Acylhydrazone inhibitors of HIV-1 ribonuclease H

Summary

Principal Investigator: MICHAEL A contact PARNIAK
Abstract: DESCRIPTION (provided by applicant): The increasing prevalence of drug resistant HIV compromises the continued utility of current HIV drugs. Virtually all clinically used drugs are directed at only two HIV targets, protease and reverse transcriptase (RT), thus one approach to the problem of HIV drug resistance is to develop therapeutics directed at novel HIV targets, since therapeutics directed at novel HIV targets will almost certainly be active against current drug- resistant virus strains. One such target is RT-associated ribonuclease H (RNaseH), an essential enzyme activity for HIV replication and the only HIV enzyme not yet targeted by any clinically used or pipeline therapeutics. We have identified acylhydrazones as interesting compounds in that certain analogs are potent inhibitors of both HIV-1 RT DNA polymerase and RT-RNaseH activities. We hypothesize that this bifunctional inhibition is due to binding of the inhibitor to two distinct sites on RT, one of which is in or near the RNaseH domain. We recently obtained a crystal structure of acylhydrazones in the RT polymerase (pol) domain, so detailed understanding of the interaction of acylhydrazones with the RT RNaseH domain will be invaluable for design of both new RNaseH inhibitors (RNaseHI) and new bifunctional inhibitors. To this end, we propose four Specific Aims, (1) to determine the structure of HIV-RT RNaseH in complex with acylhydrazone inhibitors. Detailed NMR studies of the interaction of acylhydrazones with an active isolated HIV-1 RT RNaseH domain will provide precise structural information of the inhibitor binding pocket in the RNaseH domain, the microscopic conformation of RNaseH-inhibitor interaction sites, and influence of inhibitor binding on RT-RNaseH structure;(2) to validate the RNaseHI binding pocket determined in Aim 1. The RNaseHI binding pocket determined in Aim 1 will be validated in the context of intact RT and HIV-1 virus by evaluating the effect of mutation of selected residues interacting with RNaseHI, in the RNaseH fragment (biochemical and NMR structural analyses), in intact RT, and in HIV-1;(3) to optimize the inhibitory potency of acylhydrazone and analog inhibitors. Several approaches will be used to identify and develop potent monofunctional (RNaseH-specific) and bifunctional inhibitors, including screening of a library of 5000 hydrazone derivatives and synthesis of new analogs based on structures of acylhydrazones in the RNaseH and pol site binding pockets;(4) to conduct detailed biochemical and virologic characterizations of new RNaseHI. The inhibitory properties of potent RNaseHI and bifunctional acylhydrazones from Aim 3 will be characterized with purified RT and in cell-based HIV replication studies (including resistance development). We hypothesize that bifunctional inhibitors may be preferable in the context of resistance development. Selected RNaseHI will be further evaluated in NMR structural studies to better define the binding pocket for RNaseHI. This structural information along with the crystal data will provide a firm basis for rational design of new inhibitors. PUBLIC HEALTH RELEVANCE: There are more than 20 drugs for the treatment of HIV infection, yet the virus continues to spread in the US and worldwide. Furthermore, drug resistant HIV is increasing, limiting treatment options for individuals infected with these strains. New drugs are essential. This project will explore compounds directed at a novel HIV target, ribonuclease H, with the goal of developing new drugs that will be active against existing drug resistant HIV.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Structural basis of the allosteric inhibitor interaction on the HIV-1 reverse transcriptase RNase H domain
    Martin T Christen
    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
    Chem Biol Drug Des 80:706-16. 2012
  2. pmc The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site
    Michael E Abram
    University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, PA 15219, USA
    Retrovirology 7:6. 2010
  3. pmc Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor
    Qingguo Gong
    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
    Chem Biol Drug Des 77:39-47. 2011
  4. pmc Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis
    Xianzhong Xu
    Department of Chemistry, University of California, Davis, California 95616, USA
    Proteins 79:1910-22. 2011
  5. pmc Identification of alternative binding sites for inhibitors of HIV-1 ribonuclease H through comparative analysis of virtual enrichment studies
    Anthony K Felts
    BioMaPS Institute for Quantitative Biology, Rutgers University, Piscataway, New Jersey 08854, USA
    J Chem Inf Model 51:1986-98. 2011
  6. ncbi Recent developments in (15)N NMR relaxation studies that probe protein backbone dynamics
    Rieko Ishima
    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
    Top Curr Chem 326:99-122. 2012
  7. pmc The effects of RNase H inhibitors and nevirapine on the susceptibility of HIV-1 to AZT and 3TC
    Caroline A Davis
    HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702 1201, USA
    Virology 419:64-71. 2011
  8. pmc Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase
    Karen A Kirby
    Christopher S Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA
    Antimicrob Agents Chemother 56:2048-61. 2012

Scientific Experts

  • Anthony K Felts
  • Rieko Ishima
  • Michael E Abram
  • Michael A Parniak
  • Karen A Kirby
  • Martin T Christen
  • Jinwoo Ahn
  • Lakshmi Menon
  • Caroline A Davis
  • Xianzhong Xu
  • Qingguo Gong
  • Daniel V Sietsema
  • Tanyaradzwa P Ndongwe
  • Kamalendra Singh
  • Eleftherios Michailidis
  • Maxwell D Leslie
  • Stefan G Sarafianos
  • Zhengqiang Wang
  • Yee Tsuey Ong
  • Bruno Marchand
  • Christopher A Dorst
  • Tracy L Fetterly
  • Atsuko Hachiya
  • Nataliya S Myshakina
  • Lena G Miller
  • James B Ames
  • Stephen H Hughes
  • Tatiana Ilina

Detail Information

Publications9

  1. pmc Structural basis of the allosteric inhibitor interaction on the HIV-1 reverse transcriptase RNase H domain
    Martin T Christen
    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
    Chem Biol Drug Des 80:706-16. 2012
    ..2011) Chem Biol Drug Des 77, 39-47], but with slight differences that reflect the characteristics of the amino acid sequences in p15-EC compared to the WT RNH...
  2. pmc The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site
    Michael E Abram
    University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, PA 15219, USA
    Retrovirology 7:6. 2010
    ..In this study we have characterized in detail the impact of the T477A mutation on intravirion processing of RT...
  3. pmc Interaction of HIV-1 reverse transcriptase ribonuclease H with an acylhydrazone inhibitor
    Qingguo Gong
    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
    Chem Biol Drug Des 77:39-47. 2011
    ..Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors...
  4. pmc Conformational dynamics of recoverin's Ca2+-myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis
    Xianzhong Xu
    Department of Chemistry, University of California, Davis, California 95616, USA
    Proteins 79:1910-22. 2011
    ..We propose that millisecond dynamics during T ↔ I may transiently increase the exposure of Ca(2+)-binding sites to initiate Ca(2+) binding that drives extrusion of the myristoyl group during I ↔ R...
  5. pmc Identification of alternative binding sites for inhibitors of HIV-1 ribonuclease H through comparative analysis of virtual enrichment studies
    Anthony K Felts
    BioMaPS Institute for Quantitative Biology, Rutgers University, Piscataway, New Jersey 08854, USA
    J Chem Inf Model 51:1986-98. 2011
    ..For a series of hydrazone compounds, a small amount of positive enrichment was obtained when active compounds were bound by induced-fit docking at the interface between the DNA:RNA substrate and the RNase H domain near residue Q500...
  6. ncbi Recent developments in (15)N NMR relaxation studies that probe protein backbone dynamics
    Rieko Ishima
    Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
    Top Curr Chem 326:99-122. 2012
    ..Here, we present an overview of recent advances in (15)N relaxation experiments to characterize protein backbone dynamics...
  7. pmc The effects of RNase H inhibitors and nevirapine on the susceptibility of HIV-1 to AZT and 3TC
    Caroline A Davis
    HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702 1201, USA
    Virology 419:64-71. 2011
    ..Nevirapine, and most RNHIs tested, had only small effects on the susceptibility of either HIV vector to AZT and 3TC. One RNHI, F0444-0019, increased the IC(50) for AZT for either vector by ~5-fold, which may be a concern...
  8. pmc Structural and inhibition studies of the RNase H function of xenotropic murine leukemia virus-related virus reverse transcriptase
    Karen A Kirby
    Christopher S Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA
    Antimicrob Agents Chemother 56:2048-61. 2012
    ..5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H...