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

Intracrythrocytic 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 beta-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 oil the detection by optical density measurement of solubilised beta-haematin remaining after contact with drugs. The assay uses a 192-muM haemin chloride solution in dimethyl sulfoxide, 96-well filtration microplates as well as normal microplatcs; it lasts 18-24 h and requires a spectrophotometer. We determined by this assay the IC50 of chloroquine phosphate (28 muM) and quinine base (324 muM) 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 muM, respectively), and an extract of plants that contains quinoline derivatives, inhibited the biomineralisation of FP regardless of their intrinsic colour. (C) 2002 Elsevier Science (USA). All rights reserved.