Use of Kinase Inhibitors to Correct ΔF508-CFTR Function

Trzcińska-Daneluti, Agata M.; Nguyen, Leo; Jiang, Chunhe; Fladd, Christopher; Uehling, David; Prakesch, Michaël; Al‐awar, Rima; Rotin, Daniela

doi:10.1074/mcp.m111.016626

Cited by 31 publications

(37 citation statements)

References 77 publications

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“…Our group previously developed a high-content functional screen to identify ⌬F508-CFTR correctors (and poten-tiators) in multiple individual cells simultaneously, using Cellomics KineticScan KSR and ArrayScan VTI platforms (28,29). The kinome esiRNA screen presented in this study complements the small-molecule kinase inhibitor screen described previously (29).…”

Section: Identification Of Kinases and Associated Signaling Proteinsmentioning

confidence: 78%

“…The cells were subjected to puromycin selection (5 g/ml), and 5 ϫ 10 5 infected cells were seeded onto 1.12 cm 2 snapwells (Corning). After 6 -7 days the inserts were mounted in Ussing chambers (Physiological Instruments, San Diego, CA) and studied under voltage clamp conditions as previously described (29). Briefly, ENaC channels were inhibited with 10 M amiloride (Sigma, St. Louis, MO) and non-CFTR chloride transporters were blocked with 250 M DNDS (4,4-dinitrostilbene-2,2-disulfonate, Sigma).…”

Section: Short-circuit Current Analysis In Mdck Cellsmentioning

confidence: 99%

“…A total number of 133 shRNA clones was screened (multiple shRNA clones per gene) (supplemental Table S2). Knockdown efficiency was validated by two-step RT-qPCR as described previously (29). Briefly, total RNA was isolated using the RNeasy 96 kit (Qiagen, Venlo, Netherlands), and cDNA was prepared using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems).…”

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

“…Immunoblotting-The appearance of band C was validated by immunoblotting, as described previously (28,29). Briefly, prior to immunoblotting ⌬F508-CFTR cells were transfected with shRNA constructs (TRC) for the identified genes or nonsilencing (scrambled) control for 48 h at 37°C, or incubated for 48 h at 27°C (positive control).…”

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

“…The Cellomics halide-exchange assay was performed as described previously (29). Briefly, 5 ϫ 10 4 ⌬F508-CFTR cells (i.e.…”

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

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RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

Trzcińska-Daneluti

Chen

Nguyen

et al. 2015

Molecular & Cellular Proteomics

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Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding the Cystic fibrosis transmembrane conductance regulator (CFTR).⌬F508-CFTR, the most common disease-causing CF mutant, exhibits folding and trafficking defects and is retained in the endoplasmic reticulum, where it is targeted for proteasomal degradation. To identify signaling pathways involved in ⌬F508-CFTR rescue, we screened a library of endoribonuclease-prepared short interfering RNAs (esiRNAs) that target ϳ750 different kinases and associated signaling proteins. We identified 20 novel suppressors of ⌬F508-CFTR maturation, including the FGFR1. These were subsequently validated by measuring channel activity by the YFP halide-sensitive assay following shRNA-mediated knockdown, immunoblotting for the mature (band C) ⌬F508-CFTR and measuring the amount of surface ⌬F508-CFTR by ELISA. The role of FGFR signaling on ⌬F508-CFTR trafficking was further elucidated by knocking down FGFRs and their downstream signaling proteins: Erk1/2, Akt, PLC␥-1, and FRS2. Interestingly, inhibition of FGFR1 with SU5402 administered to intestinal organoids (mini-guts) generated from the ileum of ⌬F508-CFTR homozygous mice resulted in a robust ⌬F508-CFTR rescue. Moreover, combination of SU5402 and VX-809 treatments in cells led to an additive enhancement of ⌬F508-CFTR rescue, suggesting these compounds operate by different mechanisms. Chaperone array analysis on human bronchial epithelial cells harvested from ⌬F508/ ⌬F508-CFTR transplant patients treated with SU5402 identified altered expression of several chaperones, an effect validated by their overexpression or knockdown experiments. We propose that FGFR signaling regulates specific chaperones that control ⌬F508-CFTR maturation, and suggest that FGFRs may serve as important targets for therapeutic intervention for the treatment of

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Section: Identification Of Kinases and Associated Signaling Proteinsmentioning

confidence: 78%

Section: Short-circuit Current Analysis In Mdck Cellsmentioning

confidence: 99%

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

“…The Cellomics halide-exchange assay was performed as described previously (29). Briefly, 5 ϫ 10 4 ⌬F508-CFTR cells (i.e.…”

Section: Cystic Fibrosis (Cf)mentioning

confidence: 99%

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RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

Trzcińska-Daneluti

Chen

Nguyen

et al. 2015

Molecular & Cellular Proteomics

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Identification of p38 MAPK and JNK as new targets for correction of Wilson disease‐causing ATP7B mutants

et al. 2016

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Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans‐Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum‐retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c‐Jun N‐terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7BH1069Q (as well as that of several other WD‐causing mutants) from the endoplasmic reticulum to the trans‐Golgi network compartment, in recovery of its Cu‐dependent trafficking, and in reduction of intracellular Cu levels. Conclusion: Our findings indicate p38 and c‐Jun N‐terminal kinase as intriguing targets for correction of WD‐causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD. (Hepatology 2016;63:1842‐1859)

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Bioactive atropisomers: Unraveling design strategies and synthetic routes for drug discovery

Liu,

Zhao,

Liu

et al. 2024

Medicinal Research Reviews

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Atropisomerism, an expression of axial chirality caused by limited bond rotation, is a prominent aspect within the field of medicinal chemistry. It has been shown that atropisomers of a wide range of compounds, including established FDA‐approved drugs and experimental molecules, display markedly different biological activities. The time‐dependent reversal of chirality in atropisomers poses complexity and obstacles in the process of drug discovery and development. Nonetheless, recent progress in understanding atropisomerism and enhanced characterization methods have greatly assisted medicinal chemists in the effective development of atropisomeric drug molecules. This article provides a comprehensive review of their special design thoughts, synthetic routes, and biological activities, serving as a reference for the synthesis and biological evaluation of bioactive atropisomers in the future.

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Use of Kinase Inhibitors to Correct ΔF508-CFTR Function

Cited by 31 publications

References 77 publications

RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

RNA Interference Screen to Identify Kinases That Suppress Rescue of ΔF508-CFTR*

Identification of p38 MAPK and JNK as new targets for correction of Wilson disease‐causing ATP7B mutants

Bioactive atropisomers: Unraveling design strategies and synthetic routes for drug discovery

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