Characterisation of small cyclic peptides with antilisterial and antimalarial activity

Leussa, Nyango-Nkeh Adrienne (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Antimicrobial peptides (AMPs) are currently the most researched group of compounds for new antimicrobial drugs especially with the rise in resistance to almost all available drugs by public health relevant pathogens. In this study we set out to characterise small cyclic AMPs in terms of their activity towards human pathogens Listeria monocytogenes, a food-borne pathogen causing listeriosis and Plasmodium falciparum, a parasite that causes malaria respectively, each a threat to public health. One of the small cyclic peptide libraries examined is the tyrocidines (Trcs) and analogues, which are cyclic decapeptides [cyclo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu] produced by the Gram-positive bacteria Bacillus aneurinolyticus as part of the tyrothricin complex which is non-ribosomally synthesised during sporulation. Previous research found that the six major Trcs were active against Listeria monocytogenes and Plasmodium falciparum and it was found that the identity of the aromatic residues in the aromatic dipeptide unit has an important role in activity. We set out to extend the qualitative structure to activity relationship (QSAR) studies using more Trc analogues and small synthetic Arg- and Trp-rich cyclic peptides (RW-peptides) in a bid to establish essential structural motifs and pre-requisites for activity. Eight natural and three synthetic Trc analogues and fifteen RW-peptides were either naturally or by chemical synthesis produced and characterised in terms of chemical character and biological activity. The Trcs were significantly more active than RW peptides, although much more haemolytic and thus toxic. Results indicated the relevance for hydrogen bonding with an aromatic amino acid residue for selective activity towards the leucocin A resistant L. monocytogenes B73-MR1. However, structural properties favouring a tighter membrane interaction hindered the Trc mode of action (MOA). We determined that Gln6 and hydroxyl group of Tyr7 may be involved in interaction with the putative target in L. monocytogenes. There was also need for an amphipathic balance between hydrophobicity and size/steric parameters for optimal activity. From our QSAR studies we predict as lead peptide for a future library of antilisterial Trcs: cyclo(VOMe3LfPWfNQY). Furthermore, the antilisterial activity of the Trcs was found to be predominantly lytic and salt tolerant while RW-peptides were non-lytic and sensitive to Ca2+. We confirmed that Ca2+ enhanced Trc antilisterial activity with Ca2+ increasing the Trc anti-metabolic activity, but conversely inducing a non-lytic mechanism of action. From model membrane studies, we propose that the calcium induced Trc non-lytic MOA could be due to detrimental lipid demixing, presence of a Trc sensitive Ca2+-induced non-membrane target in the prematurely calcium induced intracellular anaerobic form of Listeria monocytogenes, and/or the Trc-Ca2+ complexes may inhibit key components such as membrane bound electron transport system or bacterial dehydrogenases. We confirmed, as previously found, that the Trcs have potent antimalarial activity that is sequence specific and non-lytic. The RW-peptides had very weak activity, but our results again indicated that more hydrophobic and haemolytic peptides tend to be more active, particularly the RW-peptide containing the Trp analogue β-(benzothien-3-yl)-alanine (Bal). A novel finding was that one of the more polar Trc C analogues, namely tryptocidine C (Tpc C), in contrast to Trc C showed potent antimalarial activity indicating the specific sequence and the role of the Trp7 in activity. From these results a proposed lead peptide for future research is cyclo[VOLfP(Bal)fNQ(Bal)]. Furthermore, in our search for the Trc and Tpc C target(s) we employed high resolution fluorescence microscopy. Results show that Trc led to disorganisation of neutral lipid structures and chromatin halting growth in late trophozoite/early schizont stages. This indicated that membrane structures containing neutral lipids, as well as chromatin may be targeted by the Trcs. Another novel finding in our studies was that chloroquine (CQ) resistance not only correlated with resistance to Trcs, but the Trcs and CQ were found to be antagonistic towards each other’s activity. This indicated a shared target and we propose the food vacuole as another of the Trc targets in P. falciparum.

AFRIKAANSE OPSOMMING: Antimikrobiese peptiede (AMPe) is tans die mees nagevorsde groep verbindings in die soeke na nuwe antimikrobiese middels, veral weens 'n toenemende weerstandigheid van patogene in die openbare gesondheidsektor teen alle beskikbare middels. Die doel van hierdie studie was om klein, sikliese AMPe in terme van hul aktiwiteit teenoor twee menslike patogene wat 'n bedreiging vir openbare gesondheid is, Listeria monocytogenes, 'n voedsel-oordraagbare patogeen wat listeriose veroorsaak, asook Plasmodium falciparum, die parasiet verantwoordelik vir malaria, te karakteriseer. Een van die klein, sikliese peptiedbiblioteke wat ondersoek is, is die tyrocidines (Trcs) en analoë (sikliese dekapeptiede [siklo-(D-Phe-Pro-(Phe/Trp)-D-Phe/DTrp)-Asn-Gln-(Tyr/Phe/Trp)-Val- (Orn/Lys)-Leu]). Hierdie peptiede deur die Gram-positiewe bakterie Bacillus aneurinolyticus word wat nie-ribosomaal gesintetiseer as deel van die tirotrisien kompleks word tydens sporulasie. Vorige navorsing het gewys dat die ses hoof Trcs teen Listeria monocytogenes en Plasmodium falciparum aktief is en dat die identiteit van die aromatiese residue in die aromatiese dipeptiedeenheid 'n belangrike rol speel in die Trc-aktiwiteit. Ons het gepoog om die kwalitatiewe struktuur-aktiwiteit-verwantskap (QSAR) studies uit te brei deur meer Trc analoë en klein sintetiese Arg- en Trp-ryke sikliese peptiede (RW-peptiede) te gebruik en sodoende essensiële struktuur-motiewe en voorvereistes vir aktiwiteit vas te stel. Agt natuurlike en drie sintetiese Trc analoë, asook vyftien RW-peptiede is of deur natuurlike of chemiese sintese geproduseer en gekarakteriseer in terme van chemiese karakter en biologiese aktiwiteit. Die Trcs het beduidend meer aktiwiteit as RW-peptiede getoon, maar is ook meer hemolities en dus meer toksies. Die resultate dui op die belang van waterstofbinding met 'n aromatiese aminosuurresidu vir die selektiewe aktiwiteit teenoor die leucocin A weerstandige L. monocytogenes B73-MR1. Strukturele eienskappe wat tot 'n sterker membraan-interaksie lei, verhinder egter die werkingsmeganisme. Ons het vasgestel dat Gln en die hidroksielgroep van Tyr betrokke kan wees in die interaksie met die vermeende teenmiddelteiken in L. monocytogenes. 'n Balans tussen amfipatiese/hidrofobiese en grootte/steriese parameters is ook noodsaaklik vir optimale aktiwiteit. Vanuit ons QSAR studies word die peptied siklo-(VOMe3LfPWfNQY) as die voorloper vir 'n toekomstige peptiedbiblioteek van antilisteriale Trcs voorgestel. Verder is daar gevind dat die antilisteriese aktiwiteit van die Trcs oorwegend lities en sout-verdraagsaam is, terwyl die RW-peptiede nie-lities en Ca2+ sensitief is. Ons het bevestig dat Ca2+ die Trc antilisteriese aktiwiteit verbeter, deur die Trc se antimetaboliese aktiwiteit verhoog, maar terselfdertyd 'n nie-litiese werkingsmeganisme induseer. Vanuit model-membraan studies word voorgestel dat Trc se nie-litiese werkingsmeganisme, soos teweeggebring deur Ca2+, die gevolg kan wees van nadelige lipied vermenging, die teenwoordigheid van 'n kalsium geïnduseerde Trcsensitiewe nie-membraan teiken in 'n vervroegde kalsium geïnduseerde intrasellulêre anaerobiese vorm van Listeria monocytogenes, en/of dat die Trc-Ca2+ komplekse belangrike komponente soos ’n membraan-gebonde elektron transport sisteem of bakteriële dehidrogenases inhibeer. Daar is ook bevestig, soos voorheen gevind, dat die Trcs kragtige, antimalaria aktiwiteit besit wat volgorde-spesifiek en nie-lities is. Die RW-peptiede het swak aktiwiteit getoon, maar ons resultate het weereens bewys dat peptiede wat meer hidrofobies en hemolities is, meer aktief is, veral die RW-peptiede wat die Trp analoog β-(bensoteïen-3-iel)-alanien (Bal) bevat. 'n Nuwe bevinding is dat een van die meer polêre Trc C analoë, genaamd triptosidien C (Tpc C), in teenstelling met Trc C, sterk antimalaria aktiwiteit het, wat 'n aanduiding is van die spesifieke volgorde en die rol van die Trp7 in aktiwiteit. Vanuit hierdie bevindinge word die peptied siklo- (VOLfP(Bal)fNQ(Bal)) as 'n voorloper vir toekomstige navorsing aangedui. Vir ons soeke na die Trc en Tpc C teiken(s), het ons hoë resolusie fluoressensie mikroskopie aangewend. Resultate toon dat Trc tot die ontwrigting van 'n neutrale lipied strukture en chromatien lei en sodoende groei beperk in die laat trofosoïet/vroeë skisont fases. Dit het aangedui dat die membraanstrukture wat neutrale lipiede bevat, sowel as chromatien, deur die Trcs geteiken word. 'n Verdere nuwe bevinding in hierdie studie was dat chloroquine (CQ) weerstandigheid nie net korreleer met weerstandigheid teen Trcs nie, maar dat die Trcs en CQ antagonisties optree teenoor mekaar se aktiwiteite. Dit dui op 'n gemeenskaplike teiken en die kosvakuool as 'n addisionele Trc teiken in P. falciparum word voorgestel.

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