Second generation S1P receptor modulators: Research strategies and clinical developments

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds

Transcriptional regulation and functional characterization of the oxysterol / EBI2 system in primary human macrophages

Oxysterols such as 7 alpha, 25-dihydoxycholesterol (7a,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein–coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses [7;10]. Activation of EBI2 by specific oxysterols leads to directed cell migration of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adapted immune response is established by a stromal cells subset [13], here we investigate the involvement of the oxysterol / EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes necessary for ligand production such as cholesterol 25-hydroxylase (CH25H) and oxysterol 7alpha-hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharide (LPS) triggers a strong continuous up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Activation of EBI2 expressed on macrophages leads to release of intracellular calcium and to directed cell migration. Supernatants of LPS-stimulated macrophages show an autocrine activation of EBI2 indicating that an induction of CH25H and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is the first study characterizing the oxysterol / EBI2 pathway in primary human cells. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity

An oral S1P1 antagonist prodrug with efficacy in vivo: discovery, synthesis and evaluation

A prodrug approach to optimize the oral exposure of an S1P1 antagonist for chronic efficacy studies led to the discovery of (S)-2-{[3'-(4-Chloro-2,5-dimethyl-benzenesulfonylamino)-3,5-dimethyl-biphenyl-4-carbonyl]-methyl-amino}-4-dimethylamino-butyric acid methyl ester (BVM924). Due to the steric hindrance and the partial double bond character of the amide group and the resulting large rotational barrier around the amide bond two conformers of (BVM924) can be detected in solution and their equilibration was investigated by UPLC and 1H NMR. Methyl ester prodrug (BVM924) is hydrolyzed in vivo to the corresponding carboxylic acid (BVS819), a potent and selective S1P1 antagonist. Oral administration of the prodrug (BVM924) induces sustained peripheral lymphocyte depletion in rats. In a rat cardiac transplantation model co-administration of a nonefficacious dose of prodrug (BVM924) with a nonefficacious dose of sotrastaurin (AEB071), a protein kinase C inhibitor, or everolimus (RAD001), an mTOR inhibitor, effectively prolonged the survival time of rat cardiac allografts. This demonstrates that clinically useful immunomodulation mediated by the S1P1 receptor can be achieved with an S1P1 antagonist generated in vivo after oral administration of its prodrug

The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6

Ablation of the cellular prion protein PrPC leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrPC prevents the disease, suggesting that PrPC acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrPC -deficient mice is reduced, suggesting that PrPC acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrPC triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC -derived peptide (FT 23-50) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT 23-50 and the equivalent type-IV collagen peptide. We conclude that PrPC promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrPC, these observations are relevant to the pathogenesis of demyelinating polyneuropathies - common debilitating diseases for which there are limited therapeutic options

A Simple and Efficient CRISPR Technique for Protein Tagging

Genetic knock-in using homology-directed repair is an inefficient process, requiring the selection of few modified cells and hindering its application to primary cells. Here, we describe Homology independent gene Tagging (HiTag), a method to tag a protein of interest by CRISPR in up to 66% of transfected cells with one single electroporation. The technique has proven effective in various cell types and can be used to knock in a fluorescent protein for live cell imaging, to modify the cellular location of a target protein and to monitor the levels of a protein of interest by a luciferase assay in primary cells

Gai2 Signaling Promotes Skeletal Muscle Hypertrophy, Myoblast Differentiation, and Muscle Regeneration

Skeletal muscle atrophy results in loss of strength and an increased risk of mortality. We found that lysophosphatidic acid, which activates a G protein (heterotrimeric guanine nucleotide–binding protein)– coupled receptor, stimulated skeletal muscle hypertrophy through activation of Gai2. Expression of a constitutively active mutant of Gai2 stimulated myotube growth and differentiation, effects that required the transcription factor NFAT (nuclear factor of activated T cells) and protein kinase C. In addition, expression of the constitutively active Gai2 mutant inhibited atrophy caused by the cachectic cytokine TNFa (tumor necrosis factor–a) by blocking an increase in the abundance of the mRNA encoding the E3 ubiquitin ligase MuRF1 (muscle ring finger 1). Gai2 activation also enhanced muscle regeneration and caused a switch to oxidative fibers. Our study thus identifies a pathway that promotes skeletal muscle hypertrophy and differentiation and demonstrates that Gai2-induced signaling can act as a counterbalance to MuRF1-mediated atrophy, indicating that receptors that act through Gai2 might represent potential targets for preventing skeletal muscle wasting

UTS2B defines a novel enteroendocrine cell population and regulates GLP-1 secretion through SSTR5 in male mice.

The gut-pancreas axis plays a key role in the regulation of glucose homeostasis and may be therapeutically exploited to treat not only type II diabetes, but also hypoglycemia and hyperinsulinemia. We identify a novel enteroendocrine cell type expressing the peptide hormone urotensin 2B (UTS2B). UTS2B inhibits glucagon like peptide-1 (GLP-1) secretion in mouse intestinal crypts and organoids, not by signaling through its cognate receptor UTS2R but through the activation of the somatostatin receptor 5 (SSTR5). Circulating UTS2B concentrations in mice are physiologically regulated during starvation, further linking this peptide hormone to metabolism. Furthermore, administration of UTS2B to starved mice demonstrates that it is capable of regulating blood glucose and plasma concentrations of GLP-1 and insulin in vivo. Altogether, our results identify a novel cellular source of UTS2B in the gut, which acts in a paracrine manner to regulate GLP-1 secretion through SSTR5.These findings uncover a fine-tuning mechanism mediated by a ligand-receptor pair in the regulation of gut hormone secretion, which can be potentially exploited to correct metabolic unbalance caused by over-activation of the gut-pancreas axis

An Oral Sphingosine 1‑Phosphate Receptor 1 (S1P₁) Antagonist Prodrug with Efficacy in Vivo: Discovery, Synthesis, and Evaluation

A prodrug approach to optimize the oral exposure of a series of sphingosine 1-phosphate receptor 1 (S1P₁) antagonists for chronic efficacy studies led to the discovery of (<i>S</i>)-2-{[3′-(4-chloro-2,5-dimethylphenylsulfonylamino)-3,5-dimethylbiphenyl-4-carbonyl]­methylamino}-4-dimethylaminobutyric acid methyl ester <b>14</b>. Methyl ester prodrug <b>14</b> is hydrolyzed in vivo to the corresponding carboxylic acid <b>15</b>, a potent and selective S1P₁ antagonist. Oral administration of the prodrug <b>14</b> induces sustained peripheral blood lymphocyte reduction in rats. In a rat cardiac transplantation model coadministration of a nonefficacious dose of prodrug <b>14</b> with a nonefficacious dose of sotrastaurin (<b>19</b>), a protein kinase C inhibitor, or everolimus (<b>20</b>), an mTOR inhibitor, effectively prolonged the survival time of rat cardiac allografts. This demonstrates that clinically useful immunomodulation mediated by the S1P₁ receptor can be achieved with an S1P₁ antagonist generated in vivo after oral administration of its prodrug

The identification of the C3a Receptor (C3AR1) as the target of the VGF derived peptide TLQP-21 in rodent cells

TLQP-21, a peptide derived from VGF (non-acronymic) by proteolytic processing, has been shown to modulate energy metabolism, differentiation and cellular response to stress. Although extensively investigated, the receptor for this endogenous peptide has not previously been described. This report describes the use of a series of studies that show G protein-coupled receptor (GPCR)-mediated biological activity of TLQP-21as well as signalling in CHO-K1 cells. Unbiased genome wide sequencing of the transcriptome of the responsive CHO-K1 cells identified a priority list of possible GPCRs bringing about this activity. Using the biological activity generated by this peptide the possible targets of this ligand were tested by screening a series of defined receptor antagonists as well as siRNAs to inhibit expression of these putative receptors. The results of these studies are all consistent with the receptor of the TLQP-21 peptide in CHO-K1 cells being C3AR1. In addition the sensitivity of TLQP-21 signalling to pertussis toxin is also consistent with what is known about the signalling pathway of this receptor. We further demonstrate that the binding of TLQP-21 to the C3AR1 goes beyond signalling as we show thatTLQP-21 has a migratory role in RAW264.7 mouse cell

A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia

GPR15 is an orphan G protein-coupled receptor (GPCR) that is found in lymphocytes. It functions as a co-receptor of simian immunodeficiency virus and HIV-2 and plays a role in the trafficking of T cells to the lamina propria in the colon and to the skin. We describe the purification from porcine colonic tissue extracts of an agonistic ligand for GPR15 and its functional characterization. In humans, this ligand, which we named GPR15L, is encoded by the gene C10ORF99 and has some features similar to the CC family of chemokines. GPR15L was found in some human and mouse epithelia exposed to the environment, such as the colon and skin. In humans, GPR15L was also abundant in the cervix. In skin, GPR15L was readily detected after immunologic challenge and in human disease, for example, in psoriatic lesions. Allotransplantation of skin from Gpr15l-deficient mice onto wild-type mice resulted in substantial graft protection, suggesting nonredundant roles for GPR15 and GPR15L in the generation of effector T cell responses. Together, these data identify a receptor-ligand pair that is required for immune homeostasis at epithelia and whose modulation may represent an alternative approach to treating conditions affecting the skin such as psoriasis