Validation of an Automated Process for the Determination of Covalent Modifier Potency: Kinact/KI of the Btk/Ibrutinib Model System

Targeting the covalent modification of proteins with compounds containing reactive functional groups has led to the development of several new potent and selective drugs, as demonstrated by the epidermal growth factor receptor (EGFR) and Bruton’s tyrosine Kinase (BtK) active inhibitors used to treat various cancers. Recent publications highlight an “evolving toolbox” of techniques and technologies implemented in covalent inhibitor discovery and design. Though historically covalent compound discovery has been conducted via structure guided design, direct covalent ligand screening of electrophilic compound libraries has been gaining popularity as a primary approach. The generation of “hit” lists has increased demands on secondary assays defining and rank ordering compound potency. The rate constant, Kinact/KI, is the potency metric of irreversible inhibitors (akin to an IC50 value for reversible inhibitors). We have validated the use of simple and commercially available hardware and software products in an automated workflow for the determination of the rate constant Kinact/KI using the BtK protein and Ibrutinib inhibitor model system. The throughput afforded by this workflow can enable the determination of full Kinact/KI values for dozens of compounds per day. Additionally, minor modifications to plate prep protocols can enable the primary screening of electrophilic libraries of up to 10,000 compounds in 2-3 days.

 

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