The synthetic aminoglycoside ELX-02 induces readthrough of G550X-CFTR producing superfunctional protein that can be further enhanced by CFTR modulators.

Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl- conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy.NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.

Evaluation of a novel CFTR potentiator in COPD ferrets with acquired CFTR dysfunction.

RATIONALE: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure. OBJECTIVES: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis. METHODS: Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes. MEASUREMENTS AND MAIN RESULTS: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy. CONCLUSIONS: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model.

Novel Correctors and Potentiators Enhance Translational Readthrough in CFTR Nonsense Mutations.

Premature-termination codons (PTCs) in CFTR (cystic fibrosis [CF] transmembrane conductance regulator) result in nonfunctional CFTR protein and are the proximate cause of ∼11% of CF-causing alleles, for which no treatments exist. The CFTR corrector lumacaftor and the potentiator ivacaftor improve CFTR function with terminal PTC mutations and enhance the effect of readthrough agents. Novel correctors GLPG2222 (corrector 1 [C1]), GLPG3221 (corrector 2 [C2]), and potentiator GLPG1837 compare favorably with lumacaftor and ivacaftor in vitro. Here, we evaluated the effect of correctors C1a and C2a (derivatives of C1 and C2) and GLPG1837 alone or in combination with the readthrough compound G418 on CFTR function using heterologous Fischer rat thyroid (FRT) cells, the genetically engineered human bronchial epithelial (HBE) 16HBE14o- cell lines, and primary human cells with PTC mutations. In FRT lines pretreated with G418, GLPG1837 elicited dose-dependent increases in CFTR activity that exceeded those from ivacaftor in FRT-W1282X and FRT-R1162X cells. A three-mechanism strategy consisting of G418, GLPG1837, and two correctors (C1a + C2a) yielded the greatest functional improvements in FRT and 16HBE14o- PTC variants, noting that correction and potentiation without readthrough was sufficient to stimulate CFTR activity for W1282X cells. GLPG1837 + C1a + C2a restored substantial function in G542X/F508del HBE cells and restored even more function for W1282X/F508del cells, largely because of the corrector/potentiator effect, with no additional benefit from G418. In G542X/R553X or R1162X/R1162X organoids, enhanced forskolin-induced swelling was observed with G418 + GLPG1837 + C1a + C2a, although GLPG1837 + C1a + C2a alone was sufficient to improve forskolin-induced swelling in W1282X/W1282X organoids. Combination of CFTR correctors, potentiators, and readthrough compounds augments the functional repair of CFTR nonsense mutations, indicating the potential for novel correctors and potentiators to restore function to truncated W1282X CFTR.

Novel Therapy of Bicarbonate, Glutathione, and Ascorbic Acid Improves Cystic Fibrosis Mucus Transport.

Defective airway mucus clearance is a defining characteristic of cystic fibrosis lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using microoptical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in cystic fibrosis. We found that ARINA-1 significantly augmented mucociliary transport rates, both alone and with CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared with PBS vehicle control. Analysis of mucus reflectivity and particle-tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases in mucociliary transport rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o- F508del cells treated with VX-809 and primary human bronchial epithelial cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on cystic fibrosis mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in cystic fibrosis.

Discovery of Clinically Approved Agents That Promote Suppression of Cystic Fibrosis Transmembrane Conductance Regulator Nonsense Mutations.

RATIONALE: Premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Several agents are known to suppress PTCs but are poorly efficacious or toxic. OBJECTIVES: To determine whether there are clinically available agents that elicit translational readthrough and improve CFTR function sufficient to confer therapeutic benefit to patients with CF with PTCs. METHODS: Two independent screens, firefly luciferase and CFTR-mediated transepithelial chloride conductance assay, were performed on a library of 1,600 clinically approved compounds using fisher rat thyroid cells stably transfected with stop codons. Select agents were further evaluated using secondary screening assays including short circuit current analysis on primary cells from patients with CF. In addition, the effect of CFTR modulators (ivacaftor) was tested in combination with the most efficacious agents. MEASUREMENTS AND MAIN RESULTS: From the primary screen, 48 agents were selected as potentially active. Following confirmatory tests in the transepithelial chloride conductance assay and prioritizing agents based on favorable pharmacologic properties, eight agents were advanced for secondary screening. Ivacaftor significantly increased short circuit current following forskolin stimulation in cells treated with pyranoradine tetraphosphate, potassium p-aminobenzoate, and escin as compared with vehicle control. Escin, an herbal agent, consistently induced readthrough activity as demonstrated by enhanced CFTR expression and function in vitro. CONCLUSIONS: Clinically approved drugs identified as potential readthrough agents, in combination with ivacaftor, may induce nonsense suppression to restore therapeutic levels of CFTR function. One or more agents may be suitable to advance to human testing.

Globular adiponectin enhances invasion in human breast cancer cells.

Every year, a large number of women succumb to metastatic breast cancer due to a lack of curative approaches for this disease. Adiponectin (AdipoQ) is the most abundant of the adipocyte-secreted adipokines. In recent years, there has been an interest in the use of AdipoQ and AdipoQ receptor agonists as therapeutic agents for the treatment of breast cancer. However, while multiple epidemiological studies have previously indicated that low levels of circulating plasma AdipoQ portend poor prognosis in patients with breast cancer, recent studies have reported that elevated expression levels of AdipoQ in breast tissue are correlated with advanced stages of the disease. Thus, the aim of the present study was to clarify the mechanism by which AdipoQ in breast tissue acts directly on tumor cells to regulate the early steps of breast cancer metastasis. In the present study, the effects of different AdipoQ isoforms on the metastatic potential of human breast cancer cells were investigated. The results revealed that globular adiponectin (gAd) promoted invasive cell morphology and significantly increased the migration and invasion abilities of breast cancer cells, whereas full-length adiponectin (fAd) had no effect on these cells. Additionally, gAd, but not fAd, increased the expression levels of microtubule-associated protein 1 light chain 3 beta (LC3B)-II and intracellular LC3B puncta, which are indicators of autophagosome formation, thus suggesting autophagic induction by gAd. Furthermore, the inhibition of autophagic function by autophagy-related protein 7 knockdown attenuated the gAd-induced increase in invasiveness in breast cancer cells. Therefore, the results of the present study suggested that a specific AdipoQ isoform may enhance breast cancer invasion, possibly via autophagic induction. Understanding the roles of the different AdipoQ isoforms as microenvironmental regulatory molecules may aid the development of effective AdipoQ-based treatments for breast cancer.