Molecular Dynamics-Guided Design and Chemoproteomic Profiling of Covalent Kinase Activity Probes

Covalent small molecule probes can be powerful tools to interrogate protein activity state in native cellular environments. The design of familywide activity probes requires an understanding of the molecular sources of conserved and target-specific small molecule targeting across protein family members. Here, we developed and applied a multifaceted docking and molecular dynamics (MD) simulation pipeline to design cell-permeable covalent kinase activity probes from a set of hinge-binding pharmacophores. This computationally-guided approach yielded a series of cell-active indazole sulfonylfluorides that target a conserved catalytic lysine in active protein kinases. Chemoproteomic profiling of a lead probe, K60P, confirmed engagement of more than 100 unique native kinases across several cancer cell lines. Competitive profiling identified kinases as the predominant class of specific targets for K60P but also highlighted significant nonkinase targets for K60P and the established covalent kinase probe, XO44, underscoring the utility of native kinase profiling in situ to identify relevant targets of small molecule kinase inhibitors in cells. Dose-, time- and site-specific proteomic mapping with a known target kinase, ABL1, coupled with a Bayesian Metropolis Monte Carlo (BMMC) kinetic modeling method showed that key descriptors of covalent probe efficiency could be predicted with straightforward dose- and time-dependent covalent engagement studies and highlighted k_inact/K_I as a key variable to optimize for specific and broad kinase engagement. Finally, focused molecular dynamics simulations revealed that K60P, as well as the comparator probe XO44, preferentially engage with target kinases in their active, DFG-in conformations, which is driven by increasing population of reaction-ready small molecule conformation. These results together establish a computational and kinetic modeling framework for designing covalent activity probes and highlight the balance of target selectivity and kinetic efficiency as a key factor in determining their proteome-wide reactivity.