Structural and Mechanistic Basis of F227C-Mediated Hypersusceptibility to Islatravir in HIV-1 Reverse Transcriptase

Area of Science:

• Virology
• Structural Biology
• Medicinal Chemistry

Background:

• Islatravir (ISL; 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA)) is a novel nucleoside reverse transcriptase translocation inhibitor (NRTTI) for HIV-1 treatment.
• The F227C mutation in HIV-1 reverse transcriptase (RT) confers resistance to non-nucleoside RT inhibitors (NNRTIs) like doravirine (DOR).
• Unexpectedly, F227C enhances susceptibility to ISL, a phenomenon requiring mechanistic elucidation.

Purpose of the Study:

• To determine the structural basis for the hypersusceptibility of F227C mutant RT to ISL.
• To elucidate the biochemical mechanisms underlying this collateral sensitivity.
• To inform the design of resistance-informed combination antiretroviral therapies.

Main Methods:

• Determined a 1.8 angstrom crystal structure of F227C RT complexed with a primer/template and ISL-triphosphate.
• Performed complementary biochemical assays to assess ISL incorporation, RT translocation, and primer excision.

Main Results:

• The crystal structure revealed conformational changes in F227C RT affecting an allosteric cleft and ATP-mediated unblocking.
• Biochemical assays demonstrated that F227C mutation does not impede ISL incorporation but impairs RT translocation and ATP-dependent phosphorolytic excision of ISL-terminated primers.
• These alterations collectively enhance ISL susceptibility in the presence of the F227C mutation.

Conclusions:

• Established direct structural and mechanistic links between NNRTI resistance (F227C mutation) and ISL hypersusceptibility.
• Provided a structural foundation for developing combination HIV regimens that leverage collateral sensitivity to overcome NNRTI resistance.
• Highlights the potential of resistance-informed strategies for novel antiretroviral therapy design.