Review: measuring covalent inhibitor kinetics and linking them to in vivo effect
- Lead selection leans on equilibrium IC50 values, which ignore how drug and target levels shift over time; this matters most for irreversible covalent drugs.
- The review surveys assays for kinact and KI: progress curves, multi-timepoint IC50 fitting for library triage, mass spectrometry for adducts and sites, SPR/NMR, and washout experiments for reversibility and residence time.
- It then links these rates to pharmacology via protein turnover, target vulnerability and pharmacokinetics, using BTK, JAK3, KRAS G12C and EGFR cases.
💡 Points
On a covalent hit, an IC50 from a single timepoint says little, because potency drifts with incubation time and ranking by it can bury the compound with the best kinact/KI. Translation is the harder part: in vivo occupancy depends on how fast the target is resynthesized, so a fast-turnover protein can blunt even a well-optimized warhead. Measure turnover before trusting the kinetics.
Only time will tell: Modelling the kinetics of covalent inhibitors ↗