Cyclic Peptidyl Inhibitors against CAL/CFTR Interaction for Treatment of Cystic Fibrosis.
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ABSTRACT:
SUBMITTER: Dougherty PG
PROVIDER: S-EPMC8011814 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Defects in a single gene lead to the defective proteins that cause cystic fibrosis, making the disease an ideal candidate for mutation-targeted therapy. Although ivacaftor is currently the only FDA-approved CFTR modifier, others are in development.
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Project description:Peptidylprolyl isomerase Pin1 regulates the function and/or stability of phosphoproteins by altering the conformation of specific pSer/pThr-Pro peptide bonds. In this work, a cyclic peptide library was synthesized and screened against the catalytic domain of human Pin1. The selected inhibitors contained a consensus motif of D-pThr-Pip-Nal (where Pip is L-piperidine-2-carboxylic acid and Nal is L-2-naphthylalanine). Representative compounds were tested for binding to Pin1 by isothermal titration calorimetry and inhibition of Pin1 activity, and the most potent inhibitors had K(D) (and K(I)) values in the low nanomolar range. Treatment of breast cancer cells with the inhibitors, which were rendered membrane permeable by attachment of an octaarginine sequence, inhibited cell proliferation and increased the protein levels of two previously established Pin1 substrates, PML and SMRT. Finally, a second generation of cell permeable Pin1 inhibitors was designed by replacing the noncritical residues within the cyclic peptide ring with arginine residues and shown to have antiproliferative activity against the cancer cells.
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Project description:One-bead-one-compound (OBOC) libraries provide a powerful tool for drug discovery as well as biomedical research. However, screening a large number of beads/compounds (>1 million) and rank ordering the initial hits (which are covalently attached to a solid support) according to their potencies still post significant technical challenges. In this work, we have integrated some of the latest technical advances from our own as well as other laboratories to develop a general methodology for rapidly screening large OBOC libraries. The methodology has been applied to synthesize and screen a cyclic peptide library that features: (1) spatially segregated beads containing cyclic peptides on the surface layer and linear encoding peptides in their interior; (2) rapid on-bead screening of the library (>1 million) by a multistage procedure (magnetic bead sorting, enzyme-linked assay, and fluorescence based screening); (3) selective release of cyclic peptides from single positive beads for solution-phase determination of their binding affinities; and (4) hit identification by partial Edman degradation/mass spectrometry (PED/MS). Screening of the library against protein phosphatase calcineurin (Cn) identified a series of cyclic peptides that bind to the substrate-docking site for nuclear factor of activated T cells (NFAT) with K(D) values of ?1 ?M. Further improvement of the affinity and specificity of these compounds may lead to a new class of immunosuppressive agents that are more selective and therefore less toxic than cyclosporine A and FK506.
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Project description:Cystic fibrosis (CF) is an autosomal recessive genetic disease characterized by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a chloride channel responsible for ion flow across epithelial surfaces of lung, sinuses, pancreas, intestine, and liver. Researchers have grouped CFTR genetic mutations into various protein defects: reduced protein synthesis (class 1 mutations), abnormal protein folding and maturation (class 2 mutation), and abnormal gating (class 3 mutation). These mutations usually present as severe forms of CF due to complete absence of CFTR at cell surfaces. Milder forms (eg, protein maturation and conductance defects, classes 4-6) present as less severe forms of CF related to the presence of CFTR at the cell surface. Differences in severity are directly due to CFTR function which is based on the severity of CFTR mutation. This knowledge has proven useful for designing therapy for individual mutations and mutation classes. The discovery and US Food and Drug Administration approval of Kalydeco® (ivacaftor) in early 2011 marked the beginning of a new era of therapies that are focused on improving defective CFTR protein function. However, due to its specificity for the G551D mutation, ivacaftor only benefit?5% of CF patients. Approximately 50% of CF patients have two copies of the F508Del mutation, while other CF patients carry only one copy of this gene. More recently, Orkambi®, a two compound medication composed of lumacaftor and ivacaftor, has provided the foundation necessary to further build on molecular concepts of: correction of trafficking, potentiation, and amplification of defective CFTR. These new concepts will form the basis of future CF therapies and extend CFTR treatment to almost 50% of CF patients. Evolving knowledge of the molecular mechanisms responsible for defective CFTR has prompted new research focused on "repair" of each phase of CFTR expression and function, thus creating a new class of combination "CFTR correctors" referred to as "triplet CFTR compounds." This article will review how patients can be selected and treated with these newer agents that are based on specific mutations. In the future, many CF practitioners have expectations that initiation of treatment for CF patients will occur simply by use of biomarkers of CFTR expression (eg, sweat chloride, nasal potential difference, rectal organoids) rather than testing for specific mutations. As continued research identifies biomarkers with greater specificity and which predict clinical response, therapies can potentially be tailored to individual responses.
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Project description:Cystic fibrosis (CF), caused by biallelic inactivating mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, has recently been categorized as a familial colorectal cancer (CRC) syndrome. CF patients are highly susceptible to early, aggressive colorectal tumor development. Endoscopic screening studies have revealed that by the age of forty 50% of CF patients will develop adenomas, with 25% developing aggressive advanced adenomas, some of which will have already advanced to adenocarcinomas. This enhanced risk has led to new CF colorectal cancer screening recommendations, lowering the initiation of endoscopic screening to age forty in CF patients, and to age thirty in organ transplant recipients. The enhanced risk for CRC also extends to the millions of people (more than 10 million in the US) who are heterozygous carriers of CFTR gene mutations. Further, lowered expression of CFTR is reported in sporadic CRC, where downregulation of CFTR is associated with poor survival. Mechanisms underlying the actions of CFTR as a tumor suppressor are not clearly understood. Dysregulation of Wnt/?-catenin signaling and disruption of intestinal stem cell homeostasis and intestinal barrier integrity, as well as intestinal dysbiosis, immune cell infiltration, stress responses, and intestinal inflammation have all been reported in human CF patients and in animal models. Notably, the development of new drug modalities to treat non-gastrointestinal pathologies in CF patients, especially pulmonary disease, offers hope that these drugs could be repurposed for gastrointestinal cancers.
| S-EPMC7215855 | biostudies-literature
Project description:Macrocyclic peptides have proven to be highly effective inhibitors of protein-protein interactions but generally lack cell permeability to access intracellular targets. We show herein that macrocyclic peptides may be rendered highly cell-permeable and biologically active by conjugating them with a cyclic cell-penetrating peptide (CPP). A previously reported cyclic peptidyl inhibitor against the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid-2 (Nrf2) interaction (KD = 18 nM) was covalently attached to a cyclic CPP through a flexible linker. The resulting bicyclic peptide retained the Keap1-binding activity, resisted proteolytic degradation, readily entered mammalian cells, and activated the transcriptional activity of Nrf2 at nanomolar to low micromolar concentrations in cell culture. The inhibitor provides a useful tool for investigating the biological function of Keap1-Nrf2 and a potential lead for further development into a novel class of anti-inflammatory and anticancer agents. Our data suggest that other membrane-impermeable cyclic peptides may be similarly rendered cell-permeable by conjugation with a cyclic CPP.
| S-EPMC7288753 | biostudies-literature
Project description:Cystic fibrosis (CF) is a loss-of-function disease caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, a chloride ion channel that localizes to the apical plasma membrane of epithelial cells. The most common form of the disease results from the deletion of phenylalanine-508 (ΔF508), leading to the accumulation of CFTR in the endoplasmic reticulum with a concomitant loss of chloride flux. We discovered that cyclic tetrapeptides, such as 11, 14, and 15, are able to correct the trafficking defect and restore cell surface activity of ΔF508-CFTR. Although this class of cyclic tetrapeptides is known to contain inhibitors of certain histone deacetylase (HDAC) isoforms, their HDAC inhibitory potencies did not directly correlate with their ability to rescue ΔF508-CFTR. In full HDAC profiling, 15 strongly inhibited HDACs 1, 2, 3, 10 and 11, but not HDACs 4-9. Although 15 had less potent IC(50) values than reference agent vorinostat (2) in HDAC profiling, it was markedly more potent than 2 in rescuing ΔF508-CFTR. We suggest that specific HDACs can have a differential influence on correcting ΔF508-CFTR, which may reflect both deacetylase and protein scaffolding actions.
| S-EPMC3187562 | biostudies-literature
Project description:In a prior study, lumacaftor/ivacaftor treatment (?28 d) in patients with cystic fibrosis (CF) heterozygous for F508del-CFTR did not improve lung function.To evaluate an optimized lumacaftor/ivacaftor dosing regimen with a longer duration in a cohort of patients heterozygous for F508del-CFTR.Patients aged 18 years or older with a confirmed CF diagnosis and percent predicted FEV1 (ppFEV1) of 40 to 90 were randomized to lumacaftor/ivacaftor (400 mg/250 mg every 12 h) or placebo daily for 56 days. Primary outcomes were change in ppFEV1 at Day 56 and safety. Other disease markers were evaluated.Of 126 patients, 119 (94.4%) completed the study. Lumacaftor/ivacaftor was well tolerated, although chest tightness and dyspnea occurred more frequently with active treatment than with placebo (27.4% vs. 14.3% and 14.5% vs. 6.3%, respectively). Mean (SD) ppFEV1 values at baseline were 62.9 (14.3) in the active treatment group and 60.1 (14.0) in the placebo group. Absolute changes in ppFEV1 (least squares mean [SE]) at Day 56 were -0.6 (0.8) percentage points in the active treatment group and -1.2 (0.8) percentage points in the placebo group (P?=?0.60). CF respiratory symptom scores in the active treatment group improved by a mean of 5.7 points versus a decrease of -0.8 in the placebo group (P?<?0.01). No changes in body mass index occurred. Changes from baseline in sweat chloride (least squares mean [SE]) at Day 56 were -11.8 (1.3) mmol/L in the active treatment group and -0.8 (1.2) mmol/L in the placebo group (P?<?0.0001).Sweat chloride and respiratory symptom scores improved with lumacaftor/ivacaftor, though no meaningful benefit was seen in ppFEV1 or body mass index in patients heterozygous for F508del-CFTR. Clinical trial registered with www.clinicaltrials.gov (NCT01225211).
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Project description: Not available
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Project description:The recent cryo-electron microscopy structures of zebrafish and the human cystic fibrosis transmembrane conductance regulator (CFTR) provided unprecedented insights into putative mechanisms underlying gating of its anion channel activity. Interestingly, despite predictions based on channel activity measurements in biological membranes, the structure of the detergent purified, phosphorylated, and ATP-bound human CFTR protein did not reveal a stably open conduction pathway. This study tested the hypothesis that the functional properties of the detergent solubilized CFTR protein used for structural determinations are different from those exhibited by CFTR purified under conditions that retain associated lipids native to the membrane. It was found that CFTR purified together with phospholipids and cholesterol using amphipol: A8-35, exhibited higher rates of catalytic activity, phosphorylation dependent channel activation and potentiation by the therapeutic compound, ivacaftor, than did CFTR purified in detergent. The catalytic activity of phosphorylated CFTR detergent micelles was rescued by the addition of phospholipids plus cholesterol, but not by phospholipids alone, arguing for a specific role for cholesterol in modulating this function. In summary, these studies highlight the importance of lipid interactions in the intrinsic activities and pharmacological potentiation of CFTR.
| S-EPMC6721619 | biostudies-literature