Drug−Protein Adducts:  An Industry Perspective on Minimizing the Potential for Drug Bioactivation in Drug Discovery and Development

Although the major working hypothesis for the mechanism of idiosyncratic drug reactions (IDRs), the hapten hypothesis, has not changed since 1987, several hypotheses have been added, for example, the danger hypothesis and the pharmaceutical interaction hypothesis. Genetic studies have found that several IDRs are linked to specific HLA genes, providing additional evidence that they are immune-mediated. Evidence that most IDRs are caused by reactive metabolites has led pharmaceutical companies to avoid drug candidates that form significant amounts of reactive metabolites; however, at least one IDR, ximelagatran-induced liver toxicity, does not appear to be caused by a reactive metabolite. It is possible that there are biomarkers such as those related to cell stress that would predict that a drug candidate would cause a significant incidence of IDRs; however, there has been no systematic study of the changes in gene expression induced by drugs known to cause IDRs. A major impediment to the study of the mechanisms of IDRs is the paucity of valid animal models, and if we had a better mechanistic understanding, it should be easier to develop such models. There is growing evidence that these adverse reactions are more varied and complex than previously recognized, and it is unlikely that a quick fix will be achieved. However, IDRs are an important cause of patient morbidity and mortality and markedly increase the uncertainty of drug development; therefore, continued basic research in this area is essential.

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“…This approach has been adopted by some pharmaceutical companies (60). Unfortunately, there is no evidence yet that this has led to safer drugs.…”

Section: Decreasing Reactive Metabolitesmentioning

confidence: 99%

Idiosyncratic Drug Reactions: Past, Present, and Future

Uetrecht

2007

Chem. Res. Toxicol.

238

190

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“…Therefore, a modification of the P450 apoprotein by a reactive 17EE intermediate was believed to be the reason for the loss in enzymatic activity of the 2B enzymes. (GSH) have been employed to trap reactive electrophiles for subsequent structural analysis of the S-substituted adducts using mass spectrometry (19)(20)(21). In this study, GSH was used to trap reactive 17EE intermediates formed by P450s 2B1 and 2B6.…”

mentioning

confidence: 99%

Identification of 17-α-Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactivation of P450s 2B1 and 2B6

Kent

Lin

Mills

et al. 2006

Chem. Res. Toxicol.

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The oral contraceptive 17-α-ethynylestradiol (17EE1) is a mechanism-based inactivator of P450s 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [ 3 H]17EE resulted in labeling of the P450 apo-protein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [ 3 H]17EE and digested with CNBr. Separation of the these peptides resulted in the identification of one major labeled peptide for each enzyme. N-terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P 347 -M 376 and P 347 -M 365 in P450s 2B1 and 2B6, respectively. ESI-LC-MS and MALDI-TOF-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P 347 -M 376 peptide (3385 Da) plus a 268 Da 17EE-adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.The P450 enzymes belong to a family of microsomal cytochromes that are characterized by their unique heme absorbance spectrum at 450 nm in the reduced, carbon monoxide bound form (1). Mammalian P450s play a pivotal role in the detoxification, metabolism, and clearance of the majority of drugs as well as many carcinogens and pesticides (2). In many instances metabolism of xenobiotics by P450 enzymes results in the production of reactive intermediates. The formation of reactive intermediates that bind to cellular proteins or DNA has been implicated in the toxic or carcinogenic effects of certain environmental pollutants and drugs. Recently, it has also been suggested that protein adduction by reactive intermediates plays a significant role in the mechanisms of some neurotoxic events (3).The membrane-bound nature of mammalian P450s has made the characterization of their active sites and the elucidation of their three-dimensional structures particularly difficult. Although several crystal structures of modified P450s have been solved (4-8) much of the current 1 Abbreviations: P450, cytochrome P450; Reductase, NADPH-cytochrome P450 reductase; DLPC, dilauroyl-L-α-phosphatidylcholine; 17EE, 17-α-ethynylestradiol; BSA, bovine serum albumin; 7-EFC, 7-ethoxy-4-trifluoromethyl)coumarin; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; LC-MS, liquid chromatography-mass spectrometry; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption ionization. *To whom correspondence should be addressed at the Department of Pharmacology, Medical Science Research Bldg. III, 1150 West Medical Center Dr., Ann Arbor, MI 48109...

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“…Mass spectra were recorded on a Finnigan TSQ 7000 triple-quadrupole spectrometer under positive or negative ion mode. 1 H NMR spectra were recorded on a Bruker 300, 400, and 500 MHz NMR spectrometers using acetone-d 6 and DMSO-d 6 as solvent and internal standard. HPLC analysis was performed on Waters 1525 Binary HPLC Pump with Waters 2996 Photodiode Array Detector on the reversed phase Jupiter C18 5 µm 250 mm × 4.6 mm or 150 mm × 4.6 mm column at 1 mL/min (Phenomenex, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Teucrin A (10 mg, 0.029 mmol) was dissolved in 0.5 mL of anhydrous acetone-d 6 , and the solution was cooled in the acetone/dry ice bath. Cold DMDO-d 6 (1.2 mL, 0.034 mmol) was added under argon atmosphere.…”

Section: Methodsmentioning

confidence: 99%

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Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat

Druckova

Mernaugh

Ham

et al. 2007

Chem. Res. Toxicol.

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Covalent modification of proteins is associated with the toxicity of many electrophiles, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Here, we describe a strategy for the enrichment of adducted proteins utilizing single-chain fragment variable (ScFv) antibodies selected using phage-display technology. Teucrin A is a furan-containing diterpenoid found in the herb germander that is primarily responsible for the herb's hepatotoxicity in rodents and humans following metabolic activation by cytochrome P450 enzymes. Conjugates of the 1,4-enedial derivative of teucrin A, its presumed toxic metabolite, with lysine-and cysteine-containing peptides were synthesized and used to select ScFvs from a rodent phage-displayed library, which recognized the terpenoid moiety of the teucrin-derived adducts. Immunoaffinity isolation of adducted proteins from rat liver homogenates following administration of a toxic dose of teucrin A afforded a family of proteins that were identified by liquid chromatography/tandem mass spectrometry. Of the 46 proteins identified in this study, most were of mitochondrial and endoplasmic reticulum origin. Several cytosolic proteins were found, as well as four peroxisomal and two secreted proteins. Using Ingenuity Pathway Analysis software, two significant networks involving the target genes were identified that had major functions in gene expression, small molecule biochemistry, and cellular function and maintenance. These included proteins involved in lipid, amino acid, and drug metabolism. This study illustrates the utility of chemically synthesized biological conjugates of reactive intermediates and the potential of the phage display technology for the generation of affinity reagents for the isolation of adducted proteins.

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Drug−Protein Adducts: An Industry Perspective on Minimizing the Potential for Drug Bioactivation in Drug Discovery and Development

Cited by 690 publications

References 34 publications

Idiosyncratic Drug Reactions: Past, Present, and Future

Idiosyncratic Drug Reactions: Past, Present, and Future

Identification of 17-α-Ethynylestradiol-Modified Active Site Peptides and Glutathione Conjugates Formed during Metabolism and Inactivation of P450s 2B1 and 2B6

Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat

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