Interaction of HIV‐1 Reverse Transcriptase Ribonuclease H with an Acylhydrazone Inhibitor

Abstract

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.

Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

“…Two different RNA-DNA substrates were used: HTS-1, a blunt-ended, polymerase-independent substrate that induces nonspecific RNA cleavage, and HTS-2, a polymerase-dependent substrate that induces 3=-mediated RNA cleavage. The BHMP07 acylhydrazone and NAPHRHI naphthyridinone compounds have been previously shown to inhibit HIV-1 RT RNase H (30,95). The acylhydrazone THBNH is similar to the previously reported dihydroxy benzoyl naphthyl hydrazone (35).…”

“…Expression and purification of MoMLV and XMRV RTs were carried out similarly to previously published protocols (14,40,61). HIV-1 RNase H domain p15-Ec was expressed and purified as described previously (30).…”

“…Naphthyridinone {4-[(4=-aminomethyl-1,1=-biphenyl)methyl]-1-hydroxy-1,8-naphthyridin-2-one (NAPHRHI)} and acylhydrazone (trihydroxy benzoyl naphthyl hydrazone [THBNH] and trihydroxy benzoyl biphenyl carboxylate hydrazone [BHMP07]) RNHIs were synthesized as previously reported (23,30,35,95; U.S. patent application 20100056516). The hydroxyisoquinolinedione RNHI 7-(furan-2-yl)-2-hydroxy-isoquinoline-1,3(2H,4H)-dione (YLC2-155) was synthesized in Zhengqiang Wang's laboratory (University of Minnesota, Minneapolis, MN).…”

“…Such drugs would likely be active against all current drug-resistant viral strains, because the RNase H active site is located at the opposite end of the enzyme from the polymerase domain (ϳ50 Å away) that is currently targeted by nucleoside and nonnucleoside RT inhibitors (79). Several compounds have been found in recent years to effectively inhibit the RNase H activity of HIV-1 RT, including ␣,␥-diketo acids and derivatives (27,77,78,91), pyrimidinol carboxylic acids (43,47), hydroxytropolones (including ␤-thujaplicinol) (6,12,15,21,34), dimeric lactones (19), 1,3,4,5-tetragalloylapiitol (86), phenolic glycosides (10), vinylogous ureas (16,94), N-hydroxyimides (33,44), 2-hydroxyisoquinoline-1,3(2H,4H)-diones (8,9), acylhydrazones (11,30,35,80), and naphthyridinones (84,95). Although these inhibitors have been studied for the ability to inhibit HIV-1 RT, little is known about their effectiveness against other retroviral RTs, such as the gammaretroviral Moloney murine leukemia virus (MoMLV) and xenotropic murine leukemia virus-related virus (XMRV) RTs.…”

Inhibition of Foamy Virus Reverse Transcriptase by Human Immunodeficiency Virus Type 1 RNase H Inhibitors

“…In addition, given the fact that under nuclear magnetic resonance (NMR) conditions, the free HIV-1 RNase H domain is stable only in the presence of 20 to 80 mM MgCl 2 , it is difficult to perform binding studies with inhibitors that interact with magnesium ions (22)(23)(24). Furthermore, since the free HIV-1 RNase H domain is not active, a chimeric HIV-1 RNase H domain harboring the substrate binding loop from Escherichia coli RNase H is often used for inhibitor testing (20,(24)(25)(26).…”