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

Viral accessory proteins play critical roles in viral escape form host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, ORF10), strongly activates inflammasomedependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.This work was funded by the Spanish National Research Council (CSIC, project numbers CSIC-COV19-006, CSIC-COV-19-201, 202020E079 and 202320E187), the Catalan Agency for Management of University and Research Grants (AGAUR, 2020PANDE00048, 2021SGR1490, 2021SGR00350), the CSIC’s Global Health Platform (PTI Salud Global), The Networked Center for Biomedical Research in Liver and Digestive Diseases (CIBER-EHD), the Spanish Structures and Excellence María de Maeztu program (CEX2021-001202-M) and the European Commission-Next Generation EU (Regulation EU 2020/2094).N

Inflammasome activation by SARS-CoV-2 accessory protein: Development of novel inhibitors of the SARS-CoV-2 accessory protein ORF9b

1 p.-5 fig.SARS-CoV-2 can activate the inflammasome, which, when unbridled, contributes to pathogenic inflammatory responses and to severe COVID-19. Several SARS-CoV-2 components have been shown to participate in this activation. Here, we have systematically assayed SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7,ORF8, ORF9b, ORF9c and ORF10) for their ability to modulate inflammasome activity. We have found that among all accessory proteins, only ORF9b, a protein that locates in mitochondria, triggers a strong activation of caspase-1 activity and cytokine release in A549 lung epithelial cells and THP-1 monocyte-macrophage cells. This induction is observed both by transducing ORF9b alone or upon concomitant transduction of all accessory proteins. Based on the solved structure of ORF9b, we have conducted an in silico drug discovery effort to identify small molecules capable of disrupting the ORF9b homodimer and to attenuate its observed activity. Iterative steps of blind massive docking and molecular dynamics led to the identification of small molecules predicted to prevent ORF9b homodimeric interactions. This prediction was experimentally validated by means of surface plasmon resonance, yielding two active molecules. These molecules showed a potent inhibition of ORF9b-induced caspase-1 activation and cytokine release, and caused a remarkable eviction of ORF9b from mitochondria. These novel first-in-class ORF9b inhibitors are currently being tested for their ability to mitigate viral cytopathogenic effects.Peer reviewe