Reactive metabolite formation is a major mechanism of hepatotoxicity. Although reactive electrophiles can be soft or hard in nature, screening strategies have generally focused on the use of glutathione trapping assays to screen for soft electrophiles, with many data sets available to support their use. The use of a similar assay for hard electrophiles using cyanide as the trapping agent is far less common, and there is a lack of studies with sufficient supporting data. Using a set of 260 compounds with a defined hepatotoxicity status by the FDA, a comprehensive literature search yielded cyanide trapping data on an unbalanced set of 20 compounds that were all clinically hepatotoxic. Thus, a further set of 19 compounds was selected to generate cyanide trapping data, resulting in a more balanced data set of 39 compounds. Analysis of the data demonstrated that the cyanide trapping assay had high specificity (92%) and a positive predictive value (83%) such that hepatotoxic compounds would be confidently flagged. Structural analysis of the adducts formed revealed artifactual methylated cyanide adducts to also occur, highlighting the importance of full structural identification to confirm the nature of the adduct formed. The assay was demonstrated to add the most value for compounds containing typical structural alerts for hard electrophile formation: half of the severe hepatotoxins with these structural alerts formed cyanide adducts, while none of the severe hepatotoxins with no relevant structural alerts formed adducts. The assay conditions used included cytosolic enzymes (e.g., aldehyde oxidase) and an optimized cyanide concentration to minimize the inhibition of cytochrome P450 enzymes by cyanide. Based on the demonstrated added value of this assay, it is to be initiated for use at GSK as part of the integrated hepatotoxicity strategy, with its performance being reviewed periodically as more data is generated.

中文翻译：

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亚胺离子反应性代谢物的氰化物捕获：对临床肝毒性的影响


反应性代谢物的形成是肝毒性的主要机制。尽管反应性亲电子试剂本质上可以是软的或硬的，但筛选策略通常集中于使用谷胱甘肽捕获测定来筛选软亲电子试剂，并且有许多数据集可支持其使用。使用氰化物作为捕获剂对硬亲电子试剂进行类似的测定的情况要少得多，并且缺乏具有足够支持数据的研究。使用 FDA 明确的肝毒性状态的 260 种化合物，进行全面的文献检索，获得了 20 种不平衡化合物的氰化物捕获数据，这些化合物均具有临床肝毒性。因此，选择了另一组 19 种化合物来生成氰化物捕获数据，从而得到了更平衡的 39 种化合物数据集。数据分析表明，氰化物捕获测定具有高特异性 (92%) 和阳性预测值 (83%)，因此可以自信地标记肝毒性化合物。对所形成的加合物的结构分析表明，也存在人为的甲基化氰化物加合物，这凸显了完整结构鉴定以确认所形成的加合物的性质的重要性。该测定法被证明对含有硬亲电试剂形成的典型结构警报的化合物具有最大价值：具有这些结构警报的严重肝毒素的一半形成氰化物加合物，而没有相关结构警报的严重肝毒素均不形成加合物。使用的测定条件包括胞质酶（例如醛氧化酶）和优化的氰化物浓度，以尽量减少氰化物对细胞色素 P450 酶的抑制。 基于该检测已证明的附加价值，葛兰素史克将开始使用该检测，作为综合肝毒性策略的一部分，并随着更多数据的产生定期审查其性能。