Design, synthesis, and biological evaluation of dual-site HIV-1 inhibitors simultaneously targeting the NNRTI binding pocket and an adjacent site with favorable resistance profiles

Given high mutation rate of HIV-1 and its impact on NNRTIs' efficacy, targeting novel binding pockets has emerged as a viable strategy to overcome the drug resistance. In this study, we designed and synthesized 26 novel "dual-site" HIV-1 inhibitors that simultaneously target two distinct sites on Reverse Transcriptase (RT): the classical non-nucleoside inhibitor binding pocket (NNIBP) and a newly identified NNRTIs-adjacent site (NNIAS). Antiviral evaluation demonstrated that all compounds possessed potent activity against the wild-type (WT) HIV-1 strain, with EC₅₀ values ranging from 4.1 to 86 nM. Notably, 11× maintained potent activity against WT and seven clinically relevant HIV-1 mutant strains. ELISA confirmed its significant inhibitory effect on HIV-1 RT. Molecular docking further revealed the dual-binding mode of 11× and provided a mechanistic rationale for its activity against both WT and resistant strains. Collectively, 11× represents a promising dual-site HIV-1 Inhibitor for further development.