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受体是一种能够识别和选择性结合某种配体(信号分子)的大分子物质, 多为糖蛋白, 一般至少包括两个功能区域: 一是与配体结合的区域; 二是产生效应的区域。当受体与配体结合后, 因构象改变而产生活性, 进而启动一系列信号转导过程, 最终表现为生物学效应。受体与配体间的相互作用具有3个主要特征: ①特异性; ②饱和性; ③高度的亲和力。
配体与受体的结合是一种分子识别过程, 主要靠氢键、离子键与范德华力发挥作用。随着两种分子空间结构互补程度的增加, 相互作用基团之间的距离就会缩短, 作用力就会大大增加, 因此, 分子空间结构的互补性是受体和配体特异结合的主要因素[1]。许多药物在体内都是通过和特异性受体相互作用, 改变细胞的生理生化功能而产生效应的。
受体结合实验是一种在体外直接观察受体的实验手段, 该方法考察的是配体与受体的结合能力, 而非受体功能, 且与受体的信号通路无关。体外受体结合实验已被广泛用于阐明药物的作用机制, 探讨疾病的病因、发病机理, 提高临床合理用药和诊断水平, 探寻新的受体、受体亚型和内源性配体, 以及新药设计和药物筛选等领域[2]。该方法研究效率高、简便, 样品用量少, 毫克量的化合物即可用于研究, 大大促进了受体新药的研发。
放射性显像剂是专门用于核医学领域的一类特殊药物, 其结构中含有放射性核素, 根据核素发出的射线而达到诊断和治疗疾病的目的。很多放射性显像剂是利用放射性配体与体内受体结合的高度选择性而进行显像的, 受体结合实验是目前放射性显像剂研究中广泛使用的一种体外评价方法, 在放射性显像剂设计与筛选中发挥了重要作用[2]。
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根据Clark的占领理论, 受体与配体之间的相互作用是可逆的, 受体与配体的结合反应遵循质量作用定律[1]:
$ [{\text{R}}] + [{\text{L}}]\mathop \rightleftarrows \limits_{{k_2},{v_2}}^{{k_1},{v_1}} [{\text{RL}}] $ 上述反应式中, R和L分别为受体和配体, [R]和[L]分别为游离受体和游离配体的浓度, [RL]为受体配体复合物浓度, v1和v2分别为结合速率和解离速率, k1和k2分别为结合速率常数和解离速率常数。其中, v1=k1[R][L], v2=k2[RL]。当反应达到平衡时, v1=v2, 即k1[R][L]=k2[RL]。
k2与k1的比值为平衡解离常数, 即kD值, 表示受体与配体亲和力的大小, kD值越小, 亲和力越大, 临床所用剂量越小。对一定数量和一定亲和力的受体来说, [RL]与[L]之间为非线性关系, [L]从零开始上升时, [RL]先是上升很快, 以后逐渐变慢, 最后绝大多数受体都变为复合物, 受体达到饱和。这就是饱和曲线, 曲线的高度反映了受体数量的多少, 曲线上升的快慢则反映受体和配体的亲和力大小, 即亲和力越大, 曲线上升越快。
受体结合实验常用的方法有两种: 饱和法和竞争取代反应法。其中, 饱和法又分为单点饱和法和多点饱和法。单点饱和法操作简单、样本用量少, 一般用来计算受体结合容量, 不能得到kD值, 误差也比较大; 多点饱和法是在一定量的受体制剂中, 逐步增加配体的量, 使受体结合趋于饱和。可通过曲线拟合法或直线拟合法计算受体结合容量和配体与受体结合的亲和力(用kD表示), 结果可靠性高。但受体标本、配体用量大, 工作量大。竞争取代反应是在反应系统中加入定量的配体和受体制剂, 并逐渐增加竞争性抑制剂, 该法可用来测定抑制50%受体结合的抑制剂浓度及抑制剂与受体结合的亲和力(用抑制常数kI表示)[1-2]。在受体研究领域, 竞争取代反应法是研究受体与配体结构功能关系的重要方法。在药物研究领域, 该法则可用来研究药物的作用机理及进行药物筛选和发现新药等。
受体结合实验及其在放射性显像剂研究中的应用进展
Progress on the application of ligand receptor binding assays in radiopharmaceuticals
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摘要: 受体结合实验是一种重要的药物筛选方法,它通过体外实验来考察配体与受体的结合能力。目前,很多放射性显像剂是利用放射性配体与体内受体结合的高度选择性来进行受体显像的。因此,受体结合实验是放射性显像剂研究中广泛使用的一种体外评价方法,在放射性显像剂设计与筛选中发挥了重要作用。Abstract: Receptor binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide the information of the relative binding affinity of ligand-receptor.The imaging of many radiopharmaceuticals is based on highly selective radioligand-receptor binding.The technique plays an important role in the design and screening of receptor-targeting radiopharmaceuticals.
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Key words:
- Radiopharmaceuticals /
- Drug design /
- Ligand receptor binding assay /
- Radioligand assay
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