Development of cyclodecapeptides from the tyrothricin complex as anticancer peptides

Van Wyk, Rosalind Jeanette (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: The ability of cancer cells to become simultaneously resistant to various chemotherapeutic agents, a phenomenon known as multidrug resistance, remains the cause that prevents successful treatment of cancer. The tyrocidines and analogues, produced by soil bacterium Brevibacillus parabrevis, have been shown to be active anticancer peptides. As with many other anticancer peptides, the tyrocidines are toxic to human red blood cells which limits their use as anticancer agent. Fortunately, many different strategies exist to reduce drug toxicity and improve delivery to target sites such as targeted nanocarriers or liposomal formulations. As little research has been done on the formulation and incorporation of the tyrocidines in nanocarrier systems to target cancer cells, the formulation strategies in the present study were kept simple. We set out in this basic strategy to formulate selected tyrocidines with three lipid-based molecules, cholesterol sulfate (CS), palmitic acid (C16) and lysophosphatidylcholine (LPC), with subsequent biophysical analyses and determination of biological activity of the peptides alone and in formulation. The spherical constructs formed by the Trcs was a novel discovery. This formation of the spherical structures was hampered or completely disrupted by the formulants suggesting detergent-like effects. Decreased dimers and loss of total signal of ionic species of the C16 and LPC formulations indicated the formation of stable neutral peptide-formulant complexes. On the other hand, CS formulation maintained total ionic species signal suggesting a less stable formulation. Significant blue shifts in the fluorescence spectrums were seen for the CS formulated peptides, indicating pronounced stacking of aromatic rings in a hydrophobic environment. It was clear to us that these formulants have significant effects on the oligomerisation of the peptides, which dictates their antimicrobial activity. The CS and C16 formulations significantly decreased the haemolytic activity of the peptides which was one of the main goals of this study. We confirmed that all the tyrocidines investigated in this study are highly active against three cancerous cell lines (LNCaP, C4- 2B, HT-29) Interestingly, no direct relationship between hydrophobicity or size of the peptides and the activity of the peptides towards cancer and non-cancerous cells were observed, strongly suggesting alternative targets, possibly of internal nature. Selectivity studies revealed a promising lead formulation, TpcC:C16, that has potential as alternative anticancer agent.

AFRIKAANSE OPSOMMING: Die vermoë van kankerselle om gelyktydig weerstandig te raak teen verskillende chemoterapeutiese agente, ʼn verskynsel bekend as multimedikament-weerstand, is die oorsaak wat suksesvolle kanker behandeling verhoed. Die tirosidiene en analoë, vervaardig deur die grondbakterium Brevibacillus parabrevis, is aktiewe antikanker peptiede. Soos baie ander antikanker peptiede, is die tirosidiene toksies teen menslike rooibloedselle, wat hul gebruik as antikanker agente beperk. Gelukkig bestaan daar strategieë om middel-toksisiteit te verlaag en die aflewering by teikenselle te verbeter soos byvoorbeeld nanodraers of liposomale formulasies. Omdat daar min navorsing is oor die formulering en gebruik van tirosidiene in nanodraer sisteme om kankerselle te teiken, was die formuleringstrategieë in die huidige studie eenvoudig gehou. Ons het in die eenvoudige strategie geselekteerde tirosidiene geformuleer met drie lipied- gebaseerde molekules, cholesterolsulfaat (CS), palmitiensuur (C16) en lisofosfatidielcholien (LPC), gevolg deur biofisiese analises en bepaling van biologiese aktiwiteit van die peptiede alleen en in formulasies. Die sferiese konstrukte wat deur die tirosidiene gevorm is, is ʼn nuwe ontdekking. Hierdie vorming van die sferiese konstrukte was verminder of heeltemal ontwrig deur die formulante, wat dui op ‘n detergent-agtige effek. Afname in dimere en verlies van totale sein van ioniese spesies in die C16 en LPC formulasies het die vorming van stabiele neutrale peptied-formulant komplekse aangedui. Daarteenoor is die totale sein van ioniese spesies in die CS formulasies behou wat dui op minder stabiele formulasies. Beduidende verskuiwings in die fluoressensie spektra is vir die CS formulasies waargeneem, wat uitgesproke pakking van aromatiese ringe in ʼn hidrofobiese omgewing aandui. Dit was duidelik dat die formulante beduidende effekte op die oligomerisasie van die peptiede het, wat hulle antimikrobiese aktiwiteit dikteer. Die CS en C16 formulasies verlaag hemolitiese aktiwiteit van die peptiede wat een van die hoofdoelwitte van die studie was. Ons het bevestig dat al die bestudeerde tirosidiene hoogs aktief is teen drie kankersellyne (LNCaP, C4-2B en HT-29). Geen direkte verhouding tussen die hidrofobisiteit of grootte van die peptiede en hul aktiwiteit teenoor kankerselle en normale menslike selle kon gevind word nie, wat op alternatiewe teikens dui, waarskynlik intern van natuur. Selektiwiteit studies het ʼn belowende voorloper formulasie, TpcC:C16, met potentiaal as alternatiewe antikanker agent, uitgewys.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105712
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