Dempsey Enda, Prudêncio Miguel, Fennell Brian J, Gomes-Santos Carina S, Barlow James W, Bell Angus
Department of Microbiology, School of Genetics & Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland.
Mol Biochem Parasitol. 2013 Apr;188(2):116-27. doi: 10.1016/j.molbiopara.2013.03.001. Epub 2013 Mar 21.
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.
疟原虫对多种微管抑制剂极为敏感，但这些化合物中的大多数也会影响人类微管。然而，二硝基苯胺类和硫代氨基磷酸酯类除草剂会影响一些植物和原生动物细胞，但不会影响哺乳动物细胞。我们之前已经表明，这些除草剂会阻断最致命的人类疟疾——恶性疟原虫红细胞内期的裂体增殖，破坏其有丝分裂纺锤体，并选择性地与疟原虫微管蛋白结合。在此，我们首次表明，抗有丝分裂除草剂也会阻断疟原虫在肝脏阶段的发育。基于结构设计与除草剂结合位点结合的新型抗疟药，该位点可能存在于（某些）疟原虫和植物微管蛋白上，而不存在于哺乳动物微管蛋白上，因此可以构成一种重要的传播阻断方法。这种结合位点的性质存在争议，文献中提出了α-微管蛋白上三个重叠但不完全相同的位置。我们通过以下方式测试了这三个位点的有效性：（i）使用定点诱变引入六个氨基酸变化以阻断这些位点；（ii）在大肠杆菌中重组表达所得的微管蛋白；（iii）通过荧光猝灭测量除草剂胺丙畏和安磺灵与这些蛋白质相比野生型微管蛋白的亲和力。这些变化几乎没有影响，解离常数（Kd）比野生型高不超过1.3倍（胺丙畏）或1.6倍（安磺灵）。我们得出结论，除草剂会损害疟原虫肝脏阶段以及血液阶段的发育，但它们在疟原虫微管蛋白上结合位点的位置仍有待证实。
