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

Abstract

The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 Å resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNAprimed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 Å away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.Human immunodeficiency virus, type 1 (HIV-1), reverse transcriptase (RT) is essential for HIV replication. RT converts the single-stranded viral genomic RNA into a linear doublestranded DNA that can be integrated into the host chromosomes (reviewed in ref 1). The enzyme has two activities, (i) a DNA polymerase that can use either RNA or DNA as a template and (ii) an RNase H (RNH) that selectively degrades the RNA strand of an RNA-DNA heteroduplex. The RNH activity of RT is required for virus replication; cellular RNH cannot substitute for the retroviral enzyme (2). The RNH activity degrades the genomic RNA during first-strand ("minus-strand") DNA synthesis, which allows the newly synthesized DNA to be used as a template for second-strand ("plus-strand") DNA synthesis.HIV-1 RT is a heterodimer consisting of 66 kDa (p66) and 51 kDa (p51) subunits. The two polypeptide chains have 440 N-terminal amino acid residues in common. These comprise four polymerase subdomains: the thumb, palm, fingers, and connection (3,4). The C-terminus of p66 contains an additional 120 amino acid residues that form the bulk of the RNH domain. Despite having identical N-terminal sequences, the arrangement of the subdomains in the two subunits differs dramatically. The p66 subunit contains a large cleft formed by the fingers, palm, and thumb subdo-mains that can accommodate double-stranded nucleic acid templateprimers (3-6). Although the p51 subunit contains the same four subdomains, it does not form a nucleic acid binding cleft.Because of its pivotal role in the HIV life cycle, HIV RT is a primary target for antiretroviral agents. All RT inhibitors currently approved for the treatment of acquired immune deficiency syndrome (AIDS) inhibit...

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

“…Crystal structures of HIV-1 RT RNase H with a bound active site inhibitor confirmed that these compounds are anchored at the RNase H active site and interact with the bound divalent metal ions (22)(23)(24)(25). Allosteric RNase H inhibitors can bind in the vicinity of the polymerase active site as shown for N-acyl hydrazones (26,27), or in the vicinity of the p51 thumb subdomain as shown for vinylogous ureas (28,29).…”

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

“…Since the RNase H function is essential for viral replication (5), it has been explored as a drug target, and a number of RNase H inhibitors (RHIs) have been reported (6)(7)(8). RHIs can be classified based on their binding sites, i.e., (i) RHIs that coordinate the two Mg 2ϩ catalytic cofactors at the RNase H active site, such as N-hydroxyimides (9), hydroxytropolones (10), hydroxypyrimidines (11), naphthyridinones (12), nitrofuran-2-carboxylic acid carbamoylmethyl esters (13), hydroxyquinolinones (14), and thiocarbamates and triazoles (15), or (ii) allosteric RHIs, such as vinylogous ureas (16), thienopyrimidinones (17), hydrazones (18), anthraquinones (19), isatines (20,21), and propenones (22).…”

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

“…During the last 10 years, several classes of HIV-1 RHIs have been identified (12)(13)(14)(15). The design of potent RHIs has proven difficult due to the open morphology of the RNase H active site (16) and the limited number of available RNase H crystal structures in complex with inhibitors (17)(18)(19)(20)(21). 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).…”