A non-radiolabelled ferriprotoporphyrin IX biomineralisation inhibition test for the high throughput screening of antimalarial compounds

Intraerythrocytic malaria parasites produce large amounts of toxic ferriprotoporphyrin IX (FP) during their digestion of host cell haemoglobin. The inhibition of biomineralisation of FP to haemozoin (or β-haematin) by antimalarial drugs underlies their mode of action. We have developed an in vitro microassay for testing the inhibition of biomineralisation by drugs. It is based on the detection by optical density measurement of solubilised β-haematin remaining after contact with drugs. The assay uses a 192-μM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplates; it lasts 18-24h and requires a spectrophotometer. We determined by this assay the IC₅₀ of chloroquine phosphate (28 μM) and quinine base (324 μM) and showed that unlike previous methods it is insensitive to inorganic anions. We also determined the activity of synthetic dyes and plant extract to determinate the interference of coloured compounds on the accuracy of the test. We found that methylene blue, thionine (IC₅₀ 38 and 87 μM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour.