A highly potent, stable, and safe dePEGylated lipopeptide against Nipah virus and related henipaviruses.

Nipah virus (NiV) poses a significant threat owing to its high mortality and the lack of approved therapeutics. Targeting the conserved heptad repeat 1 (HR1) domain of the viral fusion (F) protein constitutes a promising antiviral strategy. Also, lipopeptides derived from the human parainfluenza virus 3 (HPIV3) heptad repeat 2 (HR2) regions inhibit NiV fusion by blocking formation of the critical six-helix bundle (6-HB); however, their efficacy has been impeded by the controversial use of PEGylation. To resolve these limitations, we employed our proprietary heptad repeat 2 C-terminal fragment (HR2-CF) peptide displacement strategy that eliminates PEG and overcomes the resultant steric hindrance. The lead dePEGylated lipopeptide, VQ-P1-C16, exhibited antiviral activity comparable to that of PEGylated lipopeptide, VIKI-PEG4-C16. Further introducing Glu (E) or Lys (K) mutations yielded VQ-P1-EK3-C16, which exhibited ultra-potent pseudotyped henipavirus inhibition at pM concentrations and increased fusion inhibition. Meanwhile, it protected newborn mice from pseudotyped NiV-Malaysia and NiV-Bangladesh infection in lungs and brains. Structural simulations and mechanistic studies demonstrated that VQ-P1-EK3-C16 adopts a helical conformation, exhibits high affinity for NiV-HR1, and inhibits NiV 6-HB formation. Moreover, VQ-P1-EK3-C16 showed significantly increased solubility, improved thermal stability, strengthened resistance to proteases, and extended serum half-life. These findings establish VQ-P1-EK3-C16 as a highly potent, stable, and safe fusion inhibitor with promising potential for the clinical development of therapeutics against NiV and related henipaviruses.