Download Inhibitors of Factor VIIa/Tissue Factor

ATVB In Focus
Emerging Anticoagulant Drugs
Series Editor:
Jeffrey I. Weitz
Previous Brief Reviews in this Series:
• Turpie AGG. Oral, direct factor Xa inhibitors in development for the prevention and treatment of thromboembolic
diseases. Arterioscler Thromb Vasc Biol. 2007;27:1238 –1247.
• Howard EL, Becker KCD, Rusconi CP, Becker RC. Factor IXa inhibitors as novel anticoagulants. Arterioscler
Thromb Vasc Biol. 2007;27:722–727.
• Weitz J. Emerging anticoagulant drugs. Arterioscler Thromb Vasc Biol. 2007;27:721.
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Inhibitors of Factor VIIa/Tissue Factor
Rebecca A. Shirk, George P. Vlasuk
Abstract—The formation of the proteolytic complex composed of the serine protease Factor VIIa and the cell-associated
glycoprotein tissue factor (FVIIa/TF) initiates a cascade of amplified zymogen activation reactions leading to thrombus
formation. The critical role of the coagulation cascade in pathological thrombosis has been the basis for significant
efforts to design selective inhibitors of the protease components as new anticoagulant alternatives for the treatment of
thrombotic diseases. However, for the new generation of anticoagulant drugs in development that primarily target
protease complexes distal from FVIIa/TF, the differential between efficacy and safety as defined by bleeding is
unresolved. Targeting the FVIIa/TF complex has several theoretical advantages that exploit the amplified nature of the
coagulation cascade. However, progress on the development of clinical-stage FVIIa/TF-based anticoagulants has not
been as successful to date. This review summarizes recent efforts in the discovery of synthetic inhibitors of FVIIa/TF.
(Arterioscler Thromb Vasc Biol. 2007;27:1895-1900.)
Key Words: anticoagulants 䡲 factor VIIa (FVIIa) 䡲 tissue factor 䡲 serine protease inhibitors 䡲 coagulation
H
emostasis is achieved through a highly regulated and
coordinated interplay of protein and cellular components designed to respond quickly to vascular insult.
Critical in this rapid response is the initiation of blood
coagulation by a proteolytic complex composed of the
serine protease factor VIIa (FVIIa) and its cell-associated
cofactor tissue factor (TF). Tissue factor is a transmembrane glycoprotein that is normally found in subendothelial
structures throughout the vasculature. Exposure of TF to
blood, through physical damage to the lining of the blood vessel
or increased expression in endothelial and monocytic cells in
response to certain inflammatory signals, permits an interaction with FVIIa, which is present at trace levels in the
circulation. The formation of the FVIIa/TF complex is the
critical step that initiates the sequentially amplified cascade of
proteolytic activation steps that ultimately leads to the generation
of the protease thrombin and thrombus formation.1 The critical
nature of the FVIIa/TF complex in the process of blood
coagulation makes it an attractive candidate for the development of antithrombotic agents that can inhibit complex
formation or catalytic activity. An antithrombotic agent based
on the inhibition of FVIIa/TF may have theoretical advantages over the inhibition of downstream coagulation proteases
such as factor Xa (FXa) or thrombin based on the several
thousand–fold amplification that occurs after initiation by
FVIIa/TF.
Original received May 18, 2007; final version accepted June 15, 2007.
From the Department of Cardiovascular and Metabolic Diseases Research, Wyeth Research, Collegeville, Pa.
Correspondence to Rebecca A. Shirk, PhD, Wyeth Research, P.O. Box 42528, Philadelphia, PA 19101-2528. E-mail [email protected]
© 2007 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://atvb.ahajournals.org
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DOI: 10.1161/ATVBAHA.107.148304
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The success of several small protein- and antibody-based
inhibitors of the FVIIa/TF pathway in preclinical models and
clinical trials1,2 has led to substantial interest in the development of orally active small molecule inhibitors of the enzyme
active site that could be used for the long-term prevention or
treatment of thrombosis. Vitamin K antagonists such as
warfarin, which have been used clinically for more than 50
years and target multiple vitamin K– dependent coagulation
proteases, are the only orally active anticoagulants in clinical
use today. Although vitamin K antagonists are inexpensive,
their use is complicated by a slow onset of action, food and
drug interactions that make it difficult to maintain stable
exposure, and thus requiring regular monitoring. Therefore
there is a critical unmet medical need for improved oral
anticoagulants with a better therapeutic window. Direct
comparisons of specific FVIIa/TF inhibitors with other anticoagulants in preclinical thrombosis models have shown a
reduced bleeding tendency in several studies,3– 8 suggesting
that targeting the upstream proteolytic complex FVIIa/TF
may provide a safety advantage. Such a drug would need to
be potent, orally bioavailable, and highly selective for
FVIIa/TF versus other closely related serine proteases involved in coagulation and fibrinolysis, as well as demonstrate
other optimal pharmacokinetic parameters. Despite considerable research effort over the past decade or so, to our
knowledge, no small molecule FVIIa/TF inhibitors have
advanced to the clinical trial testing phase to date.
A number of research groups have reported the successful
discovery of small molecule active-site FVIIa/TF inhibitors,
the best of which show excellent potency (single digit or low
double digit nmol/L Ki or IC50) in in vitro activity assays with
purified enzyme complex and demonstrate greater than several hundred– to thousand-fold selectivity versus thrombin,
FXa and trypsin. Several excellent reviews have been published that detail the diverse chemical scaffolds, in vitro
potency, and protease selectivity (when available) of inhibitors reported in the patent and research literature.9 –11 The goal
of the current review will be to summarize general strategies
and describe recent advances in the field of small molecule
FVIIa/TF inhibitors. Several recent reviews have covered the
latest advances in the development of peptide and protein
inhibitors of FVIIa/TF and will not be covered here.1,2,12
State-of-the Art
The X-ray structure of the FVIIa/TF complex was first published
in 1996 by Banner et al13 and facilitated the use of a structurebased design approach to guide optimization of FVIIa/TF active
site inhibitors by using X-ray structural analysis and molecular modeling to improve binding affinity and protease
selectivity. FVIIa is a trypsin-like serine protease. It shares
with thrombin, factor Xa, factor IXa, etc the characteristic
catalytic triad residues Ser195-His57-Asp102 (chymotrypsinogen numbering is used through-out) and a high degree of
homology in amino acid sequence and spatial arrangement in
the protease domain and active site. The characteristic feature
of the trypsin-like serine proteases is a deep S1 primary
specificity binding pocket with an acidic Asp189 at the
bottom, which forms a salt bridge with the basic Arg or Lys
side chain of the scissile bond in natural substrates. The size
Figure 1. Comparison of FVIIa, thrombin, and FXa active sites
occupied by small molecule ligands. A, FVIIa complexed with
D-Phe-Phe-Arg-CMK (pdb file: 1DAN). B, Thrombin complexed
with D-Phe-Pro-Arg-CMK (pdb file: 1PPB). C, FXa complexed
with Razaxaban (pdb file: 1Z6E). To assist identification of subsites S1–S3, peptide ligands are depicted with a different color
to denote each amino acid: gray, P1 Arg; copper, P2 Phe (FVIIa/
TF, panel A) or P2 Pro (thrombin, panel B); and gold, P3 D-Phe.
Razaxaban (FXa, panel C) is color-coded by atom type. Smooth
approximations of the solvent accessible surfaces, based on
van der Waals radii, were generated using “Soft Surface”, Discovery Studio 1.6 (Accelerys). The surfaces are color coded as
follows: red, side chains of catalytic His57 and Ser195 left to
right; blue, side chain of Lys192 (FVIIa/TF, panel A), Glu192
(thrombin, panel B), or Gln192 (FXa, panel C); green, side chain
of Thr99 (FVIIa/TF, panel A), Leu99 (thrombin, panel B), or residues Tyr99, Trp215, Phe174 top to bottom (FXa, panel C); yellow, the side chain of Asp60 (FVIIa/TF, panel A), insertion loop
60A– 60D (Tyr-Pro-Pro-Trp in thrombin, panel B), or Leu60 (FXa,
panel C). The S1⬘/2⬘ subsite is located “northeast” of the catalytic Ser195.
and shape of the FVIIa S1 pocket is very similar to the S1
pocket in thrombin, factor Xa, and trypsin, yet small S1
differences are observed. However, the main differences are
in the S2 proximal and S3 distal hydrophobic pockets and the
S1⬘ site, which show size and hydrophobicity differences
between enzymes (Figure 1).
Shirk and Vlasuk
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Figure 2. Structures of selected FVIIa/TF inhibitors. L-arginine
and small molecule active site inhibitors of FVIIa/TF: PHA-927
from Suleymanov et al7; “9” from Young et al18; “22” from Zbinden et al19; “10” from Rai et al33; “17d” from Miura et al.34
To achieve potency and selectivity, researchers have exploited key residues and differences between proteases in the
S1–S4 and S1⬘ binding pockets of the FVIIa active site,
typically interacting with at least 3 of the subsites. The vast
majority of the FVIIa inhibitors reported so far have a
benzamidine or other amidine P1 moiety that mimics the Arg
side chain guanidinium group in natural substrates and can
form a strong salt bridge with Asp189 in the bottom of the S1
pocket (Figure 2). A serine at position 190 in the S1 pocket is
unique to FVIIa (versus Ala190 in thrombin and FXa), and
some FVIIa inhibitors have been shown to form key hydrogen bonds with Ser190.14,15 Interaction with the catalytic
Ser195 via addition of a ␳-amino group on the benzamidine
contributes significantly to potency in a number of inhibitors.8,16 In contrast, a series of compounds was shown to form
a unique network of hydrogen bonds to Ser195 via a hydroxyl
group on a non-P1 central ring.17,18 Another P1 substitution
reported is the addition of an o-OH on ␳-aminobenzamidine
to increase selectivity. The crystal structure shows the binding of o-OH is associated with a conformational change at
Gln217–Gly219 near the bottom of the S1 pocket that is
possibly stabilized by a hydrogen bond with the side chain of
Thr221, a residue not found in other proteases examined.8 At
the top of the S1 pocket FVIIa has a distinct binding site,
called the S1 side pocket or S1 subsite, which contains a
unique Lys at position 192 versus Glu192 in thrombin and
Gln192 in FXa (Figure 1, blue surfaces). Acid moieties have
been exploited by several groups to interact with Lys192 near
the oxyanion hole, enhancing both potency and selectivity.16,18 –22 Some FVIIa inhibitors have been shown to induce
a flipped conformation of the amide bond between Lys192
and Gly193 which results in an enzymatically incompetent
oxyanion hole.8,19,20 The 180° rotation of the Lys192-Gly193
bond is stabilized by a hydrogen bond with Gln143, but other
tyrpsin-like proteases do not appear to have a suitable partner
in that location for hydrogen bond formation, which may
explain the observed selectivity for FVIIa. Key differences in
Factor VIIa/Tissue Factor Inhibitors
1897
the S2 pocket have been used to improve selectivity as well.
FVIIa has a large S2 pocket with a unique Asp60 (Figure 1A,
yellow surface) that has been targeted by basic P2 moieties in
FVIIa inhibitors,14,22 whereas other coagulation proteases lack
an acidic residue in that position. In contrast, the thrombin and
FXa S2 pockets are markedly smaller, with the thrombin S2
pocket partially blocked by a rigid insertion loopTyr60A⫺
Trp60D (Figure 1B, yellow surface) and FXa S2 occluded by the
side chain of Tyr99 (Figure 2C, top green surface). Therefore,
large P2 moieties improve selectivity against these proteases.
And lastly, several groups have targeted the S1⬘ pocket to
improve both potency and selectivity, using hydrophobic
moieties with specific substitutions for key hydrogen bond
interactions.17,23,24
Some of these small molecule FVIIa/TF inhibitors have
been tested in vivo and shown to be efficacious in preclinical
models. The first reports emerged in 2001 and showed efficacy
in a guinea pig arterial thrombosis model and that a 2 week
parenteral dosing regimen was well tolerated in the same
species.25,26 Separation of antithrombotic efficacy and bleeding tendency with a selective small molecule FVIIa/TF
inhibitor was first reported in 2003. PHA-927F (Figure 2)
was reported to inhibit FVIIa/TF with an IC50 of 25 nmol/L
and more than 3500-fold selectivity against thrombin and
FXa.7 Intravenous administration of PHA-927F in a nonhuman primate model of arterial injury model demonstrated
dose-dependent inhibition of thrombus formation with markedly less bleeding when compared with a FXa inhibitor or
thrombin inhibitor at the minimally efficacious dose or up to
4.4-fold multiples thereof. Other reports have followed of
small molecule FVIIa/TF inhibitors demonstrating efficacy
after intravenous dosing in monkeys8,18,27 and lower species
as well.28,29 Finally, compound CRA-027483 has been shown
to have ⬇100% bioavailability after subcutaneous delivery,
inhibiting thrombus formation in a baboon arterial injury
model30 and suppressing the lipopolysaccharide (LPS)induced inflammatory response in a mouse model of
endotoxemia.31
The Challenge of Making a Drug
Although a number of potent and selective FVIIa/TF inhibitors have been reported, some demonstrating in vivo efficacy
after parenteral administration, a significant challenge remains in the discovery of an orally bioavailable drug. A
successful oral drug will require a careful balance of optimal
inhibitor characteristics and drug-like or pharmacokinetic
properties, demands that can have contradictory effects on the
molecule. The FVIIa inhibitors described so far have a
highly basic amidine or benzamidine P1 moiety that can
form a salt bridge with Asp189 in the bottom of the S1
pocket. These basic functional groups, although contributing
significantly to binding affinity, are protonated at physiological pH and thus are associated with poor oral bioavailability
because of limited absorption in the gastrointestinal tract.
Two general approaches have been taken to attempt to bypass
this problem in FVIIa/TF inhibitors. The first is to replace the
basic amidine with a less basic or neutral P1 moiety. This
approach represents a significant challenge, because the mode
of binding in the S1 pocket dramatically impacts overall
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binding. The second approach is to use a prodrug approach to
reversibly mask the basic moiety.
Two groups have recently reported efforts to find a suitable
replacement for the P1 amidine moiety. Researchers at Celera
Genomics began with potent FVIIa/TF inhibitors that contained biaryl amidine P1 moieties, such as 5-amidinoindole
and 5-amidinobenzimidazole, and replaced these scaffolds
with less basic heterocyclic scaffolds that could potentially
interact with key residues in the S1 pocket.24,32,33 These
inhibitors lose substantial potency initially but typically gained
significant selectivity against thrombin, FXa, and trypsin, even
before further modification. By further optimizing the inhibitor to interact with the S1⬘ pocket or the loop separating the
S2 and S1⬘ pockets, a highly potent (20 nmol/L Ki) and
selective inhibitor with a 5-aminopyrrolo-pyridine P1 scaffold was generated (compound “10”, Figure 2).33 Similarly, a
P1 5-azaindole inhibitor with a Ki of 800 nmol/L was further
optimized to exploit the S1⬘ pocket, and the best of these
inhibitors showed Ki values of 30 to 60 nmol/L for FVIIa/TF
and ⬎1000-fold selectivity against FXa, thrombin, and trypsin.24 Crystallography data indicate that both of these P1
scaffolds bind in the S1 pocket via multiple H-bonds and by
causing a conformational rearrangement relative to structures
obtained with amidine-containing inhibitors. The
5-aminopyrrolo-pyridine and 5-azaindole P1 scaffolds described in these reports are markedly less basic, with a
calculated pKa of ⬇8.8 and ⬇7.8, respectively, versus pKa
⬇11 to 12 for benzamidine.32,33 However, it is not clear
whether these modifications are sufficient to significantly
improve oral bioavailability, as no pharmacokinetic data are
reported. Researchers at Astellas Pharma Inc took a different
approach, replacing the amidine on the P1 benzene ring of a
90 nmol/L FVIIa/TF inhibitor with “neutral” 3-aminocarbonyl
or 4-aminomethyl groups.34 Precipitous loss of activity resulted, but after further optimization of the P2 group, the best
compound inhibited TF/FVIIa with moderate potency
(IC50⫽690 nmol/L) and ⬎300-fold selectivity (“17d”, Figure
2). In a more recent report, a P1 4-aminomethylphenyl FVIIa/TF
inhibitor with moderate potency (IC50⫽590 nmol/L) was reported to be orally available.35 After oral administration to
mice of a high dose 100 mg/kg, the inhibitor reached and
sustained plasma levels of ⬇2 ␮mol/L for 0.5 to 2 hours, as
estimated by ex vivo plasma activity against human FVIIa/
TF. The oral availability was estimated at ⬇10%. Reviewing
the results from these 2 groups, it appears that discovery of a
potent FVIIa/TF inhibitor with a neutral P1 moiety remains a
challenge.
A prodrug approach has shown the most preclinical
progress to date in the search for an orally active FVIIa/TF
inhibitor. Prodrugs are agents that are delivered in inactive or
markedly less active form and then converted to the active
form by metabolic processes in vivo; therefore good oral
bioavailability of active drug is dependent on both absorption
of the prodrug from the intestine into the circulation and
subsequent cleavage of the masking group to generate the
active parent compound. Highly basic amidine functional
groups can be masked with N-hydroxyl or N-hydroxyalkyl
groups to form amidoximes or alternatively with
N-carboxyalkyl groups to generate carbamate prodrugs.36
Amidoxime and carbamate-modified amidines are not protonated under physiological conditions and therefore show
improved oral absorption. This approach was first shown to
have promise for antithrombotic drugs in the clinical development of oral GPIIb/IIIa inhibitors and later with the direct
thrombin inhibitor ximelagatran (discussed below). Researchers at Hoffman–La Roche have shown the most progress with
amidoxime prodrugs of FVIIa/TF inhibitors. In the first
report, Zbinden et al demonstrated that masking the P1
benzamidine moiety as an amidoxime prodrug increased the
oral bioavailability of the active drug from 2% to 30% in the
rat, and a double prodrug containing both the amidoxime
modification and an ester modification of an acidic group
(“22”, Figure 2) resulted in a surprising 100% oral bioavailability.19 However, the active parent inhibitor had only
moderate in vitro potency (Ki⫽190 nmol/L) and poor activity
in plasma as measured in the PT clotting assay; no in vivo
activity was reported. In a subsequent report, Zbinden et al
demonstrated antithrombotic activity after oral administration
of a double amidoxime/ester prodrug of another FVIIa/TF
inhibitor with improved potency (81 nmol/L) and markedly
better activity in plasma.20 This prodrug showed moderate
oral bioavailability (20%) in the rat and dose-dependent
antithrombotic efficacy in a guinea pig recurrent arterial
thrombosis model at high oral doses (20 to 500 mg/kg,
necessitated by species differences in plasma activity). Antithrombotic activity was accompanied by a dose-dependent
increase in the PT clotting assay but no effect on the APTT or
bleeding time except at the highest dose tested. These
findings are quite significant because this is the first report of
an orally active, potent, and selective FVIIa/TF inhibitor,
achieved through the masking of a benzamidine P1 moiety
with an amidoxime prodrug.
However, not all FVIIa/TF inhibitor prodrug efforts have
met with success. Researchers at Celera Genomics attempted
to improve oral bioavailability of compounds containing a
biaryl P1 moiety, 5-amidinoindole.37 A series of hydroxyl-,
alkoxy-, and carbamate-amidine prodrugs were synthesized
that showed moderate to good oral absorption of the prodrug
in rat (typically 40% to 100%) but poor conversion to parent
amidine, resulting in overall oral availability of ⬍1% to 2%
for the amidine drug. The authors noted that previous successes with amidine prodrugs were obtained with mono aryl
structures (eg, benzamidine) or alkyl-amidines and hypothesized that the biaryl-amidine prodrugs were not recognized by
the enzymatic machinery required for prodrug conversion. It
remains to be seen whether other more successful FVIIa/TF
prodrugs will emerge and whether they perform well in the
clinic.
In contrast to the current status of small moleclule
FVIIa/TF inhibitors, several oral active-site thrombin and
FXa inhibitors have advanced to clinical trials.38,39 Both
amidine prodrugs and nonamidine P1 approaches have been
used. Ximelagatran is an amidoxime/ester double prodrug
that is converted in vivo to the active drug melagatran, a
potent thrombin inhibitor that contains a P1 benzamidine
moiety.40 The prodrug was rapidly absorbed and converted to
melagatran with an oral bioavailability of ⬇20% in human
subjects. Ximelagatran was initially marketed in Europe in
Shirk and Vlasuk
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2004 for short-term prevention of thrombosis after orthopedic
surgery, but the drug was withdrawn in 2006 because of
concerns over liver toxicity. Other prodrugs of amidinecontaining thrombin inhibitors include dabigatran etexilate, a
carbamate prodrug which is in phase III trials, and AZD 0837,
a follow-on to ximelagatran which contains an alkylamidoxime P1 moiety and is currently in phase II clinical trials.
Oral FXa inhibitors are being evaluated in advanced clinical
studies as well, and a number of them do not contain amidine
P1 moieties.38,39 For example, rivaroxaban (BAY 59 to 7939),
apixaban (BMS 562247), and LY-517717 have successfully
completed phase II trials with subcutaneous low molecular
weight heparin as the comparator in patients undergoing total
knee or hip replacement surgery, and they were found to be
safe and efficacious.41 Rivaroxaban and apixaban have advanced to Phase III trials in a comparable patient population
and are also being tested for additional indications in phase II
/III. The binding mode to FXa has not been disclosed for most
oral FXa inhibitors currently in development. However,
crystal structures for rivaroxaban (with a neutral P1 chlorthiophene moiety),42 razaxaban (DPC-906, halted in Phase
II),43 and other nonamidine FXa inhibitors44 – 46 demonstrate a
common binding theme. The inhibitors typically bind to FXa
in a bent or L-shaped extended conformation, making essential interactions deep in the S1 pocket with a nonbasic moiety
and in what the authors term the S4 pocket, a narrow
hydrophobic channel defined by the side chains of Tyr99,
Phe174, and Trp215 (Figure 1C, green surface). For these
inhibitors, strong binding occurs in the absence of a highly
basic P1 moiety that can interfere with oral bioavailability.
Conclusions
Despite significant efforts and some success in the discovery
of potent synthetic inhibitors of FVIIa/TF, no compounds
have advanced into clinical trials. The issues as discussed
above are difficult and will likely take considerable innovation to overcome the barriers to the successful development
of a suitable orally bioavailable drug targeting FVIIa/TF.
Despite these challenges, the FVIIa/TF complex remains a
validated and important target for the development of the next
generation of antithrombotic agents.
Acknowledgments
The authors thank Dr Bruce A. Malcolm for providing the figures,
useful discussions of the literature, and critical evaluation of
the manuscript.
Disclosures
None.
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Inhibitors of Factor VIIa/Tissue Factor
Rebecca A. Shirk and George P. Vlasuk
Arterioscler Thromb Vasc Biol. 2007;27:1895-1900; originally published online June 28, 2007;
doi: 10.1161/ATVBAHA.107.148304
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