Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes

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

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale

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

Modèles in silico de transporteurs membranaires pharmacologiquement pertinents : Focus sur Major Facilitator Superfamily

Drug pharmacodynamics (PD) and pharmacokinetics (PK) are both strongly impacted by drug membrane crossing events. Such an event often involves membrane drug transporters that are divided into two main superfamilies, namely Solute Carrier (SLC) or ATP-Binding Cassette (ABC) transporter superfamilies. Despite the importance of transporters in clinical pharmacology and recent advances in local PK/PD relationship, knowledge about their functions, but also drug-drug interactions or pharmacogenomics (PGx) involving membrane transporters is still rather limited, especially at the molecular level. There are currently no experimental methods that can give an overall dynamic picture of transporter functions at the atomic level. The atomic description of transporters and their dynamics is anticipated to result in a greater knowledge of protein structure, conformational changes, and the underlying mechanism, which in turn will enhance our understanding of substrate binding, inhibition modes, and kinetics. The effectiveness of molecular dynamics (MD) simulations in recent decades has been demonstrated in their capacity to reveal structural properties, support experimental data on transporters, and provide pictures at the nanoscale. Therefore, the present work focuses on deciphering the structural patterns of Major Facilitator Superfamily (MFS) transporters by means of MD simulations. The focus was on a clinically relevant transporter, Organic Anion Transporter 1 (SLCA22A6/OAT1), which is among a group of human membrane transporters emphasized by the International Transporter Consortium (ITC) as being of "emerging clinical importance". For sake of comparison, the interplay between lipids and OAT1 as well as MFS prototypes, (i.e., Glucose Transporter 1 and 3) was also investigated.La traversée des membranes par des médicaments influencent grandement la pharmacodynamique et la pharmacocinétique de ces derniers. Ces événements impliquent souvent des transporteurs membranaires qui sont classifiés en deux superfamilles, à savoir les Solute Carriers (SLC) et les transporteurs dits « ABC » (ATP-Binding Cassette). Malgré l'importance des transporteurs en pharmacologie clinique et les progrès récents dans la compréhension des interactions PK/PD au niveau local, les connaissances sur leurs structures et fonctions et les interactions médicamenteuses ou encore l’implication de la pharmacogénomique (PGx) restent assez limitées, surtout au niveau moléculaire. Il n'existe actuellement aucune méthode expérimentale capable de donner une vue complète dynamique et structurale des transporteurs. La description atomique des transporteurs et de leur dynamique devrait permettre une meilleure connaissance de la structure des protéines, des changements de conformation et des mécanismes sous-jacent. Ceci pourra améliorer la compréhension de la liaison des substrats, des modes d’inhibition et de la cinétique de transport. Les avancées réalisées avec les simulations de dynamique moléculaire au cours des dernières décennies a été démontrée par leur capacité à compléter les données expérimentales sur les transporteurs et à fournir des images à l'échelle nanométrique. Dans ce contexte, le présent travail se concentre sur le décryptage des modèles structuraux des transporteurs de la Superfamille des Facilitateurs Majeurs (MFS) au moyen de simulations de dynamique moléculaire. L'accent a été mis sur un transporteur d’importance clinique et pharmacologique, le transporteur OAT1 (Organic Anion Transporter 1), qui fait partie de la liste des transporteurs membranaires humains considérés par le Consortium international des transporteurs (ITC) comme ayant une "importance clinique émergente". À des fins de comparaison, l'interaction entre les lipides et OAT1 ainsi que des prototypes de MFS (transporteurs de glucose 1 et 3 -SLC2A1/GLUT1 et SLCA3/GLUT3) a également été étudiée

The influence of the cationic carbenes on the initiation kinetics of ruthenium-based metathesis catalysts; a DFT study

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

Effect of CFTR correctors on the traffic and the function of intracellularly retained ABCB4 variants

International audienceBackground & 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