Towards the development of a medium-throughput assay to investigate the kinetics of β-Haematin formation in the presence of divers inhibitors

Fitzroy, Sharne-Mare (2015-12)

Thesis (MSc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: A new, improved-throughput lipid-water interface assay was developed in which the inhibition of β-haematin formation by diverse inhibitors was investigated. Monopalmitoyl-rac-glycerol was used as a model lipid, and following the introduction of the inhibitors into the aqueous buffer layer, β-haematin inhibitory activity was investigated under biologically-relevant conditions. Clinically-relevant antimalarial drugs, namely chloroquine, amodiaquine, quinidine, quinine and mefloquine were used in order to validate the newlyoptimized assay as a means of assaying drug activity. Having developed this more efficient assay, a larger set of compounds, including short-chain chloroquine analogues and a series of benzamide non-quinoline inhibitors, were successfully introduced into the system. The IC50 values determined for the inhibition of β-haematin formation through this newly-optimized assay show good correlations with previously-determined IC50 values, also determined in the lipid-mediated system, as well as biological activities determined against chloroquine-sensitive strains of Plasmodium falciparum. Furthermore, the effect of each antimalarial drug, short-chain chloroquine analogue and benzamide nonquinoline compound on the kinetics of β-haematin formation was investigated in the lipid-mediated system. A theoretical kinetic model, which is based on the Avrami equation and the Langmuir isotherm, was used to analyse the experimental data. Importantly, it has been possible to extract equilibrium adsorption constants (Kads) for each compound, which provides a quantitative measure of the strength of interaction between an inhibitor and the surface of growing β-haematin. Thus, the experimental data support a mechanism of inhibitor action via adsorption for both quinoline- and non-quinoline inhibitors. An important observation made during this study suggests that both Kads and the rate of the proposed precipitation of an inhibitor-Fe(III)PPIX complex (k2) have an effect on the IC50 value of an inhibitor. While the latter process was not investigated in the current work, the interplay between the two parameters appears to be of uttermost importance in determining the overall activity of a family of β-haematin inhibitors. Finally, the direct adsorption of an inhibitor to preformed β-haematin crystals was investigated in independent studies in order to support the conclusions drawn from the kinetics studies. A decrease in the absorbance of a solution was observed in all cases when preformed β-haematin was added, which was attributed to the physical adsorption of the inhibitor to the crystals. From these studies an adsorption constant was extracted which indicated a good correlation with the inhibitory activity and Kads values determined in the kinetics experiments. The work in this research project provides important insight into the possible mode of β-haematin inhibition by diverse inhibitors. If the interactions that aid the adsorption of compounds to surface binding sites are identified, the insight will be invaluable in the rational design of novel haemozoin inhibitors.

AFRIKAANSE OPSOMMING: ‘n Nuwe, verbeterde-deurset lipied-water tussenfase toets is ontwikkel waarin die inhibisie van β-haematien vorming deur verskillende inhibeerders ondersoek was. Monopalmitiengliserol was gebruik as ‘n model-lipied, en deur die inhibeerders in die waterige buffer laag in te sluit, was dit moontlik om die β-haematien inhiberende aktiwiteit onder biologies-relevante kondisies the ondersoek. Klinies-relevante antimalaria-middels, naamlik chlorokien, amodiakien, kinidien, kinien en meflokien was gebruik om die nuwe geoptimaliserende toets te evalueer as ‘n middel van toetsing van inhiberende aktiwiteit. Deur die ontwikkeling van hierdie meer doeltreffende toets, was dit moontlik om ‘n groter hoeveelheid verbindings, insluitend kort-ketting chlorokien analoë en ‘n reeks bensamied nie-kinolien inhibeerders, in die sisteem te inkorporeer. Die IC50 waardes bepaal vir die inhibisie van β-haematien vorming deur hierdie nuwe geoptimaliserende toets, dui op goeie korrelasies met voorheen bepaalde IC50 waardes, óók bepaal in die lipied-beheerde sisteem, asook die biologiese aktiwiteite bepaal teen chlorokien-sensitiewe stamme van Plasmodium falciparum. Verder is die effek van elke antimalarial-middel, kort-ketting chlorokien analoog en bensamied nie-kinoline inhibeerder op die kinetika van β-haematien vorming ondersoek in die lipied-beheerde sisteem. ‘n Teoretiese kinetiese model wat gebaseer is op die Avrami-vergelyking en die Langmuir-isoterm was gebruik om die eksperimentele data te analiseer. Dit was moontlik om ‘n ewewigs-adsorpsiekonstante (Kads) vir elke inhibeerder te bepaal. Die Kads waarde is ‘n kwantitatiewe meting van die sterkte van die interaksie tussen ‘n inhibeerder en die oppervlakte van groeiende β-haematien. Dus ondersteun die eksperimentele data ‘n meganisme van inhibeerder aksie deur adsorpsie deur beide die kinolien en nie-kinolien inhibeerders. ‘n Belangrike waarneming tydens die studie dui daarop dat beide Kads en die koers van die voorgestelde neerslagreaksie van ‘n inhibeerder-Fe(III)PPIX kompleks (k2) ‘n effek op die IC50 waarde van ‘n inhibeerder het. Terwyl die laasgenoemde proses nie ondersoek was in die huidige studie nie, blyk dit dat die wisselwerking tussen hierdie twee parameters die algehele aktiwiteit van ‘n familie van inhibeerders bepaal. Die direkte adsorpsie van ‘n inhibeerder aan reeds-gevormde β-haematien kristalle was ondersoek in onafhanklike studies om die gevolgtrekkings van die kinetika studies te ondersteun. ‘n Daling in die absorbansie van ‘n oplossing was waargeneem in alle gevalle wanneer reeds-gevormde β-haematien daarby gevoeg was. Hierdie waarneming is toegeskryf aan die fisiese adsorpsie van die inhibeerder aan die kristalle. Vanuit hierdie studies was ‘n adsorpsiekonstante bepaal wat goeie korrelasies met die inhiberende aktiwiteit en Kads waardes, verkry in die kinetika eksperimente, aandui. Die werk in hierdie navorsings projek verleen belangrike insig in die moontlike manier van β-haematien inhibisie deur verskillende inhibeerders. As die interaksies wat bydra tot die adsorpie van verbindings aan die oppervlak-bindings punte geïdentifiseer word, sal die insig van hierdie werk belangrik wees in die ontwikkeling van nuwe haemasoïen inhibeerders.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/97737
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