USE OF A SMALL MOLECULE INHIBITOR DRUG TO TREAT HUMAN
PAPILLOMAVIRUS INDUCED CANCER
Inderdeep Singh Atwal
B.S., University of California, Los Angeles, 2004
J.D., University of the Pacific, McGeorge School of Law, 2010
M.B.A., California State University, Sacramento, 2010
PROJECT
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
BIOLOGICAL SCIENCES
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SPRING
2011
USE OF A SMALL MOLECULE INHIBITOR DRUG TO TREAT HUMAN
PAPILLOMAVIRUS INDUCED CANCER
A Project
by
Inderdeep Singh Atwal
Approved by:
__________________________________, Committee Chair
Thomas R. Peavy, Ph. D.
__________________________________, Second Reader
Hao Nguyen, Ph. D.
____________________________
Date
ii
Student: Inderdeep Singh Atwal
I certify that this student has met the requirements for format contained in the University format
manual, and that this project is suitable for shelving in the Library and credit is to be awarded for
the Project.
__________________________, Graduate Coordinator
Susanne Lindgren, Ph. D.
Department of Biological Sciences
iii
________________
Date
Abstract
of
USE OF A SMALL MOLECULE INHIBITOR DRUG TO TREAT HUMAN
PAPILLOMAVIRUS INDUCED CANCER
by
Inderdeep Singh Atwal
Human Papillomavirus (HPV) afflicts millions of individuals throughout the
world and is the most prevalent sexually transmitted disease in the United States. Several
strains of HPV have been linked to the formation of cancer, manifesting itself in a variety
of locations (e.g; the vulva/vaginal, penis, anus, head and neck). Current cancer
treatments have been limited to three established methods: chemotherapy, radiation and
surgery. Each of these treatments have significant issues and provide mixed results for
patients.
Recent developments in the use of computer molecular modeling and
advancements in the knowledge of cancer pathways have opened numerous new avenues
for cancer treatment. A team led by Dr. Shaomeng Wang at the University of Michigan,
Ann Arbor have used crystalline structure studies of the MDM2-p53 interaction to
synthesize a molecule that has a higher affinity to MDM2 than p53, thus when the
molecule is introduced into the cell it outcompetes p53 for MDM2 binding sites which
frees p53. The freed p53 is then able to act as a tumor suppressor. The development of
specific inhibitors as a potential cancer treatment has great promise for the battle against
iv
cancer. These small molecule inhibitors would have the potential to be a highly effective
and specific treatment of cancer lacking many of the drawbacks of current cancer
treatments.
The goal of this grant proposal is to use the promising approach utilized by Dr.
Wang’s team to treat HPV induced cancer. The hypothesis of this proposed study is that
a small molecule designed de novo using bioinformatics software will be able to bind to
E7 protein derived from Human Papillomavirus, inhibiting E7 from binding to the cell
cycle regulatory protein pRb, and thereby inhibiting cancerous growth caused by HPV.
The specific aims of this study are to:
I. Utilize structure data on the E7-pRb binding pocket to design small molecule inhibitor
candidates using computer modeling software.
II. Utilize a Competitive Fluorescence Binding Assay to identify candidate molecules
with the ability to outcompete pRb for the E7 binding pocket.
III. Utilize a Cell Permeability Assay to determine whether candidate molecules can enter
cells.
IV. Utilize HPV cell cultures to test the ability of the small molecule inhibitors to halt
cancerogenesis in vitro.
v
V. Evaluation of the effects of the small molecule inhibitors on HPV cells.
The development of a small molecule inhibitor to block the binding of pRb and
HPV protein E7 is a key step in the development of a successful non-invasive therapy for
HPV induced cancer. The use of the AMBER software suite and performing the series of
steps above to evaluate their effectiveness to prevent the E7-pRb complex and inhibiting
cell proliferation will be a critical step towards developing a new drug to treat HPVinduced cancer.
_______________________, Committee Chair
Thomas R Peavy, Ph. D.
_______________________
Date
vi
TABLE OF CONTENTS
Page
List of Tables ........................................................................................................................ viii
List of Figures .......................................................................................................................... ix
SPECIFIC AIMS ...................................................................................................................... 1
BACKGROUND AND SIGNIFICANCE ................................................................................ 4
HPV Prevalence ................................................................................................................ 4
Novel Approach to Cancer Treatment: Small Molecule Inhibitors ................................. 6
Human Papillomavirus .................................................................................................... 11
HPV and Carcinogenesis ................................................................................................ 16
RESEARCH DESIGN AND METHODS .............................................................................. 19
Overview ....................................................................................................................... 19
Specific Methods............................................................................................................. 21
I. Design and Synthesis of Small Molecule Inhibitors .................................................... 21
Region of Focus for Design of a Small Molecule Inhibitor ........................................ 21
AMBER Program-Design of de novo Small Molecule Inhibitors .............................. 25
Synthesis of Small Molecule Inhibitor Candidates ..................................................... 26
II. Competitive Fluorescence Binding Assay................................................................... 26
III. Cell Permeability Assay ............................................................................................. 30
PAMPA-Parallel Artificial Membrane Permeability Assay ....................................... 30
Possible Outcomes of Permeability Studies ............................................................... 31
IV. Effects of Small Molecule Inhibitor Candidates on HPV Cell Cultures..................... 32
Promega CellTiter 96 Non-Radioactive Cell Proliferation Assay .............................. 32
V. Evaluation of the Effects of Small Molecule Inhibitor Candidates on HPV Cells...... 33
Double-labeling Native Gel Immunoblot Assay ......................................................... 33
Cell Lysates for Detection of Apoptosis ..................................................................... 35
TUNEL (Terminal Transferase dUTP Nick End Labeling) Assay ................... 36
Live/Dead Viability & Cytotoxicity Assay................................................................. 37
BUDGET………. .................................................................................................................... 38
CONCLUSION ....................................................................................................................... 42
Literature Cited ....................................................................................................................... 43
vii
LIST OF TABLES
Page
Table 1 List of HPV Strains and the resulting disease induced by the
strain ……………………………………….……………………………………...12
viii
LIST OF FIGURES
Page
Figure 1 Illustration depicting the interaction between MDM2 and
p53…….…………………..………….………………………………………. 8
Figure 2 Illustration showing Small Molecule Inhibitors interacting with
MDM2…………………….…………………………………………………...10
Figure 3 Model Image of HPV capsid arrangement……………….……………………14
Figure 4 HPV genome…………………………………………….…………………….15
Figure 5 Illustration of LXCXE region of HPV E7……………………………………..17
Figure 6 Mechanism of HPV-Induced Oncogenesis………………….………………...18
Figure 7 Structure of CR3 domain in HPV E7 Protein…………………..……………...23
Figure 8 Illustration of fluorescence binding assay…………………………....………..29
ix
1
SPECIFIC AIMS
Cancer treatment has been mostly limited to three established methods:
chemotherapy, radiation and surgery. However, each of these treatments has significant
problems and provides mixed results for patients. Recent developments in the use of
computer molecular modeling and advancements in the knowledge of cancer pathways
have opened numerous new avenues for cancer treatment. One such avenue is the use of
molecular modeling software and 3D database searches to develop small molecule
inhibitors which have the ability to neutralize oncogene products within cells and halt
cancer growth. Recently, a team led by Dr. Shaomeng Wang at the University of
Michigan, Ann Arbor have used crystalline structure studies of the MDM2-p53
interaction to synthesize a molecule that has a higher affinity to MDM2 than p53, thus
when the molecule is introduced into the cell it outcompetes p53 for MDM2 binding sites
which frees p53 (Shangary & Wang, 2008). The freed p53 is then able to act as a tumor
suppressor. The development of specific inhibitors as a potential cancer treatment has
great promise for the battle against cancer. These small molecule inhibitors would have
the potential to be highly effective and specific treatment of cancer resulting in a therapy
lacking many of the drawbacks of current cancer treatments.
The goal of the current study is to use the promising approach utilized by Dr.
Wang’s team to treat Human Papillomavirus (HPV) induced cancer. HPV infection is
widespread throughout the world; affecting individuals of both sexes with high
prevalence, and can manifest itself in a variety of locations (e.g. the vulva/vagina, penus,
anus, head and neck). The hypothesis of this proposed study is that a small molecule
2
designed de novo using bioinformatics software will be able to bind to the E7 protein
derived from Human Papillomavirus, inhibit E7 from binding to the cell cycle regulatory
protein pRb, and thereby inhibit cancerous growth caused by HPV. The development of
a small molecule inhibitor promises to create a drug-based therapy for individuals
suffering from HPV induced cancers which is highly effective and specific to HPV
induced cancer without the side effects of current treatments. Furthermore, development
of such a cancer therapy would provide a strong foundation for the treatment of other
cancers.
The specific aims of this study are set forth below:
I) Utilize structure data on the E7-pRb binding pocket to design small molecule inhibitor
candidates using computer modeling software.
II) Utilize a Competitive Fluorescence Binding Assay to identify candidate molecules
with the ability to outcompete pRb for the E7 binding pocket.
III) Utilize a Cell Permeability Assay to determine whether candidate molecules can enter
cells.
IV) Utilize HPV cell cultures to test the ability of the small molecule inhibitors to halt
cancerogenesis in vitro.
V) Evaluation of the effects of the small molecule inhibitors on HPV cells.
The development of a small molecule inhibitor to block the binding of pRb and
HPV protein E7 is a key step in the development of a successful non-invasive therapy for
HPV induced cancer. The use of the AMBER software suite and performing the series of
3
steps above to evaluate their effectiveness to prevent the E7-pRb complex and inhibiting
cell proliferation will be a critical step towards developing a new drug to treat HPVinduced cancer.
4
BACKGROUND AND SIGNIFICANCE
HPV Prevalence
HPV strains account for the most common sexually transmitted disease in the
United States (Dunne et al., 2007). The American Social Health Association estimates
that about 75-80% of sexually active Americans will be infected with HPV at some point
in their lifetime (American Social Health Association, 2011). It was estimated that in
2000, nearly 6.2 million new HPV infections occurred in Americans aged 15-44
(Weinstock et al., 2000). Studies estimate that the prevalence of high risk HPV (HPV
that has the potential to cause cancer in individuals) is around 15.2% (Dunne et al.,
2007). Estimates of HPV prevalence vary widely because of the difficulty in detecting
strains of HPV and the fact that a large portion of HPV infections are cleared by the
immune system and are never detected. Regardless of the accuracy of these estimates, it
is clear that HPV infection poses a significant health threat and is wide spread in the
population.
Human Papillomavirus is one of the most common sexually transmitted diseases
in both men and women. The virus is widely transmitted and often causes a multitude of
complications in patients, particularly in women. Persistent HPV infections in women
are the primary cause of cervical cancer (Munge & Baldwin, 2004). Furthermore, HPV
infections in women can cause cancer of the anus, vulva, vagina, penis and oropharyngeal
cancer. Nearly 11,000 women are diagnosed a year with cervical cancer and 4000
patients die annually from the disease. Of these cases, Human Papillomavirus has been
5
implicated in nearly 99.7% of cervical squamous cell cancers and is present in nearly
89% of adenocarcinomas of the cervix in women under 40 (Burd, 2003).
Although, HPV related cancer presents a significant threat to the population,
treatment and prevention options have been scarce. In 2006, the US Food and Drug
Administration approved Gardasil, a vaccine that is highly effective in preventing
infection by two strains of Human Papillomavirus; but it does not cover all strains of
HPV and does not help to treat individuals already infected with HPV. In addition,
Gardasil has only been approved for use in women. A study looking at HPV infections in
women for the period of 2003-2004 found that 15.2% of women aged 14-59 were
infected with a strain of HPV that led to an increased risk of cancer. But only 3.4% of
women aged 14-59 were infected with a type of strain of HPV protected against by the
Gardasil vaccine, which was significantly lower than previous estimates casting doubt on
the vaccines effectiveness in dealing with HPV induced cancer (Dunne et al., 2007). For
these reasons, the development of vaccines for high-risk HPV strains is promising but
falls short of being a solution to HPV induced cancer.
As far as cancer treatments for HPV induced cancer, the treatments have been
limited to traditional therapies. These options include radiation, chemotherapy and
surgery which all have significant weaknesses and limitations. Surgery can range from a
small incursion to remove a cone-shaped piece of tissue to an invasive hysterectomy.
Radiation therapy is a cancer treatment that utilizes high-energy x-rays or other types of
radiation to kill cancer cells. Chemotherapy cancer treatment utilizes drugs to stop the
growth of cancer cells, either by killing the cells or by halting cellular division. These
6
three treatments are often used in conjunction with each other to develop a treatment plan
tailored to the particular type of cancer and stage. Current cancer treatments are plagued
by a lack of consistent effectiveness and severe side effects which range from harsh
sicknesses associated with chemotherapy to trauma associated with invasive surgery. In
general, current treatments are lacking and there is a dire need for new innovative
treatments that minimize side effects to patients and have increased effectiveness. A new
technique using small molecule inhibitors could provide the key to dealing with HPV
induced cancer.
Novel Approach to Cancer Treatment: Small Molecule Inhibitors
A team led by Shaomeng Wang, Ph.D at the University of Michigan
Comprehensive Cancer Center, has had significant success fighting cancer through the
use of de novo created small molecules to inhibit cancer pathways (Shangary & Wang,
2008). The group primarily focused on the p53 pathway in the cell cycle to inhibit cancer
cell growth caused by p53 inactivity which is a leading cause of a number of different
cancers. p53 acts as a tumor suppressor and thus proved to be an attractive therapeutic
target. The team was able to coordinate extensive knowledge about the p53 pathway in
conjunction with computer modeling to design a new small-molecule inhibitor drug.
Specifically, the group focused on the p53 and the MDM2 binding site. This
interaction is critical in controlling the amount of free p53 in the cell because of the role
of MDM2 as a key regulator of p53 (see Figure 1). A MDM2 antagonist would be able
7
to release p53 from the inhibitory hold of MDM2 and would therefore stabilize and
activate the p53 tumor suppressor protein leading to cell cycle arrest or apoptosis
(programmed cell death) of cancer cells.
8
Figure 1. Illustration depicting the interaction between MDM2 and p53 (Shangary &
Wang, 2008).
9
Dr. Wang and his team were able to synthesize a non-peptidic small molecule
MDM2 antagonist from their computer aided molecular design program. Based on
crystalline structure studies of the MDM2-p53 interaction, the group was able to design
and synthesize a molecule that had a higher affinity to MDM2 than p53(see Figure 2).
Thus, when the molecule was introduced into the cell, it out competed p53 for MDM2
binding sites which freed up p53. The unbound p53 was then able to act as a tumor
suppressor. Dr. Wang has been able to test this new molecule in mice and it has been
found to have a high degree of efficacy and safety.
10
Figure 2. Illustration showing Small Molecule Inhibitors interacting with MDM2. The
small molecules (Nutlin-2 and MI-219) inhibit the binding region on MDM2 which leads
to the release of p53 (Shangary & Wang, 2008).
11
Human Papillomavirus
Human Papillomavirus is a member of the Papovaviridae family of viruses,
which also include polyomavirus and simian vacuolating virus (Burd, 2003). HPV
infection and viral replication occurs within the stratified epithelium of the skin or
mucous membranes. All strains of HPV are predominantly transmitted through direct
skin-to-skin contact between an infected individual and an uninfected individual. Over
120 types of HPV have been identified and are identified by a number nomenclature
system (see Table 1). HPV types 16, 18, 31, 33, 35, 39 ,45, 51, 52, 56, 58, and 59 are
strains of HPV that create a high risk factor for cancer development in infected patients.
These HPV strains have been connected to cancers of the vulva/vagina, penis, anus, head
and neck.
12
Table 1. List of HPV strains and the resulting disease induced by the strain (Burd, 2003).
13
HPV is a relatively small (55nm in diameter) non-enveloped virus. It is made up
of an isosahedral capsid composed of 72 capsomers (see Figure 3), which contain at least
two capsid proteins (L1 and L2). Each capsomer is a pentamer of the major capsid
protein, L1. Each virion capsid contains about 12 copies of the minor capsid protein, L2.
The HPV genome is a circular double-stranded DNA molecule (~7900 bp) that interacts
with histones(see Figure 4). The genome of HPV is divided into three segments: 1) a
non-coding upstream regulatory region of 400-100 bp, 2) an Early Region consisting of
ORFs E1, E2, E4, E5, E6 and E7 which are involved in viral replication, and 3) a Late
Region which encodes the L1 and L2 structural proteins for the viral capsid.
14
Figure 3. Model image of HPV capsid arrangement (Burd, 2003).
15
Figure 4. HPV genome (Burd, 2003).
16
HPV and Carcinogenesis
HPV infection creates oncogene products in a pathway that is susceptible to
inhibition using the method developed by Dr. Wang. HPV hijacks the cell and begins
synthesizing two proteins: E7 and E6. E7 binds with pRb activating the E2F growth
factor. Activation of E2F results in viral DNA replication by the host cell and which
results in the initiation of an unchecked S-phase growth cycle. The goal of this research
is to use information about the E7 and pRb binding site to design an inhibitor molecule
that would outcompete pRb for the E7 binding site, thereby preventing the oncogene
effect of the viral protein (see Figures 5 and 6).
The E7-pRb binding pocket is centered on a highly conserved region of pRb that
contains a common motif known as the LXCXE region. This motif and binding region
have been extensively studied and structurally mapped by x-ray crystallography. These
crystalline structure studies will be used to design a de novo molecule which would be
able to outcompete the pRb binding site and neutralize the E7 protein thereby hindering
cancerogenesis. It is essential that this de novo generated small molecule inhibitor have
a higher binding affinity to the E7 binding pocket than pRb does so that it can
outcompete pRb. The E7-small molecule binding complex will free pRb to act in
accordance with it’s normal cellular function, which is to act as a tumor suppressor by
binding to E2F-1 and preventing uncontrolled cell cycle growth (see Figure 5).
17
Figure 5. Illustration of LXCXE region of HPV E7. Studies on the LXCXE region of
HPV E7 have shown that mutations in this region affects binding to pRb (Munge &
Baldwin, 2004).
18
Figure 6. Mechanism of HPV-Induced Oncogenesis. HPV E7 gene product binds to the
hypo-phosphorylated form of pRb which leads to a disruption of the complex between
pRb and E2F-1 (a transcription factor). Unbound E2F-1 initiates transcription of genes
whose products are required for the cell to enter the S phase of the cell cycle, and it also
hinders apoptosis (Munge & Baldwin, 2004).
19
RESEARCH DESIGN AND METHODS
Overview
The development of a small molecule inhibitor with the properties to outcompete
pRb for the E7 binding site begins with identifying potential inhibitor candidates.
Developing potential candidates that fit the requirements of being an E7-pRb binding
antagonist requires use of information from high-resolution x-ray crystallographic studies
and experimentation conducted on the E7-pRb binding pocket. This data provides the
foundation for the structure-based design of a small-molecule antagonist. Studies have
indicated that disruption of genes in the LXCXE binding cleft region and an adjacent
CR3 zinc domain on E7 hinders its ability to bind to pRb. The LXCXE binding cleft of
E7 is a peptide region that has a series of conserved lysines that are central to binding
with pRb via ionic interactions. The CR3 region of HPV E7 assembles as a roughly
globular and obligate dimer of approximate dimensions 30Å x 20Å x 25Å. The CR3 zinc
domain on E7 binds with the C-terminal region of pRb and also binds with E2F-1. E7
temporarily binds a portion of E2F-1 releasing pRb from E2F-1 and then strongly binds
with pRb leading the cell toward cancer formation. The key role this CR3 zinc domain
region plays in leading to HPV induced carcinogenesis and studies indicating no
detectable homology to this region make it an ideal candidate for drug development (Liu
et al., 2006).
Using this data and a structure-based de novo design approach, the goal will be to
develop a small molecule antagonist that fits the CR3 region on E7 with greater affinity
20
to E7 than pRb. This is accomplished by inputting the crystal structure information for
the binding complex into a computer modeling software program (AMBER Program
Suite, www.ambermd.org) which can then be used to develop de novo molecules that can
theoretically bind to the E7 binding pocket by calculating bond energies and structural fit.
The AMBER software utilizes the Poisson–Boltzmann equation, which is a differential
equation that describes electrostatic interactions between molecules in ionic solutions, to
model molecular binding. Once antagonist candidates have been identified, the next step
is to have these compounds synthesized by a commercial vendor for subsequent empirical
studies.
The second phase of the project will consist of testing the efficacy of these de
novo designed small molecules to inhibit E7 and pRb binding. This will be accomplished
utilizing a competitive fluorescence binding assay to ascertain the affinity of the de novo
designed small molecule candidates to the E7 binding pocket and to ensure that the de
novo designed small molecule will outcompete pRb for the E7 binding site (halting the
carcinogenic pathway). Candidates with greater binding affinity to E7 than pRb will be
utilized for further testing.
The third phase of the project will be to determine the capability of delivering
these candidates in vitro as a therapeutic agent. Thus, it will be necessary to test whether
the small molecule inhibitor candidates can enter into the cell (cell permeability studies)
so that it can inhibit E7's ability to bind to pRb.
Successful candidates would then be used for the fourth phase of testing to
determine whether these candidate molecules can indeed halt uncontrolled cell growth of
21
HPV cancer cell lines in cell culture studies. A cell growth assay will be utilized to
determine the dose response of HPV cell lines to the de novo small molecule candidates
to determine the potential of the candidates to halt cancerous cell growth in vitro.
The final phase of testing will examine the effects of the small molecule inhibitor
on HPV cells. First, a double labeling immunoblot experiment will be utilized on cells
treated with the small molecule inhibitor to determine whether E7-pRb complexes are
being formed or whether the binding complex is blocked from forming by the small
molecule inhibitor. Second, inhibitors will be tested to examine the level of apoptosis or
the potential to cause cytotoxic effects on cells. This will be accomplished by utilizing a
Terminal Transferase dUTP Nick End Labeling (TUNEL) Assay, which will determine
the level of apoptosis in the treated cells. A Live/Dead Assay will determine relative
ratios of live to dead cells so as to determine whether the small molecule inhibitor
compound is causing cytotoxicity and death to the cells.
Specific Methods
I. Design and Synthesis of Small Molecule Inhibitors
Region of Focus for Design of a Small Molecule Inhibitor
The binding between pRb and E7 occurs in two conserved regions of the E7
protein: LXCXE and CR3 regions. The LXCXE region is a binding cleft that interacts
with a group of lysine amino acids within pRb through ionic bonds. Studies on this
region have indicated that mutations within this region which alter the protein
composition disrupt the E7-pRb binding complex. The LXCXE protein motif is
22
commonly found in several organisms and is relatively conserved across species. The
commonality of this region creates potential difficulties in using this as a binding site
when designing a de novo inhibitor drug.
The second region that plays a critical role in binding between pRb and E7 occurs
at the CR3 zinc domain within E7. The E7 CR3 domain folds in a roughly globular
conformation and assembles into a homodimer with approximate dimensions of 30Å x
20Å x 25Å (see Figure 7).
23
Figure 7. Structure of CR3 domain in HPV E7 Protein. The two red spheres at opposite
ends of the dimer complex represent the area where the inhibitor must bind to effectively
disrupt E7 binding with pRb and E2F-1 (Liu et al., 2006).
24
Each subunit of the dimer contains a two-stranded antiparallel ß-sheet formed by
residues 44-52 (ß1) and 58-65 (ß2), followed by a sharp U-turn leading to helix α 1
(residues 67-79) that sits on one side of the sheet with its axis roughly parallel to the
sheet. From the α1 helix, a 90 degree bend leads to ß3 (residues 81-83) followed by an
extended strand that cuts across the ß1-ß2 sheet and leads to a final short α 2 helix
(residues 89-91). The α 2 helix sits on the opposite side of the ß1-ß2 sheet relative to the
α 1 helix. A structural zinc ion is coordinated by four cysteine residues, two from the
hairpin loop between the ß1-ß2 strands, one from the loop connecting the α 1 and α 2
helices, and one from the α 2 helix. The observed dimer is a result of two subunit ß1-ß2α 1 faces and ß-sheet interactions between ß2-ß3 strands of the opposing subunits.
The zinc-bound E7-CR3 homodimer contains two surface patches that are highly
conserved. Mutation experiments focusing on these conserved regions reveal that one
region is required for pRb binding, whereas the other is required for E2F-1 binding. E7
temporarily binds a portion of E2F-1 releasing pRb from E2F-1 and then E7 strongly
binds with pRb leading the cell toward cancer formation. Both interactions are a direct
result of the zinc-bound E7-CR3 homodimer. The first region is specific for pRB binding
and consists of the outer edges of the two α 1 helices of the dimer residues consisting of
the following residues: Ile-70, Arg-71, Glu-74, Glu-75, and Lue-78. This region has been
shown to have a high degree of conservation among E7 proteins from different HPV
genotypes. The second region is specific for the E2F-1 region and consists of ß1 strands
within the pronounced groove at the dimer interface. The amino acid residues Arg-60 and
Leu-61 in this region are also highly conserved across the different strains of HPV.
25
Electrostatic surface mapping of the HPV E7 CR3 domain reveals that the regions
around the α 1 helix (patch 1) and ß1 strand (patch 2) harbor the most electronegative and
electropositive surfaces of the protein, respectively. The electrostatic properties and
structure of the region make this region an ideal target for inhibition and this information
will be the foundation for design of a de novo small molecule inhibitor.
AMBER Program-Design of de novo Small Molecule Inhibitors
AMBER (Assisted Model Building with Energy Refinement) is a software
package with the capability to utilize information about proteins and binding regions to
design molecules with high affinity for these pockets. The AMBER tool was effectively
used by Shaomeng Wang, Ph.D at the University of Michigan Comprehensive Cancer
Center to develop a small molecule inhibitor to block p53 and MDM2 binding (Shangary
& Wang, 2008). The software package will utilize the information about the CR3 region
to generate several small molecules that are projected to have strong affinity to bind that
region.
Based on the molecule designed by the software, properties indentified as
required for inhibition will be used to search structural databases for possible matches.
The search would utilize the National Cancer Institute (NCI) Drug Information System
3D database of 450,000 primarily organic compounds which have been tested by NCI for
anti-cancer activity.
26
Synthesis of Small Molecule Inhibitor Candidates
A commercial organic synthesis company will be contracted to make the chemical
structures for five de novo small inhibitor candidates generated from the AMBER
program since these molecules are unlikely to be readily available. There are a number
of reputable companies employing experienced organic chemists that specialize in
custom synthesis of molecules (e.g. The Chemistry Research Solution LLC,
www.tcrs.us).
II. Competitive Fluorescence Binding Assays
Utilizing the molecules generated by commercial vendors, the next phase of
experimentation must determine the effectiveness and viability of these small molecules
to inhibit E7 and pRb binding. A fluorescence based competitive binding assay will be
used to determine relative binding affinities of the molecular interactions. Fluorescence
binding assays have been widely used to determine and quantify interactions between
proteins and ligands (Nasir & Jolley, 1999).
A competitive binding assay will be used
to determine whether the candidate molecules have a level of affinity to E7 that will
outcompete pRb for the E7 binding pocket and thus have the ability to hinder the
formation of E7-pRb complexes. As with most fluorescence binding assays, it is both
sensitive and specific. In addition, it allows for high-throughput since it is performed
using a 96 well plate format.
In order to perform the assay, the E7 protein will be fluorescently labeled
whereas pRb will be biotinylated(see Figure 8). Biotin-pRb will then subsequently be
27
immobilized to wells using streptavidin-conjugated plates. In the binding assay,
fluorescently labeled E7 and serially diluted small molecule inhibitors will be mixed in
wells containing immobilized biotin-pRb. After incubating to allow binding, each well is
washed leaving fluorescently labeled E7 bound to biotin-pRb if not inhibited (small
molecule inhibitor and inhibitor-bound E7 are removed) and then the fluorescence is
measured using a fluorescence plate reader. For each dilution of the small molecule
inhibitor used in the plate assay, the fluorescence will be measured. The fluorescence
value is plotted versus small molecule inhibitor concentration. If the small molecule is
able to outcompete pRb for the fluorescently tagged E7, the fluorescence measured in the
assay should reduce as concentrations of the small molecule inhibitor increase. The Kd
(equilibrium constant) can be calculated using the measurement of fluorescence versus
the small molecule inhibitor dilution. The Kd value will be important in determining the
doses used for in vitro testing and the potential efficacy of the small molecule inhibitors.
This experiment will be performed in triplicate to account for statistical differences. The
average and standard deviation will be calculated, and the averages will be used to
calculate the Kd for the small molecule inhibitor.
Thermo Scientific EZ-Link Sulfo-NHS-Biotin is a system used to conjugate biotin
to proteins and will be used to label pRb. This system uses a short chain, water-soluble
biotinylation reagent to label the protein with biotin. A variety of biotinylation reagents
are available that target different functional groups like primary amines, sulfhydryls,
carboxyls, carbohydrates, tyrosine and histidine side chains. The use of a biotinylation
reagent that attaches biotin to primary amines will be appropriate for labeling pRb. The
28
next step will be to bind biotin-pRb to the well plate. Biotin binds to streptavidin with
great affinity and can be used to fix biotinlyated molecules to surfaces. Thermo
Scientific Immobilized Streptavidin plates have streptavidin coated on the well surfaces
which can be used to affix the biotin-pRB to the plate.
29
Figure 8. Illustration of fluorescence binding assay. A) If fluorescently labeled E7 binds
with immobilized E7, high fluorescence readings will be detected. B) If fluorescently
labeled E7 binds with the small molecule inhibitor, then when the well is washed,
reduced or no fluorescence is left on the plate.
30
III. Cell Permeability Assay
The process to determine cell permeability and viability of a potential drug
candidate requires a series of tests that determine the compound's ability to permeate
through the cell membrane so as to assess the inhibitor's potential to be found in the
correct location for binding activity. Passive permeability is the common mode of
delivery of small molecules into cells (e.g. naprozen, metoprolol, etc) (Edward & Kerns,
2008).
PAMPA-Parallel Artificial Membrane Permeability Assay
Parallel Artificial Membrane Permeability Assay utilizes a lipid filled membrane
to simulate the lipid bilayer of various cell types. PAMPA is a cost effective and highthroughput cell permeability assay, which allows for a quick determination of a
compound's ability to passively diffuse across the cell membrane. The assay utilizes a 96
well plate (donor well plate) that is filled with a solution of the test compound (i.e. small
molecule inhibitors) diluted in an aqueous buffer which is overlaid with a simulated lipid
bilayer followed by another 96 well filter plate (which has a porous filter on the bottom
of the plate) which is filled with a buffer solution (the acceptor well plate). The
sandwich of the two 96 well plates separated by the simulated bilayer is incubated at
constant temperature of 37 degrees Celsius for between 1 to 18 hours. After this period,
the plates are separated, the lipid bilayer removed, and samples taken from both the donor
and acceptor wells. The concentration of compound in the wells are to be quantified by
UV if the small molecules can be detected in this manner (e.g. plate reader) or
31
alternatively by Liquid Chromatography/Mass Spectrometry. A known concentration of
the de novo small molecule candidate will be used as a standard to calibrate quantitation.
In this way, the effective cell permeability of the test compound can be predicted since
the bilayers used are similar to the constitution of eukaryotic cell plasma membranes. The
cell permeability assay will be done in triplicate so as to assess the reproducibility. The
average and standard deviation will be calculated, and the averages used to determine the
ability of small molecule inhibitor candidates to passively permeate cell membranes.
Possible Outcomes of Permeability Studies
Compounds that are identified to have passive permeability will be used as the
best possible candidates for further testing. If no small molecule inhibitors have the
ability to passively permeate the cell membrane as determined by the PAMPA procedure,
drug delivery systems will be investigated as a method of delivering the small molecule
inhibitor into the cell. Examples of delivery systems include liposome vesicles or
polymer-based nanoparticles (Whittlesey & Shea, 2004). Liposome drug delivery
systems use a liposome to encapsulate a drug. The liposome will then fuse with the cell
membrane, releasing the drug into the cell. Liposomes make an ideal drug delivery
system because they can deliver nearly any hydrophilic drug and can be designed to
target specific tissues. Alternatively, polymer based nanoparticles could also encapsulate
or bind a target molecule (i.e; small molecule inhibitor) that is then used to bind and fuse
with cell membranes. Both liposomes and nanoparticle drug delivery systems have been
extensively used for the delivery of molecules.
32
IV. Effects of Small Molecule Inhibitor Candidates on HPV Cell Cultures
The de novo designed molecules with the highest affinity to the E7 binding
pocket and ability to permeate cells will be used for the in vitro testing phase. The de
novo candidates will be incubated with the HPV cancer cell lines during cell culture to
determine their ability to halt uncontrolled cell growth which is assumed to be due to the
inhibition of the formation of the E7-pRb binding complex. Utilizing HPV cancer cell
lines in conjunction with the Promega CellTiter Cell Proliferation Assay, it will be
possible to determine the in vitro effect of the small molecule inhibitor candidates and
whether they can inhibit cell growth.
Promega CellTiter 96 Non-Radioactive Cell Proliferation Assay
Cell proliferation assays are widely used in cell biology to study growth factors,
cytokines, nutrients and cytotoxic agents. The CellTiter 96 Non-Radioactive Cell
Proliferation Assay provides a rapid and convenient method to determine viable cell
number in proliferation, cytoxicity, cell attachment, chemotaxis and apoptosis assays.
The assay is performed by adding a premixed optimized Dye Solution to culture wells of
a 96-well plate (HPV cell culture) containing a test substance (e.g. small molecule
inhibitors). During a 4-hour incubation, living cells convert the tetrazolium component
of the Dye Solution into a formazan product. The Solubilization Solution/Stop Mix is
then added to the culture wells to solubilize the formazan product, and the absorbance at
570 nm is recorded using a 96-well plate reader. The 570nm absorbance reading is
33
directly proportional to the number of cells in the well. Wells containing a series of
dilutions of inhibitor candidates will be evaluated for their ability to inhibit cell growth
and determine the most effective molecule.
Such evidence would indicate that the small
molecule inhibitor has bound E7 and returned pRb to normal cellular activity. The cell
proliferation assay will be conducted in triplicate to generate an average and standard
deviation. The averages will be used to determine whether the small molecule inhibitors
have the ability to return the cell to normal cellular activity levels.
V. Evaluation of the Effects of the Small Molecule Inhibitors on HPV Cells
Double-labeling Native Gel Immunoblot Assay
A double labeling immunoblot experiment will be utilized on cells treated with
the small molecule inhibitor to determine whether E7-pRb complexes are being formed
or the binding complex is blocked by the small molecule inhibitor. The inhibitors that
were found effective to inhibit HPV cell growth in the previous cell proliferation assays
will be assessed as to whether they indeed bind to E7 within the cell lysate of the HPV
cells as determined by immunoblotting.
Antibodies for E7 and pRb will be used in combination with horseradish
peroxidase-linked secondary antibodies to determine whether E7-pRb complexes are
formed. When these antibodies bind to the appropriate protein, they can be detected
using enhanced chemiluminescence since the peroxidase enzyme causes the
chemiluminescence substrate to produce detectable photons of light.
34
Native polyacryarylamide gels will be used for this western blotting technique.
Native gel electrophoresis ensures that protein complexes and proteins are in their native
conformation since it does not use denaturing or reducing agents. Thus, this technique
will allow the detection of pRb and E7 subunits (unbound) and the E7-pRb complex by
observing their mobility in the gel after immunoreaction. Cells will be lysed using nonionic detergents and passage through a syringe (sheer force) to ensure that binding
complexes are not disrupted. A low percentage (4%) acrylamide gel will be used since
this will allow the resolution of larger protein complexes on the gel. Also, a high
percentage (15%) acrylamide gel will be used since this will allow the resolution of
smaller protein complexes on the gel (e.g. E7 protein). The respective molecular
weights of E7 and pRb are 17 kDa and 300 kDa, so we would expect a upward shift in
their electrphoretic mobility when the two proteins bind to each other.
After electrophoresing the protein extracts, the native gel will be western blotted
onto a membrane (e.g. nitrocellulose); subsequently probed with an antibody for E7; and
incubated with the appropriate secondary antibody conjugated to horseradish peroxidase.
Immunoreactive bands will be detected using chemiluminescence and images captured
using either a gel documentation system with CCD camera or using standard X-ray film.
After this first round of labeling is performed, a second round of antibody detection using
the pRb antibody will be performed after the blot is stripped of the previous primary and
secondary antibodies using a standard membrane stripping protocol. If E7 and pRb form
an E7-pRb protein complex, the western blot will have a single band representing this
complex (i.e. 317 kDa).
On the other hand, if E7 and pRb fail to form a complex then
35
two distinct bands will be observable one on the low percentage (4%) acrylamide gel
representing unbound pRb (300 kDa) and a band on the high percentage (15%)
acrylamide gel representing E7 (17 kDa). It is possible that there is a combination of
these bands due to their ratios in the cell. Failure to form an E7-pRb complex will be
indicative of the effectiveness of the small molecule inhibitor to disrupt the ability of E7
to bind with pRb.
Cell Lysates for Detection of Apoptosis
The small molecule inhibitors designed in this study focus on halting the
carcinogenic pathway in a method not directly related to apoptosis. But the CR3 zinc
domain region on HPV targeted by the small molecule inhibitor plays a role in
inactivation of the cyclin-dependent kinase inhibitors p27 and p21 and several
transcription factors that apparently contribute to HPV-mediated oncogenesis. Thus, the
inhibition of E7 binding to pRb by the small molecule inhibitor should result in allowing
apoptosis to occur within treated cancer cells, which will be evaluated using a TUNEL
assay. Furthermore, these inhibitors may have toxic effects on cells. A TUNEL assay
will be used to detect the level of apoptosis. As for cytotoxicity, a Live/Dead Viability
Assay will determine the relative ratio of live to dead cells. If the small molecule
inhibitor is functioning as predicted, returning cell function to normal, then the cells
should be healthy overall but will have the ability to undergo apoptosis.
36
TUNEL Assay
The Terminal Transferase dUTP Nick End Labeling (TUNEL) Assay is a method
used to detect DNA degradation in apoptotic cells and determine levels of apoptosis in
cells. TUNEL is the optimal method for determining apoptosis within cells because it is
highly reliable and works on 95% of cells and is widely used by researchers. The assay
utilizes detection of the fragmentation of nuclear chromatin due to apoptosis which
results in a multitude of 3’-hydroxyl termini of DNA ends. The presence of DNA
fragmentation can be used to identify apoptotic cells by labeling the DNA breaks with
fluorescently-labeled deoxyuridine triphosphate nucleotides (F-dUTP). The enzyme
terminal deoxynucleotidyl transferase (TdT) catalyzes a template-independent addition of
nucleotides (F-dUTP) to the 3’ hydroxyl ends of exposed double- or single-stranded
DNA ends. Non-apoptotic cells do not incorporate much of the F-dUTP because of
absence of exposed 3’-hydroxyl DNA ends. The assay will be used to quantify the
relative amount of apoptosis in populations of cells incubated with the small molecule
inhibitor and a control sample with no treatment of the small molecule inhibitor using a
fluorescence plate reader. The TUNEL assay will be conducted in triplicate and
averaged to determine the relative amount of apoptosis.
37
Live/Dead Viability & Cytotoxicity Assay
The Live/Dead Viability Cytotoxicity 96 Well Assay is produced by Invitrogen
Detection Technologies. This assay is based on the use of a two-color fluorescence cell
viability assay that is based on the simultaneous determination of live and dead cells with
two probes that measure recognized parameters of cell viability-intracellular esterase
activity and plasma membrane integrity. Live cells are distinguished by the presence of
ubiquitous intracellular esterase activity. The assay quantifies the level of esterase by
utilizing the esterase enzymatic conversion of the non-fluorescent calcein AM into the
intensely fluorescent calcein. The calcein dye is well retained in the live cells and
produces an intense uniform green fluorescence in live cells when excited. The second
probe utilizes EthD-1 which enters cells with damaged membranes (indicative of cell
death) and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic
acids producing a red fluorescent signal when excited. The EthD-1 is excluded from live
cells with intact membranes. Using a fluorescence plate reader, it will be possible to
quantify the relative ratios of live to dead cells. This experiment will be conducted in
triplicate to generate an average. The assay will be used to quantify relative live/dead
cell ratios in samples of cells mixed with the small molecule inhibitor and a control
sample with no small molecule inhibitor.
38
BUDGET
I. Design and Synthesis of Small Molecule Inhibitors
Design of Small Molecule Inhibitor
Open source program- no cost associated with design and database searches
Software: AMBER(Assisted Model Building with Energy Refinement)
Software-http://ambermd.org/
Custom Organic Synthesis
There is no fixed cost associated with custom organic synthesis and it can vary
greatly. We will be attempting to utilize existing organic compound groups as targets
and construct a molecule using intermediaries, which will greatly reduce organic
synthesis cost.
Company providing service- The Chemistry Research Solution LLC, www.tcrs.us
Cost Estimate(using building block method) $10,000 per molecule for 10 mg
$50,000.00
39
II. Competitive Fluorescence Binding Assay
Fluorescence Assay
Thermo Scientific(www.piercenet.com)
DyLight 350 NHS Ester Dyes(Amine Reactive) kit(1mg each)
$219.00
$219.00
Protein X(www.proteinx.com)
HPV E7 (type 16) purified protein(50 micro grams) x 10 vials
$380.00
$3,800.00
$341.00
$341.00
$165.00
$165.00
$206.00
$7004.00
Proteinone(www.proteinone.com)
pRb (Retinoblastoma protein) purified protein (5 mg)
Thermo Scientific(www.piercenet.com)
EZ-Link Sulfo-NHS-LC-LC-Biotin (50 mg)
Thermo Scientific(www.piercenet.com)
Streptavidin Coated Plates (5 plates) x 34
*Fluorescent plate reader equipment is readily available.
III. Effects of Small Molecule Inhibitor Candidates on HPV cell cultures
Cell Permeability-PAMPA Assay Depot(Parallel Artificial Membrane Permeability)
Service Provided by Assaydepot.com (10 assays)
$130.00
$1300.00
40
IV. Effects of Small Molecule Inhibitor Candidates on HPV cell cultures
Promega CellTiter 96 Non-Radioactive Cell Proliferation Assay
Cell Lines Service(www.cell-lines-service.de)
HeLa Cells(Human Cervical adenocarcinoma cell line-HPV)
$250.00
Promega(www.promega.com)
CellTiter 96 Non-Radioactive Cell Proliferation Assay x 2
$254.00
$508.00
V. Evaluation of the effects of Small Molecule Inhibitor Candidates on HPV cell
Double-labeling Native Gel Immunoblot Assay
Santa Cruz Biotechnology, Inc(www.scbt.com)
Human pRb goat polyclonal antibody (200 micrograms/ml) x 5 vials
$259.00
$1295.00
abcam(www.abcam.com)
HPV 16 E7 goat polyclonal antibody (200 micrograms/ml)
x 5 vials
$259.00
$1295.00
$299.00
$598.00
$449.08
$449.08
Invitrogen(www.invitrogen.com)
WesternDot Western Blotting kit (1 kit) x 2
TUNEL (Terminal Transferase dUTP Nick End Labeling) Assay
Invitrogen(www.invitrogen.com)
APO-BrdU TUNEL Assay Kit (60 assays)
41
Live/Dead Viability & Cytotoxicity Assay
Invitrogen
Live/Dead Viability/Cytotoxicity Kit for mammalian cells(1 kit) x 2
$349.00
$698.00
General Supplies(e.g. gloves, plates, agarose, culture media, culture flasks etc)
$25000.00
Total Budget----------------------------------------------------------------------------$91178.08
42
CONCLUSION
The development of a small molecule inhibitor to block the binding of pRb and
HPV protein E7 is a key step in the development of a successful non-invasive therapy for
HPV induced cancer. The use of the AMBER software suite and performing the series of
steps above to evaluate their effectiveness to prevent the E7-pRb complex and inhibiting
cell proliferation will be a critical step towards developing a new drug to treat HPVinduced cancer. Successful candidates from the above experimentation will provide a
foundation for further testing of their efficacy and safety so that human clinical trials can
be performed and eventually become the therapy of choice for HPV induced cancer.
43
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