Fig. 1: Solving k_inact and k_l using two-step model fitting and in-silico protein-ligand adduct EI^* formation kinetics simulation.

A The math expression for protein covalent occupancy \(\frac{\left[{{E}}{{{I}}}^{{{*}}}\right]}{\left[{{{E}}}^{{{o}}}\right]}\) concerning incubation time \({{\boldsymbol{t}}}\) and compound concentration \({{[}}{{I}}{{]}}\). B Two-step fitting scheme to obtain \({{{k}}}_{{{\rm{inact}}}}\) and \({{{K}}}_{{{I}}}\). Protein occupancy \(\frac{\left[{{E}}{{{I}}}^{{{*}}}\right]}{\left[{{{E}}}^{{{o}}}\right]}\) at different timepoints for a single drug concentration \({{[}}{{I}}{{]}}\) will be used to obtain the apparent \({{{k}}}_{{{\rm{obs}}}}\) value at this concentration through the first one-phase association fitting. Then each (\({{[}}{{I}}{{]}}\), \({{{k}}}_{{{\rm{obs}}}}\)) was plotted, and the second Michaelis–Menten fitting will be performed to obtain \({{{k}}}_{{{\rm{inact}}}}\) and \({{{K}}}_{{{I}}}\) value simultaneously. C MATLAB simulation conditions and fitting results confirmed the proposed method is applicable to a wide range of compound affinities (fragments or inhibitors) and reactivity in real world settings.