Investigation of Coenzyme A levels in Plasmodium falciparum to ascertain the mode of action of new antimalarial candidates

Scheepers, Melisse Sharne (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: The malaria parasite, Plasmodium falciparum, has become increasingly resistant to all commercially available drugs used in the treatment of malaria, and as such, the development of new antimalarial drugs with novel targets is of great importance. The coenzyme A (CoA) biosynthesis pathway is one such novel target since CoA and its precursor, pantothenate, have been shown to be essential for organism survival. N-phenethyl-α-methyl pantothenamide, a pantothenate analogue, has been shown in a previous study to inhibit growth in both bacteria and Plasmodium parasites, however the mode of action of this pantothenamide in Plasmodium is still unknown, and was thus investigated in this study. First, Plasmodiums’ requirement for pantothenate was investigated. We determined that parasites could survive without an extracellular source of pantothenate for up to eight days, however this contradicted what was found in literature, and was likely to be due to a Mycoplasma infection found late in the study. Secondly, it was investigated whether N-phenethyl-α-methyl pantothenamide can be metabolized to its CoA antimetabolites by the CoA biosynthetic enzymes present in P. falciparum. This was done by investigating the metabolism of the compound in both cell lysates and in in vivo P. falciparum cell cultures and it was found that PfPanK and PfDPCK is active in parasite lysates, while PfPPAT is inactive in parasite lysates. We could therefore not determine if the pantothenamide under investigation is being metabolized in the parasite by using lysates, but this is the first demonstration of the activity of PfDPCK in parasites lysate. Finally, we wanted to investigate the effect of tricyclic methylthiophenyl propanamide (TMP), a non-pantothenate analogue that inhibits the CoA biosynthesis pathway in other organisms, on P. falciparum proliferation. TMP was synthesized to use as a tool to investigate the mechanism of action of N-phenethyl-α-methyl pantothenamide to support that pantothenamides do not inhibit pantothenate kinase, as is known for TMP, but are rather metabolized downstream in the pathway. TMP was successfully synthesized and purified, however yields were too low to test TMP as an inhibitor of P. falciparum proliferation. Not only did the work done in this study shed more light on the mode of action of pantothenamides in P. falciparum, but also gave valuable insight into parasite biochemistry.

AFRIKAANSE OPSOMMING: Die malaria parasite, Plasmodium falciparum, het tot dus ver weerstand opgebou teen alle kommersiële beskikbare middele vir die behandeling van malaria. Die ontwikkelling van nuwe middele teen malaria wat ‘n uitwerking het op nuwe teikens in the parasite is van die uiterste belang. Die koënsiem A (KoA) biosintese padweg is een so 'n spesifieke teiken, aangesien KoA en sy voorloper, pantoteensuur, essensieël is vir die organisme se oorlewing. N-fenetiel-α-metiel-pantoteenamied, ‘n pantoteensuur-analoog wat reeds in vorige studies inhibisie van bakteriële- en Plasmodium parasiet-groei tot gevolg gehad het is in hierdie studie ondersoek. Meer spesifiek, die metode van werking van hierdie Eerstens het ons P. falciparum se pantoteensuur vereistes ondersoek. Ons het bepaal dat die parasiete tot en met agt dae kan oorleef sonder 'n ekstrasellulêre bron van pantoteensuur, maar dit is teenstrydig met wat ons in die literatuur vind, en is waarskynlik as gevolg van ‘n Mikoplasma infeksie wat ons eers laat in die studie ontdek het. Tweendens het ons bepaal of N-fenetiel-α-metiel-pantoteenamied omgesit word na die ooreenstemmende KoA-antimetaboliete deur die ensieme van die KoA biosintese padweg teenwoordig in P. falciparum. Die bepaling is gedoen deur die aktiwiteit van die ensieme te ondersoek in beide sellisaat en in in vivo P. falciparum selkulture. Daar is bevind dat PfPanK en PfDPCK in parasiet sellisaat aktief is, terwyl PfPPAT onaktief is. Ons kon dus nie bepaal of die pantoteenamied wat ons ondersoek deur die parasiet lisaat gemetaboliseer word nie, maar dit was die eerste bewyse van PfDPCK in parasiet sellisaat. Laastens, is die effek van trisikliese metieltiofeniel-propaanamied (TMP), 'n nie-pantoteensuur analoog wat die KoA biosintese padweg inhibeer in ander organismes, ondersoek as inhibitor van P. falciparum. TMP is gesintetiseer ter ondersteuning van die meganisme van aksie van N-fenetiel-α-metiel-pantoteenamied. Spesifiek wou ons toon dat the pantoteenamiede nie pantoteensuurkinase inhibeer soos TMP nie, maar eerder verder af in die padweg gemetaboliseer word. Die sintese en suiwering van TMP was suksesvol, maar die opbrengs was te laag om TMP te toets as ‘n inhibitor van P. Falciparum groei. Hierdie studie sal meer lig werp op die metode van werking wat van toepassing is op pantoteenamiede en meer inligting verskaf omtrent die biochemie van die parasite.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101312
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