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West Nile virus (WNV) NS2B-NS3 protease is an important drug target since it is an essential protein for the replication of the virus. In order to determine the minimum pharmacophore for protease inhibition, a series of dipeptide aldehydes were synthesized. The 50% inhibitory concentration (IC(50)) measurements revealed that a simple acetyl-KR-aldehyde was only threefold less active than 4-phenyl-phenylacetyl-KKR-aldehyde (1) (Stoermer et al., 2008) that was used as the reference compound. The ligand efficiency of 0.40 kcal/mol/HA (HA=heavy atom) for acetyl-KR-aldehyde is much improved compared to the reference compound 1 (0.23 kcal/mol/HA). The binding of the inhibitors was examined using (1)H-(15)N-HSQC experiments and differential chemical shifts were used to map the ligand binding sites. The biophysical studies show that the conformational mobility of WNV protease has a major impact on the design of novel inhibitors, since the protein conformation changes profoundly depending on the structure of the bound ligand., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A series of tripeptide aldehyde inhibitors were synthesized and their inhibitory effect against dengue virus type 2 (DENV2) and West Nile virus (WNV) NS3 protease was evaluated side by side with the aim to discover potent flaviviral protease inhibitors and to examine differences in specificity of the two proteases. The synthesized inhibitors feature a varied N-terminal cap group and side chain modifications of a P2-lysine residue. In general a much stronger inhibitory effect of the tripeptide inhibitors was observed toward WNV protease. The inhibitory concentrations against DENV2 protease were in the micromolar range while they were submicromolar against WNV. The data suggest that a P2-arginine shifts the specificity toward DENV2 protease while WNV protease favors a lysine in the P2 position. Peptides with an extended P2-lysine failed to inhibit DENV2 protease suggesting a size-constrained S2 pocket. Our results generally encourage the investigation of di- and tripeptide aldehydes as inhibitors of DENV and WNV protease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

An in silico fragment-based drug design approach was devised and applied towards the identification of small molecule inhibitors of the dengue virus (DENV) NS2B-NS3 protease. Currently, no DENV protease co-crystal structure with bound inhibitor and fully formed substrate binding site is available. Therefore a homology model of DENV NS2B-NS3 protease was generated employing a multiple template spatial restraints method and used for structure-based design. A library of molecular fragments was derived from the ZINC screening database with help of the retrosynthetic combinatorial analysis procedure (RECAP). 150,000 molecular fragments were docked to the DENV protease homology model and the docking poses were rescored using a target-specific scoring function. High scoring fragments were assembled to small molecule candidates by an implicit linking cascade. The cascade included substructure searching and structural filters focusing on interactions with the S1 and S2 pockets of the protease. The chemical space adjacent to the promising candidates was further explored by neighborhood searching. A total of 23 compounds were tested experimentally and two compounds were discovered to inhibit dengue protease (IC(50) = 7.7 μM and 37.9 μM, respectively) and the related West Nile virus protease (IC(50) = 6.3 μM and 39.0 μM, respectively). This study demonstrates the successful application of a structure-guided fragment-based in silico drug design approach for dengue protease inhibitors providing straightforward hit generation using a combination of homology modeling, fragment docking, chemical similarity and structural filters.