Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma
Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma
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DOI：
10.1073/pnas.091431798
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发表时间：
2001-05-08
影响因子：
11.1
通讯作者：
Kelly, JW
中科院分区：
文献类型：
--
作者：
Purkey, HE;Dorrell, MI;Kelly, JW
转甲状腺素(TTR)四聚体的解离和错误折叠有助于组装成淀粉样纤维，从而可能导致老年性系统性淀粉样变性和家族性淀粉样多发性神经病。我们之前已经发现了50多个小分子，它们结合并稳定四聚体TTR，在体外抑制淀粉样原纤维的形成。本文介绍了一种评价这些抑制剂与人血浆中TTR结合选择性的方法。人血浆是一种由60多种蛋白质和大量小分子组成的复杂生物液。我们的免疫沉淀方法从生物液体(如血浆)中分离TTR和结合的小分子，并通过高效液相分析定量结合到蛋白质上的小分子的数量。这种方法表明，只有一小部分在体外饱和TTR结合位点的抑制剂在血浆中做到这一点。这些选择性抑制剂现在可以在TTR淀粉样病的动物模型中进行测试，以探索淀粉样蛋白假说的有效性。这种方法可以很容易地扩展到评估小分子与给定生物液体中任何蛋白质的结合选择性，而不需要确定或猜测哪些其他蛋白质成分可能是竞争对手。这是了解潜在药物的分布、代谢、活性和毒性的中心问题。
Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.