Rational design of dual-site NNRTIs based on 2,4,5-trisubstituted pyrimidine scaffold for potent inhibition of resistant HIV-1.

To overcome HIV-1 drug resistance, we developed a series of novel dual-site NNRTIs by introducing a rigid sp-hybridized alkyne linker, enabling simultaneous occupation of both the NNIBP and NNIAS of HIV-1 RT. Accordingly, 19 exhibited potent antiviral activity against the wild-type strain (EC50 = 4.9 nM) and retained strong activity against clinically relevant mutants, including K103 N (EC50 = 3.5 nM), E138K (EC50 = 7.8 nM), and the K103 N/Y181C double mutant (EC50 = 130 nM). Molecular dynamics simulations revealed stable occupancy of both binding sites, offering a structural basis for its potent antiviral activity. These findings highlight the potential of dual-site targeting for the development of next-generation HIV-1 NNRTIs.