Journal: bioRxiv
Article Title: A macrocyclic peptide-based fusion inhibitor targeting SARS-CoV-2 Spike S2 subunit
doi: 10.64898/2026.03.04.703990
Figure Lengend Snippet: (A) Binding of PA-001 to recombinant SARS-CoV-2 Spike protein of the full length (S1+S2), S1, or S2 regions. Each recombinant Spike protein was immobilized and incubated with HA-tagged PA-001 at various concentrations up to 100 nM, followed by washing out and detection with horseradish peroxidase (HRP)-conjugated anti-HA antibody. (B) Schematic model for the SARS-CoV-2 entry process, in which viral attachment to cells via Spike-ACE2 binding is followed by the fusion of viral envelope with cellular membrane either on the cell surface or in the endosome/lysosome. (C) (a) Virus-cell attachment assay. VeroE6/TMPRSS2 cells were exposed to SARS-CoV-2 at an MOI of 0.01 at 4°C for 15 min in the presence or absence of casirivimab (CAS) + imdevimab (IMD) (50, 500, or 5,000 ng/mL), mefloquine (MFQ) (0.4, 2, or 10 μM), or PA-001 (40, 200, or 1,000 nM), and were then washed out to quantify cell-bound viral RNA by real-time RT-PCR. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons with the DMSO control was performed (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001). (b) Cell-cell fusion assay. Spike-overexpressing HEK293T cells carrying the LgBiT gene were co-cultured with ACE2-expressing VeroE6/TMPRSS2 cells carrying the HiBiT gene upon treatment with or without varying concentrations (up to 200 nM) of PA-001 for 1 h to detect luciferase activity to evaluate fusion activity mediated by Spike and ACE2. IC 50 of PA-001 is also indicated. (D) Induction of PA-001-resistant SARS-CoV-2 variants. (a) Schematic representation of the infection assay for inducing PA-001-resistant SARS-CoV-2 clones. SARS-CoV-2-infected VeroE6/TMPRSS2 cells were continuously cultured with PA-001 at 1,000 nM for 24-48 h (Passage 0). The resultant culture supernatants were collected and diluted to 10-fold, which were then inoculated to naive VeroE6/TMPRSS2 cells in the presence of 1,000 nM PA-001 and cultured for 24 h (Passage 1). The culture supernatants were repeatedly collected and then inoculated to the cells upon 1,000 nM PA-001 for the next passage. At passage 7, the culture supernatants were extracted from eight independent cultures (A8 – H8). (b) PA-001 sensitivity of the collected supernatant clones. VeroE6/TMPRSS2 cells were inoculated with these collected supernatants or the parental virus (WT) upon treatment with varying concentrations of PA-001 (left) or RDV as a reference (right) to determine virus RNA levels in the culture supernatants as shown in . (E) Amino acid sequence of the PA-001-resistant clones. (a) Upper, schematic structure for the SARS-CoV-2 Spike protein. Lower, amino acid sequence of the clones, A8–H8, as well as the parental virus (WT). The substituted amino acids are highlighted by red. The amino acid numbers are indicated. (b) Structure of Spike trimer in the pre-fusion (left, PDB: 6XR8), intermediation (center, PDB: 8Z7P), and post-fusion (right, PDB: 8FDW) forms. Red color indicates the mutated amino acids shown in (a). Blue color indicates FP. (F) Binding capacity of recombinant SARS-CoV-2 Spike carrying the L841R and P862Q substitutions to PA-001. (a) Each recombinant Spike (WT, L841R, and P862Q) was immobilized and incubated with HA-tagged PA-001 (PA-001-HA) at varying concentrations in the presence (gray, competition) or absence (red, S-PA001 binding) of non-tagged PA-001 at 10 μM. After washing out, bound HA-PA-001 was visualized with HRP-conjugated anti-HA antibody. Samples without Spike-immobilization were also prepared to define the background signal (green). (b) Binding signals with Spike WT (left), Spike L841R (center), and Spike P862Q (right) are plotted against the concentrations of treated PA-001-HA. Ordinary two-way ANOVA followed by tukey’s multiple comparisons was performed (*: p<0.05, **: p<0.01, ***: p<0.001). (G) Cell fusion assay performed as described in using the cells overexpressing either Spike WT, L841R, or P862Q co-cultured with ACE2-expressing cells upon PA-001 treatment at various concentrations.
Article Snippet: Binding of HA-tagged PA-001 to SARS-CoV-2 Spike proteins was quantified by ELISA as follows: Recombinant wild type (from Sino biological, 40589-V08B1, 40591-V08B1, and 40590-V08B) or mutated Spike proteins were immobilized on the plates, and then incubated with PA-001-HA at various concentrations together with or without excess amount of non-tagged PA-001, used as a binding competitor.
Techniques: Binding Assay, Recombinant, Incubation, Membrane, Virus, Cell Attachment Assay, Quantitative RT-PCR, Control, Cell-Cell Fusion Assay, Cell Culture, Expressing, Luciferase, Activity Assay, Infection, Clone Assay, Sequencing, Cell Fusion Assay
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