Computer-aided design of a factor Xa inhibitor by using MCSS functionality maps and a CAVEAT linker search

We have investigated a new approach to efficiently find a novel inhibitor against a serine protease (i.e. an activated coagulation factor X, FXa) by using de novo design programs and the X-ray crystal structure of the target enzyme. FXa is a coagulant enzyme that generates thrombin (a serine protease) and participates in both intrinsic and extrinsic coagulation pathways. We adopted multiple copy simultaneous search (MCSS) and CAVEAT linker search techniques, which disclosed a novel FXa inhibitor (T01312) consisting of two binding moieties (the benzamidinyl and adamantyl groups) and a linker unit (the carboxybenzylamine group). The inhibitory activity of T01312 against FXa was determined to be a small K(i)-value of 48nM, which is two orders of magnitude smaller than that against thrombin. An X-ray crystal analysis of T01312 complexed with trypsin (an analogue of FXa) and docking studies of T01312 with trypsin and FXa showed that: (i) the benzamidinyl group is a predominant binding moiety in the anionic pocket (S1 site) with an asparatic acid residue; (ii) a hydrophobic pocket (S4 site) is the binding site of the adamantyl group; (iii) the carboxylate group of the linker contributes to the selectivity for FXa against thrombin. Thus, the combination of the knowledge of the X-ray crystal structure of the target molecule with MCSS and CAVEAT linker search techniques proved to be an effective hit-finding method that does not require the screening of huge compound libraries.