Azvudine (FNC), a next-generation cytidine analog with broad inhibitory activity against drug-resistant HIV-1 strains via stabilization of the reverse transcriptase catalytic complex.

Azvudine (FNC) is a 2'-β-fluoro-4'-azidocytidine analog being investigated as a next-generation nucleoside reverse transcriptase inhibitor (NRTI). To address the treatment failure and rapid resistance emergence posed by HIV-1 variants, we evaluated FNC's resistance profile and structural rationale. Using site-directed mutagenesis, a panel of clinically relevant HIV-1 RT mutants was constructed to assess FNC's resistance profile and mechanistic behavior. FNC exhibited sub-nanomolar potency (EC50 = 0.0812-0.1596 nM) across diverse HIV-1 subtypes, achieving up to 3000-fold higher antiviral activity than lamivudine (3TC) and emtricitabine (FTC). Unlike clinically used NRTIs, FNC retained or enhanced potency against most strains harboring key resistance mutations (K65R, Y115F, TAMs, Q151M, and T69 insertion mutations) except for M184V and M184I amino acid substitutions. FNC also showed superior intracellular persistence and, accordingly, reduced both the proportion of HIV-infected cells and the copy number of intact proviral DNA more effectively than 3TC and FTC. Furthermore, FNC showed more pronounced synergy in the common clinical combination therapies via replacing their nucleoside drugs. Structural modeling and molecular dynamics simulations suggest that the 2'-fluoro and 4'-azido substituents formed extra interactions with the catalytic site via hydrogen bonding, hydrophobic contacts, and Mg2+ coordination. These interactions provide a plausible structural basis for the retained potency against resistant mutants by stabilizing the catalytic complex, reducing excision, and impeding strand translocation. Collectively, these findings identify FNC as a structurally optimized cytidine analogue with broad activity against clinically relevant resistance variants, highlighting its potential as a promising candidate for next-generation HIV-1 therapy.