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Title: High-throughput kinetic analysis for target-directed covalent ligand discovery
Author: Craven, Gregory
ISNI:       0000 0004 7657 7498
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Cysteine-reactive small molecules are used as chemical probes of biological systems and as medicines. By forming covalent bonds with target proteins they achieve distinct potency and selectivity profiles that are redefining the scope of potentially druggable targets. Identifying high-quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan-reactive compounds. This thesis describes quantitative irreversible tethering, a general method for screening cysteine-reactive small molecules based upon the maximisation of kinetic selectivity. 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 two distinct regions of the clinically important cell cycle regulator cyclin-dependent kinase 2. Crystal structures of the inhibitor complexes validate the approach and guide further optimisation. The power of this technique is highlighted by the identification of cyclin-dependent kinase 2-selective probes whose novel mode-of-action may offer future therapeutic potential.
Supervisor: Armstrong, Alan ; Mann, David ; Tate, Edward Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral