CFTR Correctors Research Articles

Synergy in Cystic Fibrosis Therapies: Targeting SLC26A9

SLC26A9, a constitutively active Cl− transporter, has gained interest over the past years as a relevant disease modifier in several respiratory disorders including Cystic Fibrosis (CF), asthma, and non-CF bronchiectasis. SLC26A9 contributes to epithelial Cl− secretion, thus preventing mucus obstruction under inflammatory conditions. Additionally, SLC26A9 was identified as a CF gene modifier, and its polymorphisms were shown to correlate with the response to drugs modulating CFTR, the defective protein in CF. Here, we aimed to investigate the relationship between SLC26A9 and CFTR, and its role in CF pathogenesis. Our data show that SLC26A9 expression contributes to enhanced CFTR expression and function. While knocking-down SLC26A9 in human bronchial cells leads to lower wt- and F508del-CFTR expression, function, and response to CFTR correctors, the opposite occurs upon its overexpression, highlighting SLC26A9 relevance for CF. Accordingly, F508del-CFTR rescue by the most efficient correctors available is further enhanced by increasing SLC26A9 expression. Interestingly, SLC26A9 overexpression does not increase the PM expression of non-F508del CFTR traffic mutants, namely those unresponsive to corrector drugs. Altogether, our data indicate that SLC26A9 stabilizes CFTR at the ER level and that the efficacy of CFTR modulator drugs may be further enhanced by increasing its expression.

678: Design and validation of luminescent HTS tool for discovery and optimization of novel combination of CFTR correctors and modifiers

678: Design and validation of luminescent HTS tool for discovery and optimization of novel combination of CFTR correctors and modifiers

Evaluation of Fused Pyrrolothiazole Systems as Correctors of Mutant CFTR Protein.

Cystic fibrosis (CF) is a genetic disease caused by mutations that impair the function of the CFTR chloride channel. The most frequent mutation, F508del, causes misfolding and premature degradation of CFTR protein. This defect can be overcome with pharmacological agents named “correctors”. So far, at least three different classes of correctors have been identified based on the additive/synergistic effects that are obtained when compounds of different classes are combined together. The development of class 2 correctors has lagged behind that of compounds belonging to the other classes. It was shown that the efficacy of the prototypical class 2 corrector, the bithiazole corr-4a, could be improved by generating conformationally-locked bithiazoles. In the present study, we investigated the effect of tricyclic pyrrolothiazoles as analogues of constrained bithiazoles. Thirty-five compounds were tested using the functional assay based on the halide-sensitive yellow fluorescent protein (HS-YFP) that measured CFTR activity. One compound, having a six atom carbocyle central ring in the tricyclic pyrrolothiazole system and bearing a pivalamide group at the thiazole moiety and a 5-chloro-2-methoxyphenyl carboxamide at the pyrrole ring, significantly increased F508del-CFTR activity. This compound could lead to the synthesis of a novel class of CFTR correctors.

P041 Effect of the triple combination of CFTR correctors and potentiator on lung function, BMI and the Brasfield score in adolescents with cystic fibrosis

P041 Effect of the triple combination of CFTR correctors and potentiator on lung function, BMI and the Brasfield score in adolescents with cystic fibrosis

Functional Profiling of CFTR-Directed Therapeutics Using Pediatric Patient-Derived Nasal Epithelial Cell Models.

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiXTM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (ΔICFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20–80 μA/cm2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.

CFTR‐mediated transepithelial bicarbonate conductance is affected differently than chloride conductance by CFTR correctors and potentiators

The advent of targeted CFTR therapeutics allows us to assess differences in epithelial Cl− and HCO3− conductances that might be attributed to unique combinations of CFTR variants (mutations), CFTR modulators (corrector(s) and potentiators) as well as secretory agonists. To address these questions, Fisher rat thyroid cells expressing wt or mutant CFTR were cultured on permeable supports for the measurement of transepithelial conductance (Gt) in defined ionic conditions. Initial experiments demonstrated stepwise reduction in the forskolin (Forsk)+ivacaftor (VX770)‐stimulated change in Gt as bath NaCl was partially replaced isosmotically with mannitol. Subsequent experiments were conducted in buffers containing 100 mEq/l Cl− with the addition of either 25 mM alternative anions (Na+ salts) or 50 mM mannitol. Outcomes showed forskolin‐induced Gt changes consistent with reported CFTR permselectivity: Cl− > NO3− ≥ Br− > isethionate ≥ gluconate > mannitol. All subsequent experiments were conducted to assess Gt with 100 mEq/l Cl− plus either 25 mEq/l HCO3−, 50 mM mannitol or 25 mEq/l Cl−, in a 5% CO2 atmosphere. Epithelia expressing wtCFTR respond to forskolin with a substantial increase in Gt. The response in HCO3− was ~75% of that observed with Cl−. Exposure to VX770 increased conductance, but a disproportionately lesser change was observed in HCO3− (vs. Cl−), providing an initial suggestion that VX770 promotes differential ion selectivity.Exposure of cells expressing F508del CFTR to correctors, either C17 or C18, allowed for forskolin‐induced stimulation of Gt with a modest increment of HCO3− conductance. In these conditions, VX770 induced additional Cl− conductance, but no enhancement of HCO3− conductance could be detected. Alternatively, cells exposed to the corrector combination (C17+C18) showed a substantial forskolin‐stimulated increase in anion conductance and a modest increase in CFTR‐associated HCO3− conductance. Thus, the combination of C17 and C18 gives a different outcome regarding F508del‐CFTR HCO3− conductance than either corrector alone.Cells expressing G551D CFTR exhibited little response to forskolin, but substantial responses to VX770. Responses to VX770 in HCO3− approached the magnitude of the responses observed in Cl−.Taken together, the results suggest that CFTR correctors and potentiators can enhance CFTR‐mediated anion conductance, but that significant differences in the conductances of HCO3− relative to Cl− are observed between CFTR variants and that responses can be influenced by the correctors employed. Ultimately, these results suggest that CFTR‐associated pH regulation will be impacted by both the patient’s mutation and the CFTR modulator treatment. Furthermore, additional studies are required to evaluate the CFTR modulators for their effects on Cl− and HCO3− conductances on the many CF causing CFTR variants.Support or Funding InformationCFF BRIDGE18XX0; K‐State CVM SMILE

Gene and Base Editing as a Therapeutic Option for Cystic Fibrosis-Learning from Other Diseases.

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the CFTR gene. There are at least 346 disease-causing variants in the CFTR gene, but effective small-molecule therapies exist for only ~10% of them. One option to treat all mutations is CFTR cDNA-based therapy, but clinical trials to date have only been able to stabilise rather than improve lung function disease in patients. While cDNA-based therapy is already a clinical reality for a number of diseases, some animal studies have clearly established that precision genome editing can be significantly more effective than cDNA addition. These observations have led to a number of gene-editing clinical trials for a small number of such genetic disorders. To date, gene-editing strategies to correct CFTR mutations have been conducted exclusively in cell models, with no in vivo gene-editing studies yet described. Here, we highlight some of the key breakthroughs in in vivo and ex vivo gene and base editing in animal models for other diseases and discuss what might be learned from these studies in the development of editing strategies that may be applied to cystic fibrosis as a potential therapeutic approach. There are many hurdles that need to be overcome, including the in vivo delivery of editing machinery or successful engraftment of ex vivo-edited cells, as well as minimising potential off-target effects. However, a successful proof-of-concept study for gene or base editing in one or more of the available CF animal models could pave the way towards a long-term therapeutic strategy for this disease.

Clinical development of triple-combination CFTR modulators for cystic fibrosis patients with one or two F508del alleles.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator gene (CFTR) that result in diminished quantity and/or function of the CFTR anion channel. F508del-CFTR, the most common CF-causing mutation (found in ∼90% of patients), causes severe processing and trafficking defects, resulting in decreased CFTR quantity and function. CFTR modulators are medications that increase the amount of mature CFTR protein (correctors) or enhance channel function (potentiators) at the cell surface.Combinations of CFTR correctors and potentiators (i.e. lumacaftor/ivacaftor, tezacaftor/ivacaftor) have demonstrated clinical benefit in subsets of patients. However, none are approved for patients with CF heterozygous for F508del-CFTR and a minimal function mutation, i.e. a mutation that produces either no protein or protein that is unresponsive to currently approved CFTR modulators. Next-generation CFTR correctors VX-659 and VX-445, each in triple combination with tezacaftor and ivacaftor, improve CFTR processing, trafficking and function in vitro and have demonstrated clinical improvements in phase 2 studies in patients with CF with one or two F508del-CFTR alleles.Here, we present the rationale and design of four randomised phase 3 studies, and their open-label extensions, evaluating VX-659 (ECLIPSE) or VX-445 (AURORA) plus tezacaftor and ivacaftor in patients with one or two F508del-CFTR alleles.

Repairing folding-defective α-sarcoglycan mutants by CFTR correctors, a potential therapy for limb-girdle muscular dystrophy 2D.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is a rare autosomal-recessive disease, affecting striated muscle, due to mutation of SGCA, the gene coding for α-sarcoglycan. Nowadays, more than 50 different SGCA missense mutations have been reported. They are supposed to impact folding and trafficking of α-sarcoglycan because the defective polypeptide, although potentially functional, is recognized and disposed of by the quality control of the cell. The secondary reduction of α-sarcoglycan partners, β-, γ- and δ-sarcoglycan, disrupts a key membrane complex that, associated to dystrophin, contributes to assure sarcolemma stability during muscle contraction. The complex deficiency is responsible for muscle wasting and the development of a severe form of dystrophy. Here, we show that the application of small molecules developed to rescue ΔF508-CFTR trafficking, and known as CFTR correctors, also improved the maturation of several α-sarcoglycan mutants that were consequently rescued at the plasma membrane. Remarkably, in myotubes from a patient with LGMD2D, treatment with CFTR correctors induced the proper re-localization of the whole sarcoglycan complex, with a consequent reduction of sarcolemma fragility. Although the mechanism of action of CFTR correctors on defective α-sarcoglycan needs further investigation, this is the first report showing a quantitative and functional recovery of the sarcoglycan-complex in human pathologic samples, upon small molecule treatment. It represents the proof of principle of a pharmacological strategy that acts on the sarcoglycan maturation process and we believe it has a great potential to develop as a cure for most of the patients with LGMD2D.

The plant cytokine kinetin and its analogues as potential therapeutic agents to correct CFTR splicing defects

The plant cytokine kinetin and its analogues as potential therapeutic agents to correct CFTR splicing defects

Human pluripotent stem cell-derived exocrine/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling

Exocrine/ductal pancreatic differentiation from human pluripotent stem cells is a poorly understood process albeit various diseases arise from this compartment. We designed a straightforward approach to direct human pluripotent stem cells (PSC) toward pancreatic organoids resembling exocrine and ductal progeny. Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultra-structural and functional hallmarks of human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without any evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model for pancreatic facets of cystic fibrosis (CF) but also other inherited pancreatic disorders. We provide for the first time evidence that pancreatic commitment occurs generally unhindered in CF. Importantly, CFTR-activation in mutated pancreatic organoids mirrors the CF-phenotype in a series of functional assays. We also conducted a scalable proof-of concept screen in CF-pancreatic organoids using a set of CFTR correctors and activators. Finally, we did orthotopic transplantation of CF-organoids to generate diseased human pancreata in mice and established a mRNA-mediated gene therapy approach in CF-organoids. Similar assays were applied to another inherited pancreatic disorder. Thus, our platform provides novel opportunities to model pancreatic disease and development but also to screen for disease rescuing agents.

THU-452 - Defective Plasma Membrane Targeting of P.I661T-ATP8B1, Associated with Familial Intrahepatic Cholestasis, Can be Rescued in Vitro by CFTR Correctors

THU-452 - Defective Plasma Membrane Targeting of P.I661T-ATP8B1, Associated with Familial Intrahepatic Cholestasis, Can be Rescued in Vitro by CFTR Correctors

A synonymous codon change alters the drug sensitivity of ΔF508 cystic fibrosis transmembrane conductance regulator.

Synonymous mutations, such as I507-ATC→ATT, in deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR), the most frequent disease-associated mutant of CFTR, may affect protein biogenesis, structure, and function and contribute to an altered disease phenotype. Small-molecule drugs are being developed to correct ΔF508 CFTR. To understand correction mechanisms and the consequences of synonymous mutations, we analyzed the effect of mechanistically distinct correctors, corrector 4a (C4) and lumacaftor (VX-809), on I507-ATT and I507-ATC ΔF508 CFTR biogenesis and function. C4 stabilized I507-ATT ΔF508 CFTR band B, but without considerable biochemical and functional correction. VX-809 biochemically corrected ∼10% of both of the variants, leading to stable, forskolin+3-isobutyl-1-methylxanthine (IBMX)-activated whole-cell currents in the presence of the corrector. Omitting VX-809 during whole-cell recordings led to a spontaneous decline of the currents, suggesting posttranslational stabilization by VX-809. Treatment of cells with the C4+VX-809 combination resulted in enhanced rescue and 2-fold higher forskolin+IBMX-activated currents of both I507-ATT and I507-ATC ΔF508 CFTR, compared with VX-809 treatment alone. The lack of an effect of C4 on I507-ATC ΔF508 CFTR, but its additive effect in combination with VX-809, implies that C4 acted on VX-809-modified I507-ATC ΔF508 CFTR. Our results suggest that binding of C4 and VX-809 to ΔF508 CFTR is conformation specific and provide evidence that synonymous mutations can alter the drug sensitivity of proteins.

In Vivo Screening for Cystic Fibrosis Drugs: Transfer of Clinical Read‐Out Parameters in CF Patients to the delF508 CFTR CF Mice

Background and Objectives Cystic fibrosis (CF) is a life threating disease with no approved, disease modifying therapy for the most common delF508 CFTR mutation. We aim to identify new drugs able to correct delF508 CFTR. Therefore, we established in vivo read outs in CF mice, which reflect the basic characteristics as decreased sweat secretion, increased sweat chloride concentration and decreased nasal potential difference (NPD) of CF patients. Study design & Measurements: Use of CFTR knockout mice (CFTRtm1Unc), two delF508 CFTR mice strains (CFTRtm1Kth and CFTRtm1Eur) and wild type (WT) mice. Establishment of the measurements for: A) the isoprenaline-stimulated salivary secretion in the presence of atropine B) the pilocarpine-stimulated salivary chloride concentration and C) the forskolin-stimulated nasal potential difference (NPD) in the presence of amiloride and zero-chloride. Results & Conclusion We found a) a decrease in salivary secretion, b) a decrease in salivary chloride concentration and c) a decrease in nasal potential difference of delF508 CF mice. These changes correlate with a) a decrease in sweat production, b) a decrease in sweat chloride concentration and c) a decrease in nasal potential difference in delF508 CF patients. Therefore, the good correlation of our preclinical read outs in CF mice to the validated clinical read-out parameters, could help to identify new and effective causal treatment options for delF508 CF patients.

Multi-physiopathological consequences of the c.1392G>T CFTR mutation revealed by clinical and cellular investigations.

This study combines a clinical approach and multiple level cellular analyses to determine the physiopathological consequences of the c.1392G>T (p.Lys464Asn) CFTR exon 10 mutation, detected in a CF patient with a frameshift deletion in trans and a TG(11)T(5) in cis. Minigene experiment, with different TG(m)T(n) alleles, and nasal cell mRNA extracts were used to study the impact of c.1392G>T on splicing in both in cellulo and in vivo studies. The processing and localization of p.Lys464Asn protein were evaluated, in cellulo, by western blotting analyses and confocal microscopy. Clinical and channel exploration tests were performed on the patient to determine the exact CF phenotype profile and the CFTR chloride transport activity. c.1392G>T affects exon 10 splicing by inducing its complete deletion and encoding a frameshift transcript. The polymorphism TG(11)T(5) aggravates the effects of this mutation on aberrant splicing. Analysis of mRNA obtained from parental airway epithelial cells confirmed these in cellulo results. At the protein level the p.Lys464Asn protein showed neither maturated form nor membrane localization. Furthermore, the in vivo channel tests confirmed the absence of CFTR activity. Thus, the c.1392G>T mutation alone or in association with the TG repeats and the poly T tract revealed obvious impacts on splicing and CFTR protein processing and functionality. The c.[T(5); 1392G>T] complex allele contributes to the CF phenotype by affecting splicing and inducing a severe misprocessing defect. These results demonstrate that the classical CFTR mutations classification is not sufficient: in vivo and in cellulo studies of a possible complex allele in a patient are required to provide correct CFTR mutation classification, adequate medical counseling, and adapted therapeutic strategies.

Marked repression of CFTR mRNA in the transgenic Cftrtm1kth mouse model

Primary nasal septal epithelia cultures allow mechanistic comparisons that correspond well with the in vivo nasal potential difference assay, a mainstay of cystic fibrosis transmembrane conductance regulator detection in vivo, and have been viewed as suitable models for testing the activity of CFTR correctors and potentiators in “personalized medicine” type approaches to cystic fibrosis (CF) therapeutics [1–3]. Primary nasal septal epithelia from mice (MNSE) have substantially greater CFTR activity in Ussing chamber analysis than comparably processed tracheal monolayers, and allow direct analysis of otherwise genetically identical (congenic) animals without the confounding effects attributable to mixed genetic backgrounds [4]. There are three widely utilized F508del transgenic mouse models—Cftrtm1Kth (Utah), Cftrtm2Cam (Rotterdam), and Cftrtm1Eur (Cambridge). The Cftrtm1Kth mutated allele contains a 3-bp (CTT) deletion between bases 1656 and 1660, which results in a loss of a phenylalanine residue in exon 10 at a position which corresponds to human position 508 [5]. This exon 10 replacement strategy left a selective marker gene inserted in one of the introns. This was a similar strategy to the creation of the Cftrtm2Cam mouse model [6]. This marker gene is likely to have an effect on transcription activity or mRNA levels of the mutant allele, but does not affect the protein product. The other widely utilized F508del mouse model is Cftrtm1Eur, which was made with a “hit and run” procedure that resulted in a mutant exon from which transcriptional activity of the mutant allele is identical to the normal allele [7]. While the Cftrtm2Cam mouse was noted to have 1/3 of the transcriptional activity in all tissues tested, prior studies concerning Cftrtm1Kth transgenic mice have indicated tissue specific differences in CFTR expression—specifically lower levels of F508del CFTR mRNA in the upper intestine compared to wild type animals. Normal levels of CFTR mRNA were reported previously in F508del murine lung, testes, and submaxillary glands in the same strain [5]. Given the high interest in evaluating misprocessing correctors of F508del CFTR in a relevant in vivo and in vitro model of disease, proper F508del CFTR expression is critical to promote maturation of a similar pool of misprocessed protein. After the University of Alabama at Birmingham Institutional and Animal Care and Use Committee approval, MNSE tissues from wild type (C57BL/6), heterozygous F508del, and homozygous F508del/F508del transgenic (Cftrtm1Kth) were cultured on permeable supports for 14 days to confluence and full differentiation. This F508del mouse strain was obtained from Jackson Labs (stock # 002515) and backcrossed to the C57BL/6 line 6 times. The mice are greater than 98% on the BL/6 background. Quantitative RT-PCR analysis (reported as relative mRNA ± S.D.) showed dramatic differences in CFTR mRNA among homozygous, heterozygous, and wild type both in MNSE cultures and tissues (Fig. 1A). Whether these differences are mediated by higher expressivity in a pure epithelial cell population (compared to mixed cells from native tissues), decreased CFTR transcription, or message instability will require further investigation. In addition, there was minimal F508del CFTR-mediated anion transport following 24 hour low-temperature correction (27 °C) in Ussing chambers (Fig. 1B). Fig. 1 Relative CFTR mRNA expression in wild type, F508del heterozygous, and homozygous murine nasal tissues and cell cultures (A). CFTR mRNA expression in F508del homozygous murine nasal tissues (n = 6 per condition) and cultures (n = 9 per condition) were ... Our findings suggest significant limitations of nasal airway cultures from Cftrtm1Kth mice, and previous studies of F508del CFTR from the tm1 Kth background should be re-evaluated in this light [8]. For example, potentiator activation profiles observed in F508del CF mice may underestimate potency as a result of diminished levels of mRNA in this murine genotype. While murine CFTR is insensitive to many agents that enhance the activity of human CFTR (e.g. ivacaftor) [9], agents (e.g. flavonoids) known to increase channel opening in both murine and human CFTR should be appraised in this light. Furthermore, this mouse model should not be utilized for evaluating F508del misprocessing correctors with upper airway experiments in vivo (e.g. NPD measurements) or in vitro (primary nasal cell culture electrophysiology) endpoints. The findings also provide a cautionary note indicating the potential for highly discordant levels of CFTR mRNA from certain CFTR genotypes should nasal cells be developed as a universal screen meant to “personalize” CFTR modulation therapy in patients with rare genetic defects.

Carrier screening for cystic fibrosis in the new era of medications that restore CFTR function

2 and has been established in the USA, Australia, and parts of Europe. 3 Carrier screening has been shown to reduce the pro- portion of livebirths with cystic fi brosis by 50% in two US states, 50% in Edinburgh, and 75% in northeastern Italy. 4-7 In these series, most of the remaining cystic fi brosis births have resulted from couples choosing to continue aff ected pregnancies to term, rather than from aff ected fetuses missed by screening. The widespread introduction of carrier screening for cystic fi brosis has been limited by several issues for health-care providers and policy makers, including variable phenotype of patients with the same genotype, the numerous muta- tions associated with cystic fi brosis, low public awareness of the disease, infrastructure for preconception and pre- natal care, genetic counselling resources, and health economic considerations. 3,8 Resolution of these issues has been slow. Furthermore, a new issue has emerged— namely, the development of CFTR restorative therapy that challenges the idea that cystic fi brosis is not curable. We discuss the implications of CFTR restorative therapy on carrier screening for cystic fi brosis. After decades of solely supportive therapies, several therapeutic advances have been made targeting the basic CFTR defect. 9,10 These developments include a CFTR suppressor that promotes ribosomal read-through for patients with nonsense (stop-codon) class I mutations (atarulen, PTC124; PTC Therapeutics, South Plainfi eld, NJ, USA), a CFTR potentiator that promotes chloride- channel gating (ivacaftor; Vertex Pharmaceuticals, Cambridge, MA, USA) for patients with class III-V mutations, and two CFTR correctors that promote traffi cking through the cytosol to the epithelial apical membrane for patients with the class II mutation Phe508del (lumacaftor and VX-661; Vertex Pharma- ceuticals). 9

Actualités dans la mucoviscidose

The improvement over the last two decades in the treatment of cystic fibrosis led to an increase in life expectancy approaching 40 years at birth. Logically, the population of adult patients has been increasing and is currently 50% of patients followed in France. These therapeutic advances have justified the establishment in 2003 of a generalized neonatal screening for cystic fibrosis. The latest data of this screening show an incidence of CF of 1/5359 live births, far below the incidence of 1/2500 which was widely accepted twenty years ago. The performance of this screening is currently based on the dosage of trypsin immuno reactive, followed in case of exceeding the threshold of a search of the 30 most common mutations, can detect around 96% of 150 to 200 CF cases every year. Therefore, the possibility of a false negative of the screening cannot be excluded and evocative symptoms of cystic fibrosis, even for children born after 2003, will lead to prescribe a sweat test. While treatments available so far goal consequences of cystic fibrosis, a new therapeutic class to correct the functional defect of the mutated protein, called CFTR modulators, is emerging. Ivacaftor, leader of this new class, belonging to the category of "CFTR potentiator" got its access on the market in September 2012 for patients carrying the G551D mutation. New other molecules, named "CFTR correctors" which can have synergistic effect with ivacaftor and concern patients carrying the most common mutation--DF 508--are under development.

Abstract 897: Role of intracellular loops 1 and 3 in folding and cell surface expression of human P-glycoprotein (ABCB1).

Abstract P-glycoprotein (ABCB1) is an ATP-Binding Cassette (ABC) transporter that effluxes a variety of structurally diverse compounds including anticancer drugs out of the cell. Known crystal structures of ABC transporters show that the transmembrane domain (TMD) is connected to the nucleotide-binding domain (NBD) through a ball and socket joint. Also, the intracellular loops (ICLs) 1 and 3 are shown to be in close proximity to the NBDs, creating an extensive interaction surface between the TMDs and NBDs. Computational models of human P-gp in the apo and nucleotide-bound conformation have been developed based on structures of mouse mdr1a, C. elegans P-gp, Sav1866 and MsbA transporters. In these models, the adenine group of ATP forms hydrogen bonds with conserved D164 and D805 on ICLs 1 and 3, respectively, which are located at the interface between the NBDs and TMDs. We investigated the role of D164 and D805 by substituting these residues with cysteine in a cys-less background. The D164C/D805C mutant P-gp was expressed in High-Five insect cells as well as in HeLa cells using the BacMam baculovirus expression system. In the insect cells grown at 27°C, the expression of the mutant P-gp was comparable to that of cysless-WT protein and exhibited the same ATP-binding affinity but a lower Vmax for ATP hydrolysis. However, when the mutant protein was expressed in HeLa cells at 37°C, we found that the D164C/D805C P-gp was mislocalized and failed to transport almost all tested drug substrates. HeLa cells transduced with D164C/D805C mutant baculovirus were grown for 16-18 hrs in the presence of cyclosporine A (CsA), a substrate that is also known to function as a chemical chaperone. This treatment resulted in expression of mutant P-gp at the cell surface to the same extent as that of cysless-WT protein; similarly, the transport was also restored to the level of cysless-WT P-gp. A partial rescue of the mutant P-gp expression and function was observed in the presence of the CFTR correctors VRT-325 and compound-4a. In addition, when BacMam baculovirus-transduced HeLa cells were cultured at 27°C for 40 hrs, the mutant protein rescued to the cell surface with transport activity to the same extent as cysless-WT P-gp. Studies of each mutant form separately revealed that the D164C mutant had intermediate cell surface expression and was rescued completely to the plasma membrane in the presence of CsA. On the other hand, the expression of D805C at the cell surface was drastically decreased and even when HeLa cells were grown in the presence of CsA, only 10-15% of mutant protein was expressed at the cell surface compared to cysless-WT protein. These findings in aggregate illustrate the role of residues in ICLs 1 and 3 in the proper folding of P-gp and specifically show that the D164 and D805 residues are not critical for ATP binding or hydrolysis as proposed earlier, but are instead important for inter-domain interactions and assembly of a functional transporter. Citation Format: Khyati Kapoor, Jaya Bhatnagar, Eduardo E. Chufan, Suresh V. Ambudkar. Role of intracellular loops 1 and 3 in folding and cell surface expression of human P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2013-897

Rescuing Mutant CFTR: A Multi-task Approach to a Better Outcome in Treating Cystic Fibrosis

Correcting multiple defects of mutant CFTR with small molecule compounds has been the goal of an increasing number of recent Cystic Fibrosis (CF) drug discovery programmes. However, the mechanism of action (MoA) by which these molecules restore mutant CFTR is still poorly understood, in particular of CFTR correctors, i.e., compounds rescuing to the cells surface the most prevalent mutant in CF patients--F508del-CFTR. However, there is increasing evidence that to fully restore the multiple defects associated with F508del-CFTR, different small molecules with distinct corrective properties may be required. Towards this goal, a better insight into MoA of correctors is needed and several constraints should be addressed. The methodological approaches to achieve this include: 1) testing the combined effect of compounds with that of other (non-pharmacological) rescuing strategies (e.g., revertants or low temperature); 2) assessing effects in multiple cellular models (non-epithelial vs epithelial, non-human vs human, immortalized vs primary cultures, polarized vs non polarized, cells vs tissues); 3) assessing compound effects on isolated CFTR domains (e.g., compound binding by surface plasmon resonance, assessing effects on domain folding and aggregation); and finally 4) assessing compounds specificity in rescuing different CFTR mutants and other mutant proteins. These topics are reviewed and discussed here so as to provide a state-of-the art review on how to combine multiple ways of rescuing mutant CFTR to the ultimate benefit of CF patients.