Summary Cytokine storm during viral infection is a prospective predictor of morbidity and mortality, yet the cellular sources remain undefined. Here, using genetic and chemical tools to probe functions of the S1P1 receptor, we elucidate cellular and signaling mechanisms important in initiating cytokine storm. While S1P1 receptor is expressed on endothelial cells and lymphocytes within lung tissue, S1P1 agonism suppresses cytokines and innate immune cell recruitment in wild-type and lymphocyte deficient mice, identifying endothelial cells as central regulators of cytokine storm. Furthermore, our data reveal immune cell infiltration and cytokine production as distinct events both orchestrated by endothelial cells. Moreover, we demonstrate that suppression of early innate immune responses through S1P1 signaling results in reduced mortality during infection with a human pathogenic strain of influenza virus. Modulation of endothelium with a specific agonist suggests that diseases where amplification of cytokine storm is a significant pathological component could be chemically tractable.

BACKGROUND AND PURPOSESphingosine1-phosphate (S1P) receptors mediate multiple events including lymphocyte trafficking, cardiac function, and endothelial barrier integrity. Stimulation of S1P 1 receptors sequesters lymphocyte subsets in peripheral lymphoid organs, preventing their trafficking to inflamed tissue sites, modulating immunity. Targeting S1P receptors for treating autoimmune disease has been established in clinical studies with the non-selective S1P modulator, FTY720 (fingolimod, Gilenya ™ ). The purpose of this study was to assess RPC1063 for its therapeutic utility in autoimmune diseases. EXPERIMENTAL APPROACHThe specificity and potency of RPC1063 (ozanimod) was evaluated for all five S1P receptors, and its effect on cell surface S1P 1 receptor expression, was characterized in vitro. The oral pharmacokinetic (PK) parameters and pharmacodynamic effects were established in rodents, and its activity in three models of autoimmune disease (experimental autoimmune encephalitis, 2,4,6-trinitrobenzenesulfonic acid colitis and CD4 + CD45RB hi T cell adoptive transfer colitis) was assessed. KEY RESULTSRPC1063 was specific for S1P 1 and S1P 5 receptors, induced S1P 1 receptor internalization and induced a reversible reduction in circulating B and CCR7 + T lymphocytes in vivo. RPC1063 showed high oral bioavailability and volume of distribution, and a circulatory half-life that supports once daily dosing. Oral RPC1063 reduced inflammation and disease parameters in all three autoimmune disease models. CONCLUSIONS AND IMPLICATIONSS1P receptor selectivity, favourable PK properties and efficacy in three distinct disease models supports the clinical development of RPC1063 for the treatment of relapsing multiple sclerosis and inflammatory bowel disease, differentiates RPC1063 from other S1P receptor agonists, and could result in improved safety outcomes in the clinic.

G protein-coupled receptors play a pivotal role in many physiological signaling pathways. Mounting evidence suggests that G protein-coupled receptors, including opioid receptors, form dimers, and dimerization is necessary for receptor maturation, signaling, and trafficking. However, the physiological role of dimerization in vivo has not been well-explored because of the lack of tools to study these dimers in endogenous systems. To address this problem, we previously generated antibodies to μ-δ opioid receptor (μOR-δOR) dimers and used them to study the pharmacology and signaling by this heteromer. We also showed that the heteromer exhibits restricted distribution in the brain and that its abundance is increased in response to chronic morphine administration. Thus, the μOR-δOR heteromer represents a potentially unique target for the development of therapeutics to treat pain. Here, we report the identification of compounds targeting μOR-δOR heteromers through high-throughput screening of a small-molecule library. These compounds exhibit activity in μOR-δOR cells but not μOR or δOR cells alone. Among them, CYM51010 was found to be a μOR-δOR–biased ligand, because its activity is blocked by the μOR-δOR heteromer antibody. Notably, systemic administration of CYM51010 induced antinociceptive activity similar to morphine, and chronic administration of CYM51010 resulted in lesser antinociceptive tolerance compared with morphine. Taken together, these results suggest that CYM51010, a μOR-δOR–biased ligand, could serve as a scaffold for the development of a unique type (heteromer-biased) of drug that is more potent and without the severe side effects associated with conventional clinical opioids.

The sphingosine-1-phosphate (S1P) receptor signaling system has biological and medical importance and is the first lipid G protein-coupled receptor (GPCR) structure to be solved to 2.8-Å resolution. S1P binds to five high-affinity GPCRs generating multiple downstream signals that play essential roles in vascular development and endothelial integrity, control of cardiac rhythm, and routine oral treatment of multiple sclerosis. Genetics, chemistry, and now structural biology have advanced this integrated biochemical system. The S1P receptors have a novel N-terminal fold that occludes access to the binding pocket from the extracellular environment as well as orthosteric and bitopic ligands with very different physicochemical properties. S1P receptors and metabolizing enzymes have been deleted, inducibly deleted, and knocked in as tagged or altered receptors in mice. An array of genetic models allows analysis of integrated receptor function in vivo. We can now directly understand causal relationships among protein expression, signal, and control points in physiology and pathology.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the U.S. National Institutes of Health launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines, but also highlight the need to innovate the science of therapeutic discovery.