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Antimicrobial Agents and Chemotherapy, September 2008, p. 3385-3393, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.01508-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification and Biochemical Characterization of Small-Molecule Inhibitors of West Nile Virus Serine Protease by a High-Throughput Screen{triangledown}

Niklaus H. Mueller,1 Nagarajan Pattabiraman,2 Camilo Ansarah-Sobrinho,3 Prasanth Viswanathan,1 Theodore C. Pierson,3 and R. Padmanabhan1*

Department of Microbiology and Immunology,1 Department of Oncology, Georgetown University Medical Center, Washington, DC 20057,2 Viral Pathogenesis Section, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, Maryland 208923

Received 21 November 2007/ Returned for modification 9 January 2008/ Accepted 30 May 2008

West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen ~32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with Ki values of 3.2 ± 0.3 µM and 3.4 ± 0.6 µM, respectively. These compounds inhibited the dengue virus type 2 protease with Ki values of 28.6 ± 5.1 µM and 30.2 ± 8.6 µM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 ± 0.4 µM; selectivity index, 100), presumably by inhibition of polyprotein processing.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Georgetown University School of Medicine, 3900 Reservoir Rd. NW, Med-Dent SW309, Washington, DC 20057. Phone: (202) 687-2092. Fax: (202) 687-1800. E-mail: rp55{at}georgetown.edu

{triangledown} Published ahead of print on 7 July 2008.

Antimicrobial Agents and Chemotherapy, September 2008, p. 3385-3393, Vol. 52, No. 9
0066-4804/08/$08.00+0     doi:10.1128/AAC.01508-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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