Wang Rong, Wang Jixia, Liu Yanfang, Zhang Xiuli, Liang Xinmiao
Key Lab of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences Dalian 116023 China
University of Chinese Academy of Sciences Beijing 100049 China.
RSC Adv. 2019 Nov 21;9(65):38055-38064. doi: 10.1039/c9ra06466d. eCollection 2019 Nov 19.
Small molecules, including natural compounds, in aqueous buffer that self-associate into colloidal aggregates is the main cause of false results in the early stage of drug discovery. Here we reported resonant waveguide grating (RWG) based assays to identify natural compound aggregation and characterize its influence on membrane receptors in living cells. We first applied a cell-free aggregation assay to determine compound critical aggregation concentration (CAC) values. Then we characterized the aggregators' influence on membrane receptors using three types of dynamic mass redistribution (DMR) assays. Results showed that colloidal aggregates may cause false activity in DMR desensitization assays; some of the false activities can be implied by the large response in DMR agonism assays and can further be identified by DMR antagonism assays. Furthermore, the aggregation mechanism was confirmed by addition of 0.025% tween-80, with cell signals attenuated and potency decreased. Finally, these observations were used for aggregate examination and promiscuity investigation of a traditional herbal medicine, , which ultimately led to the revealing of the true target and reduced the risk of a bioactivity tracking process at the very first stage. This study highlights that the RWG based assays can be used as practical tools to distinguish between real and false hits to provide reliable results in the early stage of drug discovery.
在水性缓冲液中能自缔合形成胶体聚集体的小分子，包括天然化合物，是药物发现早期阶段产生错误结果的主要原因。在此，我们报道了基于共振波导光栅（RWG）的分析方法，用于识别天然化合物的聚集情况，并表征其对活细胞中膜受体的影响。我们首先应用无细胞聚集分析来确定化合物的临界聚集浓度（CAC）值。然后，我们使用三种类型的动态质量重分布（DMR）分析来表征聚集剂对膜受体的影响。结果表明，胶体聚集体可能在DMR脱敏分析中导致假活性；DMR激动分析中的大响应可能暗示了一些假活性，并且可以通过DMR拮抗分析进一步识别。此外，通过添加0.025%吐温-80证实了聚集机制，细胞信号减弱且效力降低。最后，这些观察结果被用于对一种传统草药进行聚集体检查和混杂性研究，最终揭示了真正的靶点，并在生物活性追踪过程的最初阶段降低了风险。这项研究强调，基于RWG的分析方法可作为实用工具，区分真实和虚假的命中结果，以便在药物发现的早期阶段提供可靠的结果。
