A general glycomimetic strategy yields non-carbohydrate inhibitors of DC-SIGN

Article abstract of DOI:10.1039/c0cc00830c

Shikimic acid can be transformed into monovalent and multivalent glycomimetics that target different members of the C-type lectin class, including DC-SIGN, a dendritic cell lectin that facilitates HIV transmission.

Full text of DOI:10.1039/c0cc00830c

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A general glycomimetic strategy yields non-carbohydrate inhibitors
of DC-SIGNw
Kathleen C. A. Garber,a Kittikhun Wangkanont,a Erin E. Carlsona and Laura L. Kiessling*ab
Received 8th April 2010, Accepted 21st June 2010
DOI: 10.1039/c0cc00830c
Shikimic acid can be transformed into monovalent and multivalent
glycomimetics that target different members of the C-type lectin
class, including DC-SIGN, a dendritic cell lectin that facilitates
HIV transmission.
resides on the surface of dendritic cells, which are critical
antigen-presenting cells.11 DC-SIGN facilitates dendritic
cell–T cell interactions, but it is its involvement in the dis-
semination of infectious human pathogens that led us to seek
inhibitors. DC-SIGN can interact with viruses, such as HIV-1
or Ebola virus, and bacterial species, such as Mycobacterium
tuberculosis, to facilitate infection.11 Moreover, several pathogens
that bind DC-SIGN subvert normal immune system function.
Thus, DC-SIGN ligands could serve as probes of the underlying
mechanisms and as therapeutic leads. MBP-A and DC-SIGN are
both mannose-binding C-type lectins; therefore, our objective—to
generate agents that block DC-SIGN selectively—serves as a
challenging test of our design strategy.
Carbohydrates act in conjunction with carbohydrate-binding
proteins (lectins) to govern numerous cellular processes,
including cell adhesion, recognition, and signaling.1 Despite
the importance of lectins, ligands that can be used to inter-
rogate or mitigate their function are scarce.2–4 Carbohydrates
themselves can serve as probes, but they have liabilities. First,
they tend to bind with low affinity. Second, optimizing carbo-
hydrate leads can be difficult because it typically requires the
production of complex oligosaccharide analogs via multistep
routes. Finally, since the specificity of lectins can overlap,
natural oligosaccharides often bind multiple lectins, thereby
complicating their use as probes.4 Non-carbohydrate antagonists
are an appealing alternative, but few have been reported.2–5
Although screens of large compound libraries are beginning
to yield inhibitors,2–5 general approaches to block lectins are
lacking. Here, we describe a privileged glycomimetic scaffold
that can give rise to effective and selective inhibitors of the
C-type lectin class. Moreover, we show that such glyco-
mimetics can be incorporated into multivalent displays to
generate potent inhibitors. To date, the targeting of lectins
with multivalent glycomimetics is underexplored;6,7 our data
indicate it can serve as a powerful strategy.
DC-SIGN binds weakly to monosaccharide ligands such
as N-acetyl mannosamine (ManNAc, Kd = 8.7 mM) and
L-fucose (Kd = 6.7 mM).12 The affinity for oligosaccharides
is slightly higher (Kd = 0.21 mM for Man9GlcNAc).12 To test
whether glycomimetics with similar affinities could be found,
we used solid-phase synthesis to assemble a collection of
putative mannose mimics that vary at three positions (Fig. 2).
Aside from the triol-substituted 6-membered ring that we
anticipate would mimic mannose, the glycomimetic scaffold
differs structurally from the natural ligands. We envisioned
substituents at the points of variation could endow ligands
with lectin affinity and specificity. Our synthetic approach was
designed to utilize building blocks that are readily accessible
(e.g., either commercially available or synthesized in a few
steps). For example, we varied the amino acid substituent to
explore how changes in R1 influence binding. Glycine serves as
a small, flexible amino acid, while phenylalanine is larger,
more hydrophobic, and the aryl group can engage in a range
C-type lectins are a large class of proteins that are integral to
immune system function.8 Named for their dependence on
calcium ions for carbohydrate complexation, these lectins
often bind mannosides. In these complexes, the 2-, 3- and
4-hydroxyl groups of mannose contribute to binding
(Fig. 1A).9 We therefore used scaffold 1, which mimics this
hydroxyl group arrangement (Fig. 1B), to afford glyco-
mimetics. The natural product shikimic acid was transformed
into compounds with the necessary arrangement of hydroxyl
groups,10 from which inhibitors were identified of a prototype
C-type lectin, mannose-binding protein A (MBP-A).
A key feature of the aforementioned approach is that it has
the potential to be general. One attractive target for testing
this possibility is dendritic cell-specific intercellular adhesion
molecule 3-grabbing non-integrin (DC-SIGN). DC-SIGN
a Department of Chemistry, University of Wisconsin—Madison,
1101 University Avenue, Madison, WI 53706, USA.
E-mail: kiessling@chem.wisc.edu; Fax: +1 608 265 0764
b Department of Biochemistry, University of Wisconsin—Madison,
433 Babcock Drive, Madison, WI 53706, USA
w Electronic supplementary information (ESI) available: Additional
figures, including binding curves, and detailed biochemical and
synthetic experimental procedures. See DOI: 10.1039/c0cc00830c
Fig. 1 Strategy for inhibitor design. (A) D-Mannose (top) and a
substructure of the binding site of a complex of mannose and MBP-A
(bottom) (PDB accession code 1kwy9). The hydroxyl groups necessary
for lectin recognition and binding are shown in red, Ca2+ is yellow.
(B) Glycomimetics 1 resemble mannose.
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Fig. 4 Percent probe binding to DC-SIGN/ECD and MBP-A in the
presence of ManNAc or 2 determined in fluorescence assay.
Fig. 2 Features of the glycomimetic library targeting DC-SIGN.
than the monosaccharide ligand a-methyl-D-mannopyranoside,10
so it is gratifying that the DC-SIGN inhibitors are superior
to known monosaccharide ligands. The results, therefore,
provide a starting point for optimizing ligand potency. Most
significantly, they support the generality of our design—the
same scaffold can be used to generate ligands for two lectins
with different binding sites.
of interactions. Glutamic acid and lysine were chosen to test
the influence of anionic or cationic substituents, respectively.
The R3 substituent was varied using a collection of alkylating
agents. We tested some aliphatic R3 groups, but we focused
on benzyl substituents because aromatic side chains often line
carbohydrate binding sites.4 The dithiol linkers (R2) used
are commercially available and capable of positioning the R3
substituents near the carbohydrate or secondary binding sites.
These building blocks were employed10 to synthesize 192
compounds. The compounds were cleaved from resin and
screened without further purification to estimate their activity.
The screen involved a fluorescence-based high-throughput
competition assay that assessed the ability of compounds to
compete with immobilized mannan for binding to the fluoro-
phore-labeled tetrameric extracellular domain of DC-SIGN
(DC-SIGN/ECD). In this way, we identified several compounds
that block DC-SIGN binding (Fig. 3). Trends in the inhibition
data were readily apparent. The identity of the R1 moiety
affected binding most significantly; all of the hits contain
glutamic acid at this position, but compounds with lysine at
this position were largely ineffective. At the R2 position, the
DTT-derived linker was most prevalent, followed by the
butanedithiol-derived linker. At the R3 position, there was a
preference for aromatic groups with electron withdrawing
substituents. To confirm the validity of the initial binding
data, we purified several library members and determined their
IC50 values. The relative values parallel those determined from
the initial screen.
We postulated that the glycomimetics identified could exhibit
selectivity for their protein target. Such selectivity is desirable
for a probe yet difficult to achieve using natural glycans. A
comparison of the results from our screens using MBP-A
and DC-SIGN reveals that several of our best inhibitors of
DC-SIGN exhibited no ability to block MBP-A.10 To examine
this issue directly, we tested 2 for binding to MBP-A. Even
at high concentration (15 mM) (Fig. 4), compound 2 was
ineffective.
We envisioned augmenting the potency of our selective
glycomimetic through multivalency. DC-SIGN is tetrameric,
and multiple copies of the protein are displayed on the
dendritic cell surface.11,12 As with other protein–carbohydrate
interactions,4 DC-SIGN could bind antigens with the requisite
functional affinity by exploiting multivalent binding.7,13–15 The
majority of multivalent lectin inhibitors consist of scaffolds
decorated with mono- or oligosaccharide epitopes. In these
cases, selectivity is a concern because the carbohydrate epitope
can interact with many lectins. Because glycomimetics can
display higher selectivity for their target lectin,4 multivalent
glycomimetics may serve as useful probes, although few have
been described.
Based on its activity in the initial screen, compound 2
(Fig. 3), was characterized further. To determine its IC50
value, we measured its ability to compete with probe, a
fluorescein-labeled bovine serum albumin displaying 20–25
copies of mannose, for the immobilized extracellular domain
of DC-SIGN.5 The IC50 value of 2 (3.2 Æ 0.6 mM) is better
than that of N-acetylmannosamine (IC50 = 11.2 Æ 0.7 mM). The
MBP-A inhibitors identified previously were no more potent
Our strategy for multivalent ligand synthesis involves
the application of ring-opening metathesis polymerization
(ROMP). This method can yield polymers of defined lengths
that function as highly effective biological probes.4,16–20
Additionally, ROMP-generated polymers can promote receptor
clustering,19 and this property could facilitate investigations of
DC-SIGN-mediated internalization. Finally, ROMP can give
rise to polymers of defined length with polydispersity indices
(PDIs) near unity. In summary, ROMP affords products with
valuable attributes.
The multivalent glycomimetics were generated from poly-
mer backbones bearing succinimide esters, which could be
modified post-polymerization21 to append the glycomimetic
epitopes (Scheme 1). To generate polymers that bind avidly to
DC-SIGN, the length of the polymer must be sufficient for it
to engage in multivalent binding, i.e., either occupy multiple
binding sites within the tetrameric DC-SIGN or cluster
Fig. 3 The most potent hits identified from library screen.
6748 Chem. Commun., 2010, 46, 6747–6749
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This research was supported by the NIGMS (R01
GM049975). We thank M. J. Borrok for preliminary studies,
J. Grim for helpful conversations, Prof. J. Frost and Dr
K. Draths for a donation of shikimic acid, and Prof.
K. Drickamer for supplying the DC-SIGN expression vector.
KCAG was supported by a National Science Foundation
Graduate Research Fellowship and the NIH Chemistry–
Biology Interface Training Grant (T32 GM008505), EEC
was supported by the NIH Biotechnology Training Program
(T32GM08349), and KW was a Hilldale Undergraduate
Research Fellow.
Scheme 1 Synthetic route to ROMP-derived multivalent scaffold 3,
with a degree of polymerization of 29. The ratio of ethanolamine
spacer to DC-SIGN inhibitor is approximately 1 : 3.
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multiple copies of DC-SIGN on the cell surface. We used the
structure of the monomeric DC-SIGN carbohydrate-recognition
domain (PDB accession code 1sl422) to estimate the distances
between sub-units of DC-SIGN. The width of the DC-SIGN
CRD is roughly 40 A. When extended, we estimate that each
monomer within a ROMP-derived polymer spans approxi-
mately 5 A.23 For polymers to engage multiple copies of
DC-SIGN, our analysis indicates that they must have a
minimum length of 40 A. We generated polymers with a length
of 25 monomeric units, as these should be capable of multi-
valent binding. Accordingly, the degree of polymerization (29)
was controlled by using a ratio of ruthenium initiator to
norbornene monomer of 1 : 25.24
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The ratio of glycomimetic to ethanolamine was chosen
to balance epitope density, which has been shown to be
important for biological function,19 with water solubility.
The glycomimetic was conjugated to the polymer through
reaction of the amine-terminated ethylene glycol linker with
the succinimidyl ester moiety on each backbone unit. This
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monomer, the polymer also inhibits DC-SIGN/ECD binding;
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In summary, the shikimic acid-derived glycomimetic scaffold
can be used to generate ligands for C-type lectins. This
approach can yield inhibitors that are selective and potent.
Both MBP-A and DC-SIGN possess significantly different
binding sites, revealing the general utility of our approach
for targeting diverse lectins. Additionally, our conversion of a
lead glycomimetic into a multivalent ligand yields a potent
non-carbohydrate inhibitor of DC-SIGN. In this way, multi-
valency can be exploited to generate highly effective non-
carbohydrate inhibitors of lectins. Thus, the strategy we have
outlined will be useful in developing probes not only of DC-SIGN
function but also of the many other C-type lectins whose
function or dysfunction are critical for human health.
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