进入人体血液循环后,小分子药物与各种血浆蛋白广泛相互作用,如人血清白蛋白和α1-酸性糖蛋白。这些相互作用深刻地影响药物在体内的分布和药物与靶标的结合,从而影响药物的疗效。深入研究药物-血浆蛋白相互作用对优化药物性质具有重要意义,新药的开发,风险评估,和药物联合治疗。因此,发展高效至关重要,敏感,以及阐明药物-血浆蛋白相互作用的准确方法。色谱是一个强大的工具,具有高通量,高分离性能,以及在表征药物-蛋白质相互作用方面的高灵敏度。高效亲和色谱(HPAC)和毛细管电泳(CE)已被广泛用于该领域。这些方法包括测定蛋白质的翻译后修饰对多种药物的结合和竞争性结合的影响。此外,各种色谱方法用于获得相互作用信息,如结合常数,绑定位点编号,和解离速率常数。在这次审查中,简要综述了HPAC和CE在药物-血浆蛋白相互作用研究中的常用策略和最新进展。蛋白质的固定方法,正面分析的原理和应用,带状洗脱,超快亲和提取,峰值分析,讨论了HPAC的峰衰减分析以及CE的亲和毛细管电泳(ACE)和毛细管电泳前沿分析(CE-FA)。HPAC依靠通过共价连接或物理吸附将蛋白质固定在色谱固定相的表面上,然后通过多种色谱方法获得药物-蛋白质相互作用信息。在正面色谱分析中,不同浓度药物的流动相通过HPAC色谱柱以获得不同的突破时间。该过程可以高精度地确定亲和蛋白中药物结合位点的数量和每个位点的结合常数。带状洗脱方法可以使用位点特异性探针检测蛋白质上的药物结合位点,以确定药物和探针之间是否存在竞争。区带洗脱法的样品消耗和分析时间远少于正面色谱分析中的样品消耗和分析时间。超快亲和提取方法可以注入复杂的样品,如血清,进入亲和柱以确定游离的药物成分。它可以通过改变色谱流速来测量药物-蛋白质相互作用的组合和解离常数。峰谱分析和峰衰减分析都是研究药物和蛋白质解离的有效方法。在CE分析中,将药物和蛋白质样品溶解在电泳缓冲液中,在电泳过程中测量它们的相互作用,并且具有高精度和低样品消耗。然而,蛋白质在毛细管壁上的吸附会损害CE性能。药物-蛋白质相互作用分析中常用的CE方法是ACE和CE-FA。ACE通常通过添加不同浓度的蛋白质来改变药物的有效流动性来进行。这种方法得到了广泛的应用,最近开发了几种变体技术。CE-FA涉及以已知浓度与靶蛋白预混合的药物的取样。与其他CE方法相比,CE-FA具有高通量的独特优势,自动在线分析,以及确定药物-蛋白质相互作用的能力。最后,总结了目前色谱法的不足,探讨了色谱技术在药物-血浆蛋白相互作用研究领域的应用前景和发展方向。
After entering human blood circulation, small-molecule drugs interact extensively with various plasma proteins, such as human serum albumin and α1-acid glycoprotein. These interactions profoundly affect the distribution of drugs in vivo and the binding of drugs to targets, thus affecting the efficacy of drugs. In-depth investigation of drug-plasma protein interactions is of great significance for the optimization of drug properties, the development of new drugs, risk assessment, and combination therapy of drugs. Therefore, it is essential to develop highly efficient, sensitive, and accurate methods for elucidating drug-plasma protein interactions. Chromatography is a powerful tool with high throughput, high separation performance, and high sensitivity in the characterization of drug-protein interactions. High-performance affinity chromatography (HPAC) and capillary electrophoresis (CE) have been widely utilized in this field. These methods include the determination of the effects of the posttranslational modification of proteins on binding and the competitive binding of multiple drugs. In addition, various chromatographic methods are used to obtain interaction information such as the binding constant, binding-site number, and dissociation rate constant. In this review, the common strategies and recent advances in HPAC and CE in the
study of drug-plasma protein interactions are briefly reviewed. The immobilization methods of proteins, the principles and applications of frontal analysis, zonal elution, ultrafast affinity extraction, peak profiling, and peak decay analysis are discussed for HPAC and affinity capillary electrophoresis (ACE) and capillary electrophoresis frontal analysis (CE-FA) for CE. HPAC relies on the fixation of proteins on the surfaces of chromatographic stationary phases by covalent linking or physical adsorption, followed by obtaining the drug-protein interaction information through a variety of chromatographic methods. In the frontal chromatography analysis, mobile phases with different concentrations of drugs are passed through the HPAC column to obtain different breakthrough times. The process can determine the number of drug binding sites and the binding constant of each site in the affinity protein with high accuracy. The zonal elution method can detect the drug binding sites on proteins using site-specific probes to determine whether there is competition between drugs and probes. The sample consumption and analysis time of the zonal elution method are much less than those in frontal chromatography analysis. The ultrafast affinity extraction method can inject complex samples, such as serum, into affinity columns to determine the free drug components. It can measure the combination and dissociation constants of drug-protein interactions by changing the chromatography flow rate. Peak profiling and peak decay analyses are both effective methods for investigating the dissociation of drugs and proteins. In CE analysis, the drug and protein samples are dissolved in an electrophoresis buffer, and their interactions are measured during electrophoresis with high accuracy and low sample consumption. However, the adsorption of proteins on the capillary wall can compromise CE performance. Common CE methods in drug-protein interaction analysis are ACE and CE-FA. ACE is usually performed by changing the effective mobility of drugs via the addition of different concentrations of proteins. This method has been widely used, and several variant techniques have been developed recently. CE-FA involves the sampling of a drug premixed at a known concentration with a target protein. Compared with other CE methods, CE-FA exhibits the unique advantages of high throughput, automatic online analysis, and the ability to determine high-order drug-protein interactions. Finally, the shortcomings of current chromatography methods are summarized, and the application prospects and development direction of chromatography technology in the field of drug-plasma protein interaction research are discussed.