Mechanistic Characterization of Covalent Enzyme Inhibition by Isothermal Titration Calorimetry Kinetic Competition (ITC-KC)

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 3CLpro 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.