Category: Assay Development and Screening
Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. A high-throughput assay is described that facilitates robust kinetic analysis of electrophile-thiol conjugation, effective for both small molecule- and protein-derived thiols in combination with any irreversibly thiol-reactive ligand. Crucial to its success is its ability to account for variations in intrinsic reactivity observed within electrophilic fragment libraries by comparing reactivity between target- and control-thiols. The method is applied prospectively to discover covalent fragments that target a clinically important kinase. Crystal structures of the inhibitor complexes validate the approach and guide further optimisation. The power of this technique is highlighted by the identification of kinase-selective probes whose novel mode-of-action may offer future therapeutic potential.
Gregory Craven– Dr, Imperial College London, London, England, United Kingdom