Rapid Determination of the Specificity Constant of Irreversible Inhibitors (kinact/KI) by Means of an Endpoint Competition Assay

Owing to their covalent target occupancy, irreversible inhibitors require low exposures and offer long duration, and their use thus represents a powerful strategy for achieving pharmacological efficacy. Importantly, the potency metric of irreversible inhibitors is k(inact)/K-I not IC50. A simple approach to measuring k(inact)/K-I was developed that makes use of an irreversible probe for competitive assays run to completion against test compounds. In this system, the k(inact)/K-I value of the test compound is equal to (k(inact)/K-I) probe x [probe]/IC50. The advantages of this method include simplicity, high throughput, and application to all target classes, and it only requires an indepth kinetic evaluation of the probe.