Investigation of the antifungal activity of tryptophan-rich cyclic peptides

Mamhende, Precious Gamuchirai Muchaneta (2019-01)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: The global population of immunocompromised individuals has been rising due to the immunosuppressive nature of advanced medical interventions such as chemotherapy for cancer and immunosuppressive conditions such as HIV infection. Concurrent with this is the increasing incidence of life threatening systemic fungal infections as well as the growing resistance towards existing antifungal drugs. As a result, there is an urgent need for the discovery and development of novel drugs of antifungal therapy. Antimicrobial peptides present as promising candidates for this role due to their broad-spectrum antimicrobial activity and the limited potential for the evolution of resistance against them. In this study, the potential of small AMPs to serve as therapeutic agents against fungal infections was investigated by characterising the antifungal activity and mode of action of natural cyclodecapeptides and synthetic RW-hexapeptides against a human fungal pathogen, Aspergillus fumigatus. In addition, biophysical characterisation of their structures was conducted to evaluate the role of structure in antifungal activity. Biophysical characterisation of the secondary structure of cyclodecapeptides confirmed their conserved β-sheet structure. Structural characterisation of cyclic RW-hexapeptides highlighted their secondary structure to be quite stable and to consist of β-sheet and β-turns. On the other hand, the secondary structure of the linear analogues was shown to be flexible, changing from highly irregular (undefined) structures to more defined structures as the polarity of the environment was altered. The cyclodecapeptides and RW-hexapeptides potently inhibited the germination and growth of A. fumigatus spores highlighting the potential of both peptide groups as antifungal agents. However, the cyclodecapeptides exhibited pronounced haemolytic activity which compromised their selectivity for the fungal target. In contrast, the RW-hexapeptides had little to no haemolytic activity which translated to a greater selectivity for the fungal pathogen. A partial characterisation of the mode action employed by the cyclodecapeptides and the RWhexapeptides was done. The cyclodecapeptide, tryptocidine C (WC), rapidly induced the uptake of the membrane impermeable dye, SYTOX Green (SG) in A. fumigatus hyphae. This result confirmed the previously described membranolytic mode of action of the cyclodecapeptides. In contrast, RW-hexapeptides were not as effective at inducing the uptake of SG, indicating that membrane lysis is not their principal mode of action. Thus, the mode of action of RW-hexapeptides remains unknown but ROS could potentially be involved in the antifungal mechanism of action as one of the cyclohexapeptides, cWWW, significantly increased the accumulation of endogenous ROS in A. fumigatus hyphae. Furthermore, the slightly antagonistic interactions between WC and the most active RW-hexapeptide analogues, especially cWWW, in the peptide combination studies could indicate that the two groups of peptides may share a target but act on it differently. While RW-hexapeptides may not be lytic, the membrane could still be their target meaning that the lytic activity of WC prevents them from acting on their target hence the antagonistic interactions. In conclusion, the demonstrated selectivity of the RW-hexapeptides renders them very attractive for potential development as antifungal agents for systemic use.

AFRIKAANSE OPSOMMING: Die globale bevolking van individue met 'n onderdrukte immuunstelsel het gestyg as gevolg van die immunonderdrukkende aard van gevorderde mediese ingrypings soos chemoterapie vir kanker en immunonderdrukkende toestande soos MIV-infeksie. Gepaardgaande hiermee is daar 'n toename in die voorkoms van lewensbedreigende sistemiese swaminfeksies asook 'n toename weerstand teen bestaande antifungale middels. As gevolg hiervan is daar 'n dringende behoefte aan die ontdekking en ontwikkeling van nuwe middels vir antifungiese behandelings. Antimikrobiese peptiede (AMPe) is belowende kandidate vir hierdie rol weens hul breë spektrum antimikrobiese aktiwiteit en die beperkte potensiaal vir die ontwikkeling van weerstand. In hierdie studie is die potensiaal van klein AMPe om as middels teen swaminfeksies te dien, ondersoek deur die antifungale aktiwiteit en werking van natuurlike siklodekapeptiede en sintetiese RW-heksapeptiede teen 'n menslike swampatogeen, Aspergillus fumigatus, te karakteriseer. Daarbenewens is die biofisiese karakterisering van hul strukture uitgevoer om die rol van struktuur in antifungale aktiwiteit te evalueer. Biofisiese karakterisering van die sekondêre struktuur van siklodekapapiede het hul bewaarde β-plaatstruktuur bevestig. Strukturele karakterisering van die RW-heksapeptiede het beklemtoon dat die sekondêre struktuur van die sikliese analoë redelik stabiel is en uit reëlmatige sekondêre struktuurelemente (β-plaat en β-draaie) bestaan. Daarteenoor is die sekondêre strukture van die lineêre analoë meer buigsaam, wat vanaf hoogs onreëlmatige (ongedefinieerde) strukture tot meer gedefinieerde strukture omsskakel, soos die polariteit van die omgewing verander. Die siklodekapeptiede en RW-heksapeptiede verhinder moontlik die ontkieming en groei van A. fumigatus-spore, wat die potensiaal van beide peptiedgroepe as antifungale middels beklemtoon. Die siklodekapeptiede het egter uitgesproke hemolitiese aktiwiteit getoon, wat hul selektiwiteit vir die swamteiken benadeel het. Daarteenoor het die RW-heksapeptiede min of geen hemolitiese aktiwiteit getoon wat 'n groter selektiwiteit vir die swampatogeen beteken. 'n Gedeeltelike karakterisering van die meganisme van aksie wat deur die siklodekapeptiede en die RW-heksapeptiede gebruik word, is gedoen. Die siklodekapeptiede, triptosidien C (WC), het 'n vinnige opname van die membraan-deurdringbare kleurstof, SYTOX Groen (SG) in A. fumigatus hife veroorsaak. Die resultaat het die voorheen beskryfde litiese membraan werking van die siklodekapeptiede bevestig. In teenstelling hiermee het RW-heksapeptiede nie effektief die opname van SG veroorsaak nie, wat aandui dat membraanlise nie hul hoofmeganisme van aksie is nie. Die werkingsmeganisme van RW-heksapeptiede bly dus onbekend, maar ROS (reaktiwe suurstof spesies) kan moontlik by die antifungale aktiwiteit betrokke wees, aangesien die sikloheksapeptied, cWWW, die toename van endogene ROS in A. fumigatus hife aansienlik verhoog het. Verder kan die effense antagonistiese interaksies tussen WC en die twee aktiefste sikoheksapeptiede, spesifiek cWWW, in die peptiedkombinasie studies aandui dat die twee groepe peptiede 'n teiken kan deel, maar verskil in interaksie. Terwyl RW-heksapeptiede nie lities is nie, kan die membraan steeds hul teiken wees, wat beteken dat die litiese aktiwiteit van WC hulle verhoed om op hul teiken te reageer, wat die antagonistiese interaksies tot gevolg het. As afsluitende gevolgtrekking maak gedemonstreerde selektiwiteit van die RW-heksapeptiede hulle baie aantreklik vir die potensiële ontwikkeling vir sistemiese gebruik as antifungiese middels.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105718
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