High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE
High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE
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DOI：
10.1006/abio.2001.5314
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发表时间：
2001-09-15
影响因子：
2.9
通讯作者：
Myszka, DG
中科院分区：
文献类型：
--
作者：
Rich, RL;Day, YSN;Myszka, DG
表征化合物如何与人血清白蛋白（HSA）结合是评价候选药物的关键。使用华法林作为测试系统，我们验证了BIACORE SPR生物传感器的应用，以可靠地确定药物/HSA相互作用的结合常数。华法林在HSA表面的结合反应是非常可重复的，即使华法林是小的相比，固定化的蛋白质的大小。在高浓度下，华法林结合在HSA上的一个以上位点，这与其已知的结合特性一致。我们测定的高亲和力位点的亲和力（Kd（26 ℃）= 3.7 +/- 1.2 μ M）以及解离速率常数（k（d）（25 ℃）= 1.2 s（-1））也与先前测定的结合常数一致。这些结果验证了生物传感器技术，并说明了如何BIACORE可用于研究药物/HSA的相互作用，在一个高分辨率的模式。使用一组10个测试化合物，我们提出了一个协议，用于确定平衡解离常数的HSA在高通量模式。我们的方法涉及在低化合物浓度下工作，并同时拟合所有化合物的平衡数据。我们表明，SPR确定的%结合值与基于溶液的方法确定的值相关。直接检测小分子（130-800 Da）结合的能力，加上最低的样品要求和自动化仪器，使BIACORE技术适用于评价药物/HSA相互作用。(C)北京：科学出版社.
Characterizing how chemical compounds bind to human serum albumin (HSA) is essential in evaluating drug candidates. Using warfarin as a test system, we validate the application of BIACORE SPR biosensors to reliably determine binding constants for drug/HSA interactions. The binding responses for warfarin over HSA surfaces were extremely reproducible even though warfarin is small compared to the size of the immobilized protein. At high concentrations, warfarin bound at more than one site on HSA, which is consistent with its known binding properties. The affinity we determined for the high-affinity site (K-d(26 degreesC) = 3.7 +/- 1.2 muM), as well as the dissociation rate constant (k(d)(25 degreesC) = 1.2 s(-1)), are also consistent with binding constants determined previously. These results validate the biosensor technology and illustrate how BIACORE can be used to study drug/HSA interactions in a high-resolution mode. Using a set of 10 test compounds, we present a protocol for determining equilibrium dissociation constants for HSA in a high-throughput mode. Our method involves working at low compound concentrations and fitting the equilibrium data for all compounds simultaneously. We show that the % bound values determined by SPR correlate with the values determined by solution-based methods. The ability to examine directly the binding of small molecules (130-800 Da), coupled with minimal sample requirements and automated instrumentation, makes BIACORE technology applicable for evaluating drug/HSA interactions. (C) 2001 Academic Press.