Kinetic Insights into the Enhanced Antiviral Activity of Islatravir against Doravirine Resistance-Associated Substitution Mutations in HIV-1 Reverse Transcriptase

Islatravir (ISL, EFdA) is a nucleoside analog that inhibits HIV-1 reverse transcriptase (RT) translocation during viral replication. Its high potency stems from unique structural features: a 4'-ethynyl group that interacts with the hydrophobic pocket (containing A114, Y115, F160, M184, and D185) in HIV-1 RT, hindering translocation, and a 3'-hydroxyl group that mimics natural nucleosides for efficient incorporation. Recent phase 3 clinical trials, combining ISL with Doravirine (DOR), a non-nucleoside reverse transcriptase inhibitor, show that it is noninferior to existing treatments, offering a unique advantage due to their distinct resistance profiles. For instance, DOR-associated mutations, V106I/F227C, which confer >105-fold DOR resistance in clinics, unexpectedly boost ISL's potency by 2.3-fold in published cell-based resistance selection assays. In contrast, V106I alone does not affect Islatravir's potency, while F227C alone enhances it by 5.6-fold. To kinetically understand these findings, we used presteady-state kinetic assays to determine the kinetic parameters for EFdA 5'-triphosphate (EFdA-TP) and dATP incorporation. We found that the incorporation efficiency of EFdA-TP was 1.4-fold higher than that of dATP on an RNA template and 1.7-fold higher on a DNA template with the F227C mutant. However, this difference was only 1.1- to 1.3-fold higher with the F227C/V106I mutant. Our energy-minimized modeling revealed that these mutations remotely alter the hydrophobic 4'-ethynyl group-binding pocket's structure, surprisingly strengthening the pocket's binding interactions with EFdA-TP. Alongside this, the F227C mutation decreased dATP's binding affinity with both templates. Our data established a kinetic basis for the published cell-based resistance selection assay results, underscoring the significant potential of the ISL/DOR combination therapy in treating HIV-1 infected patients.