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

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

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)

No description available.

No description available.

Quaternary ammonium compounds (QACs) have long served as lead disinfectants in residential, industrial, and hospital settings. Their simple yet effective amphiphilic nature makes them an ideal class of compounds through which to explore antibacterial activity. We have developed novel multiQAC scaffolds through simple and cost-efficient syntheses, yielding hundreds of diverse compounds strategically designed to examine various aspects of antibacterial and anti-biofilm activity, as well as toxicity. Many of these bis-, tris-, and tetraQACs display antibacterial activity 10 to 100 times greater than conventional monoQACs, and are among the most potent biofilm eradicators to date. Through analyzing their activity against several strains, we have uncovered and provided further evidence for key tenets of amphiphilic QAC bioactivity: a balance of hydrophobic side chains with cationic head groups generates optimal antibacterial activity, though toxicity to eukaryotic cells needs to be mitigated. Given their ubiquitous nature and chemical robustness, the overuse of QACs has led to the development of QAC resistance genes that are spreading throughout the microbial world at an alarming rate. These resistant strains, when found in bacterial biofilms, are able to persist in the presence of lead commercial QAC disinfectants, warranting the development of next-generation biocides. Several of our scaffolds were designed with QAC resistance machinery in mind; thus, we utilized these compounds not only as antibacterial agents but also as chemical probes to better understand and characterize QAC-resistance in methicillin-resistant Staphylococcus aureus (MRSA). Our findings support previous postulations that triscationic QACs would retain potency against QAC-resistant strains. Furthermore, we have identified monocationic and aromatic moieties, as well as conformational rigidity, as being more prone to recognition by the resistance machinery. Using our chemical toolbox comprised of QACs of various charge state and scaffold, we explored both the mechanism and scope of QAC-resistance by examining their structure-resistance relationship. Our holistic findings have allowed us to better understand the dynamics of this system towards the design and development of next-generation QACs that will: (1) allow us to better probe the resistance machinery, and (2) remain efficacious against a variety of microbial pathogens. / Chemistry

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.