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Current HIV Research

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ISSN (Print): 1570-162X
ISSN (Online): 1873-4251

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Research Article

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

Author(s): Zsigmond Benko, Jiantao Zhang and Richard Y. Zhao*

Volume 17, Issue 6, 2019

Page: [429 - 440] Pages: 12

DOI: 10.2174/1570162X17666191128102839

Price: $65

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).

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