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Exosomal inhibitory activity of clinically approved tertiary amines for the treatment of Kaposi’s sarcoma tumorigenesis

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Exosomal inhibitory activity of clinically approved tertiary amines for the treatment of Kaposi’s sarcoma tumorigenesis

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Content Copyright 2024 Kaitlyn Allyson Trinh
EXOSOMAL INHIBITORY ACTIVITY OF CLINICALLY APPROVED TERTIARY
AMINES FOR THE TREATMENT OF KAPOSI’S SARCOMA TUMORIGENESIS
by
Kaitlyn Allyson Trinh
A Thesis Presented to the
FACULTY OF THE USC MANN SCHOOL OF PHARMACY AND PHARMACEUTICAL
SCIENCES
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
[CLINICAL AND EXPERIMENTAL THERAPEUTICS]
May 2024

ii
ACKNOWLEDGEMENTS
To the Stan Louie Lab: Dr. Stan Louie, Dr. Isaac Asante, Aditya Naik, Angela Lu, Brandon
Ebright, Catherine Chester, Priyal Dave, Weiyuan Hu
and
to my cherished family and friends for their unwavering support

iii
TABLE OF CONTENTS
Acknowledgements......................................................................................................................... ii
List of Tables ................................................................................................................................. iv
List of Figures..................................................................................................................................v
Abstract.......................................................................................................................................... vi
Chapter 1: Introduction to Kaposi’s Sarcoma-Associated Herpesvirus and Exosomes..................1
1.1 KSHV, Kaposi’s Sarcoma, and Exosomes.............................................................................1
1.2 Exosome-mediated KS Tumorigenesis..................................................................................4
1.3 Exosome Biogenesis and Cargo-Loading .............................................................................5
1.3.1 ESCRT-dependent Pathway ............................................................................................7
1.3.2 ESCRT-independent Pathway.........................................................................................8
1.4 Known Exosome Inhibitors ...................................................................................................9
Chapter 2: In Silico Drug Candidate Selection..............................................................................12
2.1 Screening of Clinically Approved Tertiary Amines as Drug Candidates............................12
2.1.1 Methods: Autodock-Vina ..............................................................................................13
2.1.2 Results: In Silico Screening of Compounds with a Tertiary Amine ............................14
2.2 Drug Candidates...................................................................................................................17
Chapter 3: In Vitro Preclinical Development of Selective Exosome Biogenesis Inhibitors .........20
3.1 Vector Validation of vGPCR-expressing B16-F10 Melanoma Cells ..................................20
3.1.1 Materials and Methods: Antibiotic Selection................................................................22
3.1.2 Results: Transfected Model...........................................................................................23
3.2 Drug Screening Cell Viability to Determine IC50...............................................................25
3.2.1 Materials and Methods: Resazurin Reduction Cell Viability Assay .............................25
3.2.2 Results: IC50 of Drug Candidates.................................................................................26
3.3 Gene Expression of Exosome Biogenesis and Cargo-loading .............................................29
3.3.1 Materials and Methods: Gene Expression Analysis of Exosomal Inhibitory Activity .31
3.2.2 Results: Gene Expression Analysis of Exosomal Inhibitory Activity...........................32
Chapter 4: Structure-Activity Relationship of Compounds Targeting Farnesyltransferase ..........39
4.1 Binding Interactions of Fluconazole to FTase .....................................................................39
4.1.1 Materials and Methods: Ligplus....................................................................................39
4.1.2 Results: Fluconazole Targeting FTase .........................................................................39
Discussion......................................................................................................................................43
Conclusion .....................................................................................................................................48
References......................................................................................................................................50

iv
LIST OF TABLES
Table 1. Human Farnesyltransferase (FTase) Binding Affinity and Dissociation Constant .........14
Table 2. Neutral Sphingomyelinase (nSMase) Binding Affinity and Dissociation Constant........15
Table 3. Rank Sum of In Silico Drug Candidates..........................................................................16
Table 4. IC50 Cell Viability Summary Across 24- and 48-hours Drug Exposure ........................28

v
LIST OF FIGURES
Figure 1. Exosome Biogenesis and Cargo-Loading via ESCRT-dependent and -independent
Pathway............................................................................................................................................7
Figure 2. Induction of Downstream Pathways by Viral GPCR.....................................................21
Figure 3. vGPCR Vector Map Using pEF6/V5-HIS TOPO Expression Vector ...........................23
Figure 4. Blasticidin Antibiotic Selection of B16-F10 and vGPCR-expressing Cells Day 5........24
Figure 5. Percentage Cell Viability of Manumycin A Based on Fluorometric Analysis ..............26
Figure 6. Percentage Cell Viability of Drug Candidates Based on Fluorometric Analysis...........27
Figure 7. Percentage Cell Viability of 24-hours Treatment of Drug Candidates ..........................28
Figure 8. Effect of Tetracycline Analogues Treatment of Exosome Biogenesis...........................33
Figure 9. Effect of Azole Drug Candidates on Exosome Biogenesis............................................34
Figure 10. Effect of Drug Candidates Imipramine and Sitafloxacin on Exosome Biogenesis......35
Figure 11. Evaluation of Cargo-loading Mechanisms due to Drug Treatment on vGPCR
Transfected Cells. ..........................................................................................................................37
Figure 12. Binding Orientations of Known Exosome Inhibitors Manumycin A and Tipifarnib to
Farnesyltransferase ........................................................................................................................40
Figure 13. Shared Binding Interactions of Fluconazole to Known Exosome Inhibitors on
Farnesyltransferase ........................................................................................................................41

vi
ABSTRACT
As the causative agent of Kaposi’s sarcoma (KS), Kaposi’s sarcoma-associated herpesvirus
(KSHV) facilitates the immunosuppressive tumor microenvironment and modulates signaling
pathways by hijacking exosome-mediated cell-to-cell communication. These exosomes are loaded
with oncogenic factors, such as microRNAs and proteins, that infect recipient cells and promote
cell transformation via paracrine mechanisms. As these exosomes play an important role in
persistent viral infection and tumor progression, exosome inhibition is a potential avenue for the
treatment of KS tumorigenesis. Therefore, we evaluated clinically approved drug compounds for
exosomal inhibitory activity and defined the structure-activity relationship of compounds targeting
farnesyltransferase to inhibit exosome biogenesis. Using Autodock-Vina, compounds with a
tertiary amine moiety were screened for potential binding affinity to target enzymes of known
exosome inhibitors, farnesyltransferase (FTase) and neutral sphingomyelinase. Drug treatment on
B16-F10 melanoma cells transfected with the oncogenic virally encoded G-protein coupled
receptor evaluated modulation of exosome biogenesis and cargo-loading mechanisms.
Downregulation of Rab27A and Rab31 affirmed the exosomal inhibitory activity of ketoconazole
and imipramine. Interestingly, 24-hour treatment of 200µM fluconazole resulted in the
downregulation of Unc13D, Rab27A, Rab31, and Akt1, suggesting that fluconazole could
potentially target FTase to inhibit exosome biogenesis. Additionally, a comparison of the binding
interactions of fluconazole and known compounds targeting FTase via molecular modeling
demonstrated a hydrophobic binding region essential to their structure-activity relationship for
exosome inhibition. These results define fluconazole as a potential exosome biogenesis inhibitor
for the treatment of KS tumorigenesis.

1
Chapter 1: Introduction to Kaposi’s Sarcoma-Associated
Herpesvirus and Exosomes
1.1 KSHV, Kaposi’s Sarcoma, and Exosomes
Human herpesvirus 8 (HHV-8), also known as Kaposi’s Sarcoma-associated Herpesvirus
(KSHV), is an oncogenic gamma herpesvirus associated with lymphoproliferative disorders, such
as Kaposi’s sarcoma (KS), primary effusion lymphoma, multicentric Castleman’s disease, and
KSHV-associated inflammatory cytokine syndrome (He, 2019). KSHV is highly prevalent in subSaharan Africa (>90%), followed by regions surrounding the Mediterranean Sea (20-30%) and
Northern Europe, Asia, and North America (<10%). The variation in infection prevalence across
geographic regions suggests that there are underlying factors that influence the incidence and
prevalence of KSHV. A suggested environmental factor is coinfection with other parasitic
infections, which increases shedding of KSHV in saliva. As it is currently thought that KSHV is
mainly transmitted through saliva, there could be cultural factors that promote saliva exchange and
viral spread (Cesarman, 2019; Wakeham, 2011). Additionally, intimate contact has been thought
to be a factor for viral spread as KSHV has been found in patients with human immunodeficiency
virus (HIV). It is also predicted that host genetic polymorphisms contribute to the endemic of
KSHV in certain populations (Plancoulaine, 2003).
KSHV is a double-stranded DNA herpetic virus with a 165-170kb genome which encodes
several genes important for cellular proliferation, cytokine production, and angiogenesis (Neipel,
1998). The board cellular tropism of KSHV allows cellular entry in various cell types, including
B lymphocytes, dendritic cells, endothelial cells, macrophages, mesenchymal stem cells, and

2
neurons (Hussein, 2019; van der Meulen, 2021). KSHV is thought to be a causative pathogenic
infectious agent promoting clinical manifestation of KS because all cases of the disease harbor the
virus. While having a heterogenous histopathology with inflammatory components, KS is found
primarily in the mucocutaneous membranes of the skin, and oral mucosa cavities, where
progressive disease may involve the lymph nodes, and visceral organs. As an angio-proliferative
disease, cells isolated from KS lesions reveal spindle-shaped endothelial tumor cells. Tumor
presentation in humans is characterized by high vascularity, inflammatory infiltration, and fibrosis
(Bishop, 2023; Moore, 2001).
KS can be categorized into various types, such as classic, endemic, acquired
immunodeficiency syndrome (AIDS) -related, and iatrogenic KS. The classic form of KS, or
sporadic KS, occurs in primarily in elderly men living in the Mediterranean or eastern European
who are of the Ashkenazi Jewish ancestry. This form was suggested to be a genetic predisposition
to the disease. Endemic KS emerged in African countries during the 1950s, infecting both children
and adults. While the progression of the lymphadenopathic form of KS in children is often very
aggressive, adults present lesions that resemble classic KS (Cesarman, 2019; Giffin, 2014). Once
considered a rare malignancy, KS prevalence dramatically rose with the HIV/AIDS epidemic, and
became one of the defining illnesses associated with AIDS. AIDS-related KS is the most
aggressive form of the disease, where its progression is thought to be mediated by cytokines
elaborated by virally infected cells (Mariggiò, 2017; Robey, 2015). Iatrogenic KS present in
immunodeficient individual, namely recipients of solid-organ transplant requiring prolong
immunosuppressive therapy such as corticosteroids. Most cases of iatrogenic KS are thought to
occur as a result of KSHV viral reactivation (Lebbé, 2008). Interestingly, the withdrawal of steroid
therapy can halt and even cause tumor regression with iatrogenic KS (Saxena, 2015). These

3
findings suggest that KS may be modulated by steroids. This also explains why KS is
predominantly found in males, where female patients with KS are very rare.
FDA-approved treatment strategies for KS have remained unchanged for the last 20 years.
Due to its heterogeneous manifestation, there is no standard therapy regimen for the treatment of
KS. Current therapeutic options range from surgical removal, cytotoxic chemotherapy, radiation
therapy, anti-retroviral therapy, and withdrawal of immunosuppressive therapy (Schneider, 2018).
For new targeted therapy, bevacizumab, an anti-VEGF-A monoclonal antibody, has been
prescribed to patients with HIV-associated KS in a phase II clinical trial. Bevacizumab was well
tolerated, with some patients achieving partial or complete response (Uldrick, 2012). Therefore,
there is room for innovation in new therapeutics for the treatment of KSHV infection and KS
tumorigenesis.
A unique feature of KS is its highly metastatic nature because its spread is not contiguous
to adjacent tissues, but rather jumps from one region to the next. These findings suggest that the
disease progression is not associated with tissue invasion but promoted by a mechanism that is yet
to be unveiled. Although cytokines, such as interleukin-8 (IL-8), IL-1 and oncostatin M, have all
been found to promote KS proliferation and spread, the findings themselves have not been
definitive (Bhatt, 2013). Therefore, the importance of a soluble factor continues to loom as the
promoting factor for KS. We hypothesize that KS malignant cells derived exosomes can instruct
disease progression including regulating the metastatic nature of KS via paracrine mechanisms.
Most recently, the emergence of extracellular vesicles (EV) research, specifically
exosomes, have highlighted their importance in intracellular crosstalk to promote cellular events
and instruct neighboring and distant cells through proteins, lipids, nucleic acids, and metabolites
found in EVs. Modern drug delivery system development has taken advantage of their high

4
transport efficiency, crafting exosomes as drug carriers with low immunogenicity and
physicochemical stability (Sadeghi, 2023). In cancer, this intercellular communication mechanism
also plays a role in disease progression as tumor-derived exosomes (TDEs) promote tumor growth,
metastasis, and immune regulation. These TDEs can carry angiogenesis-stimulating and antiapoptotic factors (Zeng, 2023). Due to their involvement in tumor formation, exosomes have been
explored as biomarkers for disease diagnosis and prognosis (Zhang Yi, 2020). Additionally, cancer
therapeutic research has explored the significance of inhibiting TDEs and neglecting this cell-tocell communication as a therapeutic for metastatic diseases (Wu, 2019). However, there has been
little to no success in the clinical development of therapies capable of interfering with the exosomemediated intercellular communication process. To this end, we investigate the critical role of EVs,
specifically exosomes, in regulating KS tumorigenesis and disease progress, where disruption of
this process can form the basis of therapeutic platforms to effectively treat KS. To this end, we
propose to evaluate the ability of Food and Drug Administration (FDA) approved drugs to disrupt
KS-mediated exosomal biosynthesis and elaboration. The ability to block this complicate pathway
require the central understanding of the cellular and molecular mechanism(s) that provoke cellular
transformation in KS tumorigenesis.
1.2 Exosome-mediated KS Tumorigenesis
In tumor progression, cellular constituents in the tumor microenvironment, such as
malignant cells, fibroblasts, and immune cells, are known to secrete exosomes, a subtype of
extracellular vesicles with a size range of 40 to 100 nm in diameter. These tumor-derived exosomes
(TDEs) mediate intercellular communication, which is crucial in promoting tumor progression,
angiogenic switch, and immunosuppression. These paracrinic processes can subvert local and
distant microenvironments (Zhang, 2020). Additionally, exosomes/EV evolving from the primary

5
tumor has been described to act as potential mediators capable of priming the pre-metastatic niche
leading to eventual metastasis and inducing migration and invasion (Lobb, 2017; Guo, 2017).
With the ability to facilitate tumor microenvironment immunosuppression, tumorigenesis, and
upregulation of mechanisms capable of inducing chemoresistance, thus targeting the inhibition of
TDEs maybe a promising strategy for cancer therapy (Hessvik, 2017; Milman, 2019).
Like other herpesviruses, KSHV exploits exosomes-mediated intercellular communication
to modulate the tumor microenvironment and facilitate cell transformation without alarming innate
immune sensors. As exosome biogenesis and virus replication share overlapping pathways, it is
thought that viruses hijack the exosome biogenesis pathways to spread and infect neighboring cells
(Mardi, 2023). During the viral latent phase of the viral infection, KSHV-associated exosomes,
containing high levels of viral-encoded microRNAs (miRNA), that may play regulate KS
tumorigenesis (Mahmoudvad, 2022; Qin, 2019). MicroRNA is a family of small non-coding RNAs
capable of regulating a wide array of processes including tumorigenesis. More important, the types
of miRNAs are dysregulated in cancers. These miRNAs are involved in cellular transformation,
targeting signaling pathways associated with cell proliferation, inflammation, angiogenesis, and
tumorigenesis (Chugh, 2013; Yogev, 2014). Furthermore, KSHV-associated exosomes/EVs can
regulate metabolism of host cells and exert metabolic reprogramming of neighboring cells with
persistent infection (Meckes, 2013; Yogev, 2017). Therefore, as exosomes and their oncogenic
cargo play a significant role in disease initiation and progression, where the ability to disrupt
KSHV-associated exosomes is a potential avenue in blocking KS tumorigenesis.
1.3 Exosome Biogenesis and Cargo Loading
Exosomes are formed from the intracellular transformation of endosomes to multivesicular
endosomes, which are trafficked to the plasma membrane and released into the extracellular space

6
(Johnstone, 1987). The classical pathway of exosome generation involves the budding of the
plasma membrane and the formation of intracellular multivesicular endosomes (MVEs), or
multivesicular bodies, containing intraluminal vesicles (ILVs). The first invagination occurs as the
budding of the plasma membrane, which leads to the formation of early endosomes (EEs). The
trans-Golgi network and endoplasmic reticulum contribute to the formation and contents of these
EEs. From there, EEs give rise to late endosomes (LEs) through various maturation processes. The
second invagination of LEs results in the generation of MVEs containing ILVs. These ILVs are
the precursors for future exosomes. These MVEs can be shuttled into either autophagosomes,
lysosomes, or the plasma membrane. MVEs directed to autophagosomes, or lysosomes result in
degradation of its contents and release into the cytoplasm. MVE docking to the plasma membrane
leads to exocytosis and the release of exosomes (Kalluri, 2020; Wei, 2021).
An important feature of exosome biogenesis is the cargo-loading of exosomal contents,
which dictates the modulation of signaling pathways due to exosome-mediated intercellular
communication between cells once released into the extracellular space. Cargo-loading of
exosomes occurs during the formation of ILVs and MVEs. There are several mechanisms in which
exosome cargo-loading occurs (Figure 1). The classical mechanism is the endosomal sorting
complex required for transport (ESCRT) machinery, which is recruited by LEs for exosome
formation and release. Alternatively, there are other cargo sorting mechanisms that are
independent of the ESCRT machinery, which instead relies on lipid raft, tetraspanin, and ceramidemediated mechanisms.
Although TDEs follow the same mechanism of biogenesis, their biogenesis and secretion
activity are different. Due to the hypoxic and acidic tumor microenvironment, key regulators of
biogenesis and secretion are overexpressed and/or hyperactivated. Additionally, genomic

7
mutations in cancer cells result in modulation of exosome biogenesis. The Ras oncogene is the
most frequently mutated oncogene found in cancer. Activation of the Ras pathway, as a result of
mutation, is associated with modulation of the ESCRT-dependent pathway of exosome biogenesis
(Sexton, 2019). Also, mutation of epidermal growth factor receptor (EGFR) gene found in cancers
result in increased exosome biogenesis and secretion (Jouida, 2021; Li, 2022).
Figure 1. Exosome Biogenesis and Cargo-Loading via ESCRT-dependent and -independent
Pathway
The first plasma membrane invagination will form early endosomes. The cargo is assembled into
exosome precursors occurs during the formation of late endosomes and multivesicular endosomes
(MVEs). Cargo-loading occurs in two distinct mechanisms. The ESCRT-dependent pathway relies
on the ESCRT complex to sort cargo. Alternatively, the ESCRT-independent pathway uses lipids
rafts, such as sphingomyelin and ceramide, to sort cargo. Docking of MVEs to the plasma
membrane results in excretion of exosomes. Created with BioRender.com
1.3.1 ESCRT-dependent Pathway
The endosomal sorting complex required for transport (ESCRT) consists of four subunits
(ESCRT-0, -I, -II, and -III), and accessory proteins such as vacuolar protein sorting 4-vesicle

8
trafficking 1 (VPS4) and ALG-2-interacting protein X (ALIX). As a main cargo sorting
mechanism, the ESCRT complex recognizes and sorts ubiquitinated cargo. Firstly, ESCRT-0
recognizes ubiquitinated cargo with its ubiquitin-binding domains, binding itself to the endosomal
membrane and initiating cargo sorting. Then, ESCRT-I and -II are recruited to the endosomal
membrane to initiate enclosure of the endosomal membrane around the cargos and form the
membrane neck. ESCRT-III further drives the budding and cleaves the membrane neck to form
ILVs within MVEs (Henne, 2011; Ju, 2021). For TDEs, key regulators, such as ESCRT-associated
proteins and accessory proteins (ALIX) are overexpressed and contributed to increased exosome
secretion (Li, 2022). Thus, the ability to target TDE-mediated activities may be selective in disrupt
cancer related mechanisms.
1.3.2 ESCRT-independent Pathway
Alternatively, cargo-loading and ILV formation can occur independent of the ESCRT
mechanism and instead depend on lipid raft-based microdomains for sorting cargo into endosomal
membrane. The neutral sphingomyelinase 2 (nSMase2) is a ceramide-dependent mechanism and
is the most studied ESCRT-independent pathways, yet it remains not fully understood.
Sphingomyelin is a sphingolipid, mainly localized to the plasma membrane and intracellular
organelle membranes. In these microdomains, nSMase2 hydrolyzes sphingomyelin to liberate the
bioactive lipids phosphorylcholine and ceramide (Airola, 2013; Hofmann, 2000).
Phosphorylcholine is part of platelet-activating factor that has immunomodulatory activity.
Phosphorylcholine is a precursor headgroup to phospholipids like phosphatidylcholine and
sphingomyelin, which are the only membrane phospholipids not built with a glycerol
backbone. Ceramide is a bioactive sphingolipid which is critical for exosome formation. It is
proposed that ceramides merge cholesterol-rich microdomains and bend the phospholipid

9
membrane to facilitate ILV and MVE formation (Trajkovic, 2008). Furthermore, nSMase2 activity
has been shown to regulate exosome secretion (Choezom, 2022).
1.4 Known Exosome Inhibitors
There has been extensive research exploring the development of pharmacological
approaches to inhibit or disrupt exosome formation and secretion. These compounds are initially
used as research tool. As exosome biogenesis consist of multiple complex pathways, there has not
been a drug that can singularly completely block exosome biogenesis. These current exosome
inhibitors can be classified based on their mechanism of action. The molecular effects of these
compounds include blockade of lipid metabolism versus inhibitors of exosome release (Catalano,
2020). Known exosome inhibitors further referred to in this study are tipifarnib, manumycin A,
and GW4869, which are often used as reference tool compounds. However, their utility to prevent
exosome-mediated activities have been hampered by their off-target activities leading to
intolerable adverse effects.
Tipifarnib and manumycin A are potent and selective inhibitors of Ras farnesyltransferase
(FTase). Ras is a superfamily of small GTP-binding proteins, such as Rab, Ran, Ras, Rho, and Arf,
that regulate diverse cellular functions, including differentiation, adhesion, proliferation, exosome
release, and apoptosis (Song, 2019). Additionally, Ras proteins are known to play a crucial role in
in cellular transformation and ultimately tumorigenesis. Ras precursor proteins are activated by
FTase (Yang, 1997). Tipifarnib, also known as R115777 or Zarnestra, is a heterocyclic non
peptidomimetic drug. As the first FTase inhibitor to enter clinical trials, the antitumor activity of
tipifarnib includes the ability to alter tumor-host cell transformation and tumorigenic cell
proliferation (End, 2001). Tipifarnib was found to also inhibit exosome biogenesis and secretion
by the ESCRT-dependent and independent pathways (Datta, 2018). Current research explores how

10
tipifarnib effectively inhibits oncogenic HRAS-driven tumorigenesis (Gilardi, 2021; Untch, 2018).
Manumycin A, an antimicrobial agent extracted from Steptomyces parvulus Tu64, is a selective
and potent Ras farnesyltransferase inhibitor (Ali, 1999; Silva, 2022). Manumycin A was found to
inhibit Ras/Raf/ERK1/2 signaling and suppress exosome biogenesis and release through the
ESCRT-dependent pathway (Datta, 2017; Li, 2013).
One of the most used inhibitors of TDEs, GW4869 is a dihydroimidazole amide compound
that is a selective exosome biogenesis inhibitor by targeting nSMase, which is responsible for the
hydrolysis of membrane lipid sphingomyelin to form ceramides, which is important in the ESCRTindependent pathway (Li, 2013). Therefore, GW4869 acts as an exosome biogenesis inhibitor via
disruption of lipid metabolism. GW4869 inhibition of vesicles has been studied in various cancer
cell lines, such as ovarian cancer cells, fibroblast cancer stem cells, and melanoma cells (Cao,
2017; Hu, 2015; Matsumoto, 2017). A current limitation to GW4869 is its adverse side effects in
vivo because its noncompetitive targeting of nSMase, disrupting the multiple biological processes
the enzyme is involved in (Yoo, 2020).
Exosomes play a vital role in the physiological and pathological processes through the
mediation of intercellular communication and cell signaling. As TDEs facilitate angiogenesis and
metastasis, inhibition of exosome biogenesis pathways is a viable approach in cancer treatment.
Although there are known exosome inhibitors, each of these compounds face different challenges
in their preclinical development and fail to translate into human clinical studies. Therefore, we aim
to evaluate clinically approved drug compounds to disrupt KS-mediated exosomal biosynthesis.
Blockade of this complex pathway provides further understanding of the cellular and molecular
mechanism(s) that mediate cellular transformation in KS tumorigenesis. Viral G-protein coupled
receptor (vGPCR), encoded in the KSHV genome as ORF74, is suggested initiate KS

11
tumorigenesis and paracrine transformation via exosome-mediated mechanisms. The study of
exosome inhibition may provide an understanding of how vGPCR initiates production of
exosomes and trigger KS tumorigenesis. Furthermore, this may impact our understanding of
cancer initiation, progression, and metastasis, guiding therapeutic foundation against KS
tumorigenesis while setting the stage for investigation of the presence of equivalent nonautonomous transformation in other viral and non-viral oncogenesis, such as breast and colon
cancers.
Aim 1 of the study identified at least eight potential drug candidates among the plethora of
clinically approved compounds with a tertiary amine and high therapeutic safety prolife. Using in
silico validation via Autodock-Vina, drug candidates were selected based on their affinity to target
enzymes of known exosome inhibitors, FTase and nSMase. These target enzymes have a critical
role in exosome formation in distinct pathways. As Aim 2, in vitro validation investigated the
effects of drug candidate treatment on exosome biogenesis and cargo-loading mechanisms. Firstly,
sufficient transfection to the formation of the KS cell model was verified by antibiotic selection.
The appropriate drug treatment concentrations for the KS cell model were determined by a cell
viability assay and IC50 concentrations. While identifying efficient exosome inhibitors among the
drug candidates, the effects of drug treatment on exosome biogenesis were evaluated to distinguish
how these drug candidates impacted different molecular processes and their potential mechanism
of action. Therefore, in this study, we propose potential exosome inhibitors suitable for the
treatment of KS tumorigenesis and investigate the structure-activity relationship (SAR) required
for exosome inhibitory activity.

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Chapter 2: In Silico Drug Candidate Selection
2.1 Screening of Clinically Approved Tertiary Amines as Drug Candidates
To develop exosome inhibitor, it is critical that we understand the molecular mechanism(s)
as to how exosomes are made and the impact of disrupting this process. In silico methods have
been used to cast a wide net to identify potential pharmacophores. To this end, our approach was
to use compounds that enter the cells and become sequestered into the vesicles, such as the
lysosomes. Drugs that are classified as tertiary amines drugs, such as triazoles, allylamines,
chloroquinolines, and fluoroquinolones, will become protonated with the change the pH in
lysosomes and other vesicles. This has the potential to narrow the compounds that may have
potential exosomal inhibitory activity.
We hypothesized that compounds with a tertiary amine are potential inhibitors of exosome
formation since these compounds can have high membrane permeability and intracellular
accumulation. Uncharged tertiary amines have a lower hydrogen-bonding capacity, allowing it to
cross cellular membranes. In an acidic environment, protonation of the tertiary amine reduces its
membrane permeability, resulting in intracellular accumulation, especially in the lysosomes
(Andrew, 1997). Additionally, it can be inferred that, from the screening of selective inhibitors of
exosome biogenesis and secretion by Datta et al, the tertiary amine structure is important for the
pharmacophore of exosome inhibitors (Datta, 2018). Currently, the major obstacle for clinical
development of known exosome biogenesis inhibitors, such as GW4869 and manumycin A, is
their cytotoxic adverse events. Therefore, repurposing FDA approved drugs with excellent safety
profile is an advantageous approach in drug development to rapidly identify candidates to inhibit
exosomes. Drug repurposing is a process in which alternative indications can be explored, thus

13
currently approved agents for other indications can be used to modulate exosome production. We
identified clinically approved tertiary amine compounds that have high therapeutic index, which
correspond to a strong safety profile, and excellent tissue penetration.
We used the in silico software, Autodock Tools and Scripps Institute Vina, to provide
predictive insight to the optimal ligand-receptor orientation between compounds and the target
enzyme. For the selection of drug candidates, in silico screening was performed against enzymes
FTase and nSMase, which are targets of known exosome biogenesis inhibitors, tipifarnib,
manumycin A, and GW4869. The optimal ligand-protein interaction between compounds with a
tertiary amine and the target enzymes were quantified by their dissociation constant (Kd). From
there, drug candidates were identified for further in vitro preclinical development.
2.1.1 Methods: Autodock-Vina
For in silico screening, Autodock Tools 1.5.7 and Scripps Institute Vina 1.1.2 (The Scripps
Research Institute) determined the binding affinity from the optimal ligand-protein interaction.
The crystal structure of human protein FTase (1SA4) and nSMase2 (5UVG) were obtained from
the Protein Data Bank. In Autodock Tools, protein receptors were prepared by the removal of
water and addition of polar hydrogens and Kollman charges. The chemical structures of ligand
compounds were also constructed in Autodock Tools from their simplified molecular-input line
entry system (SMILES) representation. The Scripps Institute Vina generated optimal ligandprotein interactions and determined their binding affinity (kcal/mol), which was converted to
Gibbs free energy (ΔG) and dissociation constant by the following formula:
Kd = 109
/e(-ΔG/RT)
R = 1.9872036 cal/(K*mol)
T = 298oK

14
From there, the dissociation constant values of each ligand compound for FTase and nSMase2
were ranked by highest to lowest binding affinity. The rank values across the two target enzymes
were summarized to a rank sum value for each ligand compound. Ligand compounds with similar
rank sum values to the known exosome inhibitors were selected as drug candidates for in vitro
experiments.
2.1.2 Results: In Silico Screening of Compounds with a Tertiary Amine
Autodock Tools-Vina determined the binding affinity of the optimal ligand-protein
interaction, which was converted to the dissociation constants. Dissociation constant and binding
affinity of the ligand to the protein receptor have an inverse relationship, thus a lower dissociation
constant indicates a stronger association of the compound to the target enzyme. As the dissociation
Table 1. Human Farnesyltransferase (FTase) Binding Affinity and Dissociation Constant
Drug Affinity (kcal/mol) Kd (µM) Rank
Tipifarnib -10.0 0.046 1
Ketoconazole -9.8 0.065 2
SB 218795 -9.5 0.108 3
Itraconazole -9.5 0.108 4
Manumycin A -9.0 0.251 5
Sitafloxacin -9.0 0.251 6
Minocycline -8.8 0.352 7
Doxycycline -8.8 0.352 8
Caroverine -8.1 1.147 9
GW4869 -7.9 1.608 10
Imipramine -7.5 3.160 11
Fluconazole -7.4 3.741 12
Nitrefazole -7.0 7.352 13
Pentylenetetrazol -7.0 7.352 14
(S)-Climbazole -6.7 12.201 15
Triadimenol -6.7 12.201 16
(S)-Climbazole -6.5 17.103 17
Neticonazole -5.9 47.107 18
Known selective inhibitors of FTase are highlighted in yellow

15
constants were ranked, a lower ranking indicated a lower potential for the ligand compound to
bind on the target enzyme and exert exosome inhibition activity. The dissociation constants of
ligand-FTase interactions were compared to manumycin A, which is known to target FTase for its
exosomal inhibitory activity (Table 1). The compound with the highest binding affinity for FTase
was tipifarnib, a highly selective inhibitor of FTase. The positive standard for FTase throughout
the study, manumycin A, also had a relatively high binding affinity among the ligands. The
dissociation constants using ligand-nSMase interactions were compared to GW4869, the reference
compound, which also had the highest binding affinity among the ligands as it is known to act on
the enzyme to inhibit exosome biogenesis (Table 2).
Table 2. Neutral Sphingomyelinase (nSMase) Binding Affinity and Dissociation Constant
Drug Affinity (kcal/mol) Kd (µM) Rank
GW4869 -8.9 0.297 1
Ketoconazole -8.4 0.691 2
SB 218795 -8.1 1.147 3
Tipifarnib -7.8 1.904 4
Doxycycline -7.7 2.254 5
Itraconazole -7.7 2.254 6
Sitafloxacin -7.5 3.160 7
Minocycline -7.1 6.209 8
Manumycin A -6.7 12.201 9
Nitrefazole -6.4 20.249 10
Caroverine -6.3 23.974 11
Imipramine -6.1 33.606 12
Neticonazole -6.1 33.606 13
Fluconazole -6.0 39.788 14
Triadimenol -6.0 39.788 15
(S)-Climbazole -5.9 47.107 16
Pentylenetetrazol -5.7 66.033 17
(R)-Climbazole -5.6 78.181 18
Known selective inhibitor of nSMase are highlighted in yellow
The dissociation constants against FTase and nSMase were ranked from lowest to highest
dissociation constants with one being the lowest value. We also combined ranking for each ligand

16
across the two target enzymes was calculated as the rank sum values (Table 3). This was to show
whether there is selectivity of these ligands for a specific target. As we aimed to screen potential
exosome biogenesis inhibition through different cargo-loading mechanisms, the ESCRTdependent or independent pathway, evaluation of the rank sum value encompassed targeting
potential across the two pathways.
Table 3. Rank Sum of In Silico Drug Candidates
Drug Rank Sum
Ketoconazole 4
Tipifarnib 5
SB 218795 7
Itraconazole 9
GW4869 11
Doxycycline 12
Sitafloxacin 13
Manumycin A 14
Minocycline 16
Caroverine 20
Imipramine 23
Nitrefazole 23
Fluconazole 26
Triadimenol 30
(S)-Climbazole 31
Neticonazole 31
Pentylenetetrazol 31
(R)-Climbazole 35
Positive standards are highlighted in yellow
Selected drug candidates are highlighted in blue
In silico screening via Autodock Tools-Vina is an effective predictor to ligand-receptor
interactions. This was evident based on the dissociation constants values for the known inhibitors
tipifarnib, manumycin A, and GW4869. Tipifarnib and manumycin A are known exosome
biogenesis inhibitors, targeting FTase. Therefore, it was expected that these compounds would
have a high binding affinity FTase. The dissociation constant values of these compounds reflected
expectations as they had low dissociation constant values to FTase among ligand compounds

17
screened. Furthermore, Scripps Institute Vina aligned with expectations of the binding affinity of
GW4869 to nSMase. As GW4869 is known to act on nSMase to inhibit exosome biogenesis, it
would have a high binding affinity to this enzyme. The determined dissociation constant of
GW4968 on nSMase reflected expectations as GW4869 had the lowest dissociation constant,
which indicated that it had the highest affinity to nSMase compared to all the ligand compounds
screened.
Firstly, imidazoles, like ketoconazole, fluconazole, and climbazole, were selected as drug
candidates due to the potentiation. Ketoconazole had the lowest rank sum value among all the
ligands as it had relatively low dissociation constant values for both FTase and nSMase. Due to
their structural similarities to ketoconazole, other azoles may also have an affinity to the target
enzymes that are not captured from in silico predictions. Therefore, other azoles, fluconazole and
climbazole, were selected as drug candidates. The rank sum values of these two drug candidates
were not relatively low, which indicated an affinity to target enzymes but not the other. In
particular, triazoles like fluconazole has higher Kd as compared to the imidazoles (e.g. climbazole
and itraconazole) for FTase and nSMase. Despite this, their structural commonality with
ketoconazole suggests that they could have similar binding interactions to the target enzymes.
Additionally, doxycycline, sitafloxacin, minocycline, and imipramine were selected as drug
candidates because their rank sum values were adjacent to the rank sums of the known standards,
manumycin A and GW4869.
2.2 Drug Candidates
In silico drug candidates were further evaluated using in vitro screening, such as
ketoconazole, fluconazole, climbazole, imipramine, sitafloxacin, minocycline, and doxycycline,
which were compared with known exosome biogenesis inhibitor, manumycin A, as reference

18
standard (Later sections). The selection of drug candidates proved promising as some candidates
have already been explored as exosome inhibitors, disrupting at different stages of biogenesis and
extracellular release.
Current works have investigated the effects of climbazole, ketoconazole, imipramine, and
sitafloxacin on exosome biogenesis. Climbazole is an imidazole agent with potent antifungal and
preservative properties. It acts to inhibit the synthesis of ergosterol, an essential component for
fungal plasma membranes (Paz-Alvarez, 2018). In a study conducted by Datta et al, metastatic
castration-resistant prostate cancer C4-2B cells treated with climbazole showed decreased levels
of both Alix and Rab27A, but not nSMase2. These findings indicated that climbazole selectively
acts on the ESCRT-dependent pathway to decrease exosome release (Datta, 2018). Ketoconazole
is an anti-fungal imidazole through inhibition of ergosterol biosynthesis, which is necessary for
maintenance of the membrane of fungi (Van, 1984). Ketoconazole has been shown to inhibit
exosome release in a dose-dependent matter as it decreased levels of exosome biogenesis
biomarkers Rab27A, Alix, and nSMase2 (Datta, 2018; Zhang, 2020). Studies have explored the
application of ketoconazole in combination cancer therapy as an exosome release inhibitor. In a
clinical trial, ketoconazole was prescribed in combination with doxorubicin for patients with
prostate cancer (Sella, 1994). It is important to note that the use of ketoconazole was used to reduce
steroidal hormones such as testosterone in prostate cancers and estrogens in breast and ovarian
cancers. In renal cell carcinoma, ketoconazole was used to enhance the efficacy of sunitinib
(Greenberg, 2021). Imipramine is a tricyclic anti-depressant that inhibits reuptake of
norepinephrine and serotonin (Hillhouse 2015). Imipramine has been shown to prevent exosome
secretion through its inhibitory effect on acid sphingomyelinase (aSMase), which is responsible
for increased membrane fluidity, multivesicular generation, and exosome release (Bianco, 2009).

19
Sitafloxacin is a fluoroquinolone with anti-microbial activity by inhibition of DNA gyrase and
topoisomerase IV in a broad range of bacteria (He, 2022). While the mechanism of action remains
unknown, Datta et al. has identified sitafloxacin as an activator of exosome biogenesis in C4-2B
cells (Datta, 2018).
Other selected drug candidates that do not have current works exploring their effects of
exosome biogenesis include fluconazole, minocycline, and doxycycline. Fluconazole is a synthetic
triazole with antifungal properties as an inhibitor of lanosterol 14-α-demethylase (Turner, 2012).
Minocycline and doxycycline are semi-synthetic tetracyclines with anti-microbial and antiinflammatory properties (Garrido-Mesa 2013; Jantratid 2010). These antibiotic compounds were
developed with ring D modifications to improve the efficacy of the antibiotic properties of
tetracycline, which bind to bacterial 30S ribosomal subunit and inhibit protein synthesis (Nelson,
1998).

20
Chapter 3: In Vitro Preclinical Development of Selective Exosome
Biogenesis Inhibitors
3.1 Vector Validation of vGPCR-expressing B16-F10 Melanoma Cells
The pathogenesis of KSHV-mediated malignancies involves factors associated with the
lytic and latent phases of the viral life cycle (Staskus, 1997). During latency, viral genomes are
replicated in synchrony with cell division and only a few KSHV genes are expressed. During the
lytic phase, nearly all viral genes are expressed, resulting in infectious virions and apoptosis. While
KSHV latent genes are essential for genome maintenance, lytic genes play a critical role in driving
tumorigenesis via direct, as well as paracrine, mechanisms. Early lytic genes, such as virus G
protein-coupled receptor, viral interleukin-6, K1, and K15, subvert host signaling pathways and
lead to expression and secretion of angiogenic, inflammatory, and proliferative factors (Cesarman,
2022; Liu, 2021; Mesri, 2010).
As an oncoprotein known for promoting angiogenesis and tumorigenesis, virus-encoded G
protein-coupled receptor (vGPCR) is suggested to play an essential role in KS pathogenesis and
KSHV regulation (Sodhi, 2000). The KSHV genome encodes vGPCR by the lytic open reading
frame 74 gene (ORF74), which resembles a seven trans-membrane CXCR2 chemokine G proteinlined IL-8 receptor. While the function of vGPCR is not fully understood, its homology to the
chemokine receptor suggests a role in immune response regulation (Grisotto, 2006; Smit, 2002).
There is evidence supports that vGPCR is a constitutively activated receptor that triggers a broad
spectrum of signaling pathways via PI3K/Akt/TOR activation, which signals downstream
pathways promoting vascular endothelial growth factor (VEGF)-driven angiogenesis and

21
inflammatory cytokines due to Rac1-NOX-ROS oxidative stress (Bais, 1998; Martin, 2011; Wu,
2015). Additionally, the role of vGPCR in KS tumorigenesis is evident as vGPCR transgenic mice
model present angiogenic lesions that resemble KS in humans. The oncogenic protein contributes
to KS pathogenesis because vGPCR provides a positive feedback mechanism to the KSHV
replication and transcription activator gene ORF50. This results in sustained OFR50-dependent
lytic transcription. Thus, disruption of lytic replication can impair KSHV infection and KS tumor
development (Bottero, 2009; Moore, 2001).
Figure 2. Induction of Downstream Pathways by Viral GPCR
vGPCR is a lytic gene associated with KSHV. Expression of vGPCR in the infected cell leads to
activation of the PI3K/Akt/TOR pathway, promoting angiogenic factors and inflammatory
cytokines elaboration. Angiogenic growth factors include vascular endothelial growth factor
(VEGF) and platelet-derived growth factor (PDGF). Inflammatory cytokines include interleukin -
1β/-8/-10 and tumor necrosis factor alpha (TNFα) (Jham, 2011). Created with BioRender.com
The mechanism of vGPCR as to how a lytic gene can play a role in cellular transformation
and immortalization continues to be poorly delineated. There are several theories suggesting its
participation in tumor initiation and maintenance. Interestingly, vGPCR is expressed in a small

22
subset of cells within the KS lesions. This supports the concept that vGPCR contributes to
paracrine transformation of endothelial-derived cells as KSHV lytic genes promote cytokine and
chemokine expression and induce paracrine-acting factors, such as VEGF and PDGF (Jham, 2013;
Mesri, 2010). The oncogenic event associated with paracrine transformation occurs when one cell
causes the transformation of neighboring cells. We hypothesis that paracrine transformation is
mediated through vGPCR-loaded exosomes secreted by neighboring KSHV-infected cells. This
non-autonomous oncogenic mechanism is the initiates KS tumorigenesis, where the ability to
dissect this transformation may set the stage for treatment strategy development that can
effectively treat and/or prevent KS. Therefore, we explore KSHV tumorigenesis with a vGPCR
transgenic cellular model.
3.1.1 Materials and Methods: Antibiotic Selection
From collaborators Dr. Young-Kwon Hong and Dr. Dong Won Choi of the USC Keck
School of Medicine, vGPCR was transfected into B16-F10 melanoma cells to express the lytic
oncogenic gene of KSHV (Figure 3). As the wild-type (WT) cell line, B16-F10 melanoma cells
are highly metastatic mouse melanoma cell line commonly used to create genetically engineered
tumor models (Potez, 2018). The transfected vector was constructed from the pEF6/V5-His TOPO
Ta expression vector with the enhancer/promoter elements from the human elongation factor 1-
alpha subunit (hEF-1a) for high-level expression in mammalian cells (Invitrogen K961020). The
vector possess ampicillin and blasticidin resistance gene in the construct, which allows for
blasticidin (Invivogen ant-bl-1) selection of cells expressing vGPCR.

23
Figure 3. vGPCR Vector Map Using pEF6/V5-HIS TOPO Expression Vector
WT B16-F10 and vGPCR-transfected B16-F10 cells were maintained in RPMI-1640
medium (Gibco 11875085)supplemented with L-glutamine, 10% fetal bovine serum (FBS) (Gibco
16140071), and Antibiotic-antimycotic (Gibco 15240062). Antibiotic-Antimycotic is comprised
on penicillin and streptomycin to prevent bacterial contamination. For antibiotic selection, B16-
F10 and vGPCR-expressing cells were plated at high confluency with 2 x 106
cells in sterile treated
10 cm plates with 5% FBS medium. Both cell lines were grown as a monolayer at 37°C with 5%
carbon dioxide. Blasticidin was introduced into the medium as treatments using concentrations
ranging from 1 to 10 µg/mL. The selection stabilized in approximately one week.
3.1.2 Results: Transfected Model
Blasticidin antibiotic selection was performed to select for vGPCR-expressing cells which
are resistant to blasticidin from the non-transfected wild-type B16-F10 cells which are sensitive

24
towards the antibiotic. B16-F10 cells were treated with 1 µg/mL blasticidin and vGPCR expressing
cells were treated with 1-10 µg/mL blasticidin. Images were taken to monitor the exposure to
different concentrations of blasticidin antibiotic (Figure 4). Comparison of blasticidin antibiotic
selection across B16-F10 and vGPCR-expressing cells determined the success of the antibiotic
selection. On day 5 of the experiment, B16-F10 cells treated with 1µg/mL blasticidin were highly
apoptotic, where these epithelial cells rounded up and detached from the cell plate. In contrast,
vGPCR-B16-F10 were able to maintain anchorage onto the plate (Figure4B), and even proliferated
in the presence blasticidin (1 µg/mL) with 10 µg/mL (Figure 4D).
Figure 4. Blasticidin Antibiotic Selection of B16-F10 and vGPCR-expressing Cells Day 5
Treatments were given as (A) B16-F10 with 1µg/mL (B) vGPCR with 1µg/mL (C) vGPCR with
5µg/mL (D) vGPCR with 10µg/mL. These images show that WT B16-F10 are sensitivity toward
blasticidin treatment. Images were taken after cell passage with new medium.
A B
C D

25
In contrast, vGPCR-expressing cells survived across the different concentrations of blasticidin, 1,
5 and 10µg/mL. After five days of exposure to the antibiotic, dead cells were removed with cell
passage and revealed vGPCR-expressing cells were selected out and survived in the plate. This
demonstrated that cells with the transfection vector survive as these cells had the antibiotic
resistance gene.
3.2 Drug Screening Cell Viability to Determine IC50
Prior to evaluation of exosomal inhibitory activity of the drug candidates, cytotoxic levels
of drug candidates were determined in order to identify the concentration range in which would be
applied to the cell line without inducing cell death. A cell viability assay via resazurin sodium salt,
also known as Alamar Blue, quantifies percentage cell viability and half-maximal inhibitory
concentration (IC50) to determine drug potency. Resazurin detects the presence of live cells
through its reduction into its fluorogenic product resorufin form through NADH dehydrogenase
mediated metabolism (O’Brien, 2000). Determination of the IC50 for the positive control standard
manumycin A and drug candidates provided a concentration range to analyze potential exosome
inhibitors and determine their impact on the initiation and progression of KS.
3.2.1 Materials and Method: Resazurin Reduction Cell Viability Assay
For the cell plate preparation, B16-F10 cells were trypsinized and seeded in sterile treated
96-well plates as 1,000 cells/well in RPMI-1640 medium without phenol red and supplemented
with L-glutamine (R&D Systems M30350). Then, cells were incubated at 37°C overnight for
serum starvation and cellular adherence. Doxycycline (14422), fluconazole (11594), imipramine
(15890), manumycin A (10010497), and minocycline (14454) were purchased from Cayman
Chemical Company. Ketoconazole (UC280) was purchased from Sigma-Aldrich and climbazole
(C2025) was purchased from TCI Chemicals. The pharmaceutical drugs were dissolved in

26
dimethyl sulfoxide (DMSO) according to the manufacturer’s instruction, which were further
diluted in medium to the desired working concentration. In biological triplicates, drug treatments
of different concentrations were introduced to cells, accounting v/v% DMSO to be <5%.
Experimental controls were non-treated cells and DMSO treated cells, reflecting the amount of
v/v% DMSO used for drug treatments. Alamar blue cell viability assay was conducted according
to manufacturer’s protocol as a 1x solution. For fluorometric analysis, fluorescence was read at
excitation 560nm and emission 590nm in 24-hour intervals. All values were normalized to their
respective DMSO% control samples to account for DMSO cytotoxicity. GraphPad Prism 10
determined IC50 values from the logarithm curve.
3.2.2 Results: IC50 of Drug Candidates
The cell viability was determined at 24-hour intervals based on fluorometric analysis,
which were normalized to non-treated controls and v/v% DMSO controls to account for DMSO
cytotoxicity. Concentrations of manumycin A consisted of 0.25, 0.5, 5, and 10 µM (Figure 5).
Other drug candidates were treated as concentrations of 100, 50, 5, and 0.5 µM (Figure 6-7).
A B
Figure 5. Percentage Cell Viability of Manumycin A Based on Fluorometric Analysis
(A) 24-hour treatment (B) 48-hour treatment. Logarithmic curves with error bars. IC50 of 3.3µM
after 24 hrs and 3.1µM after 48hrs for treatment of manumycin A on B16-F10 cells.

27
A B
Figure 6. Percentage Cell Viability of Drug Candidates Based on Fluorometric Analysis
(A) 24-hour treatment (B) 48-hour treatment. Logarithmic curves with error bars. IC50 of each
treatment individualized in Figure 7. Concentrations of IC50 identified in Table 4.
Percentage cell viability was plotted against the logarithmic of drug concentration to
determine the IC50 cell viability by GraphPad Prism derivation (Table 4). IC50 determined the
concentration at which there was a 50% decrease in viable cells due to drug exposure. Therefore,
the IC50 value provided a maximal concentration at which drug candidates could exert their
therapeutic effect before inducing cytotoxicity. Manumycin A had a relatively lower IC50
compared to the other drug candidates, which aligns its known high cytotoxicity. The IC50 of the
drug candidates were relatively higher, which reflects their therapeutic safety as clinically
approved drugs. Among the drug candidates, fluconazole and minocycline demonstrated
significantly high safety profiles as the cell viability curves plateaued and their IC50 values were
not captured across the treatment concentrations. Additionally, the analysis of cell viability in 24-
hour intervals determined further trends of cell viability due to prolonged drug exposure. Across
the two different timepoints, IC50 values increased or remained the same over time, which
indicates that longer drug exposure did not result in cytotoxicity. This can be conceded as these
drug candidates are FDA-approved therapeutics with a high safety profile.

28
A B C
D E F
G
Figure 7. Percentage Cell Viability of 24-hours Treatment of Drug Candidates
(A) Climbazole (B) Doxycycline (C) Fluconazole (D) Imipramine (E) Ketoconazole (F)
Minocycline (G) Sitafloxacin. Logarithmic curves with error bars.
Table 4. IC50 Cell Viability Summary Across 24- and 48-hours Drug Exposure
Drug treatment IC50 at 24hr (µM) IC50 at 48hr (µM)
Manumycin A 3.3 3.1
Climbazole ~ 50.7 ~ 91.1
Doxycycline 27.4 156.4
Fluconazole > 194.2 > 194.2
Imipramine ~ 33.7 ~ 32.3
Ketoconazole 14.2 17.2
Minocycline > 175.9 > 175.9
Sitafloxacin ~ 49.5 ~ 90.4
~ symbolizes determined IC50 as ambiguous according to GraphPad Prism derivation

29
Fluorometric analysis was more appropriate for the assay than colorimetric analysis because both
manumycin A and minocycline have a yellow hue, which could skew absorbance-based assays.
However, fluorogenic compounds are also an obstacle to the Alamar blue assay fluorometric
approach because they can interfere with fluorescence assays. Particularly, minocycline is an issue
as it is inherently fluorescent from 580nm, which closely intersects with the reading wavelengths
for fluorometric analysis (Carlotti, 2010). This could have resulted in an overestimation of the
IC50 value of minocycline. Therefore, further in vitro experiments of drug treatments were based
on IC50 value and lower concentrations as greater than the IC50 may induce significant cell death.
Also, concentrations of drug treatments were adjusted for any significant cytotoxicity observed
after 24-hour treatment.
3.3 Gene Expression of Exosome Biogenesis and Cargo-loading
Exosomal inhibitory activity of the drug candidates were evaluated based on gene
expression analysis of exosome biogenesis and exosomal cargo-loading mechanisms. As
exosomes serve as a mechanism for cell-to-cell communication, they also function as mediators
for disease progression, especially various types of cancers. Therefore, inhibition of exosome
release is a target of interest for cancer therapy. However, due to the complexity and heterogeneity
of exosome biogenesis, it is difficult for a single drug to completely negate the process. Exosome
inhibitors evaluated thus far have been shown to target certain pathways to obstruct exosome
biogenesis. Some categorize these compounds based on their mechanism of action, those that
affect either endosomal maturation or extracellular release (Catalano, 2019). Therefore, evaluation
of certain pathways of exosome biogenesis can provide insight to both the exosomal inhibitory
activity and mechanism of action of the drug candidates.

30
Endosome maturation involves the conversion of EEs to LEs and formation of ILVs within
MVEs. During the transition from EEs to LEs, a Rab GTPase switch occurs between Rab5 and
Rab7 endosome markers (Feng, 1995; Klöpper, 2012). Rabs proteins are a Ras-related GTPase
superfamily which are important regulators for intracellular membrane trafficking. Rab7 has been
shown to contribute to endocytic trafficking. Inactivation of Rab7 results in suppression of MVE
fusion with lysosomes, this indicates its role in regulating the trafficking of endosomes to the
lysosome (Vanlandingham, 2009; Wozniak, 2016). Additionally, phosphoinositide conversion
participates in endosome maturation and endocytic cargo transport. Localized to LEs,
phosphoinositide 5-kinase (PIKfyve) generates phosphoinositides and sorts them into ILVs. These
phosphoinositides recruit some parts of the ESCRT machinery for ILV formation (Huotari, 2011;
Rivero-Rios, 2022). Furthermore, inhibition of PIKfyve prevents LEs fusion with MVEs (de
Lartigue, 2009).
Rab27A and Unc13D are master regulators of exosome secretion. Rab27A, with its paired
protein Rab27B, facilitates MVE docking to the plasma membrane for exosome secretion
(Ostrowski, 2009). In tumor cell lines, depletion of Rab27A/B resulted in decreased exosome
secretion (Bobrie, 2012; Peinado, 2012). Unc13D, or Munc13-4, is a calcium sensor protein that
regulates vesicle tethering and membrane fusion. Unc13D plays an essential role in exosome
release in cancer cells as it generates MVEs via a Rab11-dependent pathway (Messenger, 2018).
While they both regulate common secretory pathways, the mechanism in which Rab27A and
Unc13D coexist in regulating transport and docking fusion is not fully understood. While Unc13D
is a Rab27A effector, Unc13D also regulates discrete molecular events of exosome secretion
independent of Rab27A (Johnson, 2016; Zhang, 2019).

31
Cargo-loading of exosomes plays a vital role in KS tumorigenesis, as they package
oncogenic factors into these exosomes, which are secreted to non-infected cells and drive disease
progression. Therefore, we aim to distinguish how successful drug candidates impact different
mechanisms of cargo-loading, either by the ESCRT-dependent or -independent pathway. There
are distinct mechanics for these different exosomal cargo-loading pathways that allow for
identification of their modulation. The ESCRT-dependent mechanism relies on the ESCRT
complex and its associated proteins. One of those associated protein, apoptosis-linked gene-2
product interacting protein X (ALIX), encoded by PDCD6IP, is involved in both exosome
biogenesis and release (Larios, 2020). Alix plays a critical role in MVE cargo sorting as it attaches
ubiquitinated membrane receptors to ESCRT-III and recruits ESCRT-III to LEs. Therefore, cargo
is prepared for the membrane invagination to form MVEs with sorted cargo (Sun, 2015). On the
other hand, the ESCRT-independent mechanism depends on lipid raft-based microdomains, where
certain lipids, such as lysobisphosphatidic acid and ceramides, exosomal cargo-loading. These
lipids will organize into endosomal regions around the cargo and invaginate the membrane for ILV
formation (Babst, 2011). Rab31 was identified to drive ILV formation in a matter independent of
the ESCRT-complex and promotes exosome secretion as it prevents MVE fusion with lysosomes
by inactivation of Rab7 (Wei, 2021).
3.3.1 Materials and Method: Gene Expression Analysis of Exosomal Inhibitory Activity
B16-F10 and vGPCR-expressing cells were maintained in RPMI-1640 medium
supplemented with L-glutamine, 10% exosome-free FBS (Neuromics FBS002) and their
respective selective anti-biotic, 1% Antibiotic-Anti-mycotic (Gibco 15240062) or 1 µg/mL
Blasticidin (Invivogen ant-bl-1). For serum starvation, cells were trypsinized and plated in sterile
treated 6-well plates at 1,000,000 cells/well in 2.9mL RPMI-1640 without FBS. After 2-3 hours

32
for cellular adherence, drug treatments were introduced, accounting for v/v% DMSO to be <5%.
In biological triplicates, drug treatments concentrations were based on their determined IC50.
After 24 hours of drug exposure, cells were collected for RNA isolation and the media supernatant
was reserved for exosome isolation. RNAs were extracted from whole cell lysis using Quick-RNA
Purification Miniprep Kit (Zymo Research R1055). cDNAs were synthesized from total RNAs
using SuperScript VILO cDNA Synthesis Kit (ThermoFisher Scientific 11754250) according to
the manufacturer’s protocol. With 30ng of cDNA sample, gene expression levels were quantified
by real-time polymerase chain reaction (RT-PCR) analysis using SYBR Green Master Mix
(PowerUp A25778). Mean Ct values were measured from technical triplicate PCR analyses of
each sample. Gene expression Ct was normalized to the housekeeping gene β-Actin. Fold change
values were normalized to the non-treated group of the respective cell line. Quantification and
statistical analysis for each experiment was performed on GraphPad Prism. Statistical significance
used one-way ANOVA multiple comparison test.
3.3.2 Results: Gene Expression Analysis of Exosomal Inhibitory Activity
Firstly, gene expression analysis evaluated the mRNA levels of several key regulators
associated with exosome maturation, Rab7 and PIKfyve, and exosome release, Unc13D and
Rab27A. Exosomal inhibitory activity was defined based on comparison to drug exposure of the
known exosome inhibitor manumycin A. Overall gene expression trends of WT cells due to drug
exposure were not reflected in vGPCR-expressing cells. As the transfected cell line resembles the
metastasis and angiogenesis of KS, analysis focused on the exosomal inhibitory activity exhibited
by drug candidates on vGPCR-expressing cells.
Among the two tetracycline drug candidates, doxycycline showed a significant
upregulation across all biomarkers of exosome biogenesis (Figure 8). While upregulation trends

33
was observed in both cell types, vGPCR-expressing cells showed a larger induction, especially in
the exosome release marker Rab27A. Therefore, doxycycline exhibited properties of inducing
exosome biogenesis, which countered the intended screening for exosomal inhibitory activity.
Meanwhile, minocycline did not present inductive properties to the same degree. While
minocycline showed a slight upregulation of exosome maturation, there was a significant
downregulation of exosome release marker Unc13D. As Unc13D is significant to the formation of
MVEs and docking of exosomes independent of Rab27A, further investigation evaluated the
effects of minocycline in exosomal cargo-loading to understand its complete effect.
Figure 8. Effect of Tetracycline Analogues Treatment on Exosome Biogenesis
Exosome biogenesis was represented by Rab7 and PIKfyve expression and exosome secretion was
represented by Unc13D and Rab27A expression. The heatmap on the left depicts drug treatment
of WT cell line and the one on the right represents the transfected vGPCR-expressing cell line. 24-
hour drug treatment concentrations were based their determined IC50. Drug treatments groups
were normalized to their corresponding non-treated group of the same cell line. Treatment of
tetracycline analogues were compared to the positive standard manumycin A. Each treatment
group was performed in biological triplicates and technical triplicates.
The three azoles evaluated for their effects of exosome biogenesis (Figure 9). Climbazole
showed an upregulation of early exosome maturation and exosome release biomarkers, except

34
Unc13D. Therefore, climbazole did not present exosomal inhibitory activity. While this contrasted
the study conducted by Datta et al. with prostate cancer cells, disparities could be due to the
different cell type representing different diseased states (Datta, 2018). Fluconazole presented a
downregulation of exosome maturation and release across both the WT and transfected cell lines.
Interestingly, in the transfected cell line, fluconazole reflected similar gene expression trends
observed for manumycin A. Therefore, further analysis investigated how fluconazole affected
exosomal cargo-loading and whether it possessed a mechanism of exosomal inhibitory activity
similar to manumycin A. Ketoconazole showed an upregulation of exosome maturation, but
demonstrated a slight reduction in exosome secretion via Unc13D. Thus, investigation of cargoloading modulation provided further insight into the exosomal inhibitory activity of ketoconazole.
Figure 9. Effect of Azole Drug Candidates on Exosome Biogenesis
The heatmap on the left depicts drug treatment of WT cell line and the one on the right represents
the transfected vGPCR-expressing cell line. Concentrations of climbazole, fluconazole, and
ketoconazole were based their determined IC50. Drug treatments groups were normalized to their
corresponding non-treated group of the same cell line. Treatment of azoles were compared to the
positive standard manumycin A. Each treatment group was performed in biological triplicates and
technical triplicates.

35
Other drug candidates, imipramine and sitafloxacin, aligned with current research of their
effects of exosome biogenesis (Figure 10). Imipramine has been identified to target aSMase to
inhibit exosome secretion (Bianco, 2019). Thus, exosome inhibition due to treatment of
imipramine was reflected by the downregulation of exosome release biomarker via Unc13D and
Rab27A. Thus, imipramine was further evaluated for its effects on exosomal cargo-loading. While
its mechanism of action is unknown, sitafloxacin was determined to be an activator of exosome
biogenesis (Datta, 2018). Activation of exosome biogenesis due to sitafloxacin was evident by
significant upregulation of the exosome release biomarker Unc13D. Additionally, early exosome
biogenesis markers Rab7 and PIKfyve were slightly upregulated in the presence of sitafloxacin.
Figure 10. Effect of Drug Candidates Imipramine and Sitafloxacin on Exosome Biogenesis
The heatmap on the left depicts drug treatment of WT cell line and the one on the right represents
the transfected vGPCR-expressing cell line. Concentrations of imipramine and sitafloxacin were
based their determined IC50. Drug treatments groups were normalized to their corresponding nontreated group of the same cell line. Treatment of imipramine and sitafloxacin were compared to
the positive standard manumycin A. Each treatment group was performed in biological triplicates
and technical triplicates.

36
Based on the evaluation of exosome biogenesis and release biomarkers, fluconazole,
imipramine, ketoconazole, and minocycline demonstrated preliminary properties of exosomal
inhibitory activity through different pathways of exosome biogenesis. Therefore, further gene
expression analysis investigated whether these drug candidates specifically impacted cargoloading pathway as part of their exosomal inhibitory activity (Figure 11). Modulation of exosomal
cargo-loading is essential as this is the proposed mechanism in which KSHV expresses vGPCR to
facilitate KS tumorigenesis. The ESCRT-dependent pathway was represented by Alix, the
ESCRT-independent pathway was represented by Rab31. Additionally, we evaluated how drug
treatment impacted downstream processes induced by vGPCR, particularly cell proliferation via
Akt1 expression. As concentrations greater than the IC50 would induce higher cytotoxicity, two
different concentrations of drug treatment, one at the IC50 and one less than the IC50, evaluated a
dose-dependent effect.
Manumycin A is known to inhibit exosome biogenesis by targeting FTase and preventing
activation of Rab proteins involved in exosome biogenesis. This was evident by the
downregulation of Rab gene expression, Rab27A and Rab31. Inhibition of exosome secretion was
demonstrated by the reduction of exosome release biomarkers Unc13D and Rab27A. While
manumycin A affects the ESCRT-dependent pathway, there was no significant difference in Alix
expression compared to the non-treated group. While the reason for this disparity is not clear, the
mechanism of action for manumycin A remained distinct by the downregulation of Rab gene
expression across the different pathways of exosome biogenesis.
Ketoconazole did not demonstrate modulation of exosome secretion, but statistically
significantly downregulated Rab31 in a dose-dependent manner. As there was no reduction across
all Rab genes, this indicates that ketoconazole is not targeting inactivation of Rab proteins via

37
Figure 11. Evaluation of Cargo-loading Mechanisms due to Drug Treatment on vGPCR
Transfected Cells
Unc13D and Rab27A are important for exosome secretion. To determine the effect of drug
exposure to the exosomal cargo-loading mechanisms, Alix was correlated to the ESCRTdependent pathway and Rab31 was correlated to the ESCRT-independent pathway. Cell
proliferation via Akt1 represented one of the downstream processes which vGPCR modulates in
KSHV-infected cells. Statistical significance was performed by one-way ANOVA multiple
comparison to the non-treated group. *p < 0.05, **p < 0.005, p*** < 0.0005.

38
FTase inhibition. Thus, it may be inferred that ketoconazole acts on the ESCRT-independent
pathway to inhibit exosome maturation and cargo-loading. With treatment of imipramine, there
was a statistically significant reduction across both the cargo-loading markers Alix and Rab31 in
a dose-dependent manner. This demonstrates the effect of imipramine on exosome maturation,
separate from exosome release. At 10µM minocycline, there was a statistically significant
downregulation of Rab31 and upregulation of Rab27A. Then, at higher concentration of
minocycline, exosome release biomarkers were reduced, but Rab31 expression increased. As there
were contrasting trends across the different exosome biogenesis and cargo-loading pathways,
minocycline was no longer considered a drug candidate of interest.
Interestingly, fluconazole demonstrated reduction in exosome biogenesis and cargoloading which were comparable to manumycin A. There was a significant reduction in exosome
secretion in a dose-dependent manner, with 200µM fluconazole reflecting treatment of 1.65µM
manumycin A. For exosomal cargo-loading, fluconazole downregulated Rab31 expression, which
indicate that it acts on the ESCRT-independent pathway. However, based on the significant
downregulation of Rab genes across the pathways and the gene expression trends which were
similar to manumycin A, there is a stronger suggestion that fluconazole acts on FTase to inhibit
activation of Rab genes as its mechanism of exosome inhibition. Additionally, fluconazole was
the only drug candidate that reduced cell proliferation biomarker Akt1 similar to manumycin A.
Therefore, fluconazole presents exosomal inhibitory activity comparable to manumycin A without
inducing the same cytotoxicity. Thus, comparison of known exosome inhibitors, tipifarnib and
manumycin A, to fluconazole provides further insight to structure-activity relationship important
for inhibiting FTase activity and exosome biogenesis.

39
Chapter 4: Structure-Activity Relationship of Compounds Targeting
Farnesyltransferase
4.1 Binding Interactions of Fluconazole to FTase
Gene expression analysis suggests that the drug candidate fluconazole has a similar
mechanism of exosome inhibit to manumycin A. Therefore, further in silico modeling compared
this drug candidate to the known standard manumycin A to allude to the structure-activity
relationship required for a selective exosome inhibitor targeting FTase and preventing activation
of Rab genes involved in exosome biogenesis.
3.4.1 Materials and Methods: Ligplus
As mentioned previously, in silico screening with Autodock-Vina derived the optimal
binding orientation of ligands to the target enzymes of interest, FTase and nSMase. The ligandreceptor outputs from Vina of tipifarnib-FTase, manumycin A-FTase and fluconazole-FTase were
constructed into a 3D molecular structure model via UCSF Chimera (1.17.3). Ligplus (v.2.2.8)
was used to plot the specific binding interactions of these ligand to the receptor, demonstrating
their orientation in the binding site of FTase.
3.4.2 Results: Fluconazole Targeting FTase
Firstly, the binding interactions shared between known exosome inhibitors manumycin A
and tipifarnib were identified to demonstrate binding interactions indicative of successful
inhibition of FTase activity (Figure 12). Both compounds were oriented at the binding site
surrounded by hydrophobic interactions, interacting with three tyrosine (Tyr) and three tryptophan

40
(Trp) side chains. Additionally, they have polar interactions with a serine (Ser) and arginine (Arg)
side chain.

Figure 12. Binding Orientations of Known Exosome Inhibitors Manumycin A and Tipifarnib to
Farnesyltransferase
(A) Manumycin A in the binding site of FTase (B) Tipifarnib was overlayed onto the same region
as manumycin A, grey structure in the background. The red circles indicate the side chain
interaction of tipifarnib which shared with manumycin A, highlighting the binding interactions
necessary to achieve exosome inhibitory activity by FTase. Figure created in Ligplus.
A
B

41
Figure 13. Shared Binding Interactions of Fluconazole to Known Exosome Inhibitors on
Farnesyltransferase
(A) Fluconazole overlayed on manumycin A. (B) Fluconazole overlayed on tipifarnib. The
overlayed structure is illustrated as the grey structure in the background. The red circles indicate
the binding of fluconazole which are similar to the exosome inhibitors. Figure created in Ligplus.
The binding interactions of fluconazole were compared to the known exosome inhibitor
manumycin A and tipifarnib (Figure 13). Based on the shared binding interactions, there is
hydrophobic region that is important for the structure-activity relationship targeting the inhibition
A
B

42
of FTase. The hydrophobic region is suggested to consist of Trp102, Trp303, Tyr361, and Try251
as all the compounds formed binding interactions with these side chains. Additionally, all the
compounds bind to Arg202 and Tyr166, forming a polar and non-polar binding interaction region.
As fluconazole shares these side chains binding interactions with both known exosome inhibitors
acting on FTase, this supports that fluconazole potentially targets FTase to exert exosomal
inhibitory activity. Therefore, these side chains highlight binding interactions important to the
structure-activity relationship of exosome inhibitors targeting FTase.

43
Discussion
KSHV is an oncogenic virus that is capable of promoting various lymphoproliferative
disorders, most commonly Kaposi’s sarcoma (KS). Like other herpesviruses, KSHV can hijack
cellular activity through exosome-mediated cell-to-cell communication. This can occur through
loading exosomes with viral-encoded oncogenic factors, such as miRNAs, proteins, lipids, and
other metabolites. Exosomal content is transferred to recipient cells through membrane-membrane
fusion, gap junctions, or extracellular drainage. These KSHV-associated exosomes modulate
signaling pathways and facilitate the tumor microenvironment without alarming innate immune
responders. As with other cancers, exosomal miRNAs and proteins are involved in KS
tumorigenesis and metastasis (Spugnini, 2018). Furthermore, it is proposed that vGPCR, encoded
by the lytic oncogene ORF74, initiates KS tumorigenesis by exosome-mediated secretion of
paracrine factors. While ORF74 is only expressed in a subset of KS tumor cells, these ORF74-
expressing cells induce apoptosis of adjacent cells that do not express ORF74 (de Munnik, 2015;
Martin, 2009; Pati, 2001). Thus, disruption of exosome biogenesis may interfere with viral cargoloading of exosomes, which can serve as a potential avenue to understanding the underlying
molecular mechanism associated with KS tumorigenesis and paracrine transformation and the
modulation of KSHV infection.
Cancer therapeutic research has extensively studied the inhibition of TDEs as an approach
to suppress tumor progression and disrupt drug therapy resistance. There are established exosome
inhibitors, such as tipifarnib, manumycin A, and GW4869, which have been used extensively to
study exosome inhibition in various diseased. However, their utility as an exosome inhibitor have
been reduced due to their endogenous cytotoxic activities. Ongoing research continues to identify

44
new pharmacological inhibitors that are non-cytotoxic at effective doses, such as Y27632,
calpeptin, pantethine, and spiroepoxide, for the treatment of highly metastatic cancers. For
example, Datta et al. utilized the drug-repurposing approach and proposed an extensive list of
potential selective exosome inhibitors for the treatment of prostate cancer (Datta, 2018). Most
recently, McNamee et al. found successful reduction of exosome release with treatment of the
inhibitors calpeptin, Y27632, manumycin A, and GW4869 in triple-negative breast cancer cells
(McNamee, 2023). Also targeting breast cancer models, Andreu et al. proposed docetaxel,
biscurcumin, primaquine, and doxorubicin as potential exosome release inhibitors (Andreu, 2023).
Unfortunately, these compounds also have cytotoxic activity at the concentrations that are
employed. In contrast, Ma et al. identified activators of exosome secretion, such as cucurbitacin
B, gossypol, and obatoclax, which revealed some regulators of exosome secretion in glioblastoma
and human mesenchymal stem cells (Ma, 2021).
While the inhibition of TDEs has been investigated across various cancer models, our
investigation provides a new perspective to viral oncogenesis through the inhibition of KSHVassociated exosomes for the treatment of KS tumorigenesis. The highly metastatic nature of KS
suggests that KSHV-associated exosomes initiate tumorigenesis via paracrine mechanisms.
Therefore, inhibition of exosome biogenesis could provide further insight to the molecular
mechanism(s) behind paracrine transformation.
In silico modeling screened a variety of clinically approved compounds with a tertiary
amine to predict their binding affinity to FTase and nSMase. The predictability and reliability of
Autodock-Vina to determine binding affinity values was evident by the exosome inhibitors known
to target these enzymes. Tipifarnib and manumycin A target FTase to inhibit Rab protein activation
for exosome biogenesis. These two compounds ranked highly for their affinity to FTase. As

45
GW4869 is known to target nSMase to exert its exosome inhibitory activity, Autodock-Vina
valued GW4869 with the highest binding affinity among the screened compounds. Therefore, in
silico screening selected in silico drug candidates for further investigation in KS cell models.
Our investigation affirmed previous research works, demonstrating the potential of
ketoconazole and imipramine as exosome inhibitors. Ketoconazole was predicted to have
exceptional binding affinity to target enzymes from Autodock-Vina in silico modeling. At the
concentration employed (5 to 10 µM), this imidazole had marginal effect on the exosome assemble
proteins. At 10 µM IC50, ketoconazole was able to significantly reduce Rab31 expression. Rab31
is a protein involved in the ESCRT-independent mechanism or nSMase mediated pathway. In
contrast, ketoconazole did not demonstrate changes to exosome secretion biomarkers Rab27A and
Unc13D, which may suggest the primary mechanism of action for this imidazole may be to target
exosome formation rather than secretion. Further investigation of prolonged exposure to drug
treatment may allow the impact of disruption of exosome biogenesis to be reflected in exosome
secretion. At the concentration range from 20 to 40 µM, imipramine caused reduced expression of
exosome biogenesis and release biomarkers. There was statistically significant reduction of cargoloading markers of both Rab31 and Alix, which suggests there is a large effect on cargo-loading
mechanisms. This may suggest that the imipramine may exert exosome inhibitory activity by
disruption of exosome biogenesis rather than secretion.
Conversely, tetracyclines, like minocycline and doxycycline, demonstrated a significant
upregulation of exosome biogenesis and release markers. These findings suggest there may be an
inducible mechanism that is largely impacted with tetracycline compounds. The upregulation trend
was observed in both the wild-type and transfected cell line, but significantly higher in the
transfected cell line. Therefore, the transfected expression vector may have a tetracycline inducible

46
factor embedded. Further investigation would require sequencing to identify the tetracycline
inducible factor.
Interestingly, our findings showed the unexplored potential of fluconazole as an exosomal
inhibitor. When employed at 100 to 200µM of fluconazole, which are therapeutically achievable,
there was reduction in exosome biogenesis and release biomarker expression, such as Rab27A,
Unc13D, and Rab31. Expression levels of these biomarkers were highly comparable to the positive
standard manumycin A at 1.65µM. Their similar effect to exosome biogenesis markers of the two
compounds suggests that fluconazole has a similar mechanism of action as that of manumycin A.
Unlike manumycin A, fluconazole is not cytotoxic at >179 µM. At 200 µM fluconazole, all of the
targeted genes like Rab7, PIKfyve, Unc13D and Rab27A were all downregulated. Between the
two different concentrations of fluconazole, 200µM is suggested to be the approximate effective
dose for exosome inhibitory activity compared to 100µM. Additional studies of lower and higher
concentrations may clarify the concentration range at which fluconazole effectively induces
reduction in gene expression of the exosome assembly proteins. Importantly, the reduction of these
gene targets was significantly lower in vGPCR transfected cells as compared to WT B16-F10 cells.
This may be indicating selectivity to reduce certain biomarkers within the KS cell model.
Fluconazole is interesting compound in that it has a high therapeutic index as compared to
manumycin A.
Our investigation highlights the molecular structure of exosome inhibitors via FTase
targeting through tipifarnib, manumycin A, and fluconazole. Their binding interactions to FTase
reveals the hydrophobic side chain interactions required for their structure-activity relationship
(SAR), resulting in the inhibition of FTase and inactivation of Rab proteins involved in exosome
biogenesis. Therefore, prospects of the study could quantify the inhibition of enzymatic activity

47
due to treatment of fluconazole to characterize its mechanism of action targeting FTase.
Additionally, characterization of isolated exosomes from drug exposure could provide further
understanding of the molecular mechanism behind vGPCR-loaded exosomes in paracrine
transformation. From there, in vivo preclinical development can establish the safety and efficiency
of exosome inhibitors using transgenic K14-Cre/vGPCR mice models.
The current challenge to the clinical development of exosome inhibitors is their highly
cytotoxic prolife. Thus, it is crucial to develop exosome inhibitors without inducing many adverse
side effects. Exosomes biogenesis is necessary for normal intercellular communication; therefore,
they are ubiquitous across various cell types. It is important to address tumor cell-specific targeting
of these pharmacological inhibitors to reduce many side effects. Nonetheless, targeting exosomes
biogenesis in metastatic cancers shows significant potential in cancer patients in the future.

48
Conclusion
Kaposi’s sarcoma is an angioproliferative disease linked to infection with KSHV. One of
the virally encoded lytic genes, ORF74, encodes vGPCR, which is homologous to the human IL8
receptor CXCR2, which concurs with findings that IL-8 promotes KS progression while its
inhibition blocks its disease progression. Similarly, we have previously shown that IL-1 which is
the primordial inflammatory cytokine that promotes IL-8 is critical in promoting KS proliferation.
Similarly, blockade of IL-1 using IL-1ra was able to block KS proliferation. It is proposed that
vGPCR initiates paracrine transformation and tumorigenesis as vGPCR-expressing cells secrete
exosomes loaded with angiogenic and paracrine factors. Exosomes have been a growing area of
research as they are key to cell-to-cell communication. Therapeutic approaches are exploring how
to exploit exosomes for drug delivery and disrupt cell signaling carried out by exosome
mechanisms. For highly metastatic diseases, such as KS, exosome inhibition is a significant
therapeutic approach of interest. In this study, we have identified several exosome inhibitors in KS
cell models and propose the exosome inhibitory activity. In particular, the triazole fluconazole was
predicted to have strong affinity for FTase and nSMase. Its inhibitory activities of exosomes may
be associated with its ability to inhibit the expression of PIKfyve, Rab isoforms, and Unc13D.
Since PIKfyve plays an important role in the metabolism of phosphoinositide lipids (PPIs), to
produce lipid signaling molecules important in regulating cell processes. PIKfyve phosphorylates
position 5 of PI(3)P, is the sole source of PI(3,5)P2 and responsible for the PI(5)P pool.
Interestingly, these pathways have been associated with immunomodulation which is thought to
an important oncogenesis pathway. Future studies should explore the importance of fluconazole
mediated effects on PIKfyve metabolism. Additionally, the potential exosome inhibitory activity

49
of fluconazole may contribute to further understanding of the molecular mechanism behind
exosome-mediated paracrine transformation and tumorigenesis. Along with establishing a
potential therapeutic approach against KS tumorigenesis, this may extend to understanding the
initiation and progression of other metastatic cancers, such as breast and colon cancers. Further
investigation of the exosome inhibitory activity of fluconazole may impact our understanding of
the role of exosomes in highly metastatic cancers.

50
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Abstract (if available)

Abstract As the causative agent of Kaposi’s sarcoma (KS), Kaposi’s sarcoma-associated herpesvirus (KSHV) facilitates the immunosuppressive tumor microenvironment and modulates signaling pathways by hijacking exosome-mediated cell-to-cell communication. These exosomes are loaded with oncogenic factors, such as microRNAs and proteins, that infect recipient cells and promote cell transformation via paracrine mechanisms. As these exosomes play an important role in persistent viral infection and tumor progression, exosome inhibition is a potential avenue for the treatment of KS tumorigenesis. Therefore, we evaluated clinically approved drug compounds for exosomal inhibitory activity and defined the structure-activity relationship of compounds targeting farnesyltransferase to inhibit exosome biogenesis. Using Autodock-Vina, compounds with a tertiary amine moiety were screened for potential binding affinity to target enzymes of known exosome inhibitors, farnesyltransferase (FTase) and neutral sphingomyelinase. Drug treatment on B16-F10 melanoma cells transfected with the oncogenic virally encoded G-protein coupled receptor evaluated modulation of exosome biogenesis and cargo-loading mechanisms. Downregulation of Rab27A and Rab31 affirmed the exosomal inhibitory activity of ketoconazole and imipramine. Interestingly, 24-hour treatment of 200µM fluconazole resulted in the downregulation of Unc13D, Rab27A, Rab31, and Akt1, suggesting that fluconazole could potentially target FTase to inhibit exosome biogenesis. Additionally, a comparison of the binding interactions of fluconazole and known compounds targeting FTase via molecular modeling demonstrated a hydrophobic binding region essential to their structure-activity relationship for exosome inhibition. These results define fluconazole as a potential exosome biogenesis inhibitor for the treatment of KS tumorigenesis.

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Creator Trinh, Kaitlyn Allyson (author)

Core Title Exosomal inhibitory activity of clinically approved tertiary amines for the treatment of Kaposi’s sarcoma tumorigenesis

School School of Pharmacy

Degree Master of Science

Degree Program Clinical and Experimental Therapeutics

Degree Conferral Date 2024-05

Publication Date 04/25/2024

Defense Date 04/18/2024

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

Tag ESCRT-complex,exosomal cargo-loading,exosome,exosome biogenesis,HHV-8,Kaposi's sarcoma,KSHV,manumycin A,OAI-PMH Harvest

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