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ItemDiscovery of natural product scaffolds with anthelmintic activity against Haemonchus contortusHerath, Hetti Mudiyanselage Pushpamali Dilrukshi ( 2019)The present thesis focused on discovering new candidate compounds in natural products against economically important parasitic worms of livestock to circumvent current problems associated with resistance to commercially-available drugs (anthelmintics) (cf. Chapter 1). One of the most pathogenic parasites of small ruminants, Haemonchus contortus (barber’s pole worm), was used as the screening tool. This whole-organism bioassay was used to identify active compounds/extracts that reduced motility and/or development or altered the morphology (phenotype) of H. contortus larvae in vitro. Selected compounds were purified from active extracts using a bioassay-guided fractionation approach. The screening of two compound libraries containing a total of 1,000 natural products, natural product-inspired or synthetic compounds identified 34 compounds with notable activity against H. contortus (Chapters 2 and 3). Most of the ‘hits’ (n=32) identified from the first library were analogues of arylpyrrole (a natural product scaffold), while deguelin and rotenone (two plant rotenoids) were identified from the second library. Encouraged by these findings, a library of 7,500 extracts from different plant species was screened (Chapter 4). This screen identified three active extracts from the leaves and roots of Cryptocarya novoguineensis and the roots of Piper methysticum. Bioassay-guided fractionation of active extracts yielded four known alpha-pyrones, namely goniothalamin from C. novoguineensis, and dihydrokavain, desmethoxyyangonin and yangonin (= kavalactones) from P. methysticum. Three kavalactones induced a lethal ‘evisceration’ phenotype in treated larvae of H. contortus, and had limited toxicity on mammalian epithelial (MCF10A) cells. This is the first report on the activity of such natural compounds on a parasitic nematode of animals. The work was extended to explore compounds from Australian marine species. A library of 2,000 extracts from marine invertebrates was screened (Chapter 5) and identified three active extracts from marine sponges - one from Monanchora unguiculata and two extracts from Haliclona sp. The compounds with moderate activity against H. contortus were fomiamycalin from Monanchora unguiculata and halaminol A from Haliclona sp., although the activity of these compounds was not entirely selective. Subsequently, a structure-activity relationship (SAR) investigation of one of the identified natural product scaffolds, alpha-pyrone was conducted (Chapter 6). This work identified three analogues (designated W-408, W-415 and W-417) with anthelmintic activity similar to, or greater than the synthetic parent kavalactones (desmethoxyyangonin or yangonin), with the most potent analogue W-408 achieving a marked increase in potency (7-fold) against and selectivity (> 21) for H. contortus. Taken together, this drug discovery effort identified or purified 40 natural products representing distinct chemical classes (Chapters 2-5), and the SAR investigation of the alpha-pyrone scaffold identified three key analogues with enhanced potency and/or selectivity (Chapter 6). Collectively, the findings of this thesis indicate that some of the identified natural product scaffolds have the potential to be developed as ‘lead’ candidates. Developing such anthelmintic candidates via future chemical optimisation, efficacy and safety assessments, broad spectrum activity assessments, and target identification represents an exciting prospect (cf. Chapter 7) and, if successful, could pave the way to subsequent pre-clinical and clinical evaluations.
ItemElucidating the developmental biology of Haemonchus contortus and other nematodes using a multi-omics approachMa, Guangxu ( 2019)An appraisal of current literature (Chapter 1) revealed that many parasitic worms are pathogens of animals, causing major diseases and socioeconomic losses worldwide. Efforts to control roundworms (nematodes) are often compromised by widespread resistance to currently used treatments. Thus, there is a clear need to work toward new interventions, preferably based on a deep understanding of the molecular biology of nematodes and/or the relationship that they have with their host animals. The predominant focus of the present thesis was on exploring aspects of the developmental biology of parasitic nematodes using advanced molecular (‘omic) and bioinformatic technologies, with an emphasis on the barber’s pole worm (Haemonchus contortus) - one of the most economically important parasites of ruminant livestock. The specific aims were: (1) to establish molecular data sets (resources) for H. contortus; (2) to explore RNA transcription and protein expression profiles in the developmental transition from free-living to parasitic larvae of H. contortus under well-defined conditions in vitro; (3) to construct dauer-like signalling pathways in H. contortus and some other nematodes (ascaridoids); and (4) to elucidate dauer-like signalling and the involvement of bile acid-like dafachronic acids during nematode development. All of these aims were achieved. Addressing aim 1, comprehensive molecular (transcriptomic, proteomic and lipidomic) resources were established for H. contortus using advanced nucleic acid sequencing or mass spectrometry techniques (Chapters 2-4). Addressing aim 2, these resources were utilised for in-depth explorations of molecular changes during the developmental switch from the free-living to the parasitic stage of H. contortus (Chapter 5). This work revealed extensive alterations in transcription and protein expression. There was a discordance between the mRNA transcription and protein expression changes, which appeared to relate to microRNA regulation at the post-transcriptional level and genes involved in signal transduction and signalling molecule interactions. Comparative studies with C. elegans indicated that molecules and canonical dauer signalling pathways integrate environmental cues and developmental processes in H. contortus. Addressing aim 3, gene homologues involved in the dauer signalling pathways were inferred for H. contortus using genomic, transcriptomic and proteomic data sets (Chapter 6). Transcriptomic and proteomic studies of such homologues indicated similar gene transcription and protein phosphorylation profiles between the infective stage of H. contortus and the dauer stage of Caenorhabditis elegans. Although reduced sets of genes encoding G protein-coupled receptors, insulin-like peptides and cholesterol transporters were identified in H. contortus, similar functional roles of the signalling pathways were proposed for parasitic and free-living nematodes. Addressing aim 4, the “dauer hypothesis” was tested in H. contortus using an integrated ‘omics approach (Chapter 7). The dauer-like signalling pathways were shown to be activated during the developmental transition from the free-living to the parasitic stages of H. contortus, and were linked to an amplification of a 3-keto bile acid-like steroid hormone (i.e., dafachronic acid). This hormone bound to a nuclear receptor DAF-12 in vitro, and exhibited stimulatory effects on the larval activation and development of this parasite. These stimulatory effects appeared to be associated with a modulation of the dauer-like signalling cascades and lipid (glycerolipid and glycerophospholipid) metabolism. Specific chemical inhibition of dafachronic acid biosynthesis disrupted lipid metabolism and compromised larval activation and development, suggesting key roles for the hormone signalling module in the development of H. contortus. This work “opened the door” to exploring homologous signalling pathways in biologically distinct (ascaridoid) nematodes, including Toxocara canis (causing toxocariasis) and Ascaris suum (causing acariasis) (Chapters 8 and 9). In conclusion, this thesis showed that an integrated use of these resources allows detailed explorations of signalling molecules, molecular processes and pathways likely associated with nematode development, adaptation and parasitism, and provides opportunities to identify novel intervention targets (Chapter 10). Although this work was focused mainly on H. contortus and two ascaridoid species, the multi-omics approach established herein could be readily used to explore a wide range of interesting and socioeconomically significant parasitic worms (including also trematodes and cestodes) at the molecular level, and to elucidate host-parasite interactions and disease processes.