The cryopreservation of cells and tissues requires the addition of a cryoprotectant in order to prevent cellular damage caused by ice. Unfortunately, common cryoprotectants such as DMSO and glycerol exhibit significant toxicity which makes their use unfeasible for many clinical procedures. Our laboratory is interested in the development of alternative, non-toxic cryoprotectants which possess ice recrystallization inhibition (IRI) activity. Potent IRI activity has recently been discovered in certain small molecules, but the structural features required for this process are unclear. Herein we report the development of a library of O-aryl glycosides in order to probe the importance of electron density and hydrophobic moieties for IRI activity. It was found that the degree of electron density at the anomeric oxygen does not correlate with IRI ability in para-substituted aryl glycosides, nor does changing the position of the aryl substituent impart a predictable effect on activity. However, the addition of hydrophobic alkyl or acyl chains was beneficial for IRI activity; generally, increasing chain length was found to correlate with increasing activity. In some instances, an optimal alkyl chain length was identified, after which continued lengthening results in a loss of potency. We conclude from this study that a certain extent of hydrophobic character is beneficial for the IRI activity of aryl glycosides, and that a balance between hydrophobicity and hydrophilicity is required for optimum IRI ability. It is hoped that these findings will aid future efforts towards the rational design of novel cryoprotectants.

Prior work in the Durst group had generated more than forty analogs of the potent anticonvulsant isoxylitone isolated isolated from a medicinal plant Delphinium denudatum Wall. The nitrile designated as TD532 was the most potent compound generated by A. Saikaley. The starting material for the synthesis of TD532 is isophorone. The observation that TD532 showed considerable potential as an anticonvulsant suggested that other cyclohexenones might have have similar activity. During this project close to fifty derivatives of cyclohex-2-enone, focusing mainly on 3-arylcylohex-2-enones, were prepared. The synthesis of these compounds is described and structure activity relationships are discussed. Based on all the available structure activity data, we have designated the indicated portion of structure A as the pharmacophore for anticonvulsant and anti-epileptic activity. The ester designated as TD561 (compound 40) showed excellent potential in both in vitro and in vivo assays. It has been shown to be a pro-drug of the corresponding acid TD562 (compound 48). These two compounds and the sodium salt of TD562 are currently undergoing final pre-clinical studies at the Center for Drug Research and Development in Vancouver. Five analogs, including TD561 are also under investigation by the Epilepsy and Seizure Division of the US National Institutes of Health.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21716号 / 薬科博第107号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 大野 浩章, 教授 高須 清誠, 教授 竹本 佳司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an “inverse” acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (Ki = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第18221号 / 薬科博第25号 / 新制||薬科||4(附属図書館) / 31079 / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 掛谷 秀昭, 教授 高須 清誠, 准教授 大野 浩章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21153号 / 農博第2279号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5127(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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.

Everything should be ultimately explainable in terms of the components of which it is composed. In the animal kingdom well over ninety-five percent of all recognized species are invertebrates, a distinction based on connective tissues, though most of our efforts have been directed towards vertebrates for reasons of convenience and closeness to man. Investigation of the various chemicals which make up plants and animals have been diverse, allowing us to get a general picture of what molecules to expect at each state of the evolutionary progression, but for now animal is the information complete, and for invertebrates the information is particularly sketchy. It would be convenient to have certain animals and plants completely known chemically, so that biological molecules subsequently discovered could be compared to these standards. Velella lata may be a candidate for such a standard, having a number of unique advantages. It occurs in the evolutionary chain at that point where a true multicellular animal begins, and it is the simplest animal from which genetically determined internal structural materials is conveniently isolated and purified. In plants the connective tissue is considered to be almost entirely carbohydrate polymer and in vertebrates the connective tissue is considered to be almost entirely protein (amino acid polymer), while many invertebrates have almost even mixture of protein carbohydrate in an unknown relationship. Velella presents an opportunity to examine closely the nature of the protein and its relationship to the carbohydrate. The carbohydrate of various invertebrates connective tissue matrices has been extensively studies and the structure and linkages reasonably well defined. Some questions remain regarding the length of the smallest oligosaccaride and the manner in which these are built up into higher levels of organization. Inorganic material (primarily in the form of calcium), lipid in small amounts, and moisture are also present in the invertebrate matrix. While calcium is generally considered to be in the form of the carbonate, the possibility that it is not all in this form awaits investigation, and the recent report that silicon is essential in the formation of chick connective tissues is interesting.5 Lipids are not a major component in connective tissues studied to date, and this is true of Velella also. This does not preclude, however, the possibility that lipids may play some role in the formation of membrane-like sheets which are observed in the laminations of various mollusk-arthropod matrices. Velella offers an opportunity to examine invertebrate connective tissue in an intense way to gain an understanding of the morphology and developmental dynamics which hopefully would be extensible to the vertebrate matrix.

Anthracycline-based chemotherapy is the mainstay neoadjuvant therapy for breast cancer. However, it is efficacious in only 60% of patients while carrying substantial toxicity. The application of a predictive marker of response may spare predicted ‘poor responders’ from the toxicity. Previously, we demonstrated a gene expression signature that predicts chemotherapy resistance which is linked to TP53 integrity. Further investigation showed that p53 signatures predict response in only ER+ tumors. We hypothesized that the loss of p53 confers an elevated chemotherapy sensitivity in ER+ breast tumors. We engineered isogenic p53 mutant ER+ breast cancer cell lines and assayed their cell cycle kinetics and chemotherapy sensitivity. Our results demonstrated that the loss of p53 is necessary to abrogate p53-mediated cell cycle arrest and produce an increase in apoptosis. Therefore, p53 signatures may be utilized as a predictive marker of response for patients with ER+ breast tumor and spare ‘poor responders’ from toxicity. Since ER+ p53 wild-type breast tumors are associated with anthracycline resistance, new anticancer drugs against that subgroup of tumors are needed. Phenotypic drug screening approach, which do not focus on isolated targets but instead classify compounds by their impact on cell physiology, is highly suitable for this purpose. Current cell-based phenotypic assays require fixation and staining for phenotypic markers, which reduce screen throughput and introduce potential variations and artifacts. Here we describe a high-content live-cell phenotypic assay, which streamlines the process of cytological profiling and provides a consistent platform for empirically evaluating drug action. Importantly, when combined with chemical similarity clustering, the phenotypic assay provided an inference of structure-activity relationships. Finally, a small-scale phenotypic screen of natural products enabled classification of unknown compounds against the cytological profiles of commercial compounds. Hence, the phenotypic screen provides a new and robust opportunity for accelerating the evaluation of compound activity during high-throughput drug screens. / Thesis / Master of Science (MSc)

<p dir="ltr">Within the last decade, cancer immunotherapy, the therapeutic strategy of enhancing the body’s immune system to curb tumor growth, has reached the front lines in the war on cancer. Although common strategies such as adoptive cell transfer and immune checkpoint blockade have enjoyed success against some cancers, they regrettably lack durable efficacy across a broad patient population inflicted by heterogeneous and diverse cancer types. Moreover, application of these biological therapeutics is likewise limited due to various toxicities frequently encountered in the clinic. Taking these into account, the next generation of immunotherapies must exploit novel immunomodulatory targets and therapeutic strategies that can possess both enhanced efficacy compared to current options and more acceptable toxicity profiles in patients. Compared to biologics, small molecule inhibitors are desirable as they may circumvent concerns involving efficacy and toxicity, while allowing access to a broader arsenal of macromolecular targets. Recently, protein tyrosine phosphatase nonreceptor 22 (PTPN22), a key desensitization node in T cell signaling, has emerged as a systemic and translatable cancer immunotherapy target. Nonetheless, many of its precise functions in various immune cells is not fully resolved, thus there is a critical need for both novel chemical probes for biological interrogation and inhibitors with improved <i>in vivo </i>efficacy for further therapeutic development.</p><p dir="ltr">Built upon an overview of PTPN22’s structure, function, and value as an immunotherapy target, as well as a comprehensive assessment of reported inhibitors, this dissertation documents two separate medicinal chemistry campaigns on existing PTPN22 scaffolds. Herein, the structure activity relationships, design, and biological evaluation of a novel, superiorly selective and cell-active probe/ lead compound is disclosed. This dissertation also reports the design of a novel PTPN22 inhibitor with enhanced potency, selectivity, cellular efficacy, <i>in vivo </i>pharmacokinetics, and <i>in vivo </i>antitumor efficacy in mice. Our research efforts and the overall status and future directions of the field are also succinctly discussed.</p>