Abstract

Human tryptase-? (HT?) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HT? has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HT? interaction were defined in this study. Tightbinding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7×07 M -1s-1 and a relatively slow dissociation rate constant of 0.04 s-1. Bivalent inhibition was demonstrated by displacement of paminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HT?]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HT?, 24% or 53% inhibited by preincubation with an irreversible inhibitor. Two interactions were observed consistent with mono and bivalent binding; the Ki value for bivalent inhibition was at least 104-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HT? finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HT?. The potency of CRA-2059 is thus attributable to effective bivalent binding.