Systematic Tuning of Fluoro-galectin‑3 Interactions Provides Thiodigalactoside Derivatives with Single-Digit nM Affinity and High Selectivity

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 (Kd down to
1–2 nM) in symmetrically substituted thiodigalactosides as
well as unsurpassed combination of high affinity (Kd 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.