Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.
Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.
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DOI：
10.1016/j.phytochem.2015.06.016
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发表时间：
2015-09
期刊：
影响因子：
3.8
通讯作者：
Fry SC
中科院分区：
文献类型：
--
作者：
Chormova D;Franková L;Defries A;Cutler SR;Fry SC
抑制细胞壁重塑酶xylo葡聚糖内转葡萄糖酶（XET）活性的外源药物将成为“化学遗传学”中阐明其生物学作用的有价值的工具。通过对纸固定木葡聚糖进行点印迹实验，筛选4216个小分子，我们发现了45个抑制或促进XET活性的化合物。最有效的抑制剂是单线态氧气发生器。我们在滤纸为基础的点印迹试验中筛选XET调节剂的化学收集物。一些“撞击”抑制了XET在纤维素结合的木葡聚糖上的活性，另一些则促进了XET的活性。在可溶性木葡聚糖上再次进行放射化学分析时，大多数启动子抑制XET。最强的XET抑制剂是单线态造氧剂，如核黄素（IC50为29 μM）。XET抑制剂是探测XET在体内作用的潜在工具。抑制细胞壁定位酶的小分子（异种生物制剂）对于阐明酶的生物学作用是有价值的。本研究采用高通量荧光斑点杂交技术，在体外寻找Petroselinum xyloglucan endotransglucosylase （XET）活性抑制剂。在4216种以纤维素结合的木葡聚糖为供体-底物的外源药物中，18种抑制XET活性，18种促进XET活性（尤其是蒽醌类和类黄酮类）。在以（无纤维素）可溶性木葡聚糖为底物的定量分析中，没有化合物促进XET，这表明促进作用依赖于酶-纤维素的相互作用。以无纤维素木葡聚糖为底物，我们发现了22种xet抑制剂，特别是产生单线态氧（1O2）的化合物，如核黄素（IC50 29 μM）、视黄酸、伊红（IC50 27 μM）和红血素（IC50 36 μM）。核黄素的作用是光依赖性的，支持1O2参与。其他抑制剂包括单宁、磺胺试剂和三苯基甲烷。一些抑制剂（vulpinic acid和brilliant blue G）对XET相对特异，分别只影响其他9种壁酶活性中的2种或3种；其他[例如(−)-表没食子儿茶素没食子酸酯和核黄素]是非特异性的。在体内，在8种xet抑制剂中，红素（1 μM）抑制了Rosa和Zea细胞悬浮培养中的细胞扩增，而40 μM霉酚酸和(−)-表没食子儿茶素没食子酸酯抑制了Zea培养的生长。我们的工作展示了发现壁酶抑制剂的一般高通量策略，其中一些是植物生长抑制剂，具有潜在的生理工具或除草剂先导价值。
Xenobiotics that inhibit the cell-wall-remodelling enzyme activity, xyloglucan endotransglucosylase (XET), would be a valuable tool in ‘chemical genetics’ for elucidating its biological roles. By screening 4216 small molecules in a dot-blot assay on paper-immobilised xyloglucan, we discovered 45 compounds that inhibited or promoted XET activity. The most effective inhibitors were singlet oxygen generators. We screened chemical collections for XET modulators in filter-paper-based dot-blot assays. Some ‘hits’ inhibited, others promoted, XET activity on cellulose-bound xyloglucan. Most of the promoters inhibited XET when re-assayed radiochemically on soluble xyloglucan. The strongest XET inhibitors were singlet oxygen-generators e.g., riboflavin (IC50 29 μM). XET inhibitors are potentially useful as tools for probing XET’s roles in vivo. Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads.