Discovery of 3-arylpropionic acids as potent agonists of sphingosine-1-phosphate receptor-1 (S1P1) with high selectivity against all other known S1P receptor subtypes

A series of 3-arylpropionic acids were synthesized as S1P1 receptor agonists. Structure-activity relationship studies on the pendant phenyl ring revealed several structural features offering selectivity of S1P1 binding against S1P2-5. These highly selective S1P1 agonists induced peripheral blood lymphocyte lowering in mice and one of them was found to be efficacious in a rat skin transplantation model, supporting that S1P1 agonism is primarily responsible for the immunosuppressive efficacy observed in preclinical animal models.     

The Clinically-tested S1P Receptor Agonists,FTY720 and BAF312, Demonstrate Subtype-Specific Bradycardia (S1P1) and Hypertension (S1P3) in Rat

Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function in vivo. Indeed, FTY720 (non-selective S1P_X receptor agonist) produces modest hypertension in patients (2–3 mmHg in 1-yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question (e.g. bradycardia, hypertension), and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound in vitro and, in vivo, the cardiovascular effects of FTY720 and the more selective S1P_1,5 agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720 (0.1, 0.3, 1.0 mg/kg/20 min) or BAF312 (0.5, 1.5, 5.0 mg/kg/20 min) elicited acute bradycardia in anesthetized rats demonstrating an S1P₁ mediated mechanism-of-action. However, while FTY720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations (24-hr mean blood pressure = 8.4, 12.8, 16.2 mmHg above baseline vs. 3 mmHg in vehicle controls), BAF312 (0.3, 3.0, 30.0 mg/kg/d) had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P₃ receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P₁ receptors mediate bradycardia while hypertension is mediated by S1P₃ receptor activation.     

Highly selective and potent agonists of sphingosine-1-phosphate 1 (S1P1) receptor

Novel series of sphingosine-1-phosphate (S1P) receptor agonists were developed through a systematic SAR aimed to achieve high selectivity for a single member of the S1P family of receptors, S1P1. The optimized structure represents a highly S1P1-selective and efficacious agonist: S1P1/S1P2, S1P1/S1P3, S1P1/S1P4>10,000-fold, S1P1/S1P5>600-fold, while EC50 (S1P1) <0.2 nM. In vivo experiments are consistent with S1P1 receptor agonism alone being sufficient for achieving desired lymphocyte-lowering effect.     

Indole-propionic acid derivatives as potent, S1P3-sparing and EAE efficacious sphingosine-1-phosphate 1 (S1P1) receptor agonists

Novel indole-propionic acid derivatives were developed as sphingosine-1-phosphate (S1P) receptor agonists through a systematic SAR study. The optimized and S1P(3) selective S1P(1) agonist 9f induced peripheral blood lymphocyte reduction in vivo and has an excellent efficacy in mouse experimental autoimmune encephalomyelitis (EAE).     

Identification of benzoxazole analogs as novel, S1P(3) sparing S1P(1) agonists

A novel series of benzoxazole-derived S1P(1) agonists were designed based on scaffold hopping molecular design strategy combined with computational approaches. Extensive SAR studies led to the discovery of compound 17d as a selective S1P(1) agonist (over S1P(3)) with high CNS penetration and favorable DMPK properties. 17d also demonstrated in vivo pharmacological efficacy to reduce blood lymphocyte in mice after oral administration.     

Potent S1P receptor agonists replicate the pharmacologic actions of the novel immune modulator FTY720

Alteration in lymphocyte trafficking and prevention of graft rejection in rodents observed on exposure to FTY720 (1) or its corresponding phosphate ester 2 can be induced by the systemic administration of potent sphingosine-1-phosphate receptor agonists exemplified by 19. The similar S1P receptor profiles of 2 and 19 coupled with their comparable potency in vivo supports a connection between S1P receptor agonism and immunosuppressive efficacy.     

Synthesis of 4(5)-phenylimidazole-based analogues of sphingosine-1-phosphate and FTY720: discovery of potent S1P1 receptor agonists

The novel immunosuppressant FTY720 has been demonstrated to elicit immunomodulating effects via interaction with the G-protein coupled receptor S1P(1). FTY720 induced agonism at the S1P(3) receptor, however, has been shown to result in mild bradycardia, a minor side-effect of initial FTY720 therapy. This report describes the synthesis of several potent 4(5)-phenylimidazole-based S1P(1) receptor agonists that are accompanied by poor agonist activity at S1P(3). For instance, compound 20 displayed an EC(50)=4.7+/-1.3 nM at the S1P(1) receptor and EC(50)=780+/-1.3 nM at the S1P(3) receptor using a [gamma-(35)S]GTP-binding assay as compared to phospho-FTY720 (S1P(1): EC(50)=1.3+/-1.3nM, S1P(3): EC(50)=2.0+/-2.4 nM).     

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P₁R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P₁ receptor desensitization caused by synthetic S1P₁ receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P₁ receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P₁ receptors. In recombinant S1P₁ receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P₁ receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P₁ receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase – by facilitating S1P1 receptor recycling – is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.     

Mapping pathways downstream of sphingosine 1-phosphate subtype 1 by differential chemical perturbation and proteomics

Sphingosine 1-phosphate subtype 1 (S1P(1)) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P(1) down-regulation. Discovery of the in vivo active S1P(1)-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P(1) function while highlighting differences regarding agonist effect on S1P(1) fate, because SEW2871 does not degrade S1P(1). To further understand the mechanism of agonist-induced S1P(1) down-regulation, we compared signaling and fate of human S1P(1)-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P(1) to late endosomal vesicles and activated GTPgammaS(35) binding and pERK to similar maxima, only AFD-R led to significant S1P(1) down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P(1). All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3-4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P(1) and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P(1) (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.     

Prolonging Survival of Corneal Transplantation by Selective Sphingosine-1-Phosphate Receptor 1 Agonist

Corneal transplantation is the most used therapy for eye disorders. Although the cornea is somewhat an immune privileged organ, immune rejection is still the major problem that reduces the success rate. Therefore, effective chemical drugs that regulate immunoreactions are needed to improve the outcome of corneal transplantations. Here, a sphingosine-1-phosphate receptor 1 (S1P1) selective agonist was systematically evaluated in mouse allogeneic corneal transplantation and compared with the commonly used immunosuppressive agents. Compared with CsA and the non-selective sphingosine 1-phosphate (S1P) receptor agonist FTY720, the S1P1 selective agonist can prolong the survival corneal transplantation for more than 30 days with a low immune response. More importantly, the optimal dose of the S1P1 selective agonist was much less than non-selective S1P receptor agonist FTY720, which would reduce the dose-dependent toxicity in drug application. Then we analyzed the mechanisms of the selected S1P1 selective agonist on the immunosuppression. The results shown that the S1P1 selective agonist could regulate the distribution of the immune cells with less CD4+ T cells and enhanced Treg cells in the allograft, moreover the expression of anti-inflammatory cytokines TGF-β1 and IL-10 unregulated which can reduce the immunoreactions. These findings suggest that S1P1 selective agonist may be a more appropriate immunosuppressive compound to effectively prolong mouse allogeneic corneal grafts survival.     

Novel GPCR screening approach: indirect identification of S1P receptor agonists in antagonist screening using a calcium assay

To elucidate the physiological function of sphingosine 1-phosphate receptors 1-3 (S1P1-3) we aimed to identify selective ligands for these GPCRs. S1P2 and S1P3 are coupled to Gq, and are, therefore, linked to the phospholipase C/IP3/calcium pathway. S1P1 is solely coupled to Gi and was artificially linked to calcium signaling by coexpression of Galpha 16. The three receptors desensitized on challenge of cells with an agonist (i.e., agonists appeared as antagonists in a second calcium measurement). We screened a compound library for inhibitors of S1P-stimulated calcium signals, and we could identify agonists and antagonists with a single measurement. Agonism and antagonism were confirmed by recording compound-and S1P-induced calcium signals from the same assay well. For the three receptors, we found a reciprocal correlation of agonism and "apparent" antagonism of agonists. In addition, agonists indirectly discovered by this approach do not promote calcium mobilization through endogenous GPCRs.     

Synthesis of para-alkyl aryl amide analogues of sphingosine-1-phosphate: discovery of potent S1P receptor agonists

Sphingosine-1-phosphate (S1P) is a biologically active lysophospholipid with the capacity to induce a broad range of cellular responses via its interaction with the S1P family of G-protein coupled receptors. This report describes the synthesis of several potent S1P receptor agonists. For instance, compound 9c displayed an EC(50)=8.6 nM at the S1P(1) receptor using a [gamma-35S]GTP binding assay as compared to an EC(50)=4.5 nM for the endogenous ligand. We also report the effects associated with introduction of a phenyl ring between the 'linker' and 'lipophilic tail' regions of the analogues, for example total loss of activity at S1P(2) and increased agonism at S1P(5).     

Discovery of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P₁ receptor agonist with high selectivity against S1P₃ and enhanced efficacy in lowering peripheral lymphocyte counts in mice.     

Lymphopenia induced by a novel selective S1P1 antagonist structurally unrelated to S1P

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P₁) and participates in many pathological conditions. We developed a novel type S1P₁-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P₁ resulting in reduced signaling downstream of S1P₁, including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P₁-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P₁ antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P₁ agonists on lymphocyte sequestration results from their functional antagonism.     

ASP4058, a Novel Agonist for Sphingosine 1-Phosphate Receptors 1 and 5, Ameliorates Rodent Experimental Autoimmune Encephalomyelitis with a Favorable Safety Profile

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P₁–S1P₅). S1P₁ is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P₁. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P₁ and S1P₅. ASP4058 preferentially activates S1P₁ and S1P₅ compared with S1P_{2, 3, 4} in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.     

Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3

Dendritic cells (DCs) and lymphocytes are known to show a migratory response to the phospholipid mediator, sphingosine 1-phosphate (S1P). However, it is unclear whether the same S1P receptor subtype mediates the migration of lymphocytes and DCs toward S1P. In this study, we investigated the involvement of S1P receptor subtypes in S1P-induced migration of CD4 T cells and bone marrow-derived DCs in mice. A potent S1P receptor agonist, the (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P], at 0.1 nM or higher and a selective S1P receptor type 1 (S1P(1)) agonist, SEW2871, at 0.1 muM or higher induced a dose-dependent down-regulation of S1P(1). The pretreatment with these compounds resulted in a significant inhibition of mouse CD4 T cell migration toward S1P. Thus, it is revealed that CD4 T cell migration toward S1P is highly dependent on S1P(1). Mature DCs, when compared with CD4 T cells or immature DCs, expressed a relatively higher level of S1P(3) mRNA. S1P at 10-1000 nM induced a marked migration and significantly enhanced the endocytosis of FITC-dextran in mature but not immature DCs. Pretreatment with (S)-FTY720-P at 0.1 microM or higher resulted in a significant inhibition of S1P-induced migration and endocytosis in mature DCs, whereas SEW2871 up to 100 microM did not show any clear effect. Moreover, we found that S1P-induced migration and endocytosis were at an extremely low level in mature DCs prepared from S1P(3)-knockout mice. These results indicate that S1P regulates migration and endocytosis of murine mature DCs via S1P(3) but not S1P(1).     

Discovery,design and synthesis of a selective S1P3 receptor allosteric agonist

Potent and selective S1P₃ receptor (S1P₃-R) agonists may represent important proof-of-principle tools used to clarify the receptor biological function and assess the therapeutic potential of the S1P₃-R in cardiovascular, inflammatory and pulmonary diseases. N,N-Dicyclohexyl-5-propylisoxazole-3-carboxamide was identified by a high-throughput screening of MLSMR library as a promising S1P₃-R agonist. Rational chemical modifications of the hit allowed the identification of N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide, a S1P₃-R agonist endowed with submicromolar activity and exquisite selectivity over the remaining S1P_1,2,4,5-R family members. A combination of ligand competition, site-directed mutagenesis and molecular modeling studies showed that the N,N-dicyclohexyl-5-cyclopropylisoxazole-3-carboxamide is an allosteric agonist and binds to the S1P₃-R in a manner that does not disrupt the S1P₃-R–S1P binding. The lead molecule herein disclosed constitutes a valuable pharmacological tool to explore the molecular basis of the receptor function, and provides the bases for further rational design of more potent and drug-like S1P₃-R allosteric agonists.     

Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.     

Discovery of CS-2100, a potent, orally active and S1P3-sparing S1P1 agonist

S1P(3)-sparing S1P(1) agonists have attracted attention as a suppressant of autoimmunity with reduced side effects. Our synthetic efforts and extensive SAR studies led to the discovery of 10b named CS-2100 with the EC(50) value of 4.0 nM for human S1P(1) and over 5000-fold selectivity against S1P(3). The in vivo immunosuppressive efficacy was evaluated in rats on host versus graft reaction and the ID(50) value was determined at 0.407mg/kg. The docking studies of CS-2100 with the homology model of S1P(1) and S1P(3) showed that the ethyl group on the thiophene ring of CS-2100 was sterically hindered by Phe263 in S1P(3), not in the case of Leu276 in S1P(1). This observation gives an explanation for the excellent S1P(3)-sparing characteristic of CS-2100.     

Actions of a picomolar short-acting S1P₁ agonist in S1P₁-eGFP knock-in mice

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.