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. 2020 Aug 7;21(23):3417–3422. doi: 10.1002/cbic.202000457

Figure 2.

Figure 2

Comparison of previous and current applications of Quantitative irreversible tethering (qIT). a) Overview of rate determination. A cysteine‐containing biomolecule is treated with covalent fragments under pseudo‐first‐order conditions. Reaction progress is followed by discrete measurement of free cysteine concentration by using the fluorogenic probe CPM, and rate constants (k obs) are derived from exponential regression analysis. b) Comparison of the reactivity profile of each fragment with the POI and GSH typically classes them as unreactive, reactive but non‐selective or reactive and selective. c) Quantification of kinetic POI/GSH selectivity facilitates hit identification. d) The observed rate of reaction between the POI and hit fragment is determined at a range of ligand concentrations by using qIT. Subsequent hyperbolic regression analysis is used to derive K i and k inact to facilitate comprehensive covSAR analysis.