Browsing by Author "Fitzroy, Sharne-Mare"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
- ItemTowards the development of a medium-throughput assay to investigate the kinetics of β-Haematin formation in the presence of divers inhibitors(Stellenbosch : Stellenbosch University, 2015-12) Fitzroy, Sharne-Mare; De Villiers, Katherine A.; Stellenbosch University. Faculty of Science. Dept. of Chemistry and Polymer Science.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.