HIRUNORMS ARE TRUE HIRUDIN MIMETICS - THE CRYSTAL-STRUCTURE OF HUMAN ALPHA-THROMBIN HIRUNORM-V COMPLEX

A novel class of synthetic, multisite-directed thrombin inhibitors, kn own as hirunorms, has been described recently. These compounds were de signed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report t he crystal structure of the complex formed by human a-thrombin and hir unorm V, a 26-residue polypeptide containing non-natural amino acids, determined at 2.1 Angstrom resolution and refined to an R-factor of 0. 176. The structure reveals that the inhibitor binding mode is distinct ive of a true hirudin mimetic, and it highlights the molecular basis o f the high inhibitory potency (K-i is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-t erminal tetrapeptide with the thrombin active site in a nonsubstrate m ode; at the same time, this inhibitor specifically binds through the C -terminal segment to the fibrinogen recognition exosite, The backbone of the N-terminal tetrapeptide Chg(1) ''-Val(2) ''-2-Nal(3) ''-Thr(4) '' (Chg, cyclohexyl-glycine; 2-Nal, beta-(2-naphthyl)-alanine) forms a short beta-strand parallel to thrombin main-chain residues Ser(214)-G ly(219). The Chg(1) '' side chain fills the S2 subsite, Val(2) '' is l ocated at the entrance of S1, whereas 2-Nal(3) '' side chain occupies the aryl-binding site. Such backbone orientation is very close to that observed for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opp osite to the proposed binding mode of fibrinogen and of small syntheti c substrates. Hirunorm V C-terminal segment binds to the fibrinogen re cognition exosite, similarly to what observed for hirudin C-terminal t ail and related compounds. The linker polypeptide segment connecting h irunorm V Nand C-terminal regions is not observable in the electron de nsity maps. The crystallographic analysis proves the correctness of th e design and it provides a compelling proof on the interaction mechani sm for this novel class of high potency multisite-directed synthetic t hrombin inhibitors.