Identification of small molecular inhibitors targeting G13D mutant K-ras

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Content IDENTIFICATION OF SMALL MOLECULAR INHIBITORS
TARGETING G13D MUTANT K-RAS

by
Yingzhe Yu

_____________________________________________________

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2012

Copyright 2012 Yingzhe Yu
ii
ACKNOWLEDGEMENTS

Firstly, I would like to take this opportunity to thank my thesis committees for
their advice. I would also like to express my sincerest appreciation to Dr.
Neamati, Dr. Comai and Dr. Reddy for offering me the opportunity to
participate the project. Without their guidance, this thesis would not have been
completed. I also want to thank our lab mates, especially, Dr. Otake, Dr.
Millard and Dr. Shabaik for their invaluable support. Finally, I would like to
express my deepest love and gratitude to my parents and wife for their
continuous support during my study.

iii
TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
LIST OF FIGURES iv
ABSTRACT v
CHAPTER 1: INTRODUCTION 1
1.1 Ras structure 2
1.2 Post-translational modification 2
1.3 Ras activation/inactivation 3
1.4 K-ras in human tumors 4
Table 1: Prevalence of K-ras mutations in
selected human tumors 26

CHAPTER 2: MATERIALS AND METHODS 7

2.1 Plasmid constructions 7
2.2 Cell transfection 11
2.3 Small-molecules screening 13

CHAPTER 3: RESULTS 14

3.1 Plasmid constructions 14
3.2 Cell transfection 16
3.3 Small-molecules screening 16

CHAPTER 4: DISCUSSION 18
REFERENCES 22
APPENDIX: FIGURES AND TABLE 25
iv
LIST OF FIGURES

Figure 1: Schematic depiction of K-ras structure. 25

Figure 2: Post-translational modification of Ras. 25

Figure 3: Simplified overview of Ras activation and signaling cascade. 26

Figure 4: Flow chart of the experiment. 27

Figure 5: PCR amplified k-ras located at the size of 567 bps on the gel. 27

Figure 6: Extracted and purified k-ras from the agarose gel. 28

Figure 7: Confirmation of the k-ras ligation with Topo vector. 28

Figure 8: NCBI BLAST alignment report for k-ras sequence (A: wide-type; 29
B: G13D mutant).

Figure 9: Map of the recombinant pcDNA3.1/Hygro /lacZ+k-ras 30

Figure 10: Confirmation of the k-ras ligation with pcDNA3.1/Hygro/lac-Z. 30

Figure 11: NCBI BLAST alignment report for k-ras sequence fused 31
with lac-Z

Figure 12: Confirmation of the three transfected 293 HEK cell lines 32
by detecting lac-Z expression.

Figure 13: Absorbance ratio of three transfected cell lines which were 33
treated by several compounds.

v
ABSTRACT

K- ras mutations can be frequently found in various cancers and are
associated with resistance to treatment or poor prognosis. Thus far, however,
there is no effective agent for treatment of cancers with k-ras mutations. This
study was designed to identify small molecular inhibitors which may selectively
target G13D mutant k-ras. The potentially active enamine analog compounds
(n=520), having high toxicity (>55%) in the HCT 116 colon carcinoma cell lines,
were screened in 293 HEK cells expressing the k-ras-lac-Z fusion protein in
the control vector pcDNA3.1/Hygro/lac-Z or pcDNA3.1/Hygro/lac-Z+k-ras
mutant
,
or pcDNA3.1/Hygro/lac-Z+k-ras
wild-type
. The data indicated that there is no
specific binding site between the compounds and k-ras because either the
wild-type and mutated k-ras both could not be inhibited, or the wild-type and
mutated k-ras were both inhibited with synchronously impacting the non-k-ras
transfected 293 HEK cells by the potential active enamine analog compounds.
Therefore, further studies and modifications on the structures and
conformation of the active compounds are necessary in order to identify the
ideal inhibitors for G13D mutant k-ras.

1

CHAPTER 1

INTRODUCTION
K-ras, a highly homologous group with an approximate size of 21 kDa protein,
is a family member of membrane-localized GTPases. There are many
publications which have extensively discussed the structural and functional
features of k-ras [1, 2, 4]. The 21kDa ras proteins are comprised of h-, n- and
k-ras, with k-ras existing in two forms, 4A and 4B. Each of these ras proteins
has an approximately 85% sequence homology. However, these ras family
members are different in c-terminal residues which are very important for ras
proteins’ post-translational modification [1, 5]. Additionally, there is a larger ras
superfamily, including r-ras, m-ras, TC21, Rap1A, Rap1B, RalA and RalB,
which share approximately 40–50% sequence homology [9, 25]. In total, there
are over 150 small GTPases in the ras superfamily [6]. The ras genes can
transform cells and are analogous to the oncogenes of the Harvey and Kirsten
sarcoma viruses for h-ras, for k-ras and for n- ras ---an oncogene isolated from
a neuroblastoma [17, 18]. All of these proteins play their roles as ‘‘molecular
switches,’’ conducting signals from the extracellular to intracellular
environment.

2

Ras structure
There are 189 amino acids in ras proteins for h-ras, n-ras and k-ras4A,
whereas k-ras4B contains an additional amino acid [29]. From amino acids 1
to164, the sequences are highly conserved among the four ras proteins, but
the remaining c- terminal 25 amino acids display many variations [22]. The
conserved domains are important motifs which play key roles in protein
function including GTP binding and effector binding. Switch I and switch II
loops are responsible for Guanine nucleotide exchange factor (GEF) and
GTPase activating protein (GAP) interactions (Fig. 1). [11] The fact that the
protein exists in different conformations in the GDP versus GTP bound states
was discovered by investigating the crystal structure of ras. [22, 29] It is likely
important for the interaction with GEFs and GAPs. Ras point mutations, highly
prevalent in human tumors, result in the ras proteins being insensitive to
interactions with GAP which can hydrolyze GTP to GDP. Thereby the GTPase
is trapped in the ‘‘on’’ state [22, 29].

Post-translational modification
It is a multistep process for ras proteins to undergo a post-translational
modification (Fig. 2) [11]. Firstly, the proteins need to be prenylated, the
addition of a farnesyl group to the c-terminus catalyzed by farnesyltransferase
(FT). However, when farnesyltransferase is inhibited, a geranylgeranyl group
3

may be added to the c-terminus by geranylgeranyl transferase type I (GGT-1)
or geranylgeranyl transferase type II (GGT-2). Both FT and GGT-1 can
recognize a specific CAAX motif present in each of the ras proteins [11]. The
terminal X of the CAAX motif determines whether farnesylation or
geranygeranylation occurs [11, 22]. When farnesyltransferase is inhibited,
k-ras and n-ras are prenylated by GGT-1, while h-ras is not [13]. In addition,
GGT-2 can recognize proteins that contain two c-terminal cysteine residues
and plays a major role in ras prenylation according to the protein sequences
[21]. Secondly, in the presence of CAAX proteases Rce1 or Afc1, prenylated
ras proteins are cleaved at the terminal AAX motif [21]. Thirdly, the cleaved ras
proteins are methylated at the terminus via a specific methyl transferase [21].
Finally, all methylated proteins (except k-ras4B) are palmitoylated at the SH
group of cysteine residues close to the c-terminus [13, 21].

Ras activation/inactivation
It is extremely complicated for the activation of the ras pathway in terms of the
wide range of factors that can initiate ras signaling. The signals and stimulation
from various upstream effectors such as receptor tyrosine kinases integrins,
serpentine receptors, heterotrimeric G-proteins and cytokine receptors, can
initiate ras activation [11]. One of the best described pathways is that ras is
stimulated by a tyrosine kinase receptor such as EGF receptor (Fig. 3) [11].
4

When a ligand binds to EGF receptor, it can induce the oligomerization of the
receptor, causing juxtaposition of the cytoplasmic domains, which activates the
kinase activity and transphosphorylation [3]. Then the sequence homology 2
(SH2) domains can be recognized by the adaptor proteins such as Grb2
before it recruits guanine nucleotide exchange factors (GEFs) such as SOS or
CDC25 [3]. Subsequently, the GEF is able to interact with ras proteins at the
cell membrane to process a conformational change, from GDP state to GTP
state [11]. Therefore, the ras activation is significantly related to the subcellular
localization of GEFs. The event that the GTP is hydrolyzed to GDP results in
the termination of ras activation. GAPs and p120-GAP can inhibit the
intrinsically low GTPase activity of ras proteins. [28] The role of GAPs play in
regulating ras activation was demonstrated by the fact that ras mutations
almost block the interaction of GAPs and Ras [11]. In normal cells, because of
the intrinsic GTPase activity and the function of GAPs, ras signaling is
transient [11]. However, under the circumstance of ras mutations, prolonged
ras signaling is responsible for ras induced oncogenesis [3].

K-ras in human tumors
Ras mutations are prevalent in approximately 30% of various human tumors
and the k-ras mutations have been frequently and extensively found among
the mutations of different tumor types (Table 1) [11], indicating that the k-ras is
5

expressed in most human cells and could be a target for clinical therapy. For
example, k-ras mutations showed a significant tendency in human pancreatic
tumors because k-ras mutations were identified in approximately 72–90% of all
pancreatic tumors. [26]. In addition, other ras mutations such as h-ras and
n-ras were also relatively frequently found in other tumors. For example, n-ras
mutations were commonly identified in both primary melanomas and
melanoma cell lines, whereas k-ras mutations are not [27]. Therefore, even
though the different members of ras family are highly homologous, the various
ras proteins play their function in their own manners. For instance, it is fatal to
repress or delete the k-ras gene in mouse embryos, but it is not for either h-
ras or n-ras. [19] Furthermore, the various ras proteins have different ways to
undergo post-translational modifications [14]. Alternatively, even though there
is no activation of mutated k-ras, it may still be responsible for oncogenesis via
ras gene amplification, over expression or upstream activation of the pathway,
inducing activation of ras effectors, thereby developing tumors [10, 24]. It was
reported that 40% of the tumors have the amplification of the k-ras gene in
esophageal adenocarcinomas [8]. Because k-ras mutations are so critical in
human tumors that many groups were interested in determining if these
mutations have significant correlation with disease stage or prognosis. Jansik
et al reported that k-ras mutations were used to estimate risk groups in
endometrial cancer using a pathological approach [16]. The allelespecific
6

amplification technique was applied to measure the depth of myometrial
invasion according to the expression of mutant k-ras, predicting the overall
survival and relapse rate [16]. In this study, pcDNA3.1/Hygro/lac-Z and the
recombinant plasmid cDNA3.1/Hygro/lac-Z+ k-ras (wild-type or G13D mutant)
were transfected into 293 HEK cells. By using these three transfected cell lines,
we hypothesize that the potential active enamine analog compounds could
somehow specifically target G13D mutant k-ras.

7

CHAPTER 2

MATERIALS AND METHODS
Flow chart of the experiment
The entire experiment was performed as the flow chart shows (Fig. 4).

Enamine analogs compounds
The powder compounds were dissolved in DMSO and were stored at -20 ° C
for future use.

Cell lines
The 293 human embryonic kidney cells, human colorectal carcinoma HT29
and HCT116 cell lines were obtained from ATCC (American type culture
collection) and were routinely cultured in a monolayer culture in DMEM
(Mediatech Inc., VA, USA) supplemented with 10% heat-inactivated FBS
(HyClone Laboratories Inc., UT, USA), 100 units/mL penicillin (GIBCO), and
100 units/mL streptomycin (GIBCO). All cells were incubated in the presence
of 5% CO
2
at 37° C.

RNA extraction
RNAs were extracted from the HT29 and HCT116 cell line by using the RNA
8

extraction kit (Qiagen, Valencia, CA) and were reverse-transcribed to cDNA
with a 1
st
-strand cDNA synthesis kit (GE Healthcare, NJ) according to the
manufacturer’s instructions.

Amplification of cDNA
The cDNAs were amplified by PCR with two primers (forward primer:
5’-CCCAAGCTTAAAATGACTGAATATAAAC-3’ and reverse primer:
5’-CGCGGATCCATTATAATGCATTTTTTA-3’), including HindIII and BamHI
restriction sites, specifically for the k-ras gene coding region. The PCR
reaction mixture (25 ul) contained 300 ng of the template DNA, 17.45 ul of
double-distilled H
2
O, 3% of MgCl
2
, 2.5 ul of 10 x buffer, 1 ul dNTP (10 mM), 0.3
ul of taq polymerase and 5 pmol of each primer mixture. Thermal cycling was
initiated with a 2 min reaction at 95° C and denatura tion at 95° C for 2 min
followed by 35 three-step cycles at 95° C for 30 s, 40° C for 40 s, and 72° C for
60 s, and followed by a final incubation for 7 min at 72° C.

Extraction of the PCR product from agarose gel
The k-ras DNA bands were isolated from the agarose gel under the UV light
and the weights were measured. By using the gel extraction kit (Qiagen,
Valencia, CA), the k-ras DNAs were extracted and purified from the agarose
gel.
9

Topo cloning of k-ras
The amplified k-ras DNA (300 ng) was inserted into Topo vector (3956 bps, 1
ul) according to the manufacturer’s instruction (Invitrogen, CA). The plasmids
were then transformed into competent E. coli cells in LB medium. After 1 hour
incubation at 37° C, the cells were spread onto LB plate s for 16 hr incubation.

Plasmid extraction from single clone
From the LB plates, 15 single clones were selected and each clone was mixed
with 3 ml of pre-warmed LB medium which contained 0.1% Ampicillin. The 15
mixtures were incubated for 16 hr. Then, the plasmids were extracted from
each incubated mixture by following the manufacturer’s miniprep kit instruction
(Invitrogen, CA). The concentrations of the plasmids were measured by using
Nano drop spectrophotometer.

Confirmation of the k-ras ligation with Topo vector
The extracted recombinant plasmid Topo+k-ras (1 ug) was mixed with 2 ul of
the 10 x NEBuffer 4, 0.4 ul of BamHI, 0.4 ul of HindIII and double-distilled
water to get a 20 ul reaction mixture before being incubated at 37° C for 3 hr.
Then the enzyme digestion reaction was detected by gel electrophoresis to
confirm the k-ras ligation with Topo vector.

10

Sequencing analysis of the K-ras in Topo vector
Five randomly selected plasmids were purified and sequenced to identify that
the wild-type or mutant k-ras were properly inserted into the Topo vector using
M13 reverse primers with automated sequencing using a Perkin Elmer
sequencer (DNA core, Keck School of USC).

Recombination of plasmid cDNA3.1/Hygro/lac-Z+k-ras
The identified wild-type or mutant k-ras gene was digested from the
Topo+k-ras plasmid by BamHI and HindIII restriction enzymes according to the
manufacturer’s instruction (New England Biolabs, Beverly, MA). The digested
wild-type or mutant k-ras fragment was extracted and purified from the
agarose gel after the gel electrophoresis. The purified wild-type or mutant k-ras
was then ligated into the pcDNA3.1/Hygro /lacZ (8.6 kb, Invitrogen, CA) by
following the manufacturer’s instruction. Next, the plasmids were transformed
into competent E. coli cells in LB medium. After 1 hour incubation at 37° C, the
cells were spread onto LB plates for 16 hr incubation.

Plasmid extraction from selected single clone
From the LB plates, 10 single clones were selected and each clone was mixed
with 3 ml of pre-warmed LB medium which contained 0.1% of Ampicillin. The
10 mixtures were incubated for 16 hr. Then, the plasmids were extracted from
11

each incubated mixture by following the manufacturer’s miniprep kit instruction
(Invitrogen, CA). The concentrations of the plasmids were measured by using
Nano drop spectrophotometer.

Confirmation of the k-ras liagtion with pcDNA3.1/Hygro/lac-Z
The 10 identified wild-type or mutant k-ras gene were digested from the
plasmid cDNA3.1/Hygro/lac-Z+k-ras using BamHI and HindIII restriction
enzymes according to the manufacturer’s instructions (New England Biolabs,
Beverly, MA). The ligated k-ras fragment was detected by gel electrophoresis.
Sequencing analysis of the K-ras in pcDNA3.1/Hygro/lac-Z
The recombinant plasmids were purified and sequenced to identify that the
wild-type or mutant k-ras were properly inserted into the
pcDNA3.1/Hygro/lac-Z and fused with the report marker lac-Z using a newly
designed primer (5’-GAAGATGTACCTATGGTCCTAGTAG-3’). Automated
sequencing was done using a Perkin Elmer sequencer (DNA core, Keck
School of USC).

Transfection of plasmid cDNA3.1/Hygro/lac-Z+k-ras
The 293 HEK cells were coated at a density of 1x10
5
cells in each well of
six-well plates. After an overnight culture, 1 ug of the pcDNA3.1/Hygro/lac-Z, 1
ug of the pcDNA3.1/Hygro/lac-Z+k-ras
mutant
and
12

pcDNA3.1/Hygro/lac-Z+k-ras
wild-type
was in turn mixed with 2 ul of lipofectamine
2000 (Invitrogen, CA). Then the coated 293 HEK cells were transfected by
each of the above DNA-lipofectamine 2000 complex. After 48-hr incubation,
the cells were then reseeded into 10-cm dishes and stable transfectants were
selected with 100 ug/ml of Hygromycin B (Invitrogen, CA). The effective
concentration of Hygromycin B was determined for all three cell lines by plating
5 x10
5
cells per well in six-well plates in DMEM, 10% fetal calf serum. Cells
were seeded with increasing concentration (40, 60, 80, 100, 150, 200 ug/ml) of
Hygromycin B and the effective concentration was estimated by microscopy
after 3 days. The 293 HEK cells transfected with the control vector
pcDNA3.1/Hygro/lac-Z, pcDNA3.1/Hygro/lac-Z+k-ras
mutant
, and
pcDNA3.1/Hygro/lac-Z+k-ras
wild-type
were termed 293 HEK/vector, 293
HEK/k-ras
mutant
and 293 HEK/k-ras
wild-type
cells, respectively.

Confirmation of the three transfected cell lins
The fixed 293 HEK/vector, 293 HEK/k-ras
mutant
, and 293 HEK/k-ras
wild-type
cells
were washed with PBS and stained for 1 hr at 37° C in staining mixture (5 mM
potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg/ml of X-gal, 2mM
MgCl
2
). Cells were selected on the basis of the increased number and intensity
of blue-staining cells.

13

Small-molecule screening
On day 1, the three cell lines were coated at a density of 5x10
3
cells in each
well of 96-well plates. After an overnight culture, the compounds were added
into each single well of the three cell lines at a final concentration of 10 uM. On
day 5, all cells were washed with PBS and stained for 1 hr at 37° C in staining
mixture (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg/ml
of X-gal, 2mM MgCl
2
). Then the absorbance values for 293 HEK/vector, 293
HEK/k-ras
mutant
, and 293 HEK/k-ras
wild-type
cells were measured at 405 nm to
quantitate the compounds’ effects.

14

CHAPTER 3

RESULTS
Amplification of k-ras cDNA
The k-ras cDNAs were amplified by PCR with two primers. The results of gel
electrophoresis confirmed that the wild-type k-ras from HT29 and mutant k-ras
from HCT116 cell line were amplified at the size of 567 bps (Fig. 5).

Extraction of the PCR product from agarose gel
The k-ras DNA bands were isolated from the agarose gel under the UV light
and the weights were measured. By using the gel extraction kit (Qiagen,
Valencia, CA), the k-ras DNAs were extracted and purified from the agarose
gel. The results of gel electrophoresis confirmed that the wild-type k-ras from
HT29 and mutant k-ras from HCT116 cell line were extracted and purified (Fig.
6)

Confirmation of the k-ras ligation with Topo vector
The extracted plasmid Topo+k-ras
wild-type
or Topo+k-ras
mutant
(1 ug) were
digested by BamHI and HindIII to confirm the k-ras ligation with Topo vector.
The two fragments, Topo vector and k-ras, were digested by BamHI and
HindIII and separately located at the size of 4 kb and 567 bp (Fig. 7).
15

Sequencing analysis of the K-ras in Topo vector
Randomly selected plasmids were purified and sequenced to identify that the
wild-type or mutant k-ras was inserted into the Topo vector properly with M13
reverse primers. The sequence alignment report from NCBI showed that
wild-type k-ras and G13D mutant k-ras (GGCGAC) were properly inserted
into Topo vector (Fig. 8 A & B).

K-ras (wild-type and mutant) liagtion with pcDNA3.1/Hygro/lac-Z
The identified wild-type and mutant k-ras were digested from the Topo+k-ras
plasmid by BamHI and HindIII restriction enzymes and were extracted and
purified from the agarose gel after the gel electrophoresis. Then the purified
wild-type or mutant k-ras were ligated into the pcDNA3.1/Hygro /lacZ (Fig. 9).
In order to confirm the ligation, the recombinant pcDNA3.1/Hygro/lac-Z+k-ras
(wild-type or mutant) were digested by BamHI and HindIII restriction enzymes.
As figure 10 showed, the two fragments, pcDNA3.1/Hygro/lac-Z and k-ras,
were digested by BamHI and HindIII and separately located at the size of 8.6
kb and 567 bp.

Sequencing analysis of the K-ras in pcDNA3.1/Hygro/lac-Z
The sequence alignment report from NCBI showed that wild-type k-ras and
mutant k-ras (G13D) were inserted into pcDNA3.1/Hygro/lac-Z properly to fuse
16

with report marker lac-Z because there was no frame shift between k-ras and
lac-Z (Fig. 11).

Confirmation of the three transfected 293 HEK cell lines
The fixed non-transfected 293 HEK cells, 293 HEK/vector, 293 HEK/k-ras
mutant
,
and 293 HEK/k-ras
wild-type
cells were washed with PBS and stained in X-gal
staining mixture. The non-transfected 293 HEK cells were not blue stained (Fig.
12 A), whereas, the 293 HEK/vector cells, the 293 HEK/k-ras
mutant
, and 293
HEK/k-ras
wild-type
cells were blue stained because lac-Z was expressed in the
three cell lines (Fig. 12 B, C & D). This indicated that these three cell lines
were transfected successfully and were ready for compounds screening.

Compounds screening
The three transfected cell lines were coated at 5x10
3
cells in each well of
96-well plates. After an overnight culture, the compounds were added into
each single well of the three cell lines at 10 uM. On day 5, all cells were
washed with PBS and stained for 1 hr at 37° C in X-gal staining mixture. The
absorbance values for 293 HEK/vector, 293 HEK/k-ras
mutant
and 293
HEK/k-ras
wild-type
cells at 405 nm were measured and the absorbance ratio data
showed that among the 520 compounds, there was no significant difference of
inhibitory effect between either 293 HEK/vector and 293 HEK/k-ras
mutant
, or
17

293 HEK/vector and 293 HEK/k-ras
wild-type
(Fig. 13 A). Even though the
wild-type and mutated k-ras were inhibited by several compounds, the
non-transfected 293 HEK cells were also significantly impacted by these
potential active enamine analog compounds (Fig. 13 B).

18

CHAPTER 4

DISCUSSION
K-ras mutations are common in human cancers and play very important roles
in the development of various human cancers including cancer initiation,
metastasis, prognosis and response for treatment [2, 4, 8]. Therefore,
identifying a series of compounds for various k-ras mutations (G12S, G12R,
G12V, G12D, G12A, G13D) will contribute enormously to the patients with a
k-ras mutation. In this study, we present a simple assay to identify a small
molecular inhibitor for k-ras mutation (G13D) in colorectal cancer. Because
there are several stages before k-ras mutations occur, from DNA level to
protein, to develop into cancer stage. Now we hypothesize that there are a
series of small molecular inhibitors which could target the mRNAs of the
mutant k-ras to repress its translation into protein. Although none of the
currently tested 520 compounds was capable of specifically inhibiting mutant
k-ras or inhibiting mutant k-ras without impacting other domains, it is still an
encouraging clue for us to explore the detailed structural conformation of the
k-ras in order to design and modify better compounds for it. Alternatively,
instead of only focusing on codon 13 mutation, the other frequently mutated
codons, such as 12, 18, 19 and 146, can also be selected for compounds
screening [16]. Once the compounds are identified, combined with the
19

techniques of k-ras mutation detection [23], the k-ras mutation patients could
be treated according to their outcomes of k-ras sequence without wasting time.

Alternative approaches to inhibit k-ras
1. Antisense oligonucleotides
Several studies reported that an introduced antisense k-ras DNA in tumor cells
was expected to pair with their complementary ras mRNAs and to inhibit the
ras protein translation. The expression of h-ras protein was repressed by 90%
by an oligonucleotide which could target h-ras in NIH 3T3 cells transformed
with activated h-ras [20]. Additionally, after being treated with the antisense
oligonucleotide, the cells showed a weak tumorigenicity when transfected into
mice. Similarly, an adenovirus containing a 247-bp sequence was used to
inhibit the growth of human pancreatic cells in the peritoneal cavity by
complementary binding to k-ras [20]. However, the antisense method has
showed the realistic limitation on safely and effectively delivering antisense
oligonucleotide.

2. RNA interference
RNA interference is a post-transcriptional gene silencing mechanism by which
specific gene sequences can be targeted and repressed by double stranded
RNA [7]. When the short interfering RNAs (siRNA) are introduced into cells,
20

the RNase III enzyme cuts the siRNA into smaller fragments and RNA-silencer
complex is then generated to recognize and target the specific mRNA, which
have a hairpin loop structure with the complementary sequences, resulting in
degradation of the specific mRNA. Although the mismatches of single base
pair can decrease the mRNA degradation, the mutant k-ras can still be
recognized and targeted, which is better than the antisense techniques [7].
Duursma et al reported that only the mutant k-ras
V12
was targeted and
repressed by a siRNA which was introduced into tumor cell lines by virus,
without repressing wild-type k-ras [7]. Additionally, in soft agar colony forming
assays and tumor formation in mice model, the siRNA-bound k-ras
V12
showed
a decreased tumorigenicity in cells transformed with k-ras
V12
, but not wild-type
cells [15]. Therefore, it is very important to effectively deliver the specific
siRNAs into tumor cells. However, siRNAs have the same challenge as the
antisense techniques for safe and effective target delivery [15].

3. Immunological approaches
The immunological differences between wild-type and mutant k-ras are
regarded as a breakthrough for mutant k-ras inhibition. The immune response,
involving both CD4+ and CD8+ T-lymphocytes, can be initiated by the
vaccination with mutant ras peptides in both cancer patients and normal
individuals. In in vitro assays, it has been reported that the growth of human
21

cell lines containing corresponding ras mutations was recognized and inhibited
by these T-cells [12]. Therefore, future research is required to take advantage
of this for clinical application.

Based on what has been described above, k-ras clearly plays a critical role in
the development and maintenance of human cancers. The fact that ras
mutations, especially k-ras, are so prevalent that makes it an attractive
therapeutic target. However, none of the compounds showed a specifically
repressive effect on mutated k-ras. It is very possible that the antitumor
activities of these compounds are dependent on multiple signaling pathways.
Therefore, the identification of small molecular inhibitor for cancer therapy
targeting mutant k-ras is expected to find the specific pathway.

22

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25

APPENDIX: FIGURES AND TABLE

Fig 1. Schematic depiction of K-ras structure. K-ras contains a number of
defined functional domains. Multiple regions participate in GTP binding. The
Switch I domain is responsible for GAP interaction as well as some effector
interactions, while the Switch II domain interacts with GEFs. The hypervariable
domain contains non-conserved amino acid residues, a terminal CAAX box
and a polylysine domain for K-ras 4B.

Fig 2. Post-translational modification of Ras. All Ras proteins contain the
CAAX motif at the c-terminus, directing farnesylation on the cysteine residue
by farnesyltransferase. Some Ras proteins are also substrates for
geranylgeranyltransferase, particularly in the presence of farnesyl transferase
inhibitors. Next, the terminal AAX tetrapeptide is removed by the CAAX
protease RceI, followed by methylation of the terminal cysteine residue. K-ras
proteins are differentially modified at this point, with K-ras 4A receiving
palmitoyl on cysteine residues near the carboxy terminus, while K-ras 4B
utilizes a polylysine domain for efficient membrane localization.
26

Fig 3. Simplified overview of Ras activation and signaling cascade. Activation
of a receptor tyrosine kinase (RTK) by its appropriate growth factor stimulates
autophosphorylation of SH2 domains that recruit Grb2. Guanine nucleotide
exchange factors (GEF) such as SOS are localized to the membrane by Grb2,
which then stimulate Ras to exchange GDP for GTP. The activated Ras
interacts with multiple signaling pathways, including phosphoinositide
3Vkinase (PI3-K), MEKKI, Raf kinase, RalGEFs and phospholipase C (PLC) to
produce cellular responses. Ras signaling is terminated when GTPase
activating proteins such as p120 and NF-1 stimulate Ras to hydrolyze GTP to
GDP.

Table 1: Prevalence of K-ras mutations in selected human tumors

27

Fig 4: Flow chart of the experiment

Figure 5: PCR amplified k-ras located at the size of 567 bps on the gel.

28

Fig 6: extracted and purified k-ras from the agarose gel

Fig 7: confirmation of the k-ras ligation with Topo vector.

29

Fig 8: NCBI BLAST alignment report for k-ras sequence (A: wild-type; B: G13D
mutant).
30

Fig 9: map of the recombinant pcDNA3.1/Hygro /lacZ+k-ras

Fig 10: confirmation of the k-ras ligation with pcDNA3.1/Hygro/lac-Z.
31

Fig 11: NCBI BLAST alignment report for k-ras sequence fused with lac-Z.

32

Fig 12: confirmation of the three transfected 293 HEK cell lines by detecting
lac-Z expression. (A: non-transfected 293 HEK cells; B: 293 HEK cells
transfected by pcDNA3.1/Hygro/lac-Z; C: 293 HEK cells transfected by
pcDNA3.1/Hygro/lac-Z+k- ras
mutant
; D: 293 HEK cells transfected by
pcDNA3.1/Hygro/lac-Z+k- ras
wild-type
.

33

Fig 13: absorbance ratio of three transfected cell lines which were treated by
several compounds.

Asset Metadata

Creator Yu, Yingzhe (author)

Core Title Identification of small molecular inhibitors targeting G13D mutant K-ras

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/25/2012

Defense Date 06/06/2012

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

Tag G13D mutant K-ras,inhibitors,OAI-PMH Harvest,targeting

Language English

Advisor Tokes, Zoltan A. (committee chair), Lu, Wange (committee member), Neamati, Nouri (committee member)

Creator Email yy941yy941@gmail.com,yyz0886@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-66766

Unique identifier UC11289976

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Legacy Identifier etd-YuYingzhe-1001.pdf

Dmrecord 66766

Document Type Thesis

Rights Yu, Yingzhe

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

Abstract (if available)

Abstract K-ras mutations can be frequently found in various cancers and are associated with resistance to treatment or poor prognosis. Thus far, however, there is no effective agent for treatment of cancers with k-ras mutations. This study was designed to identify small molecular inhibitors which may selectively target G13D mutant k-ras. The potentially active enamine analog compounds (n=520), having high toxicity (>55%) in the HCT 116 colon carcinoma cell lines, were screened in 293 HEK cells expressing the k-ras-lac-Z fusion protein in the control vector pcDNA3.1/Hygro/lac-Z or pcDNA3.1/Hygro/lac-Z+k-rasᵐᵘᵗᵃⁿᵗ, or pcDNA3.1/Hygro/lac-Z+k-rasʷⁱˡᵈ⁻ᵗʸᵖᵉ. The data indicated that there is no specific binding site between the compounds and k-ras because either the wild-type and mutated k-ras both could not be inhibited, or the wild-type and mutated k-ras were both inhibited with synchronously impacting the non-k-ras transfected 293 HEK cells by the potential active enamine analog compounds. Therefore, further studies and modifications on the structures and conformation of the active compounds are necessary in order to identify the ideal inhibitors for G13D mutant k-ras.