On scaffold hopping: Challenges in the discovery of sulfated small molecules as mimetics of glycosaminoglycans

Article abstract of DOI:10.1016/j.bmcl.2012.10.079

The design of sulfated, small, nonsaccharide molecules as modulators of proteins is still in its infancy as standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scoring protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing nonsaccharide molecules, which severely limits the identification of new hits. Lack of a generally applicable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic mimetics of the highly sulfated glycosaminoglycans. We explored a sequential ligand-based (LBVS) and structure-based virtual screening (SBVS) approach starting from our initial discovery of monosulfated benzofurans to discover alternative scaffolds as allosteric modulators of thrombin, a key coagulation enzyme. Screening the ZINC database containing nearly 1 million nonsulfated small molecules using a pharmacophore developed from the parent sulfated benzofurans followed by a genetic algorithm-based dual-filter docking and scoring screening identified a group of 10 promising hits, of which three top-scoring hits were synthesized. Each was found to selectively inhibit human alpha-thrombin suggesting the possibility of this approach for scaffold hopping. Michaelis-Menten kinetics showed allosteric inhibition mechanism for the best molecule and human plasma studies confirmed good anticoagulation potential as expected. Our simple sequential LBVS and SBVS approach is likely to be useful as a general strategy for identification of sulfated small molecules hits as modulators of glycosaminoglycan-protein interactions.

Full text of DOI:10.1016/j.bmcl.2012.10.079

Bioorganic & Medicinal Chemistry Letters 23 (2013) 355–359
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
On scaffold hopping: Challenges in the discovery of sulfated small molecules
as mimetics of glycosaminoglycans
Preetpal S. Sidhu a, Philip D. Mosier a, Qibing Zhou b, Umesh R. Desai a,
⇑
a Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, United States
b Institute for Materia Medica, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hunan 430074, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The design of sulfated, small, nonsaccharide molecules as modulators of proteins is still in its infancy as
standard drug discovery tools such as library of diverse sulfated molecules and in silico docking and scor-
ing protocol have not been firmly established. Databases, such as ZINC, contain too few sulfate-containing
nonsaccharide molecules, which severely limits the identification of new hits. Lack of a generally appli-
cable protocol for scaffold hopping limits the development of sulfated small molecules as synthetic
mimetics of the highly sulfated glycosaminoglycans. We explored a sequential ligand-based (LBVS)
and structure-based virtual screening (SBVS) approach starting from our initial discovery of monosulfat-
ed benzofurans to discover alternative scaffolds as allosteric modulators of thrombin, a key coagulation
enzyme. Screening the ZINC database containing nearly 1 million nonsulfated small molecules using a
pharmacophore developed from the parent sulfated benzofurans followed by a genetic algorithm-based
dual-filter docking and scoring screening identified a group of 10 promising hits, of which three top-
scoring hits were synthesized. Each was found to selectively inhibit human alpha-thrombin suggesting
the possibility of this approach for scaffold hopping. Michaelis–Menten kinetics showed allosteric inhibi-
tion mechanism for the best molecule and human plasma studies confirmed good anticoagulation potential
as expected. Our simple sequential LBVS and SBVS approach is likely to be useful as a general strategy for
identification of sulfated small molecules hits as modulators of glycosaminoglycan–protein interactions.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 26 August 2012
Revised 4 October 2012
Accepted 15 October 2012
Available online 24 October 2012
Keywords:
Allosteric inhibition
Anticoagulants
Scaffold hopping
Thrombin
Virtual screening
Thrombin is a plasma serine protease that plays important roles
in blood clotting.1 It cleaves fibrinogen to fibrin resulting in clot for-
mation, which is a natural defense mechanism to prevent excessive
loss of blood from injury. Yet, under abnormal conditions, internal
clot formation can results in heart attacks and strokes, which is typ-
ically prevented through the use of anticoagulants. Heparin, a
highly sulfated glycosaminoglycan (GAG), is one such anticoagulant
and has been in use essentially unchanged since the 1930s. It is also
an anticoagulant with numerous adverse consequences, which has
catalyzed the search for better anticoagulants.2
Majority of anticoagulant search efforts have focused on the
discovery of direct inhibitors of coagulation enzymes, including
thrombin and factor Xa. Dabigatran and rivaroxaban, small pepti-
domimetics, have been realized through these discovery efforts.
Both of these are active site inhibitors. Major efforts are also in pro-
gress to discover active site inhibitors of other coagulation en-
zymes including factor VIIa and factor XIa.
and better specificity of action. Whereas active site inhibitors typ-
ically display an efficacy of 100%, allosteric inhibitors can induce
less than quantitative inhibition, which in principle can afford
greater tunability and regulatory control. With regard to the
second point, higher specificity of action is possible from allostery
because of greater structural differences in allosteric binding sites
than in catalytic active sites.
Despite these advantages, the design of small molecule, alloste-
ric inhibitors of coagulation remains uncharted. In fact, the possi-
bilities for discovery of such molecules is high because most
coagulation enzymes contain allosteric binding sites. For example,
thrombin contains two anion-binding exosites, called exosites I
and II, that are characterized by clusters of basic residues. Various
co-factors and proteins interact with these exosites and regulate
the function of thrombin.3–5 Evidence accumulated over the past
several decades indicates that the proteolytic function of thrombin
can be altered through appropriate interaction with these exo-
sites.5 For example, heparin, a natural sulfated glycosaminoglycan,
binds in exosite II and alters the active site of thrombin.6 Likewise,
thrombomodulin, an endothelial cell surface receptor, engages
both exosites I and II to alter thrombin’s specificity from fibrinogen
to protein C.1,7 Other naturally occurring allosteric modulators of
thrombin include hirudin,8 chondroitin sulfate,9 and haemadin.10
A paradigm shifting idea is to discover small molecules that
allosterically modulate coagulation enzymes. Allostery can offer
major advantages such as fine control over a protein’s activity
⇑
Corresponding author.
E-mail address: urdesai@vcu.edu (U.R. Desai).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.079

356
P. S. Sidhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 355–359
Despite the availability of this natural avenue, few small molecules
have been designed that exploit the natural allosteric mechanism
of modulation of thrombin’s activity.
results.11,12 The hits generated from the LBVS study were then
screened by docking onto thrombin to identify a smaller group of
10 hits. Of these, three top-scoring molecules were selected for
thrombin inhibition studies. All three molecules based on a scaf-
fold significantly different from that of benzofuran, except for the
presence of sulfate group(s), were found to inhibit thrombin. The
hits displayed allosteric inhibition mechanism in a manner similar
to monosulfated benzofurans and plasma anticoagulation, as
would be expected. The success achieved with sequential LBVS
and SBVS bodes well for discovery of diverse sulfated small mole-
cules as modulators of protein function.
Recently, our laboratory designed the first small molecules act-
ing as allosteric inhibitors of thrombin.11 A group of sulfated ben-
zofurans containing
inhibition of human
a
single sulfate group exhibited good
a-thrombin under physiological conditions
(Fig. 1). Enzyme kinetic studies showed an allosteric inhibition
phenomenon, while later studies with site-directed thrombin mu-
tants identified the site of binding to a hydrophobic region close to
Arg173, a site encompassed within exosite II.12 This site of binding
is a new discovery and is likely to be a promising avenue for anti-
coagulant drug discovery.
To date monosulfated benzofuran scaffold is the only scaffold
available for allosteric modulation. This is a serious limitation. It
is important to discover alternative scaffolds to enhance struc-
ture–activity studies and improve the chance of clinical viability.
Yet, how do we discover alternative sulfated small molecule scaf-
Pharmacophore query generation. A pharmacophore was
generated based on the SAR of monosulfated benzofuran library
previously studied in our laboratory (Figs. 1 and 2).11,27 Briefly,
the potency of monosulfated benzofurans increased nearly a
1000-fold from a monomeric to a dimeric scaffold. Thus, at least
two aromatic units were deemed as essential. The average distance
between these aromatic units from their centroids was found to be
8.1 0.5 Å. The SAR also indicated a strong requirement of a hydro-
phobic substituent at the 3 position of both aromatic units. These
hydrophobic substituents were found to be 6.4 1 Å from the aro-
matic centroid. Finally, the most difficult condition to be imple-
mented in LBVS was the presence of a sulfate group at the 5
position of the first benzofuran ring (see Fig. 1). Commercial li-
braries do not contain molecules with a sulfate group. Hence, the
pharmacophore query was devised with a hydroxyl group, instead
of a sulfate group, under the assumption that it can be added later,
both in silico and by synthesis. These features constituted the
pharmacophore query for LBVS, which is shown in Figure 2.
Ligand-based virtual screening: LBVS was performed using UNITY
on ZINC database (Shoichet Laboratory, USCF, CA), which contains
structural information on nearly 1 million compounds (2006 ver-
sion) that are all nonsulfated. The LBVS hits were also expected
to satisfy three of four Lipinski’s rules (MW 6500, H-Bond donors
65, H-Bond acceptors 610, and À0.4 <LogP <+5.0). In addition, no
limit was placed on the number of rotatable bonds present in a hit.
Using this 3D pharmacophore query, LBVS on the ZINC database
identified 4560 hits. These hits were then processed computation-
ally to introduce sulfate group(s) using an in-house programming
script. The script identified free phenolic group(s) on the molecule
and performed an in silico sulfation.
folds?
A traditional technology is high-throughput screening
(HTS) of a library of diverse molecules. Yet, such a library of mole-
cules containing sulfate groups has not been developed nor is not
commercially available. Another approach is virtual screening
(VS). The primary goal here is to discover novel chemical structures
that exhibit efficacy at the desired target. This is a more cost-
effective solution with a reasonable level of hit discovery.13,14
Nearly all VS approaches can be classified into either structure-
based (SBVS) or ligand-based (LBVS) methods.14–16 While a major-
ity of discovery efforts have focused on one of the approaches, a
growing trend has been to utilize both to enhance the probability
of hit discovery.17–22 We have previously explored both SBVS and
LBVS, individually, to discover sulfated small molecules and under-
stand their interaction with proteins.11,12,23,24 Whereas SBVS of
sulfated small molecules has proved to be challenging because of
lack of robust scoring functions used in the docking, LBVS has been
found to be beset with inability to consider the shape of the bind-
ing site.25 LBVS is useful when applied to large libraries but such
libraries are not available for sulfated small molecules. Likewise,
SBVS with large libraries of sulfated small molecules, if available,
is also difficult because it is computationally intensive and very
time consuming.26
Despite these difficulties, VS is likely to be a useful approach to
rapidly discover diverse sulfated small molecule scaffolds. To as-
sess whether a VS-based approach can rapidly provide alternative
allosteric thrombin inhibitors, we explored a sequential LBVS and
SBVS approach. First we performed LBVS on a library of nearly 1
million nonsulfated molecules using a pharmacophore query
developed on the basis of our monosulfated benzofuran
Structure-based virtual screening: Molecular docking studies
were performed using the genetic algorithm-based dual-filter
protocol developed earlier for heparin-based sequences.28 In this
approach, the first filter identified structures that interacted with
the allosteric site on thrombin with a high GOLDScore, while the
second filter was used to identify molecules that bound in the
Figure 1. Structures of some monosulfated benzofuran dimers discovered as first
small molecular allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem. 2011, 54,
5522). Key units of this scaffold include: two aromatic rings, hydrophobic
substituents at 3 positions and monosulfate at the 5 position. Pharmacophore
query designed on the basis of this SAR is as shown in Figure 2.
Figure 2. Three-dimensional (3D) pharmacophore query developed on the basis of
SAR study with monosulfated benzofuran dimers (see Fig. 1).11,27 The atom in red
represents an oxygen of a hydroxyl group. See text for details.

P. S. Sidhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 355–359
357
Figure 3. Virtual screening algorithm (combination of LBVS and SBVS) used in the discovery of sulfated allosteric thrombin modulators (A) and the final 10 molecules
proposed by the algorithm as the top 10 hits (B).
same site with high consistency. Briefly, a single docking run was
performed with each member of the library derived from LBVS,
i.e., the 4560 hits. The molecules were docked onto the hydropho-
bic pocket of ꢀ22 Å radius near Arg173 of thrombin (PDB: 3EQO).
This filter led to the identification of 138 molecules with a GOLD-
Score greater than 70. These molecules were then subjected to the
consistency filter, in which docking was allowed to evolve for
greater number of iterations (ꢀ100,000) in triplicate. The struc-
tures that bound consistently in the same binding pose (RMSD
<2.0 Å) between the three independent runs were considered as
the final hits of the SBVS approach. It is important to recognize that
such a dual-filter screening algorithm has not been utilized earlier
to discover sulfated nonheparin scaffolds.
Only ten molecules of 4560 screened were found to satisfy the
requirements of both the filters (Fig. 3). These molecules possessed
both high GOLDScore as well as high consistency of binding geom-
etry. Although each structure is different, the molecules can be
broadly grouped into two major categories – linear and bifur-
cated. Molecules 1, 3 and 7 can be classified as linear, while 2,
4–6, and 8–10 belong to the bifurcated category. Majority of these
molecules contain two sulfate groups, except for 2, 6 and 7, which
are monosulfated. The GOLDScores for 1, 2 and 3 were 80.4, 74.0
and 75.6, respectively, which were significantly higher than that
for the next best hits. Hence, these were targeted for synthesis and
biochemical study.
Synthesis of 1, 2 and 3. Appropriate phenolic precursors of 1, 2,
and 3, i.e., 4-(4-(benzyloxy)phenyl)-2-((2-(2,3-dihydroxyphenyl)-2-
oxoethyl)
thio)-6-nitropyridine-3,5-dicarbonitrile,1-(4-hydroxy-
phenyl)-7-methyl-2-(4-methylbenzyl)-1,2-dihydrochromeno[2,3-c]-
pyrrole-3,9-dione and N-(4-hydroxyphenyl)-2-(3-{[(4-hydroxy-
phenyl) amino]carbonyl}phenyl)-1,3-dioxo-5-isoindolinecarboxa-
mide, respectively, were purchased from Spec (Wakefield, RI),
ChemBridge Corporation (San Diego, CA) and ChemDiv (San Diego,
CA), respectively. Sulfation of these precursors was performed
using the microwave-based sulfation method previously devel-
oped in our laboratory,29 which involved using the sulfating agent
trimethylamine-sulfur trioxide complex in the presence of the base
using the microwave radiation for 40 min at 100 °C. Sephadex C-25
cation exchange resin was used to exchange the ammonium ion to
sodium cation to improve the water solubility of the molecules.
The final molecules were purified using HPLC on a C–4 column to
obtain sulfated compounds in greater than 98% purity (see Supple-
mentary data). The formation of sulfated products was confirmed
by 1H NMR spectroscopy through a characteristic downfield shift
of protons ortho to the sulfate group(s). In addition, high resolution
ESI-MS was also used to confirm the structure of molecule (see
Supplementary data).
Linear and bifurcated refer to the presence or absence of branching with regard to
the scaffold. A scaffold devoid of a major branch point is defined as linear, while that
which displays an aromatic unit as a branch is labeled as bifurcated.

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P. S. Sidhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 355–359
Inhibition of human a-thrombin: A chromogenic substrate hydro-
lysis assay was used for evaluating the inhibition potential of the
best VS hits. Briefly, the fractional decrease in the initial rate of
Spectrozyme TH hydrolysis as a function of the concentration of
the inhibitor was plotted on a semi-log plot and fitted by the
logistic dose–response Eq.
Supplementary data). All three hits showed reasonable inhibition
of -thrombin at pH 7.4 and 25 °C. Inhibitors 1, 2, and 3 exhibited
IC50 of 65.9 7.3 M, 743 66 M and 286 20 M, respectively.
1 to derive the IC50 (Fig. 4, see
a
l
l
l
µ
These potencies are somewhat lower (ꢀ5- to 10-fold) than that ob-
served for monosulfated benzofuran dimers (Fig. 1) from which the
pharmacophore query was designed. Considering that primary
objective was to derive scaffolds different from benzofurans, the
success achieved with each of three best hits is encouraging. The
primary reasons for attempting to discover scaffolds different from
the benzofuran scaffold were to study whether additional
hydrogen-bonding atoms would reduce chances of aggregation,
introduce synthetic ease and provide access to expanded struc-
ture–activity relationships. Thus, the discovery of alternative scaf-
folds bodes well for development of synthetic analogs that may
display higher potency.
It is important to note that the potency of these GAG mimetics
is not too far from that reported in the literature on potencies of
related sulfated molecules. A large number sulfated GAG mimetics
display potencies in the low to high lM range for their targets. For
example, rationally designed sulfated flavonoids and sulfated tet-
Figure 5. Michaelis–Menten kinetics of Spectrozyme TH hydrolysis by human
a-
thrombin in the presence of
Supplementary data). Solid lines represent nonlinear regression fits to the data to
yield KM and VMAX
0 (s), 20 (N), 70 (h) and 185 (ꢀ) lM 1 (see
.
Screening against other serine proteases. To assess the selectivity
of thrombin inhibition, the three sulfated inhibitors 1, 2 and 3 were
screened against a panel of related coagulation proteases including
factors VIIa, IXa, Xa, XIa and XIIa. The screening was performed at a
single high concentration (365 lM) of inhibitors using appropriate
substrates in chromogenic substrate hydrolysis assays. Figure 6
shows the fractional residual activity of each enzyme calculated
as the ratio of initial rates of hydrolysis with and without the
inhibitor. None of the inhibitors decreased the activity of the coag-
ulation factors, except for factor XIa, by more than 20%. In the case
of factor XIa, 1 and 2 were found to display higher potency than
that of 3. This implies that our sequential LBVS and SBVS may have
also led to identification of novel factor XIa inhibitors. The result
suggests that factor XIa possesses an allosteric site similar to that
near Arg173 in thrombin, an interesting possibility worth pursu-
ing. Interestingly, the order of potencies against thrombin and fac-
tor XIa is different. This implies that binding site may be similar
but subtle differences could be exploited to discover molecules
with higher level of selectivity.
Prolongation of plasma clotting time: To further test whether the
hits are useful in preventing coagulation in a more biologically rel-
evant system, we utilized prothrombin (PT) and activated partial
thromboplastin time (APTT) coagulation assays. In these assays,
the concentration of an anticoagulant required to double the clot-
ting time of human plasma is measured using a fibrometer, as de-
scribed in our earlier work.12 The best hit, 1, was selected for these
rahydroisoquinolines displayed affinities in the range of 10 to
1000
sulfated octasaccharide for glycoprotein D of herpes simplex virus
is 19
M32 and that of a dermatan sulfate hexasaccharide for hep-
arin cofactor II is 20
M.33 The only system with high potency of
l
M for their targets.24,30,31 Likewise, the affinity of a highly
l
l
smaller sulfated molecule is the antithrombin–heparin pentasac-
charide system for which the affinity is 50 nM.34 Thus, the potency
of initial hits 1–3 is reasonable in context of sulfated molecules.
The more important point is scaffold hopping, which should ex-
pand anticoagulant search.
Mechanism of thrombin inhibition: To assess whether these hits
utilize an allosteric mechanism in a manner similar to monosulfat-
ed benzofurans,11 the kinetics of Spectrozyme TH hydrolysis in the
presence of 1 was measured (Fig. 5, see Supplementary data for de-
tails). Whereas KM remained essentially invariant at 0, 20, 70 and
185
in the absence of 1 to 15.0 mAU minÀ1
185 M of 1. This suggested that the presence of 1 does not signif-
l
M of 1, VMAX decreased steadily from 35.0 mAU minÀ1
l
MÀ1
studies. A 2-fold increase in PT and APTT required 606 and 550 lM
concentrations of 1, respectively, suggesting useful anticoagulant
potency of this initial hit (Fig. 7).
l
MÀ1 in the presence of
l
icantly affect the binding of the substrate in the active site of
thrombin, while significantly reducing its forward rate constant.
This behavior is characteristic of a noncompetitive, allosteric
mechanism. However, it is important to recognize that this does
not prove that the molecules bind in the targeted site of thrombin,
as hypothesized in the design of scaffold hopping query. This con-
firmation will require co-crystal structure studies.
Virtual screening strategies that reliably yield promising small
molecule heparin mimetics are expected to be extremely useful
in the design or discovery of modulators of processes in which hep-
arin or heparan sulfate molecules play a role. The processes in
which these GAGs play critical roles are many including angiogen-
esis, immune regulation, viral invasion and others, in addition to
coagulation.35–38 Yet, few sulfated small molecules have been de-
signed or discovered to date. A key reason for this state of art is
Figure 4. Direct inhibition of human
a-thrombin by VS hits 1, 2 and 3. The
Figure 6. Residual activity of factors VIIa, IXa, Xa, XIa and XIIa at
a fixed
inhibition of thrombin was determined spectrophotometrically through Spectro-
zyme TH hydrolysis assay at pH 7.4 and 25 °C. Solid lines represent sigmoidal fits to
the data to obtain IC50, as described in the experimental section.
concentration of 365 M of inhibitors 1 (gray bars), 2 (black bars) and 3 (white
bars). Residual activity was measured in a spectrophotometric assay as described in
Supplementary data. See text for details.
l

P. S. Sidhu et al. / Bioorg. Med. Chem. Lett. 23 (2013) 355–359
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References and notes
1. Huntington, J. A.; Baglin, T. P. Trends Pharmacol. Sci. 2003, 24, 589.
2. Hirsh, J.; Anand, S. S.; Halperin, J. L.; Fuster, V. Circulation 2001, 103, 2994.
3. Monteiro, R. Q. An. Acad. Bras. Cienc. 2005, 77, 275.
4. Bock, P. E.; Panizzi, P.; Verhamme, I. M. J. Thromb. Haemost. 2007, 5, 81.
5. Di Cera, E. Mol. Aspects Med. 2008, 29, 203.
6. Hogg, P. J.; Jackson, C. M.; Labanowski, J. K.; Bock, P. E. J. Biol. Chem. 1996, 271,
26088.
7. Anastasiou, G.; Gialeraki, A.; Merkouri, E.; Politou, M.; Travlou, A. Blood Coagul.
Fibrinolysis. 2012, 23, 1.
8. Greinacher, A.; Warkentin, T. E. Thromb. Haemost. 2008, 99, 819.
9. Liu, L. W.; Rezaie, A. R.; Carson, C. W.; Esmon, N. L.; Esmon, C. T. J. Biol. Chem.
1994, 269, 11807.
10. Richardson, J. L.; Kroger, B.; Hoeffken, W.; Sadler, J. E.; Pereira, P.; Huber, R.;
Bode, W.; Fuentes-Prior, P. EMBO J. 2000, 19, 5650.
Figure 7. Prolongation of clotting time as a function of concentration of 1 in either
APTT (A) or PT (B) assay. Solid lines are trend lines from which the concentration of
1 needed to double clotting times, i.e., 2 Â APTT and 2 Â PT, was derived as shown
by the dotted lines. See Supplementary data for detailed protocol.
11. Sidhu, P. S.; Liang, A.; Mehta, A. Y.; Abdel Aziz, M. H.; Zhou, Q.; Desai, U. R. J.
Med. Chem. 2011, 54, 5522.
12. Aziz, Abdel. M. H.; Sidhu, P. S.; Liang, A.; Kim, J. Y.; Mosier, P. D.; Zhou, Q.;
Farrell, D. H.; Desai, U. R J. Med. Chem. 2012, 55, 6888.
13. Steindl, T. M.; Schuster, D.; Wolber, G.; Laggner, C.; Langer, T. J. Comput. Aided
Mol. Des. 2006, 20, 703.
14. Oprea, T. I.; Matter, H. Curr. Opin. Chem. Biol. 2004, 8, 349.
15. Lower, M.; Proschak, E. Mol. Inf. 2011, 30, 398.
16. Chen, L.; Morrow, J. K.; Tran, H. T.; Phatak, S. S.; Du-Cuny, L.; Zhang, S. Curr.
Pharm. Des. 2012, 18, 1217.
17. Waszkowycz, B.; Clark, D. E.; Gancia, E. Wiley Interdiscip. Rev. Comput. Mol. Sci.
2011, 1, 229.
that fundamental problems beset VS strategies, especially molecu-
lar docking and scoring, with highly sulfated molecules. Modeling
such sulfated molecules is challenging because their charge den-
sity is high, which results in recognition of any group of arginines
and lysines. In addition, heparin- or heparan-sulfate binding site on
proteins are surface-exposed and shallow. The strategy developed
in this work, sequential LBVS and SBVS, attempts to overcome
these challenges. The reasonable success rate achieved using this
approach bodes well for discovery of GAG and/or heparin mimet-
ics. It is possible that the sequential approach for sulfated small
molecules works because the drawbacks of one VS approach were
overcome by the strengths of the other.
18. Musmuca, I.; Caroli, A.; Mai, A.; Kaushik-Basu, N.; Arora, P.; Ragno, R. J. Chem.
Inf. Model. 2010, 50, 662.
ˇ
19. Švegelj, M. B.; Turk, S.; Brus, B.; Rizner, T. L.; Stojan, J.; Gobec, S. J. Chem. Inf.
Mod. 2011, 51, 1716.
20. Vasanthanathan, P.; Lastdrager, J.; Oostenbrink, C.; Commandeur, J. N. M.;
Vermeulen, N. P. E.; Jørgensen, F. S.; Olsen, L. Med. Chem. Commun. 2011, 2, 853.
21. Malvezzi, A.; Queiroz, R. F.; de Rezende, L.; Augusto, O.; do Amaral, A. T. Mol.
Inf. 2011, 30, 605.
The three sulfated small molecules identified by the dual VS ap-
proach may help develop more promising leads as inhibitors of
coagulation. The potency of the initial hit 1 is reasonable when
compared to highly sulfated saccharides30 and sulfated small mol-
ecules11 studied so far. The observation that hit 1 retains anticoag-
ulant activity in human plasma is highly encouraging. Additionally,
the selectivity observed against thrombin (and possibly factor XIa)
is likely to drive further structure activity studies in these novel
scaffolds, thereby expanding anticoagulant search. In fact, factor
XIa activity of these hits is being investigated in detail and will
be reported in due course.
22. Kitchen, D. B.; Decornez, H.; Furr, J. R.; Bajorath, J. Nat. Rev. Drug Discov. 2004, 3,
935.
23. Raghuraman, A.; Mosier, P. D.; Desai, U. R. ACS Med. Chem. Lett. 2010, 1, 281.
24. Raghuraman, A.; Liang, A.; Krishnasamy, C.; Lauck, T.; Gunnarsson, G. T.; Desai,
U. R. Eur. J. Med. Chem. 2009, 44, 2626.
25. Klebe, G. Drug Discov. Today 2006, 11, 580.
26. Lyne, P. D. Drug Discov. Today 2002, 7, 1047.
27. Verghese, J.; Liang, A.; Sidhu, P. S.; Hindle, M.; Zhou, Q.; Desai, U. R. Bioorg. Med.
Chem. Lett. 2009, 19, 4126.
28. Raghuraman, A.; Mosier, P. D.; Desai, U. R. J. Med. Chem. 2006, 49, 3553.
29. Raghuraman, A.; Riaz, M.; Hindle, M.; Desai, U. R. Tetrahedron Lett. 2007, 48,
6754.
30. Gunnarsson, G. T.; Desai, U. R. J. Med. Chem. 2002, 45, 1233.
31. Al-Horani, R.; Liang, A.; Desai, U. R. J. Med. Chem. 2011, 54, 6125.
32. Copeland, R.; Balasubramaniam, A.; Tiwari, V.; Zhang, F.; Bridges, A.; Linhardt,
R. J.; Shukla, D.; Liu, J. Biochemistry 2008, 47, 5774.
33. Tollefsen, D. M. Adv. Exp. Med. Biol. 1992, 313, 167.
34. Desai, U. R. Med. Res. Rev. 2004, 24, 151.
Acknowledgment
This work was supported by Grants HL090586 and HL107152
from the National Institutes of Health to U.R.D.
35. Raman, R.; Sasisekharan, V.; Sasisekharan, R. Chem. Biol. 2005, 12, 267.
36. Rouet, V.; Hamma-Kourbali, Y.; Petit, E.; Panagopoulou, P.; Katsoris, P.;
Barritault, D.; Caruelle, J. P.; Courty, J. J. Biol. Chem. 2005, 280, 32792.
37. Ihrcke, N.; Wrenshall, L. E.; Lindman, B. J.; Platt, J. L. Immunol. Today 1993, 14,
500.
Supplementary data
38. Linhardt, R. J.; Toida, T. Acc. Chem. Res. 2004, 37, 431.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
10.079.