A high-throughput fluorimetric assay for 2-hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor.
A high-throughput fluorimetric assay for 2-hydroxyglutarate identifies Zaprinast as a glutaminase inhibitor.
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DOI：
10.1158/2159-8290.cd-13-0572
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发表时间：
2014-07
期刊：
影响因子：
28.2
通讯作者：
Piwnica-Worms D
中科院分区：
文献类型：
--
作者：
Elhammali A;Ippolito JE;Collins L;Crowley J;Marasa J;Piwnica-Worms D
最近鉴定的IDH突变导致2-羟基戊二酸（2 HG）的产生，这是一种在选定癌症中异常升高的致癌代谢物。我们开发了一种简便和廉价的荧光微量板测定法定量的2 HG，并进行了公正的小分子筛选活细胞中，以确定能够干扰2 HG生产的化合物。Zaprinast是一种PDE 5抑制剂，被鉴定为2 HG产生的有效调节剂，并证实可降低体内2 HG水平。作用机制不是由于cGMP稳定，而是突变体IDH 1上游代谢物的谱分析表明对转氨酶（GLS）的靶向抑制。Zaprinast治疗逆转了IDH 1突变细胞的组蛋白超甲基化和软琼脂生长，谷氨酰胺成瘾胰腺癌细胞的治疗减少了生长并使细胞对氧化损伤敏感。因此，扎普司特对谷氨酰胺代谢有效，并进一步建立了GLS和2 HG介导的肿瘤发生之间的治疗联系。
Recently identified IDH mutations lead to the production of 2-hydroxyglutarate (2HG), an onco-metabolite aberrantly elevated in selected cancers. We developed a facile and inexpensive fluorimetric microplate assay for quantitation of 2HG and performed an unbiased small molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a PDE5 inhibitor, was identified as an efficacious modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS). Zaprinast treatment reversed histone hypermethylation and soft agar growth of IDH1 mutant cells, and treatment of glutamine-addicted pancreatic cancer cells reduced growth and sensitized cells to oxidative damage. Thus, Zaprinast is efficacious against glutamine metabolism and further establishes the therapeutic linkages between GLS and 2HG-mediated oncogenesis.