Effect of Inducers, Incubation Time and Heme Concentration on IC50 Value Variation in Anti-heme Crystallization Assay

Abstract

Heme detoxification through crystallization into hemozoin has been suggested as a good target for the development of screening assays for new antimalarials. However, comparisons among the data obtained from different experiments are difficult, and the IC50 values (the concentrations of drug that are required to inhibit 50% of hemozoin formation) for the same drug vary widely. We studied the effects of changes in heme concentration (precursor of β-hematin), incubation time and three inducers (SDS, Tween 20 and linoleic acid) on the IC50 of some antimalarials (chloroquine, quinine, amodiaquine, and clotrimazole). The results showed that increasing both inducer concentration and incubation time raised the IC50 of selected antimalarials. Any change in those factors caused the IC50 value to vary. Standardization of assay conditions is, therefore, necessary to increase reproducibility and reduce discrepancies in assay performance. Considering all of the variables, the best choice of inducers is in the order of SDS > Tween 20 > linoleic acid.

Quinoline Drug–Heme Interactions and Implications for Antimalarial Cytostatic versus Cytocidal Activities

“…Inhibition of Hz formation by CQ and other quinoline antimalarials has been demonstrated under a variety of conditions. ,,− As described above, a number of interactions of these drugs with precrystalline forms of heme that can conceivably bring about such inhibition have been characterized by various spectroscopic methods, particularly in the case of CQ. − ,,,,− ,− Several tube-based and plate-based high-throughput assays have been developed to quantify the ability of a particular compound to inhibit Hz formation in vitro ,− (Table ). Typically, solutions with varying concentrations of the drug are incubated with a fixed concentration of FPIX under conditions that produce maximal Hz yield measured in the absence of drug, with the aim of determining the drug concentration at which 50% of Hz formed under control conditions is inhibited (Hz inhibitory IC 50 or β-hematin inhibitory activity (BHIA) IC 50 ).…”

Aktivitas Antimalaria Berbasis Penghambatan β-Hematin dalam Ekstrak Air Daun Jung Rahab (Baeckea frutecens L)

“…Hemozoin secara struktur identik terhadap β-hematin yang merupakan kristal heme hasil sintesis. Menghambat pembentukan hemozoin merupakan langkah yang baik dalam upaya skrining obat antimalaria (Nhien et al 2011…”