published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 97-100).

abstract / Chemistry / Doctoral / Doctor of Philosophy

Triptolide, a diterpene triepoxide extracted from traditional Chinese medicinal herb Tripterygium wilfordii Hook. F has been shown to have profound inhibitory effects against tumor progression, pathological angiogenesis and inflammation. However, the mechanisms by which triptolide exerts these effects remain unclear. To understand its cellular mode of action, biotinylated/desthiobiotinylated and fluorophore-labeled triptolide derivatives were used as probes to identify cellular proteins that bind to triptolide. By using two different approaches for screening drug-protein interactions, the most prominent cellular protein bound to triptolide was confirmed to be peroxiredoxin 1 (PRDX1). This result was validated by demonstrating the ability of triptolide or its conjugated probes to bind recombinant human PRDX1. Specificity of the drug-protein interaction was established by competitive inhibition of binding of fluorophore-labeled triptolide to PRDX1 by triptolide itself. Two binding sites of triptolide to PRDX1 were found, one of which being Cys173 as confirmed by orbitrap LC-MS/MS analysis. Further study by size exclusive chromatography revealed that triptolide altered the oligomeric state of PRDX1. The decameric form of PRDX1 was dissociated into lower molecular weight species in the presence of triptolide. This observation was responsible for attenuation of PRDX1’s chaperone activity upon triptolide treatment, which was supported by evidence from both light scattering and native mass spectrometry studies. Functionally, triptolide’s synergistic effect on stress-induced cell apoptosis may be mediated, at least in part, by the interaction of triptolide with PRDX1 and the consequent inhibition of its chaperone activity. Several natural products, Celastrol, Withaferin A and Radiciol were discovered as new PRDX1 inhibitors and confirmed to physically interact with PRDX1 and exert similar functional effects as triptolide. The interaction between PRDX1 and those natural products may shed light on the detailed mechanism of their biological actions and render PRDX1 a potential target for cancer therapy. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Master / Master of Philosophy

The aim of these studies was to identify circulating miRNAs that can be used as biomarkers in patients with paracetamol-induced liver injury. Whether the miRNAs discovered in humans could be back-translated to zebrafish with the aim of developing a liver toxicity model to replace rodent use was also investigated. First, the miRNA signature of DILI induced by paracetamol was defined. Plasma miRNAs were quantified in paracetamol overdose patients. A signature of 16 miRNAs was discovered that best separated patients with liver injury from those without liver injury. This signature was tested in a second cohort and resulted in the detection of paracetamol-induced liver injury with high specificity and sensitivity. At first presentation to hospital miR-122-5p was the most sensitive single miRNA and superior to ALT activity in predicting liver injury. In order to further qualify miR-122-5p, three detailed studies relevant to possible clinical scenarios were performed. The effect of acute alcohol ingestion (commonly co-ingested with paracetamol overdose) on circulating concentrations of miR-122-5p in healthy volunteers was investigated. Alcohol ingestion induced a small, non-clinically relevant, increase in miR-122-5p. The effect of chronic kidney disease (CKD) and haemodialysis (HD) on circulating miR-122-5p concentrations was explored because kidney dysfunction has been associated with a reduction in the concentration of circulating miRNAs. HD patients had lower concentrations of miR- 122-5p compared to healthy volunteers and CKD patients. To facilitate miRNA measurement outwith hospitals, miR-122-5p was measured in a blood drop from a finger prick. miR-122-5p was readily measurable in finger prick samples and concentrations were significantly higher in the blood drop from DILI patients compared with healthy volunteers. To complement miR-122-5p as a marker of toxicity, circulating paracetamol metabolites were measured in plasma samples from paracetamol overdose patients. A higher percentage of circulating metabolites formed by cytochrome P450 enzymes were present in patients with liver injury and these metabolites were superior to both ALT and paracetamol concentration with regard to early patient stratification. To reduce need for rodent studies, miRNAs were back-translated into zebrafish. In order to study circulating miR-122-5p in adult zebrafish, a bloodletting method by collecting blood retro-orbitally was developed. After studying different dosing regimens of paracetamol in adult and larvae zebrafish the model was determined to be too variable with regard to liver injury. A new drug, triptolide, originating from traditional Chinese medicine and responsible for DILI in China, was tested as an alternative model for drug-induced liver injury in zebrafish larvae. miRNA-122-5p decreased in zebrafish larvae after triptolide treatment and triptolide-induced liver injury could be tracked by fluorescent microscopy. Selective plane illumination microscopy was able to track the decrease in liver volume during triptolide exposure. In order to identify the toxic pathways involved in triptolide-induced liver injury, RNA-sequencing was performed. This identified KEGG pathways including ribosome, spliceosome and notch signalling as pathways affected by triptolide. In summary, miRNAs can be used as highly sensitive biomarkers to detect acute liver injury in patients and zebrafish. Zebrafish may represent an alternative model species to study DILI, further work is needed.

No description available.

Mounting evidence indicates an involvement of inflammation in the pathogenesis of Alzheimer’s disease. While there are other mechanisms involved, it is this role of inflammatory processes that we wish to investigate. Triptolide is the major constituent in the Chinese herb, Tripterygium wilfordii Hook F, and has been used for centuries as part of Chinese herbal medicine. The four ringed structure has close homology to drugs of the steroid class and it has been shown to be beneficial as an anti-inflammatory for rheumatoid arthritis and for treatment of certain cancers. The aim of this study was to evaluate the potential therapeutic effect of Triptolide on the neuropathology and deficits of spatial 6 learning and memory in amyloid precursor protein (APP) and presenilin 1 (PS1) doubletransgenic mice, a well established Alzheimer’s disease (AD) mouse model. After treatment of APP/PS1 mice with Triptolide (40μg/kg, three times weekly,), initiated when the mice were 5 months old, for as little as 8 weeks, significant decrease in β-amyloid (Aβ) deposition and microglia activation was observed. Moreover, Triptolide treatment robustly rescued spatial memory deficits observed in APP/PS1 mice. However, APP processing, tau hyperphosphorylation, and the activities of the two major kinases involved in tau hyperphosphorylation, cyclin dependent kinase 5 (cdk5) and glycogen synthase kinase 3β (GSK3β) were not affected by the Triptolide treatment. Based on the recent finding for the inhibitory effect of Triptolide on Aβ-induced production of pro-inflammatory cytokines from microglia, we propose that Triptolide treatment may have beneficial properties in halting glial activation and help restore an immune system that fights plaque deposition. Although the exact mechanism of action has yet to be deduced, the increase in APP CTFs while having a significant decrease in amyloid plaque deposition suggests that alterations in gamma secretase activity may be a possible answer. Currently, these results support the use of Triptolide as an effective therapeutic to prevent the progression of Alzheimer’s disease.

Cancer is one of the major causes of morbidity in the world. Although the overall survival of cancer has been significantly improved by chemotherapy in the last three decades, the success of cancer chemotherapy is still severely limited by the lack of selectivity of anti-cancer drugs to malignant cells leading to dose-limiting toxicity and the resistance of cancer cells to the conventional anti-cancer drugs. Gene-directed enzyme prodrug therapy (GDEPT) was designed to direct the anti-cancer drugs to specifically target the cancer cells by using cancer specific promoter to drive the expression of enzyme which can convert prodrug into anti-cancer drug specifically in cancer cells. However, this strategy is hindered by the lack of strong cancer specific promoters to specifically express drug-converting enzymes in cancer cells. In consequence, there is not enough anti-cancer drug activated inside the cancer cells. The first part of this study was to employ NF-κB binding sites as a novel enhancer system to improve the promoter activity of carcinoembryonic antigen (CEA) and human telomerase reverse transcriptase (hTERT) for GDEPT. In this system, the basal CEA promoter sequences were placed downstream of the 4 or 8 NF-κB DNA binding sites linked in tandem (κB4 or κB8). The system was designed to serve two particular purposes: to exploit the high levels of intratumoural NF-kB expression and keep the relative tumour specificity of the CEA and hTERT promoters. The results demonstrated that κB enhancer systems increased the transcriptional activity of CEA and hTERT promoter without compromising its cancer specificity. The fidelity of the κB4-CEA enhancer-promoter system was therefore improved by the increased transcriptional contrast between the cancer and normal cells. Moreover, in comparison with CEA promoter alone, κB-CEA enhancer-promoter system expressed human thymidine phosphorylase (TP) protein at significantly higher levels which were comparable to those expressed by CMV promoter. The κBCEA- TP system transfected cells demonstrated significantly higher sensitivity to 5'-Deoxy-5-Fluorouridine (5'-DFUR), a prodrug of 5-fluorouracil (5-FU). The second part of this study was involved in using NF-κB inhibitor as a chemosensitizer to sentizise the anti-cancer drug-induced chemoresistance cells to anti-cancer drugs. The results derived from this study manifested that the anti-alcoholism drug, Disulfiram (DS), and anti-inflammatory drug, triptolide (PG490), markedly enhanced the cytotoxicity of several conventional anti-cancer drugs in colon, lung and breast cancer cell lines. PG490 induced caspase-dependent cell death accompanied by a significant decrease in Bcl-2 levels. PG490 induced the expression of p53 and down-regulated p21 expression. This study indicated that some clinically used non-cancerchemotherapeutic drugs may be developed as chemosensitizers for cancer chemotherapy

This thesis discusses the progress made towards a formal total synthesis of triptolide, a naturally occurring diterpenoid triepoxide molecule. Isolated from a Chinese vine, triptolide features some interesting structural characteristics and has demonstrated a broad range of interesting medicinal effects. It has demonstrated remarkable cytotoxicity against a number of cancer cell lines, immunosuppressive activity, and reversible male sterility. This biological activity has made it a target of a number of total syntheses spanning from 1980 to 2010. Gold-catalyzed transformations are an emerging field in synthetic organic chemistry, but their efficacy and potential uses are gaining much recognition among the synthetic organic community. Our research group is extremely interested in the applications of such gold-catalyzed organic transformations in natural product synthesis. Here, we discuss our investigations towards accessing the tetracyclic core of triptolide using a gold-catalyzed cyclization cascade reaction. We explored a number of synthetic routes towards a common linear precursor, and our successes and failures are discussed herein. We also report numerous unsuccessful efforts towards an oxidative gold-catalyzed cyclization cascade to form the tetracyclic core of triptolide. Finally, we investigated the use of a photocatalytic radical cyclization cascade to access the desired core. We report some promising preliminary results, and this study is ongoing in the Barriault group.