Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis.

(R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushing's disease and rheumatoid arthritis. We here review the data that support the investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favorably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favors its trafficking to the plasma membrane; (2) by increasing membrane targeting of the TRPC6 ion channel, it rescues acidification in phagolysosomes of CF alveolar macrophages (which show abnormally high pH) and consequently restores their bactericidal activity; (3) its effects on neutrophils (induction of apoptosis), eosinophils (inhibition of degranulation/induction of apoptosis) and lymphocytes (modification of the Th17/Treg balance in favor of the differentiation of anti-inflammatory lymphocytes and reduced production of various interleukins, notably IL-17A) contribute to the resolution of inflammation and restoration of innate immunity, and (4) roscovitine displays analgesic properties in animal pain models.

Pushing the limits of catalytic C-H amination in polyoxygenated cyclobutanes.

A synthetic route to a new class of conformationally constrained iminosugars based on a 5-azaspiro[3.4]octane skeleton has been developed by way of Rh(ii)-catalyzed C(sp(3))-H amination. The pivotal stereocontrolled formation of the quaternary C-N bond by insertion into the C-H bonds of the cyclobutane ring was explored with a series of polyoxygenated substrates.

Synthetic deoxynojirimycin derivatives bearing a thiolated, fluorinated or unsaturated N-alkyl chain: identification of potent α-glucosidase and trehalase inhibitors as well as F508del-CFTR correctors.

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.

A functional tandem between transient receptor potential canonical channels 6 and calcium-dependent chloride channels in human epithelial cells.

TRPC6 plays important human physiological functions, notably in artery and arterioles constriction, in regulation of vascular volume and in bronchial muscle constriction. It is implicated in pulmonary hypertension, cardiovascular disease, and focal segmental glomerulosclerosis and seems to play a role in cancer development. Previously, we identified Guanabenz, an α2-adrenergic agonist used for hypertension treatment (Wytensin®), as an activator of calcium-dependent chloride channels (CaCC) in human Cystic Fibrosis (CF) nasal epithelial cells by transiently increasing [Ca2+]i via an influx of extracellular Ca2+.

SERCA and PMCA pumps contribute to the deregulation of Ca2+ homeostasis in human CF epithelial cells.

Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809.

The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity.

Increase of Ca(2+) influx in Cystic Fibrosis (CF) cells has been reported to be related to Transient Receptor Potential Canonical (TRPC6) channel, which is implicated in a functional coupling with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Several members of the Transient Receptor Potential Vanilloid (TRPV) channels family have already been described as emerging target for respiratory diseases. Two specific isoforms, TRPV5 and TRPV6 are of particular interest in the context of CF Ca(2+) homeostasis as they are highly selective toward Ca(2+) and constitutively activated.

N- and C-alkylation of seven-membered iminosugars generates potent glucocerebrosidase inhibitors and F508del-CFTR correctors.

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.

Roscovitine is a proteostasis regulator that corrects the trafficking defect of F508del-CFTR by a CDK-independent mechanism.

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.

A new 9-alkyladenine-cyclic methylglyoxal diadduct activates wt- and F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in vitro and in vivo.

Cystic fibrosis transmembrane conductance regulator (CFTR) is the main chloride channel present in the apical membrane of epithelial cells and the F508 deletion (F508del-CFTR) in the CF gene is the most common cystic fibrosis-causing mutation. In the search for a pharmacotherapy of cystic fibrosis caused by the F508del-CFTR, a bi-therapy could be developed associating a corrector of F508del-CFTR trafficking and an activator of the channel activity of CFTR. Here, we report on the synthesis of 9-alkyladenine derivatives analogues of our previously discovered activator of wt-CFTR and F508del-CFTR, GPact-11a, and the identification of a new activator of these channels, GPact-26a, through various flux assays on human airway epithelial CF and non-CF cell lines and in vivo measurement of rat salivary secretion.

Cystic fibrosis bronchial epithelial cells are lipointoxicated by membrane palmitate accumulation.

The F508del-CFTR mutation, responsible for Cystic Fibrosis (CF), leads to the retention of the protein in the endoplasmic reticulum (ER). The mistrafficking of this mutant form can be corrected by pharmacological chaperones, but these molecules showed limitations in clinical trials. We therefore hypothesized that important factors in CF patients may have not been considered in the in vitro assays.

Improvement of chloride transport defect by gonadotropin-releasing hormone (GnRH) in cystic fibrosis epithelial cells.

Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is due to mutations in the CFTR gene. F508del, the most frequent mutation in patients, impairs CFTR protein folding and biosynthesis. The F508del-CFTR protein is retained in the endoplasmic reticulum (ER) and its traffic to the plasma membrane is altered.

ANO1 contributes to angiotensin-II-activated Ca2+-dependent Cl- current in human atrial fibroblasts.

Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels.

Rescue of functional CFTR channels in cystic fibrosis: a dramatic multivalent effect using iminosugar cluster-based correctors.

Cystic fibrosis is caused by a mutation in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. N-butyl 1-deoxynojirimycin (N-Bu DNJ), a clinical candidate for the treatment of cystic fibrosis, is able to act as a CFTR corrector by overcoming the processing defect of the mutant protein. To explore the potential of multivalency on CFTR correction activity, a library of twelve DNJ click clusters with valencies ranging from 3 to 14 were synthesized.

Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain.

The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508-NBD1 and housekeeping proteins prevents ΔF508-CFTR delivery to the plasma membrane.

Bronchorelaxation of the human bronchi by CFTR activators.

The airway functions are profoundly affected in many diseases including asthma, COPD and cystic fibrosis (CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR (Cystic Fibrosis transmembrane Conductance Regulator) gene, which normally encodes a multifunctional and integral membrane cAMP regulated and ATP gated Cl(-) channel expressed in airway epithelial cells. Using human lung tissues obtained from patients undergoing surgery for lung cancer, we demonstrated that CFTR participates in bronchorelaxation.

Effect of VX-770 (ivacaftor) and OAG on Ca2+ influx and CFTR activity in G551D and F508del-CFTR expressing cells.

Background: TRPC6 has been proposed to be responsible for the abnormal OAG-dependent Ca(2+) influx in cystic fibrosis (CF) cells and we hypothesized that it interacts with CFTR. Here, we investigated how this functional complex operates in CF and non-CF epithelial cells.

Methods: Chinese hamster ovary (CHO) cells stably transfected with pNut vector containing wild type CFTR (CHO-WT), F508del-CFTR (CHO-F508del) or G551D-CFTR(CHO-G551D) were used.

Missense mutations in SLC26A8, encoding a sperm-specific activator of CFTR, are associated with human asthenozoospermia.

The cystic fibrosis transmembrane conductance regulator (CFTR) is present in mature sperm and is required for sperm motility and capacitation. Both these processes are controlled by ions fluxes and are essential for fertilization. We have shown that SLC26A8, a sperm-specific member of the SLC26 family of anion exchangers, associates with the CFTR channel and strongly stimulates its activity.