The reactive metabolite target protein database (TPDB) – a web-accessible resource

BACKGROUND: The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins. DESCRIPTION: The Reactive Metabolite Target Protein Database (TPDB) is a comprehensive, curated, searchable, documented compilation of publicly available information on the protein targets of reactive metabolites of 18 well-studied chemicals and drugs of known toxicity. TPDB software enables i) string searches for author names and proteins names/synonyms, ii) more complex searches by selecting chemical compound, animal species, target tissue and protein names/synonyms from pull-down menus, and iii) commonality searches over multiple chemicals. Tabulated search results provide information, references and links to other databases. CONCLUSION: The TPDB is a unique on-line compilation of information on the covalent modification of cellular proteins by reactive metabolites of chemicals and drugs. Its comprehensiveness and searchability should facilitate the elucidation of mechanisms of reactive metabolite toxicity. The database is freely available a

Bioinformatic analysis of xenobiotic reactive metabolite target proteins and their interacting partners

<p>Abstract</p> <p>Background</p> <p>Protein covalent binding by reactive metabolites of drugs, chemicals and natural products can lead to acute cytotoxicity. Recent rapid progress in reactive metabolite target protein identification has shown that adduction is surprisingly selective and inspired the hope that analysis of target proteins might reveal protein factors that differentiate target- vs. non-target proteins and illuminate mechanisms connecting covalent binding to cytotoxicity.</p> <p>Results</p> <p>Sorting 171 known reactive metabolite target proteins revealed a number of GO categories and KEGG pathways to be significantly enriched in targets, but in most cases the classes were too large, and the "percent coverage" too small, to allow meaningful conclusions about mechanisms of toxicity. However, a similar analysis of the directlyinteracting partners of 28 common targets of multiple reactive metabolites revealed highly significant enrichments in terms likely to be highly relevant to cytotoxicity (e.g., MAP kinase pathways, apoptosis, response to unfolded protein). Machine learning was used to rank the contribution of 211 computed protein features to determining protein susceptibility to adduction. Protein lysine (but not cysteine) content and protein instability index (i.e., rate of turnover in vivo) were among the features most important to determining susceptibility.</p> <p>Conclusion</p> <p>As yet there is no good explanation for why some low-abundance proteins become heavily adducted while some abundant proteins become only lightly adducted in vivo. Analyzing the directly interacting partners of target proteins appears to yield greater insight into mechanisms of toxicity than analyzing target proteins per se. The insights provided can readily be formulated as hypotheses to test in future experimental studies.</p

Applications of Mass Spectrometry to Analysis of Prodiginines, Bioactivated Methylenedianiline Intermediates, and Hypoxia Induced Changes in the Zebrafish Skeletal Muscle Proteome

Mass spectrometry coupled with liquid chromatography and gel electrophoresis enables separation and detection of components in a complex mixture. During the last two decades, its applications were dramatically extended and remarkable progress has been made in many fields, in particular, environmental and biological analyses. This dissertation focuses on identification and characterization of biologically active compounds and comparative analysis of protein expression changes. The first two projects (Chapters 2 and 3) focus on the application of LC/MS approach to profile the bioactivated intermediates of 4, 4\u27-methylenedianiline (DAPM) from rat vascular smooth muscle cells (VSMCs) and bile. In our study, several DAPM metabolites were detected and characterized in detail by liquid chromatography-electrospray tandem mass spectrometry. The structural assignments of these metabolites from VSMCs and rat bile significantly improve our understanding of DAPM biotransformations and toxicity. The third project described in Chapter 4 focuses on using electrospray tandem mass spectrometry (ES-MS/MS) and theoretical calculation (GAUSSIAN 03 program) to investigate the unusual methyl radical loss and consecutive fragment ions that dominate the low-energy collision induced dissociation (CID) mass spectra of prodiginine compounds. Structures of the fragment ions are proposed and explanations are given to rationalize the observed competition between the formation of even-electron ions and radical ions. Our study shows that the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. In Chapter 5, hypoxia-induced changes of zebrafish skeletal muscle were studied using two-dimensional difference in-gel electrophoresis (2D-DIGE) in vivo after 48 h in hypoxia vs. normoxia. The results showed that proteins involved in mitochondrial oxidative metabolism are down-regulated, whereas glycolytic enzymes are up-regulated to compensate for the loss of ATP synthesis in aerobic metabolism. The up-regulation of two spots identified as hemoglobin variants was also observed. These protein expression changes are consistent with a hypoxic response that enhances anaerobic metabolism or O2 transport to tissues

Bioactivation of Drugs by Cytochromes P450 : New Tools and Concepts for the Characterization of Reactive Metabolites of Drugs

Vermeulen, N.P.E. [Promotor]Commandeur, J.N.M. [Copromotor

Protein adducts at critical protein sites as markers of toxicological risk

The formation of conjugates between the electrophilic reactive metabolites of drugs and nucleophilic protein sites is known to be associated with toxicological risk. At present there is no low cost and high throughput means of reliably detecting the presence of drug-protein adducts in vitro or in vivo. The development of a reliable high throughput methodology would facilitate the study of underlying mechanisms of toxicity and prove useful in early screening of potential drug molecules. Assays using liver microsomes and trapping agents such as glutathione are used to produce and detect a wide range of drug reactive metabolites which are then characterised by mass spectrometry. The glutathione trapping is effective for metabolite identifications but, the modification of proteins by means of electrophilic attack on nucleophilic centres often occurs in an enzyme independent manner and is unlikely to be analogous to the glutathione model. In order to create a more suitable model system, three short polypeptides were designed and synthesised. These peptides were incubated with clozapine and human liver microsomes. The resulting metabolite-peptide conjugates were analysed by nanoLC-MS. Results indicated that a characteristic conjugate specific ion at 359.1 Da could be detected for each of the peptides. This data was used to create a precursor ion scan specific for the presence of this characteristic ion. Protein separation techniques including SCX, Offgel IEF and 1d-gel electrophoresis, in conjunction with LC-MS (with the precursor 359 scan), were applied to microsome prep samples in order to identify modified proteins. Using these approaches some 1700 protein identifications were made, more than 1000 of these were unique hits. The precursor ion scan was found to have poor selectivity identifying roughly 1/3 as many proteins as the information dependant acquisition approach. No drug-protein adducts were identified. Further to this a novel application of saturation DIGE was applied in order to enrich for the presence of protein adducts. The DiGE approach was used to identify some 15 proteins with apparent change in abundance (fluorescence intensity) between clozapine treated and untreated samples. Spots were excised from the 2d gel digested and analysed by reversed phase liquid chromatography mass spectrometry. The IDA scans identified some 147 unique protein hits, the precursor ion scans identified 18. Again no drug-protein adducts were found. Biotinylated desmethyl clozapine was metabolised in the human liver microsome assay. Western blotting was carried out on a 2d gel run from an assay sample. The Western membrane was probed using an HRP-Streptavidin probe. Imaging of the membrane revealed the presence of several biotin bearing proteins, many of which were not present in the negative control sample. A print out of the image was used as a map for the excision of modified proteins from a duplicate gel. Digestion and LCMS analysis of the samples revealed the presence of several proteins but no protein-adducts were found

Role of genetic polymorphisms of drug metabolising enzymes in idiosyncratic drug reactions In vitro to in vivo translation

Vermeulen, N.P.E. [Promotor]Commandeur, J.N.M. [Copromotor

cii Student Papers - 2021

In this collection of papers, we, the Research Group Critical Information Infrastructures (cii) from the Karlsruhe Institute of Technology, present nine selected student research articles contributing to the design, development, and evaluation of critical information infrastructures. During our courses, students mostly work in groups and deal with problems and issues related to sociotechnical challenges in the realm of (critical) information systems. Student papers came from four different cii courses, namely Emerging Trends in Digital Health, Emerging Trends in Internet Technologies, Critical Information Infrastructures, and Digital Health in the winter term of 2020 and summer term of 2021

Filing and Mining the Reactive Metabolite Target Protein Database

The post-translational modification of proteins is a well-known endogenous mechanism for regulating protein function and activity. Cellular proteins are also susceptible to post-translational modification by xenobiotic agents that possess, or whose metabolites possess, significant electrophilic character. Such non-physiological modifications to endogenous proteins are sometimes benign, but in other cases they are strongly associated with, and are presumed to cause, lethal cytotoxic consequences via necrosis and/or apoptosis. The Reactive Metabolite Target Protein Database (TPDB) is a searchable, freely web-accessible (http://tpdb.medchem.ku.edu:8080/protein_database/) resource that attempts to provide a comprehensive, up-to-date listing of known reactive metabolite target proteins. In this report we characterize the TPDB by reviewing briefly how the information it contains came to be known. We also compare its information to that provided by other types of “-omics” studies relevant to toxicology, and we illustrate how bioinformatic analysis of target proteins may help to elucidate mechanisms of cytotoxic responses to reactive metabolites