title: Identification of Chemical Probes from Macleaya cordata (Willd.) R.Br.
Creators: name: Nguyen, Duy Thanh
Publisher: name: Griffith University
Year: 2019
Subjects: subject: Parkinson’s Disease
subject: Phenotypic assay
subject: Traditional Chinese medicine
subject: Spectroscopy

description: The lack of our understanding about Parkinson’s Disease (PD), the second most common and incurable neurodegenerative disorder, has caused a great impact in both treatment and research for this disease. Although not a few intensive studies have been conducted with the hope of gaining more knowledge about the complex biology underlying this debilitating condition, it seems that our efforts have not completely uncovered the molecular mechanisms related to the neurodegeneration, which is critical in PD. Phenotypic assay, particularly cytological profiling (CP), has recently emerged as a powerful unbiased strategy to study the mechanisms of action in many biological systems. CP is a cell-based and image-based approach that evaluates the effects of small molecules on several organelles within the cell, enabling the assessment of multidimensional phenotypic functions instead of a single interaction that is usually seen in target-based approaches. Previous research in our institute utilizing CP has identified quite a few compounds that interacted with PD patient-derived cells. It is therefore worth believing that CP could be a future approach for combating PD. This project was conducted as part of our ongoing research intending to discover molecules from nature to study the biology of PD. Our aim was to use 1H NMR guidance to discover natural products from a traditional Chinese medicinal plant. Cytological profiling on a patient-derived cell line (human olfactory neurosphere-derived (hONS) cells) was performed for the isolated compounds to determine if they are potentially qualified as chemical probes to serve the research of PD. This thesis will demonstrate the study that integrates chemistry and biology to identify small molecules and the effects they influenced to the cell model, revealing their potential value as molecular tools for a better interrogation of the disease mechanism. The thesis was initiated with an introduction that covered several concepts, including traditional Chinese medicine (TCM), PD, CP, hONS cell model and chemical probes. Starting with TCM – a multi-century health care system in China, the introduction acknowledged that TCM herbal plants are invaluable sources of bioactive small molecules with respect to the traditional experience accumulated for thousands of years. It then moved on to a brief discussion of Parkinson’s Disease ‒ an irremediable neural degenerative disorder, and some current therapeutic treatments for this disease, as well as giving rationale as to why this condition should be researched. The introduction went on with a review on CP and its application in drug discovery, followed by a description of current disease models for PD, as well as the hONS cell model used in this project. It was noted that hONS cells derived from PD patients can recapitulate some functional aspects of this disease, and thus coupling CP with hONS cell model could shed light on the biological studies of PD. The chapter continued with a description of chemical probes, their importance in medicinal research and that these small molecules are valuable tools for interrogating the pathways of PD. Finally, the durability of NMR fingerprinting in identify compounds was demonstrated in the last part of this chapter. Chapter 2 provided details of the equipment and procedures involved in this project to assist the chemical purification and biological characterization of the compounds isolated from the selected herbal plant. Detailed spectroscopic data were also present in this chapter. Chapter 3 first gave in introduction on the selected biota, Macleaya cordata (Willd.) R.Br., including how it was initially chosen in the previous PhD project and its traditional medicinal use. The chapter then moved on to the targeted isolation of natural product from this species by utilizing the robustness of 1H NMR spectroscopy and mass spectrometry (MS), which resulted in the isolation of two new compounds and fourteen known metabolites. The in-depth structural elucidation of the two new compounds, (6R)- 10-methoxybocconoline and 6-(1-Hydroxyethyl)-10-methoxy-5,6-dihydrochelerythrine, were also mentioned in this chapter, followed by the use of density functional theory (DFT) to assign the absolute configuration (AC) of one new molecule. The chapter concluded with discussion on the validity of NMR fingerprinting and future directions on the remaining work which involved AC assignment for the other new compound. Chapter 4, the final chapter, presented the intensive data analysis for the CP screening of the isolated compounds. It was revealed that four out of sixteen metabolites showed significant perturbations to the hONS cellular parameters. Those compounds included bocconoline which impacted EEA1- and mitochondria-associated features, 6- (1-hydroxyethyl)-5,6-dihydrochelerythrine, 3-O-feruloylquinic acid and ferulic acid 4-Oglucoside which influenced LC3b-related features. These compounds can potentially be used as molecular tools to probe the biological pathways of PD. The last part of the chapter was the discussion on the CP platform used to identify the above potential probes, as well as future directions on the biology cohort. It was concluded that more justifications should be made in order to verify the potency of the compounds being qualified as useful probes. Dose dependence, mechanisms of action and drug-like properties for the candidate probes were among the future follow-up research to have a better understanding in the exploration of complex molecular mechanisms underlying PD.