Assessing reversible and irreversible binding effects of kinase covalent inhibitors through ADP-Glo assays

STAR Protoc. 2021 Aug 5;2(3):100717. doi: 10.1016/j.xpro.2021.100717. eCollection 2021 Sep 17.


Typical enzymatic inhibition assays often demonstrate improved potency for kinase covalent inhibitors compared to reversible inhibitors. This can primarily be attributed to the irreversible mode of action and could affect the evaluations of the ATP-competitive nature of covalent inhibitors, hindering optimization of these compounds. Here, we describe a version of ADP-Glo assay, in which modification of inhibitor incubation time in the presence or absence of ATP enables a quick assessment of relative reversible and irreversible effects of kinase covalent inhibitors. For complete details on the use and execution of this protocol, please refer to Schröder et al. (2020).

Keywords: Chemistry; High Throughput Screening; Molecular/Chemical Probes; Protein Biochemistry; Single-molecule Assays.