HIV-1 reverse transcriptase (RT) is a bi-functional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H (RNH) activities. HIV-1 RT has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target RT DNA polymerase. However, no inhibitors of RT RNH are on the market or in preclinical development. Several drug-like small molecule inhibitors of RT RNH have been described, but little structural information is available about the interactions between RT RNH and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new RNH inhibitor, BHMP07, with a catalytically active HIV-1 RT RNH domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the RNH domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the RNH fragment. Using RNH inhibition assays and RT mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H-inhibitor interaction and are likely to be useful for further improvements of the inhibitors.

Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). Although numerous chemotypes have been reported to inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV. We report herein the design, synthesis, and biological evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety. The synthesis involved a recently reported metal-free direct benzylation between tosylhydrazone and boronic acid, which allowed the generation of structural diversity for the hydrophobic aromatic region. Biochemical studies showed that the C-6 benzyl and biarylmethyl HID analogues, previously unknown chemotypes, consistently inhibited HIV RT-associated RNase H and polymerase with IC50s in low to submicromolar range. The observed dual inhibitory activity remained uncompromised against RT mutants resistant to non-nucleoside RT inhibitors (NNRTIs), suggesting the involvement of binding site(s) other than the NNRTI binding pocket. Intriguingly, these same compounds inhibited the polymerase, but not the RNase H function of Moloney Murine Leukemia Virus (MoMLV) RT and also inhibited Escherichia coli RNase H. Additional biochemical testing revealed a substantially reduced level of inhibition against HIV integrase. Molecular docking corroborates favorable binding of these analogues to the active site of HIV RNase H. Finally, a number of these analogues also demonstrated antiviral activity at low micromolar concentrations.

Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H (RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails chemotypes capable of chelating two divalent metal ions in the RNase H active site. The hydroxypyridone carboxylic acid scaffold has been implicated in inhibiting homologous HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the design, synthesis and biological evaluations of a novel variant of the hydroxypyridone carboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety. Biochemical studies show that most analogues consistently inhibited HIV RT-associated RNase H in the low micromolar range in the absence of significant inhibition of RT polymerase or IN. One compound showed reasonable cell-based antiviral activity (EC50 = 10 µM). Docking and crystallographic studies corroborate favorable binding to the active site of HIV RNase H, providing a basis for the design of more potent analogues.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains an unvalidated antiviral target. A major challenge of specifically targeting HIV RNase H arises from the general lack of selectivity over RT polymerase (pol) and integrase (IN) strand transfer (ST) inhibitions. We report herein the synthesis and biochemical evaluations of three novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes carefully designed to achieve selective RNase H inhibition. Biochemical studies showed the two subtypes with an N-1 methyl group (9 and 10) inhibited RNase H in low micromolar range without siginificantly inhibiting RT polymerase, whereas the N-1 unsubstituted subtype 11 inhibited RNase H in submicromolar range and RT polymerase in low micromolar range. Subtype 11 also exhibited substantially reduced inhibition in the HIV-1 INST assay, and no significant cytotoxicity in the cell viability assay, suggesting that it may be amenable to further structure-activity-relationship (SAR) for identifying RNase H inhibitors with antiviral activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors. Our data indicate that while some of these subtypes inhibited both the RNase H and polymerase (pol) functions of RT, potent and selective RNase H inhibition was achieved with subtypes 8–9 as exemplified with compounds 8c and 9c.