Small Molecule Inhibitors of the PCSK9·LDLR Interaction

Article abstract of DOI:10.1021/jacs.7b09360

The protein-protein interaction between proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) is a relatively new, and extremely important, validated therapeutic target for treatment and prevention of heart disease. Experts in the area agree that the first small molecules to disrupt PCSK9·LDLR would represent a milestone in this field, yet few credible leads have been reported. This paper describes how side-chain orientations in preferred conformations of carefully designed chemotypes were compared with LDLR side chains at the PCSK9·LDLR interface to find molecules that would mimic interface regions of LDLR. This approach is an example of the procedure called EKO (Exploring Key Orientations). The guiding hypothesis on which EKO is based is that good matches indicate the chemotypes bearing the same side chains as the protein at the sites of overlay have the potential to disrupt the parent protein-protein interaction. In the event, the EKO procedure and one round of combinatorial fragment-based virtual docking led to the discovery of seven compounds that bound PCSK9 (SPR and ELISA) and had a favorable outcome in a cellular assay (hepatocyte uptake of fluorescently labeled low-density lipoprotein particles) and increased the expression LDLR on hepatocytes in culture. Three promising hit compounds in this series had dissociation constants for PCSK9 binding in the 20-40 μM range, and one of these was modified with a photoaffinity label and shown to form a covalent conjugate with PCSK9 on photolysis.

Full text of DOI:10.1021/jacs.7b09360

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Article
Small Molecule Inhibitors Of The PCSK9•LDLR Interaction
Jaru Taechalertpaisarn, Bosheng Zhao, Xiaowen Liang, and Kevin Burgess
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09360 • Publication Date (Web): 29 Jan 2018
Downloaded from http://pubs.acs.org on January 30, 2018
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Small Molecule Inhibitors Of The
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PCSK9•LDLR Interaction
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Jaru Taechalertpaisarn,1 Bosheng Zhao,1 Xiaowen Liang,2 and Kevin Burgess*1
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1 Department of Chemistry, Texas A & M University, Box 30012, College Station, TX 77842,
USA
2 Center for Infectious and Inflammatory Diseases, 2121 W. Holcombe Blvd.,
Houston, TX 77030, USA
E-mail: burgess@tamu.edu
RECEIVED DATE
TITLE RUNNING HEAD: Inhibitors of PCSK9•LDLR
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ABSTRACT
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The protein-protein interaction between proprotein convertase subtilisin/kexin type
9 (PCSK9) and low-density lipoprotein receptor (LDLR) is a relatively new, and extremely
important, validated therapeutic target for treatment and prevention of heart disease. Experts
in the area agree that the first small molecules to disrupt PCSK9•LDLR would represent a
milestone in this field, yet no credible leads have been reported. This paper describes how
side-chain orientations in preferred conformations of carefully designed chemotypes were
compared with LDLR side-chains at the PCSK9•LDLR interface to find molecules that would
mimic interface regions of LDLR. This approach is an example of the procedure called EKO
(Exploring Key Orientations). The guiding hypothesis on which EKO is based is that good
matches indicate the chemotypes bearing the same side-chains as the protein at the sites of
overlay have the potential to disrupt the parent protein-protein interaction (PPI). In the event,
the EKO procedure and one round of combinatorial fragment-based virtual docking, led to the
discovery of seven compounds that bound PCSK9 (SPR and ELISA) and had a favorable
outcome in a cellular assay (hepatocyte uptake of fluorescently labeled LDL particles) and
increased the expression LDLR on hepatocytes in culture. Three promising hit compounds in
this series had dissociation constants for PCSK9 binding in the 20 – 40 µM range, and one of
these was modified with a photoaffinity label and shown to form a covalent conjugate with
PCSK9 on photolysis.
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INTRODUCTION
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Heart disease, a leading cause of death, is frequently associated with plaque in the arteries
causing atherosclerosis, which is attributed by elevated levels of low density lipoprotein (LDL)
in the blood. LDLRs (LDL-receptors), on the surface of hepatocytes, are responsible for
capturing LDL particles, and importing them into the liver for destruction. Thus, LDLR•LDL
complexes on the surface of hepatocytes undergo clathrin-mediated endocytosis into
endosomes wherein the acidic environment triggers rearrangement of the LDLR extracellular
domain, releasing bound lipoproteins, leaving the LDLR to be recycled to the plasma
membrane (Figure 1a).1
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Removal of LDL particles by hepatocytes is negatively modulated by a chaperone called
PCSK92,3 that recognizes plasma membrane LDLR.4 PCSK9 inhibits the rearrangement of
LDLR in LDLR•LDL complexes, so that the receptor is not recycled to the plasma membrane
but is instead routed to lysosomes where LDL, LDLR and PCSK9 are degraded (Figure 1b).5-8
Plasma LDL cholesterol levels thus can be decreased by inhibiting the PCSK9•LDLR
interaction, because that increases the display of LDLR on the hepatocytes.1,9,10 This effect
was discovered after genetic studies had correlated LDL levels in some patients with
mutations associated with gain or loss of function of PCSK9;11,12 two loss of function mutations
correlate with significantly reduced risk of coronary heart disease.11 Indeed, an individual with
no detectable PCSK9, and extremely low LDL levels, was healthy, suggesting suppression of
PCSK9 for lipid lowering is safe.13 Conversely, individuals with gain-of-function mutations in
PCSK9 have a higher risk of coronary heart disease.11,12
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Figure 1. a In the absence of PCSK9, LDL•LDLR is endocytosed onto hepatocytes, then the LDLR is
recycled and LDL is digested; and, b In the presence of PCSK9, LDLR is not recycled and the whole
complex is decomposed. Thus PCSK9•LDLR interaction suppresses recycling of LDLR and diminishes
uptake of LDL particles by the liver.
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Multiple clinical studies have shown injectable antibody therapeutics that impede the
PCSK9•LDLR protein-protein interaction (PPI) significantly decrease circulating LDL levels.14
Subsequently, two antibody drugs that disrupt PCSK9•LDLR are FDA approved (Repatha from
Amgen and Praluent from Sanofi/Regeneron); they appear to be tolerated well, with no serious
side effects, and are efficacious.15-20 PCSK9•LDLR therefore is a validated target for medicinal
chemistry.
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The preferred modality for disruption of PCSK9•LDLR, however, is small molecule drugs, not
mAbs, on the basis of mode of administration, cost, shelf life, and immunogenic response
issues. Thus in 2014 GenenTech stated: “Undoubtedly, an orally available small molecule
inhibitor of PCSK9, due to lower cost and ease of administration, would be a highly desirable
alternative therapeutic agent.”21 Peptides have been used to mimic either component in the
PCSK9•LDLR interaction22-28 but they have poor efficacy in plasma. There are limited reports
of non-peptidic small molecules that effectively inhibit the PCSK9•LDLR interaction, despite of
intense efforts to find such compounds.
Portola Pharmaceuticals have reported
tetrahydroisoquinolines that increased LDL-uptake into liver cells, and LDLR cell surface
populations.29 Similarly, Park et al reported compounds that had the same types of activities
and reduced LDL in wild type mice but not the corresponding a PCSK9 knock out murine
model.30,31 The Park compounds have fragments that structurally resemble the plasticizer
BPA. Both the Portola and Park studies do not report any direct evidence that these small
molecules bind PCSK9; the compounds could act via another mechanism. However, Stucchi
et al reported an oligo N-methyl imidazole that induced concentration dependent disruption of
PCSK9•LDLR based on an ELISA assay, and increased LDL uptake in HepG2 cells.
Curiously, the IC50 reported for the binding of this compound to PCSK9 (11.2 ± 0.2 μM) was
greater than the EC50 for increased LDL uptake into HepG2 cells (6.04 μM).
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Our labs have developed a technique for accelerated discovery of small molecules that inhibit
PPIs: EKO (Exploring Key Orientations).32,33 EKO features relatively rigid, usually non-
peptidic, chemotypes with three amino acid side-chains. Preferred conformations of these
chemotypes are simulated then systematically compared, via a data mining algorithm, with
side-chain orientations of protein-protein interface amino acids in solid-state structures. A
chemotype that overlays well on one protein at the interface is a candidate to displace that
same protein from the PPI.
Research featured here illustrates how the EKO approach was used to discover small
molecules that bind PCSK9 with low micromolar affinities, which prevent the PCSK9•LDLR
interaction in LDL uptake by hepatocytes.
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RESULTS AND DISCUSSION
Small Molecule Design And Syntheses
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Several candidate chemotypes were conceived and screened using the EKO approach. In
EKO, preferred conformations of the featured chemotypes with three methyl side-chains are
simulated, but they may overlay on any three residues of a protein in a PPI. Chemotypes with
side-chains corresponding to the protein at the overlay sites must then be prepared and
assayed to test the validity of the EKO-prediction.
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In the event, molecules in series A gave several overlays on LDLR of the PCSK9•LDLR
complex (crystal structure PDB identifier 3gcx).34
Specifically, EKO indicated preferred
conformations of structures A overlaid C – C atoms well on the following sets of interface
side-chains of LDLR in the PCSK9•LDLR crystal structure: Asp299, Leu298, Asp301; Cys297,
Asn301, Asp299; Leu298, Asp299, Asn301; Val307, Cys308, Leu318; Asn309, Cys308, Leu318. Thus the
residues implicated in total were: Cys297, Leu298, Asp299, Asp301 Val307, Cys308, Asn309, and
Leu318. Based on Ala-scan studies, Horton et al conclude the LDLR side-chains implicated in
PCSK9•LDLR hot-spots are Asn295, Glu296, Asp310, Tyr315,8 while crystallographic evidence led
others to suggest Asp299, Leu318, and Asn309 were implicated.35 Throughout we have
underlined residues that have been postulated to be hot-spots that were also overlaid via
preferred conformations of A. In our view, presence, or lack of, overlap with hot-spots is
insufficient grounds for “go” or “no-go” decisions in EKO analyses. There are two reasons for
this: (i) the uncertainties of predicting hot-spots36 even with experimental data; and, (ii) the
notion of isolated hot-spots is being superseded by hot segments in which disruption of one
residue in a tightly packed arrangement alters the contributions of the others.37-39
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Several compounds in series A were prepared and tested using a commercial TR-FRET assay
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(BPS Bioscience Inc., San Diego, CA); however, the results were inconclusive (Figure S1).
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We were unsure if this negative result was because of poor affinity of the compounds A,
insufficient sensitivity in the TR-FRET assay, or both. In any event, it seemed clear that
modifications to improve the affinity of the core A towards PCSK9 would be desirable.
Consequently, an iterative docking and energy minimization procedure was used to virtually
modify chemotypes A to include additional pharmacophores that would increase binding
efficiencies. Specifically, the preferred conformations of A were overlaid on LDLR in the 3gcx
structure, LDLR was removed, then the chemotypes were docked in place using Glide within
the Schrodinger package.40-42 That procedure gave “baseline energies” for interactions of A
with PCSK9 to which data from docking virtually modified analogs could be compared;43 this
procedure was performed in the Schrodinger software package using the CombiGlide routine.
Figure 2a shows a docked and minimized structure of A•PCSK9 generated from the original
EKO overlay. We noticed that there is a conspicuous negatively charged cavity in PSCK9
comprising Ser383, Asp367 and Ser381 proximal to the C-terminus of A, but not interacting
with it. Consequently, we made that cavity a priority in CombiGlide simulations. Those
calculations indicated that negative pocket was filled by addition of His, Lys or, optimally, Arg
at the A C-terminus; structures 1 were conceived in this way. Figure 2b illustrates how that
Arg in the small molecule H-bonds to Asp367.
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LDL-Adln; ΔG = -1.15 kcal/mol
LDLL-1dlnr; ΔG = -3.87 kcal/mol
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D299
L298
N301
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negative
S381
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Figure 2. Docking of LDL-Adln (gold) and LDLL-1dlnr (silver) onto PCSK9. a Compounds LDL-Adln
and LDLL-1dlnr overlay on the LDLR (shown in magenta wire), in which six C-C atoms of
chemotypes and LDLR side-chains (green arrows) are compared; RMSD = 1.75 and 1.95 Å for LDL-
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Adln and LDLL-1dlnr, respectively. b Improving binding affinity of LDLL-1dlnr is likely due to the H-
bonds of Arg residue to the Asp367 of PCSK9 at a negative “cliff face” region as indicated in green lines.
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A solid phase synthesis of chemotypes 1 on TentaGel-NH2 resin was developed (Scheme 1).
Microwave accelerated amino acid couplings installed the R4-bearing residue, then the
protected amino acid fragment 10 that carries R3. Nosyl-removal as indicated,44 then two
more coupling-deprotection cycles using standard N-Fmoc-protected amino acids assembled
the fragments bearing the R2 and R1 side-chains. The Fmoc was removed, and the N-terminal
dipeptide was converted to a hydantoin via a two-step process.45 Finally, cyanogen bromide
was used to cleave the protected chemotypes 1 from the resin, giving the C-terminal lactone
appendix in these structures.
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Compounds in Scheme 1 and throughout this paper are numbered according to the scaffold
(or scaffold-intermediate). Lower case one-letter codes are used to delineate the amino acid
side-chains R1 – R4 and relate them to the closest amino acid; primed letters indicate
t
protected side-chains (eg d’ for the –CH2CO2 Bu of Asp and k’ for the –(CH2)4NHBoc of Lys).
Implementation of Scheme 1 gave 15 compounds for screening, but one, LLLD-1ldnq, was
surprisingly vulnerable to degradation in the air, and was not considered further. One of the
compounds that was considered, LDLL-1dl(CN)r, is a byproduct formed via dehydration of the
Asn side-chain in the cyanogen bromide cleavage step.
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Scheme 1. Solid phase syntheses of chemotypes 1.
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Binding Assays
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Surface plasmon resonance (SPR) was used to screen the library of 14 compounds indicated
in Figure 3a. Pep2-8 is a 13-residue peptide developed by GenenTech that was reported to
bind PCSK9 (Kd 0.66 ± 0.11 μM)21, and was used here as a positive control. Compound, LLLL-
1aaar, is a “partial negative control” having the same core as the EKO-implicated compounds,
but only Ala side-chains and an all-L stereochemistry that was not predicted via EKO, ie LLLL-
1aaar controls for random stereochemistry and lack of functional side-chains. In the event,
three compounds and Pep2-8 were selected (on the basis of this initial SPR data and the
cellular uptake assays described below) for more thorough SPR analyses: LDLL-1dlnr (Kd 24.8
± 9.1 μM; Figure 3b), DLDD-1nclk (41.2 ± 17.5 μM; Figure S7b), LDLL-1dl(CN)r (35.8 ± 11.4
μM; Figure S7d), and Pep2-8 (3.56 ± 0.16 μM). The Kd for Pep2-8 determined here is slightly
higher than the value reported previously;21 this discrepancy might be because of different
techniques used to study dissociation constant (the literature procedure used was biolayer
inferometry).
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Seven compounds in the library (again, selected on the basis of the SPR data and the cellular
assays described below) were subjected to an ELISA assay to obtain additional evidence that
the small molecules bind PCSK9 (Figure 3c). Error limits in this assay are higher than in the
SPR experiments. All the compounds tested showed increased inhibition of PCSK9 to the
EGF-AB domain of LDLR relative to a blank control. Inhibition by the compounds in this assay
was one to two orders of magnitude less than the positive control pep2-8.
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c80
positive control
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Figure 3. a Initial SPR screening of 14 compounds at 50 µM and pep2-8 at 5 µM over PCSK9
supported on gold via amine coupling chemistry. b Sensorgram of LDLL-1dlnr within 1-54 µM
concentration range showing the estimated Kd was 24.8 ± 9.1 µM {kon = (4.04 ± 2.20) x 103 M-1s-1, koff =
(8.74 ± 3.40) x 10-2 s-1}. c Selected compounds and pep2-8 were screened at 50 µM for inhibitory
effect against 50 ng/mL of PCSK9 using PCSK9-LDLR in vitro binding assay kit (MBLI Co., MA)
following the manufacture instructions. Results are the averages ± SD of three independent
experiments
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Photoaffinity Labeling
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Two derivatives of LDLL-1dlnr, compounds 2 and 3, were prepared to explore if binding of
these to PCSK9 could be detected via photoaffinity labeling. Thus, a protected fragment of
LDLL-1dlnr was prepared on chlorotrityl resin, cleaved with the protecting groups in place, and
coupled to a photoaffinity fragment designed in these laboratories (see supporting).
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Pre-incubation of PCSK9 with 2 and (optionally) with a large excess of the blocking ligand 3,
irradiation of some wells at 365 nm, copper-mediated click reaction with Alexa-488-azide, then
SDS-PAGE gave the data shown in Figure 4. A fluorescent band corresponding to the
molecular mass of labeled PCSK9 (~60 kDa) was observed only in the wells that were
irradiated in the absence of the blocking ligand 3.
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Figure 4. Photoaffinity labeling of human PCSK9 protein with 2. PCSK9 was incubated with PAL ligand
2, and optionally pretreated with 50-fold excess of the competition ligand 3. UV (365 nm) irradiation,
then Cu-mediated click with Alexa-488-azide gave the samples for analysis. These samples were
diluted in SDS sample buffer and subjected to SDS-PAGE. Fluorescent proteins were detected by in-
gel fluorescence (Alexa 488) and all proteins were stained with CBB (Coomassie Brilliant Blue) G250.
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Liver-cell Uptake Assays
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The seven select compounds shown in Figure 3c were tested for cytotoxicity using liver cells
(hepatocytes; HepG2). All seven of these compounds showed no toxicity up to 50 μM. The
compound with the lowest Kd in SPR studies, LDLL-1dlnr, was also checked at 100 μM and
showed no cytotoxicity even at these higher concentration (Figure S2). In another prelude to
the key cellular assays, the water solubilities of the featured compounds were measured. The
solubility concentration gradients for these materials were linear to beyond 100 μM, ie they
were soluble at the maximum concentration used in the uptake assays below (Figure S5).
Compounds A and 1 were evaluated their drug likeliness by QikProp calculations46,47 to
determine absorption, distribution, metabolism and excretion (ADME) properties, and the
results are reported in the supporting material (Table S3 and S4).
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An established assay for PCSK9•LDLR inhibition features uptake of fluorescently labeled LDL
nanoparticles (BODIPY-LDL, Invitrogen) by hepatocytes; the cells become fluorescent as the
particles are absorbed.21 Uptake of the BODIPY-containing particles is maximized in the
absence of PCSK9, and the cells become fluorescent; conversely, adding PCSK9 diminishes
that signal. Addition of PCSK9 and a compound that interferes with the PCSK9•LDLR
interaction would be expected to give cells that are more fluorescent than those to which only
PCSK9 was added but less so than cells to which none of that protein was present.
Figure 5a shows maximal uptake (calibrated to 100 %; black bar) in the absence of PCSK9,
while all the other data points correspond to 15 µg/mL of that protein; the “negative”
corresponds to only PCSK9 added (calibrated to 0 %). Pep2-8 at 30 µM restored the LDL
uptake (yellow bar) to within 80 % of its maximal value (black). Several of the featured
chemotypes 1 showed promise insofar as they, like pep2-8, also restored fluorescence; the
ones marked with a red dagger were selected for further assays on the basis of this data and
the binding studies above. Recall that LLLL-1aaar is a “partial control” as described above
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(same chemotype, just methyl side-chains, and random stereochemistry); it did not induce
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Figure 5b shows data derived from repetition of these experiments under identical conditions
except that three different doses of the test compounds were used. Overall, all the
compounds show a dose response, except LLLD-1qndr, DLLL-1qndr, and, as expected, the
partial control LLLL-1aaar. Figure 5c shows a more extensive dose-response curve for one
these compounds, LDLL-1dlnr (again, selected on the basis of the overall data); this data
shows an encouraging correspondence.
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It is curious that LDL uptake in the HepG2 cells was significantly enhanced when 100 µM of
compound was used (Fig 5c). This is consistent with the SPR binding data in which LDLL-
1dlnr showed a longer resident time (15-20 fold slower off-rate) compared with Pep2-8. More
particularly, the longer half-life of the LDLL-1dlnr • PCSK9 complex became more obvious
when higher concentration (100 µM) of compound was injected onto the PCSK9-functionalized
surface (data not shown). One explanation for these observations is that there could be a
synergistic target site for LDLL-1dlnr that only becomes significant at higher compound
concentrations.
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Figure 5. Uptake of BODIPY-LDL by hepatocytes. a Initial screen at 50 µM concentrations of the
featured compounds. b Data of select compounds at three different doses. c A more extensive dose-
response curve of one select lead: LDLL-1dlnr. All results are represented as means ± SD of three
independent experiments. Significant differences between compounds and negative control are
determined using student’ t-test (* p ≤ 0.05, ** p ≤ 0.01).
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Cell-surface LDLR Assay
Two assays were attempted to probe if increased uptake of LDL particles correlates with
increased expression of LDLR. Our initial attempts to do this featured monitoring LDLR levels
on treated and untreated hepatocytes using flow cytometry. The data obtained (Figure S3)
indicated an increase in LDLR levels upon treatment with the seven hit compounds (as shown
in Figure 5b), but the errors in the measurements were such that the increases had borderline
statistical significance. Consequently, we resorted to a semi-quantitative approach in which
the LDLRs on live hepatocytes (treated and untreated) were visualized using an LDLR-
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selective mAb in combination with an Alexa Fluor®-labeled secondary mAb. Figure 6a shows
expression of LDLR in the cells was suppressed when they were treated with PCSK9 alone.
When cells were treated with the test compound LDLL-1dlnr then they stained more brightly
(Figure 6b) though not as brilliant as the positive control culture that lacked PCSK9 and any
test compound (Figure 6c). Similar data including bright-field and merged images was
collected for another three of the featured compounds (see Supporting Figure S4).
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Figure 6. Cell-surface LDLRs of hepatocytes were determined by fluorescent imaging. HepG2 cells
were treated (a) with PCSK9 alone (negative control); (b) with PCSK9 and LDLL-1dlnr; and (c) without
PCSK9 (positive control).
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CONCLUSION
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This study was undertaken to find validated, small molecule leads that disrupt PCSK9•LDLR.
Structural modifications to chemotypes 1 are now planned to discover derivatives that retain
and improve on their encouraging binding and increased LDL uptake characteristics, while
simultaneously engineering in features to endow more favorable ADME properties. Thus, the
next steps in the process will probably involve substitution of amino acid side-chains with
bioisosteres to improve bioavailability,48 and perhaps targeting the liver, eg via attaching
ligands implicated in galactose uptake.49-54
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The chemical design component of this study involved conception of possible chemotypes,
implementation of the EKO approach, and one cycle of virtual pharmacophore screening.
Based on the two binding and three cellular assay presented, over half of the 13 test
compounds showed significant, measurable activities. Thus, the strategy brought a degree of
rationality to this process that led to a hit rate (~50%) that would be highly unlikely via high
throughput screening of random compounds against the same target. Moreover, the limitation
of EKO that requires the chemotypes considered must bear three amino acid side-chains is
also a strength insofar as it forces practitioners to explore virgin patent diversity space.
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ASSOCIATED CONTENT
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Supporting Information
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Details of the solid phase syntheses, characterization of compounds A and 1 – 3, protocols for
the biological assays (LDL uptake, binding via SPR/ELISA/TR-FRET, MTT cell viability, and
determination of relative levels of LDLR expression), determination of water solubilities,
photoaffinity labeling, computational procedures, and predicted physiochemical characteristics.
This material is available free of charge via the internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
burgess@tamu.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
We thank Dr John Kulp of Small Molecule PPI Mimics LLC for useful discussions, Mr Jiang
Zhengyang for help with the flow cytometry experiments, and Dr Jay Horton of UT
Southwestern for the gift of the PCSK9 protein used in these studies. We thank DoD BCRP
Breakthrough Award (BC141561), CPRIT (RP150559 and RP170144), The Robert A. Welch
Foundation (A-1121). The NMR instrumentation at Texas A&M University was supported by a
grant from the National Science Foundation (DBI-9970232) and the Texas A&M University
System. The SPR studies were conducted in the protein interaction core at the Institute of
Biosciences and Technology, TAMU, Houston, TX.
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