Malarial parasites are exquisitely susceptible to a number of microtubule inhibitors but most of these compounds also affect human microtubules.
Herbicides of the dinitroaniline and phosphorothioamidate classes however affect some plant and protozoal cells but not mammalian ones. We have previously shown that these
herbicides block schizogony in erythrocytic parasites of the most lethal human
malaria, Plasmodium falciparum, disrupt their mitotic spindles, and bind selectively to parasite
tubulin. Here we show for the first time that the
antimitotic herbicides also block the development of malarial parasites in the liver stage. Structure-based design of novel
antimalarial agents binding to
tubulin at the
herbicide site, which presumably exists on (some) parasite and plant tubulins but not mammalian ones, can therefore constitute an important transmission blocking approach. The nature of this binding site is controversial, with three overlapping but non-identical locations on α-
tubulin proposed in the literature. We tested the validity of the three sites by (i) using site-directed mutagenesis to introduce six
amino acid changes designed to occlude them, (ii) producing the resulting tubulins recombinantly in Escherichia coli and (iii) measuring the affinity of the
herbicides amiprophosmethyl and
oryzalin for these
proteins in comparison with wild-type tubulins by fluorescence quenching. The changes had little or no effect, with dissociation constants (Kd) no more than 1.3-fold (amiprophosmethyl) or 1.6-fold (
oryzalin) higher than wild-type. We conclude that the
herbicides impair Plasmodium liver stage as well as blood stage development but that the location of their binding site on malarial parasite
tubulin remains to be proven.