Structural characterization of HIV fusion inhibitor LP-98: Insights into antiviral and resistance mechanisms.

LP-98 is a lipopeptide-based HIV fusion inhibitor with exceptional potency and long-acting antiviral activity, currently in phase II clinical trials. In this study, we elucidated the structural basis of LP-98's antiviral activity and resistance mechanisms. Using AlphaFold3, we first predicted the six-helical bundle (6-HB) structure formed by LP-98 and the gp41-derived NHR peptide N44, identifying key residues mediating interhelical interactions. Subsequent crystallographic analysis of the LP-98/N44 complex confirmed these binding features, revealing that a cluster of hydrophobic residues in LP-98, along with a network of 15 hydrogen bonds, two electrostatic interactions and a salt bridge, critically stabilizes the 6-HB structure. Superposition analyses of the LP-98/N44 crystal structure with either the predicted 6-HB model or the LP-40/N44 crystal structure provided further mechanistic insights into LP-98's binding mode. Additionally, structural and functional characterization of the N-terminal Tyr-127 residue using a truncated variant (LP-98-Y) demonstrated its essential role in inhibitor binding and antiviral activity. Notably, LP-98 exhibited significantly reduced efficacy against T20-resistant HIV strains harboring single or double mutations in NHR. Our structural models shed light on the molecular basis of this resistance, offering critical insights for drug optimization. Collectively, these findings provide a detailed structural understanding of LP-98's antiviral mechanism, supporting its continued development as a promising next-generation HIV fusion inhibitor.