Nucleoside reverse transcriptase inhibitors (NRTIs) are among the most potent antiretrovirals used clinically, and are often used in first-line therapy for HIV infection. However, resistance is increasingly common in HIV drug experienced patients, and there is an urgent need to identify and develop new antiretrovirals active against these resistant HIV strains. All approved NRTIs act as chain terminators because they lack a 3'OH, and it has been a long standing paradigm that the absence of the 3'OH is essential for antiviral activity. However, this feature can also impart detrimental properties to the inhibitor, such as reduced affinity for RT compared to dNTP substrates, as well as reduced intracellular conversion to the active nucleoside triphosphate. We and our collaborators have obtained data with the novel nucleoside 4'-ethynyl, 2-fluoro deoxyadenosine (4'E-2FdA) that challenge this existing paradigm. 4'E-2FdA is the most potent NRTI described to date and acts as a chain terminator despite retaining an accessible 3'OH. Our preliminary data suggest that this apparent chain termination arises from difficulty of the primer 3'-terminus to translocate following incorporation of the compound. We therefore propose that 4'E-2FdA is a Translocation-Deficient Reverse Transcriptase Inhibitor (TDRTI). We hypothesize that the presence of the 3'OH, 4'E and 2F groups contribute to the high potency and result in the novel mechanism of inhibition. We propose to conduct detailed biochemical studies to better understand how these novel NRTIs work and to determine the specific characteristics of these compounds that contribute to their pronounced antiviral potency and excellent resistance profiles. To this end we will pursue the following Specific Aims: 1. Determine the biochemical mechanism of RT inhibition by TDRTIs. 2. Determine the biochemical mechanism of TDRTI excision by RT. 3. Determine inhibition of clinically relevant NRTI-resistant RTs by TDRTIs;interactions of clinically relevant RT inhibitors with TDRTIs and toxicity of combinations. 4. Determine the mechanism of HIV resistance to TDRTIs. Addressing these aims should significantly advance scientific knowledge and be invaluable in the design of new generations of highly active innovative NRTIs.

Public Health Relevance

This project will characterize the biochemical and molecular basis for the unprecedented efficiency of a novel class of compounds that suppress HIV viruses extremely efficiently, and by doing so, it will help develop anti- HIV therapeutics that are both less susceptible to current clinically significant resistance mutations as well as more refractory to the development of viral drug resistance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI076119-05
Application #
8287168
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Turk, Steven R
Project Start
2008-07-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$360,279
Indirect Cost
$87,796
Name
University of Missouri-Columbia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Michailidis, Eleftherios; Huber, Andrew D; Ryan, Emily M et al. (2014) 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) inhibits HIV-1 reverse transcriptase with multiple mechanisms. J Biol Chem 289:24533-48
Adedeji, Adeyemi O; Sarafianos, Stefan G (2014) Antiviral drugs specific for coronaviruses in preclinical development. Curr Opin Virol 8:45-53
Adedeji, Adeyemi O; Singh, Kamalendra; Kassim, Ademola et al. (2014) Evaluation of SSYA10-001 as a replication inhibitor of severe acute respiratory syndrome, mouse hepatitis, and Middle East respiratory syndrome coronaviruses. Antimicrob Agents Chemother 58:4894-8
Muftuoglu, Yagmur; Sohl, Christal D; Mislak, Andrea C et al. (2014) Probing the molecular mechanism of action of the HIV-1 reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) using pre-steady-state kinetics. Antiviral Res 106:1-4
Singh, Kamalendra; Flores, Jacqueline A; Kirby, Karen A et al. (2014) Drug resistance in non-B subtype HIV-1: impact of HIV-1 reverse transcriptase inhibitors. Viruses 6:3535-62
Zhang, Wei; Parniak, Michael A; Sarafianos, Stefan G et al. (2014) In vitro transport characteristics of EFdA, a novel nucleoside reverse transcriptase inhibitor using Caco-2 and MDCKII cell monolayers. Eur J Pharmacol 732:86-95
Zhang, Wei; Parniak, Michael A; Sarafianos, Stefan G et al. (2014) Development of a vaginal delivery film containing EFdA, a novel anti-HIV nucleoside reverse transcriptase inhibitor. Int J Pharm 461:203-13
Huber, Andrew D; Michailidis, Eleftherios; Schultz, Megan L et al. (2014) SAMHD1 has differential impact on the efficacies of HIV nucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother 58:4915-9
Tavis, John E; Cheng, Xiaohong; Hu, Yuan et al. (2013) The hepatitis B virus ribonuclease H is sensitive to inhibitors of the human immunodeficiency virus ribonuclease H and integrase enzymes. PLoS Pathog 9:e1003125
Izumi, Kazuki; Kawaji, Kumi; Miyamoto, Fusasko et al. (2013) Mechanism of resistance to S138A substituted enfuvirtide and its application to peptide design. Int J Biochem Cell Biol 45:908-15

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