A Chemical Chaperone Restores Connexin 26 Mutant Activity

Research output: Contribution to journalArticleResearchpeer review

Authors

External Research Organisations

  • Hannover Medical School (MHH)
  • Technische Universität Braunschweig
View graph of relations

Details

Original languageEnglish
Pages (from-to)997–1005
Number of pages9
JournalACS Pharmacology & Translational Science
Volume6
Issue number7
Early online date1 Jun 2023
Publication statusPublished - 14 Jul 2023

Abstract

Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds 3 and 8 were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only 3 (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ~5 µM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.

Keywords

    Cx26, hearing impairment, protein microarray, thermophoresis, viability assay

ASJC Scopus subject areas

Cite this

A Chemical Chaperone Restores Connexin 26 Mutant Activity. / Wang, Dahua; Wang, Hongling; Fan, Lu et al.
In: ACS Pharmacology & Translational Science, Vol. 6, No. 7, 14.07.2023, p. 997–1005.

Research output: Contribution to journalArticleResearchpeer review

Wang, D, Wang, H, Fan, L, Ludwig, T, Wegner, A, Stahl, F, Harre, J, Warnecke, A & Zeilinger, C 2023, 'A Chemical Chaperone Restores Connexin 26 Mutant Activity', ACS Pharmacology & Translational Science, vol. 6, no. 7, pp. 997–1005. https://doi.org/10.1021/acsptsci.3c00056
Wang, D., Wang, H., Fan, L., Ludwig, T., Wegner, A., Stahl, F., Harre, J., Warnecke, A., & Zeilinger, C. (2023). A Chemical Chaperone Restores Connexin 26 Mutant Activity. ACS Pharmacology & Translational Science, 6(7), 997–1005. https://doi.org/10.1021/acsptsci.3c00056
Wang D, Wang H, Fan L, Ludwig T, Wegner A, Stahl F et al. A Chemical Chaperone Restores Connexin 26 Mutant Activity. ACS Pharmacology & Translational Science. 2023 Jul 14;6(7):997–1005. Epub 2023 Jun 1. doi: 10.1021/acsptsci.3c00056
Wang, Dahua ; Wang, Hongling ; Fan, Lu et al. / A Chemical Chaperone Restores Connexin 26 Mutant Activity. In: ACS Pharmacology & Translational Science. 2023 ; Vol. 6, No. 7. pp. 997–1005.
Download
@article{235e16343ba446139ab1d7069523ecaa,
title = "A Chemical Chaperone Restores Connexin 26 Mutant Activity",
abstract = "Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds 3 and 8 were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only 3 (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ~5 µM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.",
keywords = "Cx26, hearing impairment, protein microarray, thermophoresis, viability assay",
author = "Dahua Wang and Hongling Wang and Lu Fan and Tobias Ludwig and Andre Wegner and Frank Stahl and Jennifer Harre and Athanasia Warnecke and Carsten Zeilinger",
note = "Parts of the project were funded by DFG Cytolabs ZE 338/16-1 and Cluster of Excellence Hearing4All (EXC 2177/1).",
year = "2023",
month = jul,
day = "14",
doi = "10.1021/acsptsci.3c00056",
language = "English",
volume = "6",
pages = "997–1005",
number = "7",

}

Download

TY - JOUR

T1 - A Chemical Chaperone Restores Connexin 26 Mutant Activity

AU - Wang, Dahua

AU - Wang, Hongling

AU - Fan, Lu

AU - Ludwig, Tobias

AU - Wegner, Andre

AU - Stahl, Frank

AU - Harre, Jennifer

AU - Warnecke, Athanasia

AU - Zeilinger, Carsten

N1 - Parts of the project were funded by DFG Cytolabs ZE 338/16-1 and Cluster of Excellence Hearing4All (EXC 2177/1).

PY - 2023/7/14

Y1 - 2023/7/14

N2 - Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds 3 and 8 were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only 3 (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ~5 µM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.

AB - Mutations in connexin 26 (Cx26) cause hearing disorders of a varying degree. Herein, to identify compounds capable of restoring the function of mutated Cx26, a novel miniaturized microarray-based screening system was developed to perform an optical assay of Cx26 functionality. These molecules were identified through a viability assay using HeLa cells expressing wild-type (WT) Cx26, which exhibited sensitivity toward the HSP90 inhibitor radicicol in the submicromolar concentration range. Open Cx26 hemichannels are assumed to mediate the passage of molecules up to 1000 Da in size. Thus, by releasing radicicol, WT Cx26 active hemichannels in HeLa cells contribute to a higher survival rate and lower cell viability when Cx26 is mutated. HeLa cells expressing Cx26 mutations exhibited reduced viability in the presence of radicicol, such as the mutants F161S or R184P. Next, molecules exhibiting chemical chaperoning activity, suspected of restoring channel function, were assessed regarding whether they induced superior sensitivity toward radicicol and increased HeLa cell viability. Through a viability assay and microarray-based flux assay that uses Lucifer yellow in HeLa cells, compounds 3 and 8 were identified to restore mutant functionality. Furthermore, thermophoresis experiments revealed that only 3 (VRT-534) exhibited dose-responsive binding to recombinant WT Cx26 and mutant Cx26K188N with half maximal effective concentration values of 19 and ~5 µM, respectively. The findings of this study reveal that repurposing compounds already being used to treat other diseases, such as cystic fibrosis, in combination with functional bioassays and binding tests can help identify novel potential candidates that can be used to treat hearing disorders.

KW - Cx26

KW - hearing impairment

KW - protein microarray

KW - thermophoresis

KW - viability assay

UR - http://www.scopus.com/inward/record.url?scp=85163335683&partnerID=8YFLogxK

U2 - 10.1021/acsptsci.3c00056

DO - 10.1021/acsptsci.3c00056

M3 - Article

VL - 6

SP - 997

EP - 1005

JO - ACS Pharmacology & Translational Science

JF - ACS Pharmacology & Translational Science

IS - 7

ER -

By the same author(s)