Dual thio-digalactoside-binding modes of human galectins as the structural basis for the design of potent and selective inhibitors.

Chun-Hung Lin,Ting-Chien Lin,Fu-Tong Liu,Tung-Ju Hsieh,Hsien-Ya Lin,Zhijay Tu,Shang-Te Danny Hsu,Yu-Yao Tseng,Shang-Chuen Wu

doi:10.1038/srep29457

Abstract

Human galectins are promising targets for cancer immunotherapeutic and fibrotic disease-related drugs. We report herein the binding interactions of three thio-digalactosides (TDGs) including TDG itself, TD139 (3,3’-deoxy-3,3’-bis-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside, recently approved for the treatment of idiopathic pulmonary fibrosis), and TAZTDG (3-deoxy-3-(4-[m-fluorophenyl]-1H-1,2,3-triazol-1-yl)-thio-digalactoside) with human galectins-1, -3 and -7 as assessed by X-ray crystallography, isothermal titration calorimetry and NMR spectroscopy. Five binding subsites (A–E) make up the carbohydrate-recognition domains of these galectins. We identified novel interactions between an arginine within subsite E of the galectins and an arene group in the ligands. In addition to the interactions contributed by the galactosyl sugar residues bound at subsites C and D, the fluorophenyl group of TAZTDG preferentially bound to subsite B in galectin-3, whereas the same group favored binding at subsite E in galectins-1 and -7. The characterised dual binding modes demonstrate how binding potency, reported as decreased Kd values of the TDG inhibitors from μM to nM, is improved and also offer insights to development of selective inhibitors for individual galectins.

Highlights

Galectins[14,17,18,19]
In silico computational studies of TD139/galectin-3, based on the X-ray crystal structures of galectin-3 in complex with TDG17,18 or 3′-(4-methoxy-2,3,5,6-tetrafluorobenzamido) -N-acetyl-lactosamine (L3)[21] (Fig. 1), indicate that the thio-digalactoside moiety is situated at subsites C and D of the galectin CRD
Multiple sequence alignments for human galectins-1 to -12 have shown that the majority contains no more than two total arginines at subsites B and E, except for galectin-10, and C-terminal CRD of galectins-4 and -12 where there are none arginines at subsites B and E (Figure S2)

Summary

Introduction

Galectins[14,17,18,19]. these TDG derivatives bear two identical or different substituents at their C3-/C3′-positions, i.e., they are C2-symmetric or C2-asymmetric compounds, respectively. In silico computational studies of TD139/galectin-3, based on the X-ray crystal structures of galectin-3 in complex with TDG17,18 or 3′-(4-methoxy-2,3,5,6-tetrafluorobenzamido) -N-acetyl-lactosamine (L3)[21] (Fig. 1), indicate that the thio-digalactoside moiety is situated at subsites C and D of the galectin CRD. According to the computational studies, the two TD139 aromatic substituents likely stack intermolecularly with adjacent arginines (Arg144hGal[3] and Arg186hGal3) at subsites B and E of galectin-3, respectively, providing π-cation interactions[22,23,24], and could account for its enhanced binding affinity. We prepare TDG, TD139 and TAZTDG (C2-asymmetric, containing one 4-fluorophenyl-triazole at C3; Fig. 1) and study their binding interactions with human galectins-1, -3 and -7 by X-ray crystallography, isothermal titration calorimetry (ITC) and NMR spectroscopy. In addition to demonstrating how the affnity can be improved >1000-fold, such information provides valuable insights for the design of potent and selective inhibitors for specific galectins

Methods

Results

Conclusion