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Development of A Fission Yeast Cell-Based Platform for High Throughput Screening of HIV-1 Protease Inhibitors

[ Vol. 17 , Issue. 6 ]

Author(s):

Zsigmond Benko, Jiantao Zhang and Richard Y. Zhao*   Pages 429 - 440 ( 12 )

Abstract:


Background: HIV-1 protease inhibitor (PI) is one of the most potent classes of drugs in combinational antiretroviral therapies (cART). When a PI is used in combination with other anti- HIV drugs, cART can often suppress HIV-1 below detection thus prolonging the patient’s lives. However, the challenge often faced by patients is the emergence of HIV-1 drug resistance. Thus, PIs with high genetic-barrier to drug-resistance are needed.

Objective: The objective of this study was to develop a novel and simple fission yeast (Schizosaccharomyces pombe) cell-based system that is suitable for high throughput screening (HTS) of small molecules against HIV-1 protease (PR).

Methods: A fission yeast RE294-GFP strain that stably expresses HIV-1 PR and green fluorescence protein (GFP) under the control of an inducible nmt1 promoter was used. Production of HIV-1 PR induces cellular growth arrest, which was used as the primary endpoint for the search of PIs and was quantified by an absorbance-based method. Levels of GFP production were used as a counter-screen control to eliminate potential transcriptional nmt1 inhibitors.

Results: Both the absorbance-based HIV-1 PR assay and the GFP-based fluorescence assay were miniaturized and optimized for HTS. A pilot study was performed using a small drug library mixed with known PI drugs and nmt1 inhibitors. With empirically adjusted and clearly defined double-selection criteria, we were able to correctly identify the PIs and to exclude all hidden nmt1 inhibitors.

Conclusion: We have successfully developed and validated a fission yeast cell-based HTS platform for the future screening and testing of HIV-1 PR inhibitors.

Keywords:

HIV-1 protease (PR), HIV-1 protease inhibitor (PI), fission yeast (Schizosaccharomyces pombe), high throughput screening (HTS), green fluorescence protein (GFP), atazanavir (ATV), transcriptional nmt1 inhibitor (TNI).

Affiliation:

Department of Pathology, University of Maryland Medical School, Baltimore, MD 21201, Department of Pathology, University of Maryland Medical School, Baltimore, MD 21201, Department of Pathology, University of Maryland Medical School, Baltimore, MD 21201

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