Employing a novel strategy, we have virtually screened a large library of compounds to identify novel inhibitors of the reverse transcriptase (RT) of HIV-1. Fifty-six top scored compounds were tested in vitro, and two of them inhibited efficiently the DNA polymerase activity of RT. The most effective compound, N-{2-[4-(aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide (NAPETA), inhibited both RNA-dependent and DNA-dependent DNA polymerase activities, with apparent IC50 values of 1.2 and 2.1 microM, respectively. This inhibition was specific to the RT-associated polymerase activity and did not affect the RNase H activity. NAPETA also inhibited two drug-resistant HIV-1 RT mutants as well as HIV-2 RT and other DNA polymerases. Kinetic analysis of RT inhibition indicated that the DNA polymerase activity of HIV-1 RT was inhibited in a classic noncompetitive manner with respect to dTTP, demonstrating a Ki value of 1.2 microM. In contrast, the inhibition with respect to the RNA.DNA template was a mixed linear type with a Ki value of 0.12 microM and was not affected by the order in which the template.primer and inhibitor were added to the reaction mixture. Gel shift and surface plasmon resonance analyses confirmed that NAPETA interfered with the formation of the RT.DNA complex (that is crucial for the polymerization activity) by reducing the affinity of RT for DNA, accounting at least partially for the inhibition. It is likely that NAPETA inhibited RT via a mechanism that is different from that of the classic non-nucleoside RT inhibitors used for treating AIDS/HIV patients and, thus, may serve as a lead compound for the development of novel anti-HIV drugs.