Molecular Recognition of the Carbonic Anhydrase Sulphonamide Inhibitor Binding Site by Combinated Approaches

A prerequisite for the structure-based drug design is an understanding of the principles of molecular recognition in protein-ligand complexes. Photoaffinity labelling (PAL), ie the 'capture' of proteins by small molecules via photoactivation-mediated irreversible cross-linking, sequentially integrated with mass spectrometric analysis, represents a powerful method for identifying sites of molecular interactions. Other experimental and computational techniques such as x-ray-crystallography and molecular modelling are well validated tools in providing structural details at atomic level. Of course, each of these methods possesses peculiar characteristics with advantages and limitations, thus the management of complementary data constitutes a major goal. Since carbonic anhydrases (CAs) are a class of enzyme involved in numerous physiological and pathological processes, and CA inhibitors (CAIs) have emerged as useful chemical platform to develop novel bioactive compounds, a series of affinity-labelled inhibitors containing sulfonamido-photoprobes were designed and synthesized, and one of them, a benzophenone-derivative, was chosen as model photoprobe/inhibitor. In this work, a PAL method followed by mass spectrometry analysis, combined with x-ray crystallography, have been used to elucidate inhibitor-binding sites of human CAII (chosen as model of protein target). Specific amino acid residues involved in the covalent (determined by PAL) and non-covalent (in the x-ray solved structure) bonds, were identified, then molecular dynamics helped us to rationalize the behavior of these ligand-CAII interactions.