Running Head: PCSK9 SMALL MOLECULE INHIBITION

Using Virtual Screening Methods to Identify a Novel and Noninvasive Method of Heart Disease
Treatment and Prevention
Rohan Arora
American High School

Contact:
ra.rohan00@gmail.com
(510)-894-5410

PCSK9 SMALL MOLECULE INHIBITION

Abstract
Heart Disease is the top killer in the United States, causing 600,000 deaths annually. This project
sought to reduce the risk of heart disease by inhibiting a key interaction between LDLR, the
receptor that lowers cholesterol levels, and PCSK9, the molecule that inhibits this receptor. In
order to be a potential drug candidate, the inhibitor had to be a small molecule that could be
easily administered. Inhibition criteria was established based on control interactions at key
residues and Autodock Vina was used to complete a series of virtual screenings beginning with a
diverse library of 1,700 small molecules in order to identify which ligand best met inhibition
criteria. Two types of inhibition were considered: allosteric and competitive. The identified
inhibitor was a competitive molecule with the ZINC identification of ZINC00990239. It met
small molecule requirements and inhibition requirements by binding to key residues (CYS378,
ILE369, PHE379) on PCSK9 with an efficient affinity of -8.0 kcal/mol. If proven to be
successful in human trials, the identified small molecule has the potential to save thousands of
lives annually and prevent the most deadly disease in the nation.
Keywords: Heart Disease, PCSK9, Inhibition, ZINC00990239

PCSK9 SMALL MOLECULE INHIBITION

Identifying Novel Small Molecule Inhibitors of the PCSK9 Enzyme Through Structure-B ased
Virtual Screening.
Heart Disease, or the restriction of arteries due to plaque buildup, is the most deadly
killer in the United States of America, leading to 600,000 deaths every year . Studies have shown
that one of the greatest contributors to heart disease is a strong prevalence of low density
lipoprotein, or LDL, in the blood stream. Low density lipoproteins carry cholesterol through the
bloodstream and are monitored by the LDL Receptor, a plasma membrane glycoprotein that is
located on the surface of multiple cells. LDLR works by transporting cholesterol-rich LDL into
the cells; where it is broken down and used by the cell, or eliminated as waste. The LDLR plays a
critical role in the regulation of cholesterol in the blood stream, eliminating what would otherwise
cause plaque buildup on artery walls and thereby restrict blood flow.
Proprotein convertase subtilisin kexin 9, or PCSK9, is a proprotein that plays a key role
in the regulation of LDLR. For example, gain-of-function mutations in the PCSK9 gene reduce
LDLR levels in the liver, thereby resulting in large levels of LDL in the blood stream. Likewise,
loss-of-function mutations in the gene increase LDLR levels, thereby decreasing LDL levels.
However, the absence of this gene altogether appears to have no adverse consequences,
indicating that the only purpose of PCSK9 is to lower LDLR levels. PCSK9 lowers LDLR levels
to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. The
EGF-A domain of LDLR binds to a patch of residues in the catalytic domain of the PCSK9
molecule, which consists of residues 153-454. This region of the LDLR is extremely crucial for
the the recycling of the LDLR to cell surface. Although binding occurs on the cell surface,
destruction of LDLR only begins when the LDLR-PCSK9 complex is internalized. PCSK9 then

PCSK9 SMALL MOLECULE INHIBITION

initiates the destruction of LDLR by diverting it to the lysosomes so that it cannot be recycled to
the surface.
Literature
There are many studies looking at the interactions between PCSK9 and the LDLR. Most of
them serve to verify and expand on the observations presented in the previous section. This
literature search focused onthe benefits of potential PCSK9 inhibitors. One method, gene
silencing, will not be reported on. This method does not directly inhibit PCSK9, but rather blocks
and unblocks genes. Because it is not a method to inhibit the protein, we will not focus on it within
this study.
Multiple studies deal with the inhibition of PCSK9 by the use of monoclonal antibodies.
This method is possible because PCSK9 operates outside cells. Monoclonal antibodies that mimic
the effects of genetic mutations by inhibiting PCSK9, have been developed and are in clinical
trials. Amegen Inc developed an anti-PCSK9 monoclonal antibody that blocked PCSK9 and
LDLR interaction. In Phase I studies, Amgens antibody (AMG145, now named Evolocumab)
lowered LDL-cholesterol by up to 64% in healthy subjects and by up to 81% in hypercholesterolemic statin-treated subjects with or without heterozygous FH. Evolocumab also significantly
reduced ApoB (~55%) and lipoprotein(a). Multiple Phase II trials demonstrated that subcutaneous
injections of evolocumab could reduce LDL-cholesterol by >50% either alone or in addition to
other LDL-cholesterol- lowering therapies. Similar monoclonal antibodies have been developed by
the following companies: Sanofi/ Regeneron, Pfizer/Rinat, Novartis/ KaloBios, and Roche/
Genentech; all of which are going through clinical trials. (4,5)

PCSK9 SMALL MOLECULE INHIBITION

A separate approach to the inhibition of PCSK9 uses peptides A study published by NCBI
reports on the identification of a 13-amino acid linear peptide,Pep 2-8, which bonds with an
affinity of 0.7 um, is the smallest PCSK9 inhibitor discovered at the the time this paper was
published- November 2013. Upon further analysis, and to our best knowledge, it has been
confirmed that these findings still stand in the current medical world. The abstract details that
Pep2-8 bound to PCSK9, but did not bind to other proprotein convertases. Further, Pep2-8fully
restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. A
competitive inhibition mechanism was suggested by the fact the Pep 2-8 contact region largely
overlapped that of the EGF-A domain of the LDLR. To our best knowledge, this is the only
publicly viewable study performed on the inhibition of PCSK9 by peptides. (3)
A third approach on the inhibition of PCSK9 inhibitors is small molecule based. Smallmolecule drugs are most effective because they can be easily administered, allowing for simple,
noninvasive treatment. However, no small molecule has yet been identified that properly inhibits
PCSK9 binding.(1,2)
Although research has been conducted in the field of PCSK9 inhibitors, it is by far not
enough. Despite their effectiveness, monoclonal antibodies are extremely invasive because they
must be administered via injection. Further, the antibodies have potential side effects ranging
from nausea to internal bleeding. Peptide-based inhibitors are promising drug candidates,
however there exists only one publicly available study on these compounds. Furthermore, the
smallest identified peptide inhibitor still does not meet the qualifications of a small molecule
(<500 Daltons), which are noninvasive and easy to administer.

PCSK9 SMALL MOLECULE INHIBITION

Definitions
First, the following terms will be used extensively terms are defined for our purposes and
may have different definition in other areas of science) :
Virtual Screening: this is the term for general process that involves screening of a large,
virtual library of compounds, in order to identify the one with the largest affinity for a
certain compound. The specific type of virtual screening used was structure-based,
meaning that each individual compound was screened. Each compound is virtually
"docked" with the receptor, and a scoring function is applied to measure the affinity.
Receptor: In receptor-ligand docking, receptor is the term for the compound that all the
other compounds are docked against. For example, in our case, PCSK9 would be the
receptor.
Ligand: each compound docked against the receptor is known as a ligand.
Small Molecule: In molecular biology and pharmacology, a small molecule is a low
molecular weight (<900 daltons[1]) organic compound that may help regulate a biological
process, with a size on the order of 109 m. Most drugs are small molecules. The upper
molecular weight limit for a small molecule is approximately 900 daltons, which allows
for the possibility to rapidly diffuse across cell membranes so that they can reach
intracellular sites of action.[1][2] In addition, this molecular weight cutoff is a necessary but
insufficient condition for oral bioavailability. Finally, a lower molecular weight cutoff of
500 daltons (as part of the "rule of five") has been recommended for small molecule drug
development candidates based on the observation that clinical attrition rates are
significantly reduced if the molecular weight is kept below this 500 dalton limit.

PCSK9 SMALL MOLECULE INHIBITION

Limitations
Auto-dock Vina, the program used to perform the screening, has an binding energy error
margin of +/- 2.5 kcal/mol.
Objective
The presence of LDLR is important to decrease cholesterol levels and thereby lower the
risk of heart disease. By destroying LDLR, PCSK9 increases the risk of atherosclerosis. Studies
have shown that this is the only purpose of PCSK9 is to do this, and that its removal will have no
adverse effects. Therefore, the inhibition of PCSK9 is a safe and effective way to reduce the risk
of heart disease in all groups. The purpose of the study is to use structure-based virtual screening
to discover novel compounds that accomplish this very purpose. These compounds should be
less than 500 Da, in order to qualify as small molecules. In addition, they should inhibit PCSK9
either competitively or non-competitively. They should have an affinity for PCSK9, but more
importantly have a structure that binds to PCSK9 in the correct locations. For example, a
competitive inhibitor would bind to residues which are important to LDLR - PCSK9 binding. If a
compound is found that meets the above qualifications, then the hypothesis -- that such a
compound exists -- is accepted. If there is no compound that meets these conditions, then the null
hypothesis is accepted. The above conditions were set based on interactions observed in the
control, which is defined as the docking of LDLR to PCSK9. The study used as the control for
this experiment can be found here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2538846/
Materials and Methods.
To begin with about 2,000 ligands from NCI Diversity set were docked with PCSK9
using Auto-dock Vina. Auto-dock Vina is a docking program available on Windows and Linux
operating systems that is used to measure the binding energy between two compounds inputed in

PCSK9 SMALL MOLECULE INHIBITION

PDBQT format. The NCI Diversity Set was used because of its well-rounded collection of
various small molecule ligands. Each of the ligand files in the set qualifies as a small molecule
by the definition. Also, the 2,000 ligand set represents about 70,000 molecules allowing for a
quick screening of a large variety of molecules.
Auto-dock Vina
Auto-dock Vina was the program used to perform docking simulations. It reported the affinity of
each molecule and 6 binding configurations. More information can be found at:
http://autodock.scripps.edu/faqs-help/tutorial/using-autodock-4-with-autodocktools/
2012_ADTtut.pdf
Identifying Potential Active Sites
An analysis of potential binding sites was found in the following paper and then verified
further using Autodock Tools to identify residues important to the binding of LDLR and the
PCSK9 macromolecule.
Kwon, H., Lagace, T., McNutt, M., Horton, J., & Deisenhofer, J. (2008, February 4).
Molecular basis for LDL receptor recognition by PCSK9. Retrieved December 5, 2015,
from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2538846/
In order to do this the LDLR protein was downloaded from the PDB database (file code 1N7D)
and then bound to PCSK9 using VINA. Autodock was used to analyze key interactions. Potential
active binding sites were found at residues in the catalytic domain.
Identifying Potential Allosteric Sites
Potential allosteric sites were found using SPACER, an online java-based program
provided by the Bioinformatics Institute. More information about this program can be provided

PCSK9 SMALL MOLECULE INHIBITION

at the following website: http://allostery.bii.a-star.edu.sg. The identified potential binding sites

were at residues Ser147 and Cys67 (Source: Student)

Location of SER147 and CYS67 residues

Ligand and Receptor Preparation

All ligand files were downloaded in PDBQT format from the ZINC database, however
PCSK9 was downloaded from the RCSB Protein Data Bank (file code 2P4E) in PDB format and
converted to PDBQT using Auto-dock Tools 4, which added partial charges. Then, using Autodock Tools graphical interface, a search zone, or the area Auto-dock Vina would look for ligand
binding sites, was identified. The search zone consisted of all the x,y, and z plane center
coordinates, as well as the size of each plane. Two different preliminary runs were done. In the
first, the search zone encompassed only the domain of PCSK9 where LDLR bound to, in order to
search for competitive inhibitors that blocked the LDLR from binding. In the second, the it
consisted of only the area surrounding the allosteric residues, in order to identify an allosteric
inhibitor.

PCSK9 SMALL MOLECULE INHIBITION

10

Competitive Binding
The purpose of the first preliminary run was to identify competitive inhibitors of PCSK9
by searching only the catalytic domain for potential ligand binding sites. After the screening was
performed, the molecules were ordered from highest binding energy to lowest binding energy.
Autodock Vina reports 6 different binding modes or orientations, for each molecule. Starting
from the molecule with highest binding energy, each domain one of the top ten molecules was
tested to see whether or not it met the structure requirements. This was done using Auto-dock
Tools, which reported the specific residues each ligand interacted with. When a suitable
configuration was found, a BLAST search was performed to find compounds at least
90% similar in structure. 62 molecules were identified. Another screening was performed using
these compounds to determine the one with the highest affinity that still met the structure
requirements. It was then tested with other compound in the proprotein convertase family to
make sure that it had a higher binding energy for PCSK9 than any other compounds.
Non-Competitive Binding
The second preliminary screenings were unchanged from the first one, except that the
2,000 compounds were tested with each potential allosteric site. The same procedures as before
were then followed for the top molecules at each site.
Experiment Specifications
For both all of the above docking scenarios, only one run was done because after a
preliminary investigation, it was found that the program reported the same values each time.The
potential small molecule inhibitor was then tested with other members of the proprotein
convertase family to ensure it only interacted with PCSK9. The data for the control

PCSK9 SMALL MOLECULE INHIBITION

11

was derived from previously accepted data and experiments.

Results
Competitive Binding
The highest affinity value in the competitive binding group was -8.0 kcal/mol, achieved
by a compound classified by the ZINC database as ZINC00990239. Following is a table with
each of the top 5 compounds average affinity values, whether or not a mode of the molecule met
structure requirements, the image of the molecule, and its SMILES identification. (Data Source:
Experimental Research; SMILES and Structure Source: ZINC Database)
Affinity Value
Average (kcal/
mol)

Structure
Requriments

SMILES ID

ZINC00990239

-8.0 Yes, CYS378,

ILE369, PHE379

Cc1cc(nc2c1c(nc3c2
cccc3)C)c4c([nH
+]c5ccccc5c4N)C

ZINC01690699

-7.7 Yes, ASP238,

ILE369, PHE379

c1ccc2c(c1)
[nH]c(n2)c3cccc(c3)
NC(=O)c4ccc(cc4)C
(=O)Nc5cccc(c5)c6[
nH]c7ccccc7n6

ZINC04824645

-7.7 Yes, ASP238,

ILE369, PHE379,
ALA239

c1cc2cccc3c2c(c1)N
C4(N3)CCC5(CC4)
Nc6cccc7c6c(ccc7)
N5

ZINC04773602

-7.6 Yes, ASP238,

ILE369, PHE379,
ALA239

CC1=CC(Nc2c1cc(c
c2)Cc3ccc4c(c3)C(=
CC(N4)(C)C)C)
(C)C

ZINC04214344

-7.6 Yes, ILE369,

PHE379

c1ccc2c(c1)
[C@]34CC[N
+]5([C@H]3C[C@
@H]6[C@@H]7[C
@@H]4N2C(=O)C[
C@@H]7OCC=C6C
5)[O-]

Image

PCSK9 SMALL MOLECULE INHIBITION

12

The molecule with the ZINC ID of ZINC00990239 was molecule with the lowest affinity value
and, by bonding to residues CYS378, ILE369, and PHE379, meets structure requirements. The
following images show ZINC00990239 bonding to
the PCSK9 molecule. (Source: Student)

ZINC00990239 bonding to the

pocket of the PCSK9 molecule

Specific residues (sites) of bonding between

ZINC00990239 and PCSK9

After this compound was identified as the most inhibiting compound, a >/= 90% similarity
search revealed 62 similar compounds. None of the compounds inhibited PCSK9 with a higher
affinity than this compound, and therefore they were disregarded.
The third remaining test was to determine whether or not this compound was able to bond to any
other ligands and therefore have the possibility of disrupting some other bodily process. When the
compound was tested with other members of the proprotein convertase family, the results did not
indicate that it held a high affinity for compounds other than PCSK9.
Allosteric Binding
The molecule with the lowest affinity to either allosteric residue of PCSK9 was
ZINC01736227, with an affinity of -4.7 kcal/mol. This affinity is much lower than that of all the
top 5 competitive inhibitors. Therefore, these allosteric residues were evaluated to be a deadends" in our experiment as all molecules had rather high affinities towards them, and it would be
pointless to pursue these specific binding sites as we had already detected a molecule with a
higher affinity.

PCSK9 SMALL MOLECULE INHIBITION

13

Discussion and Conclusions

The data indicates the presence of a small molecule inhibitor that has the ability to inhibit
PCSK9, and thereby successfully prevent heart disease. The identified inhibitor meets all
requirements established by the control. It bonds to residues CYS378, ILE369, and PHE379, all
of which are necessary active sites for LDLR-PCSK9 interactions. This makes it a competitive
inhibitor. Although ZINC00900239 inhibitor has a lower affinity for PCSK9 than LDLR, this
obstacle is easily overcome by increasing the molar concentration of the inhibitor; thereby,
decreasing required binding energy. The inhibitor meets all drug requirements is safe because it
does not interact strongly with any other similar compounds. It is a small molecule that can be
easily administered. Since the identified inhibitor meets all the criteria established by the control,
I accept my original hypothesis. However, there is one issue with the identified inhibitor, that it is
competitive. By blocking the active site, the inhibitor will cause the concentration of LDLR in
the body to increase. Eventually, the concentration will be high enough to override the inhibitor
the binding energy required for LDLR will decrease as concentration increases. A proposed
solution for this malady is constant administration of this medicine, meaning hourly, daily,
weekly, etc., so that is higher concentration of the medication is always present in the body. The
exact pattern of the medicine's administration should be established in a future experiment.
Improvements
The most important and immediate step that should be taken is verifying the results of this
experiment in a laboratory assay. This step is important because currently all results are virtual
and have not been proven to work under real-life conditions. Also, Vina has a large error value
of +/-2.0 kcal/mol, which can lead to a fair amount of uncertainty when considering the results.
Although much of this uncertainty can be alleviated using multiple trials, the only sure

PCSK9 SMALL MOLECULE INHIBITION

14

verification is the laboratory one. If such assays are successful, then the compound should be
developed into a drug which is tested on mice and eventually humans. Regarding this specific
experiment, the utilization of a computer with a higher processing power would allow more trials
to be run, which is important to confirm the integrity of the results.
Implications
The results of this experiment have strong implications in the medical world. The small molecule
inhibits PCSK9, which allows LDLR to do its job and remove "bad" cholesterol. If the use of the
inhibitor is shown to be effective human trials, the identified small molecule has the potential to
prevent heart disease, the number one cause for death in the USA, and save millions of lives.
Furthermore, since the molecule a small molecule, it can be easily administered in a non-invasive
manner, serving patients comfortably. This experiment has identified a molecule that can not only
save multiple lives, but do so in a safe subtle way.

VERIFICATION OF VIRTUAL SCREENING

Using Virtual Screening Methods to Identify a Novel and Noninvasive Method of Heart Disease
Treatment and Prevention:
Verification of Virtual Screening Results through an ELISA Assay
Rohan Arora
American High School

VERIFICATION OF VIRTUAL SCREENING

Abstract
The purpose of this step of the experiment was to verify that it is possible for a small molecule to
inhibit the interaction between LDLR and PCSK9 by conducting a de facto ELISA assay. The
molecule used was Strychnine-N-Oxide Trihydrate and it was diluted such that it matched the
binding energy of LDLR. The assay was performed through a standard protocol over the course
of 9 hours. HRP was conjugated with the strychnine molecule and TMB was added at the end for
detection. The results indicate the the molecule achieves its intended purpose 80% of the time,
leading us to believe that it is a strong inhibitor. We conclude that Strychnine-N-Oxide is an
appropriate molecule to use to stop the reaction between LDLR and PCSK9 and reduce blood
cholesterol. This has strong implications for residual and genetic CV patients and can save over
600,000 lives annually due to its effectivity as a cholesterol lowering mechanism.

VERIFICATION OF VIRTUAL SCREENING

Introduction
High throughput screening is used in biology and medical research as an efficient drug design
and discovery technique. Such screenings of a large library of chemicals require a large amount
of funding and resources. Since these were not available to us, we utilized virtual screening
methods to identify a potential drug and then use high throughput screening to conduct a de facto
inhibition test of the small molecule. This molecule was strychnine-n-oxide trihydrate. The exact
method used was a common procedure known as an ELISA assay. The purpose of the assay is to
prove that in the presence of LDLR it is still possible for the molecule to bind to PCSK9.
Materials and Methods
Procedures
The first step involved gaining access to the required chemicals. Since LDLR and ZINC were
competing to bond to PCSK9, all three chemicals were ordered. 10 ug of Human LDLR, and 10
ug of PCSK9 was ordered from Novoprotein. 50 mg of Strychnine-n-oxide was ordered from
Sigma-Aldrich. However, it may be more resourceful to request a sample of this chemical from
NCI (NCI does not deliver to high schools). We were not able to locate any other of the top
compounds to be used in the assay and therefore the strychnine was used. BSA (Bovine Serum
Albumin) was diluted in PBS (Phosphate Buffer Salt) to make a 3% BSA/PBS solution. The 10
ug of PCSK9 was diluted in 490ug of the BSA/PBS solution to make 500 ug of solution which
was then distributed equally through ten wells on the PVC plate. After the required incubation
and washing, the BSA blocking solution was added and the plate was incubated for two hours.
During this time the enzyme conjugated strychnine solution was prepared by using the attached
protocol to bond the HRP enzyme to the strychnine-n-oxide trihydrate. The basic buffer was
prepared using mono

VERIFICATION OF VIRTUAL SCREENING

basic and dibasic salts and the recommended concentrations were used. This was then diluted to
the required concentration of 0.0004g/L through a series of step dilutions. The LDLR solution
was then prepared by diluting 10ug of LDLR in 90ug of BSA/PBS to make 100ug of 10% LDLR
solution. After the two hour blocking incubation period was complete, 100 ug of each antibody
was distributed throughout each of the 10 wells with 10 ug in each. After 2 more hours, 100 ug
of TMB was added and results were taken. The incubations were done at room temperature, and
the molecules were stored based on methods stated in the MSDS. Standard laboratory procedures
were used and the strychnine was handled especially carefully. Concentrations of LDLR and
Strychnine were derived from their affinity values in order to keep the bonding energy equal for
the two molecules.(Picture by Student)

VERIFICATION OF VIRTUAL SCREENING

Calculations
In order to measure the accuracy of the binding of the PCSK9 to either molecule, the binding
energies had to be kept equivalent. Calculations were done to convert the given Kd value of
LDLR (101 nM) to a affinity value in kcal/mol. Then the molar affinity values were converted to
grams and then compared. Based on their ratio the ratio of the concentrations of each molecule
was determined and the molecules were diluted. The LDLR solution had to be 251 times more
concentrated than the strychnine solutions due to the much larger molecular weight of LDLR. For
all other solutions, recommended concentrations were used instead of performing a checkerboard
procedure (procedure to determine the ideal concentrations).

Results
The following image is a representation of the results of the ELSIA assay (Picture By Student):

VERIFICATION OF VIRTUAL SCREENING

The negative control showed no color change and continued to show clear solution. Trials
1-4,6-7, and 9-10, exhibited a visible color change and after the stop solution was added changed
yellow. Trials 5 and 8 remained clear. Therefore, in 8 out of the 10 wells HRP was detected after
TMB was added, meaning the Strychnine-N-Oxide Trihydrate was more prominent in binding to
PCSK9 in these wells.
Conclusions
As shown in the results, 80% of the time the strychnine showed a prominence over LDLR when
binding to PCSK9. This result leads us to conclude that the results shown in the virtual screening
have been verified as accurate because it is possible for the inhibition of LDLR PCSK9 binding
to occur when this molecule is present. The s molecule has been shown to work as a real life
candidate for lowering blood cholesterol. However, the assay should be conducted various times
to make sure that these results are consistent.

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Appendix A
Material Safety Information

Appendix B
Protocol Extension

Text

HRP Conjugation