Inhibition of HIV-1 Ribonuclease H by a Novel Diketo Acid, 4-[5-(Benzoylamino)thien-2-yl]-2,4-dioxobutanoic Acid

Abstract

Human immunodeficiency virus-type 1 (HIV-1) reverse transcriptase (RT) coordinates DNA polymerization and ribonuclease H (RNase H) activities using two discrete active sites embedded within a single heterodimeric polyprotein. We have identified a novel thiophene diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid, that selectively inhibits polymeraseindependent RNase H cleavage (IC 50 ‫؍‬ 3.2 M) but has no effect on DNA polymerization (IC 50 > 50 M). The activity profile of the diketo acid is shown to be distinct from previously described compounds, including the polymerase inhibitor foscarnet and the putative RNase H inhibitor 4-chlorophenylhydrazone. Both foscarnet and the hydrazone inhibit RNase H cleavage and DNA polymerization activities of RT, yet neither inhibits the RNase H activity of RT containing a mutation in the polymerase active site (D185N) or an isolated HIV-1 RNase H domain chimera containing the ␣-C helix from Escherichia coli RNase HI, suggesting these compounds affect RNase H indirectly. In contrast, the diketo acid inhibits the RNase H activity of the isolated RNase H domain as well as full-length RT, and inhibition is not affected by the polymerase active site mutation. In isothermal titration calorimetry studies using the isolated RNase H domain, binding of the diketo acid is independent of nucleic acid but strictly requires Mn 2؉ implying a direct interaction between the inhibitor and the RNase H active site. These studies demonstrate that inhibition of HIV-1 RNase H may occur by either direct or indirect mechanisms, and they provide a framework for identifying novel agents such as 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid that specifically targets RNase H.

Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives

“…DKAs have been reported to be the first active site agents that can effectively inhibit both HIV-1 RNase H and IN functions, or only one of them, in the low micromolar range and that can also inhibit HIV-1 replication in cell-based assays (30,44 (24). Starting from these results, in the present study we synthesized 6 new couples of diketo ester and DKA derivatives with various structural features, to be used as chemical tools in the characterization of RNase H binding modes.…”

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: Balance between RNase H activity and nucleotide excision

“…Another implication of these studies is that inhibitors of HIV-1 RNase H that are currently being identified as antiretroviral agents (30)(31)(32)(33)(34)(35) will enhance resistance to some NRTIs. It will be important to determine whether the anti-RNase H inhibitors under development will antagonize the activity of some NRTIs so that effective antiretroviral combination therapies can be devised.…”

In VitroCharacterization of MK-1439, a Novel HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor

“…Full-length wild-type (WT) and two mutant RT proteins (K103N and Y181C) were expressed in Escherichia coli BL21(DE3) cells and purified as described previously (33). The t500 RNA template was made by IBA GmbH (Germany), and the biotinylated DNA primer was made by Integrated DNA Technology (IDT; Coralville, IA).…”

“…The full-length WT and 2 mutant RT proteins were expressed in Escherichia coli BL21(DE3) cells and purified as described previously (33). The ECL RT biochemical assay was performed based on the protocol described previously (34).…”