Figure 1.

Reaction coordinate for a one-step binding event. Target (E) and drug (I) binding leads to the drug–target complex (E-I). The driving force for binding is given by the difference in free energy between E+I and E-I (ΔG_Kd). Experimental measurements of the equilibrium dissociation constant K_d, or parameters such as IC₅₀ values, provide a quantitative estimate of the thermodynamics for binding. The rate at which the drug-target complex forms (k_on) and dissociates (k_off) is given by the difference in free energy between the respective ground states (E+I or E-I) and the rate-limiting transition state (ΔG_kon and ΔG_koff). ΔG_Kd is related to K_d by the relationship ΔG° = −RT ln K. Assuming that parameters such as the transmission coefficient are the same for two drug molecules, then the difference in free energy for the rate of complex dissociation of the two molecules can be given by ΔΔG_koff = −RT ln(k_off¹/k_off²). The lifetime of the drug–target complex is often quantified by the residence time, t_R, where t_R = 1/k_off.⁵ The figure shows a simple one-step mechanism, although in many cases slow-binding inhibitors operate through a two-step induced-fit mechanism.^{13,15,28−31}