High-Throughput Kinetic Analysis for Target-Directed Covalent Ligand Discovery

Gregory Craven, Dominic Affron, Charlotte Allen, Stefan Matthies, Joe Greener,
Rhodri Morgan, Edward Tate, Alan Armstrong, David J. Mann

Angew. Chem. Int. Ed., 2018
doi: 10.1002/anie.201711825

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. Here we describe quantitative irreversible tethering (qIT), a general method for screening cysteine-reactive small molecules based upon the maximization of kinetic selectivity. We apply this method prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2-selective allosteric (type IV) kinase inhibitor whose novel mode-of-action could be exploited therapeutically.