Inhibition of the Ribonuclease H and DNA Polymerase Activities of HIV-1 Reverse Transcriptase by N-(4-tert-Butylbenzoyl)-2-hydroxy-1-naphthaldehyde Hydrazone

Abstract

HIV-1 reverse transcriptase (RT) is multifunctional, with RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) activities. N-(4-tert-Butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibited both the polymerase and the RNase H activities of HIV-1 RT in vitro. IC50 values for inhibition of RDDP were 0.8-3.4 microM, depending on the template/primer (T/P) used in the assay. The IC50 for DDDP inhibition was about 12 microM, while that for inhibition of RNase H was 3.5 microM. EC50 for inhibition of HIV-1 replication in cord blood mononuclear cells was 1.5 microM. BBNH inhibition of RNase H in vitro was time-dependent, whereas inhibition of RT polymerase activities was immediate. BBNH was a linear mixed-type inhibitor of RT RDDP activity with respect to both T/P and to dNTP, whereas BBNH inhibition of RT RNase H activity was linear competitive. Protection experiments using an azidonevirapine photolabel showed that BBNH binds to the non-nucleoside RT inhibitor (NNRTI) binding pocket. Importantly, the compound inhibited recombinant RT containing mutations associated with high-level resistance to other NNRTI. While BBNH did not inhibit the DNA polymerase activities of other retroviral reverse transcriptases and DNA polymerases, the compound inhibited Escherichia coli RNase HI and the RNase H activity of murine leukemia virus RT. BBNH also inhibited HIV-1 RT RNase H in the presence of high concentrations of other non-nucleoside inhibitors with higher affinities for the NNRTI binding pocket, and of RT in which the NNRTI binding pocket had been irreversibly blocked by the azidonevirapine photolabel. We conclude that BBNH may therefore bind to two sites on HIV-1 RT. One site is the polymerase non-nucleoside inhibitor binding site and the second may be located in the RNase H domain. BBNH is therefore a promising lead compound for the development of multisite inhibitors of HIV-1 RT.

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.…”

HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor Dihydroxy Benzoyl Naphthyl Hydrazone Bound at a Novel Site

“…We previously suggested, based on kinetic analysis of the inhibition, that NAHs may inhibit RT polymerase and RNH activities by binding to two different sites on the enzyme (20,21). A second DHBNH binding site is supported by the observation that DHBNH is equally effective at inhibiting RT RNH activity in the presence or the absence of 20 µM of the NNRTI nevirapine (data not shown), a concentration that would saturate the NNRTI-binding site.…”

“…HIV-1 RT RDDP activity was generally determined by a fixed time assay as previously described (20). Briefly, reaction mixtures (100 µL total volume) contained 50 mM Tris-HCl (pH 7.8, 37 °C), 60 mM KCl, 10 mM MgCl 2 , 10-25 ngof p51/p66 RT, 0.5 units of templateprimer, and 5 µM [ 3 H]-TTP substrate.…”

The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT

“…There are exceptions, however, such as K103N and V108I, which do not necessarily interact directly with the NNRTIs but nevertheless are still situated close to the inhibitor binding site. The kinetic data and the presence of the A101P mutation are consistent with MSK-076 binding to HIV-2 RT at the site equivalent to the HIV-1 (10), cannot be excluded, such a site of drug interaction may be unlikely due to the fact that resistance mutations in the RT of MSK-076-resistant virus strains are at a marked distance from the RNase H binding site. The structural basis for the mechanism of resistance to MSK-076 induced by G112E is not clear.…”