Quantitative analyses of bifunctional molecules
Quantitative analyses of bifunctional molecules
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DOI：
10.1021/bi035839g
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发表时间：
2004-05-11
期刊：
影响因子：
2.9
通讯作者：
Wandless, TJ
中科院分区：
文献类型：
--
作者：
Braun, PD;Wandless, TJ
小分子可以被发现或设计成与生物相关蛋白质紧密结合，这些分子已被证明是基础研究和治疗应用的强大工具。在许多情况下，分子间结合事件的详细生物物理分析对于改善小分子的活性是必不可少的。这些相互作用通常可以表征为直接的双分子结合事件，并且已经开发和改进了各种实验和分析技术以促进这些分析。一些研究人员最近合成了异二聚体分子，其被设计为同时与两种不同的蛋白质结合以形成三元复合物。这些异二聚体分子通常显示出引人注目的生物活性;然而，它们难以表征。一种蛋白质和异二聚体配体之间的双分子相互作用（初级解离常数）可以通过许多方法来确定。然而，由于原始蛋白质-配体复合物的非共价性质，该蛋白质-配体复合物与第二种蛋白质之间的相互作用（二级解离常数）更难以测量。因此，这些异二聚体化合物通常根据其活性来表征，这是一种依赖于实验的度量。我们已经开发了一个通用的定量数学模型，可用于测量的初级（蛋白质+配体）和二级（蛋白质-配体+蛋白质）的异二聚体小分子的解离常数。这些值在很大程度上独立于所使用的实验技术，并且还提供了所形成的三元络合物的热力学稳定性的直接测量。荧光偏振和该模型被用来表征的异二聚体分子，SLFpYEEI，它结合到FKBP 12和Fyn SH 2域，证明该模型是有用的预测以及事后分析应用。
Small molecules can be discovered or engineered to bind tightly to biologically relevant proteins, and these molecules have proven to be powerful tools for both basic research and therapeutic applications. In many cases, detailed biophysical analyses of the intermolecular binding events are essential for improving the activity of the small molecules. These interactions can often be characterized as straightforward bimolecular binding events, and a variety of experimental and analytical techniques have been developed and refined to facilitate these analyses. Several investigators have recently synthesized heterodimeric molecules that are designed to bind simultaneously with two different proteins to form ternary complexes. These heterodimeric molecules often display compelling biological activity; however, they are difficult to characterize. The bimolecular interaction between one protein and the heterodimeric ligand (primary dissociation constant) can be determined by a number of methods. However, the interaction between that protein-ligand complex and the second protein (secondary dissociation constant) is more difficult to measure due to the noncovalent nature of the original protein-ligand complex. Consequently, these heterodimeric compounds are often characterized in terms of their activity, which is an experimentally dependent metric. We have developed a general quantitative mathematical model that can be used to measure both the primary (protein + ligand) and secondary (protein-ligand + protein) dissociation constants for heterodimeric small molecules. These values are largely independent of the experimental technique used and furthermore provide a direct measure of the thermodynamic stability of the ternary complexes that are formed. Fluorescence polarization and this model were used to characterize the heterodimeric molecule, SLFpYEEI, which binds to both FKBP12 and the Fyn SH2 domain, demonstrating that the model is useful for both predictive as well as ex post facto analytical applications.