The primary goals of our research are two-fold. In the first, we endeavor to define the multiple mechanisms by which nonnucleoside reverse transcriptase (RT) inhibitors (NNRTI) inhibit HIV-1 reverse transcription. These studies are of fundamental importance because they will assist in the discovery and/or development of new NNRTI;they will help define the interactions between NNRTIs and other classes of RT inhibitors;and they will contribute to our understanding of NNRTI resistance and hyper-susceptibility. In the second, we strive to identify and characterize novel RT inhibitors that, if developed, will complement and diversify existing antiretroviral therapies and help address the need for antiviral agents that are active against multi-drug resistant HIV-1. Accordingly, in this application we propose 3 Aims that address each of these research goals. Recent data from our laboratory demonstrate that NNRTIs modulate both the DNA polymerase and ribonuclease H (RNase H) activities of RT. Whereas the mechanisms of NNRTI inhibition of DNA synthesis have been investigated in detail, the mechanisms by which NNRTIs influence the enzyme's RNase H activity are unknown.
In Aim 1 we propose to elucidate the mechanisms for this long-range allosteric effect by using state-of-the-art biophysical techniques that include single-pair fluorescence resonance energy transfer and transient kinetic analyses. Since RNase H activity is viewed as potential target for drug discovery, we will also investigate interactions between NNRTIs and a prototype RNase H inhibitor. Because modulation of both DNA polymerase and RNase H may lead to synergistically increased drug potency, we also hypothesize that NNRTIs may preferentially target steps during reverse transcription that have an absolute requirement for both activities. Therefore, in Aim 2 we will use quantitative PCR to identify the steps during HIV-1 reverse transcription that are most sensitive to inhibition by NNRTIs. Finally, our group recently developed a high throughput screening assay to identify pharmacophores that inhibit the ability of HIV-1 RT to excise chain-terminating nucleoside analogs from the 3'-end of the DNA primer. From a total of 7,265 compounds screened, we identified 3,39-[(3-carboxy-4-oxo-2,5-cyclohexadien-1- ylidene)methylene]bis[6-hydroxy-benzoic acid] (APEX-57219) as a promising "lead" compound. Preliminary mechanistic analyses demonstrate that APEX-57219 competes with the template/primer (T/P) substrate for binding to HIV-1 RT.
In Aim 3, we propose in depth analyses to determine the mechanism of action of APEX- 57219, and to identify its binding site in HIV-1 RT. These studies will provide detailed insight into the biochemical and virological properties of this novel compound which could aid in the discovery and/or development of more potent T/P competing RT inhibitors.

Public Health Relevance

The primary goal of our ongoing research is to define the molecular mechanism(s) by which NNRTI inhibit HIV-1 reverse transcription. In addition, we propose to characterize a new class of RT inhibitor termed the template/primer competing RT inhibitor (TPcRTI). Because the TPcRTIs exhibit a novel mechanism of action, it is anticipated that, if developed, they will both complement and diversify existing HIV-1 therapeutic strategies, and more importantly, provide a new avenue for the treatment of multi-drug resistant HIV-1.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068406-09
Application #
8303395
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Barski, Oleg
Project Start
2003-08-01
Project End
2014-07-31
Budget Start
2012-08-01
Budget End
2014-07-31
Support Year
9
Fiscal Year
2012
Total Cost
$273,289
Indirect Cost
$89,153
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
La, Jennifer; Latham, Catherine F; Tinetti, Ricky N et al. (2015) Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening. Proc Natl Acad Sci U S A 112:6979-84
Sluis-Cremer, Nicolas; Wainberg, Mark A; Schinazi, Raymond F (2015) Resistance to reverse transcriptase inhibitors used in the treatment and prevention of HIV-1 infection. Future Microbiol 10:1773-82
Sluis-Cremer, Nicolas; Huber, Kelly D; Brumme, Chanson J et al. (2014) Competitive fitness assays indicate that the E138A substitution in HIV-1 reverse transcriptase decreases in vitro susceptibility to emtricitabine. Antimicrob Agents Chemother 58:2430-3
Schauer, Grant D; Huber, Kelly D; Leuba, Sanford H et al. (2014) Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence. Nucleic Acids Res 42:11687-96
Sheen, Chih-Wei; Alptürk, Onur; Sluis-Cremer, Nicolas (2014) Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action. Biochem J 462:425-32
Sluis-Cremer, Nicolas; Jordan, Michael R; Huber, Kelly et al. (2014) E138A in HIV-1 reverse transcriptase is more common in subtype C than B: implications for rilpivirine use in resource-limited settings. Antiviral Res 107:31-4
Sluis-Cremer, Nicolas (2014) The emerging profile of cross-resistance among the nonnucleoside HIV-1 reverse transcriptase inhibitors. Viruses 6:2960-73
Xu, Hongzhan; Franks, Tamera; Gibson, Gregory et al. (2013) Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay. Retrovirology 10:70
Schauer, Grant; Leuba, Sanford; Sluis-Cremer, Nicolas (2013) Biophysical Insights into the Inhibitory Mechanism of Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors. Biomolecules 3:889-904
Herman, Brian D; Sluis-Cremer, Nicolas (2013) Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue. Biochem J 455:179-84

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