Polymerase inhibition assay. As described in the Materials and Methods section, the three compounds that showed inhibitory activity in the cell-based assays were tested for their ability to inhibit the polymerase activity of HIV-1 RT. A radioactive primer annealed to a long template was extended by HIV-1 RT in the presence of varying concentrations of the compounds (the amount of DMSO was constant in all of the reactions), appropriate buffer, and 0.5 μM each dNTP. Nevirapine was included as a positive control for NNRTI inhibition. The reactions were allowed to proceed at 37° for 60 min and were then halted by the addition of EDTA. The samples were fractionated by electrophoresis on a 6.0% polyacrylamide gel, and the gel was autoradiographed. Phosphoimaging was used to determine the amount of signal in each lane. Primer extension products >90 nt in length were considered full length product. The percentage of the full length produced in each of the reaction conditions was calculated, then plotted. Reactions were done in duplicate.