Morelock M M, Ingraham R H, Betageri R, Jakes S
Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877-0368, USA.
J Med Chem. 1995 Apr 14;38(8):1309-18. doi: 10.1021/jm00008a009.
Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.
开发了实验和计算方法用于表面等离子体共振（SPR）测量，该测量涉及溶液结合成分与表面固定配体之间的相互作用。这些方案用于区分lck的SH2结构域与磷酸酪氨酸肽之间亲和力的差异。表面固定配体是磷酸酪氨酸肽EPQpYEEIPIA，其包含用于结合lck SH2的共有序列（pYEEI）。在动力学实验中，在一系列谷胱甘肽 - S - 转移酶（GST） - SH2浓度的结合和解离反应过程中测量SPR现象，生成一组SPR曲线。使用A + B<==>AB模型进行全局计算分析，得到一组参数估计值和统计数据。以简化形式设计了平衡实验，以便能够快速准确地确定平衡常数（Keq）。开发了一种针对GST - SH2的竞争性平衡测定法，其中一系列磷酸酪氨酸肽（源自pYEEI序列）的Keq值在3个数量级上变化。有趣的是，这些结果突出了 +1位谷氨酸在提供与SH2结构域的高亲和力结合方面的重要性。对于大多数药物发现计划，这些Keq测定是初级筛选效力的充分衡量标准，koff和kon在二级测定中确定。因此，将这些技术应用于SPR结合现象在受体 - 配体拮抗剂的发现和设计中应具有重要价值。
