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Antimicrobial Agents and Chemotherapy, July 2004, p. 2437-2447, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2437-2447.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Model System for High-Throughput Screening of Novel Human Immunodeficiency Virus Protease Inhibitors in Escherichia coli

Ting-Jen Cheng,¹ Ashraf Brik,² Chi-Huey Wong,² and Chen-Chen Kan^1*

Keck Graduate Institute of Applied Life Sciences, Claremont, California 91711,¹ Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037²

Received 10 February 2004/ Returned for modification 28 February 2004/ Accepted 5 March 2004

Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS. To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system. This E. coli-based system involves coexpression of an engineered ß-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain. Autoprocessing of the HIV protease precursor releases the mature HIV protease. Subsequently, the HIV protease cleaves ß-galactosidase, resulting in a loss of the ß-galactosidase activity, which can be detected in high-throughput screens. Using Food and Drug Administration-approved HIV protease inhibitors, this E. coli-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity. The usefulness of the E. coli-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores. A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo E. coli-based system. This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains. This simple yet efficient E. coli system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases.

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* Corresponding author. Mailing address: Keck Graduate Institute of Applied Life Sciences, 535 Watson Dr., Claremont, CA 91711. Phone: (909) 607-8563. Fax: (909) 607-8086. E-mail: chen-chen_kan{at}kgi.edu.

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Antimicrobial Agents and Chemotherapy, July 2004, p. 2437-2447, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2437-2447.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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