Journal: bioRxiv

Article Title: Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

doi: 10.1101/2024.05.31.596900

Figure Lengend Snippet: A . Transient expression of ORF9b strongly induces caspase-1 activity in A549 cells. Parental A549 cells (A549wt) were transduced with lentiviral particles for the expression of the indicated accessory proteins and released caspase-1 activity determined 48 h after transduction, under basal conditions or after exposure to LPS+ATP for 3 h. pLex, transduction of lentiviral particles bearing the control vector pLex. B . Stable constitutive expression of ORF9b strongly induces caspase-1 activity in A549 lung carcinoma cells. A549 cells stably integrating and constitutively expressing the indicated SARS-CoV-2 accessory proteins were assayed for specific released caspase-1 activity under basal conditions or after exposure to LPS (100 ng/mL) and ATP (5 mM) for 3 h. pLex, A549 integrating the lentiviral vector pLex with no SARS-CoV-2 insert. C . A549 cells with stable integration and expression of ORF9b (A549.ORF9b) were transduced with lentiviruses for the expression of the indicated ORFs and released caspase-1 activity determined 48 h after transduction, under basal conditions or after exposure to LPS+ATP for 3h. pLex, transduction of lentiviral particles bearing the control vector pLex. D . The induction of caspase-1 activity by ORF9b is dominant among all accessory proteins when concomitantly expressed in A549wt or A549.ORF9b cells. The concomitant expression of all accessory proteins abrogates the LPS+ATP-induced additive induction of caspase- 1 activity to that induced by ORF9b alone. Parental A549wt or A549.ORF9b cells were transduced with equal titers of lentiviral particles for each accessory protein and assayed for released caspase-1 activity after 48 h. pLex, transduction of lentiviral particles bearing the control vector pLex. E . The induction of caspase-1 activity by the concomitant expression of all SARS-CoV-2 accessory proteins in A549wt cells, without or with additional boosting by LPS+ATP, is dependent on the NLRP3 inflammasome. Parental A549wt cells were transduced with equal titers of lentiviral particles for each accessory protein and assayed for released caspase-1 activity after 48 h. Cells were treated with the NLRP3 inflammasome inhibitor MCC950 for the last 3 h or vehicle (control).

Article Snippet: Recombinant SARS-CoV-2 GST-ORF9b protein (BPS Bioscience) was immobilized on a Sensor Chip CM5 channel (Cytiva).

Techniques: Expressing, Activity Assay, Transduction, Control, Plasmid Preparation, Stable Transfection

Journal: bioRxiv

Article Title: Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

doi: 10.1101/2024.05.31.596900

Figure Lengend Snippet: A, Surface plasmon resonance (SPR) determinations of ORF9b homodimerization surface-binding hit Compounds on ORF9b-ORF9b interactions. Recombinant GST-ORF9b protein was immobilized on CM5 chips and interactions with mobile-phase GST-ORF9b Compounds recorded by SPR, in the absence (vehicle) or presence of the indicated hit Compounds at 5 μM final concentration. Determinations were performed in triplicate. B, Structures and ribbon models of the two most experimentally active Compounds in inhibition of ORF9b homodimerization interaction, FECBL01015_1 (EN300) and EOS1228_1 (Y600). For the ribbon rendering, the most relevant residues for on ORF9b for its interaction with each Compound are illustrated, all located on the homodimerization surface.

Article Snippet: Recombinant SARS-CoV-2 GST-ORF9b protein (BPS Bioscience) was immobilized on a Sensor Chip CM5 channel (Cytiva).

Techniques: SPR Assay, Binding Assay, Recombinant, Concentration Assay, Inhibition

Journal: bioRxiv

Article Title: Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

doi: 10.1101/2024.05.31.596900

Figure Lengend Snippet: A , Effect of Compounds EN300, Y600 and Y203 on of caspase-1 activity induced by ORF9b in A549 lung epithelial cells. B , Effect of Compounds EN300, Y600 and Y203 on of caspase-1 activity induced by ORF9b in THP-1 lung epithelial cells. C , Compound EN300 induces mitochondrial eviction of ORF9b in A549.ORF9b cells.

Article Snippet: Recombinant SARS-CoV-2 GST-ORF9b protein (BPS Bioscience) was immobilized on a Sensor Chip CM5 channel (Cytiva).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

doi: 10.1101/2024.05.31.596900

Figure Lengend Snippet: A, Structures, ribbon models and molecular dynamics determinations for four EN300-similar compounds, in interaction with the ORF9b homodimer. A . 2D structure of the four purchased compounds similar to the EN300 initial hit. Predicted binding free energy using MMGBSA approach, as an average over the last 10 ns of molecular dynamics. B . A 3D representation of the predicted binding mode of the compounds with the ORF9B homodimer. C . Evolution of the binding free energy, obtained with the MMGBSA methodology, along the time during the molecular dynamics.

Article Snippet: Recombinant SARS-CoV-2 GST-ORF9b protein (BPS Bioscience) was immobilized on a Sensor Chip CM5 channel (Cytiva).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

doi: 10.1101/2024.05.31.596900

Figure Lengend Snippet: A , Compounds 2, 4, 5, 6 and 7, but not Compounds 3 and 8, inhibit the activation of caspase-1 induced by ORF9b in A549 lung epithelial cells at low micromolar concentrations. B , Compounds 2, 4, 5, 6 and 7, but not Compounds 3 and 8, inhibit the activation of caspase-1 induced by ORF9b in THP-1 monocyte-macrophage cells at low micromolar concentrations. C , Compounds EN300, 2 and 6 inhibit the secretion of the indicated cytokines induced by ORF9b in THP-1 monocyte-macrophage cells. Compound 3 also inhibits de secretion of several cytokines induced by ORF9b, except IL-1α. The NLRP3 inflammasome inhibitor, MCC950, inhibits the ORF9-induced secretion of cytokines in THP-1 cells, except IL-1α. D , Compounds 2, 4, 6 and 7, but not Compound 3, induce a loss of mitochondrial localization of ORF9b in A549.ORF9b cells.

Article Snippet: Recombinant SARS-CoV-2 GST-ORF9b protein (BPS Bioscience) was immobilized on a Sensor Chip CM5 channel (Cytiva).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

doi: 10.1101/2024.05.31.596900

Figure Lengend Snippet: A , The ORF9b dimerization inhibitors Compound 1 (C1), Compound 2 (C2) and Compound 6 (C6), but not Compound 3 (C3), restore the IFN-β-induced expression of OAS3 and ISG15 in A549.ORF9b cells. pLex, A549 cells stably transduced with the control vector pLex. The Y axis corresponds to fold-change values of RT-PCR ΔΔCt values for the indicated transcripts. B , The ORF9b dimerization inhibitors Compound 1 (C1), Compound 2 (C2) and Compound 6 (C6), but not Compound 3 (C3), restore the IFN-β-induced expression of OAS3 and ISG15 in THP-1 cells transiently transduced with lentiviral particles for the expression of ORF9b or with control lentiviral particles (pLex). The Y axis corresponds to fold-change values of RT-PCR ΔΔCt values for the indicated transcripts. C , The ORF9b dimerization inhibitors Compound 1 (C1) and Compound 2 (C2), but not Compound 6 (C6), upregulate IFN-I/III response genes in A549.hACE cells upon infection with SARS-CoV-2. Left panel, histogram of Log 2 FC of RT-PCR ΔΔCt values for the indicated transcripts, in triplicate samples. Right panel, row-normalized heatmap of average RT-PCR ΔΔCt values for the indicated transcripts.

Article Snippet: Recombinant SARS-CoV-2 GST-ORF9b protein (BPS Bioscience) was immobilized on a Sensor Chip CM5 channel (Cytiva).

Techniques: Expressing, Stable Transfection, Transduction, Control, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Infection