The generation, detection, and effects of reactive drug metabolites.

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.

Differential effect of covalent protein modification and glutathione depletion on the transcriptional response of Nrf2 and NF-kappaB.

Liver injury associated with exposure to therapeutic agents that undergo hepatic metabolism can involve the formation of reactive metabolites. These may cause redox perturbation which can result in oxidative stress as well as protein modification leading to activation or inhibition of cellular transcriptional responses. Nevertheless, the effects of these challenges on more than one transcriptional pathway simultaneously remain unclear. We have investigated two transcription factors known to be sensitive to electrophilic stress and redox perturbation, Nrf2 and NF-kappaB, in mouse liver cells. Cellular stress was induced by the probes: N-acetyl-p-benzoquinineimine (NAPQI), the reactive metabolite of acetaminophen; dinitrochlorobenzene (DNCB), a model electrophile; and buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase. NAPQI, DNCB and BSO can all cause glutathione (GSH) depletion; however only NAPQI and DNCB can covalently bind proteins. We also employed RNAi to manipulate Keap1 (the inhibitor of Nrf2), Nrf2 itself and NF-kappaB-p65, to understand their roles in the response to drug stress. All three chemicals induced Nrf2, but NF-kappaB binding activity was only increased after BSO treatment. In fact, NF-kappaB binding activity decreased after exposure to NAPQI and DNCB. While RNAi depletion of Keap1 led to reduced toxicity following exposure to DNCB, depletion of Nrf2 and NF-kappaB augmented toxicity. Interestingly, increased Nrf2 caused by Keap1 depletion was reversed by co-depletion of NF-kappaB. We demonstrate that Keap1/Nrf2 and NF-kappaB respond differently to electrophiles that bind proteins covalently and the redox perturbation associated with glutathione depletion, and that crosstalk may enable NF-kappaB to partly influence Nrf2 expression during cellular stress.

The hepatotoxic metabolite of acetaminophen directly activates the Keap1-Nrf2 cell defense system.

UNLABELLED: The transcription factor Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. We have demonstrated previously that acetaminophen activates Nrf2 in mouse liver following administration of non-hepatotoxic and hepatotoxic doses in vivo, implying that Nrf2 may have an important role in the protection against drug-induced liver injury. Nrf2 activation has been proposed to occur through the modification of cysteine residues within Keap1, the cytosolic repressor of Nrf2. We hypothesized that acetaminophen activates Nrf2 via the formation of its reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which may disrupt the repression of Nrf2 through the modification of cysteine residues within Keap1. Here, we show that NAPQI can directly activate the Nrf2 pathway in mouse liver cells, inducing an adaptive defense response that is antagonized by RNA interference targeted against Nrf2. Furthermore, mass spectrometric analysis shows that NAPQI selectively modifies cysteine residues in Keap1, both in recombinant protein in vitro and in cells ectopically expressing Keap1. Using this cell-based model, we demonstrate that activation of Nrf2 by NAPQI and a panel of probe molecules [dexamethasone 21-mesylate, 15-deoxy-Delta-((12,14))-prostaglandin J(2), 2,4-dinitrochlorobenzene, and iodoacetamide] correlates with the selective modification of cysteine residues located within the intervening region of Keap1. However, substantial depletion of glutathione (to less than 15% of basal levels) by buthionine sulfoximine, which does not directly modify Keap1, is also sufficient to activate Nrf2. CONCLUSION: Nrf2 can be activated via the direct modification of cysteine residues located within the intervening region of Keap1, but also via the substantial depletion of glutathione without the requirement for direct modification of Keap1. It is possible that both of these mechanisms contribute to the activation of Nrf2 by acetaminophen.