Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

Reactive oxygen species (ROS) are generated by virally-infected cells however the physiological significance of ROS generated under these conditions is unclear. Here we show that inflammation and cell death induced by exposure of mice or cells to sources of ROS is not altered in the absence of canonical ROS-sensing pathways or known cell death pathways. ROS-induced cell death signaling involves interaction between the cellular ROS sensor and antioxidant factor KEAP1, the phosphatase PGAM5 and the proapoptotic factor AIFM1. Pgam5−/− mice show exacerbated lung inflammation and proinflammatory cytokines in an ozone exposure model. Similarly, challenge with influenza A virus leads to increased virus infiltration, lymphocytic bronchiolitis and reduced survival of Pgam5−/− mice. This pathway, which we term ‘oxeiptosis’, is a ROS-sensitive, caspase independent, non-inflammatory cell death pathway and is important to protect against inflammation induced by ROS or ROS-generating agents such as viral pathogens.

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SUMMARY The mitochondrial calcium uniporter complex is essential for calcium (Ca2+) uptake into mitochondria of all mammalian tissues, where it regulates bioenergetics, cell death, and Ca2+ signal transduction. Despite its involvement in several human diseases, we currently lack pharmacological agents for targeting uniporter activity. Here we introduce a high-throughput assay that selects for human MCU-specific small-molecule modulators in primary drug screens. Using isolated yeast mitochondria, reconstituted with human MCU, its essential regulator EMRE, and aequorin, and exploiting a D-lactate-and mannitol/sucrose-based bioenergetic shunt that greatly minimizes false-positive hits, we identify mitoxantrone out of more than 600 clinically approved drugs as a direct selective inhibitor of human MCU. We validate mitoxantrone in orthogonal mammalian cell-based assays, demonstrating that our screening approach is an effective and robust tool for MCU-specific drug discovery and, more generally, for the identification of compounds that target mitochondrial functions.