School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Department of Medicine, National Jewish Health, Denver, CO 80206, USA.
G3 (Bethesda). 2021 Sep 27;11(10). doi: 10.1093/g3journal/jkab233.
The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however, the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the APCs exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of S. cerevisiae titratable-essential gene and haploid nonessential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). Fifty-eight strains out of ∼5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1Δ), an oxysterol binding protein homolog (osh2Δ), a number of ER-resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.
烷基磷酸胆碱 (APC) 类抗肿瘤和抗原生动物药物，如埃德尔福西和米替福新，是溶血磷脂酰胆碱 (lyso-PC) 的结构类似物，在低微摩尔浓度下对酵母酿酒酵母具有抑制作用。抑制磷脂合成、诱导未折叠蛋白反应、抑制氧化磷酸化和破坏脂筏等与该类药物相关的细胞毒性作用已归因于该药物类别的成员，然而，这些药物的分子作用机制仍不完全清楚。APC 的细胞抑制和细胞毒性作用因化学结构而异，导致对不同生物体或细胞类型的有效性存在差异。我们现在报告了酿酒酵母可滴定必需基因和单倍体非必需基因缺失突变体的全面鉴定，这些突变体对 APC 药物米替福新（十六烷基-O-磷酸胆碱）具有抗性。在约 5600 个测试的菌株中，有 58 个菌株对米替福新表现出稳健且可重复的抗性。该基因集在与囊泡运输步骤相关的功能中高度富集，特别是那些涉及内吞作用和从内体衍生的囊泡逆行运输到高尔基体或液泡的功能，这表明这些运输途径在将米替福新运输到内质网和线粒体的潜在作用部位中发挥作用。此外，我们还鉴定了在磷脂酰肌醇-4-磷酸合成（TetO::STT4）和水解（sac1Δ）、甾醇结合蛋白同源物（osh2Δ）、许多内质网驻留蛋白和 eisosome 的多个成分中存在缺陷的突变体。这些发现表明，内质网-质膜接触位点和逆行囊泡运输参与了溶血磷脂酰胆碱和相关溶血磷脂样类似物向其细胞内细胞毒性活性部位的细胞器间运输。
