Calcium and calmodulin antagonists inhibit human malaria parasites (Plasmodium falciparum): implications for drug design

Abstract

The malaria parasite has an obligate calcium requirement for normal intracellular growth and invasion of host erythrocytes. Calmodulin (CaM) is a vital calcium-dependent protein present in eukaryotes. We found by radioimmunoassay that free parasites contain CaM. Schizont-infected erythrocytes had CaM levels of 23.3 +/- 2.7 ng per 10(6) cells compared to normals (11.2 +/- 1.5 ng per 10(6) cells). CaM levels were proportional to parasite maturity. Immunoelectron microscopy identified CaM diffusely within the cytoplasm of mature parasites and at the apical end of merozoites within the ductule of rhoptries, which may explain the calcium requirement for invasion. Cyclosporin A (CsA) was also found by electron microscopic autoradiography to concentrate in the food vacuole, as do chloroquine and mefloquine, and to distribute within the cytoplasm of mature parasites. The binding of dansylated CsA to schizont-infected erythrocytes was higher than to normal erythrocytes as analyzed by flow cytometry. Kinetic analysis revealed that binding was saturable for normal and infected erythrocytes and possibly free parasites. Competition for binding existed between dansylated CsA and native CsA as well as the CaM inhibitor W-7 and the classic antimalarial chloroquine. The in vitro growth of Plasmodium falciparum was sensitive to CaM antagonists, and in large part inhibition of the parasite was proportional to known anti-CaM potency. Antagonism existed between combinations of these drugs in multi-drug-resistant strains of P. falciparum, suggesting possible competition for the same binding site. In addition, the malaria parasite was also susceptible to calcium antagonists.