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Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum
Thesis (PhD)--Stellenbosch University, 2022.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2022
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| _version_ | 1867613805002883072 |
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| access_status_str | Open Access |
| author | Kok, Michelle |
| author2 | De Villiers, Marianne |
| author_browse | De Villiers, Marianne Kok, Michelle |
| author_facet | De Villiers, Marianne Kok, Michelle |
| author_sort | Kok, Michelle |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2022. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/126285 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:41:58.332Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/126285 Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum Kok, Michelle De Villiers, Marianne Strauss, Erick Stellenbosch University. Faculty of Science. Dept. of Biochemistry. Plasmodium falciparum Pantothenic acid -- Metabolism Malaria -- Prevention Drug resistance Coenzymes Enzyme inhibitors UCTD Thesis (PhD)--Stellenbosch University, 2022. ENGLISH ABSTRACT: The rise in drug-resistant Plasmodium falciparum parasites necessitates novel compounds and drug targets in the quest to produce new antimalarials. Analogues of pantothenate, an essential vitamin for parasite survival, inhibit the proliferation of blood-stage parasites. One class of analogues, N- substituted pantothenamides (PanAms), have excellent potencies in the low nanomolar range (IC50 <50 nM) with low toxicity towards human cells. Some PanAms inhibit other life cycle stages of the parasite, making them effective parasite-to-host transmission blocking agents. PanAm inhibition is based on pantothenate acting as precursor of the de novo biosynthesis of coenzyme A (CoA), an essential metabolic cofactor and universal acyl carrier. Although the PanAms hold great promise for the development as antiplasmodial agents their exact mode of action is unclear. Various PanAms have been shown to act as alternative substrates to CoA biosynthesis to form CoA antimetabolites. These antimetabolites affect CoA utilising enzymes in various parasite cellular compartments, including the apicoplast, mitochondrion, cytosol, and nucleus. To pinpoint the exact point of inhibition is therefore complex. This project focusses on elucidating the mechanism of action of N-phenethyl-α-methyl- pantothenamide (N-PE-αMe-PanAm), the most potent lead compound available in our laboratory. A fluorescent probe strategy was developed to explore CoA utilization in P. falciparum metabolism. Novel dansyl and coumarin fluorescent PanAm derivatives were successfully synthesised and used as metabolic probes in parasite-infected erythrocytes to determine organelle accumulation and localisation. Similar to N-PE-αMe-PanAm, resistance towards pantetheinase (an enzyme present in serum that degrades PanAms) and low micromolar range (8.6 - 307 µM) antiplasmodial activity was observed. Confocal microscopy confirmed that the probes localise to areas of CoA utilisation, although none of the PanAms selectively accumulated to a single organelle. These findings established fluorescent αMe-PanAms as useful tools to study CoA metabolism in vivo. The PanAms tested on P. falciparum were mixtures of the α-methyl diastereomers. Therefore, the influence of the orientation of the α-methyl group on the stability and potency of the parent PanAm was investigated. The α-methyl moiety confers an important role in the mode of action of these compounds beyond improving stability, with the 2S,2′R-diastereomer displaying the best activity (23 nM) against the parasites. Encouragingly, preliminary tests indicated that the PanAms hold promise as potent dual active antimalarials by also targeting gametocyte and gamete stage parasites which is involved in the transmission of the parasite to the mosquito host. Acetyl-CoA synthetase (ACS), situated in the cytosol, is essential during the blood-stage of the parasite’s life cycle. To confirm whether our lead compound targets PfACS three novel CoA antimetabolites were produced and successfully characterised. Due to the unsuccessful expression of PfACS, the Saccharomyces cerevisiae counterpart (ScACS) was utilised as a model for the inhibition. Full kinetic characterisation of ScACS was performed for the first time followed by inhibitions studies with the CoA antimetabolites which did not appreciably inhibit the enzyme. During this study our understanding of the mode of action of αMe-PanAms and how they influence CoA availability for CoA-dependent processes in blood-stage P. falciparum parasites were expanded. The findings support the advancement of new CoA-directed therapeutics to address the current need for novel antimalarials to combat the disease. AFRIKAANSE OPSOMMING: Die toename in middel-weerstandige Plasmodium falciparum parasiete noodsaak nuwe verbindings en sellulêre teikens in die strewe na nuwe malaria teenmiddels. Analoë van pantotenaat, 'n noodsaaklike vitamien vir parasietoorlewing, inhibeer die groei van bloedstadium parasiete. Een klas analoë, N-gesubstitueerde pantoteenamiede (PanAme), het uitstekende sterkte in die lae nanomolare gebied (IC50 <50 nM) met lae toksisiteit teenoor menslike selle. Sommige PanAme inhibeer ander lewensiklusstadia van die parasiet, wat hulle effektiewe parasiet-tot-gasheer- oordrag blokkerende middels maak. PanAm-inhibisie is gebaseer op pantotenaat wat optree as voorloper van die de novo biosintese van koënsiem A (KoA), 'n noodsaaklike metaboliese kofaktor en universele asieldraer. Alhoewel die PanAme groot belofte inhou vir ontwikkeling as antiplasmodiese middels, is hul presiese werkingswyse onduidelik. Daar is getoon dat verskeie PanAme as alternatiewe substrate vir KoA- biosintese optree om KoA-antimetaboliete te vorm. Hierdie antimetaboliete beïnvloed KoA- benuttende ensieme in verskeie sellulêre kompartemente van die parasiet, insluitend die apikoplast, mitochondrium, sitosol en selkern. Om die presiese punt van inhibisie vas te stel is dus kompleks. Hierdie projek fokus op die verstaan van die werkingsmeganisme van N-fenetiel-α-metiel- pantoteenamied (N-PE-αMe-PanAm), die sterkste leier verbinding beskikbaar in ons laboratorium. 'n Fluoressensieprobe strategie is ontwikkel om KoA-benutting in P. falciparum-metabolisme te ondersoek. Nuwe dansiel- en koemarien-fluoresserende PanAm-derivate is suksesvol gesintetiseer en as verken-molekules in parasiet-geïnfekteerde eritrosiete gebruik om organel-akkumulasie en lokalisering te bepaal. Soortgelyk aan N-PE-αMe-PanAm, is weerstand teen pantetenase ('n ensiem teenwoordig in serum wat PanAme afbreek) en lae mikromolare (8.6 – 307 µM) antiplasmodiale aktiwiteit waargeneem. Konfokale mikroskopie het bevestig dat die molekules lokaliseer na areas van KoA-benutting, alhoewel nie een van die PanAme selektief tot 'n enkele organel opgehoop het nie. Hierdie bevindinge het fluoresserende αMe-PanAme gevestig as nuttige hulpmiddels om KoA- metabolisme in vivo te bestudeer. Die PanAme getoets op P. falciparum was mengsels van die α-metiel diastereomere. Dus is die invloed van die oriëntasie van die α-metielgroep op die stabiliteit en sterkte van die ouer PanAm ondersoek. Die α-metiel eenheid verleen 'n belangrike rol in die werkingswyse van hierdie verbindings, buiten die verbetering van stabiliteit, met die 2S,2'R-diastereomeer wat die beste aktiwiteit (23 nM) teen die parasiete toon. Dit is bemoedigend dat voorlopige toetse aangedui het dat die PanAme belofte inhou as kragtige dubbele aktiewe antimalariamiddels deur ook gametosiet- en gameetstadiumparasiete te teiken wat betrokke is by die oordrag van die parasiet na die muskietgasheer. Asetiel-KoA sintetase (AKS), geleë in die sitosol, is noodsaaklik tydens die bloedstadium van die parasiet se lewensiklus. Om te bevestig of ons hoofverbinding PfAKS teiken, is drie nuwe KoA- antimetaboliete geproduseer en suksesvol gekarakteriseer. As gevolg van die onsuksesvolle uitdrukking van PfAKS, is die Saccharomyces cerevisiae eweknie (ScAKS) as 'n model vir die inhibisie gebruik. Volle kinetiese karakterisering van ScAKS is vir die eerste keer uitgevoer, gevolg deur inhibisie studies met die KoA antimetaboliete wat nie die ensiem noemenswaardig geïnhibeer het nie. Tydens hierdie studie is ons begrip van die werkingswyse van αMe-PanAme en hoe hulle KoA beskikbaarheid vir KoA-afhanklike prosesse in bloedstadium P. falciparum parasiete beïnvloed, uitgebrei. Die bevindinge ondersteun die bevordering van nuwe KoA-gerigte terapieë om die huidige behoefte aan nuwe antimalariamiddels aan te spreek om die siekte te bekamp. Doctorate 2022-10-14T11:28:42Z 2023-01-23T06:50:25Z 2023-05-03T03:00:08Z 2022-12 Thesis http://hdl.handle.net/10019.1/126285 en_ZA Stellenbosch University 256 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Plasmodium falciparum Pantothenic acid -- Metabolism Malaria -- Prevention Drug resistance Coenzymes Enzyme inhibitors UCTD Kok, Michelle Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum |
| title | Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum |
| title_full | Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum |
| title_fullStr | Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum |
| title_full_unstemmed | Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum |
| title_short | Studies on the metabolism of N-substituted pantothenamides in blood-stage Plasmodium falciparum |
| title_sort | studies on the metabolism of n substituted pantothenamides in blood stage plasmodium falciparum |
| topic | Plasmodium falciparum Pantothenic acid -- Metabolism Malaria -- Prevention Drug resistance Coenzymes Enzyme inhibitors UCTD |
| url | http://hdl.handle.net/10019.1/126285 |
| work_keys_str_mv | AT kokmichelle studiesonthemetabolismofnsubstitutedpantothenamidesinbloodstageplasmodiumfalciparum |