Novel dual-functional peptides designed via NanoBiT spike pseudovirus system for real-time monitoring and inhibition of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates viral infection by binding its surface spike protein to the human angiotensin-converting enzyme 2 (hACE2) receptor. Precise delineation of spike-hACE2 engagement is essential for viral entry and a prime target for therapeutic intervention. However, the current antiviral strategies provided only endpoint readouts and delayed the prioritization against emerging variants. Here, this study introduced novel antiviral compounds designed via a NanoLuc Binary Technology-based pseudovirus [NanoBiT; a structural complementation reporter composed of a Large BiT (LgBiT) and a Small BiT (SmBiT)]. Upon Omicron BA.2-SmBiT spike pseudoviruses infecting LgBiT-hACE2 cells, reconstituted NanoLuc generated quantifiable bioluminescence for real-time spike-hACE2 interaction during viral infection. Within this framework, dual-functional theranostic Anti-spike peptide (S7; ACTPHVSPTHCS) and the Anti-hACE2 peptide (A6; WSTDPGAHLRDY) were identified that not only inhibit SARS-CoV-2 entry by targeting spike and hACE2 proteins, but also serve as diagnostic probes for real-time monitoring. Under optimized conditions, significant inhibition of virus infection was validated in both Anti-spike peptide-treated pseudovirus and Anti-hACE2 peptide-treated hACE2 cells, with a synergistic score of 17.092. Cross-variant efficacy extended to the Omicron JN.1 lineage using a newly constructed JN.1-SmBiT pseudovirus, with molecular docking supporting binding at conserved residues. LgBiT-hACE2 transgenic mice and noninvasive bioluminescence imaging verified in vivo suppression and demonstrated enhanced inhibition with combined treatment. This programmable peptide-NanoBiT framework provided multifunctional compounds that integrate therapeutic efficacy with diagnostic capability. Importantly, the modular design highlights its adaptability to other virus-receptor interactions to underscore its potential in pandemic preparedness.