Department of Molecular Medicine, Cornell University, Ithaca, NY 14853, USA.
Mol Cancer Ther. 2012 Jun;11(6):1269-78. doi: 10.1158/1535-7163.MCT-11-0942. Epub 2012 Apr 11.
One hallmark of cancer cells is their adaptation to rely upon an altered metabolic scheme that includes changes in the glycolytic pathway, known as the Warburg effect, and elevated glutamine metabolism. Glutaminase, a mitochondrial enzyme, plays a key role in the metabolism of glutamine in cancer cells, and its inhibition could significantly impact malignant transformation. The small molecule 968, a dibenzophenanthridine, was recently shown to inhibit recombinantly expressed glutaminase C, to block the proliferation and anchorage-independent colony formation of human cancer cells in culture, and to inhibit tumor formation in mouse xenograft models. Here, we examine the structure-activity relationship that leads to 968-based inhibition of glutaminase and cancer cell proliferation, focusing upon a "hot-spot" ring previously identified as critical to 968 activity. We find that the hot-spot ring must be substituted with a large, nonplanar functionality (e.g., a t-butyl group) to bestow activity to the series, leading us to a model whereby the molecule binds glutaminase at a previously undescribed allosteric site. We conduct docking studies to locate potential 968-binding sites and proceed to test a specific set of docking solutions via site-directed mutagenesis. We verify the results from our initial assay of 968 and its analogues by cellular studies using MDA-MB-231 breast cancer cells.
癌细胞的一个标志是它们适应依赖改变的代谢方案，包括糖酵解途径的改变，即众所周知的沃伯格效应，以及谷氨酰胺代谢的升高。谷氨酰胺酶是一种线粒体酶，在癌细胞的谷氨酰胺代谢中发挥关键作用，其抑制作用可能会对恶性转化产生重大影响。小分子 968 是一种二苯并菲啶，最近被证明可以抑制重组表达的谷氨酰胺酶 C，阻断人类癌细胞在培养中的增殖和非锚定依赖性集落形成，并抑制小鼠异种移植模型中的肿瘤形成。在这里，我们研究了导致基于 968 的谷氨酰胺酶和癌细胞增殖抑制的结构-活性关系，重点研究了先前被确定为 968 活性关键的“热点”环。我们发现，热点环必须用大的、非平面的官能团（例如叔丁基）取代才能使该系列具有活性，这使我们得出了一个模型，即该分子在以前未描述的变构部位与谷氨酰胺酶结合。我们进行对接研究以定位潜在的 968 结合位点，并通过定点突变进一步测试一组特定的对接解决方案。我们通过使用 MDA-MB-231 乳腺癌细胞的细胞研究验证了我们对 968 及其类似物的初始测定的结果。
