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BACKGROUND: The forskolin-induced swelling (FIS) assay measures CFTR function on patient-derived intestinal organoids (PDIOs) and may guide treatment selection for individuals with Cystic Fibrosis (CF). The aim of this study is to demonstrate the repeatability and reproducibility of the FIS assay following a detailed Standard Operating Procedure (SOP), thus advancing the validation of the assay for precision medicine (theranostic) applications. METHODS: Over a 2-year period, FIS responses to CFTR modulators were measured in four European labs. PDIOs from six subjects with CF carrying different CFTR genotypes were used to assess the repeatability and reproducibility across the dynamic range of the assay. RESULTS: Technical, intra-assay repeatability was high (Lin's concordance correlation coefficient (CCC) 0.95-0.98). Experimental, within-subject repeatability was also high within each lab (CCCs all >0.9). Longer-term repeatability (>1 year) showed more variability (CCCs from 0.67 to 0.95). The reproducibility between labs was also high (CCC ranging from 0.92 to 0.97). Exploratory analysis also found that between-lab percentage of agreement of dichotomized CFTR modulator outcomes for predefined FIS thresholds ranged between 78 and 100 %. CONCLUSIONS: The observed repeatability and reproducibility of the FIS assay within and across different labs is high and support the use of FIS as biomarker of CFTR function in the presence or absence of CFTR modulators.
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BACKGROUND: Patient-derived intestinal organoids (PDIOs) show great potential as in vitro drug testing platform for personalised medicine in Cystic Fibrosis and oncology. PDIOs can be generated by culturing adult stem cells obtained through rectal forceps biopsy or suction biopsy, but the safety of these procedures and the success rates of generating organoids after shipment to a centralized lab using these procedures has not been studied in this context. We here report the safety and success rates of both biopsy procedures and the subsequent generation of PDIOs in the international multicentre HIT-CF Organoid Study. METHODS: 502 biopsy procedures were conducted, on 489 adult people with Cystic Fibrosis from 33 different hospitals across 12 countries. Depending on the preference of the hospital, either rectal forceps biopsies or suction biopsies were obtained and internationally shipped to a central laboratory for organoid generation. RESULTS: No adverse events were reported for 280 forceps biopsy procedures, while 222 rectal suction biopsy procedures resulted in 2 adverse events, namely continued bleeding and a probably nonrelated gastroenteritis. The success rate of organoid generation from all biopsies was 95%, and the main reason for failure was insufficient sample viability (3.2%). CONCLUSION: Our results indicate that both rectal suction biopsy and forceps biopsy procedures are safe procedures. The high success rates of PDIO generation from the obtained tissue samples demonstrate the feasibility of the organoid technology for personalised in vitro testing in an international setting.
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Enterococcus faecium is an opportunistic pathogen able to colonize the intestines of hospitalized patients. This initial colonization is an important step in the downstream pathogenesis, which includes outgrowth of the intestinal microbiota and potential infection of the host. The impact of intestinal overgrowth on host-enterococcal interactions is not well understood. We therefore applied a RNAseq approach in order to unravel the transcriptional dynamics of E. faecium upon co-culturing with human derived colonic epithelium. Co-cultures of colonic epithelium with a hospital-associated vancomycin resistant (vanA-type) E. faecium (VRE) showed that VRE resided on top of the colonic epithelium when analyzed by microscopy. RNAseq revealed that exposure to the colonic epithelium resulted in upregulation of 238 VRE genes compared to the control condition, including genes implicated in pili expression, conjugation (plasmid_2), genes related to sugar uptake, and biofilm formation (chromosome). In total, 260 were downregulated, including the vanA operon located on plasmid_3. Pathway analysis revealed an overall switch in metabolism to amino acid scavenging and reduction. In summary, our study demonstrates that co-culturing of VRE with human colonic epithelium promotes an elaborate gene response in VRE, enhancing our insight in host-E. faecium interactions, which might facilitate the design of novel anti-infectivity strategies.
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Highly effective drugs modulating the defective protein encoded by the CFTR gene have revolutionized cystic fibrosis (CF) therapy. Preclinical drug-testing on human nasal epithelial (HNE) cell cultures and 3-dimensional human intestinal organoids (3D HIO) are used to address patient-specific variation in drug response and to optimize individual treatment for people with CF. This study is the first to report comparable CFTR functional responses to CFTR modulator treatment among patients with different classes of CFTR gene variants using the three methods of 2D HIO, 3D HIO, and HNE. Furthermore, 2D HIO showed good correlation to clinical outcome markers. A larger measurable CFTR functional range and access to the apical membrane were identified as advantages of 2D HIO over HNE and 3D HIO, respectively. Our study thus expands the utility of 2D intestinal monolayers as a preclinical drug testing tool for CF.
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Fibrosis Quística , Humanos , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Mutación , Intestinos , Organoides/metabolismoRESUMEN
Background: Cystic fibrosis (CF) is a rare hereditary disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recent therapies enable effective restoration of CFTR function of the most common F508del CFTR mutation. This shifts the unmet clinical need towards people with rare CFTR mutations such as nonsense mutations, of which G542X and W1282X are most prevalent. CFTR function measurements in patient-derived cell-based assays played a critical role in preclinical drug development for CF and may play an important role to identify new drugs for people with rare CFTR mutations. Methods: Here, we miniaturised the previously described forskolin-induced swelling (FIS) assay in intestinal organoids from a 96-well to a 384-well plate screening format. Using this novel assay, we tested CFTR increasing potential of a 1400-compound Food and Drug Administration (FDA)-approved drug library in organoids from donors with W1282X/W1282X CFTR nonsense mutations. Results: The 384-well FIS assay demonstrated uniformity and robustness based on coefficient of variation and Z'-factor calculations. In the primary screen, CFTR induction was limited overall, yet interestingly, the top five compound combinations that increased CFTR function all contained at least one statin. In the secondary screen, we indeed verified that four out of the five statins (mevastatin, lovastatin, simvastatin and fluvastatin) increased CFTR function when combined with CFTR modulators. Statin-induced CFTR rescue was concentration-dependent and W1282X-specific. Conclusions: Future studies should focus on elucidating genotype specificity and mode-of-action of statins in more detail. This study exemplifies proof of principle of large-scale compound screening in a functional assay using patient-derived organoids.
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RATIONALE: Cystic fibrosis (CF) is a monogenic life-shortening disease associated with highly variable individual disease progression which is difficult to predict. Here we assessed the association of forskolin-induced swelling (FIS) of patient-derived organoids with long-term CF disease progression in multiple organs and compared FIS with the golden standard biomarker sweat chloride concentration (SCC). METHODS: We retrieved 9-year longitudinal clinical data from the Dutch CF Registry of 173 people with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Individual CFTR function was defined by FIS, measured as the relative size increase of intestinal organoids after stimulation with 0.8â µM forskolin, quantified as area under the curve (AUC). We used linear mixed-effect models and multivariable logistic regression to estimate the association of FIS with long-term forced expiratory volume in 1â s % predicted (FEV1pp) decline and development of pancreatic insufficiency, CF-related liver disease and diabetes. Within these models, FIS was compared with SCC. RESULTS: FIS was strongly associated with longitudinal changes of lung function, with an estimated difference in annual FEV1pp decline of 0.32% (95% CI 0.11-0.54%; p=0.004) per 1000-point change in AUC. Moreover, increasing FIS levels were associated with lower odds of developing pancreatic insufficiency (adjusted OR 0.18, 95% CI 0.07-0.46; p<0.001), CF-related liver disease (adjusted OR 0.18, 95% CI 0.06-0.54; p=0.002) and diabetes (adjusted OR 0.34, 95% CI 0.12-0.97; p=0.044). These associations were absent for SCC. CONCLUSION: This study exemplifies the prognostic value of a patient-derived organoid-based biomarker within a clinical setting, which is especially important for people carrying rare CFTR mutations with unclear clinical consequences.
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Fibrosis Quística , Insuficiencia Pancreática Exocrina , Biomarcadores , Colforsina/farmacología , Fibrosis Quística/complicaciones , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Progresión de la Enfermedad , Insuficiencia Pancreática Exocrina/complicaciones , Humanos , Mutación , OrganoidesRESUMEN
RATIONALE: Given the vast number of cystic fibrosis transmembrane conductance regulator (CFTR) mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF). OBJECTIVES: To study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data. METHODS: Intestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from the literature. RESULTS: Across 28 genotypes, residual CFTR function correlated (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, such as E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit. CONCLUSIONS: Measurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.
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Fibrosis Quística , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Homocigoto , Humanos , Transporte Iónico , Mutación , Organoides/metabolismoRESUMEN
MOTIVATION: The forskolin-induced swelling (FIS) assay has become the preferential assay to predict the efficacy of approved and investigational CFTR-modulating drugs for individuals with cystic fibrosis (CF). Currently, no standardized quantification method of FIS data exists thereby hampering inter-laboratory reproducibility. RESULTS: We developed a complete open-source workflow for standardized high-content analysis of CFTR function measurements in intestinal organoids using raw microscopy images as input. The workflow includes tools for (i) file and metadata handling; (ii) image quantification and (iii) statistical analysis. Our workflow reproduced results generated by published proprietary analysis protocols and enables standardized CFTR function measurements in CF organoids. AVAILABILITY AND IMPLEMENTATION: All workflow components are open-source and freely available: the htmrenamer R package for file handling https://github.com/hmbotelho/htmrenamer; CellProfiler and ImageJ analysis scripts/pipelines https://github.com/hmbotelho/FIS_image_analysis; the Organoid Analyst application for statistical analysis https://github.com/hmbotelho/organoid_analyst; detailed usage instructions and a demonstration dataset https://github.com/hmbotelho/FIS_analysis. Distributed under GPL v3.0. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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This protocol describes the isolation, handling, culture of, and experiments with human colon stem cell organoids in the context of cystic fibrosis (CF). In human colon organoids, the function of cystic fibrosis transmembrane conductance regulator (CFTR) protein and its rescue by CFTR modulators can be quantified using the forskolin-induced swelling assay. Implementation procedures and validation experiments are described for six CF human colon organoid lines, and representative CFTR genotypes are tested for basal CFTR function and response to CFTR-modulating drugs. For complete details on the use and execution of this protocol, please refer to Dekkers et al (2016) and Berkers and van Mourik (2019).
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Bioensayo/métodos , Colforsina/farmacología , Colon , Fibrosis Quística/metabolismo , Organoides , Células Cultivadas , Colon/efectos de los fármacos , Colon/metabolismo , Edema , Humanos , Organoides/efectos de los fármacos , Organoides/metabolismo , Reproducibilidad de los ResultadosAsunto(s)
Aminofenoles/uso terapéutico , Aminopiridinas/uso terapéutico , Benzodioxoles/uso terapéutico , Fibrosis Quística/tratamiento farmacológico , Organoides/efectos de los fármacos , Quinolonas/uso terapéutico , Recto/efectos de los fármacos , Adolescente , Adulto , Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Niño , Cloruros/análisis , Colforsina , Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Combinación de Medicamentos , Edema , Femenino , Volumen Espiratorio Forzado , Homocigoto , Humanos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/fisiología , Persona de Mediana Edad , Mucosa Nasal/fisiopatología , Medicina de Precisión , Estudios Prospectivos , Quinolonas/farmacología , Sudor/química , Resultado del Tratamiento , Capacidad Vital , Adulto JovenRESUMEN
BACKGROUND: The natural food supplements curcumin and genistein, and the drug ivacaftor were found effective as CFTR potentiators in the organoids of individuals carrying a S1251N gating mutation, possibly in a synergistic fashion. Based on these in vitro findings, we evaluated the clinical efficacy of a treatment with curcumin, genistein and ivacaftor, in different combinations. METHODS: In three multi-center trials people with CF carrying the S1251N mutation were treated for 8 weeks with curcumin+genistein, ivacaftor and ivacaftor+genistein. We evaluated change in lung function, sweat chloride concentration, CFQ-r, BMI and fecal elastase to determine the clinical effect. We evaluated the pharmacokinetic properties of the compounds by evaluating the concentration in plasma collected after treatment and the effect of the same plasma on the intestinal organoids. RESULTS: A clear clinical effect of treatment with ivacaftor was observed, evidenced by a significant improvement in clinical parameters. In contrast we observed no clear clinical effect of curcumin and/or genistein, except for a small but significant reduction in sweat chloride and airway resistance. Plasma concentrations of the food supplements were low, as was the response of the organoids to this plasma. CONCLUSIONS: We observed a clear clinical effect of treatment with ivacaftor, which is in line with the high responsiveness of the intestinal organoids to this drug. No clear clinical effect was observed of the treatment with curcumin and/or genistein, the low plasma concentration of these compounds emphasizes that pharmacokinetic properties of a compound have to be considered when in vitro experiments are performed.
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Aminofenoles/farmacocinética , Agonistas de los Canales de Cloruro/farmacocinética , Curcumina/farmacocinética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Genisteína/farmacocinética , Quinolonas/farmacocinética , Adolescente , Adulto , Niño , Fibrosis Quística/genética , Femenino , Humanos , Masculino , Organoides/efectos de los fármacosRESUMEN
In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
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Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Organoides/ultraestructura , Fijación del Tejido/métodos , Animales , Mama/ultraestructura , Colon/ultraestructura , Femenino , Humanos , Inmunohistoquímica/métodos , Riñón/ultraestructura , Hígado/ultraestructura , RatonesRESUMEN
Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In patients with CF, abnormalities initiate in several organs before birth. However, the long-term impact of these in utero pathologies on disease pathophysiology is unclear. To address this issue, we generated ferrets harboring a VX-770 (ivacaftor)-responsive CFTR G551D mutation. In utero VX-770 administration provided partial protection from developmental pathologies in the pancreas, intestine, and male reproductive tract. Homozygous CFTR G551D/G551D animals showed the greatest VX-770-mediated protection from these pathologies. Sustained postnatal VX-770 administration led to improved pancreatic exocrine function, glucose tolerance, growth and survival, and to reduced mucus accumulation and bacterial infections in the lung. VX-770 withdrawal at any age reestablished disease, with the most rapid onset of morbidity occurring when withdrawal was initiated during the first 2 weeks after birth. The results suggest that CFTR is important for establishing organ function early in life. Moreover, this ferret model provides proof of concept for in utero pharmacologic correction of genetic disease and offers opportunities for understanding CF pathogenesis and improving treatment.
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Aminofenoles/administración & dosificación , Agonistas de los Canales de Cloruro/administración & dosificación , Fibrosis Quística/tratamiento farmacológico , Quinolonas/administración & dosificación , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Glucemia/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Hurones , Técnicas de Sustitución del Gen , Genitales Masculinos/anomalías , Genitales Masculinos/efectos de los fármacos , Edad Gestacional , Humanos , Masculino , Mutación , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/patología , Páncreas Exocrino/fisiopatología , Embarazo , Infecciones del Sistema Respiratorio/etiología , Infecciones del Sistema Respiratorio/prevención & control , Investigación Biomédica TraslacionalRESUMEN
In vitro drug tests using patient-derived stem cell cultures offer opportunities to individually select efficacious treatments. Here, we provide a study that demonstrates that in vitro drug responses in rectal organoids from individual patients with cystic fibrosis (CF) correlate with changes in two in vivo therapeutic endpoints. We measured individual in vitro efficaciousness using a functional assay in rectum-derived organoids based on forskolin-induced swelling and studied the correlation with in vivo effects. The in vitro organoid responses correlated with both change in pulmonary response and change in sweat chloride concentration. Receiver operating characteristic curves indicated good-to-excellent accuracy of the organoid-based test for defining clinical responses. This study indicates that an in vitro assay using stem cell cultures can prospectively select efficacious treatments for patients and suggests that biobanked stem cell resources can be used to tailor individual treatments in a cost-effective and patient-friendly manner.
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Fibrosis Quística/terapia , Organoides/patología , Recto/patología , Fibrosis Quística/patología , Femenino , Humanos , MasculinoRESUMEN
Cystic fibrosis is caused by mutations in the CFTR gene, which are subdivided into six classes. Mutants of classes III and IV reach the cell surface but have limited function. Most class-III and class-IV mutants respond well to the recently approved potentiator VX-770, which opens the channel. We here revisited function and folding of some class-IV mutants and discovered that R347P is the only one that leads to major defects in folding. By this criterion and by its functional response to corrector drug VX-809, R347P qualifies also as a class-II mutation. Other class-IV mutants folded like wild-type CFTR and responded similarly to VX-809, demonstrating how function and folding are connected. Studies on both types of defects complement each other in understanding how compounds improve mutant CFTR function. This provides an attractive unbiased approach for characterizing mode of action of novel therapeutic compounds and helps address which drugs are efficacious for each cystic fibrosis disease variant.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Fibrosis Quística/patología , Pliegue de Proteína/efectos de los fármacos , Alelos , Aminofenoles/farmacología , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Biopsia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/clasificación , Genotipo , Células HEK293 , Humanos , Mutación , Organoides/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Quinolonas/farmacología , Recto/patología , TransfecciónRESUMEN
BACKGROUND: New functional assays using primary human intestinal adult stem cell cultures can be valuable tools to study epithelial defects in human diseases such as cystic fibrosis. METHODS: CFTR-mediated ion transport was measured in rectal organoid-derived monolayers grown from subjects with various CFTR mutations and compared to donor-matched intestinal current measurements (ICM) in rectal biopsies and forskolin-induced swelling of rectal organoids. RESULTS: Rectal organoid-derived monolayers were generated within four days. Ion transport measurements of CFTR function using these monolayers correlated with ICM and organoid swelling (râ¯=â¯0.73 and 0.79 respectively). Culturing the monolayers under differentiation conditions enhanced the detection of mucus-secreting cells and was accompanied by reduced CFTR function. CONCLUSIONS: CFTR-dependent intestinal epithelial ion transport properties can be measured in rectal organoid-derived monolayers of subjects and correlate with donor-matched ICM and rectal organoid swelling.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Células Epiteliales/metabolismo , Transporte Iónico/fisiología , Células Cultivadas , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Modelos Biológicos , Mutación , Organoides/fisiología , RectoRESUMEN
Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 °C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.
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Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Mucosa Intestinal/efectos de los fármacos , Organoides/efectos de los fármacos , Vasodilatadores/farmacología , Bioensayo/métodos , Células Cultivadas , AMP Cíclico/metabolismo , Fibrosis Quística/patología , Humanos , Mucosa Intestinal/metabolismo , Transporte Iónico , Mutación , Organoides/metabolismoRESUMEN
The potentiator VX-770 (ivacaftor/KALYDECO™) targets defective gating of CFTR and has been approved for treatment of cystic fibrosis (CF) subjects carrying G551D, S1251N or one of 8 other mutations. Still, the current potentiator treatment does not normalize CFTR-dependent biomarkers, indicating the need for development of more effective potentiator strategies. We have recently pioneered a functional CFTR assay in primary rectal organoids and used this model to characterize interactions between VX-770, genistein and curcumin, the latter 2 being natural food components with established CFTR potentiation capacities. Results indicated that all possible combinations of VX-770, genistein and curcumin synergistically repaired CFTR-dependent forskolin-induced swelling of organoids with CFTR-S1251N or CFTR-G551D, even under suboptimal CFTR activation and compounds concentrations, conditions that may predominate in vivo. Genistein and curcumin also enhanced forskolin-induced swelling of F508del homozygous organoids that were treated with VX-770 and the prototypical CFTR corrector VX-809. These results indicate that VX-770, genistein and curcumin in double or triple combinations can synergize in restoring CFTR-dependent fluid secretion in primary CF cells and support the use of multiple potentiators for treatment of CF.
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Aminofenoles/farmacología , Curcumina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística , Genisteína/farmacología , Organoides , Quinolonas/farmacología , Agonistas de los Canales de Cloruro/farmacología , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Teóricos , Terapia Molecular Dirigida/métodos , Mutación , Organoides/efectos de los fármacos , Organoides/patología , Recto/patologíaRESUMEN
Small-molecule therapies that restore defects in cystic fibrosis transmembrane conductance regulator (CFTR) gating (potentiators) or trafficking (correctors) are being developed for cystic fibrosis (CF) in a mutation-specific fashion. Options for pharmacological correction of CFTR-p.Phe508del (F508del) are being extensively studied but correction of other trafficking mutants that may also benefit from corrector treatment remains largely unknown.We studied correction of the folding mutants CFTR-p.Phe508del, -p.Ala455Glu (A455E) and -p.Asn1303Lys (N1303K) by VX-809 and 18 other correctors (C1-C18) using a functional CFTR assay in human intestinal CF organoids.Function of both CFTR-p.Phe508del and -p.Ala455Glu was enhanced by a variety of correctors but no residual or corrector-induced activity was associated with CFTR-p.Asn1303Lys. Importantly, VX-809-induced correction was most dominant for CFTR-p.Phe508del, while correction of CFTR-p.Ala455Glu was highest by a subgroup of compounds called bithiazoles (C4, C13, C14 and C17) and C5.These data support the development of mutation-specific correctors for optimal treatment of different CFTR trafficking mutants, and identify C5 and bithiazoles as the most promising compounds for correction of CFTR-p.Ala455Glu.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Mutación , Aminopiridinas/farmacología , Benzodioxoles/farmacología , Biopsia , Genotipo , Homocigoto , Humanos , Organoides , Pliegue de Proteína , Transporte de Proteínas , Recto/patología , Tiazoles/química , Resultado del TratamientoRESUMEN
Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells.