The conversion of HIV genomic RNA into DNA comprises multiple steps catalyzed by the viral enzyme, reverse transcriptase (RT). Accordingly, RT is multifunctional, with both DNA polymerase and ribonuclease H (RNase H) activities. Many inhibitors of HIV RT DNA polymerase activity have been discovered including ten drugs in clinical use, whereas few inhibitors of RT RNase H have been identified. Certain N-acyl hydrazones (NAH) are reasonably potent inhibitors of HIV RT RNase H activity. This Program Project component will characterize and optimize the RNase H inhibitory properties of NAH. This project thus comprises three specific aims: (1) To evaluate the mechanisms of NAH inhibition of HIV RNase H. The mechanism of inhibition of target enzyme activity impacts on the development of potent inhibitors of this target. Studies will include steady-state and transient kinetic analyses, the role of metal-binding in inhibition, comparison of NAH inhibition of polymerase-dependent and polymerase-independent RNase H activities, and effect of inhibitors on strand-transfer (novel reagents prepared by Program member R. Jones will be invaluable in these studies). 'Real-time' PCR analysis of intracellular HIV DNA synthesis in the presence of NAH (in collaboration with Program member S.H. Hughes) will assess inhibitory mechanisms in situ. (2) To optimize the antiviral and inhibitory properties of NAH. This will involve the use of traditional and parallel library synthesis to prepare new NAH analogs altered in one or more of the three 'pharmacophore' regions of the molecule to improve inhibitory potency and reduce cytotoxicity. Refinement of NAH structure will involve molecular modeling/docking and quantitative structure-activity analyses, but will primarily be based on exact structural information obtained from X-ray crystallography of RT-NAH complexes (in collaboration with Program director E. Arnold). This structural information will enable rational design of new highly potent analogs. (3) To evaluate the antiviral and resistance properties of NAH inhibitors. Low toxicity inhibitors will be used to generate resistant HIV in vitro. NAH-specific mutations identified will be further characterized by X-ray crystallography of mutant recombinant protein (in collaboration with Arnold and Hughes). The proposed studies will develop novel antiviral agents against HIV RT-associated RNase H, a critical yet under-explored HIV target.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
1P01GM066671-01
Application #
6553912
Study Section
Special Emphasis Panel (ZRG1)
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
Budget End
Support Year
1
Fiscal Year
2002
Total Cost
Indirect Cost
Name
Rutgers University
Department
Type
DUNS #
038633251
City
New Brunswick
State
NJ
Country
United States
Zip Code
08901
Tu, Xiongying; Das, Kalyan; Han, Qianwei et al. (2010) Structural basis of HIV-1 resistance to AZT by excision. Nat Struct Mol Biol 17:1202-9
Hou, Xiaorong; Wang, Gang; Gaffney, Barbara L et al. (2010) Preparation of DNA and RNA fragments containing guanine N(2)-thioalkyl tethers. Curr Protoc Nucleic Acid Chem Chapter 5:Unit 5.8
Das, Kalyan; Bandwar, Rajiv P; White, Kirsten L et al. (2009) Structural basis for the role of the K65R mutation in HIV-1 reverse transcriptase polymerization, excision antagonism, and tenofovir resistance. J Biol Chem 284:35092-100
Han, Qianwei; Sarafianos, Stefan G; Arnold, Eddy et al. (2009) Synthesis of Boranoate, Selenoate, and Thioate Analogs of AZTp(4)A and Ap(4)A. Tetrahedron 65:7915-7920
Sarafianos, Stefan G; Marchand, Bruno; Das, Kalyan et al. (2009) Structure and function of HIV-1 reverse transcriptase: molecular mechanisms of polymerization and inhibition. J Mol Biol 385:693-713
Hou, Xiaorong; Wang, Gang; Gaffney, Barbara L et al. (2009) Synthesis of guanosine and deoxyguanosine phosphoramidites with cross-linkable thioalkyl tethers for direct incorporation into RNA and DNA. Nucleosides Nucleotides Nucleic Acids 28:1076-94
Arnold, Gail Ferstandig; Velasco, Paola K; Holmes, Andrew K et al. (2009) Broad neutralization of human immunodeficiency virus type 1 (HIV-1) elicited from human rhinoviruses that display the HIV-1 gp41 ELDKWA epitope. J Virol 83:5087-100
Bauman, Joseph D; Das, Kalyan; Ho, William C et al. (2008) Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design. Nucleic Acids Res 36:5083-92
Fang, Chong; Bauman, Joseph D; Das, Kalyan et al. (2008) Two-dimensional infrared spectra reveal relaxation of the nonnucleoside inhibitor TMC278 complexed with HIV-1 reverse transcriptase. Proc Natl Acad Sci U S A 105:1472-7
Das, Kalyan; Bauman, Joseph D; Clark Jr, Arthur D et al. (2008) High-resolution structures of HIV-1 reverse transcriptase/TMC278 complexes: strategic flexibility explains potency against resistance mutations. Proc Natl Acad Sci U S A 105:1466-71

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