Introduction
A large number of clinical trials over the last 30 years have firmly consolidated
the importance of lowering low density lipoprotein cholesterol (LDLc) in the prevention
of cardiovascular diseases (CVD) and its associated devastating sequelae.
1
While healthy diets and exercise are highly recommended to lower LDLc levels, in many
individuals with high baseline levels of LDLc, this is not sufficient to bring levels
down to recommended target values in order to prevent recurrent coronary heart disease
and cardiovascular complications. This is especially true for patients at high risk
of premature cardiovascular death and disability, including those with familial hypercholesterolaemia
(FH). FH is a very common inherited disease – affecting at least 30 million people
worldwide, with an overall incidence of 1:200 globally
2
– of whom ≤1% have been diagnosed. The advent of HMG-CoA reductase inhibitors, also
known as “statins”, and their first application to hypercholesterolemic patients over
30 years ago, has revolutionized the treatment of FH patients and resulted in substantial
lowering of LDLc. In addition, cholesterol–lowering drugs, such as “ezetimibe” that
blocks cholesterol absorption from the gut by inhibiting the Niemann-Pick C1-like
1 (NPC1L1) transporter, have also been successful and a 7-year IMPROVE-IT trial revealed
that a “simvastatin-ezetimibe” combination resulted in an incremental lowering of
LDLc levels and a modest 2% improved cardiovascular outcomes.
3
Therefore, it became clear that additional treatments are needed to substantially
decrease LDLc and efficiently protect against CVD.
In 2003, the identification of the proprotein convertase subtilisin-kexin # 9, and
the genetic evidence of its up-regulation of the levels of circulating LDLc
4,5
via the enhanced degradation of the LDL receptor (LDLR)
6
, was an unexpected and welcome addition to the armamentarium of drug targets aimed
to safely lower LDLc to levels never achieved before.
7,8
Indeed, the discovery of PCSK9 and its induced-degradation of the LDLR revolutionized
the field of LDLc-regulation. Amazingly, knowledge went from bench-to-bedside in less
than nine years. A new PCSK9-targeted class of medicine is emerging, representing
the biggest weapon against heart disease since the development of “statins”. The current
crop of PCSK9 inhibitors are injectable monoclonal antibodies (mAb) to treat patients
who cannot tolerate statins, or whose LDLc is not controlled by drugs. Food and Drug
Administration approval of the first of a new class of therapeutics (PCSK9 mAb) was
achieved in 2015. The present review will briefly describe the properties of PCSK9,
our current understanding of its biology and intracellular trafficking, and then discuss
the status of the various approaches that have been proposed to lower the levels of
PCSK9.
The PCSK-family of proprotein convertases subtilisin-kexin types
It became clear from the mid-1960s that many eukaryotic secretory proteins were cleaved
by proprotein convertases (PCs) to generate active peptide/protein products from their
original inactive precursors.
9,10
Cleavage occurred at specific exposed single or paired basic amino acid sites within
the consensus motif (K/R)-X0-6-(K/R)↓, where Arg is the preferred residue at the P1
cleavage site over Lys (Figure 1).
11
This involved a variety of precursors of polypeptide hormones, growth factors, receptors,
enzymes, adhesion molecules, and even cell surface proteins from infectious viruses,
parasites and bacteria. Such widespread precursor activation was found to occur in
most species in both eukaryotes and even in some prokaryotes. However, it subsequently
became apparent that such limited cellular proteolysis can also inactivate specific
bioactive proteins (Figure 1).
12
It took more than 15 years of intensive research by a number of teams in both North
America and Europe to hunt for the elusive PCs, which were estimated to be present
at <100-fold lower concentration than their substrates. Using powerful yeast genetics,
the first successful identification of a PC was reported in 1984 for the processing
of the precursor of α-mating factor (Figure 2).
13
The enzyme, named “Kexin or Kex2p”, turned be an ancient serine protease related to
the bacterial family of subtilisin-like proteases.
14,15
The ability of Kexin to precisely process mammalian precursors at the expected physiological
sites supported the hypothesis that Kexin is a prototype of the as yet unidentified
mammalian proprotein convertases.
16
The first glimpse of the properties of such mammalian proteinases was obtained in
1988 upon analysis of a human insulinoma tumor highly enriched in both hormone and
its processing proteinases.
17
It turned out that two such convertases (type 1 and 2) were needed for the generation
of active human Insulin from its inactive precursor proInsulin. However, it was not
until the advent of gene and cDNA cloning and expression that in 1990 the first 3
members, PC1,
18
PC2,
18,19
and Furin
20
of the PC-family were finally identified, cloned and their activity validated in cells
(Figure 2). The genes coding for these enzymes were named PCSK1, PCSK2 and Furin (Figure
3). From 1990–1997 four more convertases were consecutively identified and cloned,
giving a total of seven basic-residue-specific PCs (Figure 3).
10.7717/gcsp.201702/fig-1
Figure 1.
Schematic representation of the limited proteolysis of secretory precursor proteins.
Notice that such PCSK-generated cleavages can either activate the cognate precursor
by releasing bioactive products or inactivate it by removing bioactive moieties.
10.7717/gcsp.201702/fig-2
Figure 2.
History of the discovery of the proprotein convertases.
The first discovery of Kexin in 1984, led the way to the identification of its 9 mammalian
homologues from 1990–2003.
10.7717/gcsp.201702/fig-3
Figure 3.
Schematic representation of the primary structures of the human proprotein convertases.
The kexin-like basic amino acid (aa)-specific proprotein convertases, pyrolysin-like
subtilisin kexin isozyme 1 (SKI-1; encoded by the MBTPS1 gene) and proteinase K-like
proprotein convertase subtilisin kexin 9 (PCSK9) are individually grouped to emphasize
their distinct subclasses. The various domains and N-glycosylation positions are emphasized,
along with the primary (depicted using light grey arrows, and a light grey double
arrow for SKI-1) as well as the secondary autocatalytic processing sites (depicted
using dark grey arrows). The presence of a signal peptide, a prosegment and catalytic
domain is common to all convertases that exhibit the typical catalytic triad residues
Asp, His and Ser, as well as the Asn residue comprising the oxyanion hole (Asp for
PC2). The carboxy-terminal domain of each convertase contains unique sequences regulating
their cellular localization and trafficking. Thus, PCSK9 exhibits a Cys-His-rich domain
(CHRD) that is required for the trafficking of the PCSK9–LDLR (low-density lipoprotein
receptor) complex to endosomes and lysosomes. (modified from
11
).
The above enzymes differ in their tissue expression and subcellular localization.
21
Briefly, the soluble PC1 and PC2 are found exclusively in dense-core secretory granules
in endocrine and neural tissues, and are responsible for the activation of most polypeptide
hormones.
12
The type I-membrane-bound Furin and PC7 are ubiquitously expressed,
11
and sometimes share similar precursor substrates such as those of Sortilin and Brain
Derived Neurotropic Factor (proBDNF).
22
The soluble PC5A and PACE4 are widely expressed and often activate cell surface precursors,
such as cell surface receptors and growth factors.
12,23
Animals completely lacking the convertases mouse Furin, and human and mouse PC5 have
severe developmental defects, and they die before birth.
11,24,25
In contrast, mice lacking PC7 are quite healthy, and are anxiolytic and novelty seekers.
22
The various physiological and pathological functions of these 7 basic-residue-specific
PCs have been extensively reviewed elsewhere
11,12,23,26
and will not be examined any further in this paper.
In our search for other members of the PCSK-family, in 1999 we identified an eighth
member that we called subtilisin-kexin isozyme 1 (SKI-1), because it was able to cleave
proBDNF at a non-basic site within the recognition motif
R
-X-
L/V/I
-X↓ (Figure 3).
27,28
This is also a type-I membrane bound protease best related to the pyrolysin family
of subtilases
29
. Independently, it was also discovered that SKI-1 (also called site 1 protease) was
responsible for the processing of various membrane-bound transcription factors, such
as sterol regulatory element binding proteins (SREBPs)
30
and the ER stress sensor ATF6.
31
Therefore, SKI-1/S1P plays a major role in the regulation of lipogenic genes, including
those of LDLR and PCSK9, as we shall see later. Because of these activities, the gene
for SKI-1/S1P is now called Membrane-Bound Transcription Peptidase Site 1 (MBTPS1).
Our extensive studies of the properties of this enzyme revealed that it undergoes
a very unique autocatalytic activation, which is quite different from those of the
other seven basic-residue specific PCs.
28,32,33
In addition, this unique enzyme was also reported to activate the phosphorylation
of mannose residues in proteins destined to lysosomes, since it is required to activate
the α∕β-subunit precursor protein of the GlcNAc-1-phosphotransferase forming mannose
6-phosphate (M6P) targeting markers on lysosomal enzymes.
34
Interestingly, this activity is independent of the lipogenic transcription control
by cholesterol and fatty acids.
35
SKI-1/S1P was also shown to be critical for neuronal axonal growth,
36
and for bone osteoblast mineralization.
37
Thus, SKI-1/S1P may have other unsuspected functions in specific tissues that are
independent of SREBPs or ATF6. Caudal regression syndrome (sacral agenesis), which
impairs development of the caudal region of the body, occurs with a frequency of about
1 live birth per 50,000 newborns, although this incidence rises to 1 in 350 infants
born to mothers with gestational diabetes.
The complete knockout of the mouse Mbtps1 is embryonically lethal at very early developmental
stages. Thus, to better understand the role of SKI-1/S1P in osteogenic differentiation
and skeletal development, we used a tissue-specific approach to delete the expression
of SKI-1/S1P in chondrocytes. This conditional Mbtps1 loss-of-function mouse model
exhibits phenotypic changes localized to the lumbar/sacral vertebral region (decreased
vertebral number, vertebral fusion, and kinky tail) that mimic those in caudal regression
syndrome, suggesting that loss-of-function mutations in Mbtps1 may cause the etiology
of this disease.
38
The discovery of PCSK9 and its genetic relationship to LDL
During an exhaustive PCR-based homology search to the PCSKs, in mouse, rat and human
cell lines in 2002 (reported in early 2003),
5
we cloned a novel cDNA sequence encoding a 24–25% identical catalytic subunit (260
aa) to the subtilases SKI-1/S1P, PC7, and tripeptidyl peptidase II. Using a protein
BLAST program (www.ncbi.nlm.nih.gov/BLAST), similar sequences were identified in patented
databases (Millennium Pharmaceuticals, Cambridge, MA, patent no. WO 01/57081 A2; and
Eli Lilly, LP251 patent no. WO 02/14358 A2). The sequence identified by Millennium
was obtained following serum deprivation in primary cerebellar neurons leading to
the development of apoptosis. Thus, the gene product was originally called Neural
Apoptosis Regulated Convertase 1 (NARC-1).
5
Upon inspection of the putative protein sequence we immediately realized that it encoded
a new ninth member of the PCSK-family of proprotein convertases (now called PCSK9)
and showed that it localized to human chromosome 1p32. Not knowing what the function
of the enzyme was, we first defined its tissue and cellular distribution and showed
that it was highly enriched in the adult liver, small intestine, kidney cortex and
cerebellum (Figure 4).
5
In situ hybridization and Northern blot analyses of PCSK9 expression during development,
and in the adult, and after partial hepatectomy, revealed that it is expressed in
cells that have the capacity to proliferate and differentiate. These include hepatocytes,
kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic
brain telencephalon neurons.
5
It was also highly expressed in various tumor-derived cell lines (Figure 5).
10.7717/gcsp.201702/fig-4
Figure 4.
In situ hybridization histochemistry of the expression of PCSK9 mRNA in an embryonic
day 17 (E17) mouse.
Notice the high expression of PCSK9 in liver, small intestine, kidney and cerebellum.
The locus of the human PCSK9 gene on the small arm of chromosome 1p32 is emphasized.
This pattern of tissue expression of PCSK9 was the basis that led us to define its
role in liver in cholesterol regulation.
10.7717/gcsp.201702/fig-5
Figure 5.
Cellular and tissue expression of PCSK9.
Northern blot analysis of PCSK9 mRNA in 21 rat, mouse, and human cell lines (A) and
17 rat tissues (B) (Sm, smooth; Sk, skeletal; d, day). The open arrow points to the
smaller testicular mRNA. The red stars denote high expression of PCSK9 in specific
tumor derived cell lines. The tissues expressing high levels of PCSK9 are boxed in.
Modified from.
5
.
The localization of the PCSK9 gene on the short arm of chromosome 1p32, led us to
initiate a very fruitful collaboration with Catherine Boileau and Marianne Abifadel
in Paris, who were on the lookout for a gene located on chromosome 1p32-p34.1, which
by linkage analysis was thought to represent the third FH locus (FH-3) different from
the LDLR (FH-1) and its ligand apoB (FH-2). It turned out that indeed the gene coding
for PCSK9 is the one responsible for observed hypercholesterolemia phenotype observed
in two French families, with the rare gain-of-function (GOF) mutations S127R and F216L
of human PCSK9.
4
For a more extensive review of the huge amount of detective work that led to this
conclusion, the reader is referred to an excellent review of the history of the identification
of PCSK9 as the third FH locus from their genetic perspective.
39
In essence, the two GOF point mutations in PCSK9 that were identified in the two French
families (from Nantes and Bordeaux) were responsible for the 2-fold (F216L) and 4-fold
(S127R) increase of circulating LDLc in these FH-3 patients (Figure 6). Since this
seminal discovery, a number of rare missense GOF mutations in each of the 12 PCSK9
exons were identified, always leading to higher levels of LDLc (Figure 6). The most
damaging one is the Anglo-Saxon mutation D374Y, occurring in exon 7, first identified
in the Mormon Population in Utah,
40
and later on found in England and other countries.
7
The LDLc in these heterozygous patients is at least 5-fold higher than normal. The
GOF D374Y-PCSK9 causes a severe FH phenotype that is not readily reduced by statins.
41
Carriers of this mutation are typically affected by CVD 10 years earlier than other
FH patients.
10.7717/gcsp.201702/fig-6
Figure 6.
Schematic representation of the exon/intron structure of the PCSK9 gene and the identified
GOF and LOF mutations.
The first reports of GOF (above the gene) and LOF (below the gene) PCSK9 mutations
are emphasized, as well as the countries of origin of each reported mutation. Notice
that the 3 complete LOF mutations of PCSK9 are denoted with green stars with a red
border, and the 0.4 mM levels of LDLc reached.
On the other hand, the more common loss-of-function (LOF) mutations in PCSK9 result
in low levels of circulating LDLc (Figure 6).
42
In fact, complete heterozygote LOF of PCSK9 was estimated to result in an 88% reduced
risk of developing cardiovascular complications over a 15-year follow-up period.
43
Another example is the R46L mutation, which results in a partial LOF, and is associated
with a 15% reduction in LDLc, and a 47% reduction in the risk of coronary heart disease.
43
Amazingly, two women and one man were identified who completely lacked PCSK9 expression,
and all have similarly low levels of LDLc of 0.4 mM, which is about 7–8-fold lower
than normal (Figure 6). These included: (1) an African American woman presenting compound
heterozygote PCSK9 LOF deletion/truncation mutations (ΔR97 + Y142X), apparently in
good health;
44
(2) An African woman from Zimbabwe that presented a homozygote LOF truncation C679X;
45
and (3) A French diabetic Caucasian that presents compound LOF mutations (R104C +V114A).
46
These results, and the observation that patients exhibiting LOF PCSK9 mutations have
an increased LDLc catabolic rate,
46
with apparently no obvious deleterious effects associated with very low LDLc levels,
provided compelling arguments for developing inhibitors of PCSK9 to treat hypercholesterolemia.
The cellular biology of PCSK9 and its trafficking
However, before doing so, it was necessary to understand the mechanism by which high
concentrations of PCSK9 or GOF mutations are associated with high LDLc levels, with
the reverse being true for LOF mutations. Indeed, epidemiological studies suggested
that plasma PCSK9 levels correlate with high LDLc levels,
47–49
suggesting a causal relationship.
Like all of the other seven proprotein convertases (with the exception of PC2 that
has its own chaperone 7B2) PCSK9 undergoes an autocatalytic cleavage of its inhibitory
prodomain at the VFAQ152↓ site in the endoplasmic reticulum (ER), resulting in tightly-bound
heterodimer of the prosegment and the rest of the mature protein,
5,50
that in this state is proteolytically inactive.
23
Such zymogen cleavage allows the protein to exit the ER and traffic though the Golgi
apparatus and is secreted within minutes (Figure 7).
50
However, PCSK9 is in a class of its own, since it always remains in a tight enzymatically
inactive complex with its prodomain (Figure 8) unlike other PCSKs that lose their
prodomain following a second cleavage or dissociation of the prodomain along their
intracellular route before reaching their final destinations. Indeed, the crystal
structure of secreted mature PCSK9 confirmed these biochemical observations and revealed
the very tight association of the prodomain with the catalytic subunit of PCSK9.
51
These data indicated that the circulating PCSK9 is enzymatically inactive due to its
association with the inhibitory prodomain.
10.7717/gcsp.201702/fig-7
Figure 7.
Biosynthesis of PCSK9 in HK293 cells.
The V5-tagged PCSK9 or its active site mutant H226A were transiently expressed in
HEK293 cells. The next day the cells were pulse-labeled with [35S]Met/Cys for 4 h.
Cell extracts (C) and media (M) were immunoprecipitated with a V5 antibody and the
precipitates were resolved by SDS/PAGE on an 8% tricine gel. The migration positions
of molecular mass standards (kDa), proPCSK9, PCSK9 and the prosegment are emphasized,
together with the Furin cleaved product observed. Note that the active site mutant
is neither cleaved not secreted, emphasizing the necessity of prodomain autocatalytic
cleavage for PCSK9 to exit the ER and be secreted.
10.7717/gcsp.201702/fig-8
Figure 8.
Zymogen activation of the proprotein convertases.
The various strategies used by the convertases to get activated. It all starts in
the ER where the first autocatalytic cleavage occurs. Except for PCSK9 all the other
convertases get rid of their inhibitory prodomain to become enzymatically active.
However, the identification of GOF mutations F216L and R218S led to the demonstration
that these PCSK9 mutations are associated with the loss of the ability of another
proprotein convertase – Furin – to cleave and inactivate PCSK9 at the sequence
R
FH
R
218↓.
52,53
This results in the dissociation of the prodomain and segment 153–218, resulting in
a truncated inactive form of PCSK9.
52
This new concept suggested that PCSK9 is subject to inactivation prior to its secretion
from the liver (its main source in the plasma). Indeed, recent mass spectrometry evidence
revealed that up to 40% of circulating PCSK9 is Furin-inactivated.
54
This raises the question of the pertinence of measuring the circulating levels of
PCSK9 using a simple ELISA that recognizes both active and inactive forms, and may
explain the seemingly low level of correlation between circulating total concentrations
of PCSK9 and LDLc.
48
It is expected that the concentrations of the active form of PCSK9 would better correlate
with those of circulating LDLc.
The first indication of the mechanism underlying the observed PCSK9 regulation of
LDLc was reported by Maxwell and Breslow, when they demonstrated that PCSK9 targets
the LDLR towards an intracellular degradation compartment
6
We showed that this degradation occurs in an acidic compartment, and that it involves
endocytosis of the cell surface PCSK9 ≡ LDLR complex into clathrin heavy chain coated
early endosomes.
55
It was later shown by the group of Horton and Hobbs that the catalytic domain of PCSK9
binds the EGF-A domain of the LDLR,
56
and that the enzymatic activity of PCSK9 is not necessary for its induced degradation
of the LDLR
57
Interestingly, the GOF PCSK9-D374Y mutant was found to bind to the LDLR with a 6-
to 30-fold higher affinity compared with wild-type PCSK9, by reinforcing a hydrogen
bond between PCSK9 and the EGFA domain of the LDLR.
51
In fact, it seems that the negative charge of Asp374 is the critical negative factor,
as it can be replaced by Glu374, but its replacement by uncharged or hydrophobic residues
results in similar GOF as Tyr374.
58
It is now well accepted that the bioactive heterodimeric prodomain ≡ PCSK9 binds the
EGF-A domain of the LDLR,
59
and the trimeric prodomain ≡ PCSK9 ≡ LDLR complex is escorted to endosomes/lysosomes
for degradation, but that the underlying details of the trafficking mechanism remain
obscure.
7
Therefore, the 3 FH genes interact with each other as PCSK9 binds both the LDLR
6
and apparently apoB
60
(Figure 9).
10.7717/gcsp.201702/fig-9
Figure 9.
Schematic representation of the incidence of FH-1, 2 and 3 mutations and a model emphasizing
the mutual binding of the LDLR, apoB and PCSK9.
The tendon xanthomas seen in FH patients are shown on the top left corner.
However, we consistently observed that mutants of LDLR (e.g., LDLR-L339D) that cannot
be targeted to endosomes/lysosomes by extracellular PCSK9, are still degraded intracellularly
in various cell lines by co-expressed PCSK9. This led to the discovery of an intracellular
pathway of PCSK9-induced LDLR degradation that does not require PCSK9 secretion and
that drags the LDLR to lysosomes directly after exit of the trimeric prodomain ≡ PCSK9
≡ LDLR complex from the trans-Golgi network into clathrin light chain coated endosomes.
61
Such a distinct intracellular pathway, while prevalent in most cell lines transfected
with wild type PCSK9 (but not its D374Y mutant), does not seem to play a major role
in the PCSK9-enhanced degradation of the LDLR in liver hepatocytes in vivo. The reason
for this discrepancy between liver and hepatocyte cell lines is still not clear.
7
Genetic and cellular evidence revealed that the phospho-tyrosine binding protein ARH,
which recognizes
NP
X
Y
motifs in cytosolic tails (CT) of membrane-bound proteins, is required for the internalization
of the LDLR-PCSK9 complex into clathrin-coated vesicles, leading to the enhanced degradation
of the complex by the extracellular pathway
62
However, the CT of the LDLR containing an
NP
X
Y
motif is not needed for the internalization of the PCSK9 ≡ LDLR complex
63,64
, and we recently obtained evidence that even a soluble form of the LDLR lacking its
transmembrane (TM) and CT domains (sLDLR) is still well targeted by PCSK9 for intracellular
degradation. This suggests that one or more, as yet unidentified, protein(s) (X-protein;
Xp), which contains a TM and one or more
NP
X
Y
motifs binds the cell surface PCSK9 ≡ LDLR complex and directs it to endosomes/lysosomes.
In that context, we recently reported that the two proposed candidate Xps to chaperone
the PCSK9 ≡ LDLR complex to degradation via the extracellular pathway, i.e., Sortilin
(SORT1) and the amyloid β-precursor-like protein 2 (APLP2), do not physiologically
regulate PCSK9 in cells and in vivo.
65
Furthermore, our study eliminated them as candidate sec24a-binding proteins which
were seemingly required for PCSK9 to exit from the ER into COP-II vesicles.
66
Therefore, the mechanistic details and the trafficking components that regulate both
degradation pathways of the PCSK9 ≡ LDLR complex are still unknown.
Inhibition of PCSK9 reduces LDLc levels & incidence of CVD
Using animal models, it became clear that knockout of the Pcsk9 gene in mice results
in a hypocholesterolemia phenotype with an 80% reduction in LDLc,
67,68
an enhanced response to statins
67
, and a significant decrease in the development of atherosclerosis
69
The reverse is observed in transgenic mice overexpressing the wild type form of PCSK9,
69
or its D374Y GOF mutant,
70
and in transgenic pigs expressing the D374Y mutant.
71,72
Some other benefits that result from the loss of PCSK9 expression are the reduced
incidence of inflammation,
7
sepsis,
73
and tumor metastasis
74
In addition, the observed aorta and vascular calcification in FH patients was reproduced
in Ldlr KO mice and Pcsk9 KO are protected, while PCSK9 overexpression exacerbates
the phenotype.
75
This is in part due to an inflammatory response to the formation of cholesterol crystals
in hypercholesterolemic conditions that can be reversed by the administration of anti-inflammatory
mAbs to Interleukin-1β.
76
Analysis of various reagents that modulate PCSK9 levels (Figure 10), revealed that
HNF1α
77
is the strongest activator of PCSK9 transcription, while SREBP-2 upregulates the expression
of both the LDLR and PCSK9.
47
The latter regulation explains why statins, inhibitors of cholesterol synthesis, also
activate the production of both PCSK9 and its target LDLR.
47
In fact, it was recently reported that some cholesterol ester transfer protein (CETP)
small molecule inhibitors that enhance the levels of HDL can also inhibit SREBP-2
and hence PCSK9 transcription, resulting in reduced LDLc.
78
Mediterranean diet as well as estrogens and mTOR1 all reduce the levels of PCSK9,
while inflammation and high fat or fructose rich diets
79
increase the levels of PCSK9.
7
10.7717/gcsp.201702/fig-10
Figure 10.
Modulators of PCSK9 function or expression.
The activators are denoted with a red arrow, whereas inhibitors of PCSK9 are on the
left side of the green arrow.
However, the most powerful PCSK9 inhibitors are antibodies that prevent the binding
of extracellular (circulating) PCSK9 with the LDLR. The polyclonal antibodies isolated
in our group in 2007,
55,80
were found to be good inhibitors of the extracellular PCSK9 function on LDLR in HepG2
cells stably expressing PCSK9 (Figure 11). Other polyclonal antibodies that have a
similar inhibitory function were also reported by N. Hooper’s group in 2009.
81
The first proof of concept that a fully humanized PCSK9-mAb can effectively inhibit
its function on LDLR via an allosteric mechanism and reduce LDLc levels in mice and
monkeys was reported in 2009.
82
Since then, at least three pharmaceutical companies have developed humanized mAbs
against PCSK9, evolcumab, alirocumab and bococizumab, and all are in phase III clinical
trials, of which outcomes are expected to become public by 2018.
7,83
10.7717/gcsp.201702/fig-11
Figure 11.
Effect of affinity purified PCSK9 polyclonal antibody on LDLR levels in HepG2 cells.
Human PCSK9 (0.6 µg) was pre-incubated at neutral pH and 37°C for 1 h with saline
control or 5 µg of a previously reported affinity-purified polyclonal antibody to
PCSK9.
55
These solutions were then incubated with HepG2 cells for 6 h, following which the
cells were suspended and immediately analyzed by FACS for surface human LDLR levels.
58
Notice that PCSK9 reduces the cell surface LDLR by >60%, and that the PCSK9 antibody
completely reverses this activity on the LDLR.
Proposed strategies to inhibit PCSK9 function
Multiple studies in cells, animals and human LOF mutations revealed that PCSK9 inhibition
would increase the levels of the LDLR in liver and hence reduce those of circulating
LDLc. Such a mechanism of LDLc lowering is distinct and complementary to that of statins
that inhibit cholesterol synthesis, resulting in the activation of SREBP-2 and consequently
upregulating the mRNA levels of the LDLR, but also those of PCSK9 that reduces LDLR
protein concentrations. Clearly, inhibition of PCSK9 would maximize the effect of
statins and result in a drastic reduction of LDLc.
84
This hypothesis was confirmed by the application of multiple inhibitory strategies
discussed below. However, the question arose whether PCSK9 inhibition should be reversible
in case of unexpected secondary effects, or should it be permanent , as two of the
three persons lacking PCSK9 due to complete LOF mutations seem to be in relatively
good health. However, the fact that only a very limited number of people exhibit complete
loss of PCSK9, and yet the complete cardioprotective LOF C672X heterozygote mutation
is found quite frequently in black Africans (3.3% overall), but varied significantly
among ethnic groups, ranging from 0% to 6.9%,
85
begs for caution in the use of a permanent inhibition strategy.
At least four general reversible strategies were proposed to target PCSK9 in order
to reduce its circulating levels (Figure 12):
10.7717/gcsp.201702/fig-12
Figure 12.
Various strategies to inhibit PCSK9 function or levels.
1. Neutralize plasma PCSK9 activity
mAb approach
The most advanced and proof-of-concept approach is definitively the use of inhibitory
mAbs that allosterically deform the structure of the catalytic subunit of PCSK9, thereby
preventing it from interacting with the EGF-A domain of the LDLR. The rapid progression
of the knowledge and applications of PCSK9 inhibitors took <12 years and resulted
in more than 1,300 publications (Figure 13). It is clear that the ‘injection every
two-weeks’ or ‘once every month’ of an inhibitory mAb approach is the most advanced
and privileged one today,
86
as Evolocumab (Amgen) and Alirocumab (Sanofi/Regeneron) have been approved by the
FDA and should be on the market by the end of 2015 or beginning of 2016. The use of
the mAb Bococizumab (Pfizer)
87
should follow shortly thereafter. The subcutaneous injection of these mAbs results
in 50–60% reduction of LDLc and a 30–35% reduction in the highly atherogenic Lp(a)
particles.
7,86,88
The unexpected reduction in Lp(a) levels was recently rationalized by the fact that
under supra-physiological levels of the LDLR, such as is the case with the use of
inhibitory mAbs against PCSK9, the LDLR is the receptor of Lp(a).
89
10.7717/gcsp.201702/fig-13
Figure 13.
Proprotein convertase subtilisin kexin 9 (PCSK9): historical perspective from its
discovery to clinical applications and the manuscripts associated with PCSK9 since
2003.
The pace of research, from PCSK9 discovery through to clinical trials, has been rapid
starting from its discovery in 2003 and the proof-of-principle that mAbs can inhibit
its function in 2010, all the way to ongoing phase III clinical trials and the final
approval by the FDA in 2015. CVD indicates cardiovascular disease; FH, familial hypercholesterolemia;
GOF, gain-of-function; KO, knockout; LDL-C, low-density lipoprotein-cholesterol; LDLR,
LDL receptor; LOF, loss-of-function. Notice that >1,330 manuscripts were published
on the subject of PCSK9 since 2003, resulting in an H-index of 76.
Adnectin approach
Adnectins are a family of binding proteins derived from the 10th type III domain of
human fibronectin (10Fn3), which is part of the immunoglobulin superfamily and normally
binds integrin. The 10Fn3 has the potential for broad therapeutic applications given
its structural stability, ability to be manipulated, and its abundance in the human
body and lack of immune response. Screening phage libraries for PCSK9 binders identified
and led to the engineering of a high-affinity PCSK9 binder, called BMS-962476, as
a potential alternative to mAbs. This is a ∼11-kDa polypeptide conjugated to polyethylene
glycol to enhance pharmacokinetics, which binds with sub-nanomolar affinity to the
catalytic subunit of human PCSK9, thereby inhibiting its interaction with the LDLR.
90
In cynomolgus monkeys a 5 mg/kg single injection of BMS-962476, led to a ∼50% reduction
of LDLc. This was accompanied by the reduction of circulating free PCSK9 levels by
∼6- to 7-fold over baseline, which then returned to control levels by 3 weeks, in
parallel with return to baseline of LDLc levels, likely as a consequence of the Adnectin
complex dissociating over time.
90
We are awaiting the progress of this type of inhibitor in clinical trials.
2. Decrease PCSK9 expression
siRNA approach
One way to reversibly decrease PCSK9 expression would be to lower the levels of its
mRNA. A small interfering RNA (siRNA; Alnylam Pharmaceuticals, Inc.) clinical trial
involving siRNA-targeting PCSK9 has been evaluated in a randomized, single-blind,
placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with
serum LDLc of ≥3 mmol/L or higher.
91
The data showed that at a dose of 0.4 mg/kg, this relatively safe treatment resulted
in a mean 70% reduction in circulating PCSK9 plasma protein and a mean 40% reduction
in LDLc from baseline relative to placebo. This siRNA approach was shown to be generally
safe and well tolerated in this Phase I study and there were no serious adverse events
related to study drug administration. Phase II clinical trials are underway. Although
mAbs seem to block close to 100% of free circulating PCSK9, the siRNA approach left
≈30% PCSK9 in circulation, suggesting limited efficacy of the current siRNA method.
Although a direct comparison of this approach with the mAb one is yet to be tested
in humans, the efficacy of the reduction of LDLc observed with siRNA (40%)
91
is still no better than that achieved with mAbs (50%–70%).
7,86
This suggests that the intracellular pathway in liver may have a relatively minor
contribution to the overall activity of PCSK9 on LDLR, which seems to mostly act by
the extracellular pathway.
Transcriptional inhibition
PCSK9 gene expression is regulated by SREBP-2, HNF1α and other factors (Figure 10).
7
Thus, it is plausible to develop small molecules that would enter the nucleus in liver
hepatocytes and decrease the transcription of the PCSK9 gene. Interestingly, Kowa
Pharmaceuticals reported that a CETP inhibitor (K-312) that raises HDL levels, and
also inhibits PCSK9 transcription and lowers LDLc levels in rabbits. In the human
hepatocyte-derived HepG2 cells, K-312 treatment decreased the active nuclear forms
of SREBP-1 and SREBP-2 that regulate promoter activity of PCSK9. This suggests that
K-312 may regulate the SKI-1/S1P or S2P cleavage and generation of the active nuclear
forms of these SREBPs.
11
Thus, K-312 decreases LDLc and PCSK9 levels, possibly serving as a new oral therapy
for dyslipidemia and CVD. However, the SREBP target of this inhibitor makes it relatively
non-specific for PCSK9, as the levels of other proteins regulated by SREBPs will also
be affected.
Benzofurans as modulators of CVD
Tribbles pseudokinase 1 (TRIB1) is implicated in modulating the risk of CVD.
92
Active benzofurans, as well as natural products capable of TRIB1 upregulation, also
modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR mRNA
and LDLR protein, while reducing the levels of PCSK9 mRNA and secreted PCSK9 protein
and stimulating LDLc uptake.
92
The effects of benzofurans are not masked by cholesterol depletion and are independent
of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel
class of chemically tractable small-molecule modulators that shift cellular lipoprotein
metabolism in HepG2 cells from lipogenesis to scavenging. Time will tell if such an
approach, that is not specific for PCSK9, is nevertheless safe and feasible clinically.
3. Block PCSK9 functional activity on the LDLR
EGF-A mimetics
Since PCSK9 interacts with the EGF-A domain of the LDLR, it is plausible that a competitive
EGF-A mimetic can act as a decoy to block the activity of extracellular PCSK9 on the
LDLR. The interaction surface of EGF-A and PCSK9 is large and flat, with the two proteins
interacting via a 530 Å2 flat contact patch between the catalytic domain of PCSK9
and the EGF-A domain in the LDL-R.
59,93
it is thus a real challenge to find a small molecule that would inhibit LDLR binding.
Nevertheless, a number of approaches using peptidomimetics of EGF-A have been reported.
Two truncated and modified versions of the EGF-A peptide were designed and found to
be active in the low µM range to inhibit the activity of extracellular PCSK9 on the
LDLR.
94,95
Screens of phage-displayed peptide libraries led to the identification and synthesis
of a 13-amino acid linear peptide (Pep2-8) as the smallest PCSK9 inhibitor known.
95
However, much work is still necessary to stabilize such structures for in vivo applications,
and possibly their transformation into orally active compounds.
Peptide mimetics of natural inhibitors of PCSK9
The search for natural inhibitors of PCSK9 led to the identification of Annexin A2
as a candidate inhibitor of the extracellular activity of PCSK9.
96,97
The inhibitory domain was localized to be in the 70 aa N-terminal R1-repeat domain
of Annexin A2, and this was used to develop a high nM PCSK9-inhibitor that is active
in cell lines.
97
Interestingly, Annexin A2 is a cytosolic protein that is secreted and meets PCSK9
in the extracellular milieu. Recent data also revealed that the cytosolic form of
Annexin A2 also reduces PCSK9 protein levels via inhibition of its translation, likely
upon binding inhibitory motifs in the 3′ untranslated region of the PCSK9 mRNA.
98
The identification of the binding domain of cytosolic Annexin A2 to the PCSK9 mRNA
may lead to the synthesis of intracellular inhibitors of PCSK9 translation.
4. Inhibit proPCSK9 zymogen autocatalytic cleavage or secretion from cells
The precursor proPCSK9 oligomerizes in the ER in a disulfide dependent fashion,
5
and the exit of the monomeric prodomain ≡ PCSK9 from the ER (ultimately leading to
secretion) is dependent on the zymogen autocatalytic processing of proPCSK9 into PCSK9
to excise the prodomain (Figure 1).
5,50
It is thus plausible to identify an inhibitor, possibly a small molecule, that would
prevent PCSK9 from exiting the ER, either by inhibiting its autocatalytic processing,
or by enhancing its oligomerization. A number of screens have been initiated to inhibit
autocatalytic processing of PCSK9. Since this is an in cis zero kinetics reaction
it would be rather difficult, but not impossible, to block it. Indeed, novel assays
have been proposed to test potential inhibitors of proPCSK9 processing, and suggested
that the PCSK9 active site and its adjacent residues serve as an allosteric modulator
of protein secretion, independent of its role in proteolysis, revealing a new strategy
for intracellular PCSK9 inhibition.
99
At least two permanent PCSK9 inhibition strategies have been proposed:
PCSK9 vaccination
A recent report suggested the use of peptide-based anti-PCSK9 vaccines, isolated via
the generation of polyclonal high affinity and persistent antibodies that are functional
for up to one year (AFFiRiS AG, Austria).
100
In mice, they are reported to be powerful with an up to ∼50% reduction of LDLc and
∼30% decrease in total cholesterol. It was suggested that this type of vaccine is
a safe tool for long-term LDLc management, and thus may represent a novel therapeutic
approach for the prevention and/or treatment of hypercholesterolemia-related CVD in
humans. However, the potentially negative long term consequences associated with this
approach should not be underestimated, as it is also apparent that the permanent lack
of PCSK9 may reduce the ability of the liver to regenerate
68
and may enhance viral infections.
101,102
Furthermore, the extreme rarity of individuals that completely lack PCSK9 is if anything,
an indication of a potential counter-selection against this event during evolution.
Notwithstanding these caveats, Pfizer is testing an experimental PCSK9 vaccine, designed
to induce the body to produce its own PCSK9 antibodies, which should enter human testing
in 2016. It is reported that that, if successful, the vaccine might eventually be
an annual injection.
CRISPR-Cas9 gene silencing of PCSK9
Scientists hoping to alter the genome of their favorite organisms faced an arduous
task, which has been vastly improved by the ability to quickly destroy or edit a gene
with a new technology called CRISPR (clustered regularly interspaced short palindromic
repeat)/Cas9. Such RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing
platform.
103–105
In this method the Cas9 enzyme cuts DNA at a specific sequence, determined by an accompanying
bit of RNA called a guide RNA. Then, the cell’s own DNA repair machinery typically
takes over in one of two different repair modes: (1) it simply glues the two pieces
back together, but imperfectly, so the leftover scar interrupts and disables the targeted
gene; or (2) the cell can copy a nearby piece of DNA to fill in the missing sequence.
By providing their own DNA template, scientists can now induce the cell to fill in
any desired sequence, from a small mutation to a whole new gene.
A recent proof-of-principle study suggested the possibility of permanent alteration
of PCSK9 with in vivo use of CRISPR-Cas9 genome editing.
106
Here the authors injected mice with an adenovirus expressing CRISPR-Cas9 and a CRISPR
guide RNA targeting Pcsk9 in mouse liver. The data showed that in 3–4 days from the
administration of the virus, the mutagenesis rate of Pcsk9 in the liver was >50%.
This resulted in decreased plasma PCSK9 levels, increased hepatic low-density lipoprotein
receptor levels, and decreased plasma cholesterol levels (by 35–40%), similar to the
total cholesterol reduction observed in mice completely lacking PCSK9 (−40–50%).
67,68
However, the size of the commonly used Cas9 (4.1 kb) from Streptococcus pyogenes (SpCas9)
limits its utility for basic research and therapeutic applications that use the highly
versatile adeno-associated virus (AAV) delivery vehicle. Recently, it was reported
smaller Cas9 orthologues from Staphylococcus aureus (SaCas9) can edit the genome with
efficiencies similar to those of SpCas9, while being more than 1 kb shorter.
107
When SaCas9 and its single guide RNA expression cassette were packaged into a single
AAV vector the authors were able to effectively target the Pcsk9 gene in mouse liver.
Within one week of injection, >40% gene modification was observed, accompanied by
almost complete absence of immunoreactive Pcsk9 in circulation and a ∼50% reduction
in total cholesterol levels, without apparent liver toxicity at one to four weeks
after AAV administration.
107
Obviously, both studies used either adenoviral- or AAV-induced silencing technology,
and this viral approach is not yet suitable for human patients, but the reduction
of liver PCSK9 by this technology has now been proven. Other methods of delivery of
CRISPR-Cas9 and its guide using nanoparticles might become more sophisticated to allow
for clinical trials. Furthermore, both studies were short term and were only done
in mice. So, the long term benefits and safety associated with the liver-targeted
silencing PCSK9 by this technology would have to be proven beyond doubt before it
becomes widely used in clinics.
Conclusons and future directions
This small review presents the many facets of PCSK9 and its biology, concentrating
only on its ability to enhance the degradation of the LDLR. However, PCSK9 has been
shown to target other members of the LDLR-family including the VLDLR and ApoER2,
108,109
and can affect the levels of its targets in other tissues than liver, such as small
intestine, pancreas and adipose tissue.
7
Much remains to be unravelled regarding the cellular trafficking of PCSK9 together
with its targeted receptors, its possible interactome web, and its binding to other
proteins. This is a very exciting period in the field of dyslipidemia, where thanks
to new PCSK9-silencing therapies, LDLc levels were lowered to unprecedented levels,
reaching almost 0.4 mM. This is good news for hypercholesterolemic patients who do
not reach target levels of LDLc with the available medications, cannot tolerate statins,
or who experience painful side effects with statins such as myalgia. Even homozygote
FH-1 patients that have minimal LDLR activity can now be treated with PCSK9 mAbs with
a spectacular ∼30% decrease in circulating LDLc,
110
thereby giving a much better quality of life that is less dependent on the use of
bi-weekly sessions to clear LDLc from their blood using special apheresis dialysis
columns.
Although the outcomes of the various ongoing phase III clinical trials using PCSK9-mAbs
will not be known until 2018, early signs indicate that this treatment results in
a ∼50% reduction in cumulative cardiovascular events within 1 year of treatment.
111,112
Finally, the fact that PCSK9 is inactivated by some proteases, such as Furin,
52,53
might open new strategies to enhance this inactivation mechanism and thus lower the
levels of active PCSK9. The future will tell which strategies targeting PCSK9, other
than mAbs, will find their way in dyslipidemia and cardiology clinics throughout the
world and result in affordable and safe treatments.