The advantage of biosensor analysis over enzyme inhibition studies for slow dissociating inhibitors – characterization of hydroxamate-based matrix metalloproteinase-12 inhibitors

Abstract

The kinetic characteristics of hydroxamate-based inhibitors of matrix metalloproteinase (MMP)-12 were explored using an SPR biosensor-based assay and enzyme inhibition analysis. These high-affinity inhibitors were shown to dissociate very slowly from the enzyme–inhibitor complex while a carboxylate analogue had a much faster dissociation rate, verifying the importance of the hydroxamate group for the slow dissociation. Progress curve enzyme inhibition analysis confirmed that the hydroxamate compounds but not the carboxylate compound acted as time-dependent inhibitors. The slow dissociation excluded steady-state estimation of IC₅₀-values and K_i values but also made K_i values from progress curve analysis unreliable. Although a full characterization of the inhibitors using biosensor analysis was limited by slow dissociation, it provided kinetic and mechanistic information of relevance for MMP drug discovery and avoided some pitfalls of conventional enzyme inhibition assays.