Raman Krishnan

Structure of thrombin complexed with selective non-electrophilic inhibitors having cyclohexyl moieties at P1.

The crystal structures of five new non-electrophilic beta-strand-templated thrombin active-site inhibitors have been determined bound to the enzyme. Four co-crystallize with hirugen and inhibitor isomorphously to produce thrombin-hirugen crystals (monoclinic, space group C2), while one co-crystallizes in the hexagonal system, space group P6(5). A 1,4-substituted cyclohexyl moiety is conserved at the P1 position of all the inhibitors, along with a fused hetero-bicyclic five- and six-membered ring that occupies the P2 site. Amino, amidino and aminoimidazole groups are attached to the cyclohexyl ring for recognition at the S1 specificity site, while benzylsulfonyl and diphenyl groups enhance the binding at the S3 subsite. The cyclohexyl groups at the P1 positions of three of the inhibitors appear to be in the energetically favored chair conformation, while the imidazole-substituted cyclohexyl rings are in a boat conformation. Somewhat unexpectedly, the two cyclohexyl-aminoimidazole groups bind differently in the specificity site; the unique binding of one is heretofore unreported. The other inhibitors generally mimic arginyl binding at S1. This group of inhibitors combines the non-electrophilicity and selectivity of DAPA-like compounds and the more optimal binding features of the S1-S3 sites of thrombin for peptidic molecules, which results in highly potent (binding constants 12 nM-16 pM, one being 1.1 microM) and selective (ranging from 140 to 20 000 times more selective compared with trypsin) inhibitors of thrombin. The binding modes of these novel inhibitors are correlated with their binding constants, as is their selectivity, in order to provide further insight for the design of therapeutic antithrombotic agents that inhibit thrombin directly at the active site.

Design, parallel synthesis, and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF x FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF x FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC(50) value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF x FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure-activity relationship.

Probing the S2 site of factor VIIa to generate potent and selective inhibitors: the structure of BCX-3607 in complex with tissue factor-factor VIIa.

Factor VIIa (FVIIa) is a trypsin-like serine protease in the coagulation cascade. Its complex with tissue factor (TF) triggers the extrinsic pathway of the coagulation cascade, generating a blood clot. Research programs at several centers now recognize the important roles played by TF and FVIIa in both the thrombotic and inflammatory processes associated with cardiovascular diseases. Therefore, inhibition of the TF-FVIIa complex is seen as a promising target that is key to the development of clinical candidates for various cardiovascular applications. The crystal structure of the TF-FVIIa enzyme complex has been analyzed in order to design and synthesize small-molecule inhibitors. Using structure-based drug design (SBDD), a new series of inhibitors have been discovered that demonstrate high potency against the TF-FVIIa complex while maintaining substantial selectivity versus other closely related serine proteases such as trypsin, thrombin, factor Xa and plasmin.