Identification of Protein Targets of Reactive Metabolites of Tienilic Acid in Human Hepatocytes

Abstract

Tienilic acid (TA) is a uricosuric diuretic that was withdrawn from the market only months after its introduction because of reports of serious incidents of drug-induced liver injury including some fatalities.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 [14C]-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 non-redundant 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 2–4 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 human 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.

Protein Targets of Thioacetamide Metabolites in Rat Hepatocytes

“…A similar pattern of radioactive but apparently non-proteinaceous material was observed on blots of 2D gels of liver microsomes from TB-treated rats, 7 liver mitochondria from BB-treated rats, 16 and total protein from human hepatocytes incubated with tienilic acid. 22 Because this material is observed only in the membrane fractions and does not stain with Coomassie, we speculated that it might be some type of adducted lipid material. Although TASO and TB are well known to generate adducts on the amine group of PE lipids, 6, 8 it is not clear whether these adducts could account for the low MW/low pI material in the phosophorimage and we have not pursued this question further.…”

Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes

“…This amount of covalent binding is comparable or greater than that reported for other drugs that are known to undergo bioactivation followed by protein covalent binding and subsequent adverse drug reactions (ADRs), including benzbromarone (0.39 nmol Eq/mg protein) (Takakusa et al, 2008), acetaminophen (0.09 nmol Eq/mg protein) (Nakayama et al, 2009), and flutamide (0.18 nmol Eq/mg protein) (Nakayama et al, 2009). It has been proposed that the covalent binding of chemically reactive metabolites to cellular proteins could result in direct organ toxicity as well as the formation of neoantigens that result in immune or even autoimmune reactivity (Koen et al, 2012). Interestingly, our results demonstrate that TMP has higher covalent binding capabilities in vitro with HLM incubations than previously observed with SMX (0.003 nmol Eq/mg protein) (Nakayama et al, 2009).…”

Quantitative Chemical Proteomic Profiling of the in Vivo Targets of Reactive Drug Metabolites

“…It is generally appreciated that reactive drug metabolites may be a root cause of DILI, and several methods have accordingly been introduced to identify proteins that are covalently modified by drugs in the liver, including the use of radiolabeled drugs 12 and antibodies that enrich specific drug-protein adducts 35 . These methods have succeeded in providing a growing inventory of liver proteins targeted by reactive drug metabolites; but, this inventory is likely quite incomplete, given that different studies performed on the same drug often identify virtually non-overlapping lists of protein targets 14 . Thus, fundamental questions about reactive drug metabolism remain largely unanswered, such as - i) what is the shared versus unique protein target landscape for reactive metabolites generated from different drugs in vivo ; and ii) what is the stoichiometry of modification, or fractional engagement, of targets by reactive drug metabolites in vivo ?…”

“…The identification of protein targets of reactive metabolites commonly involves the use of radiolabeled drugs, which are administered to animal or cell models and drug-adducted proteins then detected by one- or two-dimensional SDS-PAGE and autoradiography. Proteins comigrating with radioactive signals are then excised from the gel and identified by mass spectrometry (MS) methods. , While this general approach has been employed to identify proteins modified by several drugs that produce DILI, , it suffers from limitations that include poor sensitivity and resolution, resulting in a bias toward detecting high-abundance proteins and ambiguity in assigning the identified proteins as targets of reactive metabolites. These deficiencies could be addressed by incorporating latent affinity handles into drugs of interest to facilitate the enrichment of reactive metabolite-modified proteins over background proteins.…”