Koen Yakov M, Galeva Nadezhda A, Metushi Imir G, Uetrecht Jack, Hanzlik Robert P
Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Ontario M5S 3M2, Canada.
Chem Res Toxicol. 2016 Jun 20;29(6):1064-72. doi: 10.1021/acs.chemrestox.6b00098. Epub 2016 May 5.
Isoniazid (INH) has been a first-line drug for the treatment of tuberculosis for more than 40 years. INH is well-tolerated by most patients, but some patients develop hepatitis that can be severe in rare cases or after overdose. The mechanisms underlying the hepatotoxicity of INH are not known, but covalent binding of reactive metabolites is known to occur in animals and is suspected in human cases. A major unresolved question is the identity of the liver proteins that are modified by INH metabolites. Treating mice with INH leads to accumulation of isonicotinoyl-lysine residues on numerous proteins in the hepatic S9 fraction. Analysis of this fraction by SDS-PAGE followed by tryptic digestion of bands and LC-MS/MS revealed a single adducted peptide derived from d-dopachrome decarboxylase. When a tryptic digest of whole S9 was applied to anti-INH antibody immobilized on beads, only 12 peptides were retained, 5 of which clearly contained isonicotinoyl-lysine adducts and could be confidently assigned to 5 liver proteins. In another experiment, undigested S9 fractions from INA-treated and untreated (UT) mice were adsorbed in parallel on anti-INA beads and the retained proteins were digested and analyzed by LC-MS/MS. The INA-S9 digest showed 1 adducted peptide that was associated with a unique protein whose identity was corroborated by numerous nonadducted peptides in the digest and 13 other proteins identified only by multiple nonadducted peptides. None of these 14 proteins was associated with any peptides present in the UT-S9 fraction. Overall, we identified 7 mouse liver proteins that became adducted by INH metabolites in vivo. Of these 7 INH target proteins, only 2 have been previously reported as targets of any reactive metabolite in vivo.
异烟肼（INH）作为治疗结核病的一线药物已逾40年。大多数患者对INH耐受性良好，但部分患者会出现肝炎，极少数情况下或过量用药后肝炎可能会很严重。INH肝毒性的潜在机制尚不清楚，但已知其活性代谢产物会在动物体内发生共价结合，在人类病例中也疑似如此。一个主要的未解决问题是被INH代谢产物修饰的肝脏蛋白的身份。用INH处理小鼠会导致肝S9组分中许多蛋白质上异烟酰赖氨酸残基的积累。通过SDS-PAGE分析该组分，随后对条带进行胰蛋白酶消化并进行LC-MS/MS分析，发现了一条源自d-多巴色素脱羧酶的单一加合肽段。当将整个S9的胰蛋白酶消化产物应用于固定在珠子上的抗INH抗体时，仅保留了12个肽段，其中5个明显含有异烟酰赖氨酸加合物，并且可以可靠地归属于5种肝脏蛋白。在另一项实验中，将来自INH处理和未处理（UT）小鼠的未消化S9组分平行吸附在抗INH珠子上，对保留的蛋白质进行消化并通过LC-MS/MS分析。INH-S9消化产物显示出1条与一种独特蛋白质相关的加合肽段，该蛋白质的身份通过消化产物中的许多非加合肽段得到证实，另外还鉴定出仅由多个非加合肽段确定的13种其他蛋白质。这14种蛋白质中没有一种与UT-S9组分中存在的任何肽段相关。总体而言，我们鉴定出7种在体内被INH代谢产物加合的小鼠肝脏蛋白。在这7种INH靶蛋白中，之前仅有2种被报道为体内任何活性代谢产物的靶标。
