Cryo electron micrographs of human cystic fibrosis transmembrane conductance regulator (CFTR) in complex with GLPG
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ABSTRACT:
SUBMITTER: Jue Chen
PROVIDER: EMPIAR-10281 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:We previously identified phenylquinoxalinone CFTRact-J027 (4) as a cystic fibrosis transmembrane conductance regulator (CFTR) activator with an EC50 of ∼200 nM and demonstrated its therapeutic efficacy in mouse models of constipation. Here, structure-activity studies were done on 36 synthesized phenylquinoxalinone analogs to identify compounds with improved potency and altered metabolic stability. Synthesis of the phenylquinoxalinone core was generally accomplished by condensation of 1,2-phenylenediamines with substituted phenyloxoacetates. Structure-activity studies established, among other features, the privileged nature of a properly positioned nitro moiety on the 3-aryl group. Synthesized analogs showed improved CFTR activation potency compared to 4 with EC50 down to 21 nM and with greater metabolic stability. CFTR activators have potential therapeutic indications in constipation, dry eye, cholestatic liver diseases, and inflammatory lung disorders.
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Project description:Despite the addition of cystic fibrosis transmembrane conductance regulator (CFTR) modulators to the cystic fibrosis (CF) treatment regimen, patients with CF continue to suffer from chronic bacterial infections that lead to progressive respiratory morbidity. Host immunity, and macrophage dysfunction specifically, has an integral role in the inability of patients with CF to clear bacterial infections. We sought to characterize macrophage responses to CFTR modulator treatment as we hypothesized that there would be differential effects based on patient genotype. Human CF and non-CF peripheral blood monocyte-derived macrophages (MDMs) were analyzed for CFTR expression, apoptosis, polarization, phagocytosis, bacterial killing, and cytokine production via microscopy, flow cytometry, and ELISA-based assays. Compared to non-CF MDMs, CF MDMs display decreased CFTR expression, increased apoptosis, and decreased phagocytosis. CFTR expression increased and apoptosis decreased in response to ivacaftor or lumacaftor/ivacaftor therapy, and phagocytosis improved with ivacaftor alone. Ivacaftor restored CF macrophage polarization responses to non-CF levels and reduced Pseudomonas aeruginosa bacterial burden, but did not reduce other bacterial loads. Macrophage inflammatory cytokine production decreased in response to ivacaftor alone. In summary, ivacaftor and lumacaftor/ivacaftor have differential impacts on macrophage function with minimal changes observed in CF patients treated with lumacaftor/ivacaftor. Overall improvements in macrophage function in ivacaftor-treated CF patients result in modestly improved macrophage-mediated bacterial killing.
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Project description:The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of "rational" approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287-288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287-288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel.
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Project description:Most cases of cystic fibrosis (CF) are caused by mutations that block the biosynthetic maturation of the CF gene product, the CF transmembrane conductance regulator (CFTR) chloride channel. CFTR-processing mutants fail to escape the endoplasmic reticulum and are rapidly degraded. Current efforts to induce the maturation of CFTR mutants target components of the biosynthetic pathway (e.g., chaperones) rather than CFTR per se. Such methods are inherently nonspecific. Here we show that the most common CF-causing mutant (DeltaF508-CFTR) can form mature, functional chloride channels that reach the cell surface when coexpressed with several other CFTR-processing mutants or with amino fragments of the wild-type CFTR protein. This transcomplementation effect required a specific match between the region flanking the disease-causing mutation and the complementing fragment; e.g., amino fragments complemented DeltaF508-CFTR but not H1085R (a carboxy-processing mutant), whereas a carboxy fragment complemented H1085R but not DeltaF508-CFTR. Transcomplementing fragments did not affect CFTR interactions with Hsc70, a chaperone previously implicated in CFTR biosynthesis. Instead, they may promote CFTR maturation by blocking nonproductive interactions between domains within the same or neighboring CFTR polypeptides that prevent normal processing. These findings indicate that it may be possible to develop CF therapies (e.g., mini-cDNA constructs for gene therapy) that are tailored to specific disease-causing mutants of CFTR.
| S-EPMC419584 | biostudies-other
Project description:Virulence of the intracellular pathogen Listeria monocytogenes (Listeria) requires escape from the phagosome into the host cytosol, where the bacteria replicate. Phagosomal escape is a multistep process characterized by perforation, which is dependent on the pore-forming toxin listeriolysin O (LLO), followed by rupture. The contribution of host factors to Listeria phagosomal escape is incompletely defined. Here we show that the cystic fibrosis transmembrane conductance regulator (CFTR) facilitates Listeria cytosolic entry. CFTR inhibition or mutation suppressed Listeria vacuolar escape in culture, and inhibition of CFTR in wild-type mice before oral inoculation of Listeria markedly decreased systemic infection. We provide evidence that high chloride concentrations may facilitate Listeria vacuolar escape by enhancing LLO oligomerization and lytic activity. We propose that CFTR transiently increases phagosomal chloride concentration after infection, potentiating LLO pore formation and vacuole lysis. Our studies suggest that Listeria exploits mechanisms of cellular ion homeostasis to escape the phagosome and emphasize host ion-channel function as a key parameter of bacterial virulence.
| S-EPMC3029685 | biostudies-literature
Project description:Cystic fibrosis (CF) is a potentially fatal genetic disease that causes serious lung damage. With time, researchers have a more complete understanding of the molecular-biological defects that underlie CF. This knowledge is leading to alternative approaches regarding the treatment of this condition. Trikafta is the third FDA-approved drug that targets the F508del mutation of the CFTR gene. The drug is a combination of three individual drugs which are elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA). This trio increases the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and reduces the mortality and morbidity rates in CF patients. The effectiveness of Trikafta, seen in clinical trials, outperforms currently available therapies in terms of lung function, quality of life, sweat chloride reduction, and pulmonary exacerbation reduction. The safety and efficacy of CFTR modulators in children with CF have also been studied. Continued evaluation of patient data is needed to confirm its long-term safety and efficacy. In this study, we will focus on reviewing data from clinical trials regarding the benefits of CFTR modulator therapy. We address the impact of Trikafta on lung function, pulmonary exacerbations, and quality of life. Adverse events of the different CFTR modulators are discussed.
| S-EPMC8266292 | biostudies-literature
Project description:In cystic fibrosis (CF), there is early destruction of the exocrine pancreas, and this results in a unique form of diabetes that affects approximately half of adult CF individuals. An animal model of cystic fibrosis-related diabetes has been developed in the ferret, which progresses through phases of glycemic abnormalities because of islet remodeling during and after exocrine destruction. Herein, we quantified the pancreatic histopathological changes that occur during these phases. There was an increase in percentage ductal, fat, and islet area in CF ferrets over time compared with age-matched wild-type controls. We also quantified islet size, shape, islet cell composition, cell proliferation (Ki-67), and expression of remodeling markers (matrix metalloprotease-7, desmin, and α-smooth muscle actin). Pancreatic ducts were dilated with scattered proliferating cells and were surrounded by activated stellate cells, indicative of tissue remodeling. The timing of islet and duct proliferation, stellate cell activation, and matrix remodeling coincided with the previously published stages of glycemic crisis and inflammation. This mapping of remodeling events in the CF ferret pancreas provides insights into early changes that control glycemic intolerance and subsequent recovery during the evolution of CF pancreatic disease.
| S-EPMC5963477 | biostudies-literature
Project description:The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1?mol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4?mol/L VPC 23019) and AMPK inhibition (80?mol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ?F508 CFTR mutant (CFTR?F508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.
| S-EPMC4469317 | biostudies-literature
Project description: Not available
| EMPIAR-11006 | biostudies-other
Project description:IntroductionIn this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age.MethodsThirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure.ResultsPatients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced.ConclusionsThis is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.
| S-EPMC3629184 | biostudies-literature