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Quantitative analyses of bifunctional molecules.

机译：双功能分子的定量分析。

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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.
机译：可以发现或改造出与生物学相关蛋白质紧密结合的小分子，这些分子已被证明是用于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至关重要。这些相互作用通常可以被描述为直接的双分子结合事件，并且已经开发和完善了各种实验和分析技术以促进这些分析。几位研究者最近合成了异源二聚体分子，该分子被设计为与两种不同的蛋白质同时结合形成三元复合物。这些异二聚体分子通常表现出引人注目的生物学活性;但是，它们很难表征。一种蛋白质和异二聚体配体之间的双分子相互作用（一级解离常数）可以通过多种方法确定。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）更加难以测量。因此，这些异二聚化合物通常根据其活性来表征，这是实验上依赖的指标。我们已经开发了一种通用的定量数学模型，可用于测量异二聚体小分子的一级（蛋白质+配体）和二级（蛋白质-配体+蛋白质）解离常数。这些值在很大程度上与所使用的实验技术无关，并且进一步提供了所形成的三元络合物的热力学稳定性的直接量度。荧光偏振和该模型用于表征与FKBP12和Fyn SH2结构域结合的异二聚体分子SLFpYEEI，表明该模型可用于预测和事后分析应用。

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