High-Content Screening Identifies Cyclosporin A as a Novel ABCA3-Specific Molecular Corrector.

ABCA3 (ATP-binding cassette subfamily A member 3) is a lipid transporter expressed in alveolar type II cells and localized in the limiting membrane of lamellar bodies. It is crucial for pulmonary surfactant storage and homeostasis. Mutations in the ABCA3 gene are the most common genetic cause of respiratory distress syndrome in mature newborns and of interstitial lung disease in children. Apart from lung transplant, there is no cure available. To address the lack of causal therapeutic options for ABCA3 deficiency, a rapid and reliable approach is needed to investigate variant-specific molecular mechanisms and to identify pharmacologic modulators for monotherapies or combination therapies. To this end, we developed a phenotypic cell-based assay to autonomously identify ABCA3 wild-type-like or mutant-like cells by using machine learning algorithms aimed at identifying morphologic differences in wild-type and mutant cells. The assay was subsequently used to identify new drug candidates for ABCA3-specific molecular correction by using high-content screening of 1,280 Food and Drug Administration-approved small molecules. Cyclosporin A was identified as a potent corrector, specific for some but not all ABCA3 variants. Results were validated by using our previously established functional small-format assays. Hence, cyclosporin A may be selected for orphan drug evaluation in controlled repurposing trials in patients.

Functional rescue of misfolding ABCA3 mutations by small molecular correctors.

Adenosine triphosphate (ATP)-binding cassette subfamily A member 3 (ABCA3), a phospholipid transporter in lung lamellar bodies (LBs), is essential for the assembly of pulmonary surfactant and LB biogenesis. Mutations in the ABCA3 gene are an important genetic cause for respiratory distress syndrome in neonates and interstitial lung disease in children and adults, for which there is currently no cure. The aim of this study was to prove that disease causing misfolding ABCA3 mutations can be corrected in vitro and to investigate available options for correction. We stably expressed hemagglutinin (HA)-tagged wild-type ABCA3 or variants p.Q215K, p.M760R, p.A1046E, p.K1388N or p.G1421R in A549 cells and assessed correction by quantitation of ABCA3 processing products, their intracellular localization, resembling LB morphological integrity and analysis of functional transport activity. We showed that all mutant proteins except for M760R ABCA3 were rescued by the bithiazole correctors C13 and C17. These variants were also corrected by the chemical chaperone trimethylamine N-oxide and by low temperature. The identification of lead molecules C13 and C17 is an important step toward pharmacotherapy of ABCA3 misfolding-induced lung disease.

Potentiation of ABCA3 lipid transport function by ivacaftor and genistein.

ABCA3 is a phospholipid transporter implicated in pulmonary surfactant homoeostasis and localized at the limiting membrane of lamellar bodies, the storage compartment for surfactant in alveolar type II cells. Mutations in ABCA3 display a common genetic cause for diseases caused by surfactant deficiency like respiratory distress in neonates and interstitial lung disease in children and adults, for which currently no causal therapy exists. In this study, we investigated the effects of ivacaftor and genistein, two potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR), on ABCA3-specific lipid transport function. Wild-type (WT) and functional ABCA3 mutations N568D, F629L, G667R, T1114M and L1580P were stably expressed in A549 cells. Three-dimensional modelling predicted functional impairment for all five mutants that was confirmed by in vitro experiments (all <14% of WT functional activity). Treatment with potentiators rescued the mutants N568D (up to 114% of WT), F629L (up to 47% of WT), and G667R (up to 60% of WT), the latter variation needing higher concentrations of genistein, showing reduced affinity of the potentiator to the mutant protein. Our results present a first proof that functional ABCA3 mutations are rescued by CFTR potentiators, making them a potential therapeutical option for patients suffering from surfactant deficiency due to ABCA3 mutations.

Quantification of volume and lipid filling of intracellular vesicles carrying the ABCA3 transporter.

The ABCA3 lipid transporter is located in the limiting membrane of lamellar bodies (LBs) in type-II-pneumocytes. Mutations within the ABCA3 gene may functionally impair the transporter, causing lung diseases in newborns, children and adults. Assays to quantify volume and lipid filling of the LBs on the level of the vesicular structures and thereby assess the function of ABCA3 are still lacking. In the present study human influenza haemagglutinin- (HA-) tagged wild type and mutant ABCA3 proteins were stably expressed in lung A549 cells. Fluorescently-labelled TopFluor phosphatidylcholine (TopF-PC) incorporated in surfactant-like liposomes was delivered to the cells and visualized by confocal microscopy. Subsequently, a comprehensive image analysis method was applied to quantify volume and fluorescence intensity of TopF-PC in ABCA3-HA-positive vesicles. TopF-PC accumulated within the vesicles in a time and concentration-dependent manner, whereas the volume remained unchanged, suggesting active transport into preformed ABCA3 containing vesicles. Furthermore, this finding was supported by a decrease of the fluorescence intensity within the vesicles when either the ATPase of the transporter was inhibited by vanadate, or when a disease-causing mutation (K1388N) close to the ABCA3-nucleotide binding domain 2 was introduced. Conversely, a mutation (E292V) located in the first cytoplasmic loop of ABCA3 did not significantly affect lipid transport, but rather resulted in smaller vesicles. In addition to these findings, the assay used in this work for analysing the PC-lipid transport into ABCA3 positive vesicles will be useful to screen for compounds susceptible to restore function in mutated ABCA3 protein.

ABCA3 missense mutations causing surfactant dysfunction disorders have distinct cellular phenotypes.

Mutations in the ATP-binding cassette subfamily A member 3 (ABCA3) gene are the most common monogenetic cause of surfactant dysfunction disorders in newborns and interstitial lung diseases in children and young adults. Although the effect of mutations resulting in truncated or incomplete proteins can be predicted, the consequences of missense variants cannot be as easily. Our aim was to investigate the intracellular handling and disturbance of the cellular surfactant system in a stable cell model with several different clinically relevant ABCA3 missense mutations. We found that the investigated missense mutations within the ABCA3 gene affect surfactant homeostasis in different ways: first by disrupting intracellular ABCA3 protein localization (c.643C > A, p.Q215K; c.2279T > G, p.M760R), second by impairing the lipid transport of ABCA3 protein (c.875A > T, p.E292V; c.4164G > C, p.K1388N), and third by yet undetermined mechanisms predisposing for the development of interstitial lung diseases despite correct localization and normal lipid transport of the variant ABCA3 protein (c.622C > T, p.R208W; c.863G > A, p.R288K; c.2891G > A, p.G964D). In conclusion, we classified cellular consequences of missense ABCA3 sequence variations leading to pulmonary disease of variable severity. The corresponding molecular pathomechanisms of such ABCA3 variants may specifically be addressed by targeted treatments.

Metabolic labelling of choline phospholipids probes ABCA3 transport in lamellar bodies.

In the metabolism of pulmonary surfactant, the ATP-binding cassette sub-family A member 3 (ABCA3) is a crucial protein in the formation of the storage compartment for surfactant, the lamellar body (LB), and the transport of phospholipids in it. Mutations in ABCA3 not only disturb surfactant metabolism but also cause chronic interstitial lung diseases. Assays for ABCA3 transport function are needed to investigate pathophysiology of the mutations and treatment options for the patients. We metabolically labeled choline (Cho) head phospholipids with the Cho analogue, propargyl-Cho. The universal incorporation of propargyl-Cho was confirmed by mass spectrometry and labeled lipids were visualized in confocal microscopy by click reaction with an azide fluorophore. After pulse-labeling propargyl-Cho labeled lipids accumulated in ABCA3+ vesicles in a time and concentration dependent manner. When treated with the choline kinase inhibitor MN58b during the first 12 h, the lipids intensity inside ABCA3+ vesicles decreased, whereas intensity was unchanged when treated after 12 h. Miltefosine, a substrate of ABCA3, decreased the incorporation of labeled lipids in ABCA3+ vesicles at all time points. The lipids intensity inside the mutated (p.N568D or p.L1580P) ABCA3+ vesicles was decreased compared to wild type, while the intensity outside of vesicles showed no difference. Propargyl-Cho can metabolically pulse-label Cho phospholipids. Visualization and quantification of fluorescence intensity of the labeled lipids inside ABCA3+ vesicles at equilibrium can specifically assess the transport function of ABCA3.

Tools to explore ABCA3 mutations causing interstitial lung disease.

BACKGROUND: Interstitial lung diseases (ILD) comprise disorders of mostly unknown cause. Among the few molecularly defined entities, mutations in the gene encoding the ATP-binding cassette (ABC), subfamily A, member 3 (ABCA3) lipid transporter represent the main cause of inherited surfactant dysfunction disorders, a subgroup of ILD. Whereas many cases are reported, specific methods to functionally define such mutations are rarely presented. MATERIALS AND METHODS: In this study, we exemplarily utilized a set of molecular tools to characterize the mutation K1388N, which had been identified in a patient suffering from ILD with lethal outcome. We also aimed to correlate in vitro and ex vivo findings. RESULTS: We found that presence of the K1388N mutation did not affect protein expression, but resulted in an altered protein processing and a functional impairment of ABCA3. This was demonstrated by decreased dipalmitoyl-phosphatidylcholine (PC 32:0) content and malformed lamellar bodies in cells transfected with the K1388N variant as compared to controls. CONCLUSIONS: Here we present a set of tools useful for categorizing different ABCA3 mutations according to their impact upon ABCA3 activity. Knowledge of the molecular defects and close correlation of in vitro and ex vivo data will allow us to define groups of mutations that can be targeted by small molecule correctors for restoring impaired ABCA3 transporter in the future. Pediatr Pulmonol. 2016;51:1284-1294. © 2016 Wiley Periodicals, Inc.