Abstract The mixed‐ligand tris(pyrazolyl)borate sandwich compound [TpmRuTp′]Cl ( 2 ) was prepared by microwave‐assisted synthesis from Tp′Ru(COD)Cl ( 1 ) [Tp = tris(pyrazolyl)borate; Tp′ = p ‐bromophenyltris(pyrazolyl)borate; Tpm = tris(pyrazolyl)methane; COD = 1,4‐cyclooctadiene]. Subsequently, 2 was converted to the azide‐functionalized [TpmRu( p ‐N 3 C 6 H 4 Tp)] ( 3 ), which can be readily coupled to biomolecules by Cu‐catalyzed azide–alkyne cycloaddition (Cu‐AAC) in solution, as exemplified by the covalent attachment to pentyne‐functionalized HC(CH 2 ) 2 –CO–Val–O t Bu ( 4 ), and an alkyne derivative of the neuropeptide enkephaline HC(CH 2 ) 2 –CO–ENK–OH (ENK = enkephaline, Tyr–Gly–Gly–Phe–Leu). The resulting triazole compounds [TpmRu({ p ‐C 2 N 3 H–(CH 2 ) 2 –CO–Val–O t Bu}Tp)]Cl ( 5 ) and [TpmRu({ p ‐C 2 N 3 H–(CH 2 ) 2 –CO–ENK–OH}Tp)]Cl ( 6 ), represent the first [3+2] cycloaddition products of azide‐functionalized Tp compounds. They were characterized by NMR spectroscopy and mass spectrometry. Furthermore, 1 , 2 , and 3 as well as the reaction intermediate [(κ 2 ‐ N , N ′‐Tpm)RuCl(Tp′)] ( 2a ) were characterized in the solid state by single‐crystal X‐ray diffraction.
Objectives: To determine the influence of cytochrome P450 2B6 (CYP2B6) genotype on the rate of oxidative efavirenz metabolism in human liver microsomes. Materials & methods: Formation rates of 8-hydroxyefavirenz, 7-hydroxyefavirenz and 8,14-dihydroxyefavirenz were determined in vitro with efavirenz as a substrate (10 µM) in a large panel of human liver microsomes (n = 87) that were genotyped for variants of the CYP2B6 gene and phenotyped for CYP2B6 protein expression and bupropion hydroxylation. Results: Efavirenz 8-hydroxylation, the major route of efavirenz clearance, was detected in all samples, exhibiting an overall interindividual variability of 44.7-fold; 8,14-dihydroxyefavirenz and 7-hydroxyefavirenz were also detected in most samples. The formation rate of 8-hydroxyefavirenz correlated significantly with CYP2B6 protein (Spearman's rS = 0.54; p < 0.0001) and bupropion hydroxylase activity (rS = 0.73; p < 0.0001). Compared with the *1/*1 genotype, efavirenz 8-hydroxylation was significantly lower in samples with *1/*6 and *6/*6 genotype, which also had significantly decreased CYP2B6 protein (Mann–Whitney test, p < 0.05). A decrease in CYP2B6 protein was also observed in samples with *1/*5 and *5/*6 genotypes, but this did not result in significant reduction of efavirenz metabolism, probably due to differences in specific activity of the protein variants. Lower CYP2B6 protein and activity, as well as efavirenz 8-hydroxylation was also found in several samples with rarer genotypes. We found no effect of gender and age on any of the phenotypes tested, but prior exposure to carbamazepine markedly increased CYP2B6 protein expression and activity as well as efavirenz 8-hydroxylation. Conclusions: We have shown that CYP2B6 genetic polymorphism markedly influences the metabolism of efavirenz in human liver microsomes. Importantly, the CYP2B6*6 allele harboring the SNPs c.516G>T [Q172H] and c.785A>G [K262R] was significantly associated with a pronounced decrease in CYP2B6 expression and activity, as well as a low rate of efavirenz 8-hydroxylation. These results represent a first step towards elucidating the mechanism by which this allele identifies patients exhibiting very high efavirenz plasma concentrations.
Valosin containing protein (p97/VCP) is an ATPase implicated in the degradation of ubiquitin-labelled proteins through the proteasome. We recently described a resistance to apoptotic cell death induced by proteasome inhibition and a hypersensitivity to autophagy-associated cell death under conditions of severe endoplasmic reticulum (ER) stress in rheumatoid arthritis synovial fibroblasts (RASF) compared to osteoarthritis synovial fibroblasts.
Objectives
To investigate the role of p97 in apoptotic and autophagy-associated cell death in RASF and in in vivo arthritis model.
Methods
RASF were transfected with siRNA targeting p97 or treated with the selective p97 inhibitor DBeQ (5 μM). To induce cell death, RASF were treated with TRAIL (100 ng/ml) for 24 hours or the ER stress inducer thapsigargin (TG, 5 nM-5 μM) for 72 hours. 3-methyladenine (5 mM) was used as an autophagy inhibitor. The distribution and amount of poly-ubiquitinated proteins were evaluated by immunofluorescence and immunoblotting. Cell death was evaluated by flow cytometry using annexin V/ propidium iodide staining and a caspase-3 activity assay (NucView 488, Biotium). Collagen-induced arthritis (CIA) was induced in Lewis rats. Scrambled or p97 siRNA-atelocollagen complexes were injected into ankle joints of rats. CIA was scored according to paw thickness and ankle diameter. Bone erosion was assessed by micro-CT. Proliferation of fibroblasts in rat synovial tissue was quantified by immunolabeling for Hsp47.
Results
siRNA-mediated knockdown of p97 in RASF increased cell death induced by TRAIL (p=0.009, n=6), accompanied by caspase-3 activation. Both siRNA-mediated knockdown and inhibition of p97 in RASF boosted cell death induced by 5 μM TG, accompanied by a massive cytoplasmic vacuolization, the formation of poly-ubiquitinated protein aggregates and the accumulation of poly-ubiquitinated proteins, and cell death was inhibited by 3-methyladenine. Smaller amounts of TG (50 or 500 nM) induced a cytoplasmic vacuolization and the formation of poly-ubiquitinated protein aggregates in p97-inhibited RASF but not in control RASF. Intra-articular injection of p97 siRNA significantly suppressed CIA (p=0.002, n=6), bone erosion (p=0.02, n=6) and proliferation of synovial fibroblasts (p=0.004, n=6) in rats.
Conclusions
Our data indicate that the inhibition of p97 promotes both apoptotic and autophagy-associated cell death in RASF and suppresses CIA, bone erosion and proliferation of synovial fibroblasts in vivo. p97 may be a new potential target in the treatment of arthritis.
References
Kato M, et al. Arthritis Rheum 2014 Jan;66(1):40-8.
Acknowledgements
This work was supported by MHLW, MEXT, IMI-BT Cure, IAR Epalinges, euroTEAM.
Abstract Metabolic dysfunction‐associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non‐parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease‐relevant endpoints, such as steatosis, inflammation and fibrosis, and multi‐omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high‐content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti‐steatotic, anti‐inflammatory, and anti‐fibrotic effects are identified. Among these, activation of the muscarinic M 1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti‐fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via G q/11 and phospholipase C‐mediated depletion of phosphatidylinositol 4,5‐bisphosphate can be demonstrated. Combined, this study presents the first patient‐derived 3D MASH model, identifies a novel signaling module with anti‐fibrotic effects, and highlights the potential of organotypic culture systems for phenotype‐based chemogenomic drug target identification at scale.
Methyleugenol is a genotoxic carcinogen in mice and rats, the liver being the primary target tissue. Methyleugenol occurs in fennel and many herbs and spices. Furthermore, methyleugenol-containing plant extracts and chemically prepared methyleugenol are used as flavoring agents. We analyzed surgical human liver samples from 30 subjects for the presence of DNA adducts originating from methyleugenol using isotope-dilution ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Twenty-nine samples unambiguously contained the N2 -( trans -methylisoeugenol-3′-yl)-2′-deoxyguanosine adduct. A second adduct, N6 -( trans -methylisoeugenol-3′-yl)-2′-deoxyadenosine, was also found in most samples, but at much lower levels, in agreement with the results from experimental models. The maximal and median levels of both adducts combined were 37 and 13 per 10 8 nucleosides (corresponding to 4700 and 1700, respectively, adducts per diploid genome). This is the first demonstration of DNA adducts formed by a xenobiotic in human liver using UPLC-MS/MS, the most reliable method available. It has been estimated for diverse rat and mouse hepatocarcinogens that 50–5500 adducts per 10 8 nucleosides are present after repeated treatment at the TD 50 (daily dose that halves the probability to stay tumor-free in long-term studies). We conclude that the exposure to methyleugenol leads to substantial levels of hepatic DNA adducts and, therefore, may pose a significant carcinogenic risk.
[FeFe]-hydrogenases are nature's fastest catalysts for the evolution or oxidation of hydrogen. Numerous synthetic model complexes for the [2Fe] subcluster (2FeH) of their active site are known, but so far none of these could compete with the enzymes. The complex Fe2[μ-(SCH2)2X](CN)2(CO)42- with X = NH was shown to integrate into the apo-form of [FeFe]-hydrogenases to yield a fully active enzyme. Here we report the first crystal structures of the apo-form of the bacterial [FeFe]-hydrogenase CpI from Clostridium pasteurianum at 1.60 Å and the active semisynthetic enzyme, CpIADT, at 1.63 Å. The structures illustrate the significant changes in ligand coordination upon integration and activation of the [2Fe] complex. These changes are induced by a rigid 2FeH cavity as revealed by the structure of apoCpI, which is remarkably similar to CpIADT. Additionally we present the high resolution crystal structures of the semisynthetic bacterial [FeFe]-hydrogenases CpIPDT (X = CH2), CpIODT (X = O) and CpISDT (X = S) with changes in the headgroup of the dithiolate bridge in the 2FeH cofactor. The structures of these inactive enzymes demonstrate that the 2FeH-subcluster and its protein environment remain largely unchanged when compared to the active enzyme CpIADT. As the active site shows an open coordination site in all structures, the absence of catalytic activity is probably not caused by steric obstruction. This demonstrates that the chemical properties of the dithiolate bridge are essential for enzyme activity.
We investigated the impact of promoter polymorphisms on transcription of the human CYP2B6 gene. In total, 98 DNA samples from white persons from a previously characterized liver bank were sequenced throughout 2.3 kilobases of upstream sequence and haplotype structures were determined using additional coding sequence information. HepG2 cells and primary rat and human hepatocytes were transfected with luciferase reporter gene constructs driven by 2033 base pairs (bp) of the most frequent promoter variants. The novel haplotype *22 (–1848C→ A, –801G→ T, –750T→ C, and –82T→ C) showed 3- to 9-fold enhanced transcriptional activity in all transfected cells. Constructs containing single mutations surprisingly revealed –82T→ C, predicted to disrupt a putative TATA box, to be alone responsible for this effect. In silico analysis and electrophoretic mobility shift assay demonstrated conversion of the putative TATA box into a functional CCAAT/enhancer-binding protein binding site. Analysis of transcriptional start sites showed the mutant promoter to be transcribed from a start site located approximately 30 bp downstream of the wild-type start site, consistent with the use of a noncanonical TATA box at –55 bp. Median CYP2B6 mRNA expression and bupropion hydroxylase activity as a selective marker of CYP2B6 catalytic activity were approximately 2-fold higher in livers genotyped –82TC as in those genotyped –82TT (20.4 versus 9.8 arbitrary units, p = 0.007, and 201.8 versus 106.7 pmol/mg/min, p = 0.042, respectively). This promoter polymorphism thus contributes to CYP2B6 functional variability and represents a novel mechanism by which mutations can enhance transcription. Furthermore, a detailed interspecies comparison of CYP2B promoters and transcriptional start sites provided novel insights into evolutionary relationships.
A first step in the enzymatic disposition of the antineoplastic drug doxorubicin (DOX) is the reduction to doxorubicinol (DOX-OL). Because DOX-OL is less antineoplastic but more cardiotoxic than the parent compound, the individual rate of this reaction may affect the antitumor effect and the risk of DOX-induced heart failure. Using purified enzymes and human tissues we determined enzymes generating DOX-OL and interindividual differences in their activities. Human tissues express at least two DOX-reducing enzymes. High-clearance organs (kidney, liver, and the gastrointestinal tract) express an enzyme with an apparent Km of ∼140 μM. Of six enzymes found to reduce DOX, Km values in this range are exhibited by carbonyl reductase 1 (CBR1) and aldo-keto reductase (AKR) 1C3. CBR1 is expressed in these three organs at higher levels than AKR1C3, whereas AKR1C3 has higher catalytic efficiency. However, inhibition constants for DOX reduction with 4-amino-1-tert-butyl-3-(2-hydroxyphenyl)pyrazolo[3,4-d]pyrimidine (an inhibitor that can discriminate between CBR1 and AKR1C3) were identical for CBR1 and human liver cytosol, but not for AKR1C3. These results suggest that CBR1 is a predominant hepatic DOX reductase. In cytosols from 80 human livers, the expression level of CBR1 and the activity of DOX reduction varied >70- and 22-fold, respectively, but showed no association with CBR1 gene variants found in these samples. Instead, the interindividual differences in CBR1 expression and activity may be mediated by environmental factors acting via recently identified xenobiotic response elements in the CBR1 promoter. The variability in the CBR1 expression may affect outcomes of therapies with DOX, as well as with other CBR1 substrates.
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