Abstract：
The riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin utilization. To determine their mechanism of action, we generated roseoflavin-resistant parasites by in vitro evolution. Relative to wild-type, these parasites were 4-fold resistant to roseoflavin and cross-resistant to 8-aminoriboflavin. Whole genome sequencing of the resistant parasites revealed a missense mutation leading to an amino acid change (L672H) in the gene coding for a putative flavokinase (PfFK), the enzyme responsible for converting riboflavin into the cofactor flavin mononucleotide (FMN). To confirm that the L672H mutation is responsible for the phenotype, we generated parasites with the missense mutation incorporated into the PfFK gene. The IC50 values for roseoflavin and 8-aminoriboflavin against the roseoflavin-resistant parasites created through in vitro evolution were indistinguishable from those against parasites in which the missense mutation was introduced into the native PfFK. We also generated two parasite lines episomally expressing GFP-tagged versions of either the wild-type or mutant forms of PfFK. We found that PfFK-GFP localizes to the parasite cytosol and that immunopurified PfFK-GFP phosphorylated riboflavin, roseoflavin, and 8-aminoriboflavin. The L672H mutation increased the KM for roseoflavin, explaining the resistance phenotype. Mutant PfFK is no longer capable of phosphorylating 8-aminoriboflavin, but its antiplasmodial activity against resistant parasites can still be antagonized by increasing the extracellular concentration of riboflavin, consistent with it also inhibiting parasite growth through competitive inhibition of PfFK. Our findings, therefore, are consistent with roseoflavin and 8-aminoriboflavin inhibiting parasite proliferation by inhibiting riboflavin phosphorylation and via the generation of toxic flavin cofactor analogues.
摘要：
核黄素类似物，玫瑰黄素和8-氨基黄素，通过靶向核黄素的利用来抑制疟原虫的增殖。为了确定它们的作用机制，我们通过体外进化产生了抗玫瑰黄素的寄生虫。相对于野生型,这些寄生虫对玫瑰黄素具有4倍的抗性，对8-氨基化黄素具有交叉抗性。抗性寄生虫的全基因组测序揭示了一个错义突变，导致编码推定的黄素激酶（PfFK）的基因中的氨基酸变化（L672H），负责将核黄素转化为辅因子黄素单核苷酸（FMN）的酶。为了确认L672H突变是表型的原因，我们产生的寄生虫具有插入PfFK基因的错义突变。通过体外进化产生的玫瑰黄素抗性寄生虫的玫瑰黄素和8-氨基黄素的IC50值与将错义突变引入天然PfFK的寄生虫的IC50值没有区别。我们还产生了两个附加型表达野生型或突变型PfFK的GFP标记版本的寄生虫系。我们发现PfFK-GFP定位于寄生虫胞质溶胶，免疫纯化的PfFK-GFP磷酸化核黄素，玫瑰黄素,和8-氨基核黄素.L672H突变增加了玫瑰黄素的KM，解释抗性表型。突变体PfFK不再能够磷酸化8-氨基核黄素，但其对抗性寄生虫的抗疟原虫活性仍然可以通过增加核黄素的细胞外浓度来拮抗，与它也通过竞争性抑制PfFK抑制寄生虫生长一致。我们的发现,因此,与通过抑制核黄素磷酸化和通过产生有毒的黄素辅因子类似物来抑制寄生虫增殖的玫瑰黄素和8-氨基核黄素一致。
