Marisa Sousa

Revertants, Low Temperature, and Correctors Reveal the Mechanism of F508del-CFTR Rescue by VX-809 and Suggest Multiple Agents for Full Correction

Cystic fibrosis is mostly caused by the F508del mutation, which impairs CFTR protein from exiting the endoplasmic reticulum due to misfolding. VX-809 is a small molecule that rescues F508del-CFTR localization, which recently went into clinical trial but with unknown mechanism of action (MoA). Herein, we assessed if VX-809 is additive or synergistic with genetic revertants of F508del-CFTR, other correctors, and low temperature to determine its MoA. We explored and integrated those various agents in combined treatments, showing how they add to each other to identify their complementary MoA upon correction of F508del-CFTR. Our experimental and modeling data, while compatible with putative binding of VX-809 to NBD1:ICL4 interface, also indicate scope for further synergistic F508del-CFTR correction by other compounds at distinct conformational sites/cellular checkpoints, thus suggesting requirement of combined therapies to fully rescue F508del-CFTR.

An Extract from the Medicinal Plant Phyllanthus acidus and Its Isolated Compounds Induce Airway Chloride Secretion: A Potential Treatment for Cystic Fibrosis

According to previous reports, flavonoids and nutraceuticals correct defective electrolyte transport in cystic fibrosis (CF) airways. Traditional medicinal plants from China and Thailand contain phytoflavonoids and other bioactive compounds. We examined herbal extracts of the common Thai medicinal euphorbiaceous plant Phyllanthus acidus for their potential effects on epithelial transport. Functional assays by Ussing chamber, patch-clamping, double-electrode voltage-clamp and Ca2+ imaging demonstrate activation of Cl- secretion and inhibition of Na+ absorption by P. acidus. No cytotoxic effects of P. acidus could be detected. Mucosal application of P. acidus to native mouse trachea suggested transient and steady-state activation of Cl- secretion by increasing both intracellular Ca2+ and cAMP. These effects were mimicked by a mix of the isolated components adenosine, kaempferol, and hypogallic acid. Additional experiments in human airway cells and CF transmembrane conductance regulator (CFTR)-expressing BHK cells and Xenopus laevis oocytes confirm the results obtained in native tissues. Cl- secretion was also induced in tracheas of CF mice homozygous for Phe508del-CFTR and in Phe508del-CFTR homozygous human airway epithelial cells. Taken together, P. acidus corrects defective electrolyte transport in CF airways by parallel mechanisms including 1) increasing the intracellular levels of second messengers cAMP and Ca2+, thereby activating Ca2+-dependent Cl- channels and residual CFTR-Cl- conductance; 2) stimulating basolateral K+ channels; 3) redistributing cellular localization of CFTR; 4) directly activating CFTR; and 5) inhibiting ENaC through activation of CFTR. These combinatorial effects on epithelial transport may provide a novel complementary nutraceutical treatment for the CF lung disease.

Measurements of CFTR-Mediated Cl- Secretion in Human Rectal Biopsies Constitute a Robust Biomarker for Cystic Fibrosis Diagnosis and Prognosis

Background Cystic Fibrosis (CF) is caused by ∼1,900 mutations in the CF transmembrane conductance regulator (CFTR) gene encoding for a cAMP-regulated chloride (Cl−) channel expressed in several epithelia. Clinical features are dominated by respiratory symptoms, but there is variable organ involvement thus causing diagnostic dilemmas, especially for non-classic cases. Methodology/Principal Findings To further establish measurement of CFTR function as a sensitive and robust biomarker for diagnosis and prognosis of CF, we herein assessed cholinergic and cAMP-CFTR-mediated Cl− secretion in 524 freshly excised rectal biopsies from 118 individuals, including patients with confirmed CF clinical diagnosis (n = 51), individuals with clinical CF suspicion (n = 49) and age-matched non-CF controls (n = 18). Conclusive measurements were obtained for 96% of cases. Patients with “Classic CF”, presenting earlier onset of symptoms, pancreatic insufficiency, severe lung disease and low Shwachman-Kulczycki scores were found to lack CFTR-mediated Cl− secretion (<5%). Individuals with milder CF disease presented residual CFTR-mediated Cl− secretion (10–57%) and non-CF controls show CFTR-mediated Cl− secretion ≥30–35% and data evidenced good correlations with various clinical parameters. Finally, comparison of these values with those in “CF suspicion” individuals allowed to confirm CF in 16/49 individuals (33%) and exclude it in 28/49 (57%). Statistical discriminant analyses showed that colonic measurements of CFTR-mediated Cl− secretion are the best discriminator among Classic/Non-Classic CF and non-CF groups. Conclusions/Significance Determination of CFTR-mediated Cl− secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.

High-Content siRNA Screen Reveals Global ENaC Regulators and Potential Cystic Fibrosis Therapy Targets

Dysfunction of ENaC, the epithelial sodium channel that regulates salt and water reabsorption in epithelia, causes several human diseases, including cystic fibrosis (CF). To develop a global understanding of molecular regulators of ENaC traffic/function and to identify of candidate CF drug targets, we performed a large-scale screen combining high-content live-cell microscopy and siRNAs in human airway epithelial cells. Screening over 6,000 genes identified over 1,500 candidates, evenly divided between channel inhibitors and activators. Genes in the phosphatidylinositol pathway were enriched on the primary candidate list, and these, along with other ENaC activators, were examined further with secondary siRNA validation. Subsequent detailed investigation revealed ciliary neurotrophic factor receptor (CNTFR) as an ENaC modulator and showed that inhibition of (diacylglycerol kinase, iota) DGKι, a protein involved in PiP2 metabolism, downgrades ENaC activity, leading to normalization of both Na+ and fluid absorption in CF airways to non-CF levels in primary human lung cells from CF patients.

HGF stimulation of Rac1 signaling enhances pharmacological correction of the most prevalent cystic fibrosis mutant F508del-CFTR.

Cystic fibrosis (CF), a major life-limiting genetic disease leading to severe respiratory symptoms, is caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride (Cl(-)) channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection, and ultimately lung failure. The most frequent mutation, F508del, causes the channel to misfold, triggering its premature degradation and preventing it from reaching the cell surface. Recently, novel small-molecule correctors rescuing plasma membrane localization of F508del-CFTR underwent clinical trials but with limited success. Plausibly, this may be due to the mutant intrinsic plasma membrane (PM) instability. Herein, we show that restoration of F508del-CFTR PM localization by correctors can be dramatically improved through a novel pathway involving stimulation of signaling by the endogenous small GTPase Rac1 via hepatocyte growth factor (HGF). We first show that CFTR anchors to apical actin cytoskeleton (via Ezrin) upon activation of Rac1 signaling through PIP5K and Arp2/3. We then found that such anchoring retains pharmacologically rescued F508del-CFTR at the cell surface, boosting functional restoration by correctors up to 30% of wild-type channel levels in human airway epithelial cells. Our findings reveal that surface anchoring and retention is a major target pathway for CF pharmacotherapy, namely, to achieve maximal restoration of F508del-CFTR in patients in combination with correctors. Moreover, this approach may also translate to other disorders caused by trafficking-deficient surface proteins.

Step Length and Individual Anaerobic Threshold Assessment in Swimming

Anaerobic threshold is widely used for diagnosis of swimming aerobic endurance but the precise incremental protocols step duration for its assessment is controversial. A physiological and biomechanical comparison between intermittent incremental protocols with different step lengths and a maximal lactate steady state (MLSS) test was conducted. 17 swimmers performed 7×200, 300 and 400 m (30 s and 24 h rest between steps and protocols) in front crawl until exhaustion and an MLSS test. The blood lactate concentration values ([La-]) at individual anaerobic threshold were 2.1±0.1, 2.2±0.2 and 1.8±0.1 mmol.l - 1 in the 200, 300 and 400 m protocols (with significant differences between 300 and 400 m tests), and 2.9±1.2 mmol.l - 1 at MLSS (higher than the incremental protocols); all these values are much lower than the traditional 4 mmol.l - 1 value. The velocities at individual anaerobic threshold obtained in incremental protocols were similar (and highly related) to the MLSS, being considerably lower than the velocity at 4 mmol.l - 1. Stroke rate increased and stroke length decreased throughout the different incremental protocols. It was concluded that it is valid to use intermittent incremental protocols of 200 and 300 m lengths to assess the swimming velocity corresponding to individual anaerobic threshold, the progressive protocols tend to underestimate the [La-] at anaerobic threshold assessed by the MLSS test, and swimmers increase velocity through stroke rate increases.

Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity

ABSTRACT Previously, the pleiotropic “master kinase” casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

Control of TMEM16A by INO-4995 and other inositolphosphates

Ca2+‐dependent Cl− secretion (CaCC) in airways and other tissues is due to activation of the Cl− channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca2+‐activated Cl− channels are regulated by membrane lipid inositol phosphates, and that 1‐O‐octyl‐2‐O‐butyryl‐myo‐inositol 3,4,5,6‐tetrakisphosphate octakis(propionoxymethyl) ester (INO‐4995) augments CaCC. Here we examined whether TMEM16A is the target for INO‐4995 and if the channel is regulated by inositol phosphates.

Increased efficacy of VX-809 in different cellular systems results from an early stabilization effect of F508del-CFTR

Cystic fibrosis (CF), the most common recessive autosomal disease among Caucasians, is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, F508del, leads to CFTR impaired plasma membrane trafficking. Therapies modulating CFTR basic defect are emerging, such as VX‐809, a corrector of F508del‐CFTR traffic which just succeeded in a Phase III clinical trial. We recently showed that VX‐809 is additive to two other correctors (VRT‐325 and compound 4a). Here, we aimed to determine whether the differential rescuing by these compounds results from cell‐specific factors or rather from distinct effects at the early biogenesis and/or processing. The rescuing efficiencies of the above three correctors were first compared in different cellular models (primary respiratory cells, cystic fibrosis bronchial epithelial and baby hamster kidney [BHK] cell lines) by functional approaches: micro‐Ussing chamber and iodide efflux. Next, biochemical methods (metabolic labeling, pulse‐chase and immunoprecipitation) were used to determine their impact on CFTR biogenesis / processing. Functional analyses revealed that VX‐809 has the greatest rescuing efficacy and that the relative efficiencies of the three compounds are essentially maintained in all three cellular models tested. Nevertheless, biochemical data show that VX‐809 significantly stabilizes F508del‐CFTR immature form, an effect that is not observed for C3 nor C4. VX‐809 and C3 also significantly increase accumulation of immature CFTR. Our data suggest that VX‐809 increases the stability of F508del‐CFTR immature form at an early phase of its biogenesis, thus explaining its increased efficacy when inducing its rescue.

Influence of Prior Exercise on VO2 Kinetics Subsequent Exhaustive Rowing Performance

Prior exercise has the potential to enhance subsequent performance by accelerating the oxygen uptake (VO2) kinetics. The present study investigated the effects of two different intensities of prior exercise on pulmonary VO2 kinetics and exercise time during subsequent exhaustive rowing exercise. It was hypothesized that in prior heavy, but not prior moderate exercise condition, overall VO2 kinetics would be faster and the VO2 primary amplitude would be higher, leading to longer exercise time at VO2max. Six subjects (mean ± SD; age: 22.9±4.5 yr; height: 181.2±7.1 cm and body mass: 75.5±3.4 kg) completed square-wave transitions to 100% of VO2max from three different conditions: without prior exercise, with prior moderate and heavy exercise. VO2 was measured using a telemetric portable gas analyser (K4b2, Cosmed, Rome, Italy) and the data were modelled using either mono or double exponential fittings. The use of prior moderate exercise resulted in a faster VO2 pulmonary kinetics response (τ1 = 13.41±3.96 s), an improved performance in the time to exhaustion (238.8±50.2 s) and similar blood lactate concentrations ([La−]) values (11.8±1.7 mmol.L−1) compared to the condition without prior exercise (16.0±5.56 s, 215.3±60.1 s and 10.7±1.2 mmol.L−1, for τ1, time sustained at VO2max and [La−], respectively). Performance of prior heavy exercise, although useful in accelerating the VO2 pulmonary kinetics response during a subsequent time to exhaustion exercise (τ1 = 9.18±1.60 s), resulted in a shorter time sustained at VO2max (155.5±46.0 s), while [La−] was similar (13.5±1.7 mmol.L−1) compared to the other two conditions. Although both prior moderate and heavy exercise resulted in a faster pulmonary VO2 kinetics response, only prior moderate exercise lead to improved rowing performance.