Chang Fang-Yuan, Kawashima Shigehiro A, Brady Sean F
Laboratory of Genetically Encoded Small Molecules, Howard Hughes Medical Institute, The Rockefeller University , 1230 York Avenue, New York, New York 10065, United States.
Biochemistry. 2014 Nov 18;53(45):7123-31. doi: 10.1021/bi501078j. Epub 2014 Oct 31.
Indolotryptoline natural products represent a small family of structurally unique chromopyrrolic acid-derived antiproliferative agents. Like many prospective anticancer agents before them, the exploration of their potential clinical utility has been hindered by the limited information known about their mechanism of action. To study the mode of action of two closely related indolotryptolines (BE-54017, cladoniamide A), we selected for drug resistant mutants using a multidrug resistance-suppressed (MDR-sup) Schizosaccharomyces pombe strain. As fission yeast maintains many of the basic cancer-relevant cellular processes present in human cells, it represents an appealing model to use in determining the potential molecular target of antiproliferative natural products through resistant mutant screening. Full genome sequencing of resistant mutants identified mutations in the c and c' subunits of the proteolipid substructure of the vacuolar H(+)-ATPase complex (V-ATPase). This collection of resistance-conferring mutations maps to a site that is distant from the nucleotide-binding sites of V-ATPase and distinct from sites found to confer resistance to known V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies, and direct c/c' subunit mutagenesis all suggest that indolotryptolines are likely a structurally novel class of V-ATPase inhibitors. This work demonstrates the general utility of resistant mutant selection using MDR-sup S. pombe as a rapid and potentially systematic approach for studying the modes of action of cytotoxic natural products.
吲哚色胺类天然产物是一类结构独特的、由色原吡咯酸衍生而来的抗增殖剂。和之前的许多潜在抗癌药物一样，由于对其作用机制了解有限，它们潜在的临床应用探索受到了阻碍。为了研究两种密切相关的吲哚色胺类化合物（BE-54017、枝瑚菌素A）的作用方式，我们使用多药耐药抑制（MDR-sup）粟酒裂殖酵母菌株筛选耐药突变体。由于裂殖酵母保留了许多人类细胞中存在的与癌症相关的基本细胞过程，它是一个有吸引力的模型，可用于通过耐药突变体筛选来确定抗增殖天然产物的潜在分子靶点。对耐药突变体进行全基因组测序，确定了液泡H(+)-ATP酶复合物（V-ATP酶）的蛋白脂质亚结构的c和c'亚基发生了突变。这些赋予抗性的突变集合映射到一个远离V-ATP酶核苷酸结合位点的位置，且与已知V-ATP酶抑制剂的抗性位点不同。酸性液泡染色、交叉耐药研究以及直接对c/c'亚基进行诱变均表明，吲哚色胺类化合物可能是一类结构新颖的V-ATP酶抑制剂。这项工作证明了使用MDR-sup粟酒裂殖酵母进行耐药突变体筛选作为一种快速且可能系统的方法来研究细胞毒性天然产物作用方式的普遍实用性。
