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.

ONC201 is a novel compound that upregulates endogenous TNF-Related Apoptosis-Inducing Ligand (TRAIL), in tumor and normal cells, restoring autocrine and paracrine anti-tumor activity within tumor cells, and upregulates the DR5 gene by activating the integrated stress response, inducing eIF2-alpha-dependent ATF4 and CHOP [1-3]. ONC201 also demonstrates potent anti-tumor effects on colorectal cancers [4, 5]. ONC201 presented a promising oral bioavailability, wide distribution throughout the body, and ability to cross the blood-brain barrier. Further, the unique ability of its TRAIL-and-DR5-based signaling to induce apoptosis in cancer cells and not normal cells adds to its appeal as an anti-cancer therapeutic and prompted clinical development [1-4, 6]. ONC201 has successfully completed an FDA advanced Phase I/II clinical trial in advanced aggressive refractory solid tumors. Results indicated that ONC201 is well-tolerated and recommended a phase II dose of 625 mg orally every 3 weeks. Several Phase I/II clinical trials are enrolling in multiple solid tumors and hematological malignancies [7, 8]. Chapter two of this study provides evidence that ONC201 dose intensification demonstrates an increased pharmacodynamic effect and an increasing anti-tumor efficacy in vivo while having a safe toxicity profile upon weekly dosing. This data influenced the Phase II clinical trials, which have now been adjusted to include weekly dosing. Given the potential anti-metastatic effects of TRAIL signaling and the role of TRAIL in the immune surveillance of cancer, we hypothesized that ONC201 would suppress metastatic tumor development and engage the immune system in its anti-cancer activity. We also establish that ONC201 provides an important anti-metastatic effect in CRC that should be pursued in both pre-clinical and clinical studies. Tail vein and surgical CRC models demonstrate that ONC201 inhibits the number and size of metastases. Evidence has shown that TRAIL can also inhibit cancer metastasis by possibly inducing cell death or TRAIL-sensitization in the primary tumor when cells undergo extravasation upon detachment from the primary tumor [9-11]. While we show that TRAIL plays a role in ONC201’s ability to inhibit migration/invasion in vitro, further investigation of the role of TRAIL in vivo is necessary. Our data indicates that ONC201 promotes a pro-immune response in CRC subcutaneous tumors with increased NK cells that play a role in ONC201’s efficacy in syngeneic models. Since activated natural killer cells express TRAIL, we established that ON201 can activate and induce TRAIL expression in NK cells [12, 13]. As we did not find any immune infiltrates in the metastases, we suggest that the effect of the micro-environment or in more clinically-relevant models with stromal environments should be pursued. Chapter 3 of this of thesis demonstrates the characterization of ONC201’s core structure and development of ONC201 analogues including their mechanistic differences and potential in vivo efficacy and safety. We have demonstrates the importance of the angular structure of ONC201 to ONC201’s anti-tumor efficacy [14]. The novel pharmacophore has now been called as imipridone and is essential for its anti-tumor activity, as the linear isomer had no anti-tumor effect. We leveraged this unique pharmacophore to synthesize ONC201 analogues with distinct therapeutic properties; namely, targeting ONC201-resistant tumor types or possessing distinct signaling properties. Imipridone R2 analogues have a lower IC50 and are more promising than their lead compound in certain tumor types. ONC212, a halide R2 analogue, demonstrates superior efficacy in vivo in melanoma xenografts, a large therapeutic window; but does have a rapid PK. Oncoceutics is currently developing ONC212 for a first-in human Phase I clinical trial. The fourth chapter of this study demonstrates the potential of a combinational therapy with ONC201 in colorectal cancer with bevacuzimab. Clinical trials in CRC and other tumor types have demonstrated that therapeutics targeting the vascular endothelial growth factor (VEGF) pathway, such as bevacizumab, are effective in combination with certain chemotherapeutic agents. ONC201 in combination with bevacuzimab led to superior results with almost no tumor growth. This result was re-capitulated in syngeneic models. Given that bevacuzimab is approved for metastatic CRC, we suggest that ONC201 in combination with bevacuzimab should be introduced into a combinatorial Phase II clinical trial. This thesis focuses on the importance of dose-intensification of ONC201 on its pre-clinical efficacy; establish an anti-metastatic effect; demonstrate an immune increase in subcutaneous models; and elucidate the role of the core angular structure in efficacy with the development novel ONC201 analogues. The importance of pre-clinical studies, ONC201’s analogues including the successful development of ONC212, and potentially advantageous combinational therapies with anti-angiogenics is explained in the chapters throughout. / Cancer Biology & Genetics

Molecular recognition is an important tool for developing tunable controlled release systems and fabricating biosensors with increased selectivity and sensitivity. The development of polymer-based materials that exploit molecular recognition events such as host-guest complexation, enzyme-substrate and enzyme-inhibitor interactions and nucleic acid hybridization was pursued in this thesis. Using polymers as an anchor for molecular recognition can enhance the affinity, selectivity, and the capacity for immobilization of recognition units, enabling the practical use of affinity-based systems in real applications. To introduce the potential for immobilization while preserving or enhancing the affinity of small molecule recognition units, the affinity of derivatized cyclodextrins for the hydrophobic drug, dexamethasone, was investigated. Cyclodextrins (CDs) are molecules that possess a hydrophilic exterior and a hydrophobic cavity capable of accommodating a wide range of small molecule guests. Analysis of the solubilization capacities, thermodynamic parameters and aggregative potentials of carboxymethyl and hydrazide derivatives of CDs established the dextran-conjugated βCD derivative as an ideal carrier of hydrophobic drugs and the hydrazide βCD derivative as an optimal solubilizer of lipophilic pharmaceuticals, both alone and when incorporated in a polymer-based drug delivery vehicle. To enable non-covalent immobilization and stabilization of biomacromolecular recognition units, a printed layer hydrogel was investigated as a selective diffusion barrier for analyte sensing and enzyme inhibitor recognition. A printable hydrogel platform was developed from an established injectable system composed of aldehyde- and hydrazide-functionalized poly(oligoethylene glycol methacrylate) polymers. The printed layer hydrogel effectively immobilized a wide range of enzymes and protected enzyme activity against time-dependent and protease-induced denaturation, while facilitating the diffusion of small molecules. Furthermore, to demonstrate the potential of the printed film hydrogel immobilization layer to enhance the selectivity of the target, the printable hydrogel platform was used to develop a microarray-based assay for the screening of inhibitors of the model enzyme, β-lactamase. The assay was able to accurately quantify dose-response relationships of a series of established inhibitors, while reducing the required reagent volumes in traditional drug screening campaigns by 95%. Most significantly, the assay demonstrated an ability to discriminate true inhibitors of β-lactamase from a class of non-specific inhibitors called promiscuous aggregating inhibitors. Finally, to enable non-covalent immobilization of DNA recognition units, the printable hydrogel-based microarray was tested for its ability to immobilize DNA recognition sites and promote the detection of DNA hybridization events. A long, concatameric DNA molecule was generated through rolling circle amplification and was used as a sensing material for the detection of a small, fluorophore labeled oligonucleotide. The printable hydrogel was able to effectively entrap the rolling circle amplification product. Properties of the printable hydrogel were investigated for their ability to support the detection of DNA hybridization events. / Thesis / Doctor of Philosophy (PhD) / This thesis describes the development of polymer-based materials that exploit molecular recognition events for drug delivery and biosensing applications. First, cyclodextrins (CDs) are molecules that are capable of binding a wide range of small molecules. A comprehensive analysis of the complexation properties of CD derivatives revealed critical insight regarding their application in polymer-based drug delivery vehicles. Second, a printable hydrogel platform was developed to support the immobilization and activity of biomolecules and establish a biosensing interface that facilitates the diffusion of small molecules but not molecular aggregates. A microarray-based assay was developed by employing the printed hydrogel interface for the screening of inhibitors of the model enzyme, β-lactamase, and the detection of DNA hybridization events.