S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling.

KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.

Metabolic bioactivation of antidepressants: advance and underlying hepatotoxicity.

Many drugs that serve as first-line medications for the treatment of depression are associated with severe side effects, including liver injury. Of the 34 antidepressants discussed in this review, four have been withdrawn from the market due to severe hepatotoxicity, and others carry boxed warnings for idiosyncratic liver toxicity. The clinical and economic implications of antidepressant-induced liver injury are substantial, but the underlying mechanisms remain elusive. Drug-induced liver injury may involve the host immune system, the parent drug, or its metabolites, and reactive drug metabolites are one of the most commonly referenced risk factors. Although the precise mechanism by which toxicity is induced may be difficult to determine, identifying reactive metabolites that cause toxicity can offer valuable insights for decreasing the bioactivation potential of candidates during the drug discovery process. A comprehensive understanding of drug metabolic pathways can mitigate adverse drug-drug interactions that may be caused by elevated formation of reactive metabolites. This review provides a comprehensive overview of the current state of knowledge on antidepressant bioactivation, the metabolizing enzymes responsible for the formation of reactive metabolites, and their potential implication in hepatotoxicity. This information can be a valuable resource for medicinal chemists, toxicologists, and clinicians engaged in the fields of antidepressant development, toxicity, and depression treatment.

Reactive metabolite of trovafloxacin activates inflammasomes: Implications for trovafloxacin-induced liver injury.

Trovafloxacin is a quinolone antibiotic drug with broad-spectrum activity, which was withdrawn from a global market relatively soon after approval because of serious liver injury. The characteristics of trovafloxacin-induced liver injury are consistent with an idiosyncratic reaction; however, the details of the mechanism have not been elucidated. We examined whether trovafloxacin induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested ciprofloxacin, levofloxacin, gatifloxacin, and grepafloxacin for their ability to activate inflammasomes. Drug bioactivation was performed with human hepatocarcinoma functional liver cell-4 (FLC-4) cells, and THP-1 cells (human monocyte cell line) were used for the detection of inflammasome activation. The supernatant from the incubation of trovafloxacin with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells that had been incubated with trovafloxacin, heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and inflammasome activation in THP-1 cells by the FLC-4 supernatant. These results suggest that reactive metabolites of trovafloxacin can cause the release of DAMPs from hepatocytes that can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by trovafloxacin, which, in some patients, can cause immune-related liver injury.

Reactive Enamines and Imines In Vivo: Lessons from the RidA Paradigm.

Metabolic networks are webs of integrated reactions organized to maximize growth and replication while minimizing the detrimental impact that reactive metabolites can have on fitness. Enamines and imines, such as 2-aminoacrylate (2AA), are reactive metabolites produced as short-lived intermediates in a number of enzymatic processes. Left unchecked, the inherent reactivity of enamines and imines may perturb the metabolic network. Genetic and biochemical studies have outlined a role for the broadly conserved reactive intermediate deaminase (Rid) (YjgF/YER057c/UK114) protein family, in particular RidA, in catalyzing the hydrolysis of enamines and imines to their ketone product. Herein, we discuss new findings regarding the biological significance of enamine and imine production and outline the importance of RidA in controlling the accumulation of reactive metabolites.

Identification of N-acyl-N-indanyl-α-phenylglycinamides as selective TRPM8 antagonists designed to mitigate the risk of adverse effects.

Transient receptor potential melastatin 8 (TRPM8), a temperature-sensitive ion channel responsible for detecting cold, is an attractive molecular target for the treatment of pain and other disorders. We have previously discovered a selective TRPM8 antagonist, KPR-2579, which inhibited bladder afferent hyperactivity induced by acetic acid instillation into the bladder. However, additional studies have revealed potential adverse effects with KPR-2579, such as the formation of a reactive metabolite, CYP3A4 induction, and convulsions. In this report, we describe the optimization of α-phenylglycinamide derivatives to mitigate the risk of these adverse effects. The optimal compound 13x exhibited potent inhibition against icilin-induced wet-dog shakes and cold-induced frequent voiding in rats, with a wide safety margin against the potential side effects.

Evaluation of covalent binding of flutamide and its risk assessment using 19F-NMR.

The formation of reactive metabolites (RMs) is a problem in drug development that sometimes results in severe hepatotoxicity. As detecting RMs themselves is difficult, a covalent binding assay using expensive radiolabelled tracers is usually performed for candidate selection. This study aimed to provide a practical approach toward the risk assessment of hepatotoxicity induced by covalent binding before candidate selection. We focused on flutamide because it contains a trifluoromethyl group that shows a strong singlet peak by 19F nuclear magnetic resonance (NMR) spectrometry. The covalent binding of flutamide was evaluated using quantitative NMR and its risk for hepatotoxicity was assessed by estimating the RM burden, an index that reflects the body burden associated with RM exposure by determining the extent of covalent binding, clinical dose and in vivo clearance. The extent of covalent binding and RM burden was 296 pmol/mg/h and 37.9 mg/day, respectively. Flutamide was categorised as high risk with an RM burden >10 mg/day consistent with its clinical hepatotoxicity. These results indicate that a combination of covalent binding assay using 19F-NMR and RM burden is useful for the risk assessment of RMs without using radiolabelled compounds.

Bioactivation of herbal constituents: mechanisms and toxicological relevance.

The increase in the application of herbal medicines and dietary products over the last decades has been accompanied with a substantial increase in case reports of herb-induced toxicities. Metabolic activation of relatively inert functional groups to chemically reactive metabolites is often considered to be an obligatory event in the etiology of drug-induced adverse reactions. Circumstantial evidence suggests that several herb-induced toxicities are a result of transformation of herbal constituents to electrophilic reactive metabolites that can covalently bind to vital macromolecules in the body, exemplified by aristolochic acids and pyrrolizidine alkaloids. At physiologically relevant concentrations, bioactivation of furanocoumarins and methylenedioxyphenyl compounds leads to mechanism-based inactivation of drug metabolizing enzymes and clinically manifested herb-drug interactions. Of particular interest is that several organic functional groups embedded in herbal constituents act as a toxicophore as well as a pharmacophore, resembling the electrophilic warheads in the development of targeted covalent inhibitors. The aim of this review is to provide a cataloging of bioactivation mechanisms of herbal substructures, structure-activity relationships, biological targets, and assist in circumventing the structural liability in the development of more effective and safer herb-based NCEs.

A potent seven-membered cyclic BTK (Bruton's tyrosine Kinase) chiral inhibitor conceived by structure-based drug design to lock its bioactive conformation.

A seven-membered cyclic chiral analog of potent lead BTK inhibitor 1 was envisioned by structure-based design to lock the molecule into its bioactive conformation. For the elaboration of the seven-membered ring, compound 1 pyridone 6-position was substituted with the purpose to prevent formation of reactive metabolites. Eventually, the cyclic chiral compound 3 maintained the high potency of 1, and most importantly showed no activity at either GSH or TDI assays suggesting no formation of reactive metabolites. The anticipated bound conformation of 3 to BTK was confirmed by X-ray crystallography. Synthetically, the crucial seven-membered ring formation was obtained by using TosMIC as a connective reagent.

Phase I and phase II metabolism simulation of antitumor-active 2-hydroxyacridinone with electrochemistry coupled on-line with mass spectrometry.

Here, we report the metabolic profile and the results of associated metabolic studies of 2-hydroxy-acridinone (2-OH-AC), the reference compound for antitumor-active imidazo- and triazoloacridinones. Electrochemistry coupled with mass spectrometry was applied to simulate the general oxidative metabolism of 2-OH-AC for the first time. The reactivity of 2-OH-AC products to biomolecules was also examined. The usefulness of the electrochemistry for studying the reactive drug metabolite trapping (conjugation reactions) was evaluated by the comparison with conventional electrochemical (controlled-potential electrolysis) and enzymatic (microsomal incubation) approaches. 2-OH-AC oxidation products were generated in an electrochemical thin-layer cell. Their tentative structures were assigned based on tandem mass spectrometry in combination with accurate mass measurements. Moreover, the electrochemical conversion of 2-OH-AC in the presence of reduced glutathione and/or N-acetylcysteine unveiled the formation of reactive metabolite-nucleophilic trapping agent conjugates (m/z 517 and m/z 373, respectively) through the thiol group. This glutathione S-conjugate was also identified after electrolysis experiment as well as was detected in liver microsomes. Summing up, the present work illustrates that the electrochemical simulation of metabolic reactions successfully supports the results of classical electrochemical and enzymatic studies. Therefore, it can be a useful tool for synthesis of drug metabolites, including reactive metabolites.

Prediction of the Formation of Reactive Metabolites by A Novel Classifier Approach Based on Enrichment Factor Optimization (EFO) as Implemented in the VEGA Program.

The study is aimed at developing linear classifiers to predict the capacity of a given substrate to yield reactive metabolites. While most of the hitherto reported predictive models are based on the occurrence of known structural alerts (e.g., the presence of toxophoric groups), the present study is focused on the generation of predictive models involving linear combinations of physicochemical and stereo-electronic descriptors. The development of these models is carried out by using a novel classification approach based on enrichment factor optimization (EFO) as implemented in the VEGA suite of programs. The study took advantage of metabolic data as collected by manually curated analysis of the primary literature and published in the years 2004⁻2009. The learning set included 977 substrates among which 138 compounds yielded reactive first-generation metabolites, plus 212 substrates generating reactive metabolites in all generations (i.e., metabolic steps). The results emphasized the possibility of developing satisfactory predictive models especially when focusing on the first-generation reactive metabolites. The extensive comparison of the classifier approach presented here using a set of well-known algorithms implemented in Weka 3.8 revealed that the proposed EFO method compares with the best available approaches and offers two relevant benefits since it involves a limited number of descriptors and provides a score-based probability thus allowing a critical evaluation of the obtained results. The last analyses on non-cheminformatics UCI datasets emphasize the general applicability of the EFO approach, which conveniently performs using both balanced and unbalanced datasets.

Mitigation of reactive metabolite formation for a series of 3-amino-2-pyridone inhibitors of Bruton's tyrosine kinase (BTK).

Reactive metabolites have been putatively linked to many adverse drug reactions including idiosyncratic toxicities for a number of drugs with black box warnings or withdrawn from the market. Therefore, it is desirable to minimize the risk of reactive metabolite formation for lead molecules in optimization, in particular for non-life threatening chronic disease, to maximize benefit to risk ratio. This article describes our effort in addressing reactive metabolite issues for a series of 3-amino-2-pyridone inhibitors of BTK, e.g. compound 1 has a value of 459pmol/mg protein in the microsomal covalent binding assay. Parallel approaches were taken to successfully resolve the issues: establishment of a predictive screening assay with correlation association of covalent binding assay, identification of the origin of reactive metabolite formation using MS/MS analysis of HLM as well as isolation and characterization of GSH adducts. This ultimately led to the discovery of compound 7 (RN941) with significantly reduced covalent binding of 26pmol/mg protein.

Identification of a novel hormone sensitive lipase inhibitor with a reduced potential of reactive metabolites formation.

Hormone sensitive lipase (HSL) has emerged as an attractive target for the treatment of dyslipidemia. We previously reported compound 1 as a potent and orally active HSL inhibitor. Although an attractive profile was demonstrated, subsequent studies revealed that compound 1 has a bioactivation liability. The oxygen-carbon linker in compound 1 was identified as being potentially responsible for reactive metabolite formation. By exchanging of this susceptible fragment was feasible, and a benzanilide derivative 6b with a decreased bioactivation liability was obtained. Further modification of the novel benzanilide scaffold resulted in the identification of compound 24b. Compound 24b exhibited potent HSL inhibitory activity (IC50=2nM) with a significantly reduced bioactivation potential. Oral administration of compound 24b exhibited an antilipolytic effect on rats at 3mg/kg.

Oxidative bioactivation of nitrofurantoin in rat liver microsomes.

1. Nitrofurantoin (NFT), a 5-nitrofuran derivative, has been widely used for the treatment of specific urinary tract infections. It has been reported that exposure to NFT was associated with various adverse effects, particularly hepatotoxicity and pneumotoxicity. The objective of the study was to identify reactive metabolites of NFT and explore the mechanisms of the toxicities. 2. An epoxide intermediate generated in microsomal incubations was trapped by glutathione (GSH) and 4-bromobenzyl mercaptan (BBM), and the resulting GSH and BBM conjugates were characterized by LC-MS/MS. A spontaneous denitration took place in the trapping reaction. 2-Nitrofuran and 2-hydroxyfuran as model compounds were employed to probe the mechanism of the denitration. 3. The oxidative activation of NFT was P450-dependent, and P450 3A5 and P450 2A6 were the principal enzymes responsible for the bioactivation. The findings facilitate the understanding of the mechanisms of NFT-induced toxicities.

Evaluation of the metabolism, bioactivation and pharmacokinetics of triaminopyrimidine analogs toward selection of a potential candidate for antimalarial therapy.

1. During the course of metabolic profiling of lead Compound 1, glutathione (GSH) conjugates were detected in rat bile, suggesting the formation of reactive intermediate precursor(s). This was confirmed by the identification of GSH and N-acetylcysteine (NAC) conjugates in microsomal incubations. 2. It was proposed that bioactivation of Compound 1 occurs via the formation of a di-iminoquinone reactive intermediate through the involvement of the C-2 and C-5 nitrogens of the pyrimidine core. 3. To further investigate this hypothesis, structural analogs with modifications at the C-5 nitrogen were studied for metabolic activation in human liver microsomes supplemented with GSH/NAC. 4. Compounds 1 and 2, which bear secondary nitrogens at the C-5 of the pyrimidine core, were observed to form significant amounts of GSH/NAC-conjugates in vitro, whereas compounds with tertiary nitrogens at C-5 (Compound 3 and 4) formed no such conjugates. 5. These observations provide evidence that electron/hydrogen abstraction is required for the bioactivation of the triaminopyrimidines, potentially via a di-iminoquinone intermediate. The lack of a hydrogen and/or steric hindrance rendered Compound 3 and 4 incapable of forming thiol conjugates. 6. This finding enabled advancement of compound 4, with a desirable potency, safety and PK profile, as a lead candidate for further development in the treatment of malaria.

Mechanism of isoniazid-induced hepatotoxicity: then and now.

Isoniazid (INH) remains a mainstay for the treatment of tuberculosis despite the fact that it can cause liver failure. Previous mechanistic hypotheses have classified this type of drug-induced liver injury (DILI) as 'metabolic idiosyncrasy' which was thought not to involve an immune response and was mainly due to the bioactivation of the acetylhydrazine metabolite. However, more recent studies support an alternative hypothesis, specifically, that INH itself is directly bioactivated to a reactive metabolite, which in some patients leads to an immune response and liver injury. Furthermore, there appear to be two phenotypes of INH-induced liver injury. Most cases involve mild liver injury, which resolves with immune tolerance, while other cases appear to have a more severe phenotype that is associated with the production of anti-drug/anti-CYP P450 antibodies and can progress to liver failure.

Development of a cell viability assay to assess drug metabolite structure-toxicity relationships.

Many adverse drug reactions are caused by the cytochrome P450 (CYP)-dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-induced toxicity and to improve the safety of drug candidates, we developed a simple cell viability assay by combining a bioactivation system (human CYP3A4, CYP2D6 and CYP2C9) with Hep3B cells. We screened a series of drugs to explore structural motifs that may be responsible for CYP450-dependent activation caused by reactive metabolite formation, which highlighted specific liabilities regarding certain phenols and anilines.

Mechanism-based inactivation of cytochrome P450 2B6 by isopsoralen.

1. Isopsoralen (IPRN) is a major component in many traditional medicinal herbs widely used in Asian countries. The objective of the present study was to investigate the inhibitory effect of IPRN on cytochrome P450 2B6 (CYP2B6) and the mechanism involved in the enzyme inactivation. 2. Pre-incubation of CYP2B6 with IPRN resulted in a time- and concentration-dependent enzyme activity loss. The values of K(I) and k(inact) were found to be 7.89 µM and 0.067 min(-1), respectively. Ticlopidine exhibited protective effect on the IPRN-induced enzyme inactivation. The estimated partition ratio of the inactivation was 122. The GSH trapping experiments indicate that an epoxide and/or γ-ketoenal intermediate were/was generated in IPRN-fortified microsomal incubations. The synthetic work verified the formation of the reactive intermediate(s). Additionally, CYPs2E1, 2C19, 2B6 and 1A2 were found to be the major enzymes participating in the bioactivation of IPRN. 3. IPRN was characterized as a mechanism-based inactivator of CYP2B6. An IPRN-derived furanoepoxide and/or γ-ketoenal intermediate(s) were/was generated and may be responsible for the inactivation of CYP2B6.

Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.

The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100µM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.

Antimalarial benzoheterocyclic 4-aminoquinolines: Structure-activity relationship, in vivo evaluation, mechanistic and bioactivation studies.

A novel class of benzoheterocyclic analogues of amodiaquine designed to avoid toxic reactive metabolite formation was synthesized and evaluated for antiplasmodial activity against K1 (multidrug resistant) and NF54 (sensitive) strains of the malaria parasite Plasmodium falciparum. Structure-activity relationship studies led to the identification of highly promising analogues, the most potent of which had IC50s in the nanomolar range against both strains. The compounds further demonstrated good in vitro microsomal metabolic stability while those subjected to in vivo pharmacokinetic studies had desirable pharmacokinetic profiles. In vivo antimalarial efficacy in Plasmodium berghei infected mice was evaluated for four compounds, all of which showed good activity following oral administration. In particular, compound 19 completely cured treated mice at a low multiple dose of 4×10mg/kg. Mechanistic and bioactivation studies suggest hemozoin formation inhibition and a low likelihood of forming quinone-imine reactive metabolites, respectively.

Carbonyl traps as potential protective agents against methimazole-induced liver injury.

Liver injury is a deleterious adverse effect associated with methimazole administration, and reactive intermediates are suspected to be involved in this complication. Glyoxal is an expected reactive intermediate produced during methimazole metabolism. Current investigation was undertaken to evaluate the role of carnosine, metformin, and N-acetyl cysteine as putative glyoxal (carbonyl) traps, against methimazole-induced hepatotoxicity. Methimazole (100 mg/kg, intraperitoneally) was administered to intact and/or glutathione (GSH)-depleted mice and the role of glyoxal trapping agents was investigated. Methimazole caused liver injury as revealed by an increase in serum alanine aminotransferase and aspartate aminotransferase. Moreover, lipid peroxidation and protein carbonylation occurred significantly in methimazole-treated animals' liver. Hepatic GSH reservoirs were decreased, and inflammatory cells infiltration was observed in liver histopathology. Methimazole-induced hepatotoxicity was severe in GSH-depleted mice and accompanied with interstitial hemorrhage and necrosis of the liver. Glyoxal trapping agents effectively diminished methimazole-induced liver injury both in intact and/or GSH-depleted animals.