A Chemoproteomics Approach to Determine the Mechanism of Testicular Toxicity for the Bruton's Tyrosine Kinase Inhibitor CC-292.

During drug development, potential safety issues can occur at any time. Understanding the cause of a toxicity can help with deciding on how to advance the drug development program. Chemoproteomics provides a way to help understand the cause of a toxicity wherein the affected tissue is accessible and can be probed with a covalently binding compound that is analogous to the offending drug. In this case, N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292), a covalently binding Bruton's tyrosine kinase inhibitor, had produced testicular toxicity in rodents. Experiments were conducted using a CC-292 analog that could be chemically modified with biotin to probe rodent testes homogenates for potential binding sites that were subsequently recovered with avidin beads. These biotin-tagged proteins undergo trypsin digest on the avidin beads to yield peptides that are identified using mass spectrometry. Two proteins were identified from the testicular homogenates of both rats and mice, namely retinal dehydrogenase 1 (ALDH1A1) and retinal dehydrogenase 2 (ALDH1A2). Literature confirmed a link between inhibition of these enzymes and testicular toxicity. Subsequently, molecular modeling was used to demonstrate that CC-292 can be docked into both the nicotinamide adenine dinucleotide and retinal binding pockets of the analogous human ALDH1A2 enzyme. These data suggest that the off-target binding site for CC-292 on retinal dehydrogenase enzymes may provide a mechanistic explanation to the testicular toxicity observed in rodents and that there may be a potential concern for human male fertility. SIGNIFICANCE STATEMENT: Biotinylated covalently binding drug analogues are used to enrich bound proteins from tissue homogenates wherein toxicity was observed in rodents. Bound proteins were subsequently identified by mass spectroscopy. Competition of the analog binding with the parent inhibitor itself and three-dimensional molecular modeling were used to establish a likely link between the off-targets of CC-292, ALDH1A1, and ALDH1A2 with potential testicular toxicity.

Next-Generation DILI Biomarkers: Prioritization of Biomarkers for Qualification and Best Practices for Biospecimen Collection in Drug Development.

The diagnosis and management of drug-induced liver injury (DILI) remains a challenge in clinical trials in drug development. The qualification of emerging biomarkers capable of predicting DILI soon after the initiation of treatment, differentiating DILI from underlying liver disease, identifying the causal entity, and assigning appropriate treatment options after DILI is diagnosed are needed. Qualification efforts have been hindered by lack of properly stored and consented biospecimens that are linked to clinical data relevant to a specific context of use. Recommendations are made for biospecimen collection procedures, with the focus on clinical trials, and for specific emerging biomarkers to focus qualification efforts.

An analysis of pharmaceutical experience with decades of rat carcinogenicity testing: support for a proposal to modify current regulatory guidelines.

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

Erratum to "Prediction of non-genotoxic carcinogenesis in rats using changes in gene expression following acute dosing".

Carcinogenicity of chemicals can currently only be evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR).We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 nongenotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 nongenotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected on both day 1 and day 5. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.

Prediction of non-genotoxic carcinogenesis in rats using changes in gene expression following acute dosing.

Non-genotoxic carcinogenicity of chemicals is currently routinely evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for non-genotoxic carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens (NGTCs) or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR). We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 non-genotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 non-genotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected at both 1 and 5 days. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.

The carboxy-terminal Neh3 domain of Nrf2 is required for transcriptional activation.

Nrf2 is a transcription factor critical for the maintenance of cellular redox homeostasis. We have previously found that Nrf2 is a labile protein, and its activation in cells under stress involves mechanisms leading to its stabilization. As a modular protein, Nrf2 possesses distinct transactivation and DNA binding domains essential for its transcriptional activity. In this study, we found that the C-terminal "Neh3" domain of Nrf2 is also important for its activity. Deletion of the last 16 amino acids of the protein completely abolishes its ability to activate both reporter and endogenous gene expression. Using site-directed mutagenesis, we have identified a stretch of amino acids within this region that are essential for its activity and that are found to be conserved across species and among other members of the CNC-bZIP family. Importantly, deletion of the final 16 amino acids of Nrf2 does not influence its dimerizing capability, DNA binding activity, or subcellular localization, although it does increase the half-life of the protein. In addition, this region was found to be important for interaction with CHD6 (a chromo-ATPase/helicase DNA binding protein) in a yeast two-hybrid screen. RNA interference-mediated knockdown of CHD6 reduced both the basal and tert-butylhydroquinone-inducible expression of NQO1, a prototypical Nrf2 target gene. These data suggest that the Neh3 domain may act as a transactivation domain and that it is possibly involved in interaction with components of the transcriptional apparatus to affect its transcriptional activity.

Nrf2 controls constitutive and inducible expression of ARE-driven genes through a dynamic pathway involving nucleocytoplasmic shuttling by Keap1.

Nrf2 regulates the expression of genes encoding antioxidant proteins involved in cellular redox homeostasis. Previous studies have suggested that activation of Nrf2 is mediated by mechanisms promoting its dissociation from Keap1, a cytosolic repressor that acts to sequester the transcription factor in the cytoplasm. As a short-lived protein, Nrf2 is also activated by mechanisms leading to its stabilization in cells under stress, and recent evidence indicates that Keap1 has an active role in the control of its stability. In this report, using immunocytochemistry, cell fractionation, and chromatin immunoprecipitation analyses, we found that Nrf2 is primarily a nuclear protein and that it is expressed and recruited to the chromatin constitutively to drive basal gene expression. Furthermore, we found evidence indicating that Keap1 may repress Nrf2 activity by transiently shuttling into the nucleus to promote its ubiquitylation. The data suggested that the steady-state level of Nrf2 is maintained by a dynamic pathway that balances its constitutive expression with a Keap1-regulated degradation process downstream of its role as a transcriptional activator. We suggest that the stabilization of Nrf2 in cells under stress represents the central regulatory response mediated by mechanisms that interfere with its interaction with Keap1, leading to the induction of antioxidant enzymes important to maintain cellular redox homeostasis.

Positive and negative regulation of prostaglandin E2 biosynthesis in human colorectal carcinoma cells by cancer chemopreventive agents.

Increased production of prostaglandin E(2) (PGE(2)) by the combined activities of cyclooxygenase-2 (COX-2) and microsomal glutathione S-transferase 1-like 1 (MGST1-L1) enhances the progression of colorectal cancer. To assess how chemopreventive agents influence colon tumorigenesis, the modulation of PGE(2) production by indolo[3,2-b]carbazole (ICZ), beta-naphthoflavone (beta-NF), and tert-butylhydroquinone (tBHQ), as well as the nonsteroidal anti-inflammatory drug Piroxicam, has been studied in the human HCA-7 colon carcinoma cell line. We have found that these xenobiotics both down-regulate and up-regulate the expression of COX-2 and MGST1-L1. They can also either inhibit or stimulate PGE(2) synthesis. COX-2 mRNA levels were increased significantly by those compounds that activate transcription through the xenobiotic responsive element (XRE) and/or the antioxidant responsive element (ARE). A possible ARE enhancer was identified in the COX-2 promoter, and reporter gene experiments demonstrated that tBHQ induction of a transgene driven by the 5'-flanking region of COX-2 was increased by co-transfection with an expression vector for the Nrf2 transcription factor. By contrast, only compounds such as ICZ and beta-NF which activate the XRE increased the mRNA levels of MGST1-L1. While the ARE-specific inducer tBHQ did not modulate the basal expression of MGST1-L1, it was found to act as an antagonist of interleukin-1 beta-stimulated MGST1-L1 overexpression. Changes in COX-2 and MGST1-L1 expression were not always coincident with a corresponding change in PGE(2) production by human colon carcinoma cells. Importantly, dietary compounds can modulate PGE(2) biosynthesis, and this is likely to influence colon tumorigenesis.

Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome.

Nrf2 (NF-E2-related factor 2) is a central transcription factor involved in the transcriptional activation of many genes encoding phase II drug-metabolizing enzymes via the antioxidant response element. Nrf2 has previously been found to undergo nuclear translocation by a phosphorylation-dependent mechanism mediated by protein kinase C in HepG2 cells treated with tert-butylhydroquinone, beta-naphthoflavone, or 12-O-tetradecanoylphorbol-13-acetate. In the present report, we have found that the levels of Nrf2 were increased in cells treated with tert-butylhydroquinone or beta-naphthoflavone by a post-transcriptional mechanism. Treatment of HepG2 cells with cycloheximide resulted in the loss of Nrf2 within 30 min. By contrast, treatment with the proteasome inhibitors (lactacystin or MG-132) caused an accumulation of Nrf2 as well as an induction of reporter gene activity in cells transfected with the GSTA2 antioxidant response element-chloramphenicol acetyl transferase construct. Similarly, the protein phosphatase inhibitor okadaic acid also caused an accumulation of Nrf2, whereas the reverse effects were observed with PD 98059 and U 0126, two compounds that block the activation of the MAPK/ERK signaling cascade. These data suggest that Nrf2 is degraded by the ubiquitin-dependent pathway and that phosphorylation of Nrf2 leads to an increase in its stability and subsequent transactivation activity.

Regulatory mechanisms controlling gene expression mediated by the antioxidant response element.

The expression of genes encoding antioxidative and Phase II detoxification enzymes is induced in cells exposed to electrophilic compounds and phenolic antioxidants. Induction of these enzymes is regulated at the transcriptional level and is mediated by a specific enhancer, the antioxidant response element or ARE, found in the promoter of the enzyme's gene. The transcription factor Nrf2 has been implicated as the central protein that interacts with the ARE to activate gene transcription constitutively or in response to an oxidative stress signal. This review focuses on the molecular mechanisms whereby the transcriptional activation mediated by the interaction between the ARE and NF-E2-related factor 2 (Nrf2) is regulated. Recent studies suggest that the sequence context of the ARE, the nature of the chemical inducers, and the cell type are important for determining the activity of the enhancer in a particular gene.

Novel homodimeric and heterodimeric rat gamma-hydroxybutyrate synthases that associate with the Golgi apparatus define a distinct subclass of aldo-keto reductase 7 family proteins.

The aldo-keto reductase (AKR) 7 family is composed of the dimeric aflatoxin B(1) aldehyde reductase (AFAR) isoenzymes. In the rat, two AFAR subunits exist, designated rAFAR1 and rAFAR2. Herein, we report the molecular cloning of rAFAR2, showing that it shares 76% sequence identity with rAFAR1. By contrast with rAFAR1, which comprises 327 amino acids, rAFAR2 contains 367 amino acids. The 40 extra residues in rAFAR2 are located at the N-terminus of the polypeptide as an Arg-rich domain that may form an amphipathic alpha-helical structure. Protein purification and Western blotting have shown that the two AFAR subunits are found in rat liver extracts as both homodimers and as a heterodimer. Reductase activity in rat liver towards 2-carboxybenzaldehyde (CBA) was resolved by anion-exchange chromatography into three peaks containing rAFAR1-1, rAFAR1-2 and rAFAR2-2 dimers. These isoenzymes are functionally distinct; with NADPH as cofactor, rAFAR1-1 has a low K(m) and high activity with CBA, whereas rAFAR2-2 exhibits a low K(m) and high activity towards succinic semialdehyde. These data suggest that rAFAR1-1 is a detoxication enzyme, while rAFAR2-2 serves to synthesize the endogenous neuromodulator gamma-hydroxybutyrate (GHB). Subcellular fractionation of liver extracts showed that rAFAR1-1 was recovered in the cytosol whereas rAFAR2-2 was associated with the Golgi apparatus. The distinct subcellular localization of the rAFAR1 and rAFAR2 subunits was confirmed by immunocytochemistry in H4IIE cells. Association of rAFAR2-2 with the Golgi apparatus presumably facilitates secretion of GHB, and the novel N-terminal domain may either determine the targeting of the enzyme to the Golgi or regulate the secretory process. A murine AKR protein of 367 residues has been identified in expressed sequence tag databases that shares 91% sequence identity with rAFAR2 and contains the Arg-rich extended N-terminus of 40 amino acids. Further bioinformatic evidence is presented that full-length human AKR7A2 is composed of 359 amino acids and also possesses an additional N-terminal domain. On the basis of these observations, we conclude that AKR7 proteins can be divided into two subfamilies, one of which is a Golgi-associated GHB synthase with a unique, previously unrecognized, N-terminal domain that is absent from other AKR proteins.

Direct comparison of the nature of mouse and human GST T1-1 and the implications on dichloromethane carcinogenicity.

Dichloromethane (DCM) is a hepatic and pulmonary carcinogen in mice exposed to high doses by inhalation. It has been shown previously that the incidence of liver and lung tumors does not increase in rats or hamsters exposed to the dihaloalkane under conditions similar to those that produced tumors in mice. The biological consequences of DCM exposure to humans is therefore uncertain. The carcinogenic effects of DCM in the mouse are caused by the interaction with DNA of a glutathione (GSH) conjugate that is produced by the class theta glutathione S-transferase T1-1 (GST T1-1). The species specificity is thought to be due to the greater amount of transferase activity in mouse target organs and specific nuclear localization of GST T1-1 in target cells. This paper directly compares the relative capacity and locality of DCM activation in mouse and human tissues. The results show that mouse GST T1-1 is more efficient in catalyzing the conjugation of DCM with GSH than the orthologous human enzyme. In addition, the mouse expresses higher levels of the transferase than humans in hepatic tissue. Histochemical analysis confirmed the presence of GST T1-1 in the nucleus of mouse liver cells. However, in human liver GST T1-1 was detected in bile duct epithelial cells and hepatocyte nuclei but was also present in the cytoplasm. Taking this information into account, it is unlikely that humans have a sufficiently high capacity to activate DCM for this compound to be considered to represent a carcinogenic risk.