Dengue virus (DENV) causes disease globally with an estimated 25 to 100 million new infections per year. At present, no effective vaccine is available and treatment is supportive. In this study, we identified a potent and selective small-molecule inhibitor, BP2109, of the DENV NS2B/NS3 protease by high-throughput screening assay using recombinant protease complex consisting of central hydrophilic portion of NS2B and N-terminus of protease domain. BP2109 inhibited DENV (serotype 1-4) but not Japanese encephalitis virus (JEV) replication and viral RNA synthesis without detectable cytotoxicity. The compound inhibited recombinant DENV-2 NS2B/NS3 protease with an IC50 value of 15.43 +/- 2.12 muM and reduced the reporter expression of the DENV-2 replicon with an EC50 value of 0.17 +/- 0.01 muM. Sequencing analyses of several individual clones derived from BP2109-resistant DENV-2 RNAs revealed that two amino acid substitutions (R55K and E80K) are found in the region of NS2B, a cofactor of NS2B/NS3 protease complex. The introduction of R55K and E80K double mutations into the dengue NS2B/NS3 protease and a dengue replicon construct conferred 10.3- and 73.8-fold resistance to BP2109, respectively. The E80K mutation was further determined as the key mutation conferring dengue replicon drug resistance (61.3-fold) to BP2109, whereas the R55K mutation alone did not affect drug resistance to BP2109. Both R55K and E80K mutations are located at the central hydrophilic portion of NS2B cofactor where extensive interactions with NS3pro domain exist. Thus, our data provide evidence that BP2109 likely inhibits DENV by a novel mechanism.
Date:
2011-01
Relation:
Antimicrobial Agents and Chemotherapy. 2011 Jan;55(1):229-238.
