Atherosclerosis 330 (2021) 52–60

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Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

The emerging landscape of peptide-based inhibitors of PCSK9

Benjamin J. Tombling 1, Yuhui Zhang 1, Yen-Hua Huang , David J. Craik , Conan K. Wang *
Institute for Molecular Bioscience, Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Queensland,
Brisbane, Qld, 4072, Australia

A R T I C L E I N F O A B S T R A C T

Keywords: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a clinically validated target for treating cardiovascular
PCSK9 disease (CVD) due to its involvement in cholesterol metabolism. Although approved monoclonal antibodies
Peptides (alirocumab and evolocumab) that inhibit PCSK9 function are very effective in lowering cholesterol, their lim­
Combinatorial libraries
itations, including high treatment costs, have so far prohibited widespread use. Accordingly, there is great in­
Disulfide-rich peptides
Protein-protein interaction
terest in alternative drug modalities to antibodies. Like antibodies, peptides are valuable therapeutics due to their
high target potency and specificity. Furthermore, being smaller than antibodies means they have access to more
drug administration options, are less likely to induce adverse immunogenic responses, and are better suited to
affordable production. This review surveys the current peptide-based landscape aimed towards PCSK9 inhibition,
covering pre-clinical to patented drug candidates and comparing them to current cholesterol lowering thera­
peutics. Classes of peptides reported to be inhibitors include nature-inspired disulfide-rich peptides, combina­
torially derived cyclic peptides, and peptidomimetics. Their functional activities have been validated in
biophysical and cellular assays, and in some cases pre-clinical mouse models. Recent efforts report peptides with
potent sub-nanomolar binding affinities to PCSK9, which highlights their potential to achieve antibody-like
potency. Studies are beginning to address pharmacokinetic properties of PCSK9-targeting peptides in more
detail. We conclude by highlighting opportunities to investigate their biological effects in pre-clinical models of
cardiovascular disease. The anticipation concerning the PCSK9-targeting peptide landscape is accelerating and it
seems likely that a peptide-based therapeutic for treating PCSK9-mediated hypercholesterolemia may be clini­
cally available in the near future.

1. Introduction
Cardiovascular disease (CVD) is the leading cause of death globally
(17.9 million deaths per year; World Health Organization) and is a major
burden on healthcare systems and economies [1,2]. Low density lipo­
protein (LDL) is known to be a causal factor in the pathophysiology of
atherosclerotic CVD [3–5]. Plasma LDL burden is commonly estimated
by measuring plasma LDL cholesterol (LDL-C) levels, and so strategies
that reduce circulating LDL-C have become a major focus for prevention
of CVD events.
To reduce LDL levels, patients are generally prescribed statins, for
example atorvastatin, which inhibit HMG-CoA reductase – the rate
limiting enzyme involved in the cholesterol biosynthetic pathway. The
CVD benefits of statin medication are associated with their LDL-C
lowering action [6]. However, recent evidence has highlighted their
undesirable properties such as side effects [7] and more alarmingly,
their inability to reduce LDL-C to desirable and safe levels for ∼ 50% of
patients [8,9], leaving patients exposed to an increased risk of CVD. The
limitations of statins have stimulated research towards discovering new
therapeutics with different modes of action for cholesterol management.
In particular, one therapeutic avenue for cholesterol regulation that has
achieved clinical validation is the development of therapeutics that
modulate proprotein convertase subtilisin/kexin type 9 (PCSK9).
In this review, we provide an overview of the molecular mechanism
driving PCSK9’s role in lipid modulation and introduce the therapeutic
modalities that are approved or under clinical development that hinder
PCSK9 function. We focus on examining peptide inhibitors of PCSK9
currently reported. Finally, we look to future experiments that will help
progress the clinical development of peptides for managing LDL levels.

* Corresponding author.
E-mail address: c.wang@imb.uq.edu.au (C.K. Wang).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.atherosclerosis.2021.06.903
Received 30 March 2021; Received in revised form 18 May 2021; Accepted 23 June 2021
Available online 26 June 2021
0021-9150/© 2021 Elsevier B.V. All rights reserved.
B.J. Tombling et al.
2. PCSK9 role in lipid metabolism
PCSK9 is a high-profile success story of modern medical genomics.
Identification of the critical role of PCSK9 in lipid metabolism started in
2003, when PCSK9 was identified to be the third locus associated with
the autosomal dominant disease familial hypercholesterolemia (FH)
[10]. FH affects ∼ 1:200 people globally [11] making it the most com­
mon inherited metabolic disorder, but it is commonly underdiagnosed
and undertreated in the general population, which accentuates its clin­
ical severity [12]. FH is attributed to mutations in the protein machinery
responsible for cholesterol metabolism, which can limit a patient’s
ability to remove LDL-C from the blood [13,14]. Genetic studies iden­
tified mutations in the PCSK9 gene that were associated with elevated
circulating LDL-C and were called gain-of-function (GOF) mutations.
Patients carrying these mutations are subject to an increased risk of
encountering premature CVD events [15,16], suggesting PCSK9 might
be a druggable target. In addition, PCSK9 loss-of-function (LOF) muta­
tions have been shown to engender patients with significantly lower
LDL-C whilst experiencing no adverse health issues [17–19], confirming
that PCSK9 is not only an effective target but also that it can be safely
inhibited.
The cholesterol-regulatory biological mechanism of PCSK9 is now
well established. PCSK9 is a major regulator of hepatocyte surface LDL
receptor (LDLR) levels by inhibiting the receptor recycling pathway
[20–22]. LDLR function is pivotal to cholesterol homeostasis as it is
responsible for the cellular uptake and subsequent degradation of LDL
(see Fig. 1A) [23]. Circulating LDL binds to the N-terminal ligand
binding domain of LDLR via apolipoprotein B100, and the LDLR:LDL
complex becomes internalized by receptor-mediated endocytosis [24].
After migrating to the endosome, the low pH environment allows LDLR
to release LDL and recycle back to the cell surface. Disassociated LDL is
transported to the lysosome where it is degraded to provide cholesterol
and amino acid building blocks to the cell. PCSK9 intervenes with
LDLR’s ability to recycle by binding to LDLR on hepatocyte surfaces (see
Fig. 1B) [25]. A recent study exposed the ability of liver heparan sulfate
proteoglycans to capture PCSK9 and present it in a favorable confor­
mation for LDLR interaction [26]. After migration of the PCSK9:LDLR
complex to the acidic endosomal compartment through clathrin-coated
pits, conformational changes to LDLR result in the formation of addi­
tional binding sites with PCSK9 [27]. As a result, PCSK9 chaperones
LDLR to the lysosome for degradation, preventing LDLR recycling and
effectively up-regulating circulating LDL.
PCSK9 is a member of the proprotein convertase family and is pre­
dominately synthesized in the liver as a ∼ 73 kDa zymogen consisting of
a signal peptide (M1-A30), prodomain (Q31-Q152), catalytic domain
(S153-Q454) and C-terminal domain (L455-Q692) (Fig. 2A) [28]. After
53
Atherosclerosis 330 (2021) 52–60
expression and cleavage of the signal peptide, PCSK9 undergoes intra­
cellular autocatalytic processing between Q152-S153 of the prodomain.
Although cleaved, the prodomain remains non-covalently bound to the
catalytic domain, preventing further catalytic activity by blocking the
catalytic site [29,30]. The mature PCSK9 heterodimeric complex is
necessary for PCSK9’s cholesterol-regulatory function as it stabilizes the
active structure for efficient LDLR interaction and likely assists with
secreting PCSK9 into the extracellular space [31].
Structural studies have revealed the most dominant PCSK9:LDLR
interaction (Fig. 2B) in the extracellular space is composed of a ∼ 500 Å2
binding interface between the PCSK9 catalytic domain and the
epidermal growth factor-like repeat-A (EGF-A) domain of LDLR (Fig. 2C)
[32–34]. EGF-A is a microdomain component of the EGF-precursor ho­
mology domain, which plays a pivotal role in LDLR recycling from the
endosomes to the hepatocyte surface [35]. The most prevalent and
active PCSK9 GOF mutation, [D374Y]PCSK9, is directly involved in the
PCSK9:EGF-A interface and results in a ∼ 25-fold improved affinity for
the LDLR at physiological conditions [36–38]. In addition, the LDLR
GOF mutation [H306Y]LDLR is located on the EGF-A domain, and re­
sults in an improved PCSK9:LDLR interaction [38]. Both mutations are
leading causes of severe FH [36,39] and highlight the clinical impor­
tance of the EGF-A interface for PCSK9 function. In summary, genetic,
functional, and structural studies on PCSK9 have provided a basis for the
development of targeted therapeutics.
3. Therapeutic strategies to inhibit PCSK9
The approval of monoclonal antibodies that inhibit the PCSK9:EGF-A
interaction was an exciting advance in the field of cardiovascular drug
development. It provided validation that inhibiting the PCSK9:EGF-A
interaction is an approach for silencing PCSK9 function and lowering
LDL levels (see Fig. 1C). Monoclonal antibodies can bind to the EGF-A
site on the catalytic domain of PCSK9 with potent affinities [40]. Mul­
tiple Phase III clinical trials have demonstrated that the FDA approved
monoclonal antibodies, evolocumab and alirocumab, can lower circu­
lating LDL-C by ∼ 60% and achieve significant reductions in the
occurrence of CVD events when administered in combination with sta­
tins [41–43]. Furthermore, the antibodies reduce lipoprotein (a) (Lp [a])
and triglyceride levels, which are both risk factors for CVD, highlighting
the benefits of targeting PCSK9 for improving the overall lipid profile
[44].
Despite the enthusiasm following the approval of evolocumab and
alirocumab, these antibodies have seen limited clinical application
because their high treatment costs financially prohibit widespread
administration [45,46]. For example, in Australia the antibodies cost
between A$7000 and A$8000 per year which exceeds the general cost
Fig. 1. PCSK9 modulates LDLR and is a thera­
peutic target for lowering LDL.
(A) LDL is removed from plasma circulation by
LDLR in a receptor-mediated endocytosis pathway.
(B) PCSK9 downregulates LDLR by binding to the
receptor at the cell surface, preventing receptor
recycling and leading the receptor to be trans­
ported to the lysosome for degradation and PCSK9
would be also degraded in lysosome with it. (C)
Therapeutic modalities that block the PCSK9:LDLR
interaction are a validated approach to restore re­
ceptor function and lower circulating LDL levels.
B.J. Tombling et al.
effectiveness threshold for therapeutics [47,48]. This economic factor is
probably partly responsible for the early discontinuation rate of
PCSK9-mediated therapy in the clinic [49]. The failure of these anti­
bodies to dominate the PCSK9 inhibition market has ultimately left the
door open for alternative drug modalities. For example, small molecules
[50–54], small interfering RNA inhibitors [55,56],
monoclonal-antibody mimetics [57,58], nucleic acid polymers [59],
vaccines [60,61], and gene-silencing CRISPR-Cas9 technology [62] are
all at different stages of clinical development [63]. Notably, inclisiran,
which is a double-stranded small RNA molecule therapeutic that acts by
blocking the transcription of PCSK9, has been approved recently in
Europe in December 2020 [64]. In addition to these therapeutic in­
terventions, peptides have attracted substantial interest as more acces­
sible PCSK9 antagonists for next-generation cholesterol-lowering
therapy.
3.1. Peptides as PCSK9-targeted therapeutics
Peptides have advantageous therapeutic properties that make them
valuable drug leads. First, peptides can achieve high target specificity
[65]. Second, their ability to mimic complex protein structures means
they can modulate protein–protein interfaces of ‘undruggable’ targets
[65,66]. Third, the smaller size of peptides engenders them with
drug-like properties that are not associated with larger proteins, such as
membrane permeability [67] and oral activity [68]. Their smaller size
also allows for facile functionalization using a wide range of chemical
design strategies that enables a detailed structure-activity relationship
(SAR) profile to be established. Altogether, these qualities have moti­
vated the increased interest regarding peptides as therapeutic modalities
they have the potential to harness the benefits of both small molecules and antibodies. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
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Atherosclerosis 330 (2021) 52–60
Fig. 2. PCSK9 binds to the EGF-A domain of LDLR.
(A) PCSK9 consists of a signal peptide, N-terminal
prodomain (pink), catalytic domain (grey) and C-
terminal domain (blue) (UniProt Q8NBP7).
Autocatalytic cleavage between Q152–S153 results
in the prodomain non-covalently binding to the
catalytic domain. The naturally occurring mutants
of PCSK9 that are associated with FH are high­
lighted in their respective domains. Residues in
bold are involved in the PCSK9:EGF-A interface.
(B) The dominant PCSK9:LDLR interaction site
(highlighted in yellow) at the cell surface is be­
tween the catalytic domain of PCSK9 and the EGF-
A domain of LDLR (orange) (PDB 3P5C). (C) The
interaction interface (yellow) between PCSK9 and
EGF-A. (For interpretation of the references to
color in this figure legend, the reader is referred to
the Web version of this article.)
that are intermediate in size between small molecules and biologics
(Fig. 3) [69,70]. This interest is demonstrated by the elevated growth of
the peptide therapeutic market [71].
Given the nature of PCSK9’s physiology and function, there are many
reasons why peptides are an ideal drug modality for its targeted inhi­
bition. The PCSK9:EGF-A interface is flat and featureless which makes
designing small molecule inhibitors with sufficient potency very chal­
lenging (as illustrated in Fig. 3A). Furthermore, the relatively low mo­
lecular weight of peptides, compared to antibodies, means they can be
manufactured more cheaply than antibodies and they do not require
cold chain management. The ability to engineer peptides to enhance oral
bioavailability is beneficial for the long-term treatment of chronic dis­
eases such as FH due to higher patient compliance [72]. Finally, peptides
are likely to have predictable safety profiles based on the monoclonal
antibody outcomes and their shared mechanism of action; however, this
idea will need to be validated in pre-clinical and clinical studies. In
addition to the advantageous properties of peptides, PCSK9’s extracel­
lular mechanism of action avoids the need for inhibitors to possess
cell-penetrating properties, which can sometimes be challenging from a
peptide drug-design perspective. In the following sections, we will re­
view literature that provide evidence for the therapeutic potential of
peptides in the PCSK9 drug development landscape.
4. Peptide inhibitors of the PCSK9:LDLR interaction
Fig. 4 shows that efforts are accelerating towards developing
peptide-based PCSK9 inhibitors for therapeutic use. Since 2008, hun­
dreds of peptides that inhibit PCSK9 function have been reported in the
literature or in patents. Notable candidates are highlighted in Table 1,
Fig. 3. Peptides have potential as new modalities
for inhibiting PCSK9.
There are three main modalities which have been
considered for disrupting PCSK9 function: small
molecules (grey), peptides (blue) and large bi­
ologics (green, PDB 3H42). Although small mole­
cules generally have good drug-like properties,
they are not suited for targeting the flat and
featureless interface between PCSK9 and EGF-A
(PCSK9 in grey; interface in yellow). Large bi­
ologics have potential for specificity but have
encountered several limitations that have stalled
their clinical use. Peptides could fill the gap be­
tween small molecules and large biologics because
B.J. Tombling et al. Atherosclerosis 330 (2021) 52–60

Fig. 4. Development of PCSK9-targeted

peptide inhibitors have attracted increasing
interest. Since the EGF-A peptide was
discovered to inhibit PCSK9 in 2008,
worldwide interest in the development of
peptide-based drugs targeting PCSK9 has
grown. This timeline highlights some of the
key discoveries over that period. These
peptides can be categorized into three clas­
ses: nature-derived peptides (blue) [74–77,
92,93], combinatorially derived peptides
(orange) [80–84], and peptidomimetics
(purple) [85,86,101]. Three-dimensional
structures of selected peptides are shown
and colored according to their assigned
class. Disulfide bonds are shown in yellow.
Arrows connecting structures provide an
indication of the progression of initial gen­
eration peptides to more recent ones with
improved therapeutic properties, such as
enhanced activity (e.g. tEGF-A to
TEX_S2-03). (For interpretation of the refer­
ences to color in this figure legend, the
reader is referred to the Web version of this
article.)

Table 1
Published peptides that inhibit the PCSK9:LDLR interaction.
Peptide Sequencea Activity (IC50; nM) b Interface area (Å2) e Ref.
c
LDLR 697 aa (UniProt P01130) 600–750 ∼ 506–525 [34,73]
Nature-derived
EGF-A GTNECLDNNGGCSHVCNDLKIGYECLCPDGFQLVAQRRCE 3000 ∼ 515 [74]
EGF66 GTNECLANLGGCSHICRKLKIGYECLCPDGFQLVAQRRCE 3 – [75]
(tEGF-A) GTNECLDNNGGCSHVCNDLKIGYECL 16000 ∼ 515 [76]
TEX-S3_01 GTNECLSSKYPWEPYWNGGCSHVCNDLKIGYECL 600 – [77]
P5 LILPKHSDAD 2000 – [78]
T9 GQEQSHQDEGVIVR 320000 – [78]
d
T9D8A_1 GQRQWKQAEGVMVR 150000 – [79]
Combinatorially derived
Pep2-8 TVFTSWEEYLDWV 800 ∼ 397 [80]
Fusion 1 TVFTSWEEYLDWVGSGCRLPWNLQRIGLPC 20 ∼ 844 [81]
Groove binding MESFPGWNLV (hR)IGLLR 30000 ∼ 674 [81]
78 Bicyclic peptide 1 ∼ 474 [82]
P9-38 CTVFTSWEEYLDWNNLHPRNSC 20 – [83]
Tetra-P9-38 4 x P9-38 (functionalized dendrimer) 0.2 – [84]
Peptidomimetics
RIm13 Polyimidazole scaffold with functional sidechains 2000 – [85]
Compound 15 ––WNLK (hR)IsLLR 750 ∼ 635 [86]

a
= 1-amino-4-phenylcyclohexane-1-carbony; hR = homo-Arg; s = D-Ser.
b
Ability to inhibit the PCSK9:LDLR interaction.
c
dissociation constant (KD).
d
Inhibitory activity against [D374Y]PCSK9:LDLR interaction.
e
Determined from analysis of the peptide:PCSK9 crystal structures using the PDB ePISA tool.

which also shows their respective inhibition activities. For ease of
reference, we have divided PCSK9 peptide inhibitors into three main
classes: nature-derived peptides, combinatorially derived peptides, and
peptidomimetics. As described in more detail below, we regard nature-
derived peptides as those that originate from natural sources (e.g.
human or plants); for example EGF-A is from the human LDLR, and P5 is
from plant lupin. Combinatorially derived peptides include peptides
discovered from phage, mRNA or other display technologies. Peptido­
mimetics refer to those that were computationally designed to mimic
structures of bound inhibitors and are predominantly non-proteinogenic
in composition.
4.1. Nature-derived peptides
4.1.1. EGF-A-derived peptides
Following the discovery that the EGF-A domain of LDLR is the pri­
mary binding site for PCSK9 [32], EGF-A-derived peptides were of in­
terest towards developing PCSK9 antagonists. Under physiological

55
B.J. Tombling et al.
conditions, the isolated EGF-A domain displays similar affinity for
PCSK9 compared to full-length LDLR (KD of 1 μM and 0.7 μM, respec­
tively) [34,73] and binds to PCSK9 in a calcium- and pH-dependent
manner [74]. Furthermore, EGF-A showed functional activity by pre­
venting PCSK9-mediated LDLR degradation which led to increased LDL
uptake in liver cells.
Zhang et al. extended these findings and discovered EGF-A analogues
with improved affinity for PCSK9 using phage display [75]. Phage
display is a powerful affinity selection technique used to discover
bioactive peptides towards a protein target of interest. A phage library
based on the EGF-A scaffold was constructed, where PCSK9 binding
residues (N295, D299, N301, H306, V307, N309, and D310 – LDLR
residue numbering) were randomized, and panned against human
PCSK9. The most potent EGF-A analogue, EGF66, inhibited the PCSK9:
LDLR interaction with an IC50 of 3 nM, substantially improved from the
μM activity of native EGF-A (see Table 1). However, due to synthesis
difficulties, EGF66 was fused to fragment crystallizable (Fc) domains for
further binding and activity analysis. EGF66-Fc restored LDLR surface
levels with 10-fold stronger activity than native EGF-A. The improved in
vitro antagonistic activity of EGF66-Fc translated to more potent in vivo
activity as LDLR levels were fully restored in mice livers when intrave­
nously administered at 60 mg/kg whereas EGF-A-Fc only partially
restored LDLR levels.
A different approach to engineering EGF-A has been investigated, in
which EGF-A was truncated between L318-C319, thereby removing the
C-terminal region. The truncated analogue, tEGF-A, showed a similar
structure, affinity, and activity to full length EGF-A [76]. Furthermore,
tEGF-A was mutated to incorporate the LDLR GOF mutation H306Y,
which showed improved affinity for PCSK9 in vitro, but did not lead to
increased LDLR function in cell-based assays. A separate recent study
showed full length EGF-A and tEGF-A to be both active against the
FH-associated [D374Y]PCSK9 [87]. Although the authors claimed that
the EGF-A peptides showed similar efficacy to the approved PCSK9 in­
hibitor alirocumab, this perhaps requires further validation given the
sub nanomolar affinity of alirocumab compared to μM affinity of EGF-A
[88].
We recently showed that EGF-like domains have high sequence and
segment length variability, despite their conserved Cys frameworks and
overall topologies [89]. The plasticity of the EGF scaffold inspired us to
re-engineer tEGF-A by extending its segments that are directly involved
in PCSK9 interaction [77]. The most potent extended tEGF-A analogue
identified from phage library screening, TEX-S2_03, displayed
>100-fold improved affinity for PCSK9 compared to parent tEGF-A,
which translated to improved functional activity in cell-based assays.
The reason for TEX-S2_03’s improved affinity has not yet been fully
understood, but binding analysis suggested the extended segment may
occupy an additional binding site which could be exploited after further
SAR.
4.1.2. Lupin protein-derived peptides
Some enzyme-digested fragments from food-derived proteins have
the ability to inhibit the PCSK9:LDLR interaction. Two peptides isolated
from pepsin and trypsin cleavage of lupin, called P5 and T9, competi­
tively bound to PCSK9 with moderate μM activity and were able to
restore cholesterol uptake (Table 1) [78]. P5 displayed the highest ac­
tivity (IC50 of 2 μM). In addition, the lupin peptides were tested against
the GOF [D374Y]PCSK9 mutant to test their application as treatments
for FH. Interestingly, the most active compound against wild-type
PCSK9, P5, was inactive against [D374Y]PCSK9 [90]. On the contrary,
T9 had slightly improved activity for the FH associated PCSK9 mutant,
inhibiting the [D374Y]PCSK9:LDLR interaction with an IC50 of 280 μM
versus 320 μM for wild-type PCSK9. Optimization of T9 by computa­
tional design (T9D8A_1) achieved a ∼ 2-fold improvement in [D374Y]
PCSK9 antagonistic activity and restored cellular LDLR function more
efficiently [79]. Studies indicated the lupin peptides can cross Caco-2
cell monolayers [91,92], which is an established model of the intestinal
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Atherosclerosis 330 (2021) 52–60
epithelial barrier, suggesting they may display oral bioavailability. It
would be interesting to further evaluate the in vivo activity of these
peptides. The activity and cell permeability of lupin-derived peptides
suggest they are promising starting points for further optimization.
4.1.3. Peptides derived from PCSK9
Most studies focusing on inhibiting the PCSK9:LDLR interactions
have designed compounds that bind to PCSK9. However, Alghamdi et al.
investigated an interesting alternative – targeting LDLR as a pathway to
achieve PCSK9 inhibition [93]. They designed a panel of peptides
derived from overlapping segments of the PCSK9 catalytic domain and
showed the peptide named CP3 (which consisted of residues
G365–G384) displayed μM activity in restoring cell surface LDLR levels.
Future designs could involve incorporating the D374Y mutation to
improve activity. Overall, that study suggested blocking the PCSK9
binding site on LDLR can achieve PCSK9 inhibition, but further studies
are required to validate the efficacy of this approach as the LDLR might
be involved in other biological functions.
4.2. Combinatorially-derived peptides
4.2.1. Pep2-8-derived peptides
Combinatorial library screening of randomized peptide sequences
has provided a foundation for de novo identification of non-EGF-A based
peptide inhibitors of PCSK9. Zhang et al. used a series of randomized
linear peptide phage libraries to identify the 13-residue PCSK9 antago­
nist Pep2-8 (see Table 1) [80]. In vitro studies showed that Pep2-8 had
moderate affinity for PCSK9 (KD of 0.7 μM) and competitively bound to
the EGF-A binding site (IC50 of 0.8 μM). Furthermore, Pep2-8 showed
good selectivity against other members of the proprotein convertase
family, including furin, PC1, PC2, PC4 and PC9. Cell-based functional
assays confirmed the ability of Pep2-8 to reduce LDLR degradation and
promote LDL internalization in the presence of PCSK9. The Pep2-8:
PCSK9 crystal structure showed that Pep2-8 forms a ß-strand-turn-he­
lix motif, with the N-terminal β-strand mimicking EGF-A binding.
Despite Pep2-8 providing proof-of-concept that small peptide-based
inhibitors of PCSK9 were achievable, limitations such as its poor solu­
bility and moderate activity have motivated interest in the design of
improved therapeutic leads. Computationally-derived analogues of
Pep2-8 were reported to display improved antagonistic activity, but
biophysical binding data were not presented [94].
A cryptic binding pocket located near the Pep2-8 binding site on
PCSK9 has been exploited for therapeutic development [81]. The au­
thors showed that the P’ helix at the N-terminus of the catalytic domain
of PCSK9 (S153–R167) is intrinsically flexible; alternating from a bound
conformation on the PCSK9 surface to being extended away in an open
conformation which exposes a binding pocket. Using a phage library
that contained a randomized peptide motif extended off the C-terminus
of Pep2-8 led to the discovery of fusion peptides that displayed potent
bivalent affinity (KD of 2–6 nM) and antagonistic activity (see Table 1).
The fusion peptides were truncated to result in peptides that solely
bound to the cryptic binding site and displayed moderate PCSK9
antagonistic activity (IC50 = 30 μM).
To improve the affinity and inhibitory function of Pep2-8, we
introduced a design strategy termed bioactive cyclization [83]. A func­
tional cyclization linker was incorporated onto the Pep2-8 binding motif
that was composed of experimentally optimized amino acids that spe­
cifically participate in favorable interactions with PCSK9. The increased
binding face achieved by the cyclic peptide identified using this
approach, P9-38, resulted in >100-fold improved affinity compared to
Pep2-8 and demonstrated significant cell-based functional activity at
low nanomolar concentrations.
Recently we used dendrimer scaffolds (branched polyvalent struc­
tures that can be chemically functionalized to display multiple copies of
a bioactive compound) to further optimize the activity of the peptide P9-
38 by increasing valency, which we hypothesized would increase the
B.J. Tombling et al.
local effective concentration at the PCSK9 binding site [84]. Bivalent
and tetravalent analogues of P9-38 inhibited the PCSK9:LDLR interac­
tion with ~60-fold (IC50 = 0.3 nM) and ~100-fold (IC50 = 0.2 nM)
improvement over the parent peptide, respectively. The
P9-38-decorated dendrimers are two of the most potent peptide-based
PCSK9:LDLR interaction inhibitors reported to date.
4.2.2. Cyclic peptide inhibitors of PCSK9
Peptides that adopt a backbone cyclic structure are often attributed
with therapeutically advantageous properties such as a reduced entropic
penalty upon target binding and increased protection against proteolytic
degradation [95,96]. As a result of these advantageous properties, cyclic
peptides are arguably the most likely peptide class to achieve approval
for clinical use against PCSK9. Recent years have witnessed a surge in
filed patents describing the invention of potent cyclic peptide inhibitors
of PCSK9. Two patents from RA Pharmaceuticals and Merck (WO
2019/246387 A1 and WO 2019/246405 A1) describe >100 cyclic
polypeptides that inhibit the PCSK9:LDLR interaction with IC50 values in
the low nanomolar range [97,98].
The SAR studies of peptides reported by Merck were detailed in a
recent paper and underpinned extensive efforts to optimize a mRNA
library derived hit peptide into smaller, highly stable and activity
enhanced lead compounds [82]. Although >1000 improvement in ac­
tivity was observed in some cases (e.g. compound 78 with Ki of 1 nM),
the authors identified undesirable off-target effects on mast cell
degranulation of some of their compounds. However, they showed the
side-effect was specific to chemical composition and could be reme­
diated through specific amino acid substitutions, suggesting this issue
might be specific to this study rather than being a broader concern for
peptide inhibitors of PCSK9. That study also provided important insight
into the pharmacokinetic properties of PCSK9-targeted peptides, with
several peptides having been evaluated in mice, rats, or monkeys. The
peptides tested had short half-lives after intravenous administration,
with half-lives ranging from 0.25 to 1.60 h in mice and 0.06–2.46 h in
rats. A bicyclic peptide, compound 75, had a half-life of 2.84 h in
monkey. These results point towards the need to consider strategies to
improve in vivo half-lives.
Two patents from Novartis described novel cyclic peptide inhibitors
of PCSK9. One of the patents (WO 2020/110011 A1) describes the in­
vention of 13-mer cyclic peptide thioesters, with the peptide P2 dis­
playing high antagonistic activity (IC50 of 40 pM) [99]. The second
patent (WO 2020/110009 A1) details a suite of cyclic tetramer com­
pounds with a peptide-like backbone functionalized with a broad range
of non-natural amino acid side chains [100]. The most potent cyclic
tetramer, P3, achieved impressive PCSK9 antagonistic activity (IC50 of
150 pM) and its low molecular weight (918 Da) is advantageous from an
oral delivery perspective. Overall, these patents demonstrate a growing
interest from pharmaceutical companies towards the development of
peptide-based PCSK9 inhibitors and suggest peptides will play a pivotal
role in next-generation cholesterol-lowering therapy.
4.3. Peptidomimetics
The interaction of EGF-A and Pep2-8 with PCSK9 highlights that
efficient PCSK9 interaction can be mediated by formation of ordered
structural motifs, specifically β-strands, by the inhibitor peptide. This
inspired research functionalizing poly-imidazole scaffolds to mimic
peptide secondary β-sheet structures and develop PCSK9 antagonists
that compete with EGF-A [85,101]. The most active compound identi­
fied so far, RIm13 which is an analogue of MeIm, inhibits the PCSK9:
LDLR interaction with a reported IC50 of 1 μM, suggesting
poly-imidazole scaffolds are promising templates for PCSK9-targeted
drug design. Future studies aimed at optimizing the scaffold functional
groups to strengthen interactions with PCSK9 may yield analogues with
improved activity.
The groove-binding peptide (compound 15) highlighted above was
57
Atherosclerosis 330 (2021) 52–60
the subject of further structure-activity guided optimization, which
identified a bipartite molecule consisting of a cryptic binding peptide
with a N-terminal organic 1-amino-4-phenylcyclohexane-1-carbonyl
moiety [86]. The functionalized N-terminus mimicked key hydropho­
bic interactions adopted by EGF-A and led to significant improvements
in antagonistic activity over the parent peptide (IC50 = 750 nM;
Table 1). Unfortunately, attempts to truncate the peptide region to
design inhibitors with low molecular weight were not successful.
However, further optimization of peptide-based compounds targeting
the cryptic binding site of PCSK9 are ongoing and it is hoped this
strategy will lead to inhibitors suited to oral delivery [102].
5. Alternative roles of PCSK9 beyond LDLR regulation
5.1. PCSK9’s role in atherosclerosis beyond lipid homeostasis
PCSK9 has been found to accelerate the formation of atherosclerosis
independently of lipid regulation [103]. For example, PCSK9 is linked to
pathways that affect the integrity of endothelial cell monolayers and
lead to advanced atherosclerosis [104,105]. Levels of PCSK9 have also
been associated with increased proliferation rate of smooth muscles
cells, which is a contributing factor to atherosclerosis [104]. In mono­
cytes and macrophages, PCSK9 increases the expression of cytokines like
IL-6, TNF-α, CXCL2, which advance the pro-inflammatory state [106].
The link between PCSK9 and inflammation is further supported by the
effect of PCSK9 monoclonal antibodies, alirocumab and evolocumab, on
reducing inflammation and increasing the synthesis of the
anti-inflammatory factor IL-10 [107].
5.2. PCSK9 is a promising therapeutic target for other diseases
Following its causal relationship with FH and CVD, PCSK9 has been
extensively studied to assess whether it is a genetic marker for other
diseases. A mendelian randomized study showed that patients with
PCSK9 LOF mutations were associated with an increased risk of type 2
diabetes [108]. So far, clinical trials investigating the PCSK9 monoclonal
antibody inhibitors have reported no significant increase in the risk of
new-onset diabetes [109]; however, long-term follow up studies are
required to fully understand the effects of PCSK9 inhibition therapy. In
addition, there is overwhelming experimental and clinical evidence
indicating increased plasma levels of PCSK9 leads to systemic inflam­
mation, and PCSK9 inhibitors can ameliorate pro-inflammatory effects
[110].
A recent study proposed PCSK9 inhibition is a safe and efficacious
strategy for treating dengue fever [111]. This high-profile infectious
disease affects ∼ 400 million people each year, highlighting an impor­
tant new use for PCSK9 inhibition. The PCSK9 monoclonal antibody
inhibitor evolocumab is currently undergoing testing in a clinical trial to
prevent cardiac allograft vasculopathy – a major cause of mortality after
heart transplant surgery [112]. Finally, PCSK9 has recently been shown
to be a promising target to enhance immune checkpoint therapy for
treating cancer [113]. Overall, the volume of research highlighting
several health benefits of targeting PCSK9 is exciting and further studies
may only accentuate the clinical importance of developing PCSK9
inhibitors.
6. Summary and outlook
There is now clear support for the therapeutic potential of peptides as
drugs targeting PCSK9, with over one dozen studies reporting a diverse
range of peptide-based inhibitors of PCSK9. Collectively, these studies
have demonstrated that peptides can inhibit the PCSK9:LDLR interface
with high potency in the pM–nM range [75,81–84] and have potential
for specificity [74], oral delivery [92], and in vivo efficacy [75]. With the
promising results reported so far, why have peptides not seen greater
clinical investigation? Perhaps it is because the focus has been too
B.J. Tombling et al.
strongly on discovery of new leads with binding affinity to PCSK9, and
not enough on other properties required of drugs. All studies have
provided in vitro characterization of binding and/or competitive activity
in biochemical, biophysical or cellular assays. However, few have
explored questions on specificity [74,82], cell permeability [92], in vivo
bioavailability [82], and in vivo activity [75]. Only peptides discovered
from mRNA display [82] and an EGF-A analogue as a Fc-fusion protein
[75] have been tested in vivo. The gap in knowledge on aspects beyond in
vitro activity might be associated with some of the general drawbacks of
peptides. Fortunately, there are clinically validated peptide design
strategies that can help propel peptides to the clinic.
Increasing the short circulating half-life of peptides perhaps repre­
sents the most important challenge that needs to be overcome for pep­
tides to be suitable therapeutics for targeting PCSK9. The small size of
peptides means they are subject to rapid renal clearance. Indeed, small
cyclic peptide inhibitors of PCSK9 were shown to be rapidly cleared in
mice [82]. One validated strategy to improve the half-life of therapeutic
peptides both in vitro and in vivo is to fuse the bioactive peptide to tags
that confer abundant serum protein binding, such as albumin
[114–117]. For example, the approved glucagon-like peptide-1 receptor
agonists liraglutide and semaglutide are engineered with albumin
binding tags and subsequently require once-weekly subcutaneous in­
jection for the treatment of type 2 diabetes and weight management
[118,119]. Increasing drug circulation time and accumulation of
PCSK9-targeting peptides would increase their in vivo efficacy and
reduce the dosing frequency required to invoke a cholesterol-reducing
response.
A major advantage of peptides over subcutaneously administered
monoclonal antibodies is their potential to be engineered for effective
oral delivery. There are several examples where approved peptide
therapeutics are orally dosed, including the immunosuppressant cyclo­
sporine [120]. However, the majority of bioactive peptides do not
achieve sufficient levels of oral bioavailability to allow safe and effective
oral administration. Instead, optimizing the formulation can be used to
achieve oral activity through the use of permeability enhancers [121].
Little work has been done in this field for peptide inhibitors of PCSK9.
By addressing the in vivo limitations of peptides there might be
greater interest in in vivo efficacy characterization, which has been
dramatically underexplored. Whilst functional assays using model
human liver cell lines, such as HepG2, have provided an excellent and
reliable experimental platform for pre-clinical activity validation, the
focus needs to progress towards in vivo efficacy characterization. Except
for EGF analogues fused to an Fc domain [75], to the best of our
knowledge no other peptide-based inhibitor has been reported with in
vivo activity to date. And even that study focused on investigating the
regulation of LDLR on liver cells rather than broader atherosclerosis
disease. With the positive results observed for many PCSK9-peptide in­
hibitors in vitro, it would be interesting to test PCSK9-peptide inhibitors
in animal models of atherosclerosis or related metabolic diseases [122,
123] as mono or combination therapies. The effects of PCSK9 are
amplified by statin treatment which induces increased activity of the
sterol regulatory element binding protein 2 (SREBP-2), resulting in
increased PCSK9 expression and secretion [124,125]. Therefore, there
are potential benefits to applying synergistic approaches to achieve
effective cholesterol-lowering therapy.
Inhibition of PCSK9 binding to LDLR is clinically validated. In this
review, we focused on the rapid growth of the discovery of competitive
peptide-based ligands of PCSK9, demonstrating the potential of peptides
as drugs. In our opinion peptides have been underutilized for the
treatment of hypercholesterolemia. A shift in focusing on optimizing the
pharmacokinetics of peptides might be key to their clinical progression.
Financial support
Work in our laboratory on PCSK9 antagonists is supported by a grant
from the National Health and Medical Research Council (APP1107403)
58
Atherosclerosis 330 (2021) 52–60
and by access to the facilities of the Australian Research Council Centre
of Excellence for Innovations in Peptide and Protein Science
(CE200100012).
CRediT authorship contribution statement
Benjamin J. Tombling: Conceptualization, Writing – original draft,
Visualization. Yuhui Zhang: Writing – original draft, Visualization.
Yen-Hua Huang: Visualization, Supervision, Writing – review & edit­
ing. David J. Craik: Supervision, Writing – review & editing, Funding
acquisition. Conan K. Wang: Supervision, Project administration,
Funding acquisition, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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