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Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

ABSTRACT: The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

SUBMITTER: Sanna MG

PROVIDER: S-EPMC4702096 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-(2,6-dichloro-4-pyridinyl)-2-[1,3-dimethyl-4-(1-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability.

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Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

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