RESUMO
Approximately 50% of cystic fibrosis (CF) patients are heterozygous with a rare mutation on at least one allele. Several mutants exhibit functional defects, correctable by gating potentiators. Long-term exposure (≥24 h) to the only available potentiator drug, VX-770, leads to the biochemical and functional downregulation of F508del-CFTR both in immortalized and primary human airway cells, and possibly other CF mutants, attenuating its beneficial effect. Based on these considerations, we wanted to determine the effect of chronic VX-770 exposure on the functional and biochemical expression of rare CF processing/gating mutants in human airway epithelia. Expression of CFTR2 mutants was monitored in the human bronchial epithelial cell line (CFBE41o-) and in patient-derived conditionally reprogrammed bronchial and nasal epithelia by short-circuit current measurements, cell surface ELISA and immunoblotting in the absence or presence of CFTR modulators. The VX-770 half-maximal effective (EC50) concentration for G551D-CFTR activation was â¼0.63 µM in human nasal epithelia, implying that comparable concentration is required in the lung to attain clinical benefit. Five of the twelve rare CFTR2 mutants were susceptible to â¼20-70% downregulation by chronic VX-770 exposure with an IC50 of â¼1-20 nM and to destabilization by other investigational potentiators, thereby diminishing the primary functional gain of CFTR modulators. Thus, chronic exposure to VX-770 and preclinical potentiators can destabilize CFTR2 mutants in human airway epithelial models in a mutation and compound specific manner. This highlights the importance of selecting potentiator drugs with minimal destabilizing effects on CF mutants, advocating a precision medicine approach.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Mutação , Quinolonas/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Brônquios/metabolismo , Linhagem Celular , Células Cultivadas , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação para Baixo , Sinergismo Farmacológico , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Ativação do Canal Iônico/genética , Pulmão/metabolismo , Modelos Moleculares , Mucosa Respiratória/metabolismoRESUMO
PD-L1 (programmed death ligand 1) and PD-L2 are cell-surface glycoproteins that interact with programmed death 1 (PD-1) on T cells to attenuate inflammation. PD-1 signaling has attracted intense interest for its role in a pathophysiological context: suppression of anti-tumor immunity. Similarly, vitamin D signaling has been increasingly investigated for its non-classical actions in stimulation of innate immunity and suppression of inflammatory responses. Here, we show that hormonal 1,25-dihydroxyvitamin D (1,25D) is a direct transcriptional inducer of the human genes encoding PD-L1 and PD-L2 through the vitamin D receptor, a ligand-regulated transcription factor. 1,25D stimulated transcription of the gene encoding PD-L1 in epithelial and myeloid cells, whereas the gene encoding the more tissue-restricted PD-L2 was regulated only in myeloid cells. We identified and characterized vitamin D response elements (VDREs) located in both genes and showed that 1,25D treatment induces cell-surface expression of PD-L1 in epithelial and myeloid cells. In co-culture experiments with primary human T cells, epithelial cells pretreated with 1,25D suppressed activation of CD4+ and CD8+ cells and inhibited inflammatory cytokine production in a manner that was abrogated by anti-PD-L1 blocking antibody. Consistent with previous observations of species-specific regulation of immunity by vitamin D, the VDREs are present in primate genes, but neither the VDREs nor the regulation by 1,25D is present in mice. These findings reinforce the physiological role of 1,25D in controlling inflammatory immune responses but may represent a double-edged sword, as they suggest that elevated vitamin D signaling in humans could suppress anti-tumor immunity.
Assuntos
Antígeno B7-H1/agonistas , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteína 2 Ligante de Morte Celular Programada 1/agonistas , Regulação para Cima/efeitos dos fármacos , Elemento de Resposta à Vitamina D/efeitos dos fármacos , Vitamina D/análogos & derivados , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Mucosa Nasal/citologia , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Especificidade de Órgãos , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vitamina D/farmacologiaRESUMO
W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR1281, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR1281, functional screens were done of â¼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR1281-transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to â¼5-fold greater efficacy than VX-809, some of which were selective for CFTR1281, whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR1281 function â¼8-fold over that of VX-770 alone, normalizing CFTR1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Mutação de Sentido Incorreto , Piperazinas/farmacologia , Quinolonas/farmacologia , Substituição de Aminoácidos , Animais , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Ratos , Ratos Endogâmicos F344RESUMO
Dopamine axons are the only axons known to grow during adolescence. Here, using rodent models, we examined how two proteins, Netrin-1 and its receptor, UNC5C, guide dopamine axons towards the prefrontal cortex and shape behaviour. We demonstrate in mice ( Mus musculus ) that dopamine axons reach the cortex through a transient gradient of Netrin-1 expressing cells - disrupting this gradient reroutes axons away from their target. Using a seasonal model (Siberian hamsters; Phodopus sungorus ) we find that mesocortical dopamine development can be regulated by a natural environmental cue (daylength) in a sexually dimorphic manner - delayed in males, but advanced in females. The timings of dopamine axon growth and UNC5C expression are always phase-locked. Adolescence is an ill-defined, transitional period; we pinpoint neurodevelopmental markers underlying this period.
RESUMO
Initiating drug use during adolescence increases the risk of developing addiction or other psychopathologies later in life, with long-term outcomes varying according to sex and exact timing of use. The cellular and molecular underpinnings explaining this differential sensitivity to detrimental drug effects remain unexplained. The Netrin-1/DCC guidance cue system segregates cortical and limbic dopamine pathways in adolescence. Here we show that amphetamine, by dysregulating Netrin-1/DCC signaling, triggers ectopic growth of mesolimbic dopamine axons to the prefrontal cortex, only in early-adolescent male mice, underlying a male-specific vulnerability to enduring cognitive deficits. In adolescent females, compensatory changes in Netrin-1 protect against the deleterious consequences of amphetamine on dopamine connectivity and cognitive outcomes. Netrin-1/DCC signaling functions as a molecular switch which can be differentially regulated by the same drug experience as function of an individual's sex and adolescent age, and lead to divergent long-term outcomes associated with vulnerable or resilient phenotypes.
Assuntos
Anfetamina , Dopamina , Feminino , Camundongos , Masculino , Animais , Anfetamina/farmacologia , Dopamina/metabolismo , Netrina-1/metabolismo , Receptor DCC/genética , Receptor DCC/metabolismo , Axônios/metabolismoRESUMO
BACKGROUND: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosis (CF) mutations (â¼10% of the patient population) associated with a gating defect of the CF transmembrane conductance regulator (CFTR). Despite the success of VX-770 treatment of patients carrying at least one allele of the most common gating mutation G551D-CFTR, some lung function decline and P. aeruginosa colonization persist. This study aims at identifying potentiator combinations that can considerably enhance the limited channel activity of a panel of CFTR gating mutants over monotherapy. METHODS: The functional response of 13 CFTR mutants to single potentiators or systematic potentiator combinations was determined in the human bronchial epithelial cell line CFBE41o- and a subset of them was confirmed in primary human nasal epithelia (HNE). RESULTS: In six out of thirteen CFTR missense mutants the fractional plasma membrane (PM) activity, a surrogate measure of CFTR channel gating, reached only â¼10-50% of WT channel activity upon VX-770 treatment, indicating incomplete gating correction. Combinatorial potentiator profiling and cluster analysis of mutant responses to 24 diverse investigational potentiators identified several compound pairs that improved the gating activity of R352Q-, S549R-, S549N-, G551D-, and G1244E-CFTR to â¼70-120% of the WT. Similarly, the potentiator combinations were able to confer WT-like function to G551D-CFTR in patient-derived human nasal epithelia. CONCLUSION: This study suggests that half of CF patients with missense mutations approved for VX-770 administration, could benefit from the development of dual potentiator therapy.
Assuntos
Aminofenóis/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística , Transporte de Íons , Mucosa Nasal , Piranos/farmacologia , Pirazóis/farmacologia , Quinolonas/farmacologia , Células Cultivadas , Agonistas dos Canais de Cloreto/classificação , Agonistas dos Canais de Cloreto/farmacologia , Análise por Conglomerados , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Fibrose Cística/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Humanos , Ativação do Canal Iônico/genética , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/genética , Mutação de Sentido Incorreto , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , Medicina de Precisão/métodosRESUMO
Available corrector drugs are unable to effectively rescue the folding defects of CFTR-ΔF508 (or CFTR-F508del), the most common disease-causing mutation of the cystic fibrosis transmembrane conductance regulator, a plasma membrane (PM) anion channel, and thus to substantially ameliorate clinical phenotypes of cystic fibrosis (CF). To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutant expression and function at the PM. High-throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at nucleotide-binding domain (NBD1), NBD2 and their membrane-spanning domain (MSD) interfaces. Although individually these compounds marginally improve ΔF508-CFTR folding efficiency, function and stability, their combinations lead to ~50-100% of wild-type-level correction in immortalized and primary human airway epithelia and in mouse nasal epithelia. Likewise, corrector combinations were effective against rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Fibrose Cística/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Regulação Alostérica/efeitos dos fármacos , Brônquios/citologia , Brônquios/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Mucosa Nasal/citologia , Mucosa Nasal/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Molecular chaperones are pivotal in folding and degradation of the cellular proteome but their impact on the conformational dynamics of near-native membrane proteins with disease relevance remains unknown. Here we report the effect of chaperone activity on the functional conformation of the temperature-sensitive mutant cystic fibrosis channel (∆F508-CFTR) at the plasma membrane and after reconstitution into phospholipid bilayer. Thermally induced unfolding at 37 °C and concomitant functional inactivation of ∆F508-CFTR are partially suppressed by constitutive activity of Hsc70 and Hsp90 chaperone/co-chaperone at the plasma membrane and post-endoplasmic reticulum compartments in vivo, and at single-molecule level in vitro, indicated by kinetic and thermodynamic remodeling of the mutant gating energetics toward its wild-type counterpart. Thus, molecular chaperones can contribute to functional maintenance of ∆F508-CFTR by reshaping the conformational energetics of its final fold, a mechanism with implication in the regulation of metastable ABC transporters and other plasma membrane proteins activity in health and diseases.The F508 deletion (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common CF causing mutation. Here the authors show that cytosolic chaperones shift the F508del channel conformation to the native fold by kinetic and thermodynamic remodelling of the gating energetics towards that of wild-type CTFR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Fibrose Cística/genética , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Chaperonas Moleculares/genética , Mutação , Dobramento de Proteína , TemperaturaRESUMO
More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Animais , Agonistas dos Canais de Cloreto/farmacologia , Agonistas dos Canais de Cloreto/uso terapêutico , Fibrose Cística/classificação , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/agonistas , Predisposição Genética para Doença , Humanos , Ativação do Canal Iônico , Mutação de Sentido IncorretoRESUMO
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane regulator (CFTR) that result in reduced anion conductance at the apical membrane of secretory epithelia. Treatment of CF patients carrying the G551D gating mutation with the potentiator VX-770 (ivacaftor) largely restores channel activity and has shown substantial clinical benefit. However, most CF patients carry the ΔF508 mutation, which impairs CFTR folding, processing, function, and stability. Studies in homozygous ΔF508 CF patients indicated little clinical benefit of monotherapy with the investigational corrector VX-809 (lumacaftor) or VX-770, whereas combination clinical trials show limited but significant improvements in lung function. We show that VX-770, as well as most other potentiators, reduces the correction efficacy of VX-809 and another investigational corrector, VX-661. To mimic the administration of VX-770 alone or in combination with VX-809, we examined its long-term effect in immortalized and primary human respiratory epithelia. VX-770 diminished the folding efficiency and the metabolic stability of ΔF508-CFTR at the endoplasmic reticulum (ER) and post-ER compartments, respectively, causing reduced cell surface ΔF508-CFTR density and function. VX-770-induced destabilization of ΔF508-CFTR was influenced by second-site suppressor mutations of the folding defect and was prevented by stabilization of the nucleotide-binding domain 1 (NBD1)-NBD2 interface. The reduced correction efficiency of ΔF508-CFTR, as well as of two other processing mutations in the presence of VX-770, suggests the need for further optimization of potentiators to maximize the clinical benefit of corrector-potentiator combination therapy in CF.