Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are an important therapeutic class of drugs that are widely used in antiretroviral therapy strategies to treat and prevent HIV-1 infection. They bind to a hydrophobic pocket in HIV-1 RT, termed the NNRTI-binding pocket (BP), which is located ~ 10 ? away from the polymerase active site of the enzyme. Our current understanding of how NNRTIs inhibit HIV-1 reverse transcription, and how mutations in the NNRTI-BP confer inhibitor resistance, has been largely inferred from crystal structures of HIV-1 RT in complex with NNRTIs. We have only limited knowledge in regard to how NNRTIs affect the catalytically relevant RT-template/primer (T/P) binary and RT-T/P-dNTP ternary complexes. Importantly, single-molecule F?ster resonance energy transfer studies revealed that NNRTI-binding to RT can impact the binding orientation and sliding dynamics of RT on the T/P substrate. Crystallography cannot provide insight into the dynamic interactions between biomolecules. Indeed, there are only 2 crystal structures available of an NNRTI-bound RT-T/P binary complex (and none for the ternary complex) - and in one of these structures RT is cross-linked to the T/P substrate. As such, there is a critical knowledge gap in regard to how NNRTI-binding to wild-type and mutant RT impacts: (i) the dynamic inter-molecular interactions between the enzyme and its substrates;and (ii) the intra-molecular protein conformational changes in RT-T/P and RT-T/P- dNTP complexes. The primary goal of this application is to address these knowledge gaps using state-of-the- art single-molecule and ensemble biophysical approaches (developed in our laboratories) that can quantitatively assess the dynamic inter-molecular interactions between HIV-1 RT and its substrates and the intra-molecular conformational changes in RT. We anticipate that the data derived from these studies will provide unprecedented mechanistic insight into the mode of action of NNRTIs and the mechanisms associated with NNRTI resistance. Collectively these studies may significantly impact future drug discovery efforts.

Public Health Relevance

The primary goal of this application is to gain insight into how NNRTI-binding to HIV-1 reverse transcriptase (RT) impacts the dynamic inter-molecular interactions between RT and the template/primer T/P and dNTP substrates;and (ii) the intra-molecular protein conformational changes in RT-T/P and RT-T/P-dNTP complexes. The information derived from these studies will provide unprecedented mechanistic insight into this class of antiviral drugs, which will significantly impact future drug discovery efforts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM068406-10
Application #
8730981
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Barski, Oleg
Project Start
2003-08-01
Project End
2018-07-31
Budget Start
2014-08-15
Budget End
2015-07-31
Support Year
10
Fiscal Year
2014
Total Cost
$339,974
Indirect Cost
$114,974
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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