𝐴 " -𝐴 𝐴 " -𝐴 ! Where, A -measured anisotropy. Af -free anisotropic value for the 8luorescent probe Ab -bound anisotropic value for the 8luorescent probe A time-dependent reduction in probe anisotropy were measured for a 2-fold serial dilution of 100 mM compound MN551 in 10 nM probe + 300 μM SBC. To determine the initial reversible binding af8inity, the initial bound fraction of the probe at time=0, Fbo (the y-intercept in Fb v/s Time plot, Supplementary Fig. ) was plotted against MN551 concentration, and the obtained IC50 is then converted to Ki as described previously (Supplementary Fig. ) . The slope from Fb versus time graph when divided by Fbo gives rate of covalency, kobs. a plot of kobs v/s MN551 concentration using Michaelis-Menten model of GraphPad Prism 9, provides the parameters kinact (maximum potential rate of inactivation) and KI (concentration of MN551 at which kobs=kinact/2) (Supplementary Fig. and). The observed 8irst order rate constant, kobs is related to KI and kinact by the equation: