Background:
Our cell-based panels of secondary screening assays were developed to screen for inhibitors of flaviviruses. Specifically, our assay could be used to examine if inhibitors of West Nile virus (WNV) could also inhibit other flaviviruses such as Dengue (DENV), Yellow fever (YFV), Saint Louis encephalitis (SLEV), Japanese encephalitis (JEV), Murray Valley encephalitis (MVEV) and Tick-borne encephalitis viruses (TBEV). Currently, there is only one type of cell-based assay available for screening flaviviral inhibitors. These assays are limited in their overall efficiency since only a single enzyme or protein can be tested and monitored for any potential assays. These assays are highly labor-intensive and impossible to use when screening compound libraries in large quantities.
Our secondary screening assay is high throughput and sensitive which makes for rapid screening and identification of potential inhibitors in libraries with a large amount of compounds possible. We use "reverse genetic systems", which are cDNA clones of RNA viruses, and developed them for use in our genetic cell-based screening assays. Comparisons were performed on three cell based high throughput screening assays for WNV drug discovery: 1) an assay with a replicon-containing cell line that allows screening for inhibitors of viral replication; 2) a replicon bearing VLP infection assay that allows screening for inhibitors of viral entry as well as replication; and 3) a full-length reporting virus infection assay that allows screening for inhibitors of any step(s) of the viral life cycle, including entry, replication, and virion assembly. All three assays were converted into a 384-well format, validated with known WNV inhibitors, and now offer speed and sensitivity which make them ideal for screening libraries with large numbers of compounds.
Secondary Screening Assays Available:
A. WNV replicon with both Renilla luciferase (Rluc) and Neomycin Phosphotransferase (Neo)
B. WNV VLPs containing a Renilla luciferase expressing replicon (Rluc-VLP)
C. BHK-21 cell line containing a WN virus replicon (Rluc-Neo-Rep)
D. Plasmid for DENV-1 replicon containing Renilla luciferase reporter
E. Vero cell line containing DENV-1 Renilla luciferase-Neomycin gene-replicon
F. BHK cell line that constitutively expresses DENV-1 structural proteins (C-preM-E)
(Unpublished - Confidential)
Applications:
- Rapid screening of large compound libraries for potential inhibitors of WNV and other flaviviruses.
- Engineering targeted mutations into WNV, resulting in attenuated viral strains that could potentially be used for vaccine development.
Advantages:
- Multiple viral protein targets can be analyzed simultaneously through large compound libraries.
- This assay is directly derived from the human pathogenic WNV and the compounds identified from such an assay may have direct relevance to human disease.
- A panel of secondary screening assays has been developed to study the mode of action of inhibitors.
- The secondary screening assays can be adapted to screen for inhibitors targeting individual viral proteins.
- The assay is cell-based and covers targets during viral replication.
Patents:
USPTO #7,355,033
State of Development:
- The assay system has been established.
- A panel of secondary assays has been developed for analysis of potential mode of action of potential inhibitors.
The Inventor:
Pei-Yong Shi, PhD
Pei-Yong Shi is currently Head of the Dengue Unit at Novartis Institute for Tropical Diseases. He received his Ph.D. in flavivirus replication in 1995 from Georgia State University, USA. After postdoctoral training at Yale University, he joined Bristol-Myers Squibb as a Principal Scientist from 1998 to 2000. He then moved to the Wadsworth Center, New York State Department of Health. His group at the Wadsworth Center developed the first infectious clone of the epidemic strain of West Nile virus, discovered two cap methylation activities of flavivirus NS5 protein, identified essential RNA elements for flavivirus replication, and established various novel platforms that have been used to screen and study inhibitors of flaviviruses. He an Editor for Journal of General Virology and a member of the Editorial Board for Journal of Virology. He joined Novartis in August 2008.
Publications:
Min-Qing, Wei Liu, Zhiming Yuan, Feng Gu and Pei-Yong Shi. A High-Throughput Assay using Dengue-1 Virus-Like Particles for Drug Discovery. Antiviral Research (2010) doi: 10.1016/j.antiviral.2010.02.313
Francesc Puig-Basagoiti, Mark Tilgner, Brett M. Forshey, Sean M. Philpott, Noel G. Espina, David E. Wentworth, Scott J. Goebel, Paul S. Masters, Barry Falgout, Ping Ren, David M. Ferguson and Pei-Yong Shi. Triaryl Pyrazoline Compound Inhibits Flavivirus RNA Replication. Antimicrobial Agents and Chemotherapy, Apr. 2006 p. 1320-1329 doi:10.1128/AAC50.4.1320-1329 2006
Francesc Puig-Basagoiti, Tia S. Deas, Ping Ren, Mark Tilgner, David M. Ferguson, and Pei-Yong Shi. High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery. Antimicrobial Agents and Chemotherapy, Dec 2005 p. 4980-4988 doi: 10.1128/AAC.49.12.4980-4988.2005
Contact:
Diane Borghoff, B.S., M.S.
Marketing & Licensing Associate – Intellectual Property
Health Research, Inc.
150 Broadway – Suite 560, Menands, New York 12204-2719 U.S.A.
Phone 518-431-1213 Fax 518-431-1234
E-mail: DLB22@healthresearch.org Website: www.healthresearch.org
