Synthesis and Biological Evaluation of Pyrazole-Pyrimidones as a New Class of Correctors of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Cystic fibrosis (CF) is caused by the functional expression defect of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Despite the recent success in CFTR modulator development, the available correctors only partially restore the F508del-CFTR channel function, and several rare CF mutations show resistance to available drugs. We previously identified compound that synergistically rescued the F508del-CFTR folding defect in combination with the existing corrector drugs VX-809 and VX-661.

Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator () gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

Unlabelled: The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum.

Blood taken immediately after fatal resuscitation attempts yields higher quality DNA for genetic studies as compared to autopsy samples.

Background: The out-of-hospital cardiac arrest (OHCA) in the young may be associated with a genetic predisposition which is relevant even for genetic counseling of relatives. The identification of genetic variants depends on the availability of intact genomic DNA. DNA from autopsy may be not available due to low autopsy frequencies or not suitable for high-throughput DNA sequencing (NGS).

Profile of the single-use, multiple-pass protein A adsorber column in immunoadsorption.

Background And Objectives: Immunoadsorptions (IA) are used to remove autoantibodies from the plasma in autoimmune disorders. In this study, we evaluated the effects of a single-use, recombinant staphylococcal protein A-based immunoadsorber on blood composition of the patient.

Materials And Methods: In a cohort of patients with myasthenia gravis or stiff-person syndrome, essential parameters of blood cell count, coagulation, clinical chemistry or plasma proteins and immunoglobulins (Ig) were measured before and after IA (n = 11).

The emergency medical service has a crucial role to unravel the genetics of sudden cardiac arrest in young, out of hospital resuscitated patients: Interim data from the MAP-IT study.

Background: Genetics of sudden cardiac deaths (SCD) remains frequently undetected. Genetic analysis is recommended in undefined selected cases in the 2021 ERC-guideline. The emergency medical service and physicians (EMS) may play a pivotal role for unraveling SCD by saving biomaterial for later molecular autopsy.

A Precision Medicine Approach to Optimize Modulator Therapy for Rare CFTR Folding Mutants.

Trikafta, a triple-combination drug, consisting of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor) and the gating potentiator VX-770 (ivacaftor) provided unprecedented clinical benefits for patients with the most common cystic fibrosis (CF) mutation, F508del. Trikafta indications were recently expanded to additional 177 mutations in the CF transmembrane conductance regulator (CFTR). To minimize life-long pharmacological and financial burden of drug administration, if possible, we determined the necessary and sufficient modulator combination that can achieve maximal benefit in preclinical setting for selected mutants.

Discovery of CFTR modulators for the treatment of cystic fibrosis.

Introduction: Cystic fibrosis (CF) is a life-threatening inherited disease caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, an anion channel expressed at the apical membrane of secretory epithelia. CF leads to multiorgan dysfunction with progressive deterioration of lung function being the major cause of untimely death. Conventional CF therapies target only symptoms and consequences downstream of the primary genetic defect and the current life expectancy and quality of life of these individuals are still very limited.

Elexacaftor co-potentiates the activity of F508del and gating mutants of CFTR.

Trikafta, the combination of elexacaftor (VX-445), tezacaftor (VX-661) and ivacaftor (VX-770), was approved for therapy of cystic fibrosis (CF) patients with at least one allele of the CFTR mutation F508del. While the corrector function of VX-445 is well established, here we investigated the putative potentiator activity of VX-445 alone and in combination with VX-770. Acute addition of VX-445 increased the VX-770-potentiated F508del- and G551D-CFTR current by ~24% and >70%, respectively, in human bronchial and nasal epithelia.

Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination.

Based on its clinical benefits, Trikafta - the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) - was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism.

Mutation-specific dual potentiators maximize rescue of CFTR gating mutants.

Background: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosis (CF) mutations (∼10% of the patient population) associated with a gating defect of the CF transmembrane conductance regulator (CFTR). Despite the success of VX-770 treatment of patients carrying at least one allele of the most common gating mutation G551D-CFTR, some lung function decline and P. aeruginosa colonization persist.

An ultrasonic guided wave excitation method at constant phase velocity using ultrasonic phased array probes.

High-order ultrasonic guided wave modes have recently been attracting interest in a variety of nondestructive testing applications, ranging from thickness gauging to bond characterization. Accurate control of the transmitted ultrasonic guided wave mode is paramount when working at frequencies above the cutoff of the first high-order mode. The high number of modes available makes this range of frequency-thickness products difficult to exploit in practice.

Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy.

Molecular autopsy and family screening in a young case of sudden cardiac death reveals an unusually severe case of FHL1 related hypertrophic cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is a genetic cardiomyopathy with a prevalence of about 1:200. It is characterized by left ventricular hypertrophy, diastolic dysfunction and interstitial fibrosis; HCM might lead to sudden cardiac death (SCD) especially in the young. Due to low autopsy frequencies of sudden unexplained deaths (SUD) the true prevalence of SCD and especially of HCM among SUD remains unclear.

Transcytosis maintains CFTR apical polarity in the face of constitutive and mutation-induced basolateral missorting.

Apical polarity of cystic fibrosis transmembrane conductance regulator (CFTR) is essential for solute and water transport in secretory epithelia and can be impaired in human diseases. Maintenance of apical polarity in the face of CFTR non-polarized delivery and inefficient apical retention of mutant CFTRs lacking PDZ-domain protein (NHERF1, also known as SLC9A3R1) interaction, remains enigmatic. Here, we show that basolateral CFTR delivery originates from biosynthetic (∼35%) and endocytic (∼65%) recycling missorting.

Structure-guided combination therapy to potently improve the function of mutant CFTRs.

Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces.

ΔF508-CFTR Modulator Screen Based on Cell Surface Targeting of a Chimeric Nucleotide Binding Domain 1 Reporter.

The most common cystic fibrosis-causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) is deletion of phenylalanine at residue 508 (∆F508). The ∆F508 mutation impairs folding of nucleotide binding domain 1 (NBD1) and interfacial interactions of NBD1 and the membrane spanning domains. Here, we report a domain-targeted screen to identify ∆F508-CFTR modulators that act on NBD1.

Chaperone-Independent Peripheral Quality Control of CFTR by RFFL E3 Ligase.

The peripheral protein quality control (QC) system removes non-native membrane proteins, including ΔF508-CFTR, the most common CFTR mutant in cystic fibrosis (CF), from the plasma membrane (PM) for lysosomal degradation by ubiquitination. It remains unclear how unfolded membrane proteins are recognized and targeted for ubiquitination and how they are removed from the apical PM. Using comprehensive siRNA screens, we identified RFFL, an E3 ubiquitin (Ub) ligase that directly and selectively recognizes unfolded ΔF508-CFTR through its disordered regions.

Extracellular oxidation in cystic fibrosis airway epithelium causes enhanced EGFR/ADAM17 activity.

The EGF receptor (EGFR)/a disintegrin and metalloproteinase 17 (ADAM17) signaling pathway mediates the shedding of growth factors and secretion of cytokines and is involved in chronic inflammation and tissue remodeling. Since these are hallmarks of cystic fibrosis (CF) lung disease, we hypothesized that CF transmembrane conductance regulator (CFTR) deficiency enhances EGFR/ADAM17 activity in human bronchial epithelial cells. In CF bronchial epithelial CFBE41o cells lacking functional CFTR (iCFTR) cultured at air-liquid interface (ALI) we found enhanced ADAM17-mediated shedding of the EGFR ligand amphiregulin (AREG) compared with genetically identical cells with induced CFTR expression (iCFTR).

Mutation-specific downregulation of CFTR2 variants by gating potentiators.

Approximately 50% of cystic fibrosis (CF) patients are heterozygous with a rare mutation on at least one allele. Several mutants exhibit functional defects, correctable by gating potentiators. Long-term exposure (≥24 h) to the only available potentiator drug, VX-770, leads to the biochemical and functional downregulation of F508del-CFTR both in immortalized and primary human airway cells, and possibly other CF mutants, attenuating its beneficial effect.

Chaperones rescue the energetic landscape of mutant CFTR at single molecule and in cell.

Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (∆F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 °C and concomitant functional inactivation of ∆F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at single-molecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart.

Epithelial Anion Transport as Modulator of Chemokine Signaling.

The pivotal role of epithelial cells is to secrete and absorb ions and water in order to allow the formation of a luminal fluid compartment that is fundamental for the epithelial function as a barrier against environmental factors. Importantly, epithelial cells also take part in the innate immune system. As a first line of defense they detect pathogens and react by secreting and responding to chemokines and cytokines, thus aggravating immune responses or resolving inflammatory states.