Mechanism of Inhibition of HIV-1 Reverse Transcriptase by the Novel Broad-Range DNA Polymerase Inhibitor N -{2-[4-(Aminosulfonyl)phenyl]ethyl}-2-(2-thienyl)acetamide † , ‡ (original) (raw)

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 RNAdependent and DNA-dependent DNA polymerase activities, with apparent IC 50 values of 1.2 and 2.1 µM, 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 K i value of 1.2 µM. In contrast, the inhibition with respect to the RNA‚DNA template was a mixed linear type with a K i value of 0.12 µM 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. The reverse transcriptase (RT) 1 of human immunodeficiency virus type-1 (HIV-1) is an essential enzyme in the life cycle of this retrovirus. Upon infecting the target cell, RT copies the viral plus sense and single-stranded genomic RNA into a double-stranded DNA. This complex reverse transcription process, which is common in all retroviruses, is catalyzed solely by RT and is mediated by the three activities of the enzyme. The two related RNA-dependent DNA polymerase (RDDP) and DNA-dependent DNA polymerase (DDDP) activities enable DNA synthesis of the viral genome, whereas the ribonuclease H (RNase H) activity concomitantly cleaves the viral RNA strand in the RNA‚ DNA heteroduplex. The resulting double-stranded DNA is transported into the infected cell nucleus, as part of a preintegration complex, and is subsequently incorporated into the cellular DNA by the viral integrase (1). Almost all inhibitors of HIV-1 RT can be grouped into two classes of potent compounds: nucleoside/nucleotide RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNR-TIs) (2). NRTIs are competitive inhibitors that are phosphorylated by cellular kinases and subsequently mimic normal nucleotides. Since NRTIs lack the 3′-OH group, their incorporation into the nascent DNA by RT blocks further addition of nucleotides and, hence, leads to termination of chain elongation. The NNRTIs are a variety of hydrophobic noncompetitive inhibitors that are presumed to bind specifically to a hydrophobic pocket located in the proximity of the DNA polymerase active site of the RT (3). Most NNRTIs are highly specific against HIV-1 RT with minimal effects on the closely related HIV-2 RT (4). Both classes of inhibitors are currently used in the therapy against HIV-1