Discovery of Icenticaftor (QBW251), a Cystic Fibrosis Transmembrane Conductance Regulator Potentiator with Clinical Efficacy in Cystic Fibrosis and Chronic Obstructive Pulmonary Disease.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel are established as the primary causative factor in the devastating lung disease cystic fibrosis (CF). More recently, cigarette smoke exposure has been shown to be associated with dysfunctional airway epithelial ion transport, suggesting a role for CFTR in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, the identification and characterization of a high throughput screening hit 6 as a potentiator of mutant human F508del and wild-type CFTR channels is reported. The design, synthesis, and biological evaluation of compounds 7-33 to establish structure-activity relationships of the scaffold are described, leading to the identification of clinical development compound icenticaftor (QBW251) 33, which has subsequently progressed to deliver two positive clinical proofs of concept in patients with CF and COPD and is now being further developed as a novel therapeutic approach for COPD patients.

Amyloid-beta(1-42) alters structure and function of retinal pigmented epithelial cells.

Age-related macular degeneration (AMD) is characterized by the formation of drusen, extracellular deposits associated with atrophy of the retinal pigmented epithelium (RPE), disturbance of the transepithelial barrier and photoreceptor death. Amyloid-beta (Abeta) is present in drusen but its role during AMD remains unknown. This study investigated the in vitro and in vivo effects of the oligomeric form of Abeta(1-42) - OAbeta(1-42) - on RPE and found that it reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures. It also disorganized the actin cytoskeleton and halved occludin expression, markedly decreasing attachment capacity and abolishing the selectivity of RPE cell transepithelial permeability. Antioxidant pretreatment partially reversed the effects of OAbeta(1-42) on mitochondrial redox potential and transepithelial permeability. Subretinally injected OAbeta(1-42) induced pigmentation loss and RPE hypertrophy but not RPE cell apoptosis in C57BL/6 J mice. Rapid OAbeta(1-42)-induced disorganization of cytoskeletal actin filaments was accompanied by decreased RPE expression of the tight junction proteins occludin and zonula occludens-1 and of the visual cycle proteins cellular retinaldehyde-binding protein and RPE65. The number of photoreceptors decreased by half within a few days. Our study pinpoints the role of Abeta in RPE alterations and dysfunctions leading to retinal degeneration and suggests that targeting Abeta may help develop selective methods for treating diseases involving retinal degeneration, such as AMD.

Prematurely senescent ARPE-19 cells display features of age-related macular degeneration.

The etiology of age-related macular degeneration (AMD), the leading cause of blindness in the developed world, remains poorly understood, but may be related to cumulative oxidative stress. The prime target of the disease is the retinal pigmented epithelium (RPE). To study the molecular mechanisms underlying RPE degeneration, we investigated whether repetitive oxidative stress induced premature senescence in RPE cells from the human ARPE-19 cell line. After exposure to 8 mM tert-butylhydroperoxide (tert-BHP) for 1 h daily for 5 days, the cells showed four well-known senescence biomarkers: hypertrophy, senescence-associated beta-galactosidase activity, growth arrest, and cell cycle arrest in G1. A specific low-density array followed by qRT-PCR validation allowed us to identify 36 senescence-associated genes differentially expressed in the prematurely senescent cells. Functional analysis demonstrated that premature senescence induced amyloid beta secretion, resistance to acute stress by tert-BHP and amyloid beta, and defects in adhesion and transepithelial permeability. Coculture assays with choroidal endothelial cells showed the proangiogenic properties of the senescent RPE cells. These results demonstrate that chronic oxidative stress induces premature senescence in RPE cells that modifies the transcriptome and substantially alters cell processes involved in the pathophysiology of AMD. Oxidative stress-induced premature senescence may represent an in vitro model for screening therapeutics against AMD and other retinal degeneration disorders.

Sustained versus transient ERK1/2 signaling underlies the anti- and proapoptotic effects of oxidative stress in human RPE cells.

PURPOSE: Oxidative stress is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which involves retinal pigmented epithelial (RPE) cell death. However, signaling pathways involved in the oxidative-stress-induced RPE cell death are poorly understood. This study was conducted to investigate the involvement of the MAP kinase pathways during the induction of RPE cell death by oxidative stress. METHODS: ARPE-19 cells were exposed to the oxidant tert-butyl hydroperoxide (t-BHP). Cell viability was assessed by cell counting and MTT-staining, and apoptosis was quantified by TUNEL and flow cytometry. Activation of JNK1/3, p38 alphabeta MAPKs and ERK1/2 and their potential targets was detected by Western blot analysis and immunochemistry with specific anti-phospho protein antibodies. Specific pharmacologic inhibitors directed against the MAPKs were used to analyze the signaling involved in cell death of RPE cells exposed to t-BHP. RESULTS: Exposure of RPE cells to t-BHP, associated with increase in reactive oxygen species and intracellular glutathione depletion, induced time- and concentration-dependent apoptosis, which was associated with the accumulation of inactive ERK1/2 in cell nuclei and a transient and weak ERK1/2 activation. This activation was accompanied by a deactivation of P90(RSK), the major target of ERK1/2 and consequently by the delayed activation of its transcription factor CREB. MEK1/2 inhibition completely suppressed the transient activation of ERK1/2 and completely blocked apoptosis, demonstrating the role of the MEK-ERK module in mediating oxidative-stress-induced RPE cell death. In contrast, neither JNKs nor p38 alphabeta MAPKs were involved in mediating t-BHP-induced apoptotic signaling in RPE cells. CONCLUSIONS: The results suggest that inhibiting the MEK-ERK module may allow the development of selective methods for treating oxidative-stress-induced RPE degeneration, such as AMD.

Extracellular signal-regulated kinase-dependent proliferation is mediated through the protein kinase A/B-Raf pathway in human uveal melanoma cells.

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf ((WT)B-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of (WT)B-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing (WT)B-Raf and (WT)Ras grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation ((V600E)B-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as (V600E)B-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in (WT)B-Raf cells, whereas it did not affect (V600E)B-Raf cells, demonstrating a key role of PKA in mediating (WT)B-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in (WT)B-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/(WT)B-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against (WT)B-Raf-expressing uveal melanoma.

Roles of stem cell factor/c-Kit and effects of Glivec/STI571 in human uveal melanoma cell tumorigenesis.

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.