Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references (leaves 152-169). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 153-173).

Thesis (Ph.D.)--University of Wollonong, 2005. / Typescript. Includes bibliographical references: leaf 206-219.

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Lakshmi P. Kotra; submitted to the Dept. of Chemistry and Biochemistry. Title from PDF t.p. (viewed May 17, 2010). Includes bibliographical references (p. 41-43).

Ph.d.

Orientador: Fabio Trindade Maranhão Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T06:37:45Z (GMT). No. of bitstreams: 1 Lopes_StefanieCostaPinto_M.pdf: 23177514 bytes, checksum: 4171205441f1112d18413fd5eed95ac3 (MD5) Previous issue date: 2008 / Resumo: A violaceína é um pigmento violeta extraído da bactéria Gram-negativa Chromobacterium violaceum. Diversos trabalhos atribuíram à violaceína diferentes atividades biológicas tais como bactericida, antiviral, fungicida e antitumoral. Os efeitos da violaceína também foram observados contra protozoários patogênicos, como Leishmania sp e Trypanossoma sp. Neste trabalho, avaliamos a atividade antimalárica da violaceína in vitro e in vivo contra Plasmodium humano e murino, respectivamente. Neste sentido, a violaceína se mostrou tão eficiente quanto o quinino em eliminar P. falciparum (3D7), mas três vezes menos eficiente que a cloroquina. Além disso, não encontramos diferença na sua atividade contra formas jovens e maduras, mostrando uma atuação similar nos diferentes estágios de desenvolvimento do parasita. Os experimentos in vivo utilizando camundongos infectados com PcchAS tratados por 11 dias consecutivos (0-10) revelaram uma potente atividade desta droga, inibindo o desenvolvimento parasitário em até 86% no pico da parasitemia. Também foi encontrado 60% de inibição quando o tratamento iniciou-se 5 dias após o estabelecimento da infecção. Quando administrada em animais infectados com uma cepa murina letal (PcchAJ), a violaceína protegeu 80% dos animais, em contraste à 100% de mortalidade dos animais não tratados. A comparação do ED50 demonstrou que a violaceína é tão eficiente quanto o artesunato e 2 vezes mais ativa que a cloroquina, sob as mesmas condições de tratamento. Finalmente, animais naive tratados com a violaceína durante 11 dias consecutivos não mostraram alterações na densidade de glóbulos vermelhos e no peso. Também não foi observado nenhum dano morfológico nos cortes histológicos. Coletivamente, estes resultados demonstram claramente o potencial antimalárico da violaceína e abre perspectivas para o entendimento dos mecanismos envolvidos na inibição parasitária por este composto. / Abstract: Violacein is a violet pigment extracted from the bacteria Gram-negative Chromobacterium violaceum. Growing bodies of evidences have implicated violacein as an antimicrobial, antifungal, antitumoral, and a moderate trypanocidal and leishmanial activity has also been observed. Herein, we evaluated the anti-malarial activity of violacein against murine and human-derived Plasmodium. Indeed, violacein showed to be efficient in killing P. falciparum (3D7 strain) as much as quinine, but 3 fold less pronounced than chloroquine. Moreover, this anti-Plasmodial activity detected was direct against both young and mature stages of this human parasite. In vivo experiments in P. c. chabaudi AS (PcchAS) infected mice treated during 10 consecutive days after infection (0-10) revealed a powerful activity of this drug, by inhibiting parasite burden up to 86%. Also, 60% of inhibition was noticed when violacein was administrated 5 days after infection establishment. When administrated in mice infected with a lethal strain of murine-derived Plasmodium (PcchAJ) violacein protected 80% of these mice, in contrast to 100% of mortality reported to the non-treated animals. ED50 comparisons demonstrated that violacein was efficient as much as artesunate and twice more active than chloroquine. Finally, naïve mice inject with violacein during 10 consecutive days did not display alterations on red blood cell density; weight and neither morphological damages were noticed in spleen and liver histological sections. Collectively, these data clearly demonstrated the anti-malarial effect of violacein and open perspectives to understating the mechanisms involved in killing this parasite by this compound. / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

M.Sc. (Chemistry) / The objective of the project was the synthesis of thiosemicarbazone-triazole hybrid compounds and evaluation of their biological activities against malaria and obesity. In achieving our objective, compound 63 was synthesized from the reaction of benzaldehyde 62 with propargyl bromide. Click chemistry reaction of compound 63 with benzyl azide provided triazole 68. Schiff’s base condensation of triazole 68 with methyl hydrazine carbodithioate 67 furnished compound 69 which then underwent nucleophilic substitution reaction and afforded thiosemicarbazone-triazole hybrids 61a-j. The structures of the compounds were characterized using NMR spectroscopy and elemental analysis. Hybrids 61a-h were then investigated for their biological activities against malaria and obesity. The antimalarial activities of the hybrids against the 3D7 strain of the malaria parasite plasmodium falciparum showed that only hybrid 61f exhibited less than 50% parasite viability (46% compared to 37% of chloroquine). The dose response of the hybrids was not carried out due to their poor activities. Hybrids 70a-h that incorporated electron donating group in their aromatic linker were synthesized similarly as hybrids 61a-h were synthesized. Their antimalarial activities showed that all except 70c and 70g exhibited less than 50% parasite viability. The test results indicated that the addition of methoxy group to the hybrids 61b, 61e and 61f decreased their percentage parasite viability which were exhibited by their corresponding hybrids 70b, 70e and 70f, respectively. Hybrid 70f was found to be marginally active with a dose response of 7.09 μM. The anti-obesity and anti-diabetic effects of hybrids 61a-h were investigated against the mitochondrial genes (Acc-1, Cpt-1 and Pgc-1) and glucose transport genes (Glut-4, Mef2a and Nrf-1), respectively. The test results against the mitochondrial genes showed that hybrids 61e and 61h consistently exhibited on the 3 genes which indicated that the presence of a non-polar short branched chain of the amine moiety might be important in the up-regulation of oxidative (Cpt-1 and Pgc-1) and down regulation of lipid accumulation (Acc-1) genes. The test results on the glucose transport genes showed that 61e followed by 61f consistently exhibited on the 3 genes which indicated that the presence of a non-polar short branched chain of the amine moiety might be important in the up-regulation of Glut-4, Mef2a and Nrf-1.Hybrids 70a-h were also tested against obesity and type 2 diabetes mellitus. Their investigation on the mitochondrial genes showed that the addition of methoxy group to the hybrids 61a-h that have a non-polar long branched chain of the amine moiety could be a reason for the expression and suppression of Cpt-1, Pgc-1 and Acc-1, respectively. The test results of hybrids 70a-h on glucose transport genes showed that the addition of methoxy group to the hybrids 61a-h that have a non-polar short (straight and branched) alkyl chain of the amine moiety might be a reason for the up-regulation of Glut-4, Mef2a and Nrf-1. Carbohydrate incorporated thiosemicarbazone-triazole hybrid compound 75 was successfully synthesized. However, the synthesis of more libraries of compound 75 and investigation of their biological activities against malaria and obesity were not carried out due to time constraints.

Despite the concerted efforts of researchers, policy makers and public health workers worldwide, malaria persists as a significant disease threat for nearly half the world’s population. Recent advances in vector control measures, diagnostics and antimalarial drug therapies have contributed greatly to reducing the incidence of clinical disease, and by extension, the number of deaths attributable to malaria in the past two decades; however, the latter remains high—over 400,000 people die each year from malaria, the vast majority of these being children under the age of five. Our ability to rapidly and effectively treat malaria has been a cornerstone of efforts to control and eradicate this devastating disease. Nonetheless, the constant evolution and spread of drug-resistant forms of the Plasmodium parasites that cause malaria—particularly the most virulent of these, Plasmodium falciparum—have historically greatly hindered these efforts, compromising the efficacy of every previous first-line treatment. Today, treatment of P. falciparum malaria relies on artemisinin derivatives, an exquisitely potent and fast-acting class of antimalarials that are deployed ubiquitously in artemisinin-based combination therapies, or ACTs. Now, emerging resistance to ACTs threatens to once again reverse the hard-fought advances made in the global fight against malaria. Resistance to artemisinin itself was first documented in western Cambodia and northwest Thailand in 2009 and has continued to spread throughout Southeast Asia at alarming rates. Reports of resistance to ACTs followed soon thereafter. Artemisinin resistance has also emerged de novo in other parts of the world. The major concern is that it will spread to Africa, where the disease burden is highest. Previous studies have provided compelling evidence that resistance to artemisinin results primarily from specific point mutations in the C-terminal Kelch propeller domain of the P. falciparum protein K13. Here, we have addressed two central aims regarding the role of this protein in mediating resistance to artemisinin. The first was to genetically dissect the contribution of a panel of K13 polymorphisms to artemisinin resistance and parasite fitness as assessed in vitro, with the latter being a key factor impacting the spread of resistance-conferring alleles in high-transmission settings. These experiments were conducted by CRISPR-Cas9-mediated gene editing, which allowed us to successfully engineer K13 mutations into a variety of strain backgrounds, including, for the first time, recently culture-adapted African parasites. These experiments clearly show that there is no genetic obstacle to the acquisition of artemisinin resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may counter-select against the spread of resistance. The second aim relating to K13 was to investigate the underlying biology of this protein. To this end, we raised monoclonal antibodies to recombinant K13 and generated transgenic lines expressing tagged versions of the protein. Using these tools, we describe the subcellular localization of K13 in wild-type and mutant parasites in the presence and absence of drug pressure, and identify potential K13-associated proteins. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13, suggesting that mutations result in a loss of protein function. In order to overcome K13-mediated artemisinin resistance, novel therapeutics with distinct modes of action will be required. In our last aim, we characterize inhibitors of a particularly promising new antimalarial drug target, the proteasome. We report that these covalent peptide vinyl sulfone inhibitors are highly potent against genetically diverse parasites, including K13-mutant, artemisinin-resistant lines. Moreover, we observe that parasites do not readily acquire resistance to these compounds, nor do related compounds select for cross-resistance to one another. We also observe strong synergy between artemisinin and related compounds with these inhibitors in both K13 mutant and wild-type parasites. These results highlight the potential for targeting the Plasmodium proteasome as a means of overcoming artemisinin-resistant malaria.

The strides made in malaria eradication efforts have been aided by a combination of vector control and chemoprevention. However, Plasmodium resistance to first-line artemisinin-based combination therapies (ACTs), and mosquito resistance to insecticides threatens the progress made. Innovative vector control measures, vaccines and antimalarial drugs with novel modes of action are key to disease eradication. High-throughput phenotypic screening of chemical libraries tested directly against all the stages of the Plasmodium lifecycle have been the mainstay of antimalarial drug discovery efforts and have identified compounds that are effective in parasite clearance. Unfortunately, these screens are handicapped in that they are unable to specify the actual compound targets in the Plasmodium parasites. As a result, many candidate hits have had to be re-screened in specific assays to determine putative mechanisms of antiplasmodial action. Predictably, this has elevated target-specific screens as the next frontier in drug discovery. This shift has been aided by a number of factors, including the cost effectiveness of these screens and the fact that target-specific screens do not always require specialized access to parasites. When combined with knowledge of the target’s structure, where known, target-specific screens have the potential to give lead compounds with impeccable potency and selectivity. This approach has already been successfully put to use, for example, in the identification of P. falciparum p-type ATPase 4 (PfATP4) and P. falciparum phosphatidylinositol 4-kinase (PfPI(4)K) inhibitors. The new challenge now is the identification of quality targets. Here, computational biology ‘omics’ tools have proved to be an invaluable resource. Two of the more commonly used of these tools are genomics and metabolomics. In-vitro evolution assays followed by whole genome sequencing analysis is a popular genomics approach and helps unveil novel target genes. Plasmodium parasites are exposed to sublethal doses of a compound until an upward shift in the half-maximal inhibitory concentration (IC50), indicative of resistant parasites, is observed in the culture. Sequenced genomes of the resistant parasite clones are compared to those of the drug-naive parent to reveal genetic changes, which include both single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). While these genomic changes may point to genes encoding actual drug targets, they often reveal mediators of drug resistance or tolerance. Follow-up assays like SNP validation through gene editing are necessary to distinguish between actual targets, resistance mechanisms and random background mutations. Expectedly, genetic changes in uncharacterized Plasmodium genes are the bottle-necks in the identification of novel druggable targets. Even so, this genomics method has uncovered or reconfirmed novel antimalarial drug targets, including the proteasome, aminophospholipid-transporting P-type ATPase (PfAT-Pase2) and cGMP-dependent protein kinase (PfPKG). Metabolomic profiling and transcriptomics narrows down a compound’s mode of action. Here, parasites are treated with a compound of interest and the metabolites extracted and analyzed using liquid chromatography-mass spectrometry (LC-MS). The metabolomics fingerprint or metaprint is then compared to that of untreated parasites. While this method rarely provides the exact drug target, it narrows down the compound’s mode of action, which is valuable for target validation and characterization. The issue of non-specific or non-viable phenotype metabolite signals is easily filtered out by treating parasites with various drug concentrations and/or over a period of time. Other areas that limit the effectiveness of this tool and need to be addressed include the analysis of compounds that do not act through metabolic pathway disruption and potential host contamination. Nonetheless, metabolomics are a key player in drug discovery and have successfully been used in the study of pantothenamides (MMV689258) where the observed CoA analog buildup helped identify their mechanism of action in sequestering coenzyme A to block acetyl-CoA anabolism. Presented herein is a culmination of my graduate research in antimalarial drug discovery. Three independent projects are presented, and they all have either been published or are currently under reviewership. Chapter 1 is an introduction to malaria, a disease that has and continues to claim hundreds of thousands of lives, especially in my home continent of Africa. In chapter 2, I detail the experimental procedures used to generate the data presented in chapters 3-5. Chapter 3 is a detailed susceptibility profiling and metabolomic fingerprinting of Plasmodium falciparum asexual blood stages (ABS) to clinical and experimental antimalarials. This work, published in Cell Chemical Biology (2020), presents to the malaria research community a medium-throughput assay that can be utilized to identify new antimalarial lead compounds and novel assayable targets. Chapter 4 presents a detailed analysis of a novel ATP-binding cassette (ABC) transporter that confers pleiotropic antimalarial drug resistance in P. falciparum and that was first identified through in vitro evolution assays. This work is currently under review in Cell Chemical Biology. Chapter 5 presents work on an promising new preclinical compound, MMV688533, that provides single-dose cure and that was discovered using an innovative orthology-based screen by the Sanofi drug discovery team. In this chapter, I also present in detail the assays performed to better understand this compound’s mode of antiplasmodial action and the potential drivers of parasite resistance. This work has been accepted, pending minor textual revisions, in Science Translational Medicine. Finally in chapter 6, I summarize chapters 3-5 and share future follow-up work needed to strengthen and contextualize some of the experimental findings presented here.

Leung Wai-hang. / "November 2002." / On t.p. "33" and "42" are subscripts following the word "MSP-1" in the title. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 162-171). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.