Fig. 3. Inhibitor potency, binding energetics and covalent residence based on SPR data.

Scheme of the 2-step reversible kinetic binding model showing the relevant kinetic binding constants for noncovalent binding via the IN-8 ATP-binding moiety (k₁ and k₂) and for warhead-mediated covalent bond formation (k₃ and k₄). The panels at the bottom show the kinetic binding plots for JNK-IN-8 on the JNK1(Cys116Ser) mutant surface injected at 100, 300, and 1000 nM concentrations (K_D ~ 200-300 nM) as well as the response curves on the wild-type JNK1 surface (WT) at three different concentrations. The analysis on the JNK1(Cys116Ser) mutant surface gives the value of k₁ and k₂ based on a one-site noncovalent binding model; these values were independently determined for each inhibitor this way (see Supplementary Table 1). In the case of the irreversible JNK-IN-8 inhibitor k₄ = 0 (and thus only k₃ was fit), while for the reversible inhibitors k₃ and k₄ were both numerically fit to the experimental kinetic curves obtained on the JNK1 WT surface (and the panel on the right with 6S,S-IN-8 shows an example of this, for further data see Supplementary Fig. 5). The experimental binding curves are shown in black and the fitted curves in red.