Reactive Metabolite Target Protein Database (TPDB)

This record contains a Narrative that explains the history of the Reactive Metabolite Target Protein Database (TPDB) developed by Robert Hanzlik and Yakov Koen. All of the appendices are zipped into a single file for easier downloading.The biotransformation of drugs and xenobiotic chemicals in living cells sometimes generates chemically reactive metabolites (CRMs) that diffuse through the cell and react covalently with cellular constituents, especially proteins. In some cases this covalent binding may impair the function of the protein(s) leading to cellular injury and even cell death. The elucidation of the chemistry involved in the bioactivation, the details of the ensuing protein modification, and the connection between protein binding and toxicity, became a major pursuit in a number of laboratories starting in the early 1970s. An attempt to collect all the relevant data about the proteins targeted by CRMs led to the construction of the Reactive Metabolite Target Protein Database (TPDB) at the University of Kansas. This curated, searchable database became freely available online in January 2006, and was maintained until July 2020, when its maintenance ceased. This Narrative describes the nature and extent of the data that was compiled and the searches that could be implemented within the TPDB. The results of analyses of that data have been published elsewhere. All of the data originally compiled, including a list of all pertinent literature references, is now available in the Narrative and the associated Appendices at http://hdl.handle.net/1808/30592. List of Appendices: 1. Home page.pdf 2. Help page.pdf 3. Distribution of studies.xlsx 4. Chemical structures and abbreviations.pdf 5. List of hits and proteins.xlsx 6. List of protein "orthologs" (≥90% similar).xlsx 7. Commonality matrix, orthologs excluded.xlsx 8. Commonality matrix, orthologs included.xlsx 9. Targets proteins ranked by hit frequency.xlsx 10. Chemicals ranked by number of targets.xlsx 11. References cited in TPDB.xls

A small plasmid for recombination-based screening

We reported recently the construction of the 4.4-kb RoK-derived pMADI plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with Co1E1 and therefore permits recombination-based screening of 2 libraries that contain Co1E1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the [pi]-encoding pir gene required for R6K replication. To supply [pi] [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans we employed pPR1[Delta]22pir 116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger 2 inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1(1991) 120-123].Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29693/1/0000025.pd

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

Sub-Micrometer-Scale Mapping of Magnetite Crystals and Sulfur Globules in Magnetotactic Bacteria Using Confocal Raman Micro-Spectrometry

The ferrimagnetic mineral magnetite Fe3O4 is biomineralized by magnetotactic microorganisms and a diverse range of animals. Here we demonstrate that confocal Raman microscopy can be used to visualize chains of magnetite crystals in magnetotactic bacteria, even though magnetite is a poor Raman scatterer and in bacteria occurs in typical grain sizes of only 35-120 nm, well below the diffraction-limited optical resolution. When using long integration times together with low laser power (<0.25 mW) to prevent laser induced damage of magnetite, we can identify and map magnetite by its characteristic Raman spectrum (303, 535, 665 cm(-1)) against a large autofluorescence background in our natural magnetotactic bacteria samples. While greigite (cubic Fe3S4; Raman lines of 253 and 351 cm(-1)) is often found in the Deltaproteobacteria class, it is not present in our samples. In intracellular sulfur globules of Candidatus Magnetobacterium bavaricum (Nitrospirae), we identified the sole presence of cyclo-octasulfur (S-8: 151, 219, 467 cm(-1)), using green (532 nm), red (638 nm) and near-infrared excitation (785 nm). The Raman-spectra of phosphorous-rich intracellular accumulations point to orthophosphate in magnetic vibrios and to polyphosphate in magnetic cocci. Under green excitation, the cell envelopes are dominated by the resonant Raman lines of the heme cofactor of the b or c-type cytochrome, which can be used as a strong marker for label-free live-cell imaging of bacterial cytoplasmic membranes, as well as an indicator for the redox state

Ocular and Systemic Safety Evaluation of Calcium Formate as a Dietary Supplement

Purpose: The objective of this study was to perform a preliminary evaluation of the ocular and systemic safety of calcium formate, a dietary calcium supplement for prevention and management of osteoporosis. Although formate is an endogenous product of metabolism, high concentrations are associated with toxicity during methanol overdose. Methods: In this prospective clinical trial, 12 healthy women ingested calcium formate (1,300 mg) three times a day for 14 days. Study evaluations included physical and ocular examination, extensive laboratory testing, serum calcium and formate levels, Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, color vision, visual fields, visual evoked potential (VEP), and full-field, pattern, and multifocal electroretinograms (MERG). Results: The mean baseline serum level of formate was 0.572 ± 0.06 mM. Peak serum levels and final serum formate did not differ significantly from baseline. The final concentration was 0.582 ± 0.091 mM. Accumulation of serum formate did not occur. There was also no evidence of toxicity with calcium formate ingestion. All examinations and tests remained normal, including optic nerve and retinal function. Three subjects had mild transient symptoms attributable to any calcium formulation. Conclusions: Calcium formate is highly bioavailable and well-tolerated. Serum formate remained at basal levels and did not accumulate with repeated dosing. Systemic and ocular safety was demonstrated by objective testing. Given its high oral bioavailability, calcium formate may be a good choice for calcium supplementation in the prevention and management of osteoporosis. Further study will be needed to evaluate its long-term safety in a larger group of subjects representing more varied age, health, dietary, and nutritional status

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

The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [14C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding–induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs

Protein Targets of Thioacetamide Metabolites in Rat Hepatocytes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx400001xThioacetamide (TA) has long been known as a hepatotoxicant whose bioactivation requires S-oxidation to thioacetamide S-oxide (TASO) and then to the very reactive S,S-dioxide (TASO2). The latter can tautomerize to form acylating species capable of covalently modifying cellular nucleophiles including phosphatidylethanolamine (PE) lipids and protein lysine side chains. Isolated hepatocytes efficiently oxidize TA to TASO but experience little covalent binding or cytotoxicity because TA is a very potent inhibitor of the oxidation of TASO to TASO2. On the other hand hepatocytes treated with TASO show extensive covalent binding to both lipids and proteins accompanied by extensive cytotoxicity. In this work, we treated rat hepatocytes with [14C]-TASO and submitted the mitochondrial, microsomal and cytosolic fractions to 2DGE which revealed a total of 321 radioactive protein spots. To facilitate the identification of target proteins and adducted peptides we also treated cells with a mixture of TASO/[13C2D3]-TASO. Using a combination of 1DGE- and 2DGE-based proteomic approaches, we identified 187 modified peptides (174 acetylated, 50 acetimidoylated and 37 in both forms) from a total of 88 non-redundant target proteins. Among the latter, 57 are also known targets of at least one other hepatotoxin. The formation of both amide- and amidine-type adducts to protein lysine side chains is in contrast to the exclusive formation of amidine-type adducts with PE phospholipids. Thiobenzamide (TB) undergoes the same two-step oxidative bioactivation as TA, and it also gives rise to both amide and amidine adducts on protein lysine side chains but only amidine adducts to PE lipids. Despite their similarity in functional group chemical reactivity, only 38 of 62 known TB target proteins are found among the 88 known targets of TASO. The potential roles of protein modification by TASO in triggering cytotoxicity are discussed in terms of enzyme inhibition, protein folding and chaperone function, and the emerging role of protein acetylation in intracellular signaling and the regulation of biochemical pathways

Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3001658Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO2). To explore possible parallels between the metabolism, covalent binding and toxicity of TA and thiobenzamide (TB) we exposed freshly isolated rat hepatocytes to [14C]-TASO or [13C2D3]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO2 is formed, whereas at least some of the TASO2 can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined

Branched amphiphilic peptide capsules: Cellular uptake and retention of encapsulated solutes

Branched amphiphilic peptide capsules (BAPCs) are peptide nanospheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bi-layer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4 °C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially active peptides and proteins