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

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.

Structure and Dynamics of Dioleoyl-Phosphatidylcholine Bilayers under the Influence of Quercetin and Rutin.

Quercetin and rutin, two widely studied flavonoids with applications foreseen in the sectors of pharmaceutical and cosmetic industries, have been chosen as model compounds for a detailed structural and dynamical investigation onto their influence on fluid lipid bilayers. Combining global small angle X-ray scattering analysis with molecular dynamics, various changes in the properties of dioleoyl-phosphatidylcholine (DOPC) bilayers have been determined. The solubility of quercetin in DOPC membranes is assured up to 12 mol %, whereas rutin, with additional glucose and rhamnose groups, are fully soluble only up to 6 mol %. Both flavonoids induce an increase in membrane undulations and thin the bilayers slightly (<1 Å) in a concentration dependent manner, wherein quercetin shows a stronger effect. Concomitantly, in the order of 2-4%, the adjacent bilayer distance increases with the flavonoid's concentration. Partial molecular areas of quercetin and rutin are determined to be 26 and 51 Å2, respectively. Simulated averaged areas per molecule confirm these estimates. A 60° tilted orientation of quercetin is observed with respect to the bilayer normal, whereas the flavonoid moiety of rutin is oriented more perpendicular (α-angle 30°) to the membrane surface. Both flavonoid moieties are located at a depth of 12 and 16 Å for quercetin and rutin, respectively, while their anionic forms display a location closer to the polar interface. Finally, at both simulated concentrations (1.5 and 12 mol %), DOPC-rutin systems induce a stronger packing of the pure DOPC lipid bilayer, mainly due to stronger attractive electrostatic interactions in the polar lipid head region.

Deciphering the Peculiar Behavior of ß-Lapachone in Lipid Monolayers and Bilayers.

ß-Lapachone (ß-Lap) is a promising anticancer drug whose applications have been limited so far because of its poor solubility and stability. Its encapsulation in liposomes has been proposed to overcome these issues. However, surface pressure measurements show that ß-Lap exhibits atypical interfacial behavior when mixed with lipids. Although the drug does not seem to be retained in lipid monolayers as deduced from the π-A isotherms, small changes in compressibility moduli suggest that ß-Lap actually interacts with lipids, either disorganizing or rigidifying their monolayers. Thermal and structural analyses of lipid bilayers confirm the existence of ß-Lap/lipid interactions and show that the drug inserts between hydrophobic chains, close to the polar headgroup in DPPC bilayers and deeper in the acyl chains in POPC bilayers. Molecular dynamics simulations allow a comprehensive description of the drug position and orientation in DOPC and POPC bilayers in the presence or absence of cholesterol.

Binding Modes and Selectivity of Ruthenium Complexes to Human Telomeric DNA G-Quadruplexes.

Metal complexes constitute an important class of DNA binders. In particular, a few ruthenium polyazaaromatic complexes are attractive as "light switches" because of their strong luminescence enhancement upon DNA binding. In this paper, a comprehensive study on the binding modes of several mononuclear and binuclear ruthenium complexes to human telomeric sequences, made of repeats of the d(TTAGGG) fragment is reported. These DNA sequences form G-quadruplexes (G4s) at the ends of chromosomes and constitute a relevant biomolecular target in cancer research. By combining spectroscopy experiments and molecular modelling simulations, several key properties are deciphered: the binding modes, the stabilization of G4 upon binding, and the selectivity of these complexes towards G4 versus double-stranded DNA. These results are rationalized by assessing the possible deformation of G4 and the binding free energies of several binding modes via modelling approaches. Altogether, this comparative study provides fundamental insights into the molecular recognition properties and selectivity of Ru complexes towards this important class of DNA G4s.

Unravelling the immunopathological mechanisms of heavy chain deposition disease with implications for clinical management.

Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by tissue deposition of a truncated monoclonal immunoglobulin heavy chain lacking the first constant domain. Pathophysiological mechanisms are unclear and management remains to be defined. Here we retrospectively studied 15 patients with biopsy-proven HCDD of whom 14 presented with stage 3 or higher chronic kidney disease, with nephrotic syndrome in 9. Renal lesions were characterized by nodular glomerulosclerosis, with linear peritubular and glomerular deposits of γ-heavy chain in 12 patients or α-heavy chain in 3 patients, without concurrent light chain staining. Only 2 patients had symptomatic myeloma. By serum protein electrophoresis/immunofixation, 13 patients had detectable monoclonal gammopathy. However, none of these techniques allowed detection of the nephrotoxic truncated heavy chain, which was achieved by immunoblot and/or bone marrow heavy chain sequencing in 14 of 15 patients. Serum-free kappa to lambda light chain ratio was abnormal in 11 of 11 patients so examined. Immunofluorescence studies of bone marrow plasma cells showed coexpression of the pathogenic heavy chain with light chain matching the abnormal serum-free light chain in all 3 tested patients. Heavy chain sequencing showed first constant domain deletion in 11 of 11 patients, with high isoelectric point values of the variable domain in 10 of 11 patients. All patients received chemotherapy, including bortezomib in 10 cases. Renal parameters improved in 11 patients who achieved a hematological response, as assessed by normalization of the free light chain ratio in 8 cases. Tissue deposition in HCDD relates to physicochemical peculiarities of both variable and constant heavy chain domains. Early diagnosis and treatment with bortezomib-based combinations appear important to preserve renal prognosis. Thus, monitoring of serum-free light chain is an indirect but useful method to evaluate the hematological response.

Role of the Bridging Group in Bis-Pyridyl Ligands: Enhancing Both the Photo- and Electroluminescent Features of Cationic (IPr)CuI Complexes.

We report on the benefits of changing the bridging group X of bis-pyridyl ligands, that is, Py-X-Py where X is NH, CH2 , C(CH3 )2 , or PPh, on the photo- and electroluminescent properties of a new family of luminescent cationic H-heterocyclic carbene (NHC) copper(I) complexes. A joint experimental and theoretical study demonstrates that the bridging group affects the molecular conformation from a planar-like structure (X is NH and CH2 ) to a boat-like structure (X is C(CH3 )2 and PPh), leading to i) four-fold enhancement of the photoluminescence quantum yield (ϕem ) without affecting the thermally activated delayed fluorescence mechanism, and ii) one order of magnitude reduction of the ionic conductivity (σ) of thin films. This leads to an overall enhancement of the device efficacy and luminance owing to the increased ϕem and the use of low applied driving currents.

Impact of lipid composition and photosensitizer hydrophobicity on the efficiency of light-triggered liposomal release.

Photo-triggerable liposomes are considered nowadays as promising drug delivery devices due to their potential to release encapsulated drugs in a spatial and temporal manner. In this work, we have investigated the photopermeation efficiency of three photosensitizers (PSs), namely verteporfin, pheophorbide a and m-THPP when incorporated into liposomes with well-defined lipid compositions (SOPC, DOPC or SLPC). By changing the nature of phospholipids and PSs, the illumination of the studied systems was shown to significantly alter their lipid bilayer properties via the formation of lipid peroxides. The system efficiency depends on the PS/phospholipid association, and the ability of the PS to peroxidize acyl chains. Our results demonstrated the possible use of these three clinically approved (or under investigation) PSs as potential candidates for photo-triggerable liposome conception.

Chemoenzymatic Preparation and Biophysical Properties of Sulfated Quercetin Metabolites.

Sulfated quercetin derivatives are important authentic standards for metabolic studies. Quercetin-3'-O-sulfate, quercetin-4'-O-sulfate, and quercetin-3-O-sulfate as well as quercetin-di-O-sulfate mixture (quercetin-7,3'-di-O-sulfate, quercetin-7,4'-di-O-sulfate, and quercetin-3',4'-di-O-sulfate) were synthetized by arylsulfotransferase from Desulfitobacterium hafniense. Purified monosulfates and disulfates were fully characterized using MS and NMR and tested for their 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and N,N-dimethyl-p-phenylenediamine (DMPD) radical scavenging, Folin-Ciocalteau reduction (FCR), ferric reducing antioxidant power (FRAP), and anti-lipoperoxidant activities in rat liver microsomes damaged by tert-butylhydroperoxide. Although, as expected, the sulfated metabolites were usually less active than quercetin, they remained still effective antiradical and reducing agents. Quercetin-3'-O-sulfate was more efficient than quercetin-4'-O-sulfate in DPPH and FCR assays. In contrast, quercetin-4'-O-sulfate was the best ferric reductant and lipoperoxidation inhibitor. The capacity to scavenge ABTS+• and DMPD was comparable for all substances, except for disulfates, which were the most efficient. Quantum calculations and molecular dynamics simulations on membrane models supported rationalization of free radical scavenging and lipid peroxidation inhibition. These results clearly showed that individual metabolites of food bioactives can markedly differ in their biological activity. Therefore, a systematic and thorough investigation of all bioavailable metabolites with respect to native compounds is needed when evaluating food health benefits.

In silico pharmacology: Drug membrane partitioning and crossing.

Over the past decade, molecular dynamics (MD) simulations have become particularly powerful to rationalize drug insertion and partitioning in lipid bilayers. MD simulations efficiently support experimental evidences, with a comprehensive understanding of molecular interactions driving insertion and crossing. Prediction of drug partitioning is discussed with respect to drug families (anesthetics; ß-blockers; non-steroidal anti-inflammatory drugs; antioxidants; antiviral drugs; antimicrobial peptides). To accurately evaluate passive permeation coefficients turned out to be a complex theoretical challenge; however the recent methodological developments based on biased MD simulations are particularly promising. Particular attention is paid to membrane composition (e.g., presence of cholesterol), which influences drug partitioning and permeation. Recent studies concerning in silico models of membrane proteins involved in drug transport (influx and efflux) are also reported here. These studies have allowed gaining insight in drug efflux by, e.g., ABC transporters at an atomic resolution, explicitly accounting for the mandatory forces induced by the surrounded lipid bilayer. Large-scale conformational changes were thoroughly analyzed.

Bottom-Up Hierarchical Self-Assembly of Chiral Porphyrins through Coordination and Hydrogen Bonds.

A series of chiral synthetic compounds is reported that shows intricate but specific hierarchical assembly because of varying positions of coordination and hydrogen bonds. The evolution of the aggregates (followed by absorption spectroscopy and temperature-dependent circular dichroism studies in solution) reveal the influence of the proportion of stereogenic centers in the side groups connected to the chromophore ring in their optical activity and the important role of pyridyl groups in the self-assembly of these chiral macrocycles. The optical activity spans 2 orders of magnitude depending on composition and constitution. Two of the aggregates show very high optical activity even though the isolated chromophores barely give a circular dichroism signal. Molecular modeling of the aggregates, starting from the pyridine-zinc(II) porphyrin interaction and working up, and calculation of the circular dichroism signal confirm the origin of this optical activity as the chiral supramolecular organization of the molecules. The aggregates show a broad absorption range, between approximately 390 and 475 nm for the transitions associated with the Soret region alone, that spans wavelengths far more than the isolated chromophore. The supramolecular assemblies of the metalloporphyrins in solution were deposited onto highly oriented pyrolitic graphite in order to study their hierarchy in assembly by atomic force microscopy. Zero and one-dimensional aggregates were observed, and a clear dependence on deposition temperature was shown, indicating that the hierarchical assembly took place largely in solution. Moreover, scanning electron microscopy images of porphyrins and metalloporphyrins precipitated under out-of-equilibrium conditions showed the dependence of the number and position of chiral amide groups in the formation of a fibrillar nanomaterial. The combination of coordination and hydrogen bonding in the complicated assembly of these molecules-where there is a clear hierarchy for zinc(II)-pyridyl interaction followed by hydrogen-bonding between amide groups, and then van der Waals interactions-paves the way for the preparation of molecular materials with multiple chromophore environments.

Binding modes of a core-extended metalloporphyrin to human telomeric DNA G-quadruplexes.

The molecular recognition of human telomeric G-quadruplexes by a novel cationic π-extended Ni(II)-porphyrin (Ni(II)-TImidP4) is studied in aqueous solutions via (chir)optical spectroscopy, Fluorescence Resonance Energy Transfer (FRET) melting assay, and computational molecular modeling. The results are systematically compared with the recognition by a conventional meso-substituted Ni(II)-porphyrin (Ni(II)-TMPyP4), which allows us to pinpoint the differences in binding modes depending on the G-quadruplex topology. Importantly, FRET melting assays show the higher selectivity of Ni(II)-TImidP4 towards human telomeric G4 than that of Ni(II)-TMPyP4.

Stereochemistry and conformation of skyllamycin, a non-ribosomally synthesized peptide from Streptomyces sp. Acta 2897.

Skyllamycin is a non-ribosomally synthesized cyclic depsipeptide from Streptomyces sp. Acta 2897 that inhibits PDGF-signaling. The peptide scaffold contains an N-terminal cinnamoyl moiety, a ß-methylation of aspartic acid, three ß-hydroxylated amino acids and one rarely occurring α-hydroxy glycine. With the exception of α-hydroxy glycine, the stereochemistry of the amino acids was assigned by comparison to synthetic reference amino acids applying chiral GC-MS and Marfey-HPLC analysis. The stereochemistry of α-hydroxy glycine, which is unstable under basic and acidic conditions, was determined by conformational analysis, employing a combination of data from NOESY-NMR spectroscopy, simulated annealing and free MD simulations. The simulation procedures were applied for both R- and S-configured α-hydroxy glycine of the skyllamycin structure and compared to the NOESY data. Both methods, simulated annealing and free MD simulations independently support S-configured α-hydroxy glycine thus enabling the assignment of all stereocenters in the structure of skyllamycin and devising the role of two-component flavin dependent monooxygenase (Sky39) as S-selective.

NHC copper(I) complexes bearing dipyridylamine ligands: synthesis, structural, and photoluminescent studies.

We describe the synthesis of new cationic tricoordinated copper complexes bearing bidentate pyridine-type ligands and N-heterocyclic carbene as ancillary ligands. These cationic copper complexes were fully characterized by NMR, electrochemistry, X-ray analysis, and photophysical studies in different environments. Density functional theory calculations were also undertaken to rationalize the assignment of the electronic structure and the photophysical properties. These tricoordinated cationic copper complexes possess a stabilizing CH-π interaction leading to high stability in both solid and liquid states. In addition, these copper complexes, bearing dipyridylamine ligands having a central nitrogen atom as potential anchoring point, exhibit very interesting luminescent properties that render them potential candidates for organic light-emitting diode applications.

cis-trans Isomerization of silybins A and B.

Methods were developed and optimized for the preparation of the 2,3-cis- and the 10,11-cis-isomers of silybin by the Lewis acid catalyzed (BF3∙OEt2) isomerization of silybins A (1a) and B (1b) (trans-isomers). The absolute configuration of all optically pure compounds was determined by using NMR and comparing their electronic circular dichroism data with model compounds of known absolute configurations. Mechanisms for cis-trans-isomerization of silybin are proposed and supported by quantum mechanical calculations.

Gut microbiota biotransformation of drug glucuronides leading to gastrointestinal toxicity: Therapeutic potential of bacterial ß-glucuronidase inhibition in mycophenolate-induced enteropathy.

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.

Early detection of liver injuries by the Serum enhanced binding test sensitive to albumin post-transcriptional modifications.

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.

Diastereoselective synthesis of C60/steroid conjugates.

The design and synthesis of fullerene-steroid hybrids by using Prato's protocol has afforded new fullerene derivatives endowed with epiandrosterone, an important naturally occurring steroid hormone. Since the formation of the pyrrolidine ring resulting from the 1,3-dipolar cyloaddition reaction takes place with generation of a new stereogenic center on the C2 of the five-membered ring, the reaction proceeds with formation of a diastereomeric mixture [compounds 6 and 7 in 70:30 ratio, 8 and 9 in 26:74 ratio (HPLC)] in which the formation of the major diasteroisomers 6 and 9 is consistent with an electrophilic attack of [60]fullerene on the Re face of the azomethine ylide directed by the steroidic unit. The chiroptical properties of these conjugates reveal typical Cotton effects in CD spectra that have been used to assign the absolute configuration of the new fulleropyrrolidines. The electrochemical study of the new compounds reveals the presence of four quasi-reversible reduction waves which are cathodically shifted in comparison with the parent C60, thus ascertaining the proposed structures.

Antioxidant properties of phenolic Schiff bases: structure-activity relationship and mechanism of action.

Phenolic Schiff bases are known for their diverse biological activities and ability to scavenge free radicals. To elucidate (1) the structure-antioxidant activity relationship of a series of thirty synthetic derivatives of 2-methoxybezohydrazide phenolic Schiff bases and (2) to determine the major mechanism involved in free radical scavenging, we used density functional theory calculations (B3P86/6-31+(d,p)) within polarizable continuum model. The results showed the importance of the bond dissociation enthalpies (BDEs) related to the first and second (BDEd) hydrogen atom transfer (intrinsic parameters) for rationalizing the antioxidant activity. In addition to the number of OH groups, the presence of a bromine substituent plays an interesting role in modulating the antioxidant activity. Theoretical thermodynamic and kinetic studies demonstrated that the free radical scavenging by these Schiff bases mainly proceeds through proton-coupled electron transfer rather than sequential proton loss electron transfer, the latter mechanism being only feasible at relatively high pH.

Free radical scavenging by natural polyphenols: atom versus electron transfer.

Polyphenols (synthetically modified or directly provided by human diet) scavenge free radicals by H-atom transfer and may thus decrease noxious effects due to oxidative stress. Free radical scavenging by polyphenols has been widely theoretically studied from the thermodynamic point of view whereas the kinetic point of view has been much less addressed. The present study describes kinetic-based structure-activity relationship for quercetin. This compound is very characteristic of the wide flavonoid subclass of polyphenols. H-atom transfer is a mechanism based on either atom or electron transfer. This is analyzed here by quantum chemical calculations, which support the knowledge acquired from experimental studies. The competition between the different processes is discussed in terms of the nature of the prereaction complexes, the pH, the formation of activated-deprotonated forms, and the atom- and electron-transfer efficiency. The role of the catechol moiety and the 3-OH group of quercetin as scavengers of different types of free radicals (CH3OO(•), CH3O(•), (•)OH, and (•)CH2OH) is rationalized. Identifying the exact mechanism and accurately evaluating kinetics is of fundamental importance to understand antioxidant behavior in physiological environments.

Regio- and stereocontrolled synthesis of oligostilbenoids: theoretical highlights at the supramolecular level.

Oligostilbenoids (e.g., ampelopsin F, viniferin, pallidol) result from homogeneous or heterogeneous coupling of monomeric stilbenoid units, leading to various chemical structures. Oligostilbenoid synthesis is regio- and stereocontrolled. To tackle this regio- and stereocontrol, a supramolecular chemistry approach is required that can be achieved by quantum chemistry. The stability of noncovalent π-stacks, formed between two stilbenoid units prior to oxidation, is accurately evaluated with density functional theory (DFT) including dispersive effects (within the DFT-D formalism). These noncovalent arrangements drive the regiocontrol. The rest of the chemical pathway is a succession of dearomatization and rearomatization stages. The thermodynamics and kinetics of the processes are calculated with classical hybrid functionals. This study allows discrimination between the two main possible chemical pathways, namely, radical-neutral and radical-radical reactions. The former appears more likely, thermodynamics and kinetics being in perfect agreement with the experimental 1:2 ratio obtained for ampelopsin F:pallidol analogues, respectively.