Abstract The clinical utility of targeting kinases with covalent drugs has been firmly established, culminating in the recent FDA approval of afatinib and ibrutinib, which irreversibly inhibit the tyrosine kinases, EGFR and BTK. More broadly, chemical probes that form a covalent bond with a nonconserved, noncatalytic cysteine often show enhanced potency, selectivity, and utility in biological studies. Despite these advantages, protein-reactive compounds are usually avoided in high-throughput screening campaigns. Motivated by a desire to explore the chemistry of reactive electrophiles and thiols under physiological conditions, we initiated a study of Michael acceptors bearing substituents with increasing electron withdrawing capacity. We hypothesized that the intrinsically labile nature of the cysteine/cyanoacrylamide bond could be exploited to yield cysteine-targeted, reversible covalent inhibitors. These concepts led to the design of ultra-selective cyanoacrylamide-based RSK inhibitors with picomolar affinity and slow dissociation kinetics, the structural basis of which was revealed by x-ray crystallography. The intrinsically reversible, yet thermodynamically favorable nature of the cysteine/cyanoacrylamide bond suggested that this ‘sweet spot’ in electrophile space could form the basis of a fragment-based approach to ligand discovery. Screening a collection of cyanoacrylamide fragments against three kinases (RSK2, NEK2, and PLK1) containing a structurally equivalent cysteine delivered unique inhibitors for all three kinases with potencies in the submicromolar range. Optimization guided by x-ray crystallography rapidly led to the first dual inhibitor of MSK and RSK, with subnanomolar affinity and >500-fold selectivity over NEK2 and PLK1. In close collaboration with Brian Shoichet and Nir London, we have employed covalent docking methods to screen large virtual libraries of electrophilic fragments. We designed a pilot virtual library of 13,000 cyanoacrylamide fragments that can be synthesized in one simple step from commercially available aldehydes. A virtual screen of this library rapidly led to novel inhibitors of NEK2, PLK-, and MSK/RSK-family kinases. Finally, I will discuss our efforts to discover and design new electrophilic chemotypes that form reversible covalent bonds with cysteine residues. Citation Format: Jack Taunton. Targeting kinases with reversible covalent fragments. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY05-02. doi:10.1158/1538-7445.AM2014-SY05-02