Development of a novel screen for protease inhibitors

Abstract

We have developed a novel plasmid-based, quantitative, in vitro screen to test the protease-inhibiting activities of existing and newly discovered agents.

FIG. 1

Plasmids used in transfection experiments. pEGFP-PC-Luciferase (responder plasmid) contains EGFP and the P. pyralis-derived luciferase gene separated by three HIV-1 protease (PR) cleavage sites; expression is driven by the cytomegalovirus (CMV) promoter. pRL-TK (normalizing plasmid) encodes the R. reniformis-derived luciferase gene driven by the thymidine kinase (TK) promoter. LTRPR, LTR2XPR, and LTR4XPR contain one, two, and four copies of the HIV-1 PR gene, respectively, driven by the HIV-1 long terminal repeat (LTR). SV40, simian virus 40.

FIG. 2

Increasing the copy number of the HIV-1 PR gene results in increasing luciferase activity from the responder plasmid. HeLa-Tat cells were cotransfected with pEGFP-PC-Luciferase, pRL-TK, and plasmids containing one, two, or four copies of the HIV-1 PR gene. Cells were assayed for luciferase activity from the responder plasmid, and the resulting luciferase activity was normalized to Renilla luciferase activity from the normalizing plasmid. Each bar represents the percent increase in luciferase activity over the luciferase activity in the absence of the PR plasmid, which was set at 100. This graph represents the results (means and standard deviations) of five repeated experiments.

FIG. 3

PR inhibitors decrease the luciferase activity in this assay in a dose-dependent manner. HeLa-Tat cells were cotransfected with pEGFP-PC-Luciferase and pRL-TK alone or with LTR4XPR in the presence of increasing concentrations of known PR inhibitors (Ritonavir and Nelfinavir). Cells were assayed for luciferase activity at 3 days posttransfection. Each bar represents the percent increase or decrease in luciferase activity compared to that seen in the absence of drug (set to 100). Each graph represents the results (means and standard deviations) of five repeated experiments.