Sphingosine-1-phosphate (S1P) is an amino-alcohol signaling molecule produced from the intracellular phosphorylation of the lipid sphingosine. Despite possessing several identified intracellular targets, the predominant signaling functionality of S1P is derived from its activation of membrane-bound G-protein coupled receptors (GPCRs). The binding of S1P to these receptors (S1P1-5) is closely associated with immune cell development and recruitment. As such, the modulation of S1P-related pathways is of particular interest for the development of immunomodulating agents. To reach its native GPCRs, S1P must be released from the cell. This process is facilitated by the transmembrane transport protein Spinster homolog 2 (Spns2) in most vertebrates. Studies in murine species have demonstrated that the protein plays a key role in directing immune cell chemotaxis and the progression of autoimmune diseases. Consequently, Spns2 represents an attractive target for the pharmaceutical induction of immunosuppression. While several drugs that act through the modulation of S1P receptor signaling have received FDA approval for the treatment of autoimmune disorders (fingolimod, siponimod, ozanimod, and ponesimod), they typically manifest on-target cardiovascular side-effects. Therefore, the development of novel Spns2 inhibitors is a prudent alternative approach to achieve S1P-mediated lymphopenia. In this dissertation, the design, synthesis, and activities of highly potent Spns2 inhibitors are disclosed. These structures spanned several scaffolds and culminated in the discovery of a phenylurea derivative 4.11i. In vitro assessment of 4.11i demonstrated that the compound possessed an IC50 value of 92 nM, making it the most potent inhibitor of Spns2 disclosed to date. Intraperitoneal administration of 4.11i (10 mg/kg dose) into mice reduced circulating lymphocyte counts and impaired the progression of experimental autoimmune encephalomyelitis (a murine model of multiple sclerosis). Taken together, these data validated the target of 4.11i in vivo and represented the first reported instance of Spns2 inhibition as a viable multiple sclerosis treatment. Additional work is currently being undertaken to further improve in vivo activity and pharmacokinetic properties of 4.11i. / Doctor of Philosophy / White blood cells comprise a significant portion of the body's natural defense mechanisms. In healthy individuals, these white blood cells identify and destroy foreign materials and organisms. However, in patients with multiple sclerosis, immune cells can become sensitized to protein fragments lining the myelin sheath of neurons. These autoreactive immune cells recognize the body's natural neuronal proteins as antigens. Damage exerted by autoreactive cells leads to the development of neurological impairments (i.e., fatigue, muscle weakness, and slurred speech) as nerve impulses are disrupted before reaching their target. First-line treatment of multiple sclerosis often centers on the administration of immunosuppressive drugs to curtail the progression of the disease and mitigate immune cell-directed demyelination. A driving factor in white blood cell localization is the lipid sphingosine-1-phosphate (S1P). Concentrations of S1P are often not static in the body, with different tissue types and fluids possessing variable levels. Immune cells, and lymphocytes in particular, use this natural S1P gradient to dictate their movement within the body. Lymphocytes will track with the S1P gradient, going from areas of lower S1P concentration (lymph tissue) to areas of higher S1P concentration where synthetic enzyme expression is upregulated (multiple sclerosis lesions). Consequently, the development of drugs that can alter this S1P gradient represents an ideal avenue to achieve immunosuppression. One key mediator of S1P release is the transmembrane transport protein Spinster homolog 2 (Spns2). This protein directs the secretion of intracellular S1P into the extracellular space and is necessary for lymphocytes to enter circulation. However, little effort has been devoted to the development of Spns2 inhibitors. As such, the inhibition of this protein represents a novel and underexplored target for the treatment of autoimmune disorders. In this disclosure, the structures of several highly potent Spns2 inhibitors are revealed. The work around these structures led to the discovery of 4.11i. This compound proved highly potent in biological assays and animal models. Mice treated with 4.11i experienced a reduction in circulating lymphocyte counts and demonstrated less symptom manifestation in multiple sclerosis disease models.

In humans, mammals, and perhaps all vertebrates, sphingolipids exist as a family of cellular signaling molecules and have been shown to be involved in a wide range of biological processes ranging from proliferation to apoptosis. As such, sphingolipid signaling has garnered the attention of numerous researchers as an attractive candidate for pharmacological manipulation. The synthetic pathway of one prominent sphingolipid, sphingosine 1-phosphate (S1P), has been implicated in a variety of disease states such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Formation of S1P is facilitated from the ATP dependent phosphorylation of sphingosine (Sph) through its generative enzyme's sphingosine kinase 1 and 2 (SphK1 and SphK2). Inhibition of SphK1 and SphK2 results in the manipulation of S1P levels, which has been shown to be therapeutic in various animal models of disease. While there are multiple examples of potent SphK1-selective and dual SphK1/2 inhibitors, SphK2-selective inhibitors are scarce. Herein, we describe the design, synthesis and biological testing of SphK2-selective inhibitors. We first describe the discovery that introducing a trifluoromethyl group onto the internal aryl ring of our inhibitor scaffold led to superior selectivity and potency towards SphK2. We demonstrate that the trifluoromethyl moiety is interacting with a previously unknown side cavity in the substrate binding site of SphK2 that is unique and could be exploited in the design of SphK2-selective inhibitors. The synthesis of 21 derivatives with various substituents spanning off the internal aryl ring was completed, therefore characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site. This work led to the development of the most potent SphK2-selective inhibitor known at the time. We then describe the transformation of our SphK2-selective inhibitors into an orally bioavailable drug. We explain how the guanidine functionality on our inhibitor scaffold hinders our compounds from being orally bioavailable. Consequently, a library of 24 derivatives with various modifications to the guanidine functionality was synthesized and evaluated for improved orally bioavailability. Highlighted in this work is the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832), which displays a hSphK2 Ki of 82 nM and 122-fold selectivity for SphK2. Chemical modification and in vivo assessment of 3.14 (SLS1081832) prodrugs was explored. / Doctor of Philosophy / In humans, sphingosine 1-phosphate (S1P) is a signaling molecule that is generated through an ATP dependent reaction of sphingosine (Sph) via sphingosine kinase 1 and 2 (SphK1 and SphK2). Furthermore, S1P has been shown to be implicated in various diseases such as cancer, sickle cell disease, multiple sclerosis, and renal fibrosis. Inhibition of SphK1 and SphK2 has been shown to be therapeutic towards the symptoms of these diseases. Therefore, in order to alleviate these disorders, the concentrations of S1P must be controlled through pharmacological inhibition of SphK1 and SphK2. There are multiple reported examples of potent SphK1-selective and dual SphK1/2 inhibitors; however, SphK2-selective inhibitors are scarce. This work describes the synthesis and biological assessment of 21 compounds for their effectiveness in selectively targeting and inhibiting SphK2. The work led to the discovery of a previously unrecognized side cavity in the binding pocket of SphK2 that enhances inhibitor potency and selectivity towards SphK2. Furthermore, studies characterizing the preferred size and chemical nature of moieties positioned in that portion of the binding site led to the development of the most potent SphK2- selective inhibitor known at the time. Building on this work, we next focused on the transformation of our SphK2-selective inhibitors into a drug that could be administered orally. We describe the synthesis of 24 compounds with various modifications to one portion of our scaffold and their effect on improved orally bioavailability. This work led to the development of the most potent SphK2-selective inhibitor currently known 3.14 (SLS1081832).

L'objectif de cette thèse était de développer et de caractériser un modèle de cellules souches cardiaques humaines dans un contexte de thérapie cellulaire. Après avoir sélectionné et caractérisé une population de cellules souches d'origine mésenchymateuse, isolée à partir d'auricules humaines, exprimant le marqueur W8B2 (CSCs W8B2+), nous nous sommes focalisés (par les techniques de RT-qPCR à haut rendement, d'immuno-marquage, de western-blot et de fluorescence calcique) sur ; 1. la caractérisation génique des canaux ioniques et des acteurs de la signalisation calcique et 2. l'étude de leur différenciation in vitro en parallèle à l'activité calcique intracellulaire. Les résultats montrent que CSCs W8B2+ tendent à se différencier en cellules pacemaker. Certains gènes spécifiques nodaux, comme Tbx3, HCN, ICaT,L, Kv, NCX, s'expriment durant la différenciation. L'enregistrement de l'activité calcique (via une sonde optogénétique) montre la présence d'oscillations calciques qui évoluent en fréquence et en intensité pendant la différenciation. Les stocks-IP3 sensibles et l'échangeur NCX joueraient un rôle fondamental.Nous avons ensuite étudié l'importance du canal BKCa et des récepteurs sphingosine 1-phosphate (S1P) dans la régulation des propriétés fondamentales des CSCs W8B2+. L'inhibition du BKCa diminue la prolifération cellulaire en accumulant les cellules à la phase G0/G1, réprime l'auto-renouvellement mais n'affecte pas la migration. Quant à la S1P elle freine la prolifération et l'auto-renouvellement via une voie différente de celles des récepteurs S1P1,2,3.Ce travail fait ressortir des cibles moléculaires fondamentales dans un contexte de thérapie cellulaire cardiaque. / The aim of this thesis was to develop and characterize a model of human heart stem cells in a context of cell therapy.A population of mesenchymal stem cells, expressing the W8B2 marker (CSCs W8B2+), was first isolated from human auricles and characterized using high-throughput RT-qPCR techniques, immuno-labeling, western-blot and calcium fluorescence imaging. These experiments were focused on 1. the gene expression of ion channels and calcium signaling proteins; and 2. the study of CSCs W8B2+ in vitro differentiation and associated intracellular calcium activity changes.The results show that CSCs W8B2+ tend to differentiate into pacemaker cells. Some nodal specific genes such as Tbx3, HCN, ICaT, L, Kv, NCX, are expressed during differentiation. The recording of calcium activity (via an optogenetic probe) shows the presence of calcium oscillations that change in frequency and intensity during differentiation. IP3 sensitive calcium stocks and the NCX exchanger would play a fundamental role in these variations.Then we studied the importance of the BKCa channel and the sphingosine 1-phosphate (S1P) receptors in the regulation of the fundamental properties of the W8B2+ CSCs. Inhibition of BKCa reduces cell proliferation by accumulating cells in the G0 / G1 phase, suppresses cell self-renewal but does not affect migration properties. Concerning S1P, it decreases proliferation and self-renewal without stimulate S1P1,2,3 receptors.This work highlights fundamental potential molecular targets in a context of cardiac cell therapy.

Les adénocarcinomes rénaux à cellules claires (ccRCC), qui représentent 70% des tumeurs rénales, sont fortement mais irrégulièrement vascularisés, ce qui les rend hypoxiques et donc résistants aux chimiothérapies. L'hypoxie favorise l'agressivité tumorale via l'activation des facteurs de transcription HIF-1alpha et HIF-2alpha (Hypoxia-Inducible Factors). Pour cette raison, le ciblage de l'hypoxie intratumorale et des facteurs HIF dans les ccRCC constitue une stratégie thérapeutique pertinente. Dans ce projet, nous montrons pour la première fois que la voie sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) régule HIF-2alpha in vitro et in vivo. Nos résultats indiquent que la SphK1 régule le taux intracellulaire et l'activité transcriptionnelle de HIF-2alpha dans des lignées de ccRCC représentatives de certains sous-groupes retrouvés en clinique humaine ; et impliquent la S1P extracellulaire, via le récepteur S1P1, dans la régulation de HIF-1alpha et HIF-2alpha. D'autre part, nous avons évalué l'impact de l'inhibition des récepteurs à S1P et de la SphK1 par le FTY720 dans un modèle de ccRCC in vivo. Nos résultats indiquent que le FTY720 entraine une diminution transitoire du taux intratumoral de HIF-1alpha et HIF-2alpha ainsi qu'un remodelage du réseau vasculaire tumoral. En effet, le FTY720 induit une normalisation vasculaire qui aboutit à une oxygénation tumorale transitoire. Enfin, nous montrons que ce traitement permet de sensibiliser un modèle murin de ccRCC à la chimiothérapie. Ces résultats valident le rôle de la voie SphK1/S1P comme régulateur de l'adaptation à l'hypoxie dans les ccRCC. Ils constituent une étape indispensable à la transposition en clinique humaine du concept selon lequel la voie SphK1/S1P peut être ciblée afin de diminuer l'hypoxie intratumorale et de chimiosensibiliser certains cancers, le FTY720 étant déjà sur le marché. / Clear cell renal cell carcinomas (ccRCC) represent 70% of renal tumors. Because of their dense and irregular vascular network, ccRCC become hypoxic and therefore resistant to chemotherapies. Hypoxia promotes tumor aggressiveness via the activation of HIF-1alpha and HIF-2alpha (Hypoxia-Inducible Factors). For this reason, the control of intratumoral hypoxia and HIF in ccRCC could be a relevant therapeutic strategy to improve the efficacy of current treatments. In this study, we show for the first time that the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway regulates HIF-2alpha in vitro and in vivo. Our results indicate that SphK1 regulates HIF-2alpha intracellular level and transcriptional activity in ccRCC cell lines that are representative of some clinical ccRCC subgroups. Our data also involve extracellular S1P, via its receptor S1P1, in the regulation of HIF-1alpha and HIF-2alpha. In addition, in a ccRCC mouse model, we show that FTY720 - an inhibitor of the SphK1/S1P pathway- transiently decreases HIF-1alpha and HIF-2alpha intratumoral level. This is associated with a transient remodeling of the tumor vascular network indicating that FTY720 induces a vascular normalization that leads to transient tumor oxygenation. Finally, we show that this treatment sensitizes a ccRCC mouse model to chemotherapy. Overall, these results validate the key role of the SphK1/S1P pathway in the adaptation to hypoxia in ccRCC cell and animal models. Our results provide a mechanistic basis to target the SphK1/S1P pathway with FTY720 by increasing the efficacy of chemotherapy in ccRCC. They are a prerequisite for clinical transposition as FTY720 is a drug approved used in human clinic.

We have demonstrated that simvastatin and sphingosine 1-phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Kruppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, S1PR1, via a KLF2-dependent mechanism. S1PR1 luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 mu M), and S1PR1 and KLF2 protein expression detected by immunoblotting. In vivo, transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both S1PR1 and KLF2 gene expression (mRNA, protein) were significantly increased by simvastatin in vitro and in vivo. S1PR1 promoter activity was significantly increased by simvastatin (P < 0.05), which was significantly attenuated by KLF2 silencing (siRNA). Simvastatin induced KLF2 recruitment to the S1PR1 promoter, and consequently, significantly augmented the effects of the S1PR1 agonist on EC barrier enhancement (P < 0.05), which was significantly attenuated by KLF2 silencing (P < 0.05). These results suggest that simvastatin upregulates S1PR1 transcription and expression via the transcription factor KLF2, and consequently augments the effects of S1PR1 agonists on preserving vascular barrier integrity. These results may lead to novel combinatorial therapeutic strategies for lung inflammatory syndromes.

Chronic inflammation in the brain results in the development of several CNS diseases, including Alzheimer’s and Parkinson’s diseases, multiple sclerosis, and tumors. IL-1, a pro-inflammatory cytokine released by activated microglia and astrocytes, instigates the expression of factors promoting the progression of these CNS disorders, including cytokines, chemokines, and components of matrix remodeling systems, such as the plasminogen activator system. IL-1 also increases the mRNA expression and activity of SphK, the enzyme that phosphorylates Sph to form S1P, a bio-active sphingolipid. This thesis demonstrates that IL-1 and S1P enhance the mRNA and protein expression of PAI-1 and uPAR, two key components of the plasminogen activator system, in glioblastoma cells. The S1P-induced mRNA expression of PAI-1 and uPAR is mediated by the S1P2 receptor, and requires Rho-kinase and MEK1. However, IL-1 regulation of PAI-1 and uPAR mRNA expression is independent of SphK, and thus S1P. IL-1- and S1P-induced mRNA expression of PAI-1 and uPAR results in the increased in vitro invasion of glioblastoma cells. Since significant amounts of IL-1 are secreted from gliomas, and it increases the production of S1P via inciting the activity and mRNA expression of SphK, we propose a mechanism by which S1P and IL-1 influence the invasion of glioblastoma cells by increasing the mRNA and protein expression of uPAR and PAI-1. IL-1 and S1P also influence the mRNA expression of chemokines implicated in the development and progression of multiple sclerosis, namely IP-10 and RANTES, in primary human astrocytes. IP-10 and RANTES attract T cells, which are the major pathological cause of multiple sclerosis. This thesis demonstrates a novel mechanism by which S1P significantly inhibits the IL-1-induced mRNA expression of these chemokines. The mechanism by which S1P reduces IL-1-induced IP-10 and RANTES mRNA expression involves the prolonged hyperphosphorylation of TAK1, as well as the inhibition of IL-1-stimulated IFN beta production and the phosphorylation of STAT1 and STAT2. In summary, this dissertation describes the mechanisms by which S1P and IL-1 control the mRNA expression of two chemokines associated with multiple sclerosis, and the components of the plasminogen activator system, which are critical for the invasion of glioblastoma cells; thus, indicating future therapeutic targets for destructive CNS disorders.

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

Cholangiocarcinoma (CCA) is a fatal primary malignancy that is formed in the bile ducts. Cancer-associated myofibroblasts play a crucial role in CCA proliferation and invasion. Furthermore, there is a growing interest in the role of the exosome in the interaction between the cancer-associated myofibroblasts and cholangiocarcinoma which lead to CCA growth. However how cholangiocarcinoma-derived exosome affect the cancer-associated myofibroblasts in the tumor microenvironment remain unknown. In this study, we examined whether exosome produced by cholangiocarcinoma could involve in the prompt of CCA cells growth by regulation of myofibroblast. We found that cholangiocarcinoma-derived exosome could prompt elevated α-smooth muscle actin and stromal cell-derived factor one expression that induces myofibroblast proliferation. We then demonstrated that cholangiocarcinoma-derived exosome upregulated periostin expression that plays an important role in cancer metastasis. In 3D organotypic rat CCA coculture model, TCA and S1P considerably increase the growth of CCA cell. Conclusion: cholangiocarcinoma-derived exosome trigger cancer-associated myofibroblasts proliferation in the tumor microenvironment that leads to prompt CCA growth.

名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程） 学位授与年月日:平成20年3月25日

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells.