title: Using Cytological Profiling to Discover Chemical Probesfrom Traditional Chinese Medicines against Parkinson's Disease
Creators: name: Wang, Chao
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Cytological profiling
subject: Traditional Chinese medicines
subject: Parkinson’s disease
subject: Chemical probe
subject: Metabolites

description: Parkinson’s disease (PD) is a chronic neurodegenerative disorder with a high prevalence rate worldwide, imposing serious burden to affected individuals, their families and society as a whole. Unfortunately, the molecular mechanisms of PD still remain poorly understood, and there are no drugs available to cure the disease. Current medications targeting the dopaminergic system only provide symptomatic relief but are not able to stop, slow or reverse the progression of neurodegeneration. Our overall aim was to use cytological profiling (CP) on a PD patient-derived cellular model (human olfactory neurosphere-derived (hONS) cells), coupled with an NMR-guided isolation strategy to identify chemical probes from traditional Chinese medicines (TCMs) for future exploration of the molecular mechanisms and innovative therapeutic opportunities in PD. The research presented in this thesis demonstrated the practicability of the integrated methodology in the chemical probe discovery against PD, and revealed the potential value of the identified candidate probes for further disease mechanism interrogation and novel therapeutic development. The thesis begins with an introduction of PD and four concepts—chemical probe, TCM, NMR fingerprinting and CP in relation to the small molecule approach to shedding light on PD biology. It was explained that TCM is a valuable source for the discovery of bioactive natural products as chemical probes against PD. The TCM-sourced chemical approach to PD biology can be pushed forward through the use of a CP technique with an NMR-guided isolation strategy. CP allows unbiased characterization of natural products and their cellular responses, while NMR-guided isolation enables precise structural dissection of chemical mixtures. The integrated methodology will facilitate a deeper understanding of PD etiology and pathogenesis, and benefit future anti-PD drug discovery. Chapter 2 describes the establishment and CP screening of a TCM library comprising 304 extracts from 152 herbs for the discovery of chemical probes against PD. Twenty percent of the extracts displayed distinct phenotypic effects on PD hONS cells. Two biotas namely Phlegmariurus carinatus and Asarum sieboldii var. seoulense were prioritized based on NMR fingerprints and Dictionary of Natural Product (DNP) database search outcomes from the active extracts for further largescale extraction and isolation. The results are presented in chapters 3 and 4. Chapter 3 describes the chemical and biological investigation of TCM Phlegmariurus carinatus. Chemical investigation of the whole plant under the guidance of 1H NMR fingerprinting resulted in the isolation of 24 compounds representing two biologically interesting structural classes: triterpenoids (3.1-3.16) and lycopodium alkaloids (3.17-3.24). Among the isolates, phlegmacaritones A (3.1) and B (3.2) were a pair of novel serratane related triterpenoid epimers possessing an unprecedented 15,30-lactone-14,15-seco skeleton, while phlegmanols G-L (3.3-3.5, 3.14-3.16) were new serratane-type triterpenoids. The structures of the novel and the new compounds were unambiguously elucidated on the basis of NMR and mass spectroscopic data. Besides, the hypothetical biosynthetic pathway of phlegmacaritones A (3.1) and B (3.2) was proposed, which suggested that their novel skeleton could be biogenetically transformed from typical serratane triterpenoids. Phenotypic responses of the metabolites in PD hONS cells revealed that the triterpenoids phlegmacaritone B (3.2), phlegmanol G (3.3) and phlegmanol E (3.9) and the lycopodium alkaloid lycoposerramine U (3.23) exhibited different patterns of significant effects on α-tubulin, mitochondria, LC3b and EEA1 related cytological parameters. These four compounds may be used as chemical probes to explore the molecular mechanisms of PD. Chapter 4 describes the targeted isolation of 20 known compounds from the roots of TCM Asarum sieboldii var. seoulense using 1H NMR fingerprinting method, and their cytological profiles against PD hONS cells. The 20 compounds represented three biologically interesting structural classes: isobutylamides (4.1-4.5), lignans (4.6-4.9) and phenolics (4.10-4.20). NMR fingerprinting proved to be an effective tool for the isolation of the major compounds (4.1, 4.2, 4.6, 4.7, 4.10 and 4.14-4.17) as well as their minor analogues (4.3-4.5, 4.8, 4.9, 4.11-4.13 and 4.18-4.20). The isobutylamide, N-isobutyl-2(E),4(E),8(Z)-decatrienamide (4.5), with misdocumented 1H NMR data in the original literature, were recharacterized by 1D and 2D NMR, MS and spectral simulation. Three major metabolites, N-isobutyl- 2(E),4(E),8(Z),10(E)-dodecatetraenamide (4.2), 3,4-methylenedioxypropiophenone (4.15) and 4,5-methylenedioxypropiophenone (4.16) caused moderate perturbation to mitochondrial and LC3b features, or strong alteration in α-tubulin features. They could be responsible for the TCM’s traditional application in the treatment of PD related symptoms, and thus used as tools for probing molecular mechanisms underlying PD. Chapter 5 presents the seven potential chemical probes identified in Chapters 3 and 4, their distinctive phenotypes and their drug-like physicochemical property. It also includes a preliminary mechanism of action (MOA) study using the neurotoxin rotenone through 1H NMR-based extracellular metabolite profiling. The candidate probes representing four different structural classes tended to exhibit different MOAs as deduced from their distinguished phenotypic effects on PD hONS cells, which indicated their value to the interrogation of different molecular pathways implicated in PD. All candidate probes passed the Lipinski’s rule-of-five criteria with four of them predicted to penetrate the blood-brain barrier. This suggested their further potential as lead compounds for the discovery of novel therapeutics for PD. The preliminary 1H NMR metabolite profiling study resulted in the identification of glucose metabolism enhancement as one of the MOAs of rotenone. It provided a proof of concept of the feasibility of using NMR-based metabolomics for future MOA studies of the candidate probes identified in this thesis. The final chapter offers details of the equipment and experimental procedures used in the isolation of 44 metabolites from two chosen biotas, cytological profiling experiment and 1H NMR-based metabolic profiling study, as well as the spectroscopic data of all isolates.