The design of small and high-affinity lectin inhibitors remains a major challenge because the natural ligand binding sites of lectin are often shallow and have polar character. Herein we report that derivatizing galactose with un-natural structural elements that form multiple non-natural lectin-ligand interactions (orthogonal multipolar fluorine-amide, phenyl-arginine, sulfur-π, and halogen bond) can provide inhibitors with extraordinary affinity (low nanomolar) for the model lectin, galectin-3, which is more than five orders of magnitude higher than the parent galactose; moreover, is selective over other galectins.
Symmetrical and asymmetrical fluorinated phenyltriazolyl-thiodigalactoside derivatives have been synthesized and evaluated as inhibitors of galectin-1 and galectin-3. Systematic tuning of the phenyltriazolyl-thiodigalactosides' fluoro-interactions with galectin-3 led to the discovery of inhibitors with exceptional affinities (K down to 1-2 nM) in symmetrically substituted thiodigalactosides as well as unsurpassed combination of high affinity (K 7.5 nM) and selectivity (46-fold) over galectin-1 for asymmetrical thiodigalactosides by carrying one trifluorphenyltriazole and one coumaryl moiety. Studies of the inhibitor-galectin complexes with isothermal titration calorimetry and X-ray crystallography revealed the importance of fluoro-amide interaction for affinity and for selectivity. Finally, the high affinity of the discovered inhibitors required two competitive titration assay tools to be developed: a new high affinity fluorescent probe for competitive fluorescent polarization and a competitive ligand optimal for analyzing high affinity galectin-3 inhibitors with competitive isothermal titration calorimetry.
Molecular recognition
is fundamental to biological signaling. A
central question is how individual interactions between molecular
moieties affect the thermodynamics of ligand binding to proteins and
how these effects might propagate beyond the immediate neighborhood
of the binding site. Here, we investigate this question by introducing
minor changes in ligand structure and characterizing the effects of
these on ligand affinity to the carbohydrate recognition domain of
galectin-3, using a combination of isothermal titration calorimetry,
X-ray crystallography, NMR relaxation, and computational approaches
including molecular dynamics (MD) simulations and grid inhomogeneous
solvation theory (GIST). We studied a congeneric series of ligands
with a fluorophenyl-triazole moiety, where the fluorine substituent
varies between the
ortho
,
meta
,
and
para
positions (denoted O, M, and P). The M and
P ligands have similar affinities, whereas the O ligand has 3-fold
lower affinity, reflecting differences in binding enthalpy and entropy.
The results reveal surprising differences in conformational and solvation
entropy among the three complexes. NMR backbone order parameters show
that the O-bound protein has reduced conformational entropy compared
to the M and P complexes. By contrast, the bound ligand is more flexible
in the O complex, as determined by
19
F NMR relaxation,
ensemble-refined X-ray diffraction data, and MD simulations. Furthermore,
GIST calculations indicate that the O
-
bound complex
has less unfavorable solvation entropy compared to the other two complexes.
Thus, the results indicate compensatory effects from ligand conformational
entropy and water entropy, on the one hand, and protein conformational
entropy, on the other hand. Taken together, these different contributions
amount to entropy–entropy compensation among the system components
involved in ligand binding to a target protein.
Galectin-3 is a carbohydrate-binding protein central
to regulating
mechanisms of diseases such as fibrosis, cancer, metabolic, inflammatory,
and heart disease. We recently found a high affinity (nM) thiodigalactoside
GB0139 which currently is in clinical development (PhIIb) as an inhaled
treatment of idiopathic pulmonary fibrosis. To enable treatment of
systemically galectin-3 driven disease, we here present the first
series of selective galectin-3 inhibitors combining high affinity
(nM) with oral bioavailability. This was achieved by optimizing galectin-3
specificity and physical chemical parameters for a series of disubstituted
monogalactosides. Further characterization showed that this class
of compounds reduced profibrotic gene expression in liver myofibroblasts
and displayed antifibrotic activity in CCl4-induced liver
fibrosis and bleomycin-induced lung fibrosis mouse models. On the
basis of the overall pharmacokinetic, pharmacodynamic, and safety
profile, GB1211 was selected as the clinical candidate and is currently
in phase IIa clinical trials as a potential therapy for liver cirrhosis
and cancer.
Multipolar fluorine–amide interactions with backbone and side‐chain amides have been described as important for protein–ligand interactions and have been used to improve the potency of synthetic inhibitors. In this study, fluorine interactions within a well‐defined binding pocket on galectin‐3 were investigated systematically using phenyltriazolyl‐thiogalactosides fluorinated singly or multiply at various positions on the phenyl ring. X‐ray structures of the C‐terminal domain of galectin‐3 in complex with eight of these ligands revealed potential orthogonal fluorine–amide interactions with backbone amides and one with a side‐chain amide. The two interactions involving main‐chain amides seem to have a strong influence on affinity as determined by fluorescence anisotropy. In contrast, the interaction with the side‐chain amide did not influence affinity. Quantum mechanics calculations were used to analyze the relative contributions of these interactions to the binding energies. No clear correlation could be found between the relative energies of the fluorine–main‐chain amide interactions and the overall binding energy. Instead, dispersion and desolvation effects play a larger role. The results confirm that the contribution of fluorine–amide interactions to protein–ligand interactions cannot simply be predicted, on geometrical considerations alone, but require careful consideration of the energetic components.
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