCL8 stimulation, CXCR2 is internalized via
clathrin-mediated endocytosis and routed for cell surface recycling or to late
endosomes/lysosomes for receptor degradation (Fan et al., 2003; Yang et al., 1999). To
assess surface CXCR2 expression, we performed a high-throughput in-cell Western
assay. CXCL8 significantly stimulated receptor internalization as indicated by a
significant decrease (56.7%) in cell surface CXCR2 expression within 30 minutes of
stimulation (Figure 3.5A and B). CX4338 dose-dependently inhibited CXCL8-mediated
receptor internalization with an IC
50
of 11.3 μM. SB265610 dose-dependently inhibited
CXCR2 internalization, with greater potency than CX4338 (IC
50
= 0.31 μM)
(approximately 30 fold difference), which is consistent with the Tango assay (Figure
3.5C). To assess receptor degradation, total CXCR2 was also assessed using a similar in-
cell Western assay (cells were permeabilized to detect both surface and intracellular
CXCR2). Treatment with CXCL8 also decreased total CXCR2 expression (22.5%) with
five hours of stimulation (Figure 3.5D and E). Pretreatment with CX4338 prior to
CXCL8 stimulation did not significantly inhibit receptor degradation after five hours of
stimulation with CXCL8, whereas SB265610 significantly inhibited receptor degradation
(Figures 3.5F). Taken together, CX4338 inhibits receptor internalization while having no
effects on receptor turnover.
83

Figure 3.5 CX4338 inhibits CXCL8-mediated CXCR2 internalization.
84
(A) CX4338 and SB265610 dose-dependently inhibits CXCL8-mediated CXCR2 internalization. CXCR2-
bla U2OS cells were seeded at 11,000 cells/well in 384-well black/clear bottom plates in assay media
(DMEM supplemented with 1% FBS). Cells pre-treated with CX4338 or SB265610 at indicated
concentrations for 30 minutes prior to stimulation with 50 nM of CXCL8 for an additional 30 minutes.
Compounds and CXCL8 was removed and cells were fixed with 4% formaldehyde for 20 minutes.
Formaldehyde was removed and plates were washed with 1X PBS. Plates were incubated with a
monoclonal antibody with an epitope targeting the first 19 amino acids of human CXCR2 at 1:1000
dilution in Licor blocking buffer overnight at 4°C. CXCR2 antibodies were removed and plates were
washed with 1X PBS three to six times. Plates were then incubated with mouse IR680 secondary antibody
at 1:1000 dilution in Licor blocking buffer for two hours at room temperature or overnight at 4°C.
Secondary antibodies was removed and plates was washed with 1X PBS three to six times. Plates were
spun upside down in centrifuge to remove excess liquid and allowed to dry at room temperature prior to
imaging on the Licor Odyssey bioimager. (B) Quantification of A showing percent CXCR2 internalized
induced by 50 nM of CXCL8. Data is normalized to unstimulated control and represents mean ± SD of a
representative experiment (n=12). ***indicates p-value >0.0001 calculated with the student t-test. (C)
Pretreatment with CX4338 and SB265610 dose-dependently inhibit CXCL8-induced receptor
internalization (same treatment as A). Data represents mean ± SEM of two independent experiments. (D)
CX4338 does not inhibit CXCL8-mediated CXCR2 turnover. Cells pre-treated with CX4338 or SB265610
at indicated concentrations for 30 minutes prior to stimulation with 50 nM of CXCL8 for an additional five
hours. Compounds and CXCL8 was removed and cells were fixed with 4% formaldehyde for 20 minutes.
Formaldehyde was removed and plates were washed with 1X PBS. Cells were permeabilized with 0.3%
Triton X100 prior to incubation with CXCR2 antibodies. CXCR2 antibodies and mouse IR680 secondary
antibodies were also diluted in Licor blocking buffer supplemented with 0.3% Triton X100. Total CXCR2
(membrane-bound and cytoplasmic) was detected. (E) Quantification of A showing percent CXCR2
turnover induced by 50 nM of CXCL8. Data is normalized to unstimulated control and represents mean ±
SD of a representative experiment (n=10). ***indicates p-value >0.0001 calculated with the student t-test.
(F) Quantification of images shown in D. CX4338 did not inhibit CXCL8-mediated receptor turnover, but
rather slightly enhance receptor turnover. SB265610 inhibited CXCL8-mediated receptor turnover above
100%, acting as inverse agonist. Data represents mean ± SEM of at least two independent experiments
performed in duplicates.
3.3. CX4338 enhances CXCR2-mediated G-protein signaling
Two main signaling cascades mediate CXCR2 signaling: β-arrestin coupling and G-
protein signaling mediated by second messengers such as calcium, kinases, and cyclic
AMP (Fan et al., 2001b; Hall et al., 1999; Su et al., 2005). Since we showed that
CX4338 inhibited CXCR2-mediated β-arrestin-2 recruitment, rearrangement, and
receptor internalization, we postulated that CX4338 would also inhibit CXCR2-mediated
G-protein signaling. Thus, we also assessed the effects of CX4338 on calcium
mobilization, ERK1/2 phosphorylation and cyclic AMP signaling.
85
Consistent with previous results, CXCL8 rapidly induced calcium mobilization,
reaching a peak at 30 seconds (Figure 3.6A and B) (Hall et al., 1999). SB265610 dose
dependently inhibited CXCL8-induced calcium mobilization when 293T-CXCR2 cells
were co-treated with SB265610 and CXCL8 (Figure 3.6A). However, co-treatment
CX4338 and CXCL8 enhanced peak calcium mobilization and prolonged calcium release
(Figure 3.6B). Pretreatment with SB265610 for 12 minutes prior to CXCL8 stimulation
shows that SB265610 alone induced slight increase in calcium release and dose-
dependently decreased CXCL8-induced calcium release at 12 minutes (Figure 3.6C).
CX4338 alone also induce slow calcium release in a time and dose-dependent fashion
(Figure 3.6D). Pretreatment with CX4338 prior to CXCL8 stimulation did not enhance
peak calcium and did not return to basal calcium levels.
86

Figure 3.6 CX4338 enhances CXCL8 induced calcium release.
(A) SB265610 inhibits CXCL8-induced calcium mobilization. 293T-CXCR2-GFP cells were seeded at
30,000 cells/well in 384-well black/clear bottom plates in complete media overnight. Cells were loaded
with Fluo-4 NW dye and 2.5 mM of probenecid and incubated at 37°C for 30 minutes and at room
temperature for an additional 30 minutes. Cells were co-stimulated with various concentrations of
SB265610 and 300 nM of CXCL8. Intracellular calcium flux was immediately read on the Envision plate
reader at 495/535 nm. Cells were also co-stimulated with 0.5% DMSO and 300 nM of CXCL8. Control
cells were not stimulated. (B) Cells were prepared as in A and co-stimulated with 300 nM of CXCL8 and
CX4338 at indicated concentrations. Cells were also co-stimulated with 0.5% DMSO and 300 nM of
CXCL8. Control cells were not stimulated. (C, D) Cells were pretreated with SB265610, CX4338, or
0.5% DMSO for 12 minutes prior to CXCL8 (300 nM) stimulation. Relative fluorescence unit (RFU) was
normalized to basal signal. Arrows indicates time of CXCL8 stimulation.
Cells stimulated with CXCL8 has been shown to transiently induce ERK1/2
phosphorylatioin, which is believed to be primarily mediated by G-protein signaling
(Nasser et al., 2009; Nasser et al., 2007). Consistent with previous studies, CXCL8
increased ERK1/2 phosphorylation, while pretreatment with SB265610 at 5 μM
completely inhibited ERK1/2 phosphorylation (Figure 3.7A). However, 30 min
pretreatment with CX4338 at 50 μM markedly enhanced CXCL8-induced ERK1/2
87
phosphorylation within five minutes and sustained up to 10 minutes (Figure 3.7B).
CX4338 without CXCL8 stimulation did not induce ERK1/2 phosphorylation.
Since previous studies showed that enhanced CXCR2-mediated G-protein signaling
in β-arrestin-2 deficient cells led to activation of stress kinases (Zhao et al., 2004), we
also assessed the effects of CX4338 on p38 and JNK activation. CX4338 alone slightly
induced p38 and JNK phosphorylation and enhanced CXCL8-mediated JNK
phosphorylation. CXCL8 did not induce p38 phosphorylation (Figure 3.7B). CX4338
and SB265610 did not induce ROS production (Figure 3.7C).

Figure 3.7 CX4338 enhances ERK1/2, p38, and JNK phosphorylation.
88
(A) CX4338 enhance ERK1/2 phosphorylation. 293T-CXCR2-GFP cells were seeded in 6-well plates at
500,000 cells/well for five hours in complete media (DMEM supplemented with 10% FBS) and serum
starved (DMEM) overnight. Cells were pre-treated with CX4338 (50, 20, 10 μM) or SB265610 (SB, 5
μM) for 30 minutes and stimulated with CXCL8 (20 nM) for 5 minutes. ERK1/2 phosphorylation was
detected using Western blot. Lanes indicated with C are control lanes for untreated and CXCL8-stimulated
cells. GAPDH was used as a loading control. Data shown is a representative blot from two independent
experiments. (B) CX4338 enhanced CXCL8-mediated MAP kinase signaling (p38, JNK and ERK1/2).
Serum starved 293T-CXCR2-GFP cells were pre-treated with CX4338 (50 μM) or 0.5% DMSO for 30
minutes prior to CXCL8 (20 nM) stimulation at the indicated times. (C) Effects of CX4338 on ROS levels.
293T-CXCR2-GFP cells were seeded in 96-well plates at 100,000 cells/well overnight. Cells were treated
with CX4338, SB265610, or 0.5% DMSO (vehicle control) with or without 25 μM of DCF dye for 30
minutes in PBS at 37°C. Cells were removed from plate and read on flow cytometer.
Using CXCR2 Glosensor cyclic AMP assay, we showed that SB265610 dose
dependently inhibited the effects of CXCL8 on forskolin induced cyclic AMP signaling
with an IC
50
of 3.8 ± 2.4 μM (Figures 3.8A and C), while CX4338 did not have any
effects on CXCL8-mediated cyclic AMP signaling at the range of concentrations tested
(Figures 3.8B and C).

Figure 3.8 CX4338 does not alter CXCL8-mediated cyclic AMP signaling.
89
293T-CXCR2-GFP-p22F cells were seeded at 30,000 cells/well in 384-well white plates in assay media
(CO2-independent media supplemented with 10% FBS). The next day, media was removed and cells were
loaded with 1% cyclic AMP reagent (Promega) diluted in assay media for two hours at room temperature
(in the dark). Cells were pre-treated with (A) SB265610 or (B) CX4338 at the indicated concentrations for
10 minutes prior to CXCL8 (50 nM) treatment for additional 10 minutes. Cells were then stimulated with
forskolin (50 μM) until max luminescence signal was reached (10-20 minutes). Data is normalized to
baseline (before compound treatment with compounds) and is a representative kinetic curve of 3
independent experiments. (C) Maximum signal was used to calculate % inhibition of CXCL8-mediated
forskolin cyclic AMP production. Data is shown as mean ± SEM of at least three independent experiments.

90
3.4. CX4338 inhibits CXCL8-mediated chemotaxis
After assessing the effects of CX4338 on CXCR2-mediated signaling, we next
assessed the cellular effects of CX4338. One of the main functions of CXCR2 and
CXCL8 is to regulate chemotaxis of neutrophils and endothelial cells during infection
and angiogenesis. CXCL8 markedly induced cell migration in both wound healing and
transwell cell migration assays in CXCR2 over-expressing cells (Figures 3.9A and B).
CX4338 and SB265610 dose-dependently inhibited CXCL8-mediated cell migration in a
wound healing assay (Figure 3.9A). Both CX4338 and SB265610 completely inhibited
cell migration at 10 μM. CX4338 also completely inhibited cell migration using
transwell inserts, at concentrations as low as 1 μM.
The anti-chemotactic effects of CX4338 on PMNs were investigated in in vitro and
in vivo models. In vitro testing using a modified boyden chamber transwell assay
measured human neutrophils chemotaxing toward 50 nM of CXCL8. We previously
determined 50 nM of CXCL8 was an optimal concentration to induce chemotaxis (data
not shown). As shown in Figures 3.9C, CX4338 inhibited CXCL8-induced chemotaxis
in a concentration-dependent manner with an IC
50
of 68.6 μM (Supplementary Figure 4).
More importantly in vivo, 5 mg/kg of CX4338 was also able to significantly inhibit PMN
migration (approximately 60 % inhibition) in a murine model of neutrophilic airway
inflammation. (Figure 3.9D). Interestingly, 3 mg/kg of SB265610 had similar efficacies
on PMN migration (p>0.05).
91

Figure 3.9 CX4338 inhibits CXCL8-mediated cell migration.
(A) CXCR2-bla U2OS were seeded in 96-well plates at 30,000 cells/well in DMEM supplemented with 1%
FBS overnight. A single wound was induced in with a sterile pipette tip and washed with 1X PBS. Cells
were treated with CX4338 or SB265610 at 10, 5 and 1 μM and stimulated with CXCL8 (100 nM) for 24
hours. Control wells (CTRL) were not stimulated with CXCL8. CXCL8 wells were treated with CXCL8
(100 nM). Representative images of two independent experiments performed in duplicates are shown here.
(B) CXCR2-bla U2OS cells were seeded on 24-well transwell inserts at 100,000 cells/well in DMEM
supplemented with 1% FBS overnight. The next day, cells were treated with 10, 5, 1 μM of CX4338 and
stimulated with CXCL8 (200 nM) for 48 hours. Cells in A and B were stained with Giesmsa stain for 45
minutes and imaged on BD Pathway bioimager at 10X magnification. (C) Concentration-dependent
inhibition of human neutrophil chemotaxis. Neutrophils were incubated with CX4338 for 1 hour, and
placed in the top wells of a chemotaxis plate containing 50 nM of CXCL8 in the bottom wells. Neutrophils
were allowed to chemotax for 2-4 hours at 37°C and 5% CO2. Inhibition of chemotaxis was evaluated
based on cell counts relative to control (untreated with CXCL8). CX4338 concentration-dependently
inhibited CXCL8-induced chemotaxis with an IC50 of 68.6 μM (95% CI 56.32 - 83.53, r2 = 0.90). Results
are mean ± SD of two to three experiments. (D) Attenuation of neutrophil recruitment in BALF at 24 hours
by pretreatment with CX4338 prior to intranasal LPS challenge of mice. CX4338 (5 mg/kg subcutaneously)
significantly inhibited neutrophil influx (*, p < 0.05). Results are mean ± SD of three animals per treatment
group (N=12).
92
3.5. CXCR2 expression in a panel of cancer cell lines
After the identification and characterization of CX4338, we sought to determine the
cellular effects of CX4338 on CXCR2-expressing cells. We performed RT-PCR and
Western blotting to determine the expression of CXCR2 in a panel of cancer cell lines at
the transcriptional and protein level. Using osteosarcoma cell line, U2OS (parental cell
line for the CXCR2-bla U2OS Tango cells), as a reference in our RT-PCR analysis, we
found that all NSCLC cell lines (H460, H2228, EKVX, H358, A549, H2347, H522) had
less CXCR2 transcript than U2OS, whereas leukemia cell lines (Jurkat and HL60)
expressed 16.4 and 8.5 times more CXCR2 mRNA than U2OS (Table 3.4). Both
CXCR2 over-expressing cell line (293T-CXCR2-GFP and CXCR2-U2OS) exhibit more
than 2000 fold increase in mRNA transcripts compared to U2OS. Using Western
blotting, we showed that 293T-CXCR2-GFP had higher CXCR2 protein expression than
CXCR2-bla U2OS cells, despite having similar transcript level (Figure 3.10). We also
showed that many NSCLC cell lines also expressed CXCR2 in the protein level despite
having low transcriptional levels. NSCLC cell lines also expressed more than 60-fold
increase in CXCL8 mRNA than U2OS (Table 3.5).
.
93
Table 3.4 CXCR2 transcriptional expression in a panel of cancer cell lines.

94
Table 3.5 CXCL8 transcriptional expression in a panel of cancer cell lines
95

Figure 3.10 CXCR2 expression in a panel of cell lines.
Cells were grown in complete growth media. Cell lysates was prepared using RIPA lysis buffer and 50 μg
of protein was loaded in a 10% SDS gel and transferred to a PVDF membrane. Membranes were incubated
with CXCR2 (E-2 Clone) from Santa Cruz Biotechnology at 1:1000 overnight at 4°C. The lower 43 kDa
band is the native CXCR2, whereas the higher 72 kDa band is GFP tagged CXCR2.
3.6. CXCR2 knockdown with siRNA reduced cell proliferation
Knockdown of CXCR2 with siRNA in CXCR2-bla U2OS and NSCLC cell lines
(H460, A549 and H1299) reduced cell proliferation when cells were grown in low serum
conditions (1% FBS) (Figure 3.11A). It is important to note that reduced cell
proliferation was not observed in normal serum conditions (10% FBS). We also found
that treatment with SB265610 in low serum conditions dose-dependently reduced cell
proliferation in CXCR2-bla U2OS, H1299 and CaOV3 (ovarian cancer) cell lines
(Figures 3.11B, C, and D). CX4338 did not reduce cell proliferation in CXCR2-bla
U2OS and H1299 cell lines when compared to DMSO controls (Figures 3.11B, C, and
D). To confirm the knockdown of CXCR2, CXCR2-bla U2OS cells treated with CXCR2
siRNA for 48 hours did not have any Tango activity when stimulated with CXCL8,
indicating the knockdown of CXCR2 (Figure 3.11E). As a control, CXCR4-bla U2OS
96
cells treated with CXCR2 siRNA retained its Tango activity when stimulated with SDF-1
(Figure 3.11F).

Figure 3.11 CXCR2 siRNA reduced cell proliferation in CXCR2-expressing cell lines.
(A) CXCR2-bla U2OS, CXCR4-bla U2OS, and NSCLC (H460, A549. H1299) cells were transfected with
30 nM of CXCR2 siRNA and scramble siRNA for 72 hours in low serum (1% FBS) conditions. Data is
normalized to lipofectamine control (cells transfected with lipofectamine only). Data is representative of
three independent experiments performed in triplicates and shown as mean ± SD. (B, C, and D) CXCR2-
bla U2OS (B) H1299 (C), and CaOV3 (D) cells were treated with SB265610, CX4338, 0.5% DMSO, 30
nM of CXCR2 siRNA, 30 nM of scramble siRNA (SRMB CTRL), and lipofectamine only (Lipo CTRL)
for 72 hours in low serum conditions (1% FBS). Data is normalized to untreated control and is
representative for two independent experiments represented as mean ± SD. (E and F) CXCR2-bla U2OS
(E) and CXCR4-bla U2OS (F) treated with lipofectamine, scramble siRNA or CXCR2 siRNA for 48 hours
were stimulated with 30 nM of CXCL8 or SDF-1 and β-arrestin-2 recruitment was assessed with the Tango
assay.
97
3.7. Discussion and Conclusions
Using a high throughput cell-based screening assay (Tango), we identified a novel
thioamide compound,

CX4338. CX4338 is structurally different from any reported
CXCR2 inhibitor (Figure 3.1). While performing similarity searches for CX4338 within
our in-house database of compounds, we found a similar compound, CX1142 (Figure
3.4). Removing the thioamide from CX4338 and replacing it with a ketone increased its
IC
50
from 6.5 μM to 32 μM in the CXCR2 Tango assay (Tables 3.1 and 3.3). It also
markedly reduced its specificity to CXCR2, suggesting the thioamide backbone structure
is essential for anti-CXCR2 activity as well as specificity. Additional optimizations of
CX4338 and structure activity relationship (SAR) analysis may be able to significantly
improve the potency and selectivity of CX4338 and further elucidate essential chemical
features.
We postulate that CX4338 and SB265610 differentially bind to CXCR2 since
CX4338 and SB265610 belong to distinct chemical classes and differentially regulate
intracellular receptor signaling as observed with cyclic AMP and ERK 1/2
phosphorylation as well as additive effects observed between SB265610 and CX4338 in
combination studies (Figure 3.2D). SB265610 acts as an allosteric, inverse agonist via
binding to an intracellular region of CXCR2 involving the C-terminus and intracellular
loop 1 (Bradley et al., 2009; Salchow et al., 2010). Though a common intracellular
allosteric site exists, studies with radiolabeled SB265610 and other CXCR2 antagonists
suggest that distinct classes of antagonists differentially bind to CXCR2, evidence of
multiple allosteric sites (de Kruijf et al., 2011; de Kruijf et al., 2009).
98
Mechanistic studies also suggest CX4338 regulates CXCR2 as a biased ligand, a
unique mechanism that has not been previously observed with CXCR2 ligands. Both
SB265610 and CX4338 inhibited CXCR2 β-arrestin-2 recruitment and subsequently
inhibited receptor internalization (Figures 3.2 & 3.5). However, CX4338 significantly
enhanced CXCR2/G-protein signaling (calcium mobilization and ERK1/2
phosphorylation) and did not affect CXCL8-mediated cyclic AMP signaling, while
SB265610 potently inhibited all G-protein signaling (Figures 3.6, 3.7, and 3.8).
Generally, most GPCRs are regulated through G-protein and β-arrestin1/2 signaling, and
it was thought that agonists or antagonists equally activate or inhibit all receptor-
mediated signaling (referred to as correlated efficacies) (Reiter et al., 2012). More
recently, a number of GPCR biased ligands have been identified. These ligands
selectively alter GPCR-mediated signaling (reviewed in (Rajagopal et al., 2010b)).
Though CXCR2 biased ligands have not been reported, biased ligands for other
chemokine receptors (CXCR4 and CXCR7) have been previously described (Balabanian
et al., 2005; Rajagopal et al., 2010a).
Biased inhibition of CXCR2/β-arrestin-2 signaling via CX4338 is consistent with
previous findings. Neutrophils derived from β-arrestin-2 knockout mice and RBL-2H3
cells expressing phosphorylation-deficient CXCR2 demonstrated reduced CXCR2-
mediated β-arrestin-2 translocation, receptor internalization, and chemotaxis (Richardson
et al., 2003; Su et al., 2005). Indirect β-arrestin1/2 inhibition via the deletion of G-
protein receptor kinase 6 (GRK6) that is required for CXCR2 phosphorylation and
subsequent β-arrestin1/2 recruitment, also showed decreased receptor internalization and
99
chemotaxis (Raghuwanshi et al., 2012). Since β-arrestin1/2 is essential for receptor
desensitization and negatively regulates G-protein signaling, enhanced CXCR2-mediated
G-protein signaling (calcium mobilization and ERK1/2, p38, JNK phosphorylation) was
also observed in CXCR2/β-arrestin-2 deficient models (Richardson et al., 2003; Su et al.,
2005). Zhao et al. also show that enhanced CXCR2/G-protein signaling in β-arrestin1/2
deficient cells led to activation of stress kinases including JNK and p38 and subsequent
NAPDH-oxidase (NOX) dependent ROS generation and cell death (Zhao et al., 2004).
Though, we also observe p38 and JNK activation upon CX4338 treatment (Figure 3.7B),
we did not observe significant increase in ROS levels (Figure 3.7C), which might explain
the lack of cell death observed during CX4338 treatment (Table 3.2). Perhaps, the effects
of ROS-mediated cell death are dependent on NOX expression and thus cell line
dependent. The proposed mechanism of action of CX4338 is represented in a schematic
diagram in Figure 3.12.
A counterscreen with CXCR4 Tango

assay supports that CX4338 interacts with the
CXCR2 receptor and not β-arrestin-2. CXCR2 and CXCR4 belong to the same
chemokine family and are activated by different ligands. Every component of the Tango
assay is identical, except for chemokine expression and ligand activation. Thus, we do
not anticipate selectivity in CXCR2/4 Tango assays if β-arrestin-2 is the target of
inhibition. CX4338 selectively inhibited CXCR2/β-arrestin recruitment over CXCR4/β-
arrestin-2 recruitment (selectivity is greater than 16, Table 3.1), which provides evidence
that CX4338 selectively interacts with CXCR2 and the effects of CX4338 observed in
CXCR2/β-arrestin-2 and G-protein signaling is not due to nonspecific β-arrestin-2
100
inhibition, but may involve the upstream phosphorylation of the CXCR2 receptor via
GRK6. Additional studies will be required to confirm this.

Figure 3.12 Proposed mechanism of CX4338.
CX4338 selectively inhibits CXCL8-induced β-arrestin-2 recruitment and subsequent receptor
internalization, recycling and degradation. Receptor recycling and degradation negatively regulates
CXCR2-mediated G-protein signaling. However, selective inhibition of CXCR2/β-arrestin signaling via
CX4338 leads to sustained and enhanced CXCR2-mediated G-protein signaling which include MAP
kinases (JNK1, p38, and ERK), calcium mobilization from the ER to the cytoplasm, and adenylyl cyclase
inhibition.
These studies also show that selective inhibition of CXCR2-mediated β-arrestin-2
signaling by CX4338 is sufficient to inhibit CXCL8-mediated in vitro chemotaxis (Figure
5), which is consistent with previous experiments with β-arrestin1/2 deficient models
(Raghuwanshi et al., 2012; Richardson et al., 2003). Importantly, CX4338 also
significantly reduced LPS-stimulated chemotaxis of neutrophils into BALF in mice
101
(Figure 3.9D), which is contrary to previous studies that showed β-arrestin-2 deficiency
up-regulated CXCL1-mediated chemotaxis in in vivo mice models (dorsal air pouch and
excisional wound healing assay) (Raghuwanshi et al., 2012; Su et al., 2005). The
observed discrepancy might be due to different in vivo models used to assess chemotaxis.
Dose groups were similar with no statistically significant difference in the
pharmacodynamics endpoint of airway PMNs. Further studies to describe the
pharmacokinetics of CX4338 and SB265610 will aid in evaluation of CX4338
therapeutic potential. Interestingly, CX4338 did not reduced cell proliferation as
observed with SB265610 and CXCR2 siRNA in CXCR2-bla U2OS, H1299, and CaOV3
cell lines, suggesting that selective inhibition of CXCR2/β-arrestin-2 is not sufficient to
alter cell proliferation (Figure 3.11).
In conclusion, we have identified a distinct class of CXCR2 antagonists with a
unique mechanism of action that shows promising therapeutic potential. CX4338 inhibits
CXCR2 as a biased antagonist that selectively targets CXCR2/β-arrestin-2 coupling and
selective inhibition of CXCR2/β-arrestin-2 is sufficient to inhibit CXCL8-mediated
chemotaxis. CX4338 can also be used as a mechanistic marker to understand the role of
CXCR2/β-arrestin-2 signaling and warrants further optimization and development.
3.8. Materials and Methods
Neutrophil Chemotaxis. Blood was collected from healthy human volunteers for
neutrophil isolation. All experiments were conducted with the approval of the University
of Southern California Institutional Review Board. Blood was collected into EDTA-
sprayed tubes (Greiner Bio-One, Monroe, NC), and neutrophils were isolated using One-
102
Step Polymorph separation media (Accurate Chemical and Scientific, Westbury, NY)
with centrifugation at 500 g for 35 minutes according to manufacturer’s
recommendations. RBC Lysis Buffer (IBI Scientific, Peosta, IA) was used to remove
residual erythrocytes. The final pellet of neutrophils was resuspended in HBSS without
Ca
2+
or Mg
2+
(Lonza, Walkersville, MD) supplemented with 1% BSA (Amresco, Solon,
OH) and kept on ice. Neutrophils were counted using Turk’s Blood Diluting Fluid (Ricca
Chemical, Arlington, TX) and a hemocytometer. Viability was assessed by tryphan blue
exclusion. All preparations consisted of approximately 95% viable neutrophils.
Neutrophils were incubated in triplicate with CX4338 over four logs of concentration
or vehicle (1% DMSO) for 1 hour. Neutrophils were then placed in the top wells of a 96-
well MultiScreen-MIC plate with 3 µm pore size (Millipore, Billerica, MA) containing
50 nM of CXCL8 in the bottom wells. Neutrophils were allowed to migrate for 2-4 hours
at 37°C and 5% CO
2
. In some experiments, the migrated neutrophils in the bottom wells
were stained with Turk’s Blood Diluting Fluid and manually counted with a
hemocytometer. In other experiments, the migrated neutrophils were labeled with
CyQuant NF dye (Invitrogen, Eugene, OR), and fluorescence intensity was measured
using a spectrofluorometer with excitation at 485 nm and emission at 530 nm (Perkin
Elmer). Neutrophil count was calculated using a standard curve. Percentage of maximal
chemotaxis was calculated using the following formula: % maximal chemotaxis = [(#
treated cells migrated with CXCL8 - # untreated cells migrated without CXCL8) / (#
untreated cells migrated with CXCL8 - # untreated cells migrated without CXCL8)] x
103
100. Data was plotted using a sigmoidal E
max
model to estimate IC
50
. the concentration
associated with a 50% inhibition of the maximal chemotactic response (IC
50
).
LPS-induced Lung Inflammation. Eight- to 10-week-old male BALB/c mice were
obtained from Charles River and were maintained in the animal facilities at Zilkha
Neurogenetic Institute (Los Angeles, CA). Twelve total mice with 3 in each treatment
group (LPS alone n=3, CX4338 n=3, and SB265610 n=3) at 24 hours were used for
pharmacodynamics evaluation of LPS induced airway inflammation. Animals had access
to food and water and were kept in controlled laboratory conditions with average
temperature of 71°F and a light–dark cycle of 12 hours.
Acute lung inflammation was induced by intranasal LPS insufflation (1 µg/20 g body
weight) under light sedation (100 µL of a ketamine (80 mg/kg) and xylazine (10 mg/kg)
solution). At 24 hours, airway leukocytes were collected by bronchoscopy.
Bronchoalveolar lavage was performed after lethal intraperitoneal administration of
Euthasol (75 mg/kg). The trachea was exposed through a midline incision with
subsequent insertion of a 26G tracheal tube. The lungs were washed three times with
three different aliquots of PBS (1 mL, 0.8 mL, 0.8 mL) with recovery via syringe
aspiration of 86%. BAL samples were placed on ice and centrifuged at 360 g and 4°C for
10 minutes. The cell pellet was used to evaluate the number of infiltrating leukocytes.
Total and differential airway cell counts were determined for each BALF sample using a
hemocytometer and Wright stain to examine nuclear morphology under the microscope.
A single 1 mL injection containing CXCR2 antagonists (CX4338, SB265610)
diluted in 5% DMSO in normal saline or vehicle only (5% DMSO in normal saline) was
104
administered subcutaneously 30 minutes prior to LPS insufflation. All compounds were
aseptically prepared and sterile filtered through a 0.22 micron filter.
All animal work was conducted with the approval of the University of Southern
California Institutional Animal Care and Use Committee (Protocol #11675). All studies
involving animals are reported in accordance with the ARRIVE guidelines (Kilkenny et
al., 2010; McGrath et al., 2010).
RT-PCR analysis. RNA was extracted from U2OS, CXCR4-bla U2OS, CXCR2-bla
U2OS, leukemia (Jurkat and HL60), ovarian cancer (CaOV3), and NSCLC (H460,
H2228, EKVX, H358, A549, H2347, and H522) cell lines with Aurum Total RNA Mini
Kit (Bio-Rad, Hercules, CA). RNA concentration was determined on the NanoDrop
instrument and one microgram of RNA was converted to cDNA with the High-Capacity
cDNA Reverse transcription kit from Applied Biosystems (Life Technologies, Carlsbad,
CA). One microliter of the cDNA reaction was used for RT-PCR with the SsoAdvanced
SYBR Green Supermix (BioRad) in a 20 μL reaction in 96-well FAST plates. 200 nM of
CXCR2, CXCL8, and GAPDH primers were used for each reaction. RT-PCR was
performed with the ABI 7900 Fast Real-Time PCR system (Life Technologies) for a total
of 50 cycles with annealing temperatures between 55-60°C. Fold change was calculated
using the delta delta C
T
method (Pfaffl). The following primer sets were used:
GAPDH FORWARD: 5’-ACGCATTTGGTCCTATTGGG-3’ (Tm=55.8)
GAPDH REVERSE: 5’-TGATTTTGGAGGGATCTCGC-3’ (Tm=54.8)
CXCL8 FORWARD: 5’-ATGACTTCCAAGCTGGCCGTG-3’ (Tm=62.2)
CXCL8 REVERSE: 5’- CATAATTTCTGTGTTGGCGCA-3’ (Tm=60.5)
105
CXCR2 FORWARD: 5’-TGCATCAGTGTGGACCCTTA-3’ (Tm=56.4)
CXCR2 REVERSE: 5’-CCGCCAGTTTGCTGTATTG-3’ (Tm=54.5)

CXCR2 siRNA transfection (cell proliferation). Cells were seeded at 12,000
cells/well in 96-well in OPTI-MEM (Life Technologies) supplemented with 1% FBS and
transfected with CXCR2 siRNA (Santa Cruz Biotechnology, Cat # SC-40028 ) and
scramble siRNA (Santa Cruz Biotechnology, Cat# Sc-37007) with Lipofectamine
RNAiMAX transfection reagent (Life Technologies) on the same day. CXCR2 siRNA
and scramble siRNA was diluted to a concentration of 600 nM in OPTI-MEM.
Lipofectamine was diluted in OPTI-MEM to a concentration of 600 nM. (The final
concentration of both reagents in the wells is 30 nM). Lipofectamine and siRNAs was
mixed together at room temperature and allowed to sit for 30 minutes prior to treatment
with cells. Lipofectamine controls was treated with lipofectamine reagent only. Cells
were allowed to grow for 72 hours and cell proliferation was measured with AlamarBlue
(ABD Serotec, Kidlington, UK) according to manufacturers protocol. siRNA’s and
lipofectamine reagents were not removed during the 72 hour incubation time. Cells were
grown in OPTI-MEM supplemented with 1% FBS. The cell proliferation effects of CX
compounds on cells in low serum condition was also performed in the same plates with
cells treated with CXCR2 siRNAs.
CXCR2 siRNA transfection (Tango assay). CXCR2-bla U2OS cells were seeded at
500,000 cells/ well in 6-well tissue culture plates

106
Statistical Analysis. Statistical and graphical analysis was done using GraphPad
Prism version 5.04 (GraphPad Software, San Diego, CA). p-values was calculated using
unpaired student t-test.

107
CHAPTER 4: IDENTIFICATION AND MECHANISTIC STUDIES OF NOVEL
PYRIMIDINE-BASED CXCR2 INHIBITORS
In these studies, we sought to identify additional classes of CXCR2 antagonists by
screening an in-house library of compounds (Russian III) in a CXCR2/G-protein-
mediated cyclic AMP assay. We have identified a class of small-molecules, CX797, that
inhibited CXCL8-mediated cyclic AMP signaling and receptor degradation, while
specifically up-regulating CXCL8-mediated β-arrestin-2 recruitment and MAP kinase
signaling (Figure 4.1). CX797 also inhibited CXCL8-mediated cell migration as well as
cell proliferation.

108
4.1. CX797 inhibits the effects of CXCL8 on cAMP signaling

Figure 4.1 Chemical structure of CX compounds.
Compounds were identified from a high throughput screen using the Glosensor cyclic AMP assay.
Using the Glosensor cyclic AMP assay previously described in section 2.2.4, we
screened a library of 1000 diverse compounds initially at 4 µg/µL. CXCL8 significantly
reduced forskolin-induced cyclic AMP production in 293T-CXCR2-GFP-p22F cells
(Figure 4.2A). CX797 showed comparable potency as SB265610 in the cyclic AMP
assay with IC
50
values of 7.8 and 3.8 μM, respectively. (Figures 4.2B and C, Table 4.1).
Next, we performed similarity searches for analogues of CX797 that were commercially
available. CX119 is the most active of this class of compound, with an IC
50
of 2.3 μM
(Figures 4.2C and D). However, analogues CX143, CX894, CX798, CX455, and CX877
were less potent at inhibiting the effects of CXCL8 on forskolin-induced cyclic AMP
signaling with IC
50
’s greater than 20 μM (Figure 4.2C). Cyclic AMP kinetic curves of
CX797, CX143, CX119, and SB265610 are shown in Figure 4.3.
109

Figure 4.2 CX compounds inhibits CXCL8-mediated cyclic AMP signaling.
(A) CXCL8 inhibits forskolin-stimulated cyclic AMP signaling. 293T-CXCR2-GFP-p22F cells were
untreated (control), treated with forskolin (FOR) at 50 μM, or pre-treated with CXCL8 at 50 nM for 10
minutes and stimulated with forskolin (CXCL8 + FOR) for 15-20 minutes until maximum signal was
reached. Data represents mean ± SD. * indicates p-value > 0.01, ** indicates p-value > 0.001 calculated
using the student t-test. (B) SB265610 and CX compounds inhibited CXCL8-mediated, forskolin-
stimulated cyclic AMP signaling. Cells were pre-treated with SB265610 or CX compounds at 20 μM for
10 minutes prior to CXCL8 (50 nM) treatment for 10 minutes. Cells were then stimulated with 50 μM of
forskolin until max signal was reached (15-30 minutes). Data represents mean ± SD of 3-7 experiments.
(C, D) Dose response of SB265610, CX797, CX119, and CX143 using treatment conditions as described in
B.

110
Table 4.1 CX Compounds inhibits CXCL8 down-regulation of forskolin-induced cyclicAMP
Compound IC
50
(µM)
1

SB265610 3.78±1.09
CX797 7.79±1.15
CX119 2.31±1.71
CX143 29.89±2.21
1
IC
50
= mean value of 3 experiments ±SD

Figure 4.3 Kinetic cyclic AMP curves of CX797, CX143, CX119, and SB265610.
293T-CXCR2-GFP-p22F cells were seeded at 30,000 cells/well in 384-well white plates in assay media
(CO2-independent media supplemented with 10% FBS). The next day, media was removed and cells were
loaded with 1% cyclic AMP reagent (Promega) in assay media for two hours at room temperature (in the
dark). Cells were treated CX797 (A), CX143 (B), CX119 (C), and SB265610 (D) at the indicated
concentrations and immediately read on the Envision plate reader (time = 0) for 10 minutes, then treated
with CXCL8 (50 nM, time = 10) for 10 minutes, and stimulated with forskolin (50 μM, time =20) for an
additional 30 minutes. Cyclic AMP kinetic curves is normalized to baseline luminescence signal (prior to
compound treatment). Control cells were untreated, FORK only cells were stimulated with forskolin and
was not pre-treated with CXCL8, FORK + CXCL8 cells were pre-treated with CXCL8 and stimulated with
forskolin at the times indicated by the arrows.
111
4.2. CX797 enhances CXCL8-mediated β-arrestin-2 recruitment
Next, we used the CXCR2 Tango

assay to detect the effects of CX797 on CXCL8-
mediated β-arrestin-2 recruitment. Interestingly, instead of inhibiting β-arrestin-2
recruitment as seen with SB265610, CX797 and its analogues synergistically increased
CXCL8-mediated β-arrestin-2 recruitment at the concentrations tested (Figure 4.4A).
CX797 does not act as an CXCR2 agonist since CX797 alone does not activate β-
arrestin-2 recruitment at 10 and 20 µM (Figure 4.4B). Next, to assess the selectivity of
CX797, we tested CX797 in the CXCR4 Tango assay and found that CX797 did not have
any effects on β-arrestin-2 recruitment upon ligand stimulation with SDF-1 (Figure
4.4C). Since CX797 and SB265610 behave differentially in the CXCR2 Tango

assay, we
next assessed the interaction of CX797 and SB265610 in combination studies. The
addition of CX797 at 10 µM reduced the activity of SB265610 (50 nM) in the CXCR2
Tango assay (Figure 4.4D).
112

Figure 4.4 CX797 selectively increases CXCL8-mediated β-arrestin-2 recruitment.
Receptor activation was measured using the CXCR2 Tango assay. (A) CXCR2-bla U2OS cells were
seeded at 11,000 cells/well in 384-well black plates in assay media (DMEM supplemented with 1% FBS).
The next day, cells were treated with CX797 or SB265610 at indicated concentrations for 30 minutes prior
to stimulation with CXCL8 (20 nM) for five hours. Β-arrestin-2 recruitment was measured using
LiveBLAzer β-lactamase substrate kit (Invitrogen). Data represents mean ± SD of three to four
independent experiments. (B) CXCR2-bla U2OS cells were prepared as in A. Cells were treated with
CX797at indicated concentration without CXCL8 stimulation. Data represents mean ± SD of one
experiment performed in triplicates. (C) CX797 is selective for CXCR2 over CXCR4. CXCR2 or
CXCR4-bla U2OS cells were pre-treated with CX797 for 30 minutes and stimulated with CXCL8 (20 nM)
or SDF-1 (30 nM]), respectively, for five hours. (D) CX797 antagonizes the inhibitory effects of
SB265610 on β-arrestin-2 recruitment. CXCR2-U2OS cells were pre-treated with various concentrations
of SB265610 or SB265610 and CX797 [10 μM] for 30 minutes, followed by CXCL8 (20 nM) stimulation
for five hours. Data shown represents mean ± SD of one experiment performed in duplicates.

113
4.3. CX797 inhibits CXCL8-mediated CXCR2 degradation
Given the ability of CX797 to enhance β-arrestin-2 recruitment in the presence of
CXCL8 stimulation, we further pursued the effects of CX797 in β-arrestin-2 mediated
pathways. As a mediator of signal termination, β-arrestin1/2 regulates CXCR2
internalization and desensitization via clathrin-mediated endocytosis (Yang et al., 1999).
Upon receptor internalization, CXCR2 is differentially routed by Rab GTPases for
recycling back onto the cell surface or to late endosomes/lysosomes for degradation when
exposed to prolong ligand stimulation (Fan et al., 2003). First, we assessed the effects of
CX797 on basal receptor turnover. 293T-CXCR2-GFP cells treated with CX797 or
SB265610 for 24 hours dose-dependently increased CXCR2 as indicated by an increased
in GFP signal (Figures 4.5A). CXCL8 induced CXCR2 degradation, while pretreatment
with CX797 dose-dependently inhibited CXCL8-mediated receptor degradation, with
complete inhibition at 20 μM (Figure 4.5B). SB265610 was more effective than CX797
at inhibiting receptor degradation (Figure 4.5B). SB265610 at 5 μM significantly and
completely inhibited CXCL8-medaited CXCR2 degradation as well as inhibited basal
receptor turnover that is indicated with greater than 100% inhibitory effects seen in all
concentrations we tested. Immunofluorescence of 293T-CXCR2-GFP cells treated with
SB265610 or CX797 also show a significant up-regulation of CXCR2 compared to
untreated control (Figure 4.5C). As a control, CX797 did not increase GFP signal in cells
expressing GFP-tagged β-arrestin-2 (HEK293-CXCR2-β-arrestin-2-GFP, Figure 4.5 D).
114

115
Figure 4.5 CX797 and SB265610 alters CXCR2 expression.
(A) 293T-CXCR2-GFP cells were seeded at 50,000 cells/well in 96-well clear tissue culture plates in
complete growth media. The next day, cells were treated with various concentration of CX797 or
SB265610 for 24 hours. Compounds were removed and cells were washed with 1X PBS. Cells were
suspended in 1X PBS and GFP signal was measured using flow cytometry in the FITC channel. Mean fold
change was normalized to untreated 293T-CXCR2-GFP cells. Data represents mean ± SD of four to five
independent experiments. * indicates p-value >0.05, ** indicates p-value >0.005, calculated using the
student t-test. (B) CX797 and SB265610 inhibit CXCL8-mediated CXCR2 degradation. Cells were
prepared as in A. Cells were pre-treated with CX797 and SB265610 for five hours and stimulated with
CXCL8 (50 nM) for additional 24 hours. Cells were collected and GFP signal was detected as in A. Data
shown represents mean ± SD of at least three independent experiments performed in triplicates. * indicates
p-value >0.05 calcuated with student t-test. (C) 293T-CXCR2-GFP cells were seeded in 384-well black
plates at 10,000 cells/well in complete growth media. The next day, cells were treated with CX797 or
SB265610 at 20 μM for five hours and imaged on the BD Pathway 435 Bioimager with a GFP filter. (D)
HEK293-CXCR2-β-arrestin-2-GFP cells were prepared and treated with CX797 as in A. Data shown
represents mean ± SD of one experiment performed in triplicates.

116
4.4. CX797 inhibits CXCL8-mediated CXCR2 internalization
Using in-cell Western, we showed that CX797 and its analogues (CX119 and
CX143) enhanced total and surface CXCR2 expression in similar fashion as SB265610
(Figure 4.6). CX compounds dose-dependently increased total CXCR2 expression within
five hours of compound treatment. CX compounds also slight increased surface CXCR2
expression. Next, we assessed the effects of CX797 on receptor internalization induced
by CXCL8 stimulation. CXCR2-U2OS Tango cells were pre-treated with CX797 or
SB265610 for 30 minutes followed by CXCL8. CX797 slightly inhibited CXCL8-
mediated receptor internalization at 20 μM (26 % inhibition, Figures 4.7A and B). In
contrast, treatment with SB265610 potently inhibited CXCL8-mediated CXCR2
internalization more than 80% at all the concentrations tested (Figures 4.7A and B).
CXCL8 stimulation not only reduced surface but also total CXCR2 expression (Figure
4.7C), consistent with our flow cytometry results. Both CX797 and SB265610
significantly inhibited CXCL8-mediated CXCR2 degradation at 5 μM (64% and 146%
inhibition, respectively, Figures 4.7C and D).
117

Figure 4.6 CX797 enhancse total and surface CXCR2 expression.
CXCR2-bla U2OS cells were seeded in 384-well black plates at 11,000 cells/well in assay media (DMEM
supplemented with 1% FBS). The next day, media was removed and cells were treated with CX119,
CX143, CX797, and SB265610 for five hours. Total (A) and surface (B) CXCR2 expression was assessed
using in-cell Western. (C, D) Quantification of (A) and (B), respectively. Data is representative of three
independent experiment performed in duplicates and expressed as mean ± SD.
118

Figure 4.7 CX797 inhibits CXCL8-mediated CXCR2 degradation in In-cell Western assays.
CXCR2-bla U2OS cells were seeded in 384-well black plates at 11,000 cells/well in assay media (DMEM
supplemented with 1% FBS). The next day, media was removed and cells were pre-treated with CX797 or
SB265610 at indicated concentrations for 30 minutes and stimulated with CXCL8 (50 nM) for additional
five hours. Cells were fixed with 4% formaldehyde. Total (A, B) and surface (C, D) CXCR2 expression
was assessed using in-cell Western. (A) Data shown represents mean ± SD of three to four independent
experiments. CTRL wells were untreated. CXCL8 wells were stimulated with CXCL8 (50 nM).
*indicates p-value >0.05, ** p-value >0.005, *** p-value > 0.0005, calculated using student t-test. (B) A
representative image of A is shown in B. (C) CX797 and SB265610 inhibit CXCL8-mediated CXCR2
degradation. Cells were treated and fixed as in A, however, cells were permeabilized with 0.3% Triton
X100 to detect total CXCR2 expression. Data shown represents mean ± SD of two independent
experiments performed in duplicates. *indicates p-value >0.05, ** p-value >0.005, *** p-value > 0.0005,
calculated using student t-test. (D) A representative image of data shown in C.
4.5. CX797 enhances CXCL8-mediated ERK1/2 phosphorylation.
CXCR2 couples to Gαi-protein and activates phosphatidylinositol-3 kinase (PI3K),
promoting Akt signaling (Knall et al., 1997). PI3K activation is coupled with the
activation of the MAP kinase pathway which promotes cell proliferation (Knall et al.,
1996). Zhao et al. also showed that upon CXCR2 activation, β-arrestin1/2 complexes
with CXCR2 and components of the ERK1/2 signaling cascade (Zhao et al., 2004). To
assess the effects of CX797 on CXCL8-mediated Akt and ERK1/2 activation, we used
119
Western blotting to detect Akt and ERK1/2 phosphorylation. Pretreatment with
SB265610 completely inhibited CXCL8-induced ERK1/2 phosphorylation, while
pretreatment with CX797 greatly enhanced CXCL8-induced ERK1/2 phosphorylation at
all doses tested (Figure 4.8). Akt phosphorylation was not increased upon CXCL8
stimulation and both CX797 and SB265610 had no detectable effects on Akt
phosphorylation.

Figure 4.8 CX797 up-regulates CXCL8-mediated ERK1/2 phosphorylation
293T-CXCR2-GFP cells pre-treated with CX797 at indicated concentrations or SB265610 (5 μM) for 30
minutes and stimulated with 20 nM of CXCL8 for five minutes. Control (C) cells were untreated with
compounds.

120
4.6. CX797 inhibits cell migration in wound healing assay
Activation of neutrophils and endothelial cells resulting in chemotaxis is one of the
main functions of CXCR2 and CXCL8 (Addison et al., 2000). CXCL8 also induce
cancer cell migration and invasion (Ning et al., 2011). Thus, we used a wound healing
assay to assess the effects of CX797 on CXCL8-induced cell migration. CXCL8
significantly induced wound healing in CXCR2-U2OS Tango cells compared to
unstimulated control (Figure 4.9). Pretreatment with CX797 or SB265610 prior to
CXCL8 stimulation dose-dependently inhibited CXCL8-mediated wound closure. No
cytotoxicity was observed in all treatment conditions.

Figure 4.9 CX797 dose dependently inhibit cell migration.
Using the wound healing scratch assay, effects of CX797 or SB265610 on cell migration was assessed. A
single wound was induced with a sterile pipette tip and Tango CXCR2-bla U2OS cells were treated with
CX797 or SB265610 at indicated concentrations and stimulated with CXCL8 (100 nM) in DMEM
supplemented with 1% FBS for 24 hours. Control wells were stimulated with CXCL8 (100 nM) alone or
media only to detect CXCL8 induced and basal cell migration. Representative images of two independent
experiments performed in duplicates are shown.
121
4.7. CX797 induces cytostatic effects on HL60, Jurkat, and NSCLC cell lines.
Since CXCR2 has been implicated in endothelial and cancer cell proliferation (Li et
al., 2003; Ning et al., 2012), we determined the effects of CX797 and SB265610 on
leukemia (Jurkat and HL60) and non-small cell lung (NSCLC) (H1299, H460, EKVX,
and A549) cancer cell proliferation using the Alamar Blue and MTT assays. CX797 and
SB265610 significantly inhibited leukemia cell proliferation at higher concentrations (50
µM) with more than 50% inhibition (Figures 4.10A and B) in Jurkat cells. CX797
inhibited less than 50% at 50 µM in H460, A549, and EKVX NSCLC cell lines (Figure
4.10C). CX797 did not inhibit colony formations in H1299 and A549 cells at the various
concentrations tested (Figure 4.10D). Next, we performed cell cycle analysis. SB265610
arrested cells in G0/G1 in both HL60 and Jurkat cells with a pronounced increase in
HL60 cells. CX797 had no effects on cell cycle in HL60 cells, while producing a modest
G0/G1 arrest in the Jurkat cells (Figures 4.10E and F). To assess whether the effects of
CX797 and SB265610 on cell proliferation were dependent on CXCR2 levels, we tested
the compounds on CXCR2-overexpressing (293T-CXCR2-GFP) and 293T cell
proliferation. Both compounds inhibited both cell lines in a similar fashion, with no
significant differences between CXCR2-overexpressing and parental 293T cell lines
(Figures 4.10 G and H).
122

Figure 4.10 CX797 induces cytostatic effects on HL60, Jurkat, and NSCLC cells.
HL60 (A) and Jurkat (B) cells were seeded at 10,000 cells/well in 384-well black plates overnight. Cells
were treated with SB265610, CX797, or DMSO at various doses for 72 hours. Alamar Blue was added for
four hours and the plates were read at 535 nm excitation and 585 nm emission on the Envision Multilabel
plate reader. (C) NSCLC cell lines were treated as in A and B, and cell proliferation was measured using
MTT. (D) CX797 does not show significant cytotoxicity at doses as high as 50 μM in H1299 and A549
cells using colony formation assay. (E, F) CX797 arrest cells in G0/G1 phase in Jurkat cells. (E) HL60 or
(F) Jurkat cells were seeded at 100,000 cells/well in 6-well plates. Cells were treated with CX797 or
SB265610 at 50 μM for 72 hours and fixed with 70% ethanol. (G) SB265610 and (H) CX797 inhibited
293T-CXCR2 (CXCR2 over-expressing) and 293T cell proliferation. Cells were seeded at 1,000 cells/well
in 96-well clear plates overnight. Cells were treated with compounds for 72 hours and cell proliferation
was measured using MTT. Data shown represents mean ±SEM of at least three independent experiments.
** indicates p-value <0.01, * indicates p-value <0.05 using student t-test.
123
4.8. Discussions and conclusions
Since compounds that can efficiently raise cyclic AMP levels have shown
effectiveness in the clinic by significantly reducing inflammation, we designed our initial
screen to exploit this aspect of the CXCR2 receptor. For example, PDE4 inhibitors, such
as Roflumilast, are currently used in the clinic for severe COPD associated with chronic
bronchitis (Rennard et al., 2011). Roflumilast up-regulates cyclic AMP levels in many
immune cell types that express PDE4 by preventing the breakdown of cyclic AMP
(Spina, 2008). In the case of CXCR2, the receptor is coupled to Gαi protein that inhibits
adenylyl cyclase, the enzyme that converts ATP to cyclic AMP (Hall et al., 1999). While
PDE4 inhibition prevents the breakdown of cyclic AMP, CXCR2 inhibition increases
cyclic AMP production, both mechanisms resulting in increased cyclic AMP.
Thus, we sought to identify inhibitors that can effectively alter cyclic AMP levels
mediated by CXCR2. We employed a high throughput, cell-based, CXCR2-specific
cyclic AMP assay utilizing the Glosensor
TM
technology from Promega to screen for
CXCR2 inhibitors in our in-house library of highly diverse compounds. We have
identified a pyrimidine-based class of compounds that has been previously synthesized
and shown to have no activity against 5-HT2A activity at 100 μM (El-Baih et al., 2006;
El-Kerdawy et al., 2010). Additionally, derivatives of this class of compounds have been
shown to exhibit CNS depressant and skeletal muscle relaxant activities in animals
treated with a dose of 5mg/kg (Gupta et al., 2009). CXCR2 is also expressed in neurons
and stimulation with CXCL1 induced calcium mobilization, enhanced neurotransmitter
release, and impaired long-term depression of synaptic strength, which suggest that the
124
inhibition of CXCR2 may have neurological effects that are observed in CX797
derivatives previously reported in Gupta et al. (Giovannelli et al., 1998; Gupta et al.,
2009).
CX797 and its analogues inhibited CXCL8-mediated down-regulation of cyclic
AMP. Similar results were observed with previously reported CXCR2 inhibitor,
SB265610. Though the breadth of CX797 analogues in our studies is limited, it provides
a platform for further optimization of CX797 and essential insights into the compound’s
activity-structure relationship. For example, removal of the chiral methyl group (CX119)
improved its IC
50
from 7.8 to 2.3 μM. Also, additional alkylation and substitution on the
sulfur group (CX143, CX984, CX982) significantly reduced activity (Figures 4.1 and
4.2C, Table 4.1).
CX797 differentially altered CXCR2 signaling by inhibiting CXCR2/CXCL8-
mediated G-protein signaling and significantly enhancing CXCL8-mediated β-arrestin-2
recruitment in the Tango assay (Figures 4.2, 4.3, and 4.4), suggesting that CX797 may act
as a biased ligand. A number of studies and reviews by the Lefkowitz group have
challenged the conventional view of correlated efficacies, in which ligand binding
equally stimulates or inhibits all receptor signaling and function (Rajagopal et al., 2010b;
Reiter et al., 2012; Violin and Lefkowitz, 2007). The most widely studied biased GPCR
is the angiotensin II type 1 receptor (AT
1
). AT
1
receptor activation via its ligand
angiotensin II activates both G-protein and β-arrestin signaling, however, stimulation
with another agonist (Sar1, Ile4, Ile8-AngII (SII)) preferentially stimulates β-arrestin
signaling, having no effects on G-protein signaling (Anborgh et al., 2000; Rajagopal et
125
al., 2006; Wei et al., 2003). Additionally, TRV120027 is the first biased AT
1
receptor
ligand in clinical trials for heart failure and kidney disease that inhibits AT
1
receptor G-
protein signaling while activating β-arrestin signaling (Boerrigter et al., 2012; Violin et
al., 2010). CX797 behaves in a similar fashion as TRV120027. Additional studies with
β-arrestin1/2 knockouts and mutants may be able to confirm the presence of CXCR2
biased signaling and further characterize the role of CX797 in β-arrestin1/2 biased
signaling.
Even though CX797 enhanced CXCL8-mediated β-arrestin-2 recruitment to CXCR2,
it did not enhance CXCL8-mediated receptor internalization as expected. Instead, it
slightly inhibited CXCL8-mediated receptor internalization (Figures 4.6 and 4.7). Given
our results, we postulate that enhanced β-arrestin-2 and receptor association occurs once
the receptor is internalized, preventing the recycling and turnover of CXCR2 and
resulting in increased total CXCR2 as observed in our studies (Figures 4.5 and 4.6).
Thus, the binding/interaction site of CX797 is most likely intracellular as with most
reported CXCR2 inhibitors to date (Bertini et al., 2012; de Kruijf et al., 2011; Nicholls et
al., 2008; Salchow et al., 2010). In addition, the differential effects of CX797 and
SB265610 on CXCR2 signaling along with the antagonistic effects of these two
compounds (Figure 4.4D) indicate CX797 and SB265610 receptor binding sites are
distinct. However, additional studies are required to confirm and further elucidate the
binding mechanism of CX797 and its precise mechanism of action on the CXCR2
receptor.
126
Previous studies show that β-arrestin complexes with components of the MAP kinase
pathway such as Raf and ERK1/2 upon CXCR2 activation. However, cells with β-
arrestin1/2 double knockout showed increased ERK1/2 phosphorylation compared to
cells expressing wild-type β-arrestin1/2, suggesting ERK1/2 activation is regulated by G-
protein signaling and not β-arrestin1/2 signaling (Zhao et al., 2004). The association of
MAP kinase components with β-arrestin1/2 remains unclear. In our studies we found
that pretreatment with CX797 enhanced β -arrestin-2/CXCR2 association and ERK1/2
phosphorylation (Figure 4.8). Contrary to previous studies, our results suggest β-arrestin-
2 coupling also activates signaling pathways such as MAP kinase, which is the case with
other GPCR receptors (Wei et al., 2003). Additionally, this phenomenon is most likely
not due to any off target modulation since CX797 does not have any effect on ERK1/2
signaling in the absence of CXCL8 stimulation, further suggesting that CX797-mediated
ERK1/2 activation is CXCR2-mediated.
Though CX797 and SB265610 differentially regulated MAPK activation, β-arrestin-
2 signaling, and receptor internalization, both compounds significantly and dose-
dependently inhibited CXCL8-mediated cell migration in CXCR2-expressing cells
(Figure 4.9). Perhaps, the increased in cyclic AMP induced by CX797 and SB265610 is
a critical signaling mechanism that contributes to the inhibition of CXCL8-mediated cell
migration. Previous studies also support the critical role of cyclcic AMP in cell
migration (Chen et al., 2008; Newman et al., 2003). For example, forskolin (adenylyl
cyclase activator) and IBMX (PDE inhibitor), which raise cyclic AMP levels,
significantly inhibited mouse embryonic fibroblast and mouse breast tumor cell migration
127
in wound healing assay (Chen et al., 2008). The concentrations in which SB265610 and
CX797 inhibited cell migration (as low as 1 µM) did not inhibit cell proliferation,
suggesting these compounds can effectively inhibit cell migration without inducing
cytotoxicity (Figure 4.10). This is particularly important when developing therapeutics
targeting COPD or lung diseases. Also, colony formation assay with NSCLC indicate
CX797 is not cytotoxic, but rather cytostatic at higher concentrations, which is further
corroborated with G0/G1 cell cycle arrest. Though this is consistent with other studies
that also show the accumulation of cells in the G0/G1 phase when treated with CXCR2-
directed shRNA, we postulate the observed cytostatic effects in our cell proliferation
assay is a nonspecific effect that may not be CXCR2-mediated (Yang et al., 2010). This
is supported by our data showing that CX797 and SB265610 inhibited both 293T-
CXCR2-GFP and 293T cell proliferation at high concentration (50 µM).
In conclusion, we have identified a novel CXCR2 biased ligand, CX797 that
differentially alter CXCR2 signaling by enhancing the β-arrestin-2 recruitment and
inhibiting G-protein signaling. CX797 also inhibited cell migration at concentrations that
show no cytotoxicity, which may be advantageous for treating inflammation-mediated
diseases. Additionally, continued optimization and characterization of CX797 will offer
important insights to CXCR2 signaling and its effects on cellular function, which will
allow us to design disease specific CXCR2 inhibitors.
128
CHAPTER 5: DESIGN AND DISCOVERY OF NOVEL COMPOUNDS BASED
ON A CXCR2 INHIBITOR PHARMACOPHORE MODEL.
A robust chemical pharmacophore model based on previously reported CXCR2
antagonists was developed to screen a database of 5 million commercially available
compounds. Small-molecule compounds identified from the pharmacophore screening
were selected for in vitro screening in the CXCR2 Tango assay. A class of sulfonamides
(CX25, CX86, and CX815) were identified as having IC
50
concentrations in the low
micromolar to submicromolar range. Further in vitro studies to assess their effects on
cancer cells show CX compounds inhibited cell migration in wound healing scratch
assay, lung and colon cancer cell proliferation (MTT-based) and colony formation.
These compounds also differentially arrested cell cycle progression at the S and G2/M
phase in lung and colon cancer cells, respectively. Structure activity relationship analysis
of these compounds show modest changes in chemical structure significantly affect its
activity.
However, CX25, CX86, and CX815 was not very selective for CXCR2 over CXCR4
and its effects on cell proliferation, colony formation, and cell cycle progression may not
be CXCR2-mediated, suggesting that these compounds may have off-target effects.
Therefore, we performed additional screening of CX25 analogues and identified CX4152
to be selective for CXCR2 over CXCR4 in the Tango assay as well as induce CXCR2
internalization, while having no effects on CXCR2-mediated G-protein signaling
(calcium mobilization). CX4125 also inhibited CXCL8-mediated PMN cell migration.
129
5.1. Development of a robust CXCR2 inhibitor-based pharmacophore model

Figure 5.1 Chemical structures of training set used to generate Hypo-1 pharmacophore hypothesis.
Compounds were derived from previously reported CXCR2 inhibitors in literature.
We have chosen three classes of previously described CXCR2 inhibitors
(squaramides, guanidines, and diarylureas) to develop harmacophore models to identify
novel compounds sharing similar chemical properties arranged in the same spatial
orientation from a database of 5 million compounds. A pharmacophore is defined as a set
of structural features in a drug molecule that is recognized at a receptor site and is
responsible for its biological activity (Dayam et al., 2006). Most common
pharmacophore features of a drug molecule are hydrogen bond donors and acceptors,
aromatic rings, hydrophobic regions and cation/anions. Using a training set of 20 known
CXCR2 inhibitors, we have generated 10 pharmacophore models with Catalyst software
(Figure 5.1). All the pharmacophore hypotheses comprise of four chemical features:
130
hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), hydrophobic (HY), and
aromatic ring (Ar). The best pharmacophore hypothesis, Hypo-1, was selected for further
investigation on the basis of its statistical parameters (Table 5.1).
Hypo-1 has excellent predictive ability (training set correlation value = 0.93), and the
lowest root mean square (RMS) deviations value of 0.83 for the training set (Table 5.1).
It also has a strong correlation value for the test set of 0.78 (Figures 5.2 and 5.3). The
value of the total cost of each hypothesis is much closer to fixed cost value, which is
expected for a good hypothesis. The difference between the null hypothesis cost and the
fixed and total cost of the best hypothesis (Hypo-1) were 39.35 and 31.91 bits,
respectively. The entropy (configuration cost) value of 13.59 for the hypothesis is also
within the allowed range of 17.00.

131

Figure 5.2 Chemical structures of test set used to validate Hypo-1 pharmacophore hypothesis.
Compounds were derived from previously reported CXCR2 inhibitors in literature and are different from
training set compounds.
132
Table 5.1 Results obtained from pharmacophore hypothesis generation using compounds from training set.
Hypothesis Total cost Error cost RMS Correlation (r)
Test set
correlation (r)
Feature
b

1 89.43 74.13 0.83 0.93 0.79 HA, HD, HY, RA
2 95.20 80.37 1.14 0.86 0.71 HA, HD, HY, RA
3 95.83 80.84 1.16 0.86 0.69 HA, HD, HY, RA
4 96.04 81.19 1.18 0.86 0.69 HA, HD, HY, RA
5 96.81 81.62 1.20 0.85 0.68 HA, HD, HY, RA
6 97.12 82.05 1.22 0.85 0.69 HA, HD, HY, RA
7 97.25 82.40 1.23 0.84 0.62 HA, HD, HY, RA
8 98.29 83.58 1.28 0.83 0.64 HA, HD, HY, RA
9 98.44 83.25 1.26 0.83 0.61 HA, HD, HY, RA
10 98.58 83.51 1.27 0.83 0.59 HA, HD, HY, RA
a
Null cost =121.34, Fixed Cost = 81.99, Configuration cost = 13.59. All costs are in units of bits.
b
HA, hydrogen-bond acceptor: HD, hydrogen-bond donor: HY, hydrophobic; RA, Ring aromatic

133

Figure 5.3 Experimental pIC
50
vs. predicted pIC
50
of training (•) and test set (о).
Table 5.2 shows the experimental biological data and the estimated IC
50
values of the
training set compounds calculated using Hypo-1. The discrepancy between the actual
and the estimated activity observed for these compounds was only about 1 order of
magnitude. Experimental and estimated activity data from both training and test set
compounds are plotted as shown in Figure 5.3.
For the validation of our pharmacophore model, a database of 252 molecules
consisting 153 actives and 99 inactives that was previously reported in literature was
screened with Hypo-1 (Aciro et al., 2010; Aki et al., 2009; Biju et al., 2009a; Chao et al.,
2007; Dwyer et al., 2006; Jin et al., 2004; McCleland et al., 2007; Nie et al., 2006;
Widdowson et al., 2004). GH score was calculated to show robustness of the model. The
best hypothesis Hypo-1 successfully retrieved 80 % of actives from the database (Table
134
5.3). In addition, the Hypo-1 also retrieved 10 inactive compounds (false positives) and
predicted 31 active compounds as inactive (false negatives). An enrichment factor of
1.52 and a GH score of 0.80 indicate the significant quality of the model.
Table 5.2 Experimental and estimated IC50 values of the training set CXCR2 inhibitors based on
Hypo-1 pharmacophore model.
Compound
Experimental
IC
50
(μM)
Estimated
IC
50
(μM)
Error Fit value
1 0.008 0.0066 -1.2 6.75
2 0.008 0.0059 -1.4 6.50
3 0.028 0.033 1.2 6.58
4 0.032 0.028 -1.1 5.72
5 0.358 0.36 1 5.35
6 0.054 0.42 7.7 5.02
7 0.004 0.0045 1.1 7.08
8 0.005 0.0086 1.7 6.39
9 0.012 0.018 1.5 6.91
10 0.012 0.02 1.6 6.82
11 0.029 0.011 -2.7 6.56
12 0.112 0.023 -4.8 6.16
13 0.006 0.013 2.1 5.42
14 0.007 0.015 2.1 6.43
15 0.114 0.44 3.9 5.26
16 0.2 0.36 1.8 4.96
17 0.906 0.46 -2 5.00
18 10.9 3.3 -3.3 3.76
19 25 13 -1.9 3.85
20 1.72 0.37 -4.7 5.13
135
Table 5.3 Hypo-1 validation using a database of previously reported CXCR2 Inhibitors from
literature

Hypo-1 was mapped onto the three highly active and structurally diverse classes of
CXCR2 inhibitors (squaramides, guanidines, and diarylureas) with an excellent fitness
value (>8) (Figure 5.4). Hypo-1 was used to screen a database of 5 million commercially
available compounds. This yielded 350 compounds with a fitness value >5 and favorable
ADMET properties. These compounds were chosen for CXCR2 inhibition screening in
the CXCR2 Tango assay.
Total compounds in database (D) 252
Total number of actives in database (A) 153
Total hits (H
t
) 132
Active hits (H
a
) 122
% Yield of actives 92.42
% Ratio of actives in the hit list 79.74
Enrichment factor (E) 1.52
False negatives 31
False positives 10
GH score (goodness of hit ) 0.80
136

Figure 5.4 Pharmacophore modeling of Hypo-1.
(A) Hypo-1 was generated using the Hypo-Gen model with Catalyst Software. Pharmacophore features are
color-coded magenta for hydrogen-bond donor (HBD), cyan for hydrophobic group (HY), orange for
aromatic group (Ar), and green for hydrogen-bond acceptor (HBA). (B) Distances between pharmacophore
features are given in angstroms. (C-E) Representatives of CXCR2 inhibitors in training set are mapped
onto the Hypo-1 model.
5.2. Identification of CX compounds in CXCR2 screening
Using CXCR2 Tango assay, we initially screened 144 compounds from 350
pharmacophore hits based on structural diversity as well as commercial availability. 13
compounds that belong to a structurally similar class of sulfonamides were identified as
hits from our initial screen. The best initial hits identified from pharmacophore screening
are represented in Table 5.4 and mapped onto Hypo-1 pharmacophore model in Figure
5.5. Our most active compound, CX25 (IC
50
=360 nM) was chosen for further structure-
activity- relationship (SAR) studies. From similarity searches, we further identified and
tested 103 additional CX25 analogues. From there, 33 compounds with IC
50
s below 10
µM were identified. Most of the tested CX25 analogs exhibited three similar scaffolds as
137
shown in Figure 5.6 and their activities are listed in Tables 5.5-7. Inactive compounds
identified from Hypo-1 are listed in Table 5.8.

Figure 5.5 Pharmacophore mapping of CX compounds and CXCR2 inhibitors.
(A) CX25, (B) CX86, (C) CX815 and CXCR2 compounds in clinical trials, (D) SB656933 and (E)
SCH527123 mapped onto Hypo-1 model.
Overall, compounds made up of scaffold A were most active and had IC
50
s in the
nanomolar and low micromolar range. Halogen substitution (Cl, Br, F and CF
3
) on the
benzene in Scaffold A enhanced activity. The polar thiadiazole ring structure on the
benzene (CX25 and CX86) also improved activity by providing a hydrogen bond
accepting group (S) in between the ortho and meta position. This observation supports
our current pharmacophore model, which predicts a hydrogen bond accepting group on
the hydrophobic benzene is essential for activity. However, other substitutions with
acetyl, acetylamine, sulfonamide, and methoxy groups in the para position on the
138
benzene significantly decreased activity. Mono-, bi-, and tri-methylation of the benzene
did not improve activity. However, isopropyl substitution improved activity by 4-fold.
We also observed that methoxy substitution on the quinoline significantly improved
activity of compounds with Scaffold A. For example, methoxy substitution on the
quinoline in CX25 improved its activity (IC
50
=360 nM) when compared to CX86
(IC
50
=1.1 µM).
All compounds with Scaffold B were inactive at 10 µM (Table 5.5). For example,
compound CX624 was inactive at 10 µM, whereas compound CX061 had an IC
50
of 2.5
µM. These data suggest that the NH group (hydrogen bond donor) on the quinoline is
essential for activity as it is a significant pharmacophore feature. Compounds with
naphthalene instead of quinoline (Scaffold C) were slightly less active than compounds
with Scaffold A, suggesting the importance of the nitrogen in the quinoline structure.

Figure 5.6 Chemical structures of compound scaffold A, B, and C.
Compounds with scaffold A were the most active in the CXCR2 Tango assay. Compounds with scaffold B
were all inactive. Compounds with naphthalene instead of quinoline (scaffold C) were slightly less active
than compounds with Scaffold A, suggesting the importance of the nitrogen in the quinoline structure.
139
Table 5.4 CXCR2 inhibition and cancer cell proliferation results of active CX compounds selected from Hypo-1 screening
Compound
Code
Structure Fit
CXCR2 Inhibition
(IC
50
, µM)
CXCR4 Inhibition
(IC
50
, µM)
MTT (IC
50
, µM)
H1299 H460
HCT116
p53+/+
HCT116
p53-/-
SB225002

6.69 0.19±0.24 >20 3.6±4.9
4.1±2.
4
4.6±3.9 5.4±4.1
SB265610

6.72 0.28±0.09 >20 >50 >50 >50 >50
CX25

6.4 0.36±0.1 0.59±0.1 8.0±6.5
3.9±1.
5
3.9±1.9 6.2±5.2
CX86

5.81 1.1±1.4 3.4±3.3 7.3±4.8
6.1±2.
3
3.8±0.5 5.5±2.2
CX815

6.15 0.4±0.1 >50 3.5±0.9
4.1±0.
2
5.0±3.7 NT
140
CXCR2 Inhibition was determined using the CXCR2 Tango assay. Cell proliferation was determined using MTT assay. NT = not tested. Fit = fitness
value for Hypo-1.
Table 5.5 CXCR2 inhibition and cancer cell proliferation results of CX compounds with Scaffold A selected from CX25, CX86 and CX815
similarity search
Compound
Code
Structure Fit
CXCR2 Inhibition
(IC
50
, µM)
CXCR4 Inhibition
(IC
50
, µM)
MTT (IC
50
, µM)
H1299 H460
HCT116p53
+/+
CX692

5.99 5.0±6.3 0.6 3.6±2.0 3.3±1.1 NT
CX122

6.01 2.1±1.7 2.8±1.7 2.3±1.7 1.7±1.4 4.5
CX595

5.83 2.1±1.5 2.6±1.8 2.4±1.6 1.4±1.0 5.0
CX295

5.84 4.0±0 5.1±2.8 2.0±1.9 1.7±2.0 NT
CX857

6.04 4.5±4.9 4.7±10.9 NT NT NT
141
CX748

6.01 3.0±1.4 3.0±2.8 1.4±1.6 1.5±2.1 1.6
CX721

5.94 4.5±0.7 21.0±26.6 >20 >20 >20
CX279

6.03 2.2 3.0±1.4 2.1±1.8 2.2±1.8 1.6
CX684

5.94 1.4±1.1 1.0±0.6 1.2±0.7 0.9±0.7 2.5
CX061

6.18 2.5±1.7 2.5±1.9 2.3±1.9 1.5±1.1 3.3
CX297

5.88 2.4±2.3 3.6±1.2 3.0±0.7 4.1±1.3 NT
142
CX296

5.82 5.6±3.2 20.7±17.2 45.0±7.3 70.8 NT
CX388

6.13 1.6 5.0 3.2 5.0 14
CX310

5.86 1.6±1.2 NT NT NT 8.2
CX324

5.86 3.1±2.7 4.8±3.2 3.0±1.4 2.6±1.4 NT
CX128

5.9 2.4±2.3 <0.2 >10 >10 NT
143
CX826

6.16 1.7±2.0 7.1 1.4±1.0 0.9±0.6 5.0
CX073

6.06 7.0±6.1 >20 20.0 17.8 NT
CX587

6.21 5.0 NT 8.5±0 >10 NT
CXCR2 Inhibition was determined using the CXCR2 Tango assay. Cell proliferation was determined using MTT assay. NT = not tested. Fit = fitness
value for Hypo-1.

144
Table 5.6 CXCR2 inhibition and results of CX compounds with Scaffold B selected from CX25,
CX86 and CX815 similarity search
Code Structure Fit CXCR2 Inhibition (IC
50
, µM)
CX258

5.56 >10
CX251

6.02 >10
CX508

6.01 >10
CX2692

6.15 >10
CX763

5.77 >10
CX624

5.58 >10
145
CX239

5.1 >10
CX616

6.12 >10
CX800

5.88 >10
CX996

5.54 >10
CX360

5.35 >10
CX987

5.02 >10
CX456

6.89 >10
146
CX260

7.56 >10
CX638

5.49 >10
CX609

5.04 >10
CX382

6.72 >10
CX168

6.25 >10
CX232

5.89 >10
CX226

5.98 >10
147
CX190

6.99 >10
CX438

7.2 >10
CXCR2 Inhibition was determined using the CXCR2 Tango

assay. NT = not tested. Fit = fitness value for
Hypo-1

148
Table 5.7 CXCR2 inhibition and cancer cell proliferation results of CX compounds with Scaffold C selected from CX25, CX86 and CX815
similarity search
Compound
Code
Structure Fit
CXCR2 Inhibition
(IC
50
, M)
CXCR4 Inhibition
(IC
50
, µM)
MTT (IC
50
, µM)
H1299 H460
CX309

6.37 16.5±1.1 8.9 6.2±5.6 >20
CX640

6.33 8.4±4.6 NT >10 >10
CX078

7.18 8.0±6.8 10.0 NT NT
CX459

6.46 3.6±6.9 11.7 8.3±8.2 17.8
149
CX910

7.05 >20 NT NT NT
CX588

7.74 3.6 NT NT NT
CX886

7.76 7.9 NT 3.6±0.6 15.8
CX887

6.57 15.4±1.1 8.9 NT NT
150
CX410

6.54 17.8 NT 5.0±0 >10
CX396

6.34 6.5±8.7 7.6 NT NT
CX404

6.34 11.1±6.8 20 NT NT
CX474

6.42 16.3±3.4 >20 20.0 >20
151
CX489

7.12 >20 NT NT NT
CX4152

6.21 7.6±6.2 64.7±18.9 31.6 20.0
CXCR2 Inhibition was determined using the CXCR2 Tango assay. Cell proliferation was determined using MTT assay. NT = not tested. Fit = fitness
value for Hypo-1.

152
Table 5.8 CXCR2 inhibition of inactive CX compounds selected from CX25, CX86 and CX815
similarity search
Compound
Code
Structure
CXCR2 Inhibition
IC
50
(µM)
CX108-S

>10
CX856-S

>10
CX951-S

>10
CX111-S

>10
CX413-S

>10
CX741-S

>10
CX458-S

>10
153
CX958-S

>10
CX036-S

>10
CX638-S

>10
CX175-S

>10
CX581-S

>10
CX025-S

>10
CX056-S

>10
CX177-S

>10
154
CX692-S

>10
CX051-S

>10
CX455-S

>10
CX800-S

>10
CX996-S

>10
CX360-S

>10
CX987-S

>10
CX022-S

>10
CX233-S

>10
155
CX103-S

>10
CX322-S

>10
CX240-S

>10
CX659-S

>10
CX216-S

>10
CX675-S

>10
CX506-S

>10
CX346-S

>10
156
CX250-S

>10
CX072-S

>10
CX039-S

>10
CX373-S

>10
CX479-S

>10
CX139-S

>10
CX307-S

>10
157
CX460-S

>10
CX575-S

>10
CX456-S

>10
CX334-S

>10
CX260-S

>10
CX460-S

>10
CX181-S

>10
CX693-S

>10
CX293-S

>10
CXCR2 Inhibition was determined using the CXCR2 Tango assay. NT = not tested
158

5.3. CX compounds inhibit cell migration in wound healing assay
Next, we assessed the effects of select CX compounds on CXCL8-induced cell
migration using wound healing scratch assay (Figures 5.7 and 5.8). CX25 and CX86
inhibited CXCL8-induced cell migration in CXCR2-bla U2OS cells at concentrations as
low as 1 nM comparable to SB225002 and SB265610, which is several folds lower than
its MTT IC
50
for cancer cell proliferation (4-8 µM ). This indicates the observed
inhibition is not due to the compounds effects on cell proliferation. CX815 also slightly
inhibits cell migration across all concentrations tested.

Figure 5.7 Chemical structures of CX compounds selected for further studies.
CX25, CX86, CX815 and CX122 were selected for further studies on cell migration, cancer cell
proliferation, colony formation, cell cycle progression, and calcium flux.
159

Figure 5.8 CX compounds inhibits cell migration.
The migratory effects of CX compounds in CXCR2-bla U2OS cells was assessed using a wound healing
scratch assay. CXCR2-bla U2OS cells were seeded in 96-well plates (30,000 cells/well) in DMEM
supplemented with 1% FBS overnight. A single scratch wound was made using a sterile pipette tip the next
day. Wells were washed with 1X PBS and treated with CX compounds, SB265610, or SB225002 at
various concentrations and stimulated with recombinant CXCL8 at 200 nM for 24 hours at 37°C. Cells
were fixed with 100% methanol for 15 minutes at RT and stained with Giemsa stain for one hour. Each
well was imaged on BD Pathway 435 Bioimager with transmitted light with 4X magnification. Control
wells (lower panel) were treated with CXCL8 (200 nM) alone or media only to detect CXCL8 induced and
basal cell migration.
5.4. CX compounds inhibit cancer cell proliferation
Next we assessed the effects of our compounds in a panel of highly proliferative
NSCLC and colon cancer cell lines expressing CXCR2. Active CX compounds from
Tango assay were tested for cytotoxicity in NSCLC cell lines (NCI-H1299 and NCI-
H460) and colon cancer cell lines (HCT116 p53
+/+
, HCT116 p53
-/-
) using MTT assay.
160
CX25 was slightly more active than CX86 at inhibiting cancer cell proliferation and with
similar IC
50
s as CXCR2 inhibitor, SB225002 (Table 5.4 and Figure 5.9). SB265610
exhibit IC
50
’s greater than 50 μM in all cell lines tested. Overall, compounds with
Scaffold A and C inhibited cancer cell proliferation with IC
50
s in the micromolar range.

Figure 5.9 CX compounds inhibit cancer cell proliferation.
Representative dose response curves of CX25, CX86, CX815, and CX122 in MTT assay. Active CX
compounds inhibited cancer cell proliferation in (A) H1299, (B) H460, (C) HCT116p53+/+, (D)
HCT116p53-/- cell lines. Cells were seeded at 1-3,000 cells/well in 96-well plates in media supplemented
with 10% FBS overnight. Cells were treated with compounds at various concentrations (in triplicates) for
72 hours.

161
5.5. CX compounds inhibit colony formation
Colony formation assays was used to assess cytotoxicity of CX compounds. CX25
was most active, completely inhibiting colony formation at 10, 5 and 2 µM in all cell
lines (Figure 5.10). CX86, CX815, and SB225002 completely inhibited colony
formation at 10 and 5 µM. CX3122 was less active, inhibiting colony formation at only
10 µM in most of the cell lines. As predicted from the MTT assay data, SB265610 did
not inhibit colony formation. In general, these colony formation data corroborated the
MTT results.

Figure 5.10 CX compounds inhibit colony formation.
Representative images of colony formation in H1229, H460, HCT116 p53+/+ and HCT116 p53-/- cell
lines. Cells were treated with CX25, CX86, CX815, CX122, SB225002, and SB265610 at various
concentrations for 24 hours. Colonies were cultured for 4-7 days and stained with crystal violet and
imaged on Chemi-Doc Imaging System (Bio-Rad, Hercules, CA).

162
5.6. CX compounds differentially arrest cell cycle progression
Since these compounds inhibit cell proliferation and colony formation, we next
determined their effects on cell cycle progression. Interestingly, all CX compounds
arrested cell cycle progression at S phase in NSCLC and arrested cells at G2/M phase in
colon cancer (Figure 5.11). SB225002 completely inhibited cells in the G2/M phase
(>90%) across all cell lines, while SB265610 had no effects on cell cycle progression.

Figure 5.11 CX compounds differentially regulate cell cycle progression.
Cells were treated with compounds at 10 µM for 24 hours. CX compounds arrested (A) H1299 and (B)
H460 cells in S phase, while arresting (C) HCT116 p53+/+ and (D) HCT116 p53-/- cells in G2/M phase.
163
5.7. CX compounds induce rapid intracellular calcium release in a dose
dependent manner
Lastly, to examine the potential mechanism of CX compounds, we assessed their
effects on intracellular calcium flux. CX86 and CX25 significantly induce calcium flux
in a dose dependent manner with micromolar concentrations (5-20 µM), whereas CX815
and CX3122 did not have any effects. SB265610 and SB225002 also induced calcium
flux with a much slower kinetic profile and lower maximum signal (Figure 5.12).

Figure 5.12 CX compounds induce intracellular calcium flux.
Intracellular calcium release was monitored following treatment with 20 µM of compounds in (A)
HCT116p53+/+ (B) and H1299 cells. Dose response of (C) CX25 and (D) CX86 was evaluated in H1299
cells. Control wells were treated with assay buffer.
5.8. CX4152 exhibit a different mechanistic profile than CX25 on CXCR2
Interestingly, a representative compound (CX4152) that exhibit scaffold C (Table
5.7) from our initial screening process showed a different mechanistic profile than CX25
and CX86. CX4152 showed more selectivity for CXCR2 (IC
50
=7.6±6.2 μM) over
164
CXCR4 (IC
50
=64.7±18.9 µM) in the Tango assays than CX25 (Figure 5.13A). CXCR2
Tango cells pretreated with CX4152 for 30 minutes prior to CXCL8 stimulation showed
greater potency than cells co-treated with CX4152 and CXCL8 at the same time (Figure
5.13B). CX4152 also fit well into the pharmacophore model with a fit value of as well as
showed no activity in calcium release (Figure 5.13C). CX4152 also did not inhibit
CXCL8-mediated calcium release that was observed with SB265610. Figure 5.13D show
that order of addition of CX4338 and CXCL8 dramatically effects the potency of
CX4152 in the CXCR2 Tango assay. Thirty minutes pretreatment with CX4152 prior to
CXCL8 sitmulation was more potent than cotreatment of CX4152 and CXCL8 at the
same time.

Figure 5.13 CX4152 exhibits a different mechanistic profile than CX25.
(A) Dose response curve of CX4152 in CXCR2 and CXCR2 Tango assay . (B) CX4152 is mapped onto
Hypo-1. (C) CX4152 alone did not alter calcium mobilization and had no effects on CXCL8-mediated
calcium mobilization. (D) Pretreatment with CX4152 prior to CXCL8 stimulation is more potent than co-
treatment with CX4152 and CXCL8 at the same time in the CXCR2 Tango assay.
165
5.9. CX4152 induce CXCR2 internalization
Next, we assessed the effects of CX4152 on receptor internalization. CX4152
induced receptor internalization in a dose and time dependent manner (Figures 5.14A and
B). We also observed a decreased in total CXCR2 at five hour treatment (Figures 5.14C
and D). Total receptor increased was observed at earlier time points. We also performed
similarity searches of CX4152 and found additional compounds that exhibit similar
chemical features. These analogs were not more potent than CX4152 and showed less
CXCR2 selectivity (Table 5.9).

Figure 5.14 CX4152 induce CXCR2 internalization and turnover.
CXCR2 surface and total expression was measured using in-cell Western. CXCR2-bla U2OS cells were
treated with CX4152 at indicated concentrations for different time points. (A) CX4152 dose and time
dependently reduced CXCR2 surface expression. (B) Quantification of A, a representative experiment of
three independent experiments. (C) CX4152 reduced total CXCR2 expression at five hour time point. (D)
Quantification of C, a representative experiment of two independent experiments.

166
Table 5.9 CXCR2/4 Tango and MTT IC50s for CX4152 Analogs
Compound
CXCR2
(IC
50
, μM)
1

CXCR4
(IC
50
, μM)
1

MTT (IC
50
, μM)
2

293T-
CXCR2
293T H460 H1299 A549

BAS 00788794
40±13.2 4 2 2.5 3.1 3.5 3.5

BAS 00788793
28.3±22.5 1 <1 <1 2 2.7 1.1

BAS 00914083
44±26.5 12 23 19 26 12 38

BAS 00788843
40±28.3 35 NT NT NT NT NT
BAS
00788788
10±0 10 30 30 25 23 40
167

BAS 00788789
28.3±7.6 6 60 >100 >100 40 70

BAS 00914138
27.5±24.7 18 36 33 22 29 39

BAS 03390965
32±39.6 1 1.1 3 3.5 4 4

BAS 00350572
52.3±44.5 30 NT NT NT NT NT

BAS 00350570
12.4±11.5 25 4.6 4.5 4.7 5 7

BAS 00350562
18.7±22.8 6 NT NT NT NT NT
168

BAS 00788787
3

6.3±1.1 30 20 55 10 55 60

BAS 03390994
>100 NT NT NT NT NT NT

BAS 09618226
>100 NT NT NT NT NT NT

BAS 09618732
>100 NT NT NT NT NT NT

BAS 05337769
>100 NT NT NT NT NT NT
169

BAS 09616232
>100 NT NT NT NT NT NT

BAS 00914093
>100 NT NT NT NT NT NT
1
IC
50
is the mean ± SD of three independent experiments in the CXCR2/4 Tango assay.
2
Cells were treated
with compounds for 72hr and cell proliferation measured using the MTT assay.
5.10. CX4152 inhibit cell proliferation and PMN cell migration
We further assessed the cellular effects of CX4152 on cell proliferation of CXCR2-
expressing cells (293T-CXCR2-GFP and CXCR2-bla U2OS) and its parental cell line
(293T, U2OS) as well as CXCR4-expressing (CXCR4-bla U2OS) and NSCLC (A549)
cell lines. CX4152 dose-dependently inhibit cell proliferation (72hr treatment) in all cell
lines tested, suggesting the effects on cell proliferation may not be CXCR2-mediated
(Figure 5.15A). CX4152 also inhibited CXCL8-mediated cell migration in PMN with an
IC
50
of 58.29 μM (Figure 5.15B).
170

Figure 5.15 CX4152 inhibit cell proliferation and PMN cell migration.
(A) 293T, 293T-CXCR2-GFP, CXCR2-bla U2OS, CXCR4-bla U2OS, U2OS, and A549 cells was treated
with CX4152 for 72hrs and cell proliferation was measured using the MTT assay. Data represents
mean±SD of two independent experiments. (B) Concentration-dependent inhibition of human neutrophil
chemotaxis. Neutrophils were incubated with CX4152 for 1 hour, and placed in the top wells of a
chemotaxis plate containing 50 nM of CXCL8 in the bottom wells. Neutrophils were allowed to chemotax
for 2-4 hours at 37°C and 5% CO2. Inhibition of chemotaxis was evaluated based on cell counts relative to
control (untreated with CXCL8). CX4338 concentration-dependently inhibited CXCL8-induced
chemotaxis with an IC50 of 58.29 μM. Results are mean ± SD of two to three experiments.
171
5.11. Discussion and conclusions
The identification of CXCR2 inhibitors in this study was facilitated by computational
studies using a CXCR2 inhibitor-based pharmacophore model, Hypo-1. Hypo-1 exhibits
high predictability power to determine CXCR2 inhibition of compounds (test set
correlation between predicted activity and experimental activity is 0.78, Figure 5.3) and
excellent GH score (0.81), indicating its ability to discriminate between actives and
inactives in a large database of compounds, successfully reducing the need to perform
large-scale screening studies (Table 5.3). For example, Hypo-1 selected 350 compounds
from a database of 5 million compounds. 144 of those compounds were procured from
commercial sources and tested for CXCR2 activity. 13 compounds show activity with
IC
50
s less than 10 µM, a hit rate of 9.7%. Of these, CX25 was the most potent, inhibiting
CXCR2 activation with an IC
50
of 360 nM, and was selected for further studies to
characterize its efficacy in cancer (Table 5.4). Hypo-1 also identified four essential
chemical features (hydrogen bond donor, aromatic ring, hydrophobic, hydrogen bond
acceptor) that are critical for CXCR2 inhibition. Modest changes in structure have
dramatic effects on activity. This is most clearly demonstrated with scaffold A and B.
When the hydrophobic group (benzene) and the aromatic group (quinoline/naphthalene)
between the sulfonamide backbone was swapped, activity was completely abolished.
CX compounds (CX25, CX86, CX815, CX122) showing nanomolar activity against
CXCR2 in the Tango assay identified from Hypo-1 also inhibited cell proliferation and
colony formation in cancer cell lines with similar potencies as SB225002. SB225002
belongs to the diarylurea class of CXCR2-selective inhibitors that was first discovered by
Glaxosmithkline via high-throughput screening endeavors (White et al., 1998). However,
SB265610, a derivative of SB225002, is just as potent at inhibiting CXCR2 in the Tango
172
assay as SB225002, but did not show cytotoxicity in all cell lines tested and inhibited cell
proliferation less than 50% at 50 μM concentration. The observations with SB265610 is
consistent with our CXCR2 siRNA studies that show knockdown of CXCR2 at most
reduced cell proliferation by 20-40% in various cell lines tested. This is consistent with
previous reported studies that show CXCR2 knockdown with shRNA in melanoma cells
at most reduced cell proliferation by 40% in low serum (Singh et al., 2010).
Additionally, we showed that SB225002 arrested cells in the G2/M phase, which is
inconsistent with previous studies that showed that CXCR2 knockdown in ovarian cancer
cell lines arrested cells in the G0/G1 phase (Yang et al., 2010). This suggests that the
toxicity observed with CX compounds and SB225002 may not be CXCR2-mediated, but
rather an off-target effect of these compounds. Indeed, SB225002 was recently shown to
exhibit microtubule destabilizing activity by binding to tubulin and inducing mitotic
arrest in p53 mutant cancer cell lines (Du et al., 2013; Goda et al., 2013). Goda et al. also
report that SB265610 did not induce G2/M cell cycle arrest at concentrations as high as
100 μM. This suggest that the slight difference in chemical structure between SB225002
and SB265610 dramatically alters the off-target effects of inhibitors belonging to the
diarylurea class.
This further suggest that Hypo-1 pharmacophore model may also capture compounds
that exhibit off-target effects that are inherent CXCR2 inhibitors used to generate Hypo-1
(training set). Thus, the cytotoxic effects observed with CX compounds may be due to
off target effects. We do not believe CX compounds have tubulin binding activity since
it did not induce G2/M cell cycle arrest but rather S-phase arrest (Figure 5.11). Also, CX
compounds induced a rapid burst of intracellular calcium release in a dose dependent
173
manner (5-20 μM) in lung and colon cancer cells (Figure 5.12). SB225002 and
SB265610 slightly induced calcium at higher concentrations (20 μM), though the
induction was not as significant compared to CX25 and CX86. Calcium signaling is also
a key component of apoptotic signaling. Agents that increase intracellular free calcium
such as thapsigargin (SERCA inhibitor) and ionomycin (calcium ionophore) have been
shown to induce apoptosis (Jiang et al., 1994; Palm et al., 1996). Another possibility is
that at higher concentrations, these compounds may behave as a partial agonist, which
was observed with another previously reported CXCR2 inhibitor, VUF10948.
VUF10948 inhibited CXCL8 binding and β-arrestin-2 recruitment in the nanomolar
range, however, at higher concentrations; it potentiated β-arrestin-2 recruitment in the
absence of ligand (de Kruijf et al., 2009).
A counter screen with another chemokine receptor, CXCR4, showed that CX
compound also inhibited CXCR4 with similar potencies suggesting that these compounds
are not very selective for CXCR2 and may target other chemokine receptors as well as
similar classes of GPCRs. Additional SAR and optimization of these compounds may
improve the selectivity of these compounds. Thus, we performed additional similarity
searches and screening. We identified CX4152 to be 8 times more selective for CXCR2
over CXCR4 in the Tango assay, with IC
50’
s of 7.6 ±6.2 and 64.7 ± 18.9 μM,
respectively. CX4152 exhibit scaffold C, a similar chemical backbone as CX25.
However, CX4152 exert very different cellular effects. CX4152 induced receptor
internalization within 10 minutes, without activating CXCR2 (calcium mobilization),
suggesting that CX4152 inhibitory effects in the CXCR2 Tango assay is due to β-
arrestin-2-independent receptor internalization. This phenomenon is most likely not due
174
to receptor down-regulation since total CXCR2 expression was not decreased until 5
hours later. Rather, at earlier time points total CXCR2 appears to increase, perhaps due
to the accumulation of intracellular CXCR2. This is further supported by studies that
show CX4152 was more potent at inhibiting β-arrestin-2 recruitment in the CXCR2
Tango assay when cells were pre-treated with CX4152 prior to CXCL8 stimulation than
when cells were treated with CX4152 and CXCL8 at the same time (Figure 5.13D).
These observations suggest that CX4152 may be acting as agonist. However, it did not
activate calcium mobilization (Figure 5.13C) nor inhibit cyclic AMP signaling (data not
shown). We postulate CX4152 inhibits CXCR2/ β-arrestin-2 association by down-
regulating surface expression of CXCR2. This mechanism of action was sufficient to
inhibit CXCL8-mediated PMN cell migration (Fig 5.15B). CX4152 also reduced cell
proliferation in a panel of cell lines that has high and low CXCR2 expression (Figure
5.15A); suggesting that the anti-proliferative effects of CX4152 is CXCR2-indepdendent,
perhaps targeting other chemokine receptors or other cellular targets.
In conclusion, the identification of potential CXCR2 inhibitors exhibiting cytotoxic
properties was facilitated by computational studies using Hypo-1, a CXCR2 inhibitor-
based pharmacophore model. CX compounds inhibit cancer cell proliferation, migration,
and cell cycle progression in a similar fashion as previously reported CXCR2 inhibitor,
SB225002. The anti-proliferative and cytotoxicity of CX compounds is most likely due
to off-target effects that has been shown with SB225002 (tubulin-binding). From our
studies, other cellular targets of CX compounds may be involved in calcium signaling.
However, additional studies will be required to confirm this. Using the same
pharmacophore model, we also identified CX4152 that showed selectivity for CXCR2
175
over CXCR4 as well as rapidly induce receptor internalization without activating the
receptor. The fact that CX compounds and CX4152 exhibit similar chemical features
show that slight chemical modifications has a dramatic effect on CXCR2 inhibition as
well as effects on non-specific cellular targets. This further leads us to postulate that the
CXCR2 inhibitors that were used to generate Hypo-1 may exhibit differential effects of
CXCR2 signaling as well as CXCR2-independent cellular effects. Thus, it is essential to
fully characterize the different classes of CXCR2 inhibitors reported in the literature to
refine and generate a more specific pharmacophore model to reduce identification of
CXCR2 inhibitors with multiple cellular targets.
5.12. Materials and Methods
5.12.1. Molecular modeling
Molecular modeling was performed on a Dell Precision T7400n Mini-Tower, Quad
Core Xeon Proc X5450 dual processor with 32 nodes computer. Catalyst software
(Accelrys Inc., San Diego, CA) was used to generate pharmacophore models.
5.12.2. Data set and modeling.
Biological activity data (binding assay performed with Chinese hamster ovary
(CHO) cell lines expressing CXCR2), represented as IC
50
(nM) were obtained from the
literature published by GlaxoSmithKline (Jin et al., 2004; McCleland et al., 2007; Nie et
al., 2006; Widdowson et al., 2004). Chemical structures of selected CXCR2 antagonists
were separated into training and test set based on structural and activity diversity (Figure
1 & 2). Three different chemical classes of CXCR2 inhibitor (N, N
'
- diarylsquaramides,
N, N
'
- diarylureas, and N, N
'
- diarylguanidines) were chosen for pharmacophore
hypothesis generation. Both training and test sets have a structurally similar set of
176
compounds and representatives from the three different classes to ensure structural
diversity. Our training set consisted of 20 compounds with significant structural diversity
and wide range of bioactivity with IC
50
ranging from 4 nM to 13 μM. After building the
required ligand structure of a specified configuration using Discovery Studio 2.0, energy
minimization was performed using open force field (OFF) methods with the steepest
descent algorithm (Accelrys Inc., San Diego, CA) . A gradient convergence value of
0.001 kcal/mol was used. Unique low-energy geometries generated were exported and
minimized further using a modified CHARMM force field in Catalyst (Accelrys. Inc.,
San Diego, CA) . All possible conformations for each compound were generated using
Poling algorithm which penalizes any newly generated conformer if it is too close to any
already found conformer (Smellie et al., 1995c). A maximum number of 250
conformations for each compound was generated using the CHARMM force field
parameters with the “best conformation generation” option with a consistent of 20
kcal/mol energy thresholds above the lowest-energy conformer to ensure maximum
coverage of the conformational space (Smellie et al., 1995a; Smellie et al., 1995b). All
other parameters were set to default settings. While generating the hypothesis using
HypoGen module from Catalyst, the minimum and maximum count of features for
hydrogen bond acceptor (HBA), hydrogen bond donor HBD), hydrophobic (HY), ring
aromatic (RA) features were set to 0 and 5. The default uncertainty value of 3 was used
for the compound activity, representing the ratio of uncertainty range of the measured
biological activity against the actual activity for each compound. An initial analysis of the
function mapping tools revealed that the hydrogen bond acceptor (HBA), hydrogen bond
donor (HBD), hydrophobic (HY), and aromatic ring (Ar) features effectively map all
177
critical features of the training set compounds. Hence, the four features were selected to
form the basis for the hypothesis generation process.
5.12.3. Pharmacophore model validation.
Hypothesis models were used to predict the activity of the test set of known CXCR2
inhibitors (Figure 2). A Güner and Henry (GH) scoring method was used to calculate the
“goodness-of-hit” (Guner and Henry, 2000). To assess the quality of pharmacophore
models, a database of active and inactive inhibitors from literature as per the equation
below:

where, D is the total compounds in database, A is the number of actives, H
a
is the
total number of actives in hit list, H
t
is the total number of compounds in hit list. For this
assessment, a database of known active and inactive inhibitors (previously published in
literature) were used to screen the pharmacophore. GH score were calculated for the top
10 hypotheses. The GH score values range from 0 to 1. A GH score value of 0.6 to 1
indicates an excellent model.
5.12.4. Database Search.
The best pharmacophore hypothesis (Hypo 1) was used as a search query to retrieve
compounds with novel structural scaffolds and desired chemical features from a multi-
conformer Catalyst-formatted database consisting of 5 million commercially available
compounds. The fast flexible search method in Catalyst was used to search the database.
ADMET properties. ADMET (Absorption, Distribution, Metabolism, Excretion and
Toxicity) risks of the CXCR2 hits were evaluated using ADMET Predictor from
178
Simulations Plus, Inc (De Buck et al., 2007). ADMET predictor calculates a number of
descriptors related to the ADME and toxicity properties from a structure file of
compounds.

179
CHAPTER 6: CONCLUDING REMARKS AND FUTURE PERSPECTIVES
The studies presented in this dissertation highlights several important aspects of
CXCR2 inhibitors. First, it is critical to thoroughly examine the effects of these
inhibitors on CXCR2 signaling, both G-protein and β-arrestin1/2 signaling. In the past,
mechanistic studies have heavily focused on G-protein signaling (calcium, cyclic AMP,
G-protein dissociation), however, we have shown along with others the importance of
receptor-mediated β-arrestin1/2 signaling and the differential effects of these inhibitors
on cellular functions such as cell proliferation. Second, the concept of CXCR2 biased
ligands as shown with CX4338 and CX797, suggest that it is possible to selectively
modulate certain CXCR2 signaling while leaving other aspects of the receptor untouched.
The ability to design biased ligands offer the potential to design disease specific CXCR2
inhibitors and perhaps reduced side effects associated with CXCR2 inhibition. For
example, CX4338 may be a more appropriate for treating COPD instead of cancer since
it had very little effects on cell proliferation, but potently inhibited cell migration.
Third, our studies with previously described CXCR2 inhibitors, SB225002 and
SB265610 as well as compounds identified from our pharmacophore models, CX25 and
CX4152, demonstrate that slight chemical modifications may significantly alter CXCR2
signaling as well as off-target effects. Therefore, extensive mechanistic studies of
previously reported inhibitors is required before one can successfully generate a more
refine pharmacophore model to screen for CXCR2 inhibitors. Though, we have shown
success with identifying compounds that modulate CXCR2 with Hypo-1 pharmacophore
model, many of these compounds were not selective for CXCR2 as well as exhibit
cellular effects that were attributed to other unidentified cellular targets. This is partly
180
due to the limited information (only binding data) that was used to generate Hpyo-1,
which exemplifies the saying, “you get what you put in”. Figure 6.1 is a schematic
summarizing the potential binding sites of different classes of CXCR2 inhibitors and its
cellular effects. Despite having similar chemical backbones and chemical features as
demonstrated in our pharmacophore studies, CXCR2 inhibitors exhibit differential
binding mechanisms as well as cellular functions.
Lastly, our studies have demonstrated the robustness of several high throughput
assays that can be used to screen for CXCR2 inhibitors and assess its effects on CXCR2
signaling. These assays include the Tango, cyclic AMP GloSensor, and in-cell Western
receptor internalization and degradation assays. In particular, the receptor internalization
and degradation assay is by far the most cost-effective receptor assay that we have come
across that can be easily optimized for other GPCRs. Of note, using the in-cell Western
assay, we showed that SB265610 significantly up-regulated CXCR2 surface and total
expression in the absence of CXCL8. This may be a undesirable effect of CXCR2
inverse agonists in CXCL8-driven disease models and should be considered prior to
clinical advancements. Thus, we strongly suggest further work to assess the effects of
current CXCR2 inhibitors on receptor internalization and degradation, which can be
easily done using the in-cell Western assay.
181

Figure 6.1 Schematic view of CXCR2 inhibition of previously reported CXCR2 inhibitors and CX25.
Site-directed mutagenesis studies show SB225002, SB265610, and SCH527123 bind to different regions of
CXCR2 and not necessarily at the ligand binding site. CX25, SCH527123, SB265610, SB225002,
AZ10397767, and CXCR2 RNA silencing demonstrate various effects on in vitro cancer models, such as
inhibiting cell migration and proliferation as well as decrease sensitivity to anticancer agents. Some of
these compounds also inhibit tumor growth, angiogenesis, and neutrophil infiltration in in vivo cancer
models.
In our studies, we have identified several promising compounds (CX4338, CX797,
CX25, and CX4152) that differentially modulate CXCR2 signaling as well as inhibit
cellular functions in various CXCR2-expressing cell lines. Though there are some
evidence that show these compounds may act via CXCR2, extensive binding studies are
still required to assess the affinity of these compounds to the receptor and to assess the
selectivity of these compounds to CXCR2. Given that most of the reported CXCR2
inhibitors are allosteric inhibitors and the differential effects of these compounds on
182
CXCR2 signaling, we strongly believe these compounds may act as allosteric inhibitors.
Thus, a direct binding assay is required to determine the binding capacity of these
compounds.
It would also be noteworthy to explore the effects of our compounds with PDE4
inhibitors. As a preliminary study, we found that 5 μM of SB265610 enhanced the
effects of a PDE4 inhibitor, ICI63197, in the cyclic AMP Glosensor assay (Figure 6.2),
suggesting that dual targeting of CXCR2 and PDE4 inhibitors may be a promising
therapeutic avenue for COPD patients. For example, roflumilast is a PDE4 inhibitor that
is approved for COPD treatment and effective at reducing neutrophil-mediated
inflammation in COPD (Rennard et al., 2011). However, it is associated with many
adverse side effects due to non-specific targeting of other PDE-expressing cells. We
postulate co-treatments with CXCR2 inhibitors may be able to reduce to dosage of
roflumilast used in the clinics and subsequently reduced adverse side effects associated
with PDE4 inhibition.
183

Figure 6.2 SB265610 enhance efficacy of PDE4 inhibitors in the cyclic AMP Glosensor assay.
293T-CXCR2-GFP-p22F cells were treated with various doses if ICI 63197 with or without 5 μM of
SB265610 for 10 minutes. Experiment was performed in duplicates. Fold change was calculated by
dividing treated over untreated controls.
Lastly, we believe to fully assess the anti-CXCR2 effects of our compounds requires
testing them in in vivo animal models with intact immune systems. Though others have
shown promising anti-tumor effects (growth and microvessel density) with CXCR2
inhibition, many of these models are performed in nude mice with compromised immune
systems. These studies may not necessarily translate to models with intact immune
systems and may not accurately assess the efficacy of CXCR2 inhibition. It is also
important to consider the expression of CXCR2 in animal models used to asses CXCR2
inhibitors. Murine and human CXCR2 share 68% homology, thus suggesting that
CXCR2 inhibitors may discriminate between human and murine CXCR2. Mihara et al.
generated a human CXCR2 knock-in and murine CXCR2 knock-out mice model that
showed that human CXCR2 is functionally equivalent to murine CXCR2 and that
CXCR2 inhibitor, SB332235, reduced CXCL8-induced neutrophil migration (Mihara et
184
al., 2005). This model might provide a more translatable in vivo model to assess the
efficacy of our compounds.
It would also be of great value to test our compounds and other CXCR2 inhibitors in
an inflammation-mediated lung cancer murine model. Moghaddam et al. developed a
inflammation-induced lung cancer model in a background of a G12D activated K-ras
allele in airway secretory cells (Moghaddam et al., 2009). Mice with the Cre
recombinase gene inserted into the Clara cell secretory protein (CCSP) gene (CCSPCre)
was crossed with LSL-K-rasG12D mouse resulting in CCSPCre/LSLK-rasG12D mice.
The CCSP gene is only expressed in airway secretory cells and thus the Cre recombinase
that will facilitate the insertion of K-rasG12D mutant allele will only be expressed in
airway secretory cells. When these mice were exposed to aerosolized NTHi lysate
(Haemophilus influenza, commonly found in lower respiratory tract of COPD patients)
resulted in neutrophil/macrophage/CD8 T cell associated COPD-like airway
inflammation. These mice also exhibited a 3.2-fold increase in lung surface tumor
number (156 ± 9 versus 45 ± 7), suggesting that COPD-like airway inflammation
promotes lung carcinogenesis in a background of an activated K-ras allele in airway
secretory cells. If CXCR2 inhibition is able to reduce inflammation and lung
carcinogenesis in this model, it will provide additional evidence that links inflammation
and cancer together and would also suggest that CXCR2 inhibitors may be developed as
chemopreventive agent in patients suffering from chronic inflammation.

185
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Abstract (if available)

Abstract Chemokine receptor, CXCR2, and its ligands are an essential component of the immune system that mediates the trafficking of neutrophils to sites of infection. It is also important in angiogenesis by activating and coordinating the assembly of endothelial cells. Given its roles in the immune system and angiogenesis, CXCR2 has been implicated in a number of inflammation-mediated diseases such as chronic obstructive pulmonary diseases (COPD) and various types of cancer. CXCR2 inhibition has shown promising anti-inflammatory effects in the clinics as well as anti-cancer effects in several in vivo animal models. And within the last two decades, several classes of small-molecules targeting CXCR2 has been developed and advanced onto clinical trials. ❧ However, current CXCR2 inhibitors are limited to four major chemical classes of compounds and are by far scarce compared to other clinically relevant therapeutic targets. Thus, we sought to identify additional classes of CXCR2 inhibitors using cell-based, functional assays rather than conventional competitive ligand binding assays. We also capitalized on the literature of previously described CXCR2 inhibitors and developed a pharmacophore model to identify important chemical features of CXCR2 inhibitors and to perform in silico screening of large databases of compounds. ❧ We have identified several classes of compounds that modulate CXCR2 signaling and cellular functions in CXCR2-expressing cells. These compounds are chemically different from any reported CXCR2 inhibitors. Mechanistic studies of these compounds showed that they differentially regulate CXCR2-mediated G-protein and β-arrestin1/2 signaling. For example, CX4338 inhibited CXCR2-mediated β-arrestin1/2 recruitment, while enhancing CXCR2-mediated G-protein signaling (calcium and ERK1/2 activation). Selective inhibition of CXCR2/β-arrestin1/2 association by CX4338 was sufficient to reduce cell migration and LPS-induced PMN migration. We also discovered CX797 in our screening endeavors using a cell-based cyclic AMP assay. CX797 inhibited the effects of CXCL8 on forskolin-induced cyclic AMP signaling, while enhancing CXCR2/β-arrestin2 association and up-regulating total CXCR2 expression. CX797 also inhibited CXCL8-mediated cell migration and cell proliferation in CXCR2-expressing cell lines. ❧ Lastly, using a pharmacophore model, we identified CX25 as a potent inhibitor of CXCR2 in the Tango assay. However, these class of compounds did not show much selectivity amongst different chemokine receptors and inhibited cancer cell proliferation in a CXCR2-independent manner. CX4152 was also identified from the pharmacophore screen and exhibit similar chemical features as CX25. Though CX4152 was not as active as CX25 against CXCR2, it showed 8-fold selectivity for CXCR2 over CXCR4 (another chemokine receptor). Mechanistic studies suggest that CX4152 inhibit CXCR2/β-arrestin-2 association via CXCR2 down-regulation. Treatment with CX4152 reduced surface CXCR2 expression within 10 minutes in a dose and time dependent manner. Taken together, compounds identified from our screens have differential effects on CXCR2 signaling and provide several novel avenues to mechanistically modulate CXCR2 function and signaling.

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Asset Metadata

Creator Ha, Helen Nen (author)

Core Title Identification of small-molecules targeting CXCR2 function and signaling

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 07/30/2013

Defense Date 05/27/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag CXCL8,CXCR2,Inflammation,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Neamati, Nouri (committee chair), Beringer, Paul (committee member), Duncan, Roger (committee member), Okamoto, Curtis Toshio (committee member)

Creator Email helenha@usc.edu,helenha83@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-308809

Unique identifier UC11294550

Identifier etd-HaHelenNen-1901.pdf (filename),usctheses-c3-308809 (legacy record id)

Legacy Identifier etd-HaHelenNen-1901.pdf

Dmrecord 308809

Document Type Dissertation

Format application/pdf (imt)

Rights Ha, Helen Nen

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA