人血清α-1-酸性糖蛋白(AAG)是一种急性期血浆蛋白,参与许多药物的结合和转运,特别是碱性和亲脂性物质。已经报道终止AAG的N-聚糖链的唾液酸基团响应于许多健康状况而改变,并且可能对药物与AAG的结合具有影响。在这项研究中,我们量化了天然和去唾液酸化的AAG与来自不同药物治疗组的7种药物之间的结合(卡维地洛,地尔硫卓,双嘧达莫,丙咪嗪,利多卡因,普萘洛尔,长春碱)使用微尺度热电泳(MST)。该方法的选择是由于其鲁棒性和高灵敏度,允许基于荧光分子的热泳运动的分子相互作用的精确量化。天然和去唾液酸化的AAG的详细聚糖分析显示,对于酶促去唾液酸化的AAG,唾液酸含量降低了98%以上。MST结果表明,去唾液酸化通常会改变AAG与药物之间的结合亲和力,导致Kd值的增加或减少,可能是由于唾液酸含量不同引起的AAG构象变化。这种作用也反映在去唾液酸化的AAG的变性温度增加中。我们的发现表明脱盐作用对游离药物浓度的影响不同,取决于药物与AAG相对于人血清白蛋白(HSA)的结合亲和力。对于像潘生丁这样的药物,利多卡因,还有卡维地洛,对AAG有更高的亲和力,脱盐显著改变游离药物浓度。相比之下,普萘洛尔等药物,丙咪嗪,还有长春碱,它们具有很强的白蛋白结合作用,只显示最小的变化。值得注意的是,双嘧达莫的游离药物浓度对AAG浓度和糖基化的变化特别敏感,观察到下降高达15%,强调个性化医疗中剂量调整的必要性。
Human serum alpha-1-acid glycoprotein (AAG) is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. The sialic acid groups that terminate the N-glycan chains of AAG have been reported to change in response to numerous health conditions and may have an impact on the binding of drugs to AAG. In this study, we quantified the binding between native and desialylated AAG and seven drugs from different pharmacotherapeutic groups (carvedilol, diltiazem, dipyridamole, imipramine, lidocaine, propranolol, vinblastine) using microscale thermophoresis (MST). This method was chosen due to its robustness and high sensitivity, allowing precise quantification of molecular interactions based on the thermophoretic movement of fluorescent molecules. Detailed glycan analysis of native and desialylated AAG showed over 98% reduction in sialic acid content for the enzymatically desialylated AAG. The MST results indicate that desialylation generally alters the binding affinity between AAG and drugs, leading to either an increase or decrease in Kd values, probably due to conformational changes of AAG caused by the different sialic acid content. This effect is also reflected in an increased denaturation temperature of desialylated AAG. Our findings indicate that desialylation impacts free drug concentrations differently, depending on the binding affinity of the drug with AAG relative to human serum albumin (HSA). For drugs such as dipyridamole, lidocaine, and carvedilol, which have a higher affinity for AAG, desialylation significantly changes free drug concentrations. In contrast, drugs such as propranolol, imipramine, and vinblastine, which have a strong albumin binding, show only minimal changes. It is noteworthy that the free drug concentration of dipyridamole is particularly sensitive to changes in AAG concentration and glycosylation, with a decrease of up to 15% being observed, underscoring the need for dosage adjustments in personalized medicine.