Casper David, Bukhtiyarova Marina, Springman Eric B
Department of Biochemistry, Locus Pharmaceuticals, Inc, Four Valley Square, 512 Township Line Road, Blue Bell, PA 19422, USA.
Anal Biochem. 2004 Feb 1;325(1):126-36. doi: 10.1016/j.ab.2003.10.025.
Protein kinases are emerging as one of the most intensely studied classes of enzymes as their central roles in physiologically and clinically important cellular signaling events become more clearly understood. We report here the development of a real-time, label-free method to study protein kinase inhibitor binding kinetics using surface plasmon resonance-based biomolecular interaction analysis (Biacore). Utilizing p38alpha mitogen-activated protein kinase as a model system, we studied the binding properties of two known small molecule p38alpha inhibitors (SB-203580 and SKF-86002). Direct coupling of p38alpha to the biosensor surface in the presence of a reversible structure-stabilizing ligand (SB-203580) consistently produced greater than 90% active protein on the biosensor surface. The dissociation and kinetic constants derived using this Biacore method are in excellent agreement with values determined by other methods. Additionally, we extend the method to study the thermodynamics of small molecule binding to p38alpha and derive a detailed thermodynamic reaction pathway for SB-203580. The Biacore method reported here provides an efficient way to directly and reproducibly examine dissociation constants, kinetics, and thermodynamics for small molecules binding to p38alpha and possibly other protein kinases. Immobilization in the presence of a stabilizing ligand may further represent a broadly applicable paradigm for creation of highly active biosensor surfaces.
蛋白激酶正成为研究最为深入的酶类之一，因为它们在生理和临床重要的细胞信号传导事件中的核心作用越来越清晰地被理解。我们在此报告一种基于表面等离子体共振的生物分子相互作用分析（Biacore）的实时、无标记方法的开发，用于研究蛋白激酶抑制剂的结合动力学。利用p38α丝裂原活化蛋白激酶作为模型系统，我们研究了两种已知的小分子p38α抑制剂（SB - 203580和SKF - 86002）的结合特性。在存在可逆结构稳定配体（SB - 203580）的情况下，将p38α直接偶联到生物传感器表面，始终能在生物传感器表面产生大于90%的活性蛋白。使用这种Biacore方法得出的解离常数和动力学常数与通过其他方法测定的值非常吻合。此外，我们扩展了该方法以研究小分子与p38α结合的热力学，并推导了SB - 203580的详细热力学反应途径。本文报道的Biacore方法提供了一种直接且可重复地检测小分子与p38α以及可能与其他蛋白激酶结合的解离常数、动力学和热力学的有效方法。在存在稳定配体的情况下进行固定化可能进一步代表了一种广泛适用的范例，用于创建高活性生物传感器表面。
