First steps in the direction of synthetic, allosteric, direct inhibitors of thrombin and factor Xa

Article abstract of DOI:10.1016/j.bmcl.2009.06.013

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that (i) se

Full text of DOI:10.1016/j.bmcl.2009.06.013

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4126–4129
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
First steps in the direction of synthetic, allosteric, direct inhibitors
of thrombin and factor Xa
Jenson Verghese a,b, Aiye Liang a,b, Preet Pal Singh Sidhu a, Michael Hindle c,
Qibing Zhou d, Umesh R. Desai a,b,
*
a Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA, United States
b Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA, United States
c Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, United States
d Department of Chemistry, Virginia Commonwealth University, Richmond, VA, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of
small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against
thrombin and factor Xa. The results reveal that (i) selected monomeric benzofuran derivatives inhibit
the two enzymes, albeit weakly; (ii) the two enzymes recognize different structural features in the ben-
zofurans studied suggesting significant selectivity of recognition; and (iii) the mechanism of inhibition is
allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 March 2009
Revised 28 May 2009
Accepted 1 June 2009
Available online 6 June 2009
Keywords:
Thrombin
Factor Xa
Inhibition
Allosteric
Synthetic small molecules
Library
Screening
Lignins
Dehydropolymers
Thrombin and factor Xa are pivotal enzymes of the coagulation
cascade. Both enzymes are the prime target of the current heparin-
based anticoagulant therapy, which has been in use for more than
seven decades. Despite its usefulness, heparin is associated with
several adverse effects including an enhanced risk for bleeding.1,2
In addition, the animal origin of the drug is a cause for concern
with respect to viral or other potentially infectious agent contam-
ination. Likewise, recent incidences of oversulfated chondroitin
sulfate contaminating unfractionated heparin (UFH) preparations
also highlight the need for new heparin-like anticoagulants.3–5
Molecules that functionally mimic heparin without its adverse
effects are highly desirable. Heparin is a polysaccharide that is dec-
orated with numerous sulfate and carboxylate groups. Designing
heparin mimics, especially not based on a saccharide scaffold, to
specifically recognize its prime targets such as antithrombin,
thrombin and factor Xa, is a major challenge. Non-sugar scaffolds
that can bear multiple sulfate and carboxylate groups and are as
large as a typical active sequence in heparin are difficult to synthe-
size. Additionally, designing such scaffolds is fraught with the
problem of poor specificity arising from surface exposed heparin
binding sites on proteins.6,7 Finally, robust computational tools
available to reliably predict the interactions of highly anionic mol-
ecules with heparin-binding proteins are not yet available.
Our effort to design large non-saccharide scaffolds that function-
ally mimic heparin resulted in the chemo-enzymatic synthesis of
sulfated DHPs. Sulfated DHPs are synthetic variants of the naturally
available lignin and a specific sulfated DHP, CDSO3 (Fig. 1), was
found to potently inhibit thrombin and factor Xa with an IC50 of
18–34 nM under physiological conditions.8 More importantly,
CDSO3 also inhibited blood clotting under ex vivo conditions with
potency comparable to the low molecular weight heparins.9
CDSO3 represents a library of structures arising from multiple
inter-monomeric linkages (e.g., b-5, b-O-4, b-b and 5-5), variable
sulfation and carboxylation of oligomeric chains, the presence of
numerous chiral centers, and variable chain length (Fig. 1).10 These
four major variables introduce phenomenal structural diversity in
a typically preparation of sulfated DHP. For example, a simple
calculation reveals that 262,144 distinct decamer structures are
possible for CDSO3.
Mechanistically, CDSO3 binds to exosite II of thrombin to allos-
* Corresponding author. Tel.: +1 804 828 7328; fax: +1 804 827 3664.
E-mail address: urdesai@vcu.edu (U.R. Desai).
terically disrupt the catalytic apparatus resulting in inhibition.8
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.013

J. Verghese et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4126–4129
4127
equivalent of H2O as the traditional hydrolytic conditions (NaOH/
EtOH and HCl/EtOH) resulted in the breakdown of the aromatic
O-sulfate group. The library of 17 small molecules so synthesized
contained members devoid of anionic groups, for example, 1E, to
those bearing two sulfate and one carboxylate groups, for example,
6AS.
CDSO3
(MR = 3,320 Da; 5 – 13-mers)
_
OSO3
_
O
O
R
O
4
β
β
5
O
_
Thrombin and factor Xa inhibition properties: Inhibition of throm-
bin and factor Xa by b-5-like benzofuran derivatives was followed
by spectrophotometric determination of the initial rate of hydroly-
sis of appropriate chromogenic substrate, as previously described
in our work.8 Spectrozyme TH and S-2772 were used as substrates
of thrombin and factor Xa, respectively. Appropriate controls to
correct for changes introduced by small volumes of organic sol-
vents, such as DMSO used for dissolving highly hydrophobic mol-
ecules, were performed. The fractional decrease in the rate of
initial hydrolysis of Spectrozyme TH (thrombin) or S-2772 (factor
Xa) in the presence of 0.4–4.3 mM benzofuran derivative as com-
pared to that in its absence corresponded to the inhibition poten-
tial of the molecule (see Supplementary data for details).
Of the 17 molecules screened, only 1A, 2A, 4AS, 5AS, 6AS, 7A
and 7AS were found to exhibit inhibitory properties (Fig. 3). This
indicates that only selected b-5-like monomeric units possess the
capability to induce direct inhibition of thrombin and factor Xa.
The minimal concentration necessary to display inhibition was
OSO3
O
_
O
phenoxy-
dihydro-
propanoic acid benzofuran
Figure 1. Oligomeric structure of
a chemo-enzymatically synthesized CDSO3
consisting of b-5 and b-O-4 inter-monomeric linkages. Substituent R can be either
–H, –OH, or –OSO3ꢀ. Chiral centers are identified using curvy bonds. CDSO3 can be
thought of as being made from dihydro-benzofuran and phenoxy-propanoic acid
monomeric units joined either in an alternative or successive manner. The average
chain length of CDSO3 is 5–13-monomer units.
This is the first example of an exclusive exosite II-mediated inhibi-
tion mechanism and represents an exciting opportunity for design-
ing new anticoagulants.
In this work, a small library of 17 b-5-like monomeric benzofu-
ran derivatives was synthesized and screened against thrombin
and factor Xa. The results reveal that (i) monomeric CDSO3-based
structures are inhibitory, albeit the potency is weak; (ii) thrombin
and factor Xa appear to recognize different structural features sug-
gesting significant selectivity of recognition; and (iii) the inhibition
mechanism is allosteric.
found to be approximately 400 lM, which corresponds to a moder-
ate level of inhibition potential. Yet, interesting structure–activity
relationships are evident. All ester containing molecules were
found to exhibit no inhibition suggesting a key role for the –COOꢀ
group. Whereas no inhibitor displays more than 40% inhibition of
thrombin at 2.9 mM, at least four out of seven (4AS, 5AS, 6AS,
and 7A) inhibit factor Xa better (>40%) at 2.6 mM (Fig. 3). This sug-
gests a preference of the benzofuran scaffold for targeting factor
Xa. More importantly, inhibitors 5AS and 6AS display 86% and
75% inhibition of factor Xa, which is much better than all other ac-
tive inhibitors, suggesting significant selectivity of recognition by
the enzyme. The common ‘pharmacophore’ present in these two
molecules is the 3-COOꢀ and 6-OSO3ꢀ unit, which is absent in all
other structures. Interestingly, factor Xa inhibition by 4AS, which
is a regioisomer of 5AS, was much weaker (42%) further supporting
preferential recognition hypothesis. Inhibitors 5AS and 6AS more
closely resemble the native CDSO3 monomeric unit than inhibitors
7A or 7AS, which possess a non-native, extended COOꢀbearing lin-
ker (see Fig. 2).
Results and discussion. Rationale for the design of the benzofuran
library: b-5 and b-O-4-linked chemo-enzymatically prepared
CDSO3 can be thought of as being made from dihydro-benzofuran
and phenoxy-propanoic acid monomeric units. These monomeric
units may be alternatively, successively or randomly linked (see
Fig. 1). Each unit may or may not bear sulfate group(s) to give
the heterogeneous CDSO3. Assessing the role of both these struc-
tural units requires the availability of a large library of sulfated
and carboxylated, aromatic molecules containing multiple stereo-
centers. As a first step, we focused on synthesizing a small, achiral
benzofuran library to assess whether the smallest structural unit of
CDSO3, that is, a b-5-like monomer, possesses thrombin and factor
Xa inhibitory property.
Synthesis and description of the benzofuran library: The synthesis
of the b-5-like benzofuran monomer library is described in detail
in the Supplementary data. Briefly, laccase-mediate oxidative cou-
pling of catechol and ethylacetoacetate was used to prepare the
parent 5,6-dihydroxy-benzofuran-3-carboxylic acid ethyl ester
monomer 1E (Fig. 1), as reported in literature.11 Monomer 1E
served as a starting point for differentially introducing the multiple
sulfate and carboxylate groups on the scaffold. Most synthetic
steps used in the construction of the library involved simple func-
tional group transformations (see Supplementary data). Yet, the
synthesis of library members containing both O-sulfate and car-
boxylate groups, especially 4AS, 5AS, 6AS and 7AS, was not trivial.
The synthesis of polysulfated small, aromatic molecules is known
to be challenging.12 Common chromatographic techniques used
to purify organic molecules fail to work well with these highly
water soluble molecules. Additionally, the stability of these highly
anionic molecules is suspect. In fact, molecule 1XS was found to be
fairly unstable in aqueous solution, the reason for which is unclear
at the present time. We utilized a microwave-based sulfating pro-
tocol developed in our laboratory13 followed by size exclusion and
cation exchange chromatography to synthesize the targeted sul-
fated benzofuran ethyl esters in good to high yields (see Schemes
I–III in Supplementary data).
RO
RO
O
O
CH3
CH3
R'O
R'O
COOEt
COOH
1 E : R= H,
2 E : R= Me,
3 E : R= H,
4 ES : R= Me,
5 ES : R= SO3¯ , R’= Me
6 ES : R= SO3¯ , R’= SO3¯
R’= H
R’= H
R’= Me
R’= SO3¯
1 A :
2 A :
4 AS : R=Me,
5 AS : R=SO3¯ , R’= Me
6 AS : R=SO3¯ , R’= SO3¯
R= H,
R= Me
R’= H
R’= H
R’= SO3¯
,
CH3O
O
MeO
O
CH3
CH3
R'O
R'O
CH2OR"
CH2CH2COOR"'
1 X :
R’= H,
R”= H
7 E :
7 ES : R’= SO3¯ , R”’= Et
7 A : R’= H, R”’= Na
7 AS : R’= SO3¯ , R”’= Na
R’= H,
R”’= Et
1 XS : R’= SO3¯ , R”= SO3¯
Conversion of the ester to the acid functionality worked only
with potassium t-butoxide in anhydrous DMSO containing one
Figure 2. Structures of 17 benzofuran derivatives present in the small synthetic
library screened against factor Xa and thrombin.

4128
J. Verghese et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4126–4129
enzyme and a 3.6-fold decrease in the catalytic rate of hydrolysis.
1A
2A
100
Thus, the presence of 5AS brings about structural changes in the
active site of thrombin, which significantly alters the formation
of the factor Xa–S-2772 Michaelis complex as well as induces dys-
function of the catalytic apparatus of the enzyme. Considering that
the parent inhibitor, that is, CDSO3, is known to bind exosite II of
thrombin,8 these results support the hypothesis that the benzofu-
ran-based synthetic small molecules also inhibit factor Xa and
thrombin through an exosite II-mediated allosteric dysfunction of
the enzymatic catalytic triad.
Thrombin
80
60
40
20
0
4AS
5AS
6AS
7A
*
7AS
*
Significance: A large number of inhibitors have been designed in
the past decade to inhibit thrombin and factor Xa. These include
peptide-based biomolecules, peptidomimetics, natural products
and small hydrophobic structures.14–16 Nearly all of these inhibi-
tors, especially the small organic molecules, target the active site
of the enzymes. Small molecules that do not function as competi-
tive inhibitors of thrombin and factor Xa are unknown and the
molecules studied here are the first example of allosteric
inhibitors.
The benzofuran-based inhibitors studied here are not potent
inhibitors of the two procoagulant enzymes. This is not too unex-
pected because (a) these first generation inhibitors are fairly small
molecules containing sub-optimal binding features, (b) only one
type of monomeric structural unit (benzofuran) is present in these
molecules as compared to the two structural units present in
CDSO3 (see Fig. 1), and (c) the CDSO3 binding site on thrombin
and factor Xa is likely to be large, possibly involving multiple ben-
zofuran and phenoxy propanoic acid structural units to generate
nanomolar affinity. It is to be expected that coupling the most po-
tent structures found here, that is, 5AS and 6AS, with phenoxy-
propanoic acid-based monomeric units will result in better inhibi-
tion profile. In fact, the average chain of CDSO3 is 5–13-mer in
length,8,10 which implies that this process may have to be opti-
mized through several iterations. Thus, the screening of the small
benzofuran-based library represents a first step in the design of
allosteric inhibitors of thrombin and factor Xa.
The design of allosteric inhibitors is typically challenging be-
cause of inadequate information on the nature of the binding site
and the mode of interaction. This inadequacy is amplified with
heparin mimics because of the highly surface exposed interaction
involving primarily electrostatic phenomenon. Yet, the novel allo-
steric mechanism of these small synthetic molecules affords an
opportunity to discover radically different molecules from the
known ligands with possibly different pharmacological and toxico-
logical profile. Thus, the synthetic molecules designed on the basis
of the CDSO3 scaffold may results in potent functional mimics of
heparin.
1A
100
Factor Xa
2A
80
60
40
20
0
4AS
5AS
6AS
7A
7AS
Figure 3. Inhibition of thrombin (upper plot) and factor Xa (lower plot) by
benzofuran derivatives 1A, 2A, 4AS, 5AS, 6AS, 7A, and 7AS. The monomers were
present at 2.9 mM (thrombin) or 2.6 mM (factor Xa) concentration, except for the
asterisk (Ã) labeled cases for which the concentration was 4.3 mM. All other
derivatives in the library were found to be essentially inactive below 4.3 mM. The
standard error in these experiments was less than 10%.
Mechanism of factor Xa inhibition: To understand the molecular
basis for the inhibitory potential of the benzofuran derivatives,
the kinetics of S-2772 hydrolysis by factor Xa at pH 7.4 and 25 °C
in the presence and absence of 5AS was determined. S-2772 is a
chromogenic substrate of factor Xa. The initial rate of hydrolysis
varied in a hyperbolic manner with the concentration of the sub-
strate, as expected (Fig. 4), from which the Michaelis constant
(KM) and maximal velocity of the reaction (VMAX) were derived
(see Supplementary data). The KM and VMAX for S-2772 in the ab-
sence of 5AS was found to be 441 86
197 22 mAU minÀ1 MÀ1, respectively. In the presence of 5AS,
these values changed to 61 13
M and 54 4 mAU minÀ1 MÀ1
respectively. This suggests that the presence of 5AS significantly
affects the binding of the chromogenic substrate to the active site
of the enzyme. More specifically, the interaction of 5AS with factor
Xa results in a sevenfold increase in affinity of the substrate for the
lM
and
l
l
l
,
Acknowledgements
120
100
80
60
40
20
0
This work was supported by the National Heart, Lung and Blood
Institute (HL069975 and HL090586), the American Heart Associa-
tion National Center (EIA 0640053N), the A. D. Williams Founda-
tion (6-46064) and the Mizutani Foundation for Glycoscience.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.013.
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