Structure based design of anticoagulants
L.H.J. Kleijn
(3404366)
Literature thesis
Drug Innovation Master Program
Thromboembolic
disorders
are
treated
with
anticoagulants. For over half a century, heparins and
coumarins have served as therapeutic anticoagulants.
They are potent antithrombotic agents, but their
numerous shortcomings inspired the search for new and
orally available anticoagulants. The emergence of 3D
structures for coagulating factors thrombin (fIIa) and
factor Xa (fXa) in the early 1990’s boosted research into
direct fXa and fIIa inhibitors. This review discusses how
the availability of these 3D structures contributed to the
development of oral anticoagulants that have since then
been developed.
Utrecht University
2013
Figure 2. Coumarin warfarin (left) and heparin (right).
exposed to the blood after damage to the vessel tissue. Serine
protease fVII binds TF to form the fVIIa-TF complex that
converts fX to fXa. Prothrombinase, which forms after fXa
binds fV, cleaves prothrombin (fII) into thrombin (fIIa), its
active form. Thrombin converts fibrinogen to fibrin and
initiates the intrinsic pathway, which promotes the
formation of more thrombin. Fibrin polymerizes to initiate
clot formation. Thrombosis seems to manifest itself when
coagulating factor fXII is activated by, for instance, bacterial
toxins or proteoglycans. Factor XIIa sets of the coagulation
cascade by activating fXI and fVII. 2-4
Thrombosis
Thrombosis is localized clotting of blood due to an imbalance
in the blood coagulation system. A blood clot (thrombus) can
block blood vessels or dislodge and travel with the
bloodstream (embolus). Thrombosis occurs in the arterial as
well as in the venous circulation, but their pathophysiology
differs. Arterial thrombosis, primarily caused by rupture of a
plaque on the arterial wall, is mainly treated with
antiplatelet drugs. Venous thrombosis, which includes deep
vein thrombosis (DVT) and pulmonary embolism (PE), is
caused by changes in the blood composition, blood flow or
changes of the vessel wall.1,2
Anticoagulants are used to treat and prevent a wide range of
thrombotic diseases. They reduce the formation of fibrin by
acting on the blood-clotting cascade (Figure 1). Under
normal conditions, the extrinsic pathway of coagulation is
initiated when glycoprotein receptor tissue factor (TF) is
Heparins and Coumarins
Anticoagulants have been used clinically since long before
the mechanisms of blood coagulation were understood
(Figure 2). Unfractured polysaccharide heparin (3,00030,000 Da) was first tested clinically in 1937. Lowmolecular-weight heparins (LMWH’s) became available in
the 1980’s and fondaparinux (1,728 Da), the active
component of heparin, obtained FDA approval in 2001. All
heparins require parenteral administration and cause major
bleeding in circa 3% of patients. Heparins induce a
conformational change in antithrombin III (ATIII) after
which ATIII is 10,000 fold more active at deactivating
thrombin, fXa and fIXa.5-7
Dicoumarol was the first coumarin to be tested as orally
available anticoagulant in 1942. Warfarin, the leading
coumarin, entered the market in 1954. Warfarin treatment
requires frequent coagulation monitoring and dose
adjustment due to its narrow therapeutic window, drug-food
interactions and delayed onset of therapeutic action.
Coumarins prevent vitamin K epoxide from being converted
to its reduced form (vitamin K hydroquinone) by inhibiting
vitamin K epoxide reductase.
A lack of vitamin K
hydroquinone prevents γ–carboxylation of glutamate
residues on the N-terminal region of prothrombin, fX and
other coagulation factors. A low degree of γ–carboxylation
prevents
coagulating
factors
from
undergoing
conformational change to achieve their active form.8,9
Direct thrombin inhibitors
Interest in antithrombotic research increased in the 1980’s
as the contribution of blood clotting to disease became more
apparent. 4 Due to its blood-coagulating role, thrombin was
considered a promising target for inhibition. During this
time, experiments with direct thrombin inhibitor (DTI)
hirudin (Ki = 22 fM) confirmed the potential of DTI’s as
therapeutic anticoagulants.10 Naturally occurring hirudin
was first isolated from leeches in 1955, but can now be
obtained via recombinant DNA techniques (lepirudin). In
1990, the crystallographic structure of a lepirudin-thrombin
complex was solved. 11 Hirudin served as inspiration for
synthetic DTI hirulog (20 AA’s) for intravenous (IV)
administration. Peptidomimetic approaches, inspired by the
Figure 1. The coagulation cascade under normal conditions (top) and
thrombotic conditions (bottom). Taken from Colman R.W.3
1
Table 1. The substrate sequence residues for fXa (fibrinopeptide A,
FPA; fibrinopeptide B, FPB) and fIIa (fibrinogen-α, FGA; fibrinogenβ, FGB).
and hydrogen bonding capacities. This information can be
used to both design potential ligands as well as to evaluate
these ligands in silico. Such molecular modeling experiments
can in return uncover essential or desired structural motifs
for competitive binders and transition state isosteres.
Transition state mimics block the activity of catalytic
proteins by occupying their active site. This is achieved by
formation of a covalent bond or via competitive binding. 16,17
Subsequently, the transition from potent binders to viable
drug candidate involves numerous structural revisions. Such
revisions, aimed at optimization of in vivo stability (e.g.
susceptibility to hydrolysis) and pharmacokinetic properties
(e.g. Lipinski’s rule of five18), demand iterative in vitro and in
vivo evaluation. Such “lead optimization” is required
regardless of how the lead was discovered, via structurebased design or for instance via high throughput screening
(HTS) and it is beyond the scope of this review to look at
these processes in detail. 17
Figure 3. The Schechter and Berger nomenclature for substrate
amino acid residues. (right).12,26
structure of fIIa substrate fibrinogen, led to the development
of DTI argatroban for IV administration.12After publication of
the fIIa crystal structure in 1989, ximelagatran (4, scheme 1)
was the first orally available DTI. 4,12 Unfortunately,
prolonged Ximelagatran treatment causes hepatic toxicity
and the drug was withdrawn during the last stages of clinical
trials in 2006. 12,13 Dabigatran etexilate (7, scheme 1), FDA
approved in 2010, remains the only clinically used oral
DTI.1,8
Direct fXa inhibitors
The scope of the search for oral anticoagulants (OAC’s) was
broadened after publication of the fXa crystal structure in
1993 (Padmanabhan et al. 14). Reports on the narrow
therapeutic window of fIIa inhibition sparked the idea that
fXa might be a better target for direct anticoagulant
inhibition. fXa possesses far less functions than thrombin,
both inside and outside the hemostatic system, and is
therefore a safer target for inhibition. 15 Rivaroxaban1,12,13
(11, scheme 2) was the first oral direct fXa inhibitor to
receive FDA approval in 2008 and Apixaban (16, scheme 3)
followed in 2012.
Thrombin and fXa
fXa (396 AA) and thrombin (295 AA’s) posses similar
trypsin-like serine protease domains (254 AA’s and 259 AA’s
respectively) with a shared sequence homology of 37%
(Figure 4). The ‘catalytic triad’ of this domain consists of
His57, Asp102 and Ser195. The serine hydroxyl moiety
initiates nucleophillic attack on the substrate and the
negatively charged transition state is stabilized through
hydrogen bonding. Elimination of the newly formed Nterminus and hydrolysis of the serine ester yield the cleavage
products. Schechter and Berger describe the nomenclature
for substrate amino acid residues (Pi) and their
corresponding binding sites (Si) (Figure 3).19
Thrombin cleaves fibrinogen-α (Gly-Gly-Val-Arg-||-Gly-Pro)
and fibrinogen-β (Phe-Ser-Ala-Arg-||-Gly-His) to form fibrin
building blocks fibrinopeptide A (16 AA’s) and
Structure-based design
Structural data of proteins, obtained via X-ray
crystallography or NMR data, can be used to study proteinligand binding sites in great detail. Such active sites are
described in terms of molecular surfaces, electrostatic fields
Figure 4. The fXa binding pocket. Picture taken from Pinto et al. 13
Figure 5. The fXa S4 “aromatic box”. Figure taken from Nar H.1
2
Scheme 1. Structures of direct fIIa inhibitors. K1 values and interactions with binding pockets, based on co-crystal structure analysis are indicated
(S1 = blue, S2 = magenta, S4 = red).
fibrinopeptide B (14 AA’s). fXa activates thrombin by
cleaving prothrombin twice (Phe-Asn-Pro-Arg-||-Thr-Phe
and Tyr-Asp-Gly-Arg-||-Ile-Val). Table 1 shows the position
of substrate amino acids during the cleavage events
mentioned above. The natural substrate sequences that bind
the protease cleavage sites are indicative of the properties of
those sites. Differences and similarities between thrombin
and fXa substrate sequences therefore reflect differences and
similarities between thrombin and fXa binding sites.
Arginine, which contains a lipophilic aliphatic chain and a
positively charged guanidine moiety, fills S1 during all
cleavage events mentioned above. A negatively charged
carboxylic acid residue (Asp189) at the bottom end of the
largely hydrophobic S1 site immobilizes the guanidine
residue through ionic and hydrogen-bond interactions. In
fXa, but not in fIIa, a “disulfide pocket” (Cys191, Gln192,
Gly218, Cys220) is located next to S1.
The fXa S2 site is selective for smaller amino acids than the
fIIa S2 site. Bulky Tyr99 limits the size of S2 in fXa, while fIIa
contains a pocket expanding hydrophobic insertion loop
(Tyr60A-Pro60B-Pro60C-Trp60D). The Gly216 backbone
allows for hydrogen-bond interactions in the flat and
exposed S3 site in both enzymes. Modeling experiments
suggest that the fIIa S3 site allows for more lipophillic
interactions than its fXa counterpart.
The fXa S4 site consists of an aromatic box (Tyr99, Phe174,
Trp215) that leads to a cation hole, consisting of the carbonyl
moieties of Lys96, Glu97 and Thr98 (figure 5). In fIIa, a
similar but mostly non-aromatic region (Asn98, Leu99,
Ile174, Trp215) provides access to the same cation hole.
However, in fIIa, the existence of Glu97A limits access to S4
and the S2 insertion loop requires thrombin binders to
assume a folded conformation.
To conclude, structural differences between the fIIa and fXa
binding sites, in particular S2 and S4, allow for selective
inhibition. Rivaroxaban is 10,000-fold selective for fXa over
other serine proteases that include fIIa and trypsin. 20
Conversely, the similarities between fIIa and fXa have been
exploited for the development of dual inhibitors (see figure
9). 12
Ximelagatran and dabigatran etexilate
Ximelagatran (4) (Scheme 1) failed to obtain FDA approval
in 2006 and its manufacturer, AstraZeneca, subsequently
retracted the drug from the European market after two years
of clinical use. The story behind its development is
interesting as it involves
“conventional”
peptidomimetic approaches
as well as structure-guided
design.
In
the
1950’s,
fibrinopeptide A had been
shown to act as competitive
inhibitor of the fibrinogen
cleavage reaction by fIIa. This
inspired the design of FPA
peptide analogues that would
be able to do the same.
Peptides D-Phe-Val-Arg, DPhe-Val-Arg-Gly-Pro and a
series of dipeptide-based
compounds seemed to be
promising starting points for
peptidomimetic based efforts
to improve upon potency and
“drug-like” properties. This
Figure 6. The co-crystal structure of dabigatran
in fIIa. The benzamidine group is positioned in
approach
led
to
the
S1 and the methylbenzimidazole occupies S2
development of DTI inogatran
behind the insertion loop. The pyridyl ring is
(1), in which the arginine
positioned in the S4 aromatic box where it
motif is preserved. However, a
…
engages in an edge-on CH π interaction. Picture
high rate of clearance and
30
taken from van Ryn et al.
poor oral bioavailability deem
inagotran unsuitable as oral
3
thrombin inhibitor.
An opportunity to improve upon inagotran in structureguided fashion presented itself after AstraZeneca obtained
the crystal structure of fIIa in complex with irreversibly
bound inhibitor PPACK (D-Phe-Pro-Arg-chloromethylketone) (2) in 1989 (Bode et al. 21). This crystal structure
placed the arginine sidechain in S1, the proline pyrrolidine in
S2 and the phenylalanine sidechain in S4. The
chloromethylketone moiety forms a hemiketal with Ser195
and alkylates His57. Based on this structural data, primitive
docking experiments and intuition, the researchers at
AstraZeneca came up with 12 to 15 structures for synthesis
and biological testing. One of these compounds, melagatran
(3), was 8 times more potent than inagotran and showed a
more favorable dose-response profile than Warfarin.
However, the oral bioavailability was still poor (3-7%).
Ultimately, AstraZeneca was able to develop the more orally
available (18-24%) double prodrug ximelagatran (4), which
is rapidly metabolized to melagatran in the body.4,12
Dabigatran etexilate (7), like ximelagatran, was developed
through a combined peptidomimetic structure-based
strategy. The central glycine was removed from starting
point NAPAP (5) to obtain a series of more rigid peptoids
with increased in vivo stability. Based on co-crystal structure
analysis, a central template-like benzamidazole core was
incorporated to provide rigidity and favorable orientation
for the binding site filling functionalities. In addition,
structural data was helpful during replacement of the
sulfonamide spacer with a carboxamide while retaining the
correct orientation for the P4 pyridine moiety. These
modifications yielded dabigatran (6), which was ultimately
marketed by Boehringer Ingelheim (BI) as its double
prodrug dabigatran etexilate (7).1,8
Scheme 2. The development of Rivaroxaban. K1 values and
interactions with binding pockets, based on co-crystal structure
analysis are indicated (S1 = blue, S2 = magenta, S4 = red).
interacting with Asp189, the P1 group displaces a structural
water molecule and engages in (Ar)C-Cl…π bonding with
Tyr228. An attractive interaction arises between the
electropositive σ hole opposite to the C-Cl bond and π
density above the tyrosine aromatic ring. 1 The non-basic P1
group was introduced independently in the fXa field in 1998,
of which clinically used rivaroxaban (13) (Bayer) and
apixaban (14) (Bristol-Myers Squibb Company) are
examples.
Bayer found several “hits”, including compound 9, via HTS of
a 200,000 compounds containing library against fXa
(Scheme2). The phosphonium group was incorrectly
assumed to act as arginine mimic without making use of
structural information. Replacement of the phosphonium
group with amidine, imidazoline and pyridine groups yielded
potent (Ki ≥ 2 nM), but poorly bioavailable compounds.
Meanwhile, the Bayer group found a steep structure-activity
relationship for the chlorothiophene moiety. The lack of
successful drug candidates prompted the researchers to look
at a less potent hit, compound 10. Substitution of the
thiophene group with the same chlorothiophene moiety that
compound 9 possesses increased the potency dramatically
(Ki = 90 nM). Further improvement of the oxazolidinone,
based on the experience that had been gained with
compound 9, yielded rivaroxaban. A co-crystal structure
with fXa explained rivaroxabans mode of binding and the
presence of the chlorothiophene group in S1 (Figure 7).
However, Bayer did not make use of fXa structural
information during the development process.1,12,13,23
Bristol-Myers Squibb Company (BMS) did make extensive
use of structural information during development of
apixaban (16). Researchers at BMS noted similarities
between the substrate sequences of glycoprotein IIb/IIIa and
fXa. They used the library that had been composed in search
of IIb/IIIa inhibitors to screen for fXa inhibitors. One of the
hits, compound 12 (Scheme 3), was optimized via structurebased design and molecular-modeling. Intended P4 groups
were designed for edge-to-face and π-π interactions with
Trp215 in the S4 aromatic box. The P1 benzamidine moiety
was replaced with less basic benzylamine, which gave
DPC423 (13). Figure 8a shows the binding mode of DPC423,
obtained by modeling the compound in the fXa binding site.
24 This revealed that the mode of binding for DPC423 is very
similar to analogs of 12 despite the single interaction with
Asp187 (versus a bidentate interaction in benzamidine
compounds like 12). Compound 13 was improved upon by
incorporation of more basic and water-soluble P4 groups.
The benzylamine moiety was replaced with an
aminobezisoxazole group to yield phase II clinical candidate
razaxaban (14).
Non-basic P1 groups in rivaroxaban and apixaban
Conventional fIIa and fXa direct inhibitors, including
melagatran and dabigatran, contain arginine-like P1
functionalities. The notion that such an arginine motif is
essential for potent binding was refuted in the late 1990’s
when several non-basic potent fIIa and fXa direct inhibitors
emerged. The co-crystal structure of compound 8 (Merck
USA) with fIIa, published in 1998, shows the chlorophenyl
moiety occupying the S1 site. 22 However, rather than
Figure 7 The co-crystal structure of rivaroxaban in fXa. The
chlorothiophene group fills S1 while S2 and S3 are unoccupied. The
morpholinone is positioned in the S4 aromatic box where it
interacts with Trp215 via edge-to-face CH…π bonding and via
sandwich-oriented interactions with Tyr99 and Phe174. Picture
taken from van H. Nar.1
4
(a)
(b)
(c)
(d)
Figure 8. (a) A binding model of DPC423 in fXa24; (b) Overlapping cocrystal structures of BMS740808 (orange) and razaxaban (white) in
the binding site of fXa31; (c,d) X-ray structures of apixaban bound to
fXa.25,32
Scheme 3. The development of apixaban with
color indicated binding motifs (S1 = blue, S2 =
magenta, S4 = red)).1
5
so far and most of the discoveries are ascribed to
serendipity. 1
On the development of oral anticoagulants
The recent successes in the field of direct oral anticoagulants
are obvious. Dabigatran etexilate, rivaroxaban and apixaban
are now used clinically. Oral fXa inhibitors edoxaban (Daiichi
Sankyo), betrixaban (Merck Direct Factor) and darexabam
(Astellas) are undergoing phase III clinical trials and four
others are in phase II trials.29It is however more difficult to
ascribe these successes to single factors. Apart from the
availability of structural information during the last two
decades, advances were made in HTS possibilities and there
was a general increase in funding in this field of research.
It is clear that there has been great interest in structural
information. So far, 318 fIIa and 117 fXa (co-)crystal
structures have been reported in the Protein Data Bank and,
most likely, many more remain undisclosed in possession of
pharmaceutical companies.1 Co-crystal structures have been
essential in unraveling the binding modes of fIIa and fXa
inhibitors. Detailed mapping of the S1/P1 interactions paved
the way for non-basic P1 groups that increase the
bioavailability of drug candidates without reducing potency.
The affinity of aromatic and cationic P4 groups for the S4 site
is now well understood. However, there seems to be a high
degree of “retrospect rationalization” of the how and why of
potent inhibitors and a more modest predictive effect. The
insights into S1 and S4 binding possibilities have been
gained from co-crystal structures with compounds that seem
not to have been designed to bind the respective sites in the
manner that they apparently did. Of course, this does not
diminish the value of such structural insights as they provide
researchers with starting valid starting points for new drug
candidates. It does question the necessity of knowing how
certain structural motifs contribute to binding potency after
binding assays have already demonstrated that they do so.
In addition, the contribution of peptidomimetic approaches,
which were initiated before structural information on
coagulating factors was available, should not be
underestimated. Those peptidomimetic strategies, at times
aided by structural data, provided the basis for the oral
anticoagulants that have been and continue to be developed.
Figure 9. fIIa and fXa dual inhibitors with color indicated binding
motifs (S1 = blue, S4 = red, S2/S4 = purple).1
The crystal structure of razaxaban in complex with fXa
(Figure 8b) shows that the aminobezisoxazolic amine
interacts strongly with Asp189 and the carbonyl of Gly218 in
the S1 site. The imidazole group occupies S4 where the N-3
nitrogen forms a hydrogen bond with the N-H of Gly216.
Razaxaban was improved further by enforcing the possibly
scissile amide bond with a cyclic motif. This yielded
compound 15 (BMS740808) that binds fXa in similar fashion
as razaxaban (Figure 8b).
Synthetic analogues of 15 showed that potency could be
improved further by variation of the P1 and P4 groups.
Implementation of the P4 lactam and the p-methoxyphenyl
non-basic P1 group yielded apixaban (16) and ensured a
favorable pharmacokinetic profile. The trifluoromethyl
group had been replaced with a carbamoyl moiety to
decrease undesired protein binding. The apixaban co-crystal
structure confirms the presence of the p-methoxyphenyl
group in S1, where, interestingly, it doesn’t seem to interact
with any specific residue (Figure 8a). Hydrogen bonds exist
between the central carboxamide carbonyl and Gly216 as
well as between the N-2 pyrazole nitrogen and Gly192. The
aryllactam is positioned between Tyr99 and Phe174 in
S4.1,13,25,26
Dual fIIa and fXa inhibitors
Dual inhibition of thrombin and fXa by multiple inhibitors at
low doses has demonstrated a synergistic antithrombotic
effect in animal models.27Single inhibitors that show equal
affinity for fII as for fX are therefore a promising new class of
anticoagulants.28 No dual inhibitors have been improved for
clinical use, but Boehringer Ingelheim, Sanofi-Aventis, Merck
and GlaxoSmithKline (GSK) have published on potent
compounds. Tanogitran (17) (Figure 9), an IV administered
phase II clinical trial candidate, is derived from BI’s
dabigatran project. Co-crystal structure analysis revealed
that tanogatran binds fIIa and fXa in different conformations,
but with identical S1 and S4 interactions. Compound 18,
which shows identical affinity for thrombin and fXa, was
developed by GSK. Their researchers found a steep SAR for
the unsaturated linker between the chlorothienyl and
sulfonamide groups. The unsaturated compound, without
methyl alkylation, is 92-fold more selective for fXa over fIIa,
while saturation of that double bond led to 9-fold preference
for fIIa over fXa. Finally, methyl substitution of the ethenyl
group in compound 18 led to equal affinity for both
coagulating factors. Compound 18 binds thrombin and fXa in
very distinct manners as the terminal morpholino occupies
S2 in fIIa and S4 in fXa.1,12
All advanced dual inhibitors posses aliphatic P4 groups,
which seems to be a good strategy to circumvent the
differences between the fIIa and fXa S4 sites. Interestingly,
structural information was barely used during the
development of the dual inhibitors that have been published
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