Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes
Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes
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DOI：
10.1021/tx300103j
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发表时间：
2012-05-01
影响因子：
4.1
通讯作者：
Hanzlik, Robert P.
中科院分区：
文献类型：
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作者：
Koen, Yakov M.;Sarma, Diganta;Hanzlik, Robert P.
替尼酸（TA）是一种尿酸利尿剂，由于有严重的药物性肝损伤事件（包括一些死亡事件）的报告，其在上市仅几个月后就被撤出市场。认为其肝毒性本质上主要是免疫过敏性的。像其他噻吩化合物一样，TA经历生物转化为S-氧化物代谢物，然后与细胞蛋白质共价反应。为了鉴定TA代谢产物的蛋白质靶点，我们将[C-14]-TA TA与人肝细胞孵育，通过2D凝胶电泳分离细胞蛋白，并通过胰蛋白酶消化然后通过LC-MS/MS分析36个放射性斑点中的蛋白质。31个斑点含有至少一种可鉴定的蛋白质。16个斑点仅含有14种非冗余蛋白中的一种，因此认为其是TA代谢物的靶标。14个中的6个也在含有1至3个额外蛋白质的其他放射性斑点中发现。14个中的8个未报告为除TA以外的任何反应性代谢物的靶点。其他15个斑点各含有2至4种可鉴别的蛋白质，其中许多是其他化学反应性代谢物的已知靶标，但由于未观察到加合肽，因此这些斑点中加合蛋白的鉴别不明确。有趣的是，所有的放射性斑点都对应于低丰度的蛋白质，而混合物中许多高丰度的蛋白质没有显示出放射性。此外，在大鼠肝脏中TA的约16种先前报道的蛋白质靶标中（Methogo，R.，Dansette，P.，和Klarskov，K.（2007）Int.J.Mass Spectrom.，268，284 - 295），在这项工作中鉴定的14个靶标中只有一个（延胡索酰乙酰乙酸酶）。这种差异的一个原因可能是统计学上的，因为每项研究都从肝细胞中存在的数千个靶点中鉴定了少量靶点。另一个可能是物种差异（即，大鼠对人），还有一种方法可以是检测加合蛋白的方法（即，蛋白质印迹相对于C-14）。人类靶蛋白的知识非常有限。在350多种已知的反应性代谢物的蛋白质靶标中，只有42种已知来自人类，其中只有21种已知是一种以上化学物质的靶标。尽管如此，人类靶蛋白可以在体外使用分离的肝细胞进行鉴定的证明，应使物种差异的问题在未来得到更充分的解决。
Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities. Its hepatotoxicity is considered to be primarily immunoallergic in nature. Like other thiophene compounds, TA undergoes biotransformation to a S-oxide metabolite which then reacts covalently with cellular proteins. To identify protein targets of TA metabolites, we incubated [C-14]-TA TA with human hepatocytes, separated cellular proteins by 2D gel electrophoresis, and analyzed proteins in 36 radioactive spots by tryptic digestion followed by LC-MS/MS. Thirty-one spots contained at least one identifiable protein. Sixteen spots contained only one of 14 nonredundant proteins which were thus considered to be targets of TA metabolites. Six of the 14 were also found in other radioactive spots that contained from 1 to 3 additional proteins. Eight of the 14 had not been reported to be targets for any reactive metabolite other than TA. The other 15 spots each contained from 2 to 4 identifiable proteins, many of which are known targets of other chemically reactive metabolites, but since adducted peptides were not observed, the identity of the adducted protein(s) in these spots is ambiguous. Interestingly, all the radioactive spots corresponded to proteins of low abundance, while many highly abundant proteins in the mixture showed no radioactivity. Furthermore, of approximately 16 previously reported protein targets of TA in rat liver (Methogo, R., Dansette, P., and Klarskov, K. (2007) Int. J. Mass Spectrom., 268, 284-295), only one (fumarylacetoacetase) is among the 14 targets identified in this work. One reason for this difference may be statistical, given that each study identified a small number of targets from among thousands present in hepatocytes. Another may be the species difference (i.e., rat vs human), and still another may be the method of detection of adducted proteins (i.e., Western blot vs C-14). Knowledge of human target proteins is very limited. Of more than 350 known protein targets of reactive metabolites, only 42 are known from humans, and only 21 of these are known to be targets for more than one chemical. Nevertheless, the demonstration that human target proteins can be identified using isolated hepatocytes in vitro should enable the question of species differences to be addressed more fully in the future.