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.
A fission yeast cell-based system for multidrug resistant HIV-1 proteases
