Experiments comprising a “pre-incubation” phase, where enzyme is incubated with inhibitor prior to the addition of assay substrate, are commonly used to evaluate covalent inhibitors, often via discontinuous or “endpoint” IC50 assays. However, due to the lack of mathematical tools to describe its biphasic time-dependent nature, this experiment has thus far been unable to provide kinact and KI values. Herein we report EPIC-Fit, a new method to determine kinact and KI values from global fitting of Endpoint Pre-incubation IC50 data that can be implemented using Microsoft Excel. Experimental characterization of a known tissue transglutaminase inhibitor, AA9, using EPIC-Fit provided kinact and KI values with strong correlations to the values determined by other, previously established methods of evaluation. This unprecedented method serves to finally include time-dependent pre-incubation endpoint assays in the medicinal chemist’s toolbox for rigorous characterization of irreversible inhibitors.
A blog highlighting recent publications in the area of covalent modification of proteins, particularly relating to covalent-modifier drugs. @CovalentMod on Twitter, @covalentmod@mstdn.science on Mastodon, and @covalentmod.bsky.social on BlueSky
Covalent adduct Grob fragmentation underlies LSD1 demethylase-specific inhibitor mechanism of action and resistance
Amanda L. Waterbury, Jonatan Caroli, Olivia Zhang, Paloma R. Tuttle, Chao Liu, Jiaming Li, Ji Sung Park, Samuel M. Hoenig, Marco Barone, Air...

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DOI Ansgar Oberheide, Maxime van den Oetelaar, Jakob Scheele, Jan Borggräfe, Semmy Engelen, Michael Sattler, Christian Ottmann, ...
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Özge Ünsal, Z. Selin Bacaksiz, Vladislav Khamraev, Vittorio Montanari, Martin Beinborn, and Krishna Kumar ACS Chemical Biology 2024 DOI: ...
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Klett, T., Schwer, M., Ernst, L. N., Engelhardt, M. U., Jaag, S. J., Masberg, B., … Boeckler, F. M. Drug Design, Development and Therapy, 20...