No description available.

No description available.

Malaria afflicts about 500 million people worldwide thus causing significant global economic toll. The drugs available to treat the disease are rapidly losing their efficacy because of widespread prevalence of drug resistant parasites. Thus there is an urgent need to discover novel malaria therapeutics. This research is focused on to study the properties of a novel naturallike synthetic scaffold and analyze its selectivity, and cellular mechanism of action in Plasmodium falciparum. We have identified a novel compound, 3-amino-2-piperidinequinoline (APQ), which we termed UCF401. APQ demonstrated IC[sub50] at submicromolar concentrations against Plasmodium falciparum using the SYBR Green-I fluorescence assay measuring cellular proliferation. This compound also demonstrated low cytotoxicity against the NIH3T3 and HEPG2 cells using MTS assays, showing an IC50 of 174 [micro]M and 125 [micro]M respectively, suggesting of excellent selectivity. We evaluated the compliance of APQ with Lipinski's parameters and determined the in vitro physicochemical profiles of the compound. Our results show that APQ is a Lipinski parameter compliant and has good physicochemical properties. The cellular mechanism of action of APQ was characterized through the assessment of the effects of the compound at different stages of the parasite's intraerythrocytic life cycle. This assay was done by treating a synchronized cell line with the compound at 5X the IC50 value and then imaging the cells at 12-hour intervals. We found that APQ arrests parasite development at the trophozoite stage. In addition we determined that APQ is parasitocidal after a 96 h exposure. These results demonstrate that APQ can be considered as a validated hit and/or early lead.

No description available.

As malaria, caused by Plasmodium spp., continues to afflict millions of people worldwide, there is a dire need for the discovery of novel, inexpensive antimalarial drugs. Although there are effective drugs on the market, the consistent development of drug resistant species has decreased their efficacy, further emphasizing that novel therapeutic measures are urgently needed. Natural products provide the most diverse reservoir for the discovery of unique chemical scaffolds with the potential to effectively combat malarial infections, but, due to their complex structures, they often pose extreme challenges to medicinal chemists during pharmacokinetic optimization. In our laboratory we have performed unbiased, cell-based assays of numerous synthetic compounds from chemical libraries enriched with nature-like elements. This screening has led to the discovery of many original chemical scaffolds with promising antimalarial properties. In an attempt to further characterize these scaffolds, the most promising compounds were assayed in order to determine their cytotoxic effects on mammalian cells. In addition, the development of a drug resistant parasite line of Plasmodium falciparum to the most promising compound was done in order to determine the relative probability for parasite resistance development.

No description available.

学位プログラム名: 京都大学大学院思修館 / 京都大学 / 新制・課程博士 / 博士(総合学術) / 甲第23345号 / 総総博第18号 / 新制||総総||3(附属図書館) / 京都大学大学院総合生存学館総合生存学専攻 / (主査)教授 山敷 庸亮, 教授 杉山 弘, 教授 積山 薫 / 学位規則第4条第1項該当 / Doctor of Philosophy / Kyoto University / DGAM

Gene therapy has emerged as a revolutionary approach for treating genetic disorders, holding great promise for improving patient outcomes. Among the various viral vectors used for delivery of therapeutic transgenes, Adeno-Associated Viruses (AAVs) have gained prominence due to their favorable characteristics including low immunogenicity, long-term gene expression, and the ability to target both dividing and non-dividing cells. However, AAV’s are associated with the high costs of production and challenges with production of a high-quality virus, limiting AAV’s utilization and widespread use. In this study, we aimed to develop a high-throughput screening assay targeting AAV production enhancers, thus addressing the manufacturing obstacles and advancing the affordability and accessibility of gene therapies. To help overcome the limitations and expenses associated with AAV manufacturing, an innovative high-throughput screening assay was developed with the intent to identify cell culture additives/conditions which maximize AAV production. We optimized various parameters, including the transgene, producer and reporter cell lines, harvest timings and methods, and transduction techniques. The optimized screening assay was employed to evaluate novel compounds across several timings of addition, for their ability to enhance AAV production. Notably, several compounds indicated transfection enhancing capabilities up to 3.4-fold and the developed assays final variability was below 14%. Additionally, compound combinations were assessed to uncover potential additive and synergistic effects that could further enhance AAV productivity. In conclusion, our study presents a significant advancement in targeting the manufacturing challenges associated with AAV. By utilizing an optimized high-throughput screening assay, researchers and manufacturers can identify compounds that enhance AAV production, paving the way for cost-effective and scalable manufacturing processes. Ultimately, this progress holds the potential to improve the affordability, accessibility, and impact of gene therapies for patients worldwide.

In 2020, cancer impacted an estimated 1.8 million people and result in over 600,000 deaths in the United States. Some cancer treatments options are limited due to drug resistance, requiring additional drug development to improve patient survival rates. It is necessary to continuously develop new therapeutic approaches and identify novel targets, as cancer is ever-growing and adapting. Experimental research strategies have limitations when exploring how to target certain protein classes, including membrane-embedded or protein-protein bound, due to the complexity of their environments. These two domains of research are experimentally challenging to explore, and in silico research practices provide insight that would otherwise take years to study. Computer-aided drug design (CADD) routes can support the areas of drug discovery that are considered difficult to explore with experimental techniques. In this work, we provide research practices that are easily adaptable and translatable to other difficult protein targets and interfaces. First, we identified the morphological impact of a single-site mutation in the G-protein coupled receptor (GPCR), OR2T7, which had been identified as a novel prognostic marker for glioblastoma. Next, we explored the blockbuster target, Programmed Cell Death Protein 1 – (PD-1) and the agonistic vs antagonistic response that can be exploited for Non-Small Cell Lung Cancer (NSCLC) therapeutic development. Last, we explored the sphingolipid transport protein, Spns2, which has been demonstrated to be important in regulating the metastatic cancer enabling microenvironment. This work utilized molecular dynamics simulations (MDS) to explore the protein structure-function relationship for each protein of interest, allowing for the exploration of biophysical properties and protein dynamics. We identified that the D125V mutation in OR2T7 likely influences activation of the MAPK pathway by impacting G-protein binding via reducing the helical plasticity in the TM6 and TM7 regions. PD-1 was identified to have a domain near the PD-L1 binding interface that increases β-sheet stability and increases residue-residue distances with the membrane-proximal region within PD-1, thus leading to an active conformation. Lastly, Spns2 was identified to follow a rocker-switch transport model and provided preliminary insight into sphingolipid-Spns2 channel binding, interacting with residues Thr216, Arg227, and Met230, as well as highlighting the role of Arg119 in a salt-bridge network of interactions essential in substrate translocation. Collectively, this work illustrates the advantages of computational workflows in the drug discovery process and provides a framework that can be applied for additional GCPRs, transport proteins, or protein-protein interfaces to enhance and accelerate the CADD research. / Doctor of Philosophy / Cancer is an ever-evolving disease that requires continuous development of new treatment options. Experimental research strategies can be timely, expensive, or lack atomistic insight into drug development processes. Computer-aided drug design (CADD) routes provide research strategies to support areas of drug discovery that can be difficult to explore with experimental techniques. Membrane-bound proteins and protein-protein interfaces are two domains of research that are typically difficult to explore, and computational research practices provide insight that would otherwise take years to study. In this work, we provide research practices that are easily adaptable and translatable to other difficult protein targets and interfaces. First, we identified the impact of a single-site mutation in the G-protein coupled receptor (GPCR), OR2T7, which had been identified as a novel prognostic marker for glioblastoma. Next, we explored the blockbuster target, Programmed Cell Death Protein 1 – (PD-1) and active vs inactive states that can be exploited for Non-Small Cell Lung Cancer (NSCLC) therapeutic development. Last, we explored the sphingolipid transport protein, Spns2, which has been demonstrated to be important in metastatic cancer growth. This work utilized molecular dynamics simulations (MDS) to explore the protein structure-function relationship for each protein of interest, allowing for the exploration of biophysical properties and protein movement. We identified that the D125V mutation in OR2T7 likely influences activation of the MAPK pathway, which supports multiple cancer-regulation pathways, by impacting G-protein binding via reducing the structural flexibility. PD-1 was identified to have a domain near the PD-L1 binding interface that increases structural stability, thus leading to an upregulation of cancer survival pathways. Lastly, Spns2 analysis provided insight into movement involved in sphingolipid transport, provided preliminary insight into sphingolipid-Spns2 binding, as well as highlighting the role of Arg119 in a network of interactions essential in substrate translocation. Collectively, this work highlights the usefulness of computational workflows in the drug discovery process and provides a framework that can be utilized for additional GPCRs, transport proteins, or protein-protein interfaces to enhance and accelerate the CADD research.

No description available.