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

Abstract

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.

Structure and Energetics of Ligand–Fluorine Interactions with Galectin‐3 Backbone and Side‐Chain Amides: Insight into Solvation Effects and Multipolar Interactions

“…Fluorine–amide C−F⋅⋅⋅C=O interactions with the backbone amides of Arg144, Ile145, and Ser237, as well as a multipolar C−F⋅⋅⋅H−N and C−F⋅⋅⋅H−Cα interaction with the amide group of Gly238 were identified in the crystal structures. These correspond well to interactions previously observed and postulated to be important for ligand binding . QM calculations confirm an approximate linear dependence between the binding free energies and interaction energies between the substituted benzene rings and the Ser237‐Glu238 backbone.…”

“…The meta and para analogues 3 and 4 had similar affinities: K d =22 μ m for 3 and 31 μ m for 4 . On the other hand, the affinity of the ortho analogue 2 was 3‐ to 4‐fold lower ( K d =92 μ m ), in fact no better than that of the unsubstituted phenyl compound ( 1 , K d =88 μ m ), which suggests a lack of a favorable fluorine–amide interaction with the side‐chain amide of Asn160. Figure f compares the binding of 2 and 9 (equivalent to 1 , except for the solubility‐enhancing glucose group distant from the binding pocket).…”

“…Compound 9 was of >95 % purity according to UPLC analysis (Waters Acquity UPLC system, Waters Acquity CSH C18 column, 0.5 mL min −1 flow rate, H 2 O/MeCN gradient 5–95 %, 10 min with 0.1 % formic acid). The synthesis of compounds 1 – 8 was described by Peterson et al …”

“…In particular, orthogonal multipolar C−F⋅⋅⋅C=O interactions with both peptide backbone and side‐chain carbonyl groups have been described as important for fluorine. Amides are abundant in proteins, and orthogonal multipolar fluorine–amide interactions between ligand fluorines and backbone amides (Figure a) have been reported . The fluorine–amide interaction is proposed to arise from an attractive dipole interaction between the C−F and C=O groups, and the geometric preferences have also been determined in a model system using chemical double‐mutant cycles.…”

“…Galectin‐3 is a β‐ d ‐galactopyranoside‐binding protein that, by cross‐linking glycoconjugates, plays important roles in modulating the behavior of proteins in, for example, the immune system, as well as in tumor growth and metastasis . Fluorinated phenyltriazolyl‐thiogalactosides have been reported with varying galectin‐3 affinities, depending on the position of the fluorine atom . A binding pocket for the aryl moiety of 3‐substituted galactosyl inhibitors is created by displacement of the side chain of Arg144 from a water‐mediated salt bridge on the protein surface such that it stacks on top of the aryl moiety.…”