A new approach to explore the binding space of polysaccharide-based ligands: Selectin antagonists

Article abstract of DOI:10.1021/ml300263x

The discovery of molecules that interfere with the binding of a ligand to a receptor remains a topic of great interest in medicinal chemistry. Herein, we report that a monosaccharide unit of a polysaccharide ligand can be replaced advantageously by a conformationally locked acyclic molecular entity. A cyclic component of the selectin ligand Sialyl Lewis^x, GlcNAc, is replaced by an acyclic tether, tartaric esters, which link two saccharide units. The conformational bias of this acyclic tether originates from the minimization of intramolecular dipole-dipole interaction and the gauche effect. The evaluation of the binding of these derivatives to P-selectin was measured by surface plasmon resonance spectroscopy. The results obtained in our pilot study suggest that the discovery of tunable tethers could facilitate the exploration of the carbohydrate recognition domain of various receptors.

Full text of DOI:10.1021/ml300263x

Letter
pubs.acs.org/acsmedchemlett
A New Approach to Explore the Binding Space of Polysaccharide-
Based Ligands: Selectin Antagonists
Mickael Calosso,† Daniel Charpentier,† Marc Vaillancourt,† Mohammed Bencheqroun,†
Gabrielle St-Pierre,† Brian C. Wilkes,‡ and Yvan Guindon*,†,§,∥
‡
†Bio-organic Chemistry and Chemical Biology and Peptide Research, Institut de recherches cliniques de Montrea
́
l (IRCM),
Montrea
§Dep
artement de Chimie, Universite
∥Department of Chemistry, McGill University, Montrea
́
l, Queb
́
ec H2W 1R7, Canada
́
́
de Montrea
́
l, C.P. 6128, succursale Centre-ville, Montrea
l, Quebec H3A 2K6, Canada
́ ́
l, Quebec H3C 3J7, Canada
́
́
S
* Supporting Information
ABSTRACT: The discovery of molecules that interfere with the binding of a ligand to
a receptor remains a topic of great interest in medicinal chemistry. Herein, we report
that a monosaccharide unit of a polysaccharide ligand can be replaced advantageously
by a conformationally locked acyclic molecular entity. A cyclic component of the
selectin ligand Sialyl Lewisx, GlcNAc, is replaced by an acyclic tether, tartaric esters,
which link two saccharide units. The conformational bias of this acyclic tether
originates from the minimization of intramolecular dipole−dipole interaction and the
gauche effect. The evaluation of the binding of these derivatives to P-selectin was
measured by surface plasmon resonance spectroscopy. The results obtained in our
pilot study suggest that the discovery of tunable tethers could facilitate the exploration of the carbohydrate recognition domain of
various receptors.
KEYWORDS: polysaccharide-based ligands, selectin antagonists, Sialyl Lewisx, surface plasmon resonance spectroscopy,
carbohydrate recognition domain
Binding to E-selectin is also considered as a target for drug
he identification of deregulated molecular events
Tassociated with disease and the discovery of medicinal delivery and molecular imaging.4
At the tactical level, the design of a competitive ligand is
prototypes aimed at restoring the molecular homeostasis are
topics of intense multidisciplinary research. Interfering with
ligand−receptor has been one of the strategies successfully used
in this regard. Of particular interest to us are cases where O-
and N-linked carbohydrate motifs on the ligand are involved in
the binding of a receptor to a carbohydrate recognition domain
(CRD), such as identified in the binding of E- and P-selectins
and their ligands.1 P-selectins interact with P-selectin
glycoprotein ligand-1 (PSGL-1), while E-selectins interact
with both E-selectin ligand-1 (ESL-1) and PSGL-1. These
interactions are involved in the capture and rolling of
circulating leukocytes on the vascular wall at the site of
inflammation. E- and P-selectins both, albeit with different
kinetics, transmigrate to the surface of the vascular endothelium
in response to inflammatory stimuli. ESL-1 and PSGL-1 are
expressed on the surface of most circulating leukocytes and
have been shown to play a key role in leukocytes−endothelial
cell interactions. Displayed at the ESL-1 and PSGL-1 terminus
is a sialylated and fucosylated tetrasaccharide termed Sialyl
Lewis X (sLex) that is recognized by E-, P-, and L-selectins
(Figure 1a). Interfering with these molecular binding events
was suggested to represent an interesting drug target for
inflammatory diseases and tumors metastasis.2 Recently,
selectin antagonists have been shown to reverse acute vascular
occlusions in sickle cell3 and atherothrombosis disease models.
often based on the finding of the bioactive conformation of the
natural ligand interacting with a receptor. Determining the
chemical functionalities of the ligand involved in the binding to
the receptor and other binding sites on the receptor
surrounding the ligand are also both critical. Ultimately, we
aim at identifying a molecular scaffold locked in the bioactive
conformation bearing a sufficient number of pharmacophores
to have a better affinity to the receptor than the natural ligand.
These molecules should also have druglike physicochemical
properties to achieve acceptable pharmacokinetic and pharma-
codynamic profiles.
X-ray diffraction analysis of complexes involving E- and P-
selectin fragments was realized with sLex or the N-terminal
domain of human PSGL-1 (hPSGL-1) modified by both
tyrosine sulfatation and glycosylation (Figure 1a).5 These
studies indicate that the acid group on the NeuAc is the only
functionality showing interaction with P-selectins and that no
interactions are found with E-selectin. From a drug design
standpoint, the removal of the NeuAc subunit could therefore
be envisaged. Furthermore, no interaction with the selectins
Received: August 31, 2012
Accepted: October 11, 2012
Published: October 11, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
and the ligand occur. The first step was to find acyclic tether
molecules that have a population of conformers similar to the
ones induced by the cyclic tether (Figure 1d), which in the
present study would require that the two oxygens linked to the
carbohydrate be gauche to each other. Ideally, these acyclic
tethers should be tunable. Conservative modification of the
factors at the origin of the conformational bias (e.g., X and Y in
Figure 1d) should lead to different populations of conformers,
thus allowing for a more extensive exploration of the
surrounding chemical space. The conformation of those
molecules should also be verifiable using spectroscopic
techniques.
The use of a polysaccharide as a starting point for the
development of a ligand is challenging. These complex
molecules have often low affinity and poor druglike properties.
The presence of cyclic monosaccharides, which are quite rigid
from a conformation standpoint, also complicates the
exploration of the surrounding binding space of the ligand.
Replacing some of these monosaccharides, not directly involved
in the binding to the receptor, by simple semirigid tethers with
a given conformational bias should facilitate the exploration of
the receptor, while decreasing the carbohydrate character of the
competing putative ligand.
Figure 1. Strategy. (a) Structure of sLex. The functionalities involved
in binding to E- and P-selectin proteins and the amino acid residues
implicated are illustrated. (b) Example of a cyclic tether. (c) Example
of an acyclic tether. (d) Acyclic tether with a defined conformational
bias.
The interest of developing an acyclic tether to replace the
GlcNAc unit stemmed from our research program in synthetic
methodologies. X-ray analysis of tartrate esters derivatives
shows that the two carbon−oxygen bonds at C2 and C3 are
gauche to each other, while the esters are anti.18,19 Two factors
could be at the origin of this conformational bias: the
minimization of the dipole−dipole effect induced by the esters
and the gauche effect resulting from the vicinal diol. To further
maximize the biological activity of our pseudodisaccharide, we
also considered optimizing the galactose subunit. Sialyl Lewisx
analogues having a cyclic tether replacing GlcNAc, wherein one
or more hydroxyl groups of the galactose have been replaced by
benzoate esters, was shown to have, in certain cases, improved
potencies.17 To examine this phenomenon with our analogues,
we decided to evaluate in this pilot study a compound bearing a
benzoate at C4, a docking experiment suggesting a favorable
interaction with the Tyr 94 of P-selectine. An analogue bearing
a C4-naphtoate group was also examined. While having similar
physicochemical properties, its binding to Tyr 94 of the P-
selectin might be precluded for steric encumbrance reasons.
The synthesis of sLex analogues bearing an acyclic tartrate
unit offers significant challenges, including the stereochemical
control of the O-glycosidation reactions at the anomeric
position of the galactose and the fucose subunits. The first
series of analogues were prepared by introducing the tartrate
tether on benzylated thioethyl fucoside 1 by a kinetic α-
selective glycosylation in presence of NIS to give 3 (Scheme
1).20 The latter was coupled to thioethyl galactoside 4 with an
anchimeric C2 group assistance to generate 5 with high 1,2-
trans selectivity on the galactose unit.21 After Zemplen
deacetylation of 5, the C4-Gal and C6-Gal alcohols were
protected with a benzylidene acetal yielding 6. C3-regioselec-
tive alkylations through in situ formation of organotin oxides
derivatives of 6 in the presence of 7 or 8 and CsF led to sLex
analogues 9 and 10, after Pd/C-catalyzed hydrogenations.
The synthesis of sLex analogues 18 and 19 with benzoate or
naphtoate at the C4-Gal position was more challenging because
the use of a C2-Gal ester protecting group, which would allow
anchimeric participation favoring the β-attack of the incoming
nucleophile, was precluded (Scheme 2). Another protecting
was noted for the GlcNAc moiety, confirming its role as a
tether holding in space the fucose and galactose. Similar
conclusions were reached independently through the synthesis
of sLex analogues.6−10 The pharmacophores of sLex being
identified, the modification of the GlcNAc, and the addition of
supplementary binding sites on the galactose and the fucose
were considered by us and others.
Two scenarios are classically explored to replace a cyclic
structural subunit, not directly involved in binding, of a given
biologically active molecule, such as sLex. The first consists of
replacing the cyclic GlcNAc by another cyclic tether.11−14 This
strategy has the advantage of allowing the rapid generation of
molecules with activity similar to the original ligand (e.g., sLex).
Although used successfully to achieve potent antagonists
(Figure 1b),12 this approach renders difficult the full
exploration of the chemical space of the CRD, because the
cyclic tether that holds the two sugars imposes a conforma-
tional bias that cannot be easily modified. Notwithstanding the
positive impact of these modifications on the overall biological
profile of a given analogue (stability), we are facing the same
lack of molecular plasticity that was present with the original
ligand. A second scenario consists of linking the fucose and
galactose derivatives by an acyclic tether, which could enable a
better probing of the CRD (Figure 1c).15,16 Obviously, with
this strategy, the molecule could reach potentially new binding
sites because of its plasticity. However, the lack of a
preorganized “bioactive”17 conformation in the ground state
could lead to entropic penalties and a decrease in potency.
Herein, we propose a novel and complementary strategy that
involves the replacement of the cyclic subunit of the motif by
an acyclic tether with a defined conformational bias (the ATC-
B strategy). This strategy, as depicted in (Figure 1d), aims at
taking advantage of the two approaches previously described.
On one hand, we will benefit from an alignment of the
pharmacophores similar to the one induced by GlcNAc (panels
a and b). On the other hand, we could benefit from the
increased plasticity of the acyclic tether. This could be
advantageous in the context of the induced fit occurring during
binding, whereas conformational changes in both the receptor
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ACS Medicinal Chemistry Letters
Letter
a
a
Scheme 1. Synthesis of sLex Analogues 9 and 10
Scheme 2. Synthesis of sLex Analogues 18 and 19
a
(a) (i) NaH, PMBCl, DMF, 74%; (ii) AcOH/H2O (80/20), 73%.
(b) Bu2SnO, MeOH, then CsF and 8 in THF, 85%. (c) (i) BzCl,
DMAP, DCM; (ii) DDQ, DCM, H2O; (iii) CBZCl, DMAP, DCM
(47% over three steps for 14). (d) (i) 1-Naphthoyl chloride, DMAP,
DCM; (ii) DDQ, DCM, H2O; (iii) CBZCl, DMAP, DCM (40% over
three steps for 15). (e) Br2, AgOTf, 3, DCM (64% for 16 and 58% for
17). (f) Pd(C)/H2, THF:MeOH (30% for 18 and 13% for 19).
a
(a) NIS/CF3SO3H, CH2Cl2, −30 °C, 4 Å mol sieves, 88%. (b) NIS/
CF3SO3H, CH2Cl2, −30 °C, 4 Å mol sieves, 60%. (c) (i) MeONa,
MeOH; (ii) PhCH(OMe)2, MeCN, CSA (60% over two steps). (d)
Bu2SnO, MeOH, then CsF and 7 in THF, 100%. (e) Pd(C)/H2,
dioxane, 75%. (f) Bu2SnO, MeOH, then CsF and 8 in THF, 75%. (g)
Pd(C)/H2, dioxane, 77%.
adhesions and represent potential targets to disrupt the cell−
cell adhesion phenotypes. They could be at the origin of false-
positive results when cell-based assays are used for the
evaluation of putative selectin antagonists, a concern that was
alleviated in this study by using direct biophysical measure-
ments with surface plasmon resonance (SPR) spectroscopy.27
In our first assay, reported herein, we immobilized a
monomeric hPSGL-1 fused with the Fc portion of a human
lgG (chimeric protein termed rhPSGL-lg) covalently attached
to the sensor chip (Figure 2). Its binding to soluble P-selectin
was evaluated. The kinetic parameters measured by SPR were
similar to kinetic data previously reported for various forms of
PSGL-1 interacting with sP-selectin (Table 1).28−30
group allowing anchimeric assistance that could be cleaved in
the presence of an ester group had to be identified. We thus
turn our attention to the use of a carbonate protecting
group.22,23 The reaction sequence was elaborated from
previously reported β-thioethyl galactoside 11 (Scheme 2).
The C2-Gal alcohol group was protected with a PMB group,
and the acetonide was cleaved under mild acidic condition to
furnish 12. The selective introduction of an oxyacetic chain was
achieved through the displacement of 8 by a tin acetal
intermediate derived from 12. An excellent 85% yield of the
O3-alkylated product was obtained. The hydroxyl at C4-Gal
was then either benzoylated or naphtolated, the PMB was
cleaved by an oxidative process (DDQ), and the CBZ group
was installed to provide compound 14 or 15. These
thioglycosides were then respectively coupled with 3 in
presence of Br2 and AgOTf at −78 °C to give sLex analogues
18 and 19, after Pd/C catalytic debenzylation. The high
selectivity obtained for the β-anomers formation represents a
remarkable improvement that we will be exploring in a separate
study.
A plethora of downstream events resulting from the selectin-
dependent rolling of leukocytes on the vascular endothelium
are observed. For instance, a dynamic clustering of PSGL-1 at
the surface of leukocytes24 and the reorganization of their
cytoskeleton and intracellular signaling25,26 are noted. These
molecular events play a critical role in regulating the transient
Figure 2. SPR approach, where rhPSGL-Ig is fixed to the sensor chip
in the flow cell. (a) Affinity and kinetic constants determination for the
binding to sP-selectin. (b) IC50 determination of sLex analogues.
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naphtoate derivative 19 was not active in our assay (Table 3,
entry 4), suggesting that bulky proximal functionality at O4-Gal
interferes with binding at the CRD of the selectine receptor.
In conclusion, we have demonstrated in our pilot study that
an acyclic tether with a conformational bias could replace
GlucNAc to furnish effective Sialyl Lewisx mimetics. We also
have shown that a derivative of the fucose subunit can improve
significantly the potency of these pseudo disaccharides. We are
now studying the effects of altering the substituents of the
tartrate moiety to modulate the conformation, plasticity, and
resulting biological activity of sLex analogues.
Table 1. Affinity and Kinetic Constants of the Binding of sP-
Selectin to rPSGL-Ig
KA SD
KD SD
kon SD
koff SD
(1.69 0.19) × 106 M−1
(591 73) × 10−9
M
(1.1 0.1) × 106 M−1 s−1
0.65 0.07 s−1
These calcium-dependent interactions were abrogated by
blocking antibodies to either hPSGL-1 or hP-selectin, and their
dependency on post-translational modifications was supported
by the loss of any significant interaction when rhPSGL-lg was
treated with sialylidase or sulphatase (Table 2).
ASSOCIATED CONTENT
* Supporting Information
■
S
Table 2. Relative Response (After Treatment/Before
Treatment) Obtained Using SPR Following Treatment with
Sialidase, Sulfatase, Blocking Anti-hPSGL-1, or Blocking
Anti-hP-selectin
Details for surface plasmon resonace assays, methods of organic
synthesis, and spectroscopic data of synthesized compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
treatment
relative response (%)
AUTHOR INFORMATION
Corresponding Author
*Tel: 514-987-5785. Fax: 514-987-5789. E-mail: yvan.
guindon@ircm.qc.ca.
■
sialidase treatment
sulfatase treatment
anti-hPSGL-1
3.8 0.3
7.4 0.4
0.9 0.3
0.4 0.2
anti-hP-selectin
Funding
Funding for this research has been granted from Natural
Sciences and Engineering Research Council (NSERC) and
Fonds Quebecois de la Recherche sur la Nature et les
́ ́
Technologies (FQRNT).
We next evaluated our compounds by SPR in a competition
assay. Various concentrations of our analogues in a solution
containing a constant amount of sP-selectin were injected on
the flow cell containing rhPSGL-lg linked to the sensor chip. In
this assay, the sialyl Lewisx analogues competed with the
carbohydrate motifs attached on the immobilized proteins for
binding to sP-selectin (Figure 2b). The objective of this study
was therefore to evaluate the biological and pharmacological
effect of the replacement of the GlcNAc moiety by the tartaric
ester as a strategy to develop P-selectin antagonist.
The first tartrate analogue 9 was inactive, but analogue 10
bearing a benzyl on the oxyacetic chain at O3-Gal position
showed an IC50 comparable to the one obtained with sLex
(Table 3, entries 1 and 2). Interestingly, both compounds
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are grateful to our colleague Dr. Michel Prev
and suggestions in writing this manuscript.
■
́
ost for advice
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