PROJECT SUMMARY
Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) efficiently suppress HIV and serve as backbone
of antiretroviral therapies. However, new therapeutics are needed for continued control of HIV infection. 4’-
Ethynyl-2-fluoro-2’-deoxyadenosine (EFdA) is an NRTI with exceptional potency, stability, and unique
mechanism of action against HIV, which has been licensed by Merck. EFdA (also known as MK-8591 or Islatavir)
has been successfully used in Phase I and recently introduced in Phase II clinical trials. EFdA has generated
“compelling early results for both treatment and prevention” in patients, tested at remarkably low doses (up to
>10,000-fold lower doses than current NRTI drugs) aiming at once-weekly oral and once-yearly slow-release
dosing, unprecedented modalities for HIV therapies. Hence, this work is directly relevant to NIH guidelines for
high priority AIDS funding (NOT-OD-15-137) for “next generation therapies…that are longer acting.”
We have shown that a) EFdA has high potency in vitro, in cell culture (EC50=50 pM in PBMCs), mice and non-
human primate animal models. Although EFdA retains the 3’-OH, it blocks HIV reverse transcriptase (RT) in
vitro, primarily as an immediate/obligate and at times delayed chain terminator due to difficulty of EFdA-
terminated viral DNA to translocate. Thus, EFdA is termed a nucleotide reverse transcriptase translocation
inhibitor (NRTTI). However, the inhibition mechanism of EFdA in HIV-infected cells (primary or cell lines) remains
unknown. Toward that end, Co-I Malim has established an innovative assay that enables study of the EFdA
antiviral mechanism in HIV-infected cells. Additionally, in vitro passage experiments have identified mutations
that impart EFdA resistance through two paradigm-shifting dual mechanisms or resistance: decreased
incorporation/enhanced excision and decreased incorporation/enhanced translocation. Data on an EFdA analog
suggest efficient inhibition of EFdA-resistant HIV, although the mechanism of this phenomenon is not
understood. Notably, treatment with key new generation NNRTI doravirine (DOR) leads to resistance mutations
that impart hypersusceptibility to EFdA, paving the way for EFdA/DOR combination therapies. We hypothesize
that the structural attributes of EFdA and its analogs impact interactions with diverse RTs (wild-type, drug-
resistant, from diverse clades, viruses), leading to clinically important differences in efficiency of inhibition,
resistance, hypersusceptibility, and biochemical mechanism of action. This hypothesis will be tested through
three specific aims, which endeavor to answer the above questions using a combination of novel sequencing,
biochemical, structural, and virological approaches. This work will help optimize combination therapies that
reduce drug burden and have exceptional long-acting potential, addressing adherence challenges of chronic HIV
treatment.