C2-symmetrical thiodigalactoside bis-benzamido derivatives as high-affinity inhibitors of galectin-3: Efficient lectin inhibition through double arginine-arene interactions

Article abstract of DOI:10.1002/anie.200500627

Inhibitors of galectin-3, which has been implicated in cancer- and immunity-related processes, can be synthesized from thiodigalactoside derivatives with aromatic amide substituents at both C3 positions (see structure). Dissociation constant (K_d

Full text of DOI:10.1002/anie.200500627

Communications
Lectin Inhibitors
DOI: 10.1002/anie.200500627
C2-Symmetrical Thiodigalactoside Bis-Benzamido
Derivatives as High-Affinity Inhibitors of
Galectin-3: Efficient Lectin Inhibition through
Double Arginine–Arene Interactions**
Ian Cumpstey, Anders Sundin, Hakon Leffler, and
Ulf J. Nilsson*
Galectin-3 is a b-galactoside-binding protein[1] that has been
implicated as playing an important role in cancer and
inflammation processes.[2] Recently, a dominant negative C-
terminal fragment of galectin-3 was shown to slow the growth
of breast cancer in mice by blocking galectin-3 endogenous
ligands.[3] Although many intriguing biological activities have
been demonstrated,[2] the precise mode of action of galectin-3
remains unknown. The synthesis of potent, low-molecular-
weight inhibitors of galectin-3 is therefore an important goal,
as such molecules could be used both as tools to investigate
the molecular biology of the protein and as lead compounds
for drugs.
The b-galactoside binding site (termed subsite C in
Ref. [1b]) is highly conserved across the galectins, whereas
the neighboring sites on either side of the Gal group (B,
toward the nonreducing terminus; D, toward the reducing
terminus) are more variable, albeit important, contributors to
the interaction. N-Acetyllactosamine (LacNAc, 1), which
binds in subsites C and D, is the best natural disaccharide
ligand for galectin-3 (Figure 1a). We have previously
reported the synthesis of potent inhibitors of galectin-3
based on modification of LacNAc at C3 of galactose to
target subsite B.[5] The best inhibitors were aromatic amides
of general structure 2 (Figure 1c) and according to the crystal
structure of an inhibitor–galectin-3 complex, the increase in
binding affinity results from particularly favorable interac-
tions of the aromatic amide moiety with an arginine side chain
(Arg144).[5b] Arene–guanidinium interactions[6] have
attracted considerable interest in recent years, as they can
be as strong as cation–anion interactions[6c] and up to 70% of
all arginine side chains are involved in such interactions.[6b]
[*] Dr. I. Cumpstey, A. Sundin, Dr. U. J. Nilsson
Organic Chemistry, Lund University
Box 124, 221 00 Lund (Sweden)
Fax: (+46)46-222-8209
E-mail: ulf.nilsson@organic.lu.se
Dr. H. Leffler
Section MIG, Department of Laboratory Medicine
Lund University
Solvegatan 23, 223 62 Lund (Sweden)
[**] This work was supported by the Swedish Research Council and the
programs “Chemistry for the Life Sciences” and “Glycoconjugates
in Biological Systems” sponsored by the Swedish Foundation for
Strategic Research.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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(Figure 1c). The resulting molecules 4 are C2-symmetrical,
which simplifies the synthesis and could also lead to interest-
ing thermodynamic properties as there are two degenerate
binding modes.
Computer modeling[9] of the complex of galectin-3 with a
thiodigalactoside bis-benzamido derivative 4a (Table 1),
which was performed by starting from the reported crystal
Table 1: Kd values for the inhibitors 4a–d established by fluorescence
polarization.[11]
Compound
Kd [nm]
Relative
activity[a]
9
69000[5b]
43000
1
1.6
10
3
2a
6700[5b]
2b
2c
2d
4a
4b
4c
4d
2500[5b]
1100[5b]
950[5b]
3000
61
28
63
Figure 1. a) Representation of the complex of N-acetyllactosamine 1
with galectin-3 in which dashed lines represent hydrogen bonds.[4] The
areas known as subsites C and D are indicated, and the key polar inter-
acting groups on the saccharide are shown in bold.[1b] b) Thiodigalacto-
side 3 with key polar groups for interaction with galectin-1 shown in
bold.[8b] c) Lactosamine-based galectin inhibitors 2 and proposed
thiodigalactoside-based inhibitors 4, showing the similarity in structure
and conformation.
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23
The guanidinium ion is positively charged, has a p system, and
is poorly solvated in water,[7] which makes it ideal for
interactions with aromatic p systems. Consequently, the
targeting of protein arginine residues with aromatic structures
is emerging as an attractive strategy for protein inhibitor
development.
Thiodigalactoside 3 has been shown to bind to galectins
with about the same affinity as LacNAc,[8] and it was
hypothesized that it bound in a mode similar to that of
LacNAc,[8a] that is, in subsites C and D, with a similar
conformation and hydrogen-bonding network(Figure 1b).
This notion has recently been confirmed for galectin-1, for
which X-ray structures have been solved both with LacNAc 1
and with thiodigalactoside 3 as ligands.[8b] The galactose
residues bind identically in subsite C for the two disacchar-
ides, and in subsite D, the Gal of thiodigalactoside and
GlcNAc of LacNAc show identical patterns of interaction
with the protein.
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1380
2090
50
33
[a] Compounds 9, 2a–d, and 3 are included for reference.
structure of galectin-3 complexed with a C3-benzamido
LacNAc-based inhibitor,[5b] gave an energy minimum with
the two galactose units bound in subsites C and D (Figure 2).
Moreover, the two aromatic rings of the benzamides were
involved in stacking interactions with the two arginine
residues, Arg144 and Arg186.
To test the potential of 3,3’-bis-amide-derivatized thiodi-
galactosides as inhibitors of galectin-3, we synthesized a
selection of these compounds. The amide structures were
We reasoned that derivatization with aromatic amides at
C3 of galactose in thiodigalactoside, as for 2, could increase
the affinity for galectin-3 through favorable interactions with
Arg144 in subsite B. Furthermore, similar derivatization at
the second galactose C3 of 2 would give an amide that could
interact with an arginine residue (Arg186) in subsite D
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Communications
ments implies that systematic targeting of such interactions
could find wider use in drug design.
Received: February 21, 2005
Published online: July 11, 2005
Keywords: arenes · drug design · inhibitors · proteins ·
.
stacking interactions
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chosen based on the best results from our investigation into
C3-benzamido LacNAc derivatives 2.[5] Consequently, the bis-
amide structures 4a–d were synthesized starting from the
known 1,2,4,6-tetra-O-acetyl-3-azido-3-deoxy-d-galactopyra-
nose[10] (Supporting Information).
Bis-amides 4a–d were tested for binding to galectin-3 with
a fluorescence polarization assay[11] (Table 1). Pleasingly, our
strategy was successful; bis-amides 4a–d all have dissociation
constant values (Kd = 33–3000 nm) significantly better than
those of nonderivatized thiodigalactoside 3 and the methyl b-
glycoside of N-acetyllactosamine 9, both of which were
included in the test as reference compounds. The substituted
benzamides 4b–d bind much better than the unsubstituted
benzamide 4a, as would be expected if the thiodigalactoside
derivatives 4 bound in the galactose binding site of galectin-3
(subsite C) similarly to the LacNAc-derived amides 2.[5b]
Furthermore, these thiodigalactosides 4 have much higher
affinities than the corresponding LacNAc derivatives 2
(compare 2a–d and 4a–d), and it is logical to conclude that
this finding is the consequence of a favorable interaction
between the second aromatic amide and Arg186, as suggested
by our docking experiments (Figure 2). An alternative
interpretation would be that compounds 4a–d cross-linkby
binding to subsites B and C of two galectin-3 molecules.
However, such a binding mode is unlikely as, for steric
reasons, it would require a much longer galactose–galactose
distance than that present in 4a–d.
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In conclusion, the targeting of arginine side chains with
aromatic structures has been demonstrated as an efficient
approach toward the discovery of monovalent high-affinity
lectin inhibitors. The 3,3’-bis-benzamido-thiodigalactosides
4a–d are the best monovalent galectin-3 inhibitors reported
to date and, as such, constitute promising lead structures for
the development of galectin-targeting drugs. In general, the
targeting of arginine–arene interactions may be under-
exploited in drug design relative to targeting, for example,
hydrogen-bonding and ionic interactions. Our observation
that both of the arginine–arene interactions between galectin-
3 and the inhibitors 4a–d provide substantial affinity enhance-
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