Analytical Methods for Bioactive Substances Based on Cellular Signal Transduction Mechanisms

在大多数化学分析方法中，分析物对干扰物质的选择性主要取决于分析物A与其分子识别试剂B之间的结合常数K_f，K_f=[AB]/[A][B]。典型的例子是免疫测定。最近，我们一直在研究生物活性物质的分析方法，不仅基于与受体的结合，而且还部分考虑了随后的信号转导的生物过程。这一想法使我们探索一些新的分析方法，可以评估生理上更相关的选择性分析物（激动剂或拮抗剂）。典型的例子是离子通道、转运蛋白和包埋在脂质双层膜中的激酶型蛋白。与结合测定和相关技术相比，这些传感器利用相应的下游信号，例如离子通道电流或磷酸化程度， 关于我们 f受体本身及其靶蛋白，以产生与结合测定方法相比生理学上更相关的信号。这是通过使用表面等离子体共振（SPR）和荧光探针试剂的体外和体内分析方法来实现的，所述荧光探针试剂涉及这些细胞信号传导机制的一部分。对于细胞内受体蛋白，例如基于钙调蛋白的Ca ^2+依赖性开/关的Ca ^2+信号传导途径，已经通过SPR评估了在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Ca^2+离子，而且Sr^2+、Pb^2+、Cd^2+和许多镧离子也被发现依赖于“Ca^2+信号”，并且表现为Ca^2+信号的强激动剂。激动剂的筛选与其生理学相关的选择性对于生物学研究以及制药需求是非常重要的。传统的结合试验既不能给出关于激动剂活性的足够信息，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测定程序似乎成为生物活性分析物的通用分析方法。我们对这一途径的进一步研究包括基于细胞内Ca^2+信号转导的抗原特异性IgE的筛选，以及基于涉及多药耐药蛋白的主动转运的抗癌药物的筛选。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