NYAD-201 inhibits HIV-1 infectivity. (A) Five hours post transfection with pNL4-3, 293T cells were treated with 12.5, 25, and 50 μM NYAD-201 for 2 or 4 days and supernatant were collected and monitored for RT activity. Approximately 7 μl RT-normalized virus stocks were used to infect the TZM-bl cells for 2 h in a total volume of 200 μl. This approach led to a dilution of peptide concentration from the producer cell supernatant of ~30-fold. Two days after infection, cells were washed, lysed and assayed for luciferase activity. (B) Cells or virions were treated with 5, 20 or 50 μM NYAD-201 before, during, or after infection as described in Methods. Infection of TZM-bl cells was carried out for 2 h and two days after infection cells were washed, lysed and assayed for luciferase activity. (C) NYAD-201 or NYAD-233 were added to TZM-bl cells at indicated concentrations during the 2 h infection. Two days after infection cells were washed and luciferase activity was measured as in A. (D) HIV-1 virions were pseudotyped with VSV-G by cotransfecting the Env-defective pNL4-3 derivative (pNL4-3/KFS) with the VSV-G expression vector pHCMV-G. RT-normalized WT and VSV-G pseudotyped virions were used to infect TZM-bl cells in the presence of the indicated concentrations of NYAD-201 for 2 h. Two days after infection, luciferase activity was measured in cell lysates. N = 4 for A, B, D; n = 3 for C; ± SD.