A detailed understanding of the binding site and the interactions of a bioactive compound with its biotarget is important to make structural modifications to overcome obstacles in potency and selectivity. The light-induced covalent cross-linking of a photoreactive ligand with a biological macromolecule, known as photoaffinity labeling (PAL), is a powerful biochemical technique to study protein–ligand interactions. An increasing number of literature reports suggests an association of the aberrant enzymatic activity of the NAD+-dependent human lysine deacylase SIRT2 with diseases such as cancer and neurodegenerative disorders. As such, the development of potent and selective SIRT2 modulators is of great interest. We have applied a PAL approach in an effort to locate the binding site of our substituted 2-alkyl-chroman-4-one based SIRT2 inhibitors. We have developed a diazirine-based PAL probe based on SAR data around a parent ligand. The application of cross-linking experiments in combination with tandem mass spectrometry identified a modified tryptic peptide arising from the covalent attachment of the probe to SIRT2. The peptide covers both the active site of SIRT2 and the proposed binding site of the chroman-4-one based inhibitors. Currently, the work focuses on the development of bifunctional probes carrying the photophore and a functional group serving as handle for post-labeling modifications with biotin for affinity enrichment of modified peptides or fluorescent dyes for visualisation purposes.

Photoaffinity labeling approach towards binding site identification of chroman-4- one based sirtuin 2 inhibitors

255th National Meeting (2018)

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