The bioactive lipid mediator sphingosine-1-phosphate (S1P) has emerged as a key regulator of a variety of important physiological functions, including cell growth, cell survival, cell motility, angiogenesis, lymphocyte trafficking, and mast cell function. S1P is formed by two different sphingosine kinases (SphKs) and binds to a family of 5 differentially expressed G-protein coupled receptors (S1PRs). The majority of research to date has focused on the activation of these receptors, but there is compelling evidence to suggest that S1P exerts intracellular functions independent of S1PRs. However no bona fide intracellular targets of S1P have been identified. In my dissertation, I have identified a novel intracellular binding protein for S1P. This finding has important implications for the pleiotropic actions of S1P.

La Sphingosine 1-phosphate (S1P) est un lipide bioactif qui joue un rôle majeur dans de nombreux processus tels que le développement vasculaire, la circulation des cellules immunitaires et la régulation de l’intégrité vasculaire. L’absence de S1P circulante engendre une augmentation constitutive de la perméabilité vasculaire ainsi qu’une sensibilité accrue à l’anaphylaxie systémique passive, cependant l’implication respective des différentes sources de la S1P au cours de ces processus, demeure méconnue. Ceci est plus particulièrement vrai pour les plaquettes qui constituent une réserve importante de S1P. La S1P d’origine plaquettaire ne contribue pas au pool de S1P plasmatique, il n’est libéré que lors de l’activation plaquettaire. Nous avons étudié le rôle de la S1P d’origine plaquettaire au sein des vaisseaux sanguins, au cours du développement embryonnaire et chez des souris adultes, en présence ou en absence d’autres sources de S1P circulante, notamment des globules rouges. Par délétion des gènes codant pour les sphingosine kinases 1&2 au sein des megakaryocytes, nous avons généré des souris dont les plaquettes sont incapables de synthétiser et donc de sécréter la S1P. Ces plaquettes sont par ailleurs incapables, in vitro, de promouvoir le maintien de la barrière endothéliale. Néanmoins, une déficience en S1P d’origine plaquettaire n’augmente pas le risque d’hémorragie au cours du développement embryonnaire, dans un contexte d’inflammation, d’irradiation, ou de traumatisme ; cela suggère que la S1P d’origine plaquettaire n’est pas indispensable au cours de ces processus. Cependant, nous avons découvert que la résistance au choc anaphylactique est compromise en l’absence de S1P d’origine plaquettaire. L’aspirine bloquant la sécrétion de S1P par les plaquettes, un traitement à l’aspirine induit également une sensibilisation au choc anaphylactique de façon similaire à celle observée dans le cas d’une déficience en S1P d’origine plaquettaire. Il semblerait que dans ce contexte, la S1P d’origine plaquettaire joue plutôt un rôle pour promouvoir le tonus vasculaire via S1P2, que de maintenir l’intégrité vasculaire. / Sphingosine 1-phosphate (S1P) is a bioactive lipid that plays key roles in vascular development, immune cell trafficking and regulation of vascular integrity. Lack of circulating S1P leads to constitutive vascular leak and sensitivity to passive systemic anaphylaxis, but the relative roles of different sources of S1P in these processes is unclear. This is especially true for platelets, which carry large amounts of S1P but do not contribute S1P to plasma unless activated. We have addressed roles of platelet S1P in developing and mature murine vessels in the presence or absence of other circulating S1P sources, notably red blood cells. By deletion of the genes encoding sphingosine kinases 1&2 in megakaryocytes, we generated mice with platelets that were greatly impaired in their ability to secrete S1P. These platelets were also unable to promote endothelial barrier function in vitro. Nevertheless, platelet S1P deficiency did not sensitize to bleeding during embryonic development, or in association with inflammation, irradiation or traumatic injury, suggesting that platelet S1P is dispensable for these processes. Instead, we find that recovery from anaphylactic shock is impaired in the absence of platelet-derived S1P. Consistent with the ability of aspirin to block S1P release from platelets, it impaired recovery from anaphylactic shock to a similar degree as platelet S1P deficiency. Instead of protecting vascular integrity, platelet-derived S1P appeared to act via the promotion of vascular tone through S1P2 in this context. Collectively, we find that S1P is necessary for the capacity of platelets to promote endothelial barrier function in vitro, but that this capacity becomes redundant in the presence of red blood cell-derived S1P in vivo. Nevertheless, during anaphylactic shock, both sources of S1P are necessary for complete recovery. Blocking S1P release with aspirin, an over-the-counter anti-inflammatory drug, may impair the protective functions of platelet S1P.

Steroidogenic factor (SF1) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Recent studies have demonstrated that phospholipids are ligands for SF1. In the present study our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using mass spectrometry we show that in H295R adrenocortical cells SF1 is bound to sphingosine (SPH) under basal conditions and that cAMP stimulation decreases the amount of SPH bound to the receptor. We also show that silencing both acid and neutral ceramidases using siRNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. In vitro analysis of ligand binding using scintillation proximity assays show that several sphingolipids and phospholipids, including phosphatidic acid (PA), can compete with [3H]SPH for binding to SF1, suggesting that SF1 may have more than one ligand and binding specificity may change with the changes in intracellular fluxes of phospholipids. Further, phosphatidic acid (PA) induces SF1-dependent transcription of CYP17 reporter constructs. Inhibition of diacyglycerol kinase (DAGK) activity using R59949 and silencing DAGK- expression attenuates SF1-dependent CYP17 transcriptional. We propose that PA is an activating ligand for SF1 and that cAMP-stimulated activation of SF1 takes place by displacement of SPH.

Les surcharges en fer, qu’elles soient d’origine génétique ou secondaire, favorisent la baisse de densité minérale osseuse et par conséquent l’apparition d’une ostéoporose. Des liens entre surcharge en fer et perte osseuse ont pu être établis in vivo et in vitro, néanmoins les mécanismes mis en jeu, notamment sur la cellule ostéoblastique, restent incomplètement caractérisés. Notre objectif a donc été de préciser les mécanismes cellulaires conduisant à l’altération du phénotype et de l’activité ostéoblastique observée en présence d’un excès de fer. La réalisation préalable d’une étude transcriptomique sur la lignée ostéoblastique humaine MG-63 nous a permis d’identifier plusieurs gènes susceptibles de voir leur niveau d’ARNm régulé par le fer. Il a été fait l’hypothèse que ces différents gènes pouvaient être impliqués dans la survenue des pertes osseuses observées au cours des surcharges en fer. Ainsi l’expression du gène SPNS2, dont la protéine permet l’export de la Sphingosine-1-Phosphate (S1P), a été identifiée comme potentiellement induite par un excès de fer. Les relations entre l’expression du gène SPNS2 et un excès de fer ont ainsi été investiguées et les résultats obtenus ont mis en évidence une augmentation fer-dépendante de l’ARNm du gène SPNS2 dans la lignée MG-63, non retrouvée dans d’autres types cellulaires. Cette caractérisation nous a ainsi conduits à déterminer, dans la lignée MG-63, l’impact fonctionnel d’une exposition au fer sur l’export cellulaire de la S1P. Nous avons donc pour cela mis au point une méthode d’étude basée sur une stratégie « fluxomique » nous permettant d’évaluer l’efflux de la S1P au moyen d’un outil de spectrométrie de masse. Nos résultats objectivent une diminution des capacités de synthèse et d’export de la S1P en présence de fer et ceci malgré la surexpression du gène SPNS2. La diminution concomitante de l’expression du récepteur S1PR1 et du gène COL1A1 codant pour la chaîne α du collagène de type I suggère un impact fonctionnel de la baisse de concentration en S1P extracellulaire sur la cellule MG-63. La mise en évidence, dans un modèle ostéoblastique, d’une altération fer-dépendante de l’axe de signalisation S1P/S1PR ouvre de nouvelles perspectives quant à la compréhension des mécanismes mis en jeu lors des pertes osseuses associées aux surcharges en fer. / Osteoporosis may complicate genetic or secondary iron overload as reported in clinical and animal studies. However, the mechanisms leading to disrupted bone homeostasis are still to be fully elucidated. In vitro, iron exposure of both osteoblast and osteoclast cell models induces phenotypic and functional impairment, but the molecular mechanisms of iron excess on bone cell physiology are not well characterized, particularly in osteoblast. Our objective was to study the impact of iron overload on osteoblast biology and characterize the molecular mechanisms involved. Transcriptomic analysis previously performed by our group on MG-63 osteoblast-like cell-line to identify iron-modulated genes revealed that expression of SPNS2 gene, which encodes a transporter for the signaling lipid sphingosine 1-phosphate (S1P), is potentially induced by iron. The purpose of this work was to characterize the SPNS2 iron-related regulation and analyze its potential impact on S1P efflux and the S1P/S1PR signaling pathway in MG-63 cells. Our findings showed that iron exposure induces a dose-dependent increase of SPNS2 mRNA levels in MG-63 osteoblast-like cells that was not observed in hepatocyte and enterocyte cell models. We then performed a fluxomic assay on MG-63 cells to investigate iron potential impact on S1P efflux. Unexpectedly, our data showed that extracellular S1P levels were decreased in presence of iron excess and its associated SPNS2 upregulation. Furthermore, based on the observed iron associated S1PR1 and COL1A1 decrease, the defect in S1P export system seems to have functional consequence on MG-63 cells. These results suggest that iron may affect osteoblast S1P/SPR signaling and potentially alter a wide range of bone processes, thus participating in bone impairment in situations of chronic iron overload. These data open a new door for the understanding of mechanisms involved in iron-induced osteoporosis.

Sphingosine kinase (SphK) has emerged as an attractive target for cancer therapeutics due to its role in cell proliferation. SphK phosphorylates sphingosine to form sphingosine-1-phosphate (S1P) which has been implicated as a major player in cancer growth and survival. SphK exists as two different isoforms, namely SphK1 and SphK2, which play different roles inside the cell. The dearth of isoenzyme-selective inhibitors has been a stumbling block for probing the exact roles of these two isoforms in disease progression. This report documents our efforts in developing SphK2-selective inhibitors. We provide the first demonstration of a SphK inhibitor containing a quaternary ammonium salt. We developed highly potent and moderately selective inhibitors that were cell permeable and interfered with S1P signaling inside the cell. In an effort to improve the selectivity of our inhibitors and enhance their in vivo stability, we designed and synthesized second generation inhibitors containing a heteroaromatic linker and a guanidine headgroup. These inhibitors were more potent and selective towards SphK2 and affected S1P signaling in cell cultures and various animal models. / Ph. D.

Sphingosine 1-phosphate (S1P) has become a prevalent drug discovery target due to studies implicating it to several disease pathologies such as fibrosis, sickle cell disease, inflammation, diabetes, and cancer. S1P functions to induce cell proliferation and migration. S1P signaling occurs through intracellular targets or transport outside of the cell via ABC transporters, where it acts as a ligand to G-protein coupled receptors (S1P1-5). Sphingosine kinase (SphK) 1 and 2 phosphorylate sphingosine to S1P; these are the only enzymes known to mediate the phosphoryl transfer. Inhibiting either or both SphKs helps to modulate S1P, which may be useful as a therapeutic avenue for disease states where S1P signaling has gone awry. Herein, we document our efforts in profiling the structure-activity relationships (SAR) of SphK2 through an iterative process of synthesis and biological testing. First, an SAR structured around the head and linker region of our lead molecule, SLR080811, was performed. SLR080811 has a Ki of 1.3 µM and is 5-fold selective for SphK2. The modifications performed on SLR080811 yielded two promising inhibitors: SLP120701 (SphK2 selective with a Ki of 1.2 µM) and SLP7111228 (>200 fold selective for SphK1 with a Ki of 48 nM). In vitro studies in U937 cells yielded a decrease in S1P levels with the introduction of inhibitors. Mouse studies provided insight into the pharmacokinetic effect of our SphK2-selective inhibitors, revealing an increase in S1P levels in the blood. When in vivo studies were performed with the SphK1 selective inhibitor, S1P levels in blood decreased. These molecules provide the chemical biology tools to determine the effect of modulating S1P levels in vivo. We also focused our investigation on the tail region of the pharmacophore. From this study, SLM6031434 and SLM6041418 were discovered and both proved to be more potent and selective SphK2 inhibitors than SLR080811. SLM6031434 has a Ki of 370 nM and is 23-fold selective for SphK2. SLM6041418 has a Ki of 430 nM and is 24-fold selective for SphK2. Consistent with our previous observations, in vitro studies showed a decrease in S1P levels when inhibitor was introduced. Similarly, in vivo studies resulted in an increase of S1P levels in the blood. These compounds are positioned towards animal models of disease. / Master of Science

Influenza A virus (IAV) is generally associated with the seasonal malady that causes brief respiratory illness during the winter months, known simply as "the flu." Most otherwise healthy individuals will suffer from mild fever, congestion, headaches and myalgia that are resolved within 5-7 days of onset. However, there are nearly 500,000 influenza-related deaths that occur world-wide every year. Many of these casualties and patients hospitalized with influenza also test positive for bacterial pneumonia, the most common agent being Streptococcus pneumoniae. Although all individuals are subject to this viral:bacterial synergistic disease, the young, elderly, and immunocompromised are the most susceptible. Previous studies have shown that viral infection creates a prolonged hyper-responsive pro-inflammatory state in the lungs, which increases susceptibility to secondary bacterial infection. Lethality is due to detrimental pulmonary damage from a dysregulated host inflammatory response, known as the "cytokine storm." However, the nature of dual infection has not been well-studied in the elderly demographic. Therefore, we aim to better define this disease synergy in an aged mouse model and explore potential therapeutic alternatives that could be beneficial for the aged and other vulnerable populations. Sphingolipid modulation has emerged as a potential target to ameliorate the excessive inflammation (cytokine storm) elicited by highly pathogenic influenza. There is particular emphasis on sphingosine 1-phosphate (S1P) signaling, as well as control of intracellular S1P levels via sphingosine kinases (SK). Sphingolipids are involved in a multitude of cellular processes, and are tightly regulated by their metabolizing enzymes. We hypothesize that manipulation of sphingolipid signaling and alteration of the internal sphingolipid milieu will diminish the inflammatory response elicited by IAV infection. Using fluorescence-activated cell sorting (FACS), real-time PCR and cytometric bead array (CBA) analysis, we evaluated the immunomodulatory effects of systemic sphingosine analog treatment within the lung microenvironment under homeostatic and influenza-infected conditions. FTY720 treatment caused transient, but significant lymphopenia, influx of neutrophils and efflux of macrophages in the lungs, which was enhanced during a mild influenza infectionGene expression in the lungs was generally unaltered, but protein levels showed increases in specific influenza-induced cytokines, suggesting these treatments may have post-transcriptional effects on cytokine expression. To evaluate sphingolipid modulation in specific pulmonary cell types, we next observed the effects of these compounds and sphingosine kinase (SK) inhibitors in epithelial and alveolar macrophage-like cell lines. SK inhibitors and Enigmol demonstrated anti-viral effects in A549 cells, decreasing viral loads by up to 1.5 logs. Real-time PCR and CBA analysis further demonstrated that these effects were associated with alterations in key cytokine expression, including CCL2, CCL5, CXCL10, IL-6, and IL-8. Collectively, these findings indicate that therapeutic sphingolipid modulation has the potential for creating a protective microenvironment in the lungs that could alleviate or even prevent viral:bacterial synergistic disease. / Master of Science

Sphingosine 1-phosphate (S1P) is a cellular signaling molecule that has been implicated in a variety of diseases including cancer, fibrosis, Alzheimer's, and sickle cell disease. It is formed from the phosphorylation of sphingosine (Sph) by sphingosine kinase (SphK) and SphK exists as two isoforms-"SphK1 and SphK2, which differ with respect to their cellular activity and localization. As the key mediators in the synthesis of S1P, SphKs have attracted attention as viable targets for pharmaceutical inhibition. To validate their potential as therapeutic targets, we aimed to develop potent, selective, and in vivo active inhibitors of SphK. Herein, we describe the design, synthesis and biological evaluation of SphK2 inhibitors. We first describe the development of six SphK2 inhibitors that assess the utility of replacing lipophilic tail groups with heterocyclic rings. These six compounds demonstrate that the lipid binding pocket for SphK2 cannot accommodate compounds with tail groups that are conformationally restricted or positively charged. We then describe the development of aminothiazole-based analogues of an SphK1-selective inhibitor. A library of 37 aryl-substituted aminothiazole tail groups were synthesized, revealing a structure-activity relationship study that examines electronic effects on the aryl-substituted aminothiazoles and the effect of modifying the amino portion of the aminothiazole. These molecules show surprisingly good potency and selectivity for SphK2. In particular, we highlight 3.20dd (SLC4101431), a biphenyl aminothiazole that is the post potent and selective SphK2 inhibitor to date, with an SphK2 Ki of 90 nM and 100-fold selectivity for SphK2. This molecule's in vivo activity will also be discussed. Mitochondrial uncouplers are small molecules that shuttle protons from the inter membrane space to the mitochondrial matrix independent of ATP synthase, which disrupts oxidative phosphorylation and promotes increased nutrient metabolism for homeostasis to be maintained. Consequently, small molecule mitochondrial uncouplers have been pursued as probes for mitochondrial function and as potential therapeutics for the treatment of obesity and type 2 diabetes. Herein, we describe the design, synthesis, and biological evaluation of small molecule mitochondrial uncouplers. We report a library of 52 compounds that have good mitochondrial uncoupling activity over a wide therapeutic range, including 5.16t (SHC4111522) and 5.17i (SHC4091665), which have EC50 values of 0.63 uM and 1.53 uM, respectively, and achieve at least 2-fold increase in oxygen consumption rates relative to basal levels. With these molecules, we demonstrate that pKa and cLogP significantly contribute to uncoupling activity and must be accounted for when developing new generation small molecule mitochondrial uncouplers. / Ph. D.

Metastasis remains the largest contributor for ovarian cancer mortality. The five-year survival rate decreases dramatically as the disease advances from the primary tumor site to other organ sites within the peritoneal cavity. Thus, characterizing the mechanisms behind this metastatic potential may better elucidate the molecular mechanisms of ovarian cancer progression and may reveal novel targets for preventative and therapeutic treatments. Sphingosine-1-phosphate (S1P) is a critical secondary messenger responsible for many pro-cancer signals, e.g., proliferation, angiogenesis, inflammation, anti-apoptosis, and others. While S1P's role in the aggressive profile of many other cancers is well defined, its function in ovarian cancer development is less understood. The concentration of S1P is significantly increased in the ascites of women with malignant ovarian cancer, suggesting a role in ovarian cancer progression. This study aims to understand the importance of S1P in ovarian cancer metastasis. Using our well-characterized murine cell model for progressive ovarian cancer, we investigate the impact of S1P on ovarian cells and their interactions with the stromal vascular fraction recruited from the adipose tissue in culture conditions that mimic the physiologic environment of the peritoneal cavity. These studies will provide a mechanistic link of obesity, inflammation, and the increased risk of obese women to develop and die from ovarian cancer and identify signaling events as targets for interventions. / Master of Science

Excessive microvascular permeability is a serious complication involved in traumatic injury and inflammatory diseases. Alcohol intoxication can exacerbate the physiological derangements produced by microvascular endothelial barrier dysfunction in such disease conditions. Sphingosine-1-phosphate (S1P) has known endothelial barrier-protective properties, and has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and hemorrhagic shock and resuscitation (HSR). However, whether the barrier-protective properties of S1P extend to endothelial cells of the blood-brain barrier (BBB) is unclear. The mechanisms of S1P-induced barrier protection during alcohol intoxication or HSR are also unknown. In the current study, we tested the hypothesis that S1P could enhance endothelial barrier during alcohol intoxication or hemorrhagic shock by preserving the integrity of junction proteins and the endothelial glycocalyx, and protecting mitochondrial function. Cultured primary human brain microvascular endothelial cell (HBMEC) monolayers were used to characterize endothelial-specific mechanisms of S1P protection of the BBB during alcohol treatment. Transendothelial electrical resistance (TER) and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization was determined by immunofluorescence confocal microscopy. We also used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Cultured rat intestinal microvascular endothelial cell (RIMEC) monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that alcohol significantly impaired HBMEC TER and increased solute permeability, which was reversed with application of S1P after alcohol treatment. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization. Our in vivo results show that S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. Besides that, S1P protects RIMECs against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III. Our results indicate that S1P may be useful for restoring BBB function during alcohol intoxication. Moreover, S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to prevention of excess microvascular permeability.