Identification of Small Molecule Modulators of the RIG-I and MDA5 Pathways

Abstract

The vertebrate immune system consists of cytosolic receptors that recognize a wide range of viral nucleic acids during an infection and elicit a robust antiviral response to clear the infection. Retinoic acid-inducible gene-I (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5) are two such pattern recognition receptors (PRRs) that stimulate a type I interferon (IFN) response against a variety of double stranded ribonucleic acid (dsRNA). While the proper functioning of these receptors is crucial for curbing viral egress, mis-regulation of MDA5 or RIG-I can result in auto-inflammatory and autoimmune conditions such as Systemic Lupus Erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and Singleton-Merten syndrome (SMS). Previous research suggests that the ATPase active site of these receptors acts as an allosteric modulation site, playing a role in altering the confirmation of RIG-I and MDA5 and thereby affecting their signaling activity. In fact, our lab has examined several mutations in the ATPase site that result in loss of the ATPase activity. While the dsRNA binding capabilities remained intact, these mutants displayed significant changes in signaling activity. Half of these mutant proteins showed a hyper-signaling phenotype, while the other half displayed a complete loss of signaling. This finding has been our rationale for targeting the ATPase site with small molecule intervention. In this study, we have used a biochemical and functional assay to screen for thousands of small molecules to identify potential hits that inhibit the ATP hydrolysis capabilities of RIG-I and/or MDA5. These hits were then further characterized using cell-signaling and cell-free assays to delve into the mechanism of action. More specifically, these assays served to identify whether downstream mediators in the RIG-I and MDA5 pathways were inhibited or activated by the compounds. Through these assays, we have triaged several candidates that selectively modulate the activity of RIG-I and/or MDA5. The identification and confirmation of such compounds greatly increases our understanding of the biochemistry of antiviral immunity and raises the potential for treatment of a broad range of immune disorders.