A practical method for determining the rate of covalent modification of fragments and leads

The clinical success of covalent drugs such as sotorasib has renewed interest in covalency for rational drug design. The most rigorous potency metric for covalent modifiers is the second-order rate constant k_inact/K_I. However, existing methods for measuring k_inact/K_I are resource-intensive and involve complex data interpretation. We describe the diagonal dose-response time-course (dDRTC), an efficient mass spectrometry-based method for determining k_inact/K_I, enabling routine k_inact/K_I quantification earlier in programs and accelerating SAR interpretation for lead discovery. We apply dDRTC to a dozen covalent fragment and lead-like modifiers for three targets, KRAS^G12C and two E3 ligase complexes. Kinetic simulations comparing a range of k_inact and K_I values establish recommended parameters for dDRTC and reveal that the approach is particularly suited for covalent fragments and leads. Our results demonstrate accurate determination of k_inact/K_I values across three orders of magnitude with eight-fold increased throughput, reduced protein consumption, and simplified data analysis.