Dataset Information

Photoaffinity ligand of Cystic Fibrosis corrector VX-445 identifies SCCPDH as an off-target

ABSTRACT: Cystic fibrosis (CF) pharmacological correctors such as VX-445 are used in combinations as highly effective modulator therapy (HEMT) at CF clinics due to their efficacy in improving Cystic Fibrosis transmembrane conductance regulator (CFTR) protein trafficking and function. Yet, several side-effects from these correctors and adverse drug interactions have been reported, emphasizing the need to understand the etiology of these effects that potentially arise from off-targets. We synthesized VU439, a functionalized photoaffinity ligand (PAL) of VX-445 that harbors a diazirine-moiety for in situ photocrosslinking upon UV radiation and an alkyne handle for click chemistry modifications to visualize and enrich crosslinked proteins. The PAL probe retained corrector function with mild efficacy in improving CFTR trafficking and function. Chemoproteomics analysis by mass spectrometry (MS) was used to identify crosslinked proteins in CF bronchial epithelial cells expressing F508del CFTR, identifying saccharopine dehydrogenase-like oxidoreductase (SCCPDH), an uncharacterized putative oxidoreductase, as a VX-445 specific off-target. We then characterized changes in the metabolomic profiles of cells overexpressing SCCPDH to determine the consequence of VX-445 binding to SCCPDH. These data show dysregulation of amino acid metabolism and a potential inhibitory activity of VX-445 on SCCPDH. The identified off-target may explain exacerbation of psychological symptoms observed in patients on HEMT, thus emphasizing the need for further optimization of corrector combinations.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Minsoo Kim

LAB HEAD: Lars Plate

PROVIDER: PXD060898 | Pride | 2025-07-28

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20240415_MK_3_140_NN445_DIA_6well_manual_DMSO_1.raw	Raw
	20240415_MK_3_140_NN445_DIA_6well_manual_DMSO_10.raw	Raw
	20240415_MK_3_140_NN445_DIA_6well_manual_DMSO_4.raw	Raw
	20240415_MK_3_140_NN445_DIA_6well_manual_DMSO_7.raw	Raw
	20240415_MK_3_140_NN445_DIA_6well_manual_NN445_11.raw	Raw

Items per page:

1 - 5 of 39

Publications

Photoaffinity Ligand of Cystic Fibrosis Corrector VX-445 Identifies SCCPDH as an Off-Target.

Kim Minsoo M Kim Kwangho K Lee Jesun J Barny Lea A LA Scaggs Toya D TD Romaine Ian M IM Jeon KyuOk K Codreanu Simona G SG Sherrod Stacy D SD McLean John A JA Naren Anjaparavanda P AP Sulikowski Gary A GA Plate Lars L

ACS chemical biology 20250620 7

Cystic fibrosis (CF) pharmacological correctors improve the cystic fibrosis transmembrane conductance regulator (CFTR) protein trafficking and function. Several side effects of these correctors and adverse drug interactions have been reported, emphasizing the need to understand off-targets. We synthesized VU439, a functionalized photoaffinity ligand (PAL) of VX-445. Chemoproteomics analysis by mass spectrometry (MS) was used to identify cross-linked proteins in CF bronchial epithelial cells expr ...[more]

PMID: 40539870

Similar Datasets

Project description:Cystic Fibrosis (CF) is a genetic disorder CF is caused by mutations of the gene encoding for the cystic fibrosis transmembrane conductance regulator protein (CFTR), a transmembrane anion channel expressed at the apical membrane of several organs, including the epithelial cells of the airway. CFTR mutations result in dysfunctional ion transport across the apical membrane at the surface of the epithelia, generating thickened and dehydrated secretions. In the lung, this leads to a decrease in the mucociliary clearance, favoring bacterial colonization and progressive obstruction of the duct. Although over 2000 CFTR variants have been identified so far, the most common mutation is a deletion of the phenylalanine in position 508 (F508del), which shows an allelic frequency of around 90% among CF patients. F508del-CFTR is incorrectly folded, causing its retention at the endoplasmic reticulum (ER) and subsequent proteasomal degradation. Among the several drugs available in CF pharmacological treatment, VX-809 (commercial name Lumacaftor) is the most used drug for patients carrying F508del-CFTR mutation. This drug corrects the aberrant folding of F508del-CFTR by favoring the correct intramolecular interactions, thus enabling a higher number of copies of the defective protein to reach the plasma membrane. Localization of Organelle Proteins by Isotope Tagging after Differential ultra-Centrifugation (LOPIT-DC, https://doi.org/10.1038/s41467-018-08191-w) workflow was used to study the spatial localization of proteins in the CFBE41o- cells and how it is impacted by CFTR rescue triggered by VX-809. LOPIT-DC is a powerful and well-established tool for the investigation of the spatial distribution of global proteome within cells, which combines subcellular fractionation based on differential centrifugation, quantitative mass spectrometry, and machine learning. Its application provides a global snapshot of the steady-state subcelluler distribution of proteins. The aim of this project is to identify proteins that change their cellular localization in association with the treatment, to uncover molecules that play a role in the trafficking of the defective CFTR to the plasma membrane.

Project description:Background: Cystic fibrosis (CF) is caused by mutations in the CFTR gene that impair function of this cAMP-regulated Cl- channel. In the small intestine, loss of CFTR function creates a dehydrated, acidic luminal environment which is believed to cause an accumulation of mucus, a phenotype characteristic of CF. CF mice have an innate immune response and impaired intestinal transit as well. We investigated whether lubiprostone, which activates the CLC2 Cl- channel, would improve the CF intestinal phenotype. Methods: Cftrtm1UNC (CF) and wildtype (WT) littermate mice on the C57BL/6 background were used. Lubiprostone (10ug/kg-day) was administered by gavage for two weeks. Mucus accumulation was estimated from crypt lumen widths in Carnoy's-fixed, PAS/AB stained sections. Luminal bacterial load was measured by qPCR for the bacterial 16S gene. Gastric emptying and small intestinal transit were assessed by gavage of rhodamine dextran. Gene expression was evaluated by Affymetrix Mouse430 2.0 microarray. Results: Crypt width in control CF mice was 700% that of WT mice (P<0.001). Lubiprostone did not affect WT crypt width but, unexpectedly, increased CF crypt width 22% (P=0.001). Lubiprostone increased bacterial load in WT mice to 490% of WT control levels (P=0.008). Conversely, lubiprostone decreased bacterial overgrowth in CF mice by 60% (P=0.005). Lubiprostone significantly increased gastric emptying at 20 min postgavage in both WT (P<0.001; control=57±3%, treated=81±4%) and CF mice (P<0.001; control=48±4%, treated=75±5%). After lubiprostone small intestinal transit was significantly enhanced in WT mice (P=0.024) but the effect was not significant in CF mice (P=0.377). Among other innate immune markers, expression of mast cell genes was elevated ~20-fold in the control CF intestine and lubiprostone decreased expression to WT control levels. Conclusions: These results indicate that lubiprostone has some benefits for the CF intestinal phenotype, especially on bacterial overgrowth and the innate immune response. The unexpected observation of increased mucus accumulation in the crypts of lubiprostone-treated CF mice suggests the possibility that lubiprostone increases mucus secretion. For each group (control wild type, lubiprostone-treated wild type, contol Cftr null, and lubiprostone-treated cftr null), equal amounts of total RNA extracted from the entire small intestine were pooled for analysis.