Nanomolar inhibitor of the galectin-8 N-terminal domain binds via a non-canonical cation-π interaction

Abstract

Galectin-8 is a tandem-repeat galectin consisting of two distinct carbohydrate recognition domains and is a potential drug target. We have developed a library of galectin-8N inhibitors that exhibit high nanomolar K_d values as determined by a competitive fluorescence polarization assay. A detailed thermodynamic analysis of the binding of D-galactosides to galectin-8N by isothermal titration calorimetry reveals important differences in enthalpic and/or entropic contributions to binding. Contrary to expectations, the binding of 2-O-propargyl-D-galactoside was found to strongly increase the binding enthalpy, whereas the binding of 2-O-carboxymethylene-D-galactoside was surprisingly less enthalpy-driven. The results of our work suggest that the ethynyl group can successfully replace the carboxylate group when targeting the water-exposed guanidine moiety of a critical arginine residue. This results in only a minor loss of affinity and an adjusted enthalpic contribution to the overall binding due to non-canonical cation-π interactions, as evidenced by the obtained crystal structure of 2-O-propargyl-D-galactoside in complex with the N-terminal domain of galectin-8. Such an interaction has neither been identified nor discussed to date in a small-molecule ligand-protein complex.

Fig. 1. Structures of selected galectin-8N inhibitors from our research groups.

Carboxybenzimidazole-d-galactoside 1 showed substantial potency and served as a lead compound, whereas 3-lactoyl-d-galactoside 2 and Carboxybenzimidazole-d-galactal 3 showed improved selectivity compared to the lead 1^,,.

Fig. 2. Schematic representation of the design of 2-O-substituted galectin-8N inhibitors.

a 3D structure of Gal-8N in complex with d-galactal inhibitor 3 (PDB ID:7P1M: grey cartoon representation of Gal-8N, 3 in sticks: carbon in green, oxygen in red, and nitrogen in blue). For clarity, only residues forming hydrogen bonds (presented as black dashed lines) are shown as grey sticks. b Schematic representation of design of new d-galactosides that relies on introduction of new moieties at position 2-O of the d-galactoside 1. c Docking binding mode of the designed analogue 8a with benzyl substitution at 2-OH of the d-galactose. The cation-π interaction between the phenyl ring and the Arg45 side chain is shown as a red dashed line. d Docking binding mode of the designed analogue 16b with triazolebenzoic acid at 2-OH of the d-galactose. The cation-π interaction between the triazole ring and the Arg69 side chain is shown as red dashed line. The hydrogen bond between the carboxylic acid group and Arg69 is shown as a black dashed line. e Docking binding mode of the designed analogue 22 with cycloalkyl substituent at 2-OH of the d-galactose. f Docking binding mode of the designed analogue 29 with methylenecarboxylic acid at 2-OH of the d-galactose. Interactions with Arg45 and Arg69 are presented as black dashed lines.

Fig. 3. ITC analysis.

a Calorimetric titration curves measured at 25 °C by injections of compounds 1 (black squares), 11 (red), and 29 (blue) into the Gal-8N solution. Number of replicate titrations: n = 2. b Bar chart and c the table with standard thermodynamic parameters of 1, 11 and 29 binding to Gal-8N determined by fitting of 1:1 binding model to the titration curves presented in (a). ± Values are standard deviation obtained by Monte Carlo error analysis.

Fig. 4. Electron density map, crystal structure and quantum mechanical calculations of 11 in complex with galectin-8N.

a 2m|Fo|–D|Fc| electron density map. b Crystal structure of 11 in complex with Gal-8N (PDB ID: 9FYJ): green cartoon representation of Gal-8N, 11 in sticks: carbon in light blue, oxygen in red, and nitrogen in blue with amino acid residues that from binding pocket exposed (in sticks: carbon in grey, oxygen in red, and nitrogen in blue). c Quantum mechanical calculations on the same crystal structure of the Gal-8N–11 complex using Jaguar (Schrödinger suite). The calculations revealed that the LUMO of Arg45 of Gal-8N (depicted in blue) does not overlap with the HOMO of the alkyne from 11 (depicted in red) indicating that the true origin of the interaction is most probably electrostatic.

Fig. 5. Energy Decomposition Analysis (EDA).

a The two fragments chosen for EDA analysis. The first fragment is depicted as ball and stick, the second fragment as stick bonds. b The Molecular Orbital Energy-Level Diagram obtained by the EDA analysis indicates that the acetylene and Arg45 fragments have a bonding interaction as the bonding interaction between the two fragments is generated by a new molecular orbital. c A natural orbitals for chemical valence (NOCV) deformation density plot describes the charge flow between acetylene and Arg45 when the two fragments are combined. Electrons flow from red to blue, i.e. from acetylene towards the guanidinium group.