Laboratory of Metals in Medicine (MetMed), Department of Chemistry "Ugo Schiff", University of Florence, Via della Lastruccia 3-13, 50019 Sesto Fiorentino, Italy.
Dalton Trans. 2020 May 14;49(18):5906-5913. doi: 10.1039/d0dt00283f. Epub 2020 Apr 21.
The clinically established gold drug Auranofin was reacted individually with a group of representative proteins, namely ubiquitin, ribonuclease A, carbonic anhydrase, haemoglobin and superoxide dismutase, and adduct formation was monitored in the various cases by ESI-MS analysis. We found that the reaction is highly selective for solvent exposed free cysteines that are modified through coordination of the AuPEt fragment. Indeed, ESI-Q-TOF MS spectra carried out on protein samples incubated with a three fold molar excess of Auranofin allowed direct detection of the native proteins bearing bound AuPEt fragments in the cases of carbonic anhydrase and haemoglobin. At variance, the two proteins that do not possess any free cysteine residue, i.e. ubiquitin and ribonuclease A, were unable to bind the gold fragment. In the case of superoxide dismutase, adduct formation is hindered by the scarce solvent accessibility of the free cysteine residue. These findings were further confirmed by a series of competition binding experiments with ebselen, a potent and selective cysteine-modifying reagent; we observed that pre-treatment with ebselen prevents the binding of the AuPEt fragment to both carbonic anhydrase and haemoglobin.
临床上已确立的金药物金诺芬与一组有代表性的蛋白质（即泛素、核糖核酸酶 A、碳酸酐酶、血红蛋白和超氧化物歧化酶）分别进行反应，并通过 ESI-MS 分析监测各种情况下的加合物形成。我们发现，该反应对溶剂暴露的游离半胱氨酸具有高度选择性，这些半胱氨酸通过 AuPEt 片段的配位而被修饰。实际上，对用金诺芬的三倍摩尔过量孵育的蛋白质样品进行 ESI-Q-TOF MS 谱分析，允许在碳酸酐酶和血红蛋白的情况下直接检测结合有 AuPEt 片段的天然蛋白质。相比之下，不含任何游离半胱氨酸残基的两种蛋白质（即泛素和核糖核酸酶 A）无法结合金片段。在超氧化物歧化酶的情况下，由于自由半胱氨酸残基的溶剂可及性较差，加合物的形成受到阻碍。通过与 ebselen（一种强效且选择性的半胱氨酸修饰试剂）进行的一系列竞争结合实验进一步证实了这些发现；我们观察到，用 ebselen 预处理可防止 AuPEt 片段与碳酸酐酶和血红蛋白结合。
