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1.
Org Biomol Chem ; 13(43): 10734-44, 2015 Nov 21.
Article in English | MEDLINE | ID: mdl-26356422

ABSTRACT

The synthesis of eleven 1-deoxynojirimycin (DNJ) derivatives presenting either a monofluoro, difluoro, thiolated or unsaturated N-alkyl chain of various length is described. Exploiting the unsaturated moiety on the nitrogen, fluorine has been introduced through a HF/SbF5 superacid catalysed hydrofluorination and thiol-ene click chemistry allowed introduction of sulfur. The synthetic derivatives have been tested for their ability to inhibit glycosidases and correct F508del-CFTR. Two of the unsaturated iminosugars exhibited potency similar to Miglustat as F508del-CFTR correctors. The thioalkyl iminosugars as well as the corresponding alkyl iminosugars demonstrated low micromolar α-glucosidases and trehalases inhibition. Introduction of fluorine abolished F508del-CFTR correction and trehalase inhibition.


Subject(s)
1-Deoxynojirimycin/analogs & derivatives , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Enzyme Inhibitors/chemistry , Glycoside Hydrolase Inhibitors/chemistry , Trehalase/antagonists & inhibitors , 1-Deoxynojirimycin/pharmacology , Animals , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Enzyme Inhibitors/pharmacology , Glycoside Hydrolase Inhibitors/pharmacology , Halogenation , Humans , Insecta , Mutation , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/pharmacology , Swine , Trehalase/metabolism , alpha-Glucosidases/metabolism
2.
Org Biomol Chem ; 12(44): 8977-96, 2014 Nov 28.
Article in English | MEDLINE | ID: mdl-25277226

ABSTRACT

The glycosidase inhibitory properties of synthetic C-alkyl and N-alkyl six-membered iminosugars have been extensively studied leading to therapeutic candidates. The related seven-membered iminocyclitols have been less examined despite the report of promising structures. Using an in house ring enlargement/C-alkylation as well as cross-metathesis methodologies as the key steps, we have undertaken the synthesis and biological evaluation of a library of fourteen 2C- and eight N-alkyl tetrahydroxylated azepanes starting from an easily available glucopyranose-derived azidolactol. Four, six, nine and twelve carbon atom alkyl chains have been introduced. The study of two distinct D-gluco and L-ido stereochemistries for the tetrol pattern as well as R and S configurations for the C-2 carbon bearing the C-alkyl chain is reported. We observed that C-alkylation of the L-ido tetrahydroxylated azepane converts it from an α-L-fucosidase to a ß-glucosidase and ß-galactosidase inhibitor while N-alkylation of the D-gluco iminosugar significantly improves its inhibition profile leading to potent ß-glucosidase, ß-galactosidase, α-L-rhamnosidase and ß-glucuronidase inhibitors whatever the stereochemistry of the alkyl chain. Interestingly, the N-alkyl chain length usually parallels the azepane inhibitor potency as exemplified by the identification of a potent glucocerebrosidase inhibitor (Ki 1 µM) bearing a twelve carbon atom chain. Additionally, several C-alkyl azepanes demonstrated promising F508del-CFTR correction unlike the parent tetrahydroxyazepanes. None of the C-alkyl and N-alkyl azepanes did inhibit ER α-glucosidases I or II.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Glucosylceramidase/antagonists & inhibitors , Imino Sugars/pharmacology , Alkylation , Crystallography, X-Ray , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Glucosylceramidase/metabolism , Humans , Imino Sugars/chemical synthesis , Imino Sugars/chemistry , Models, Molecular , Molecular Conformation , Structure-Activity Relationship
3.
Br J Pharmacol ; 171(21): 4831-49, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25065395

ABSTRACT

BACKGROUND AND PURPOSE: The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. EXPERIMENTAL APPROACH: Human CF airway epithelial cells were treated with roscovitine 100 µM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. KEY RESULTS: Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. CONCLUSIONS AND IMPLICATIONS: Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Calcium/metabolism , Cell Line , Cyclin-Dependent Kinases/antagonists & inhibitors , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Endoplasmic Reticulum/metabolism , Epithelial Cells , HEK293 Cells , Humans , Mutation , NAV1.5 Voltage-Gated Sodium Channel/genetics , NAV1.5 Voltage-Gated Sodium Channel/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Transport/drug effects , Roscovitine
5.
Eur Respir J ; 36(2): 311-22, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20110398

ABSTRACT

One of the major therapeutic strategy in cystic fibrosis aims at developing modulators of cystic fibrosis transmembrane conductance regulator (CFTR) channels. We recently discovered methylglyoxal alpha-aminoazaheterocycle adducts, as a new family of CFTR inhibitors. In a structure-activity relationship study, we have now identified GPact-11a, a compound able not to inhibit but to activate CFTR. Here, we present the effect of GPact-11a on CFTR activity using in vitro (iodide efflux, fluorescence imaging and patch-clamp recordings), ex vivo (short-circuit current measurements) and in vivo (salivary secretion) experiments. We report that GPact-11a: 1) is an activator of CFTR in several airway epithelial cell lines; 2) activates rescued F508del-CFTR in nasal, tracheal, bronchial, pancreatic cell lines and in human CF ciliated epithelial cells, freshly dissociated from lung samples; 3) stimulates ex vivo the colonic chloride secretion and increases in vivo the salivary secretion in cftr(+/+) but not cftr(-/-) mice; and 4) is selective for CFTR because its effect is inhibited by CFTR(inh)-172, GlyH-101, glibenclamide and GPinh-5a. To conclude, this work identifies a selective activator of wild-type and rescued F508del-CFTR. This nontoxic and water-soluble agent represents a good candidate, alone or in combination with a F508del-CFTR corrector, for the development of a CFTR modulator in cystic fibrosis.


Subject(s)
Adenine/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Purines/pharmacology , Pyrimidines/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , Iodides/chemistry , Lung/metabolism , Mice , Mice, Transgenic , Microscopy, Fluorescence/methods , Patch-Clamp Techniques , Purines/chemistry , Pyrimidines/chemistry , Saliva/metabolism , Solubility , Water/chemistry
6.
Br J Pharmacol ; 153(8): 1697-705, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18297105

ABSTRACT

BACKGROUND AND PURPOSE: TRPM4 and TRPM5 are calcium-activated non-selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5. EXPERIMENTAL APPROACH: Experiments were conducted using HEK-293 cells permanently transfected to express human TRPM4 or TRPM5. Currents were recorded using the whole-cell and inside-out variants of the patch-clamp technique. KEY RESULTS: The CFTR channel activator benzo[c]quinolizinium MPB-104 inhibited TRPM4 current with an IC(50) in the range of 2 x 10(-5) M, with no effect on single-channel conductance. In addition, 9-phenanthrol, lacking the chemical groups necessary for CFTR activation, also reversibly inhibited TRPM4 with a similar IC(50). Channel inhibition was voltage independent. The IC(50) determined in the whole-cell and inside-out experiments were similar, suggesting a direct effect of the molecule. However, 9-phenanthrol was ineffective on TRPM5, the most closely related channel within the TRP protein family. CONCLUSIONS AND IMPLICATIONS: We identify 9-phenanthrol as a TRPM4 inhibitor, without effects on TRPM5. It could be valuable in investigating the physiological functions of TRPM4, as distinct from those of TRPM5.


Subject(s)
Phenanthrenes/pharmacology , TRPM Cation Channels/antagonists & inhibitors , TRPM Cation Channels/drug effects , Animals , CHO Cells , Cell Line , Cricetinae , Cricetulus , Humans , Inhibitory Concentration 50 , Patch-Clamp Techniques/methods , Phenanthrenes/administration & dosage , Quinolizines/administration & dosage , Quinolizines/pharmacology , Transfection
7.
Eur Respir J ; 30(5): 857-64, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17596272

ABSTRACT

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cyclic adenosine monophosphate (cAMP)-dependent chloride channel located mainly at the apical membrane of epithelial cells. In myocytes of pulmonary arteries, numerous chloride channels have been identified and described, but not the CFTR. Thus the presence and function of the CFTR was investigated in rat intrapulmonary arteries. CFTR expression, localisation and function were analysed in cultured smooth muscle cells using Reverse transcriptase (RT)-PCR and immunoprecipitation followed by protein kinase A phosphorylation, immunolocalisation and an iodide efflux assay, respectively. The role of the CFTR in pulmonary vasoreactivity was determined in arterial rings using an organ bath system. RT-PCR and immunoprecipitation analyses, as well as the immunolocalisation study, revealed the expression of CFTR gene transcripts and protein. The iodide efflux assay showed the existence of functional cAMP-, calcium- and volume-dependent chloride channels. Furthermore, the following effects were found: 1) inhibition of forskolin/genistein-activated iodide efflux by glibenclamide, diphenylamine-2-carboxylic acid and CFTR-specific inhibitor (CFTR(inh))-172; 2) activation of iodide efflux by the benzoquinolizinium derivative CFTR activators MPB-07 and MPB-91; and 3) inhibition of MPB-dependent efflux by CFTR(inh)-172. Finally, CFTR activators induced concentration-dependent vasorelaxation in rings preconstricted with phenylephrine, in the presence or absence of endothelium. The present results are the first to reveal functional cyclic adenosine monophosphate-regulated cystic fibrosis transmembrane conductance regulator contributing to endothelium-independent vasorelaxation in rat intrapulmonary arterial myocytes.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiology , Pulmonary Artery , Analysis of Variance , Animals , Cells, Cultured , Chloride Channels/metabolism , Chloride Channels/physiology , Colforsin/pharmacology , Cyclic AMP/pharmacology , Genistein/pharmacology , Glyburide/pharmacology , Immunoprecipitation , Iodides/metabolism , Male , Phosphorylation , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Vasodilation/physiology
8.
Thorax ; 60(1): 55-9, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15618584

ABSTRACT

BACKGROUND: Most patients with cystic fibrosis (CF) have a DeltaF508 mutation resulting in abnormal retention of mutant gene protein (DeltaF508-CFTR) within the cell. This study was undertaken to investigate DeltaF508-CFTR trafficking in native cells from patients with CF with the aim of discovering pharmacological agents that can move DeltaF508-CFTR to its correct location in the apical cell membrane. METHOD: Nasal epithelial cells were obtained by brushing from individuals with CF. CFTR location was determined using immunofluorescence and confocal imaging in untreated cells and cells treated with sildenafil. The effect of sildenafil treatment on CFTR chloride transport function was measured in CF15 cells using an iodide efflux assay. RESULTS: In most untreated CF cells DeltaF508-CFTR was mislocalised within the cell at a site close to the nucleus. Exposure of cells to sildenafil (2 hours at 37 degrees C) resulted in recruitment of DeltaF508-CFTR to the apical membrane and the appearance of chloride transport activity. Sildenafil also increased DeltaF508-CFTR trafficking in cells from individuals with CF with a single copy DeltaF508 (DeltaF508/4016ins) or with a newly described CF trafficking mutation (R1283M). CONCLUSIONS: The findings provide proof of principle for sildenafil as a DeltaF508-CFTR trafficking drug and give encouragement for future testing of sildenafil and related PDE5 inhibitors in patients with CF.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/drug effects , Cystic Fibrosis/metabolism , Phosphodiesterase Inhibitors/pharmacology , Piperazines/pharmacology , Adolescent , Adult , Biological Transport/drug effects , Biological Transport/genetics , Child , Child, Preschool , Chlorides/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/metabolism , Fluorescent Antibody Technique , Humans , Infant , Mutation/genetics , Nose , Purines , Respiratory Mucosa , Sildenafil Citrate , Sulfones
9.
Am J Physiol Cell Physiol ; 281(5): C1657-66, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11600430

ABSTRACT

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC(50) = 85 microM) and activated CFTR in Calu-3 cells (EC(50) = 47 microM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.


Subject(s)
Adenosine Triphosphatases/physiology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Enzyme Activators/pharmacology , Quinolizines/pharmacology , Adenosine Triphosphatases/metabolism , Animals , CHO Cells , Chloride Channels/drug effects , Chloride Channels/metabolism , Cricetinae , Electrophysiology , Iodides/metabolism , Patch-Clamp Techniques , Phosphorylation , Rats , Rats, Inbred F344 , Thyroid Gland/cytology , Thyroid Gland/drug effects , Thyroid Gland/metabolism
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