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 kinact/KI. However, existing methods for measuring kinact/KI 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 kinact/KI, enabling routine kinact/KI 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, KRASG12C and two E3 ligase complexes. Kinetic simulations comparing a range of kinact and KI 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 kinact/KI values across three orders of magnitude with eight-fold increased throughput, reduced protein consumption, and simplified data analysis. In this work, Jeon et al. introduce the diagonal dose-response time-course (dDRTC), a fast and operationally simple mass spectrometry-based method for determining the efficiency of covalent modification (kinact/KI) across a wide dynamic range.