Journal: Scientific Reports

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1038/s41598-025-31755-y

Figure Lengend Snippet: X-ray crystal structure of the PDZ2 domain of ZO-1 bound to the SARS-CoV-2 E protein PBM . ( A ) Ribbon diagram of the final crystal structure model of the ZO-1 PDZ2 domain in complex with the SARS-CoV-2 E protein PBM peptide. The structure forms a swapped dimer, with one monomer colored light blue and the other dark blue. The bound peptides are shown in orange. Secondary structure elements are labeled according to the canonical PDZ domain scheme. ( B ) Close-up view of the SARS-CoV-2 E protein PBM bound within the PDZ domain. Key interacting residues are shown as sticks and labeled. Intermolecular hydrogen bonds and polar interactions are indicated by cyan dashed lines.

Article Snippet: The pGEX-2T plasmid encoding amino acids 186–262 of human ZO-1 was obtained from Addgene (59735, Addgene ) .

Techniques: Labeling

Journal: Scientific Reports

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1038/s41598-025-31755-y

Figure Lengend Snippet: HTRF-based screening of compounds targeting the interaction between ZO-1 PDZ2 and the SARS-CoV-2 E protein PBM. The assay was conducted in 96-well, white, low-volume HTRF plates using 5 µL of purified GST-ZO-1-PDZ2 at a final concentration of 3 nM, 5 µL of purified His 6 -Ecyto at a final concentration of 60 nM, and 1 µL of each of the 1000 compounds from a focused-PPI chemical library. The histogram shows the % residual HTRF ratio. Among the 1,000 compounds screened, only the 36 compounds with % residual HTRF ratio below 30% were classified as primary hits (C1 to C36). Horizontal lines indicate the mean with SD ( n = 2).

Article Snippet: The pGEX-2T plasmid encoding amino acids 186–262 of human ZO-1 was obtained from Addgene (59735, Addgene ) .

Techniques: Purification, Concentration Assay

Journal: Scientific Reports

Article Title: HTRF-based identification of small molecules targeting SARS-CoV-2 E protein interaction with ZO-1 PDZ2

doi: 10.1038/s41598-025-31755-y

Figure Lengend Snippet: Characterization of C19, a promising antiviral compound. ( A ) Chemical structure of C19, generated using ChemDraw software. ( B,C ) Docking poses of C19 targeting the ZO-1 PDZ2 swapped dimer. Panels show the best predicted binding poses of C19 when docked to the ZO-1 PDZ2 dimer either in its free form ( B ) or in complex with the E protein PBM ( C ), using DOCK6 simulations. The PDZ domains are shown in cartoon representation. The compound is positioned within key binding regions of the ZO-1 PDZ2 dimer, preferentially occupying the PBM-binding site. ( D ) HTRF inhibition dose-response curve of C19. The assay was performed in 96-well, white, low-volume HTRF plates with 5 µL of purified GST-ZO-1-PDZ2 (final concentration: 3 nM), 5 µL of purified His₆-Ecyto (final concentration: 60 nM), and 1 µL of C19, tested in a 10-point, twofold serial dilution (100 µM to 195 nM). The resulting curve represents the percentage of residual interaction (%R) based on the normalized FRET signal. A sigmoidal curve was generated using nonlinear regression in Prism software, plotting C19 concentration against the normalized response ( n = 1). ( E,F ) Antiviral activity of C19. The assay was conducted in 96-well culture plates using Vero-E6 cells (40,000 cells/well) infected with a NanoLuciferase-expressing recombinant SARS-CoV-2 virus at an MOI of 0.001. Cells were incubated for 72 h in the presence of C19, applied as a 9-point, twofold serial dilution ranging from 20 µM to 78 nM. Viral replication was assessed by measuring luminescence in the cell culture supernatant using the Nano-Glo assay kit ( E ). Data were normalized to both infected untreated and uninfected untreated control conditions. Sigmoidal curve was generated using nonlinear regression in Prism software, plotting C19 concentration against the normalized luminescence signal ( n =3). Viral quantification was performed using a plaque assay on Vero-E6 cells ( F ). Plaques were observed at 3 days post-infection (dpi) following infection with serial dilutions of supernatants collected from infected non-treated (INT) or infected treated (IT) cells, treated for 72 h with 20 µM of compound C19. Viral titers are reported as plaque-forming units per milliliter (PFU/mL). Horizontal lines represent the mean ± SEM, and dashed lines indicate the limit of detection ( n =3; nd=not detected). Statistical significance was determined by a t -test ( p < 0.005).

Article Snippet: The pGEX-2T plasmid encoding amino acids 186–262 of human ZO-1 was obtained from Addgene (59735, Addgene ) .

Techniques: Generated, Software, Binding Assay, Inhibition, Purification, Concentration Assay, Serial Dilution, Activity Assay, Infection, Expressing, Recombinant, Virus, Incubation, Cell Culture, Glo Assay, Control, Plaque Assay