Abstract 2061: A proven activity-based workflow for the identification and characterization of time-dependent kinase inhibitors using a continuous assay format
Authors: May Earl , Urul Daniel , Huynh Khanh , Cornell-Kennon Susan , Nemmara Venkatesh , Lu Zhibing , Hoare Samuel , Lyles Michelle , Schaefer Erik 
Published: March 2024

Time-dependent inhibitors (TDIs) of enzyme targets offer distinct advantages for the development of potent and selective compounds with favorable pharmacokinetic and pharmacodynamic properties. Such inhibitors are characterized by non-linear progress curves: after an initial inhibited velocity, a rate constant governs the transition to a final steady-state reaction rate of the inhibited enzyme. A final rate of zero indicates irreversible inhibition, whereas a non-zero final rate indicates slow-binding inhibition. Characterizing these inhibitory modes of action is enabled with a continuous assay format that avoids the common pitfalls and misleading results seen with end-point assays. A continuous assay format enables efficient and robust determination of the kinetic parameters required to drive structure-activity relationship optimization to streamline the development of more effective drugs. It is important to note that simple IC50s for TDIs will not suffice, and can, indeed, also be misleading. We have developed a robust three-step workflow based on kinetic catalytic activity measurements to quickly identify and characterize TDIs. First, dose-response experiments are conducted with and without an enzyme-inhibitor preincubation step. The curvature of the reaction progress curve in the non-preincubated experiment and a shift in IC50 from the preincubated experiment are indicative of TDI. In the absence of TDI, simple IC50s are reported with, if possible, Ki values. If TDI is present, a second experiment is conducted to assess compound reversibility using either a jump-dilution protocol or a novel free-compound clearance method that uses gel filtration spin columns or spin plates. In either protocol, forward progress curve analysis is used to monitor the recovery of enzymatic activity after dilution of inhibitor in solution. Lastly, the potency of the inhibitor is evaluated using kinetic experiments tailored to the nature of the inhibition – either reversible or irreversible. If reversible, then the rate constant from the reversibility experiment is used to determine the residence time of the molecule. If irreversible, then a 24-point dose-response experiment with serial 1.5-fold dilutions is performed, and all the progress curves are globally fit to determine kinact/KI, and, if possible, kinact and KI separately. The method will be fully described through the characterization of known EGFR inhibitors of three inhibition types: fast-off (Gefitinib), slow binding (Lapatinib), and irreversible (Osimertinib).

Citation Format: Earl May, Daniel Urul, Khanh Huynh, Susan Cornell-Kennon, Venkatesh Nemmara, Zhibing Lu, Samuel Hoare, Michelle Lyles, Erik Schaefer. A proven activity-based workflow for the identification and characterization of time-dependent kinase inhibitors using a continuous assay format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2061.