京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13167号 / 論医博第2154号 / 新制||医||1029(附属図書館) / (主査)教授 宮本 享, 教授 小泉 昭夫, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13535号 / 論医博第2275号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 湊谷 謙司, 教授 小林 恭 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Sphingosine 1-phosphate (S1P) is a lipid chemoattractant molecule. Once formed, S1P can be transported extracellularly by S1P transporters spinster homolog 2 (Spns2) or major facilitator domain containing 2B (mfsd2b). In the extracellular space, S1P can bind to S1P-specific G-protein coupled receptors (S1PR), which initiate many signaling pathways. A critical role of extracellular S1P is its ability to cause lymphocyte egress, which can have implications for inflammatory and autoimmune diseases. For this reason, there has been a growing interest in exploring potential spns2 inhibitors to further elucidate their therapeutic potential. Initial screenings confirmed that fused heterocycles, including phthalimide and benzoxazoles, demonstrated moderate inhibition of Spns2 using a HeLa cell assay. An extensive structure-activity relationship (SAR) study of these scaffolds was performed to analyze the impact of various amine head groups, regioisomers, and alkyl tails on performance. It was determined that 2-aminobenzoxazoles with secondary amines were potent inhibitors of the transporter. Additionally, the position of the lipophilic tail moiety played a large role in activity. From these modifications, SLB1122168 (2.44p) was found to be our lead compound. It was determined that (2.44p) had an IC50 of 94 ± 6 nM and was shown to be efficacious in decreasing lymphocyte count by 55% in a dose-dependent manner in both rat and mice models. The discovery of (2.44p) can serve as a novel chemical tool to investigate Spns2 biology and use it as a probe to determine the potential of Spns2 as a drug target. Organoboron compounds are useful synthetic intermediates in forming C-X, C-C, and C-H bonds. One way to synthesize these compounds is through copper catalysis. Copper is favorable to other transition metals because it is an Earth-abundant, low-cost metal that can be utilized in regio- and stereoselective reactions. Conjugated 1,3-enynes are important functional groups that iii are found in active natural products, organic synthetic intermediates, and materials. Previous methods used rare transition metals, designer ligands, or harsh acidic conditions to synthesize such compounds. In this dissertation, we developed a stereoselective one-pot copper-catalyzed semi-reduction of 1,3-diynes to produce (Z)-1,3-enynes. This method uses Cu(OAc)2, HBpin and Xantphos to successfully synthesize (Z)-1,3-enynes that were tolerated well over a broad substrate scope, including heterocyclic, alkyl, and aryl substituents. It was determined that this reaction went through a 2-boryl intermediate which was facilitated by a CuH species. / Doctor of Philosophy / Autoimmune diseases are caused by immune cells attacking healthy cells. The signaling lipid sphingosine-1-phosphate (S1P) plays a major role in trafficking immune cells, in which immune cells follow the S1P gradient from low concentrations (secondary lymphoid tissues) to high concentrations (lymph). In the case of multiple sclerosis, immune cells can attack healthy neurons that cause a myriad of symptoms. Currently, there are four drugs approved by the Food and Drug Administration (FDA) targeting the S1P pathway for multiple sclerosis. In all cases, these drugs act as S1P-receptor (S1PR) functional antagonists, which decreases the amount of extracellular S1P, which in turn decreases the immune cells in the lymph that can attack healthy cells. Unfortunately, all four drugs exhibit on-target cardiovascular side effects. To circumvent the on-target side effects seen in current FDA-approved drugs, other nodes of the S1P pathway have been assessed for multiple sclerosis. One node of interest is spinster homolog 2 (Spns2), a transporter of S1P, whose inhibition has also been shown to decrease extracellular S1P. In this dissertation, we will be assessing various inhibitors for their in vitro and in vivo properties. 1,3-Enynes are a functional group found in medicinally relevant compounds and can be used as intermediates to make more complex compounds. Current methods to make this functional group use expensive rare metals or harsh acidic conditions. We developed new methods that utilized copper, an abundant metal, and boron, an atom whose empty p orbital allows for unique reactivity. Utilizing a copper-hydride species allowed us to semi-reduce 1,3-diynes to (Z)-1,3-enynes, where water was used instead of acid to allow for the semi-reduction to occur. This reaction was shown to tolerate a wide range of substrates and gave good to excellent yield.

Doctor of Philosophy / Autoimmune diseases are caused when a person's immune system attacks its own healthy cells. In a person with multiple sclerosis, their immune system becomes sensitized to the myelin sheath that covers their neurons in the central nervous system. This results in the degradation of the myelin sheath and irreversible degradation of the nerve cell axons. This damage leads to the development of several neurological impairments, such as pain, fatigue, mobility problems, and numbness. While there is no cure for multiple sclerosis, disease-modifying therapies are typically taken by patients to suppress their immune system and slow disease progression. Sphingsoine-1-phosphate (S1P) is a lipid that is important for the trafficking of lymphocytes into a person's central nervous system. This trafficking is largely due to the natural gradient of S1P which is high levels in blood but low in tissues. Lymphocytes will follow this gradient from areas of low S1P concentration (lymphatic tissue) to areas with higher S1P concentrations. Modulation of S1P levels is the mechanism of action for several FDA approved drugs as they target primarily S1P1 receptors to achieve lower levels of circulating lymphocytes. However, targeting this receptor also results in cardiovascular side effects such as first-dose bradycardia. The transporter for S1P, spinster homolog 2 (Spns2), which is upstream of the S1P receptors, is another viable target that our lab has recently been targeting. Spns2 inhibition decreases extracellular S1P levels and result in reduced lymphocytes in mice models. In this dissertation, several inhibitors were developed and assessed for their in vitro and in vivo ability to inhibit Spns2.

A variety of diseases including Alzheimer's disease, asthma, cancer, fibrosis, multiple sclerosis, and sickle cell disease have been associated with elevated levels of sphingosine-1-phosphate (S1P). S1P, a pleiotropic lipid mediator involved in a broad range of cellular processes, is synthesized solely by the phosphorylation of sphingosine (Sph) and is catalyzed by the two isoforms of sphingosine kinase (SphK1 and SphK2). Therefore, SphKs are a potential therapeutic target; however, the physiological role of SphK2 is still emerging. In order to determine the role of SphK2 in vivo, more potent and selective small molecule inhibitors of SphK2, as well as dual inhibitors are necessary. Herein, explorations and advancements on the second generation SphK2 selective inhibitor SLR080811 are disclosed. Investigations into the lipophilic tail region of the hSphK2 inhibitor SLR080811 are detailed. This investigation highlights the dependency of SphK2 selectivity and potency on overall compound length. More importantly, this study identified the internal aryl ring of SLR080811 as a key pharmacophore of the scaffold. To further probe the significance of the aromatic region, the phenyl ring was replaced by a 2,6-naphthyl ether skeleton. Investigations into the tail region of this scaffold are described in detail. Key discoveries from this structure-activity relationship study include SLC5111312 (hSphK2 Ki = 0.90 μM, dual hSphK inhibitor), SLC5091592 (hSphK2 Ki = 1.02 μM, > 20-fold hSphK2 selective) and SLC5121591 (hSphK2 Ki = 0.61 μM, >16-fold hSphK2 selective). Molecular modeling studies with hSphK2 indicate that the extended aromatic group is able to participate in π-π stacking interactions with Phe548. In silico docking studies indicate that a guanidine hydrogen bond to Asp211 is key for SphK2 selectivity, and incorporation of a 3'-hydroxyl group on the pyrrolidine ring increases hydrogen bonding to Asp308, thereby increasing SphK1 potency and reducing selectivity. Additionally, biological studies employing SLC5111312 have helped to further elucidate the role of SphK2, suggesting that SphK2 has a catalytic role in the regulation of blood S1P levels. The shape of the hSphK2 binding pocket was probed by introducing an indole moiety in place of the naphthyl ring and varying its substitution pattern. One key discovery from this study is SLC5101465 (hSphK2 Ki = 0.09 μM, > 111 fold SphK2 selective), which has a 1,5-indole substitution pattern with an N-nonyl "tail". Molecular docking simulations highlight the importance of rotatable bonds and a relatively linear orientation between the "head group" and "tail group" to maintain essential hydrogen bond interactions to Asp residues with the guanidine moiety while minimizing steric interactions in the middle of the binding pocket. Expanding upon the 1,5-indole scaffold of SLC5101465, a series of aryl tail derivatives are examined. This study confirms the necessity of electron withdrawing groups located at the end of the inhibitor scaffold to optimize binding in the tail region of the SphK2 binding pocket. / Ph. D.

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