Non-equilibrium modalities of inhibition: Characterizing irreversible inhibition for the ErbB receptor family members.

Abstract

Most drug target interactions for clinically approved small-molecules are non-equilibrium slow-onset, tight-binding or irreversible in nature, with pronounced element of time-dependence of inhibition. Analysis of such modality of inhibition requires a continuous enzyme kinetic measurement that can yield complete progress curves and an automated high-throughput analysis pipeline. Given the increasing emphasis on designing non-equilibrium modes of inhibiting an enzyme target (especially irreversible), the above specified pipeline for data generation and analysis is essential for extracting parameters to guide decisions in early drug discovery. In this manuscript, the methodology and data analysis protocol from our irreversible inhibitor characterization campaigns for the ErbB receptor family members is presented. Guidance is provided on the appropriate design of assay to generate quality data, setting up the analysis and estimation of inactivation rate (kinact) and the pseudo-equilibrium binding affinity (KI) constant (or their ratio kinact/KI) in a high-throughput manner for the inhibitor interacting with the protein target of interest.