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
Salie, Zhe Li; Kirby, Karen A; Michailidis, Eleftherios et al. (2016) Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA). Proc Natl Acad Sci U S A 113:9274-9
Stoddart, Cheryl A; Galkina, Sofiya A; Joshi, Pheroze et al. (2015) Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque. Antimicrob Agents Chemother 59:4190-8
Zhang, Wei; Hu, Minlu; Shi, Yuan et al. (2015) Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission. Pharm Res 32:2960-72
Gres, Anna T; Kirby, Karen A; KewalRamani, Vineet N et al. (2015) STRUCTURAL VIROLOGY. X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability. Science 349:99-103
Yamada, Ken; Wahba, Alexander S; Bernatchez, Jean A et al. (2015) Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT. ACS Chem Biol 10:2024-33
Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko et al. (2015) Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247. FASEB J 29:70-80
Takamatsu, Yuki; Tanaka, Yasuhito; Kohgo, Satoru et al. (2015) 4'-modified nucleoside analogs: potent inhibitors active against entecavir-resistant hepatitis B virus. Hepatology 62:1024-36
Liu, Dandan; Ji, Juan; Ndongwe, Tanya P et al. (2015) Fast hepatitis C virus RNA elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach. Antimicrob Agents Chemother 59:3482-92
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

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