Mechanistic Characterization of Covalent Enzyme Inhibition by Isothermal Titration Calorimetry Kinetic Competition...

Covalent enzyme inhibitors can offer high potency and specificity and are increasingly sought after in drug discovery. They typically inhibit in two steps: noncovalent binding followed by covalent bond formation. Rational optimization requires quantitative information on both steps. Current methods for measuring these steps are technically demanding, time-consuming, and are not well suited for routine insertion into drug discovery pipelines. We have developed a new approach, using isothermal titration calorimetry kinetic competition (ITC-KC), that overcomes many of these challenges. ITC-KC measures enzyme activity directly, via the heat flow generated during catalysis, making it a sensitive and nearly universal approach. We performed extensive numerical simulations in which ITC-KC outperformed current methods with 3- to 10-fold greater accuracy. We applied ITC-KC to a library of 19 inhibitors of the protease 3CL^pro from SARS-CoV-2 and found that the reactive warheads and noncovalent binding portions of these molecules influenced the two-step inhibition mechanism in complex and unpredictable ways. This highlights the need for detailed mechanistic information in the development of covalent inhibitors, information that ITC-KC can provide rapidly, accurately, and essentially universally.