Foster Celia K, Thorpe Colin
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States.
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, United States.
Free Radic Biol Med. 2017 Jul;108:741-749. doi: 10.1016/j.freeradbiomed.2017.04.367. Epub 2017 Apr 30.
This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide Isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5-10nM, can utilize a range of thiol substrates (including 5µM dithiothreitol, 10µM reduced RNase thiols, and 5mM glutathione; GSH), and can operate from pH 6-9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-β-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (K values of 7 and <0.1µM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.
本文探讨了如何在含有潜在竞争性含硫醇肽和蛋白质的实际浓度下，评估哺乳动物蛋白质二硫键异构酶（PDI）酶的硫醇反应性抑制剂库存不断增加的情况下的功效。为此，我们通过使用一种市售的半胱氨酸衍生物（BODIPY FL L-胱氨酸；BD-SS）引入了一种广泛使用的还原酶测定法的变体，该衍生物在二硫键断裂时荧光增加55倍（激发/发射；490/513nm）。这种与酶标仪兼容的方法可检测低至5-10nM的人PDI，可使用一系列硫醇底物（包括5µM二硫苏糖醇、10µM还原型核糖核酸酶硫醇和5mM谷胱甘肽；GSH），并且可以在各种缓冲液中在pH 6-9.5的条件下运行。PDI测定通常采用低微摩尔浓度的底物，这在评估硫醇导向抑制剂时会导致歧义。本研究在预孵育和测定阶段均使用5mM GSH，以更真实地反映抑制剂在细胞内可能遇到的高浓度硫醇。细胞外PDI面临的潜在竞争性硫醇浓度要低得多；为了评估这些条件下的还原酶活性，将预还原的PDI用抑制剂处理，然后在不存在额外竞争性硫醇的情况下跟踪BD-SS还原时的荧光增加。两种测定模式都用四种机制不同的PDI抑制剂进行了测试。发现两种可逆试剂，3,4-亚甲二氧基-β-硝基苯乙烯（MNS）和砷化合物APAO，在不存在竞争性硫醇的情况下是PDI的强抑制剂，但在存在5mM GSH时无效。对硝基苯乙烯的进一步研究表明，MNS不仅与GSH形成容易的迈克尔加合物，还与未折叠蛋白质的硫醇形成加合物（K值分别为7和<0.1µM），这表明这种膜渗透性试剂存在多个潜在的细胞内靶点。发现不可逆烷基化剂氯乙酰胺16F16对PDI的抑制作用仅被5mM GSH适度减弱。最后，发现硫醇非依赖性黄酮类抑制剂槲皮素-3-O-芸香糖苷在再氧化和周转测定类型中显示出相同的功效。这项工作提供了一个框架来评估可能靶向PDI的CxxC基序的抑制剂，并解决了体内硫醇导向试剂行为解释中的一些复杂性。
