Sulindac inhibits canonical Wnt signaling by blocking the PDZ domain of the protein Dishevelled.
Sulindac inhibits canonical Wnt signaling by blocking the PDZ domain of the protein Dishevelled.
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DOI：
10.1002/anie.200902981
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发表时间：
2009
影响因子：
16.6
通讯作者：
Zheng, Jie J.
中科院分区：
文献类型：
--
作者：
Lee, Ho-Jin;Wang, Nick X.;Shi, De-Li;Zheng, Jie J.
非甾体抗炎药（NSAIDs）的保护性抗癌作用引起了广泛关注，并且正在进行几种NSAIDs用于治疗或预防各种癌症的临床试验。[1]由于NSAID以其抑制环氧合酶1和2（考克斯-1/2）而闻名，因此假设它们通过阻断前列腺素合成来抑制肿瘤生长。[2]然而，这一假设并不能解释所有可用的数据。[3-5]累积的证据表明，一些NSAID还靶向Wnt/β连环蛋白信号通路，这是人类癌细胞中细胞发育和生长的关键调节通路。[4，5a-c]例如，舒林酸（Clinoril）已显示在乳腺癌、肺癌和结肠癌细胞系中抑制典型的β-连环蛋白相关Wnt信号传导。[4e]然而，这种效应的分子机制尚不清楚。Wnt信号在胚胎发育和成年组织的维持中起着至关重要的作用。[5]在几种类型的癌症中观察到Wnt信号传导的异常激活。[5，6] Dishevelled（Dvl）是Wnt途径中的关键分子，通过其PDZ结构域，将Wnt信号从膜结合的Wnt受体传递到下游组分。[5，7]我们和其他研究小组一直致力于开发Dvl PDZ蛋白质-蛋白质相互作用的小分子抑制剂，用于阐明生物学过程，并作为潜在的癌症治疗和预防剂。[8]在此，我们显示舒林酸和舒林酸砜均结合Dvl的PDZ结构域，并且舒林酸在Dvl水平抑制Wnt3A诱导的β-连环蛋白信号传导。我们的研究结果表明，舒林酸（及其代谢物）的抗癌保护作用不仅反映了考克斯-1/2抑制，而且还反映了通过阻断Dvl PDZ结构域对异常典型Wnt信号传导的抑制。为了测试舒林酸和舒林砜与Dvl PDZ结构域的结合，我们用NMR光谱进行了化学位移扰动实验;该方法广泛用于表征蛋白质-配体相互作用。[9]当添加到15N标记的Dvl PDZ结构域的溶液中时，两种化合物均产生化学位移扰动，表明与Dvl PDZ结构域的相同区域结合（支持信息中的图1a和图S1）。Dvl PDZ结构域的结构包含六个β链（β A-β F）和两个α螺旋（α A和α B）。结合诱导的化学位移扰动表明，两种化合物与Dvl PDZ结构域在其α B和β B结构之间结合，该结构域的天然配体也是如此。[7，10]我们接下来检查舒林酸和舒林砜是否仅与Dvl PDZ结构域结合，因为在人蛋白质组中存在许多PDZ结构域。[8c我们测试了代表大多数人PDZ结构域的三个其他PDZ结构域：PSD-95蛋白的第一和第二PDZ结构域（分别为PDZ 1和PDZ 2; 1类PDZ结构域）和GRIP 1蛋白的第七PDZ结构域（PDZ 7; 2类PDZ结构域）。Dvl PDZ结构域既不属于类别1也不属于类别2。[7，11]值得注意的是，NMR滴定实验显示舒林酸或舒林酸砜与这三个PDZ结构域之间几乎没有或没有相互作用（参见支持信息中的图S2和表S1），该结果表明舒林酸和舒林酸砜特异性结合Dvl PDZ结构域。为了进一步研究舒林酸和舒林酸砜识别Dvl PDZ结构域的特异性，我们确定了与舒林酸复合的Dvl PDZ结构域的结构（图1b）。2D（15N，13C）-双重过滤核奥弗豪泽效应（NOE）光谱实验表明...
The protective anticancer effect of nonsteroidal anti-inflammatory drugs (NSAIDs) has attracted much attention, and clinical trials of several NSAIDs are under way for treatment or prevention of various cancers.[1] As NSAIDs are best known for their inhibition of cyclooxygenase 1 and 2 (COX-1/2), they are hypothesized to suppress tumor growth by blocking prostaglandin synthesis.[2] However, this hypothesis does not explain all of the available data.[3–5] Accumulated evidence suggests that some NSAIDs also target the Wnt/βcatenin signaling pathway, a key regulatory pathway for cell development and growth, in human cancer cells.[4, 5a–c] For example, sulindac (Clinoril) has been shown to suppress canonical β-catenin-related Wnt signaling in breast cancer, lung cancer, and colon cancer cell lines.[4e] However, the molecular mechanism of this effect is not clear. Wnt signaling plays crucial roles in embryonic development and in tissue maintenance in adults.[5] Abnormal activation of Wnt signaling is observed in several types of cancers.[5, 6] Dishevelled (Dvl) is a key molecule in the Wnt pathways that, through its PDZ domain, relays Wnt signals from membrane-bound Wnt receptors to downstream components.[5, 7] We and other research groups have worked to develop small-molecule inhibitors of the Dvl PDZ protein–protein interaction for use in the elucidation of biological processes and as potential cancer-treatment and prevention agents.[8] Herein, we show that both sulindac and sulindac sulfone bind to the PDZ domain of Dvl and sulindac suppresses Wnt3A induced β-catenin signaling at the level of Dvl. Our results suggest that the anticancer protective effect of sulindac (and its metabolite) reflect not only COX-1/2 inhibition but also the inhibition of abnormal canonical Wnt signaling by blockade of the Dvl PDZ domain. To test the binding of sulindac and sulindac sulfone to the Dvl PDZ domain, we conducted chemical shift perturbation experiments with NMR spectroscopy; this method is widely used to characterize protein–ligand interactions.[9] When added to a solution of 15N-labeled Dvl PDZ domain, both compounds generated chemical shift perturbations that indicated binding to the same region of the Dvl PDZ domain (Figure 1a and Figure S1 in the Supporting Information). The structure of the Dvl PDZ domain comprises six β strands (βA–βF) and two α helices (αA and αB). The chemical shift perturbations induced by binding indicated that both compounds bind to the Dvl PDZ domain between its αB and βB structures, as do the native ligands of the domain.[7, 10] We next examined whether sulindac and sulindac sulfone bind exclusively to the Dvl PDZ domain, as there are many PDZ domains in the human proteome.[8c, 11] We tested three other PDZ domains that are representative of most human PDZ domains: the first and second PDZ domains of PSD-95 protein (PDZ1 and PDZ2, respectively; class 1 PDZ domains) and the seventh PDZ domain of GRIP1 protein (PDZ7; a class2 PDZ domain). The Dvl PDZ domain belongs to neither class 1 nor class 2.[7, 11] Remarkably, NMR titration experiments showed little or no interaction between sulindac or sulindac sulfone and these three PDZ domains (see Figure S2 and Table S1 in the Supporting Information), this result indicates that sulindac and sulindac sulfone bind specifically to the Dvl PDZ domain. To further investigate the specificity with which sulindac and sulindac sulfone recognize the Dvl PDZ domain, we determined the structure of Dvl PDZ domain in complex with sulindac (Figure1b). 2D (15N, 13C)-double-filtered nuclear Overhauser effect (NOE) spectroscopy experiments showed …
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