pubs.acs.

org/jcim Article

Discovery of Novel Small Molecule Inhibitors Disrupting the PCSK9-

LDLR Interaction
Hengzhi Sun,§ Jinzheng Wang,§ Shengjie Liu,§ Xinyu Zhou, Liang Dai, Caiping Chen, Qinglong Xu,
Xiaoan Wen, Keguang Cheng, Hongbin Sun,* and Haoliang Yuan*
Cite This: J. Chem. Inf. Model. 2021, 61, 5269−5279 Read Online

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ABSTRACT: Proprotein convertase subtilisin kexin 9 (PCSK9)

has been identified as a reliable therapeutic target for hyper-
cholesterolemia and coronary artery heart diseases since the
monoclonal antibodies of PCSK9 have launched. Disrupting the
protein−protein interaction (PPI) between PCSK9 and the low-
density lipoprotein receptor (LDLR) has been considered as a
promising approach for developing PCSK9 inhibitors. However,
PPIs have been traditionally considered difficult to target by small
molecules since the PPI surface is usually large, flat, featureless, and
without a “pocket” or “groove” for ligand binding. The PCSK9-
LDLR PPI interface is such a typical case. In this study, a potential
binding pocket was generated on the PCSK9-LDLR PPI surface of
PCSK9 through induced-fit docking. On the basis of this induced binding pocket, virtual screening, molecular dynamics (MD)
simulation, and biological evaluations have been applied for the identification of novel small molecule inhibitors of PCSK9-LDLR
PPI. Among the selected compounds, compound 13 exhibited certain PCSK9-LDLR PPI inhibitory activity (IC50: 7.57 ± 1.40 μM).
The direct binding affinity between 13 and PCSK9 was determined with a KD value of 2.50 ± 0.73 μM. The LDLR uptake function
could be also restored to a certain extent by 13 in HepG2 cells. This well-characterized hit compound will facilitate the further
development of novel small molecule inhibitors of PCSK9-LDLR PPI.

■ INTRODUCTION
Low-density lipoprotein receptors (LDLRs) are the liver
surface receptors of LDL and responsible for eliminating
LDL cholesterol (LDL-c) from human blood.1 After binding to
a LDL particle, the LDLR is internalized into clathrin-coated
pits and subsequently convey LDL from cytoplasm to
lysosome for degradation. While in the endosomes, LDLR
separates with LDL and recycles back to the plasma member.2
Proprotein convertase subtilisin kexin 9 (PCSK9), the last
identified member of the proprotein convertase family, is
mainly expressed in hepatocytes and subsequently secreted
into blood.3 It is initially synthesized as a 692-aa zymogen, and
then, the signal peptide (residues 1−30) is excised from the
protein precursor. In the endoplasmic reticulum, proPCSK9
undergoes an ensuing autocatalytic cleavage of the peptide
bond between Gln152 and Ser153, resulting in an N-terminal
prosegment that still attaches to the PCSK9 catalytic site by
noncovalent interactions and blocks the subsequent serine
protease activity (Figure 1A).4−6 The mature PCSK9 consists
of three main parts: a pro-domain (residues 31−152), a
catalytic domain (residues 153−451), and a C-terminal
domain (residues 452−692).7 It has been well interpreted
that PCSK9 is an endogenous intracellular and extracellular
negative-regular chaperone of LDLR on liver cells. Extracellular
PCSK9 binds to LDLR by a protein−protein interaction, and
the PCSK9-LDLR complex directly routes to lysosome for
destruction, which impairs the LDLR recycle and the plasma
LDL-c elimination (Figure 1B).8−10 The concept of blocking
PCSK9-LDLR PPI as a novel cholesterol-lowering therapy has
been solidly proved by the monoclonal antibodies launched on
the market. Alirocumab11 and evolocumab,12 developed by
Sanofi and Amgen, respectively, both recognize and bind
directly to the PCSK9 surface that interacts with the LDLR-
EGF(A) domain. This competitive inhibition could cause a
robust reduction (>50%) of the plasma LDL-c level and a
modest decrease (∼15%) in major cardiovascular events in the
long term with no significant adverse events reported in clinical
trials.13−15 Despite the success of PCSK9 antibodies as
cholesterol-lowering therapeutics, high cost and injection-
only administration have limited their clinical accessibility.
Received: May 11, 2021
Published: September 23, 2021

© 2021 American Chemical Society https://doi.org/10.1021/acs.jcim.1c00521

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Figure 1. (A) Autocatalytic process drives PCSK9 precursor to mature. (B) PCSK9 plays an important role in the regulation of LDLR degradation.
ER, endoplasmic reticulum; TGN, trans-Golgi network.
Figure 2. (A) 3D structure model of PCSK9-LDLR complex (PDB ID: 3BPS). (B) Key amino acid residues on PCSK9 surface involved in PCSK9-
LDLR PPI.
Therefore, it is highly desirable to develop small molecule
inhibitors of PCSK9-LDLR PPI.
Protein−protein interactions (PPIs) play vital roles in
physical and pathological processes such as metabolism,16
immunity,17 inflammation,18 tumor proliferation, and meta-
stasis.19 Great efforts have been made in discovering drugs
targeting and modulating key PPIs.20,21 However, the
characteristics of PPIs are distinct from the traditional
druggable targets such as enzymes, ion channels, and
GPCRs.22 PPIs interfaces are generally large, flat, often
hydrophobic, and more flexible. Also, suitable pockets or
grooves are usually lacking for ligand binding on the PPIs
interfaces,23,24 resulting in difficulties for molecular design and
drug discovery.25,26 Nevertheless, some encouraging progress
has been made in developing PPIs inhibitors targeting PD1-
PDL1,27 IL2-IL2R,28 MDM2-p53,29−31 and Bcl2-BclXL.32,33
PCSK9-LDLR PPI has been well understood based on the
detailed analysis of crystal structures.34−36 The interface of
PCSK9 is a ∼500 Å2, solvent-accessible, flat surface on the
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catalytic domain, which was made up of a series of
hydrophobic amino acid residues centered on Phe379 and
surrounded by several polar residues (Figure 2).34 The direct
interactions between PCSK9 and LDLR include a β-sheet-like
contact unit formed by residues 377−379 of PCSK9 and 308−
310 of LDLR-EGF(A) domains, hydrogen bond interactions
contributed by Ser153, Arg194, and Asp238, and hydrophobic
interactions involved in Ile369, Cys378, and Phe379 of
PCSK9. Currently, only a handful cases of PCSK9-LDLR
PPI inhibitors have been reported, including small peptides,
peptidomimetics, and small molecules (Figure 3). Pep2-8,37 a
13-peptide discovered by phage-displayed peptide libraries
screening, showed potent PCSK9-LDLR PPI inhibitory
activity, PCSK9 binding affinity, and LDL uptake restoration.
CB_3638 was a small molecule exhibiting certain PCSK9-
LDLR PPI inhibitory activity in vitro, but the direct binding
affinity to PCSK9 was unknown. LDLL-1dnlr39 was a
peptidomimetic compound with micromolar PCSK9 binding
affinity and capable of disturbing the targeted PPI and
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Figure 3. Chemical structures of some reported PCSK9-LDLR PPI inhibitors.
increasing LDL uptake from extracellular medium. The
tetraimidazole derivative RIm1340 exhibited certain inhibition
on PCSK9-LDLR binding and a cholesterol-lowering effect on
HepG2 cells. Compound 3f,41 which was derived from its lead
compound nilotinib, showed PPI disturbing efficacy without
kinases inhibitory activity. It was hypothesized that 3f was
bound in the catalytic site of PCSK9 which was occupied by
the self-inhibitory peptide from the Pro-domain N-terminal42
and known to be inaccessible for small molecule antagonists.43
Therefore, the binding region of 3f has yet to be clarified
experimentally. Compounds SBC-115,076 44 and SBC-
110,42445 were reported as PCSK9 inhibitors, which were
discovered from PPI surface modeling and high-throughput
virtual screening. They exhibited lipid-lowering effects in cell
and animal model tests. However, their direct binding affinity
to PCSK9 was not reported. Together, there is still a lack of
reliable small molecule inhibitors of PCSK9-LDLR PPI.
Virtual screening is a reliable tool in the process of hit
identification and lead discovery.46 Molecular dynamics (MD)
simulation is prevalently applied in the study of protein−ligand
interactions.47 In this study, virtual screening, MD simulation,
and biological evaluations have been applied to identify novel
small molecule inhibitors of PCSK9-LDLR PPI. Our
experimental results confirmed that compound 13 was a
promising hit for further study.
■
MATERIALS AND METHODS
Virtual Screening Protocol. The crystal structure of
PCSK9 (PDB ID: 2P4E) obtained from the protein data bank
(http://www.rcsb.org) was refined with the protein preparation
wizard module, including adding hydrogen atoms, assigning
partial charges, and determining the protonation state. SBC-
115,076 was prepared using the Ligand preparation module
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and then docked to PCSK9 according to the reported
methods44 using the Induced-Fit Docking module implemented
in Schrödinger 2009. The obtained structure of PCSK9 in
complex with SBC-115,076 was used for virtual screening
against the prepared Specs compound library containing about
250,000 compounds. The binding site was defined based on
the docking conformation of SBC-115,076. The virtual
screening workflow was performed using the three different
precision modes in sequence, high-throughput virtual screen-
ing (HTVS), standard-precision (SP), and extra-precision
(XP). The final results were output on the basis of Glide
docking score.
Molecular Dynamics Simulation and Analysis. The
protein−ligand interactions between PCSK9 and compound
13 were investigated by molecular dynamics simulation. The
complex structure obtained by molecular docking was used for
setting up the model of MD simulation. The AM1-BCC charge
model was applied for the calculation of atomic point charges
of the compound. The missing residues in the crystal structure
were complemented according to the amino acid residue
sequence of PCSK9. The Pro-domain was removed because of
the self-cleavage in the protein mature process. The complex
structure, including the protein, compound 13, counterions,
and water molecules, was immersed in a cubic box of a TIP3P
water model with a 10 Å minimum solute-wall distance. The
procedures and detailed parameter settings were in accordance
with our previous studies.48 The simulation time of 500 ns was
set for the complex system.
The trajectory was analyzed by using cpptraj in AmberTools
15. The stability of the protein−ligand complex was judged by
calculating the root-mean-square deviation (RMSD), root-
mean-square fluctuation (RMSF), and radius of gyration (Rg).
The close contacts and hydrogen bond occupancy for the
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Figure 4. (A) Original flat PCSK9-LDLR interface on PCSK9. (B) Binding pocket was generated by induced-fit docking of SBC-115,076 to the
PPI interface on PCSK9. (C) Binding mode of SBC-115,076 was predicted with induced-fit docking.
equilibrated trajectory were analyzed. The binding free energy
(ΔGpbsa) was calculated with the method of molecular
mechanics Poisson−Boltzmann surface area (MM/PBSA).
The binding free energy decomposition analysis was also
performed to estimate the contribution of each residue in the
pocket for ligand binding.
ELISA Assay for PCSK9-LDLR Binding. The well-
identified PCSK9-LDLR peptidic inhibitor pep2-8 was
selected as the positive control in all our biological evaluations.
It was synthesized by Synpeptide Inc. with the HPLC purity of
>98%, the exact sequence of which is Ac-
TVFTSWEEYLDWV-NH2. To test the PCSK9-LDLR PPI
inhibitory activity, ELISA assays were conducted using
PCSK9-LDLR the in vitro binding assay kit (CycLex Co.,
Japan) according to the manufacturer’s protocol with minor
modifications. Briefly, the compounds were dissolved in
dimethyl sulfoxide (DMSO) as 20 mM stocking solutions
while the positive control pep2-8 was prepared to a 2 mM
solution using dd H2O and then serially diluted to final
concentrations with a reaction buffer that contained recombi-
nant His-tagged PCSK9 at 80 ng/mL. After a gentle shake and
preincubated at room temperature for 1 h, a 100 μL reaction
mixture was added to a microplate that was coated with
EGF(AB) peptide of LDLR. Subsequent procedures were
performed in strict accordance with the instructions. The
absorbance of a test well was measured at dual wavelengths of
450/540 nm, respectively, using an EnVision Multimode Plate
Reader (EnSpire, PerkinElmer). The inhibition percentage of
test compounds was calculated from the standard His-tagged
PCSK9 dose−absorbance curve after vehicle control correc-
tion. To determine the IC50 of test compounds, the serial
inhibition percentage (the Y-coordinate) was plotted as the
function of a test concentration (the X-coordinate). The
results were analyzed with GraphPad Prism version 5.0
(GraphPad, La Jolla, CA, USA). All the tests were duplicated
three times, and data were denoted as mean ± standard
deviation (s.d.) (n = 3).
Binding Assay by Surface Plasmon Resonance (SPR).
The His-tagged human PCSK9 (residues 1−692) was obtained
as lyophilized powder with SDS-PAGE purity of >95% from a
commercial source (Sino Biological; #29698-H08H).
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Direct binding of each compound to PCSK9 was tested by
SPR using Biacore T200 (GE Healthcare) at room temper-
ature. The CM5 chip was immobilized with the PCSK9
protein in 10 mM sodium acetate (pH 4.5) according to a
standard amine-coupling procedure and remaining coupling
sites were blocked with 1 M ethanolamine, pH 8.5. Each
compound for binding affinity determination was initially
dissolved in dimethyl sulfoxide to 20 mM as a stocking
solution and serially diluted with a PBS buffer containing
0.05% (v/v) Tween 20 and 5% (v/v) DMSO to certain
concentrations (0.39−200 μM) when tested. Then, the test
sample was injected at a flow rate of 30 μL/min for 90 s to
contact with the proteins immobilized on the chip, followed by
dissociation from the chip for another 90 s. The SPR
sensorgrams were solvent corrected, reference and buffer
subtracted, and analyzed with Biacore T200 evaluation
software (GE Healthcare) to determine the kinetic parameters
and KD values.
Cell Viability Assay. The HepG2 cell line was obtained
from the Cell Bank of Shanghai Institute of Cell Biology,
Chinese Academy of Sciences, and authenticated by STR
profiling (Guangzhou Cellcook Biotech Co., Ltd.). Cells were
maintained in a 5% CO2-humidified incubator at 37 °C and
cultured in modified Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum
(FBS), 1% (v/v) penicillin-streptomycin, and 1% (v/v) sodium
pyruvate (Gibco). Medium and supplements were obtained
from Biological Industries except where indicated.
For cell viability test, HepG2 cells were seeded at 4000 cells/
well in the 96-well plate for 24 h. Then, the medium containing
test compounds was added to the final concentrations (3.12−
200 μL), and cells were incubated for another 48 h. Viable cells
were determined by MTT (Promega; #G7570) treatment for 3
h and fluorescence detection using an EnVision Multimode
Plate Reader (EnSpire, PerkinElmer). Analysis and IC50 values
were assessed by GraphPad Prism version 5.0. Data were
presented as mean ± s.d. (n = 3).
BODIPY-LDL Uptake Assay. HepG2 cells were used in
the fluorescent-conjugated LDL uptake assay. They were
seeded at 30,000 cells/well in a 96-well microplate with a clear
bottom and black wall (Corning; #CLS3603). Following
overnight incubation, the medium was changed to DMEM
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without supplements for another 24 h. The positive peptide

pep2-8 (10 μM) or compound 13 at various concentrations
was preincubated with 10 μg/mL of PCSK9 in DMEM
containing 0.5% (v/v) DMSO for 0.5 h before being added to
the cells. The vehicle wells were only treated with PCSK9 in a
serum-free medium without inhibitors. After 2 h, BODIPY FL
LDL (Thermo Fisher Scientific; #L3483) was added with a
final concentration of 10 μg/mL per well and incubated for
another 3.5 h. Each test well was washed three times with cold
PBS, and cellular uptake of fluorescence was evaluated by an
EnVision Multimode Plate Reader (EnSpire, PerkinElmer)
using excitation and emission wavelengths of 515 and 520 nm,
respectively. Data were presented as means ± s.d. (n = 3).
Statistical significance of differences was determined by one-
way ANOVA, and the levels were indicated as *P < 0.05, **P
< 0.01, and ***P < 0.001, compared with the PCSK9-treated
DMSO group.
RNA Isolation and Real Time PCR Analysis. HepG2
cells were seeded at 2 × 105 cells/well in a 12-well plate for 24
h. Then, each test well was treated with berberine at 10 μM, Figure 5. Virtual screening workflow.
pep2-8 at 10 μM, or compound 13 at several concentrations,
respectively, and incubated for 6 h. Total RNA was isolated
from HepG2 cell lysate using the Trizol reagent (Invitrogen,
Carlsbad, CA). The lysate was well mixed with chloroform and
centrifuged for 15 min at 13,000 rpm at 4 °C. The top aqueous
phase containing RNA was isolated and mixed with
isopropanol to precipitate the total RNA. After collection
and concentration measurement, 1 μg of total RNA was used
to synthesize the cDNA with a commercial-sourced HiScript II
Q RT SuperMix kit for qPCR (Vazyme Biotech Co.) by
following the manufacturer’s instructions. Real-time PCR was
performed using a Power SYBR Green Master Mix with the Figure 6. Preliminary screening results (inhibitory rate) of the 30
primers in Table S1. Expressions of PCSK9 and LDLR mRNAs selected compounds by the PCSK9-LDLR in vitro binding assay with
in each sample were normalized to the corresponding GAPDH pep2-8 as the positive control.
mRNA. The relative mRNA expression levels were determined
using the 2−ΔΔCt, method and each cDNA sample was run in
triplicate. Data were presented as mean ± s.d. (n = 3).
Statistical significance of differences was determined by one-
way ANOVA, and the levels were indicated as *P < 0.05, **P
< 0.01, and ***P < 0.001 compared with DMSO group.

■ RESULTS AND DISCUSSION

Virtual Screening. The PCSK9-LDLR PPI interface was
initially large and flat (Figure 4A), which was difficult and not
suitable for discovering small molecule inhibitors. The virtual
screening attempts directly and rigidly based on the original
surface failed to identify a PCSK9-LDLR inhibitor in the
reported study41 and our previous attempts. Usually, a
“pocket” is needed for the binding of small molecules to the
target protein. Compound SBC-115,076 was identified as a
PCSK9-LDLR PPI inhibitor by induced-fit docking. Although
the direct binding affinity was not reported, the cellular and in
vivo experiments confirmed its bioactivity in lipid regulation.
The discovery of SBC-115,076 gave us the inspiration. The
PPI interface on PCSK9 consisted of several loops, including
Ser153-Trp156, His193-Glu197, Arg237-Ala239, and Ser376-
Phe379. Taking the flexibility of PPI interface into account,
SBC-115,076 was docked to the PPI interface on PCSK9 by
referring to the reported methods of induced-fit docking. The
induced pocket was long and narrow, passing through the
whole PPI interface (Figure 4B). It consisted of Ile154,
Trp156, Glu159, Arg194, Glu195, Arg237, Asp238, Thr377,
and Phe379 (Figure 4C). Hydrogen bond interactions were
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Figure 7. (A) Calculated IC50 value of compound 13 in the PCSK9-
LDLR in vitro binding assay. (B) Chemical structure of compound 13.
found between SBC-115,076 and the residues of Arg237 and
Thr377. This predicted complex structure was used in the
subsequent virtual screening. After the prepared Specs
compounds were docked into the pocket with the three
precision modes successively, the retained compounds were
visually inspected by considering their binding modes, docking
scores, and structural diversity. The final selected 30 molecules
(Table S2) from the virtual screening workflow (Figure 5)
were purchased from the vendor. Biological evaluations,
including PCSK9-LDLR PPI inhibitory activity, PCSK9
binding affinity, and cellular BODIPY-LDL uptake, were
performed to identify the hit compounds.
ELISA Assay for PCSK9-LDLR Binding. Subsequently,
their preliminary PCSK9-LDLR PPI inhibitory rates of the 30
selected compounds at the concentration of 25 μM were
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Figure 8. Binding affinity values of (A) 13 and (B) pep2-8 to PCSK9 were determined using SPR.
Figure 9. Binding mode of 13 was predicted with molecular docking. (A) Binding pocket is shown as a white surface. (B) Amino acid residues
constituting the binding pocket are shown as sticks.
Figure 10. RMSD values of PCSK9−13 complex in the first 170 ns of
the entire molecular dynamics simulation.
measured by the ELISA assay, while pep2-8 at the
concentration of 10 μM was applied as the positive control.
Among them, compounds 6 and 13 at 25 μM displayed
inhibitory rates of 82.8 ± 1.7% and 64.2 ± 0.7%, respectively.
The inhibitory rates of compounds 2 and 7 were also more
than 40%, 41.97 ± 6.77%, and 45.93 ± 1.55%, respectively
Figure 11. (A) Rg and (B) RMSF values of protein Cα atoms are calculated.
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(Figure 6, Table S3). However, compounds 2 did not pass the
Pan-Assay Interference Compounds (PAINS) filter. Therefore,
the remaining three compounds, 6, 7, and 13, were picked out
for further determination of IC50 values. Compounds 6 and 7
did not show considerable and does-dependent inhibitory
activity (IC50 > 50 μM) with ascending concentrations. But
compound 13 exhibited a dose-dependent inhibitory response
against the PCSK9-LDLR interaction with an IC50 value of
7.57 ± 1.40 μM (Figure 7, Figure S1). We have also performed
mass spectrometry analysis of compound 13 (Figure S2), and
the result was in accordance with the provided structure
information, confirming its molecular weight.
Binding Assay by SPR. The direct binding affinity
between PCSK9 and compound 13 was determined using
SPR assay.49 The protein−ligand binding affinity was dose-
dependent with a KD value of 2.50 ± 0.73 μM (Figure 8A,
Figure S3), suggesting the direct binding between 13 and
PCSK9. The KD value of pep2-8 binding to PCSK9 in our
assay was 3.93 μM (Figure 8B), consistent with the reported
value.39
MD Simulation. Molecular dynamics simulation was
performed to investigate the binding mode of 13 and
PCSK9. This hit compound was docked into the pocket that
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Figure 12. Close contact analysis of the PCSK9−13 complex.

Table 1. Hydrogen Bond Analysis with the Equilibrium Trajectory

Ligand Acceptor Donor Occupancy (%) Average distance (Å) Average angle (deg)
13 Ile154@O MOL@N3H7 55.09 2.93 159.17
MOL@O3 Ser153@OH 18.53 2.77 155.81
MOL@O2 Ser153@NH3 12.30 2.88 140.25
MOL@O2 Ser153@NH2 10.89 2.87 139.60
MOL@O2 Ser153@NH1 10.85 2.88 140.30

Table 2. Binding Free Energy Calculated Using the MM/ 175 ns (Figure S4). By checking the trajectory with VMD and
PBSA Method extracting the representative snapshots, it was found that the
ligand disassociated from the binding pocket from about 170
PCSK9−13
ns. Therefore, the trajectory of the first 170 ns was used for
Energy (kcal mol−1) Mean Std. analysis. The RMSD values (Figure 10) reflecting the stability
ΔGvdW −33.89 3.66 of the protein−ligand system were calculated by using the
ΔGele −43.05 7.68 initial conformation as a reference. The protein stayed stable
ΔGgas −76.94 9.02 all through the process around an average RMSD value of 2.0
ΔGpol, sol 60.59 8.17 Å. There were some fluctuations happening to the ligand
ΔGnonpol, sol −5.16 0.24 before 50 ns, while it then converged to an equilibrium state
ΔGsol 55.43 8.05 with an average RMSD value of 2.5 Å. The calculated Rg values
ΔGpbsa −21.51 4.02 (Figure 11A) showed that the protein−ligand system stayed
compact in the whole process with Rg values fluctuating
uniformly between 25.0 and 25.5 Å. The stability of residues in
the process of simulation was reflected by the RMSF values.
The residues constituting the binding site, including those
forming hydrogen bond interactions with 13, kept a small
RMSF value (Figure 11B), indicating the stable state of the
binding site in the whole process of simulation.
Considering the equilibrium state of the simulation, the MD
trajectory of the last 50 ns was used for calculation of close
contacts, hydrogen bond occupancy, and binding free energy.
The close contacts were defined as the protein−ligand
interactions between the ligand and residues within 4.5 Å
around it. The residues, Ser153, Ile154, Pro155, Trp156,
Glu159, Asp238, and Thr377 (Figure 12), showed a relatively
high percentage of close contacts. In combination with the
hydrogen bond occupancy analysis (Table 1), both of the
hydrophobic and hydrogen bond interactions contributed
greatly to the close contacts of Ser153 and Ile154, while
Figure 13. Binding free energy decomposition was performed for the hydrophobicity was important for the contribution of Pro155.
PCSK9−13 complex. Although some hydrogen bonds were found between 13 and
the pocket initially, only the one with Ile154 was stable in the
induced by SBC-115,076 (Figure 9A). Some residues process of simulation. These results indicated the important
constituting the binding pocket, including Ser153, Ile154, role these residues played for ligand binding.
Glu195, Asp238, and Thr377, formed hydrogen bond The binding free energy calculated with the MM/PBSA
interactions with compound 13 (Figure 9B). This docking method was −21.51 kcal/mol. The ΔGele and ΔGvdw terms
result was used as the initial conformation for the unrestrained contributed greatly to the binding free energy (Table 2).
molecular dynamics simulation of 500 ns. It seemed that some Binding free energy decomposition was also performed to
fluctuations happened to the protein−ligand system around investigate the contribution of each residue to ligand binding.
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Figure 14. Cellular effects of 13 on restoring LDL uptake function. (A) LDL uptake RFU values of each group. (B) Relative restored rates of LDL
uptake of each group.
Figure 15. Real-time PCR analysis of (A) LDLR mRNA and (B) PCSK9 mRNA in HepG2 cells after the treatment with 13.
The threshold value of an absolute decomposed energy for
judging the importance of a residue is 1.0 kcal/mol. The
residues, Ile154, Pro155, Trp156, and Phe379, made great
contributions to the binding of 13 (Figure 13). Some other
residues, Glu195 and Asp238, were not favorable for ligand
binding. Both of these residues located on the same side of the
binding site (Figure 9B), indicating the direction for further
structural optimization.
Cellular Effects of Compound 13. As PCSK9 is mainly
expressed from hepatocyte and responsible for the degradation
of LDLR on the surface of liver, it is recommended to use liver
cells in the cell functional tests of PCSK9 inhibitors. HepG2 is
an immortalized cell line consisting of human liver carcinoma
cells and has been widely used in the studies of LDL uptake
assays of PCSK9-LDLR PPI inhibitors mentioned
above.37,39,41 In this study, cellular effects of compound 13
were then investigated in HepG2 cells. The MTT assay was
first conducted to guarantee the cell viability for the following
experiments. As shown in Figure S5, this hit compound
exhibited no obvious inhibitory effects on HepG2 cell
proliferation at all the test concentrations. The fluorescent-
labeled LDL uptake assay is indispensable for the test of
cholesterol-lowering effect of drugs. We employed the
prevalently used BODIPY-LDL37,39 as the fluorescence probe
due to its stability under physiological condition and high
quantum yields. In the BODIPY-LDL uptake assay, the LDL
uptake level was reduced by 35.6% after adding PCSK9 to
HepG2 cells (Figure 14A). Theoretically, inhibition of PCSK9-
LDLR PPI could restore impaired hepatocyte LDLR function
and LDL uptake. Treatment with pep2-8 at the concentration
of 10 μM restored almost 100% LDL uptake level (Figure
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14B). Although compound 13 did not restore the LDLR
function at the concentration of 10 μM, it restored LDL uptake
of PCSK9-treated HepG2 cells to 36.0 ± 5.3% at 44 μM
compared with the DMSO control. At the concentration of
200 μM, it did not show improved potency. Therefore,
compound 13 could restore the LDL uptake level to a certain
extent.
The effects of compound 13 on transcriptions of PCSK9 and
LDLR genes were then investigated. Berberine was reported as
a lipid-lowering molecule that could upregulate hepatic
LDLR50 and downregulate plasma PCSK951 transcriptionally.
In our assay, HepG2 cells were treated with pep2-8 and 13
under the same conditions of the LDL uptake assay with
berberine (10 μM) as the positive control. Compared with
berberine, compound 13 showed minor effects on LDLR and
PCSK9 mRNA levels at the concentrations of 10 and 44 μM
(Figure 15). The LDLR mRNA level was obviously lower than
the control group by treating with 13 at the higher
concentration of 200 μM. The results indicated the LDL
uptake recovery effect of 13 at low concentrations in HepG2
was independent of transcriptional regulation of PCSK9 and
LDLR genes.
■
CONCLUSION
Although with great value as reliable targets, PPIs are usually
characterized as “undruggable” since most of the PPI interface
is large, flat, and featureless, without a “pocket” or “ groove” for
the binding of small molecules. The PCSK9-LDLR PPI
interface is such a typical case. Therefore, it has been
considered as a challenging work for developing small molecule
https://doi.org/10.1021/acs.jcim.1c00521
J. Chem. Inf. Model. 2021, 61, 5269−5279
Journal of Chemical Information and Modeling
inhibitors of PCSK9-LDLR PPI. In this study, a potential
binding pocket on the PCSK9-LDLR PPI interface of PCSK9
was generated by induced-fit docking of SBC-115,076. This
induced binding pocket was subsequently used for virtual
screening to identify novel small molecule inhibitors of
PCSK9-LDLR PPI. The selected 30 compounds were
purchased from the vendor. Among them, compound 13
exhibited potent and dose-dependent inhibitory activity against
PCSK9-LDLR interaction in the ELISA assay. Also, the SPR
result showed direct binding between 13 and PCSK9. The
molecular dynamics simulation revealed the stable binding
mode of 13 in the induced pocket. The results of the cellular
study showed that 13 could restore the LDL uptake function of
PCSK9-treated HepG2 cells to some extent. This well-
characterized hit compound validated the reliability of our
method. It is presumed that better results would be obtained
by performing induced-fit docking against the entire
compound library if abundant computational resources are
available. We hope this study will facilitate the further
development of novel small molecule PCSK9-LDLR PPI
inhibitors.
■
DATA AND SOFTWARE AVAILABILITY
The screening compound library was downloaded from Specs
(https://specs.net). The PDB files were downloaded from the
RCSB protein data bank (https://www.rcsb.org). All the
output data and structures of purchased compounds have been
provided in the article and Supporting Information. The free
software package of PyMOL1.5.0.5 (https://pymol.org) was
used in this study. The MD simulation was performed and
analyzed with the licensed Amber14 and publicly available
AmberTools15 (https://ambermd.org). Virtual screening was
performed using Schrödinger2009 (https://www.schrödinger.
com) purchased by China Pharmaceutical University.
■
ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jcim.1c00521.
Additional experimental materials, structural informa-
tion, and experimental and computational results (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
Hongbin Sun − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China; orcid.org/0000-0002-3452-7674;
Phone: +86-25-83271050; Email: hongbinsun@
cpu.edu.cn
Haoliang Yuan − Jiangsu Key Laboratory of Drug Discovery
for Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China; orcid.org/0000-0003-0248-4347;
Phone: +86-25-83271050; Email: yhl@cpu.edu.cn
Authors
Hengzhi Sun − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China
5277
pubs.acs.org/jcim Article
Jinzheng Wang − Jiangsu Key Laboratory of Drug Discovery
for Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China
Shengjie Liu − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China
Xinyu Zhou − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China
Liang Dai − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China; orcid.org/0000-0003-0172-4759
Caiping Chen − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China
Qinglong Xu − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China; orcid.org/0000-0003-4450-6866
Xiaoan Wen − Jiangsu Key Laboratory of Drug Discovery for
Metabolic Disease and State Key Laboratory of Natural
Medicines, China Pharmaceutical University, Nanjing
210009, P. R. China; orcid.org/0000-0001-7852-1154
Keguang Cheng − State Key Laboratory for Chemistry and
Molecular Engineering of Medicinal Resources, School of
Chemistry and Pharmacy, Guangxi Normal University,
Guilin 541004, P. R. China; orcid.org/0000-0002-2883-
0845
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jcim.1c00521
Author Contributions
§
H.S., J.W., and S.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Financial support from the National Natural Science
Foundation of China (82073707, 91853125, 81730094,
81773584, 21977118, and 21971256) and Natural Science
Foundation of Jiangsu Province (BK20202009), BAGUI
Scholar Program of Guangxi Province of China (2016A13),
and Key R&D Program of Jiangsu Province (BE2018710) are
gratefully acknowledged. This project was also supported by
the “111” Project from the Ministry of Education of China and
State Administration of Foreign Expert Affairs of China (111-
2-07).
■
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