RESUMO
Formation of sterically hindered C-C double bonds via catalytic olefin metathesis is considered a very challenging task for Ru catalysts. This limitation led to the development of specialised catalysts bearing sterically reduced N-heterocyclic carbene (NHC) ligands that are very active in such transformations, yet significantly less stable as compared to general purpose catalysts. To decrease the small-size NHC catalysts susceptibility to decomposition, a new NHC ligand was designed, in which two sterically reduced aryl arms were tied together by a C-8 alkyl chain. The installation of this macrocyclic ligand on the ruthenium centre led to the formation of an olefin metathesis catalyst (trans-Ru6). Interestingly, this complex undergoes transformation into an isomer bearing two Cl ligands in the cis-arrangement (cis-Ru6). These two isomeric complexes exhibit similarly high thermodynamic stability, yet different application profiles in catalysis.
Assuntos
Rutênio , Alcenos/química , Catálise , Ligantes , Metano/análogos & derivados , Rutênio/químicaRESUMO
BACKGROUND & AIM: ABCB4 is expressed at the canalicular membrane of hepatocytes. This ATP-binding cassette (ABC) transporter is responsible for the secretion of phosphatidylcholine into bile canaliculi. Missense genetic variations of ABCB4 are correlated with several rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). In a repurposing strategy to correct intracellularly retained ABCB4 variants, we tested 16 compounds previously validated as cystic fibrosis transmembrane conductance regulator (CFTR) correctors. METHODS: The maturation, intracellular localization and activity of intracellularly retained ABCB4 variants were analyzed in cell models after treatment with CFTR correctors. In addition, in silico molecular docking calculations were performed to test the potential interaction of CFTR correctors with ABCB4. RESULTS: We observed that the correctors C10, C13, and C17, as well as the combinations of C3 + C18 and C4 + C18, allowed the rescue of maturation and canalicular localization of four distinct traffic-defective ABCB4 variants. However, such treatments did not permit a rescue of the phosphatidylcholine secretion activity of these defective variants and were also inhibitory of the activity of wild type ABCB4. In silico molecular docking analyses suggest that these CFTR correctors might directly interact with transmembrane domains and/or ATP-binding sites of the transporter. CONCLUSION: Our results illustrate the uncoupling between the traffic and the activity of ABCB4 because the same molecules can rescue the traffic of defective variants while they inhibit the secretion activity of the transporter. We expect that this study will help to design new pharmacological tools with potential clinical interest.
Assuntos
Colestase Intra-Hepática , Colestase , Subfamília B de Transportador de Cassetes de Ligação de ATP , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Simulação de Acoplamento Molecular , FosfatidilcolinasRESUMO
Cationic carbenes are a relatively new and rare group of ancillary ligands, which have shown their superior activity in a number of challenging catalytic reactions. In ruthenium-based metathesis catalysis they are often used as ammonium tags, to provide water-soluble, environment-friendly catalysts. In this work we performed computational studies on three cationic carbenes with the formal positive charge located at different distances from the carbene carbon. We show that the predicted initiation rates of Grubbs, indenylidene, and Hoveyda-Grubbs-like complexes incorporating these carbenes show little variance and are similar to initiation rates of standard Grubbs, indenylidene, and Hoveyda-Grubbs catalysts. In all investigated cases the partial charge of the carbene carbon atom is similar, resulting in comparable Ccarbene-Ru bond strengths and Ru-P/O dissociation Gibbs free energies.
RESUMO
AIMS: Drug-induced enteropathy is often associated with the therapeutic use of certain glucuronidated drugs. One such drug is mycophenolic acid (MPA), a well-established immunosuppressant of which gastrointestinal adverse effects are a major concern. The role of bacterial ß-glucuronidase (ß-G) from the gut microbiota in MPA-induced enteropathy has recently been discovered. Bacterial ß-G hydrolyzes MPAG, the glucuronide metabolite of MPA excreted in the bile, leading to the digestive accumulation of MPA that would favor in turn these adverse events. We therefore hypothesized that taming bacterial ß-G activity might reduce MPA digestive exposure and prevent its toxicity. MAIN METHODS: By using a multiscale approach, we evaluated the effect of increasing concentrations of MPA on intestinal epithelial cells (Caco-2 cell line) viability, proliferation, and migration. Then, we investigated the inhibitory properties of amoxapine, a previously described bacterial ß-G inhibitor, by using molecular dynamics simulations, and evaluated its efficiency in blocking MPAG hydrolysis in an Escherichia coli-based ß-G activity assay. The pharmacological effect of amoxapine was evaluated in a mouse model. KEY FINDINGS: We observed that MPA impairs intestinal epithelial cell homeostasis. Amoxapine efficiently blocks the hydrolysis of MPAG to MPA and significantly reduces digestive exposure to MPA in mice. As a result, administration of amoxapine in MPA-treated mice significantly attenuated gastrointestinal lesions. SIGNIFICANCE: Collectively, these results suggest that the digestive accumulation of MPA is involved in the pathophysiology of MPA-gastrointestinal adverse effects. This study provides a proof-of-concept of the therapeutic potential of bacterial ß-G inhibitors in glucuronidated drug-induced enteropathy.
Assuntos
Biotransformação , Microbioma Gastrointestinal , Glucuronidase , Glucuronídeos , Ácido Micofenólico , Ácido Micofenólico/metabolismo , Ácido Micofenólico/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Glucuronidase/metabolismo , Glucuronidase/antagonistas & inibidores , Humanos , Animais , Camundongos , Glucuronídeos/metabolismo , Células CACO-2 , Masculino , Imunossupressores/farmacologia , Imunossupressores/toxicidade , Imunossupressores/metabolismo , Enteropatias/induzido quimicamente , Enteropatias/tratamento farmacológico , Enteropatias/metabolismo , Enteropatias/microbiologia , Proliferação de Células/efeitos dos fármacos , GlicoproteínasRESUMO
Early and sensitive biomarkers of liver dysfunction and drug-induced liver injury (DILI) are still needed, both for patient care and drug development. We developed the Serum Enhanced Binding (SEB) test to reveal post-transcriptional modifications (PTMs) of human serum albumin resulting from hepatocyte dysfunctions and further evaluated its performance in an animal model. The SEB test consists in spiking serum ex-vivo with ligands having specific binding sites related to the most relevant albumin PTMs and measuring their unbound fraction. To explore the hypothesis that albumin PTMs occur early during liver injury and can also be detected by the SEB test, we induced hepatotoxicity in male albino Wistar rats by administering high daily doses of ethanol and CCl4 over several days. Blood was collected for characterization and quantification of albumin isoforms by high-resolution mass spectrometry, for classical biochemical analyses as well as to apply the SEB test. In the exposed rats, the appearance of albumin isoforms paralleled the positivity of the SEB test ligands and histological injuries. These were observed as early as D3 in the Ethanol and CCl4 groups, whereas the classical liver tests (ALT, AST, PAL) significantly increased only at D7. The behavior of several ligands was supported by structural and molecular simulation analysis. The SEB test and albumin isoforms revealed hepatocyte damage early, before the current biochemical biomarkers. The SEB test should be easier to implement in the clinics than albumin isoform profiling.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Fígado , Ratos , Masculino , Humanos , Animais , Fígado/metabolismo , Ratos Wistar , Doença Hepática Induzida por Substâncias e Drogas/patologia , Albuminas/metabolismo , Etanol/metabolismo , Biomarcadores/metabolismo , Isoformas de Proteínas/metabolismo , Tetracloreto de Carbono/toxicidadeRESUMO
ATP-binding cassette C-family drug membrane transporters play an important role in local pharmacokinetics, that is, drug concentration in cellular compartments. From the structural point of view, only the bovine ortholog of the multidrug resistance-associated protein 1 (bMRP1) has been resolved. We here used µs-scaled molecular dynamics simulations to investigate the structure and dynamics of the bovine multidrug resistance-associated protein 1 in pre- and post-hydrolysis functional states. The present work aims to examine the slight but likely relevant structural differences between pre- and post-hydrolysis states of outward-facing conformations as well as the interactions between the multidrug resistance-associated protein 1 and the surrounding lipid bilayer. Global conformational dynamics show unfavourable extracellular opening associated with nucleotide-binding domain dimerization indicating that the post-hydrolysis state adopts a close-cleft conformation rather than an outward-open conformation. Our present simulations also highlight persistent interactions with annular cholesterol molecules and the expected active role of lipid bilayer in the allosteric communication between distant domains of the transporter.
RESUMO
Multidrug resistance-associated proteins are ABC C-family exporters. They are crucial in pharmacology as they transport various substrates across membranes. However, the role of the degenerate nucleotide-binding site (NBS) remains unclear likewise the interplay with the surrounding lipid environment. Here, we propose a dynamic and structural overview of MRP1 from ca. 110 µs molecular dynamics simulations. ATP binding to NBS1 is likely maintained along several transport cycles. Asymmetric NBD behaviour is ensured by lower signal transduction from NBD1 to the rest of the protein owing to the absence of ball-and-socket conformation between NBD1 and coupling helices. Even though surrounding lipids play an active role in the allosteric communication between the substrate-binding pocket and NBDs, our results suggest that lipid composition has a limited impact, mostly by affecting transport kinetics. We believe that our work can be extended to other degenerate NBS ABC proteins and provide hints for deciphering mechanistic differences among ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Nucleotídeos , Nucleotídeos/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , LipídeosRESUMO
The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for a-ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. µs-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate α-ketoglutarate were also investigated suggesting that it may bind to highly conserved intracellular motifs. We here hypothesise that α-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations.
Assuntos
Ácidos Cetoglutáricos , Proteína 1 Transportadora de Ânions Orgânicos , Humanos , Proteína 1 Transportadora de Ânions Orgânicos/genética , Proteínas de Membrana Transportadoras , LipídeosRESUMO
The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by µs-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called "charge-relay system" that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.