Biased agonists of the chemokine receptor CXCR3 differentially signal through Gαi:ß-arrestin complexes.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and signal through the proximal effectors, G proteins and ß-arrestins, to influence nearly every biological process. The G protein and ß-arrestin signaling pathways have largely been considered separable; however, direct interactions between Gα proteins and ß-arrestins have been described that appear to be part of a distinct GPCR signaling pathway. Within these complexes, Gαi/o, but not other Gα protein subtypes, directly interacts with ß-arrestin, regardless of the canonical Gα protein that is coupled to the GPCR. Here, we report that the endogenous biased chemokine agonists of CXCR3 (CXCL9, CXCL10, and CXCL11), together with two small-molecule biased agonists, differentially formed Gαi:ß-arrestin complexes. Formation of the Gαi:ß-arrestin complexes did not correlate well with either G protein activation or ß-arrestin recruitment. ß-arrestin biosensors demonstrated that ligands that promoted Gαi:ß-arrestin complex formation generated similar ß-arrestin conformations. We also found that Gαi:ß-arrestin complexes did not couple to the mitogen-activated protein kinase ERK, as is observed with other receptors such as the V2 vasopressin receptor, but did couple with the clathrin adaptor protein AP-2, which suggests context-dependent signaling by these complexes. These findings reinforce the notion that Gαi:ß-arrestin complex formation is a distinct GPCR signaling pathway and enhance our understanding of the spectrum of biased agonism.

Genome wide screen of RNAi molecules against SARS-CoV-2 creates a broadly potent prophylaxis.

Expanding the arsenal of prophylactic approaches against SARS-CoV-2 is of utmost importance, specifically those strategies that are resistant to antigenic drift in Spike. Here, we conducted a screen with over 16,000 RNAi triggers against the SARS-CoV-2 genome using a massively parallel assay to identify hyper-potent siRNAs. We selected 10 candidates for in vitro validation and found five siRNAs that exhibited hyper-potent activity with IC50<20pM and strong neutralisation in live virus experiments. We further enhanced the activity by combinatorial pairing of the siRNA candidates to develop siRNA cocktails and found that these cocktails are active against multiple types of variants of concern (VOC). We examined over 2,000 possible mutations to the siRNA target sites using saturation mutagenesis and identified broad protection against future variants. Finally, we demonstrated that intranasal administration of the siRNA cocktail effectively attenuates clinical signs and viral measures of disease in the Syrian hamster model. Our results pave the way to development of an additional layer of antiviral prophylaxis that is orthogonal to vaccines and monoclonal antibodies.