Nucleoside reverse transcriptase inhibitors (NRTIs) are key components of Highly Active Antiretroviral Therapies (HAART) for the treatment of HIV-infected patients. All FDA-approved anti-HIV NRTIs lack a 3'-OH, and therefore inhibit DNA polymerization by HIV RT through immediate chain termination. The absence of a 3'-OH also imparts detrimental properties to these NRTIs by reducing their binding affinity for RT compared to the natural dNTP substrates and reducing intracellular conversion to active NRTI triphosphates. We have reported during the first funding cycle that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside analog that retains the 3'-OH moiety and demonstrates exceptional antiviral properties (EC50 = 50 pM in PBMCs) and a remarkable selectivity index (>200,000). EFdA's potency and selectivity index is by orders of magnitude more favorable than all currently-approved anti-HIV drugs. Despite the presence of a 3'-OH, the incorporated EFdA-monophosphate acts primarily as an immediate chain terminator because RT has difficulty translocating on the primer possessing 3'-terminal EFdA-MP, which is a unique mechanism of action. Therefore, EFdA-TP is a Translocation-Defective RT inhibitor (TDRTI). Additionally, EFdA has also been shown to efficiently inhibit clinically-important NRTI-resistant HIV strains, and demonstrating hypersusceptibility to the tenofovir-resistant K65R HIV. Notably, in our study of SIV-infected macaques the animals that had suppressed viral loads upon treatment with EFdA carried the M184V mutation, suggesting that M184V can be effectively suppressed by EFdA. We have also shown that EFdA has a remarkably high barrier for HIV resistance, and many EFdA-resistant HIV strains have reduced replication capacity. More recently we have shown that the combination of EFdA with Rilpivirine shows a synergistic effect, which would be useful in the design of new therapeutic regimens. We will build on our work from the first funding cycle to understand the potency of EFdA at the RT and cellular levels, development of EFdA resistance, and EFdA combinations with currently-approved anti-HIV drugs. We will also use biochemical and structural approaches to characterize the inhibition mechanism of EFdA at the RT level, the activation of EFdA by deoxycytidine kinase (dCK) and deamination by adenosine deaminase (ADA). This study will provide information that will guide the design of novel NRTIs and EFdA combination therapies with other approved anti-HIV drugs that may lead to a breakthrough in the treatment of HIV infection.

Public Health Relevance

4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a very potent inhibitor of HIV, including drug-resistant viral strains, and blocks the virus in a new way unlike ll currently approved anti-HIV drugs. This project will evaluate the factors that affect EFdA's efficacy, including its unique mechanism of action, cellular activation, resistance to deamination, and high barrier of resistance to HIV. These studies will provide important insights into the design of new inhibitors of HIV and also information on which EFdA combination therapies with other approved antivirals may lead to a breakthrough in the treatment of HIV infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI076119-06A1
Application #
8748626
Study Section
Special Emphasis Panel (ZRG1-AARR-J (02))
Program Officer
Turk, Steven R
Project Start
2007-07-01
Project End
2019-04-30
Budget Start
2014-05-06
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
$392,694
Indirect Cost
$114,281
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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