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A cell based high-throughput screening approach for the discovery of new inhibitors of respiratory syncytial virus

Dong-Hoon Chung12, Blake P Moore35, Daljit S Matharu4, Jennifer E Golden4, Clinton Maddox3, Lynn Rasmussen3, Melinda I Sosa3, Subramaniam Ananthan3, E Lucile White3, Fuli Jia36, Colleen B Jonsson12 and William E Severson1*

Author Affiliations

1 Current address: Center for Predictive Medicine for Biodefense and Emerging Infectious Diseases, University of Louisville, Louisville, KY, USA

2 Current Address: Department of Microbiology and Immunology, University of Louisville, Louisville, KY, USA

3 Southern Research Specialized Biocontainment Screening Center, Southern Research Institute, Birmingham, AL, USA

4 University of Kansas Specialized Chemistry Center, University of Kansas, Lawrence, KS, USA

5 Current Address: Department of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, USA

6 Current Address: Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, TX, USA

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Virology Journal 2013, 10:19  doi:10.1186/1743-422X-10-19

Published: 10 January 2013

Abstract

Background

Human respiratory syncytial virus (hRSV) is a highly contagious pathogen and is the most common cause of bronchiolitis and pneumonia for infants and children under one year of age. Worldwide, greater than 33 million children under five years of age are affected by hRSV resulting in three million hospitalizations and 200,000 deaths. However, severe lower respiratory tract disease may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. There is no vaccine commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Thus, the discovery of new inhibitors for hRSV would be clinically beneficial.

Results

We have developed and validated a 384-well cell-based, high-throughput assay that measures the cytopathic effect of hRSV (strain Long) in HEp-2 cells using a luminescent-based detection system for signal endpoint (Cell Titer Glo®). The assay is sensitive and robust, with Z factors greater than 0.8, signal to background greater than 35, and signal to noise greater than 24. Utilizing this assay, 313,816 compounds from the Molecular Libraries Small Molecule Repository were screened at 10 μM. We identified 7,583 compounds that showed greater than 22% CPE inhibition in the primary screen. The top 2,500 compounds were selected for confirmation screening and 409 compounds showed at least 50% inhibition of CPE and were considered active. We selected fifty-one compounds, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays Several compounds had SI50 values greater than 3, while the most active compound displayed an SI50 value of 58.9.

Conclusions

A robust automated luminescent-based high throughput screen that measures the inhibition of hRSV-induced cytopathic effect in HEp-2 cells for the rapid identification of potential inhibitors from large compound libraries has been developed, optimized and validated. The active compounds identified in the screen represent different classes of molecules, including aryl sulfonylpyrrolidines which have not been previously identified as having anti-hRSV activity.

Keywords:
Aryl sulfonylpyrrolidines; Anti-infective drugs; Automation/robotics; Cell-based assays; HTS; Human respiratory syncytial virus (hRSV); Sulfonamides