A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans-(4-guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro-substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin-like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4-dichlorophenylacetyl-Lys-Lys-GCMA (K_i=0.13 μM). The inhibitor adopts a horseshoe-like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl-P3-Pro-4-amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L-configuration. Among this series, several derivatives inhibit thrombin with K_i values of less than 1 nM. Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin-like serine proteases. X-ray analysis of the most potent analogue in complex with thrombin demonstrated that the N-terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.