Fission yeast as a HTS platform for molecular probes of HIV-1 Vpr-induced cell death

Abstract

Fission yeast as a HTS platform for molecular probes of HIV-1 Vpr-induced cell death Zsigmond Benko1, Robert T Elder2, Dong Liang1, Richard Y Zhao11Department of Pathology, Department of Microbiology-Immunology, Institute of Human Virology, University of Maryland Medical School, Baltimore, MD, USA; 2Children’s Memorial Research Center, Northwestern University Feinberg School of Medicine, Chicago, IL, USAAbstract: HIV-1 viral protein R (Vpr) plays an important role in the viral life cycle and pathogenesis of HIV-1. Its activities associate with activation of viral replication, suppression of host immune responses and depletion of CD4+ T-lymphocytes. In particular, Vpr induces cell death through apoptosis that may contribute to the depletion of CD4 T cells, which is a hallmark of HIV-1 infection. Currently, there are no anti-Vpr drugs or adequate assays for high throughput screening to identify selective and potent inhibitors of Vpr activities. In this report, we developed a simple fission yeast-based High Throughput Screening (HTS) system that allows us to screen small molecule compounds that specifically inhibit HIV-1 Vpr. This HTS system includes an absorbance-based primary growth assay, a secondary assay using a semi-quantitative colony-forming dot test, and a fluorescence-based LIVE/DEAD yeast viability assay, which is used as the counter screen tests. We further present here results of a pilot study using a Microsource Spectrum Collection Library, which contains 2,000 biologically active and structurally diverse compounds of known drugs, experimental bioactives, and pure natural products. One compound Benfotiamine showed >50% inhibitory concentration (IC50) and 100% inhibition at 20 and 100 µM, respectively. Interestingly, Benfotiamine is in the same family as thiamine, which was used in this system to control Vpr production through transcriptional inhibition. Even though transcriptional suppressor is not what we are looking for, finding Benfotiamine through the described HTS system from a drug library nevertheless demonstrated the feasibility of the screening assay.Keywords: HIV-1 viral protein R (Vpr), fission yeast (Schizosaccharomyces pombe), high throughput screening (HTS), small molecules, drug library, Benfotiamine