Details
| Original language | English |
|---|---|
| Pages (from-to) | 997–1005 |
| Number of pages | 9 |
| Journal | ACS Pharmacology & Translational Science |
| Volume | 6 |
| Issue number | 7 |
| Early online date | 1 Jun 2023 |
| Publication status | Published - 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
- Medicine(all)
- Pharmacology (medical)
- Pharmacology, Toxicology and Pharmaceutics(all)
- Pharmacology
Cite this
- Standard
- Harvard
- Apa
- Vancouver
- BibTeX
- RIS
In: ACS Pharmacology & Translational Science, Vol. 6, No. 7, 14.07.2023, p. 997–1005.
Research output: Contribution to journal › Article › Research › peer review
}
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 -