Analytical Methods for Bioactive Substances Based on Cellular Signal Transduction Mechanisms

在大多数化学分析方法中，被分析物对干扰物质的选择性基本上取决于被分析物A与其分子识别试剂B之间的结合常数K_f， K_f=[AB]/[A][B]，这通常是结合分析的基础。一个典型的例子是免疫测定。近年来，我们一直在研究生物活性物质的分析方法，这些方法不仅基于与受体的结合，而且还部分考虑了信号转导的生物学过程。这个想法引导我们探索一些新的分析方法，可以评估生理上更相关的分析物（激动剂或拮抗剂）的选择性。典型的例子是离子通道，转运蛋白和激酶型蛋白嵌入脂质双层膜。与结合分析和相关技术相比，这些传感器利用相应的下游信号，如离子通道电流或磷酸化程度，以产生与结合分析方法更相关的生理信号。这是通过体外和体内的分析方法实现的，使用表面等离子体共振（SPR）和荧光探针试剂，涉及这些细胞信号传导机制的一部分。至于细胞内受体蛋白，Ca^<2+>信号通路，例如基于钙调蛋白的Ca^<2+>依赖/关闭开关，SPR已经评估了离子选择性方面的激动剂选择性，以形成Ca^<2+>钙调蛋白靶肽三元复合物。通过这种方法，不仅发现了Ca^<2+>离子，还发现了Sr^<2+>、Pb^<2+>、Cd^<2+>和许多镧离子骑乘在“Ca^<2+>信号传导”上，并对Ca^<2+>信号传导发挥了强激动剂的作用。筛选激动剂的生理相关选择性对生物学研究和制药需求非常重要。传统的结合试验既不能提供关于激动剂活性的充分信息，也不能区分激动剂和拮抗剂。因此，基于细胞信号转导机制的分析程序似乎成为生物活性分析物的一般分析方法。我们对这种方法的进一步研究包括基于细胞内Ca^<2+>信号传导筛选抗原特异性IgEs，以及基于涉及多药耐药蛋白的主动转运筛选抗癌药物。少

In most of chemical methods of analysis, selectivity of analytes against interfering substances is essentially governed by the respective binding constant, K_f, between the analyte, A, and its molecular recognition reagent, B, K_f=[AB]/[A][B] This is generally the basis for binding assay. A typical example is immunoassay. Recently, we have been studying analytical methods for bioactive substances, being based not only on binding to receptors but also the following biological process of signal transduction is, in part, taken into account. The idea has lead us to explore some new analytical methods that can evaluate physiologically more relevant selectivity of analytes (agonists or antagonists). Typical examples are ion channel, transporter proteins and kinase-type proteins embedded in lipid bilayer membranes. In contrast to the binding assay and related techniques, these sensors utilize the corresponding downstream signals, such as ion-channel currents or the extent of phosphorylation o … More f the receptor itself and its target proteins for yielding physiologically more relevant signals compared to those with the binding assay approach. This is achieved by both in vitro and in vivo analytical approaches using surface plasmon resonance (SPR) and fluorescent probe reagents involving part of these cellular signaling mechanisms. As to intracellular receptor proteins, Ca^<2+> signaling pathways for example based on a Ca^<2+>-dependent on/off switching of calmodulin, the agonist selectivity in terms of ion selectivity has been evaluated by SPR for the formation of a Ca^<2+> calmodulin target peptide ternary complex. By this method, not only Ca^<2+> ion, but also Sr^<2+>, Pb^<2+>, Cd^<2+> and many of lanthunum ions have been found to ride on "Ca^<2+> signaling" and behave as a strong agonist toward the Ca^<2+> signaling. Screening of agonists concerning their physiologically relevant selectivity is very important for biological studies as well as for pharmaceutical needs. The conventional binding assays can neither give sufficient information on the agonist activity nor discriminate between agonists and antagonists. Assay procedures based on cellular signal transduction mechanisms thus appear to become a general analytical method for bioactive analytes. Our further studies of this apporach include screening of antigen-specific IgEs based on intracellular Ca^<2+> singnaling, and also screening of anticancer drugs based on active transport involving multi-drug resistance proteins. Less