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 #
5R01AI076119-04
Application #
8096758
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
2011-07-01
Budget End
2012-06-30
Support Year
4
Fiscal Year
2011
Total Cost
$360,455
Indirect Cost
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
Puray-Chavez, Maritza; Tedbury, Philip R; Huber, Andrew D et al. (2017) Multiplex single-cell visualization of nucleic acids and protein during HIV infection. Nat Commun 8:1882
Achuthan, Vasudevan; Singh, Kamlendra; DeStefano, Jeffrey J (2017) Physiological Mg2+ Conditions Significantly Alter the Inhibition of HIV-1 and HIV-2 Reverse Transcriptases by Nucleoside and Non-Nucleoside Inhibitors in Vitro. Biochemistry 56:33-46
Hachiya, Atsuko; Kirby, Karen A; Ido, Yoko et al. (2017) Impact of HIV-1 Integrase L74F and V75I Mutations in a Clinical Isolate on Resistance to Second-Generation Integrase Strand Transfer Inhibitors. Antimicrob Agents Chemother 61:
de la Higuera, Ignacio; Ferrer-Orta, Cristina; de Ávila, Ana I et al. (2017) Molecular and Functional Bases of Selection against a Mutation Bias in an RNA Virus. Genome Biol Evol 9:1212-1228
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
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
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
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
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

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