Ligand binding affinity determined by temperature-dependent circular dichroism: Cyclin-dependent kinase 2 inhibitors
Ligand binding affinity determined by temperature-dependent circular dichroism: Cyclin-dependent kinase 2 inhibitors
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DOI：
10.1016/j.ab.2005.07.032
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发表时间：
2005-10-15
影响因子：
2.9
通讯作者：
Windsor, WT
中科院分区：
文献类型：
--
作者：
Mayhood, TW;Windsor, WT
为了支持细胞周期蛋白依赖性激酶2(CDK2)的药物开发工作，一种可以对铅抑制剂进行排序或估计亲和力的中通量结合分析已经发展起来。该方法被称为温度相关圆二色谱(TdCD)，它利用蛋白质的圆二色谱(CD)经典的依赖温度的去折叠方法来测量蛋白质在没有和存在潜在抑制剂的情况下的去折叠程度。展开的中点是T-m值。通过测量不同蛋白质-抑制剂复合体的T-m增加，对化合物的亲和力和离解常数进行了排序和预测。这是第一次描述了TdCD方法的广泛表征，用于表征药物发现模式中的铅抑制剂。该方法具有几个良好的性质。使用JASCO 810光谱旋光仪的新型六电池Peltier温度控制器，每天可以测定12-18种化合物的亲和力。该方法每个样品只需要20-40微克蛋白质，可以用来估计具有皮摩尔解离常数的化合物对微摩尔的亲和力。铅发现方法的一个重要性质是，使用低浓度的化合物，例如20微米，从一次实验中可以估计出大约5微米的离解常数，该浓度通常低到足以使大多数小分子溶解以供测试。此外，该方法不需要标记化合物或蛋白质。虽然其他方法，如等温滴定量热法(ITC)可以提供结合的完整热力学特征，但ITC要求每个样品1-2毫克蛋白质，不容易确定低于纳摩尔值的结合常数，对于可溶性化合物用途最广，每天需要进行两到三次实验。ITC方法通常不用于高通量药物发现模式；然而，使用来自几个ITC实验的热力学信息可以使TdCD方法在确定可靠的结合常数方面非常稳健。使用激酶抑制剂BMS-250595、紫藜芦醇B、AG-12275、黄烷醇和其他几种化合物，可以很好地比较TdCD和ITC获得的CDK2结合的K-d值。(C)2005 Elsevier Inc.保留所有权利。
To support drug discovery efforts for cyclin-dependent kinase 2 (CDK2), a moderate-throughput binding assay that can rank order or estimate the affinity of lead inhibitors has been developed. The method referred to as temperature-dependent circular dichroism (TdCD) uses the classical temperature-dependent unfolding of proteins by circular dichroism (CD) to measure the degree of protein unfolding in the absence and presence of potential inhibitors. The midpoint of unfolding is the T-m value. Rank ordering the affinity and predictions of the dissociation constant of compounds is obtained by measuring the increase in T-m for different protein-inhibitor complexes. This is the first time an extensive characterization of the TdCD method has been described for characterizing lead inhibitors in a drug discovery mode. The method has several favorable properties. Using the new six-cell Peltier temperature controller for the Jasco 810 spectropolarimeter, one can determine the affinity of 12-18 compounds per day. The method also requires only 20-40 mu g protein per sample and can be used to estimate the affinity of compounds with dissociation constants of picomolar to micromolar. An important property of the method for lead discovery is that dissociation constants of approximately 5 mu M can be estimated from a single experiment using a low concentration of compound such as 20 mu M, which is generally low enough for most small molecules to be soluble for testing. In addition, the method does not require labeling the compound or protein. Although other methods such as isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of binding, ITC requires 1-2 mg protein per sample, cannot readily determine binding constants below nanomolar values, is most versatile with soluble compounds, and has a throughput of two to three experiments per day. The ITC method is not usually used in a high-throughput drug discovery mode; however, using the thermodynamic information from several ITC experiments can make the TdCD method very robust in determining reliable binding constants. Using the kinase inhibitors BMS-250595, purvalanol B, AG-12275, flavopiridol, and several other compounds, it is demonstrated that one can obtain excellent comparisons between the K-d values of binding to CDK2 obtained by TdCD and ITC. (C) 2005 Elsevier Inc. All rights reserved.