Exploring Binding Sites of VRT534 at Cx26 as a Putative Chemical Chaperone for Targeted Treatment of Hereditary Hearing Disorders

Carsten Zeilinger^1*Jennifer Harre^2,3Dahua Wang¹Athanasia Warnecke^2,3

• ¹BMWZ, Centre of Biomolecular Drug Research, Leibniz University Hannover, Hanover, Lower Saxony, Germany
• ²Hannover Medical School, Hanover, Lower Saxony, Germany
• ³Department of Otorhinolaryngology, Head- and Neck-Surgery, Hannover, Germany

Hearing loss is the most common sensory disorder that significantly affects the quality of life of millions of people worldwide. The use of chemical chaperones could be a therapeutic option for hereditary forms of deafness in which protein misfolding plays a crucial role. In this context, it was recently shown that VRT534, a chemical chaperone, can influence the activity of mutated forms of human connexin 26 (Cx26), which has already been successfully used to treat cystic fibrosis. However, the exact interaction between VRT534 and Cx26 is hitherto unknown. Therefore, we investigated whether and how human Cx26 is able to bind VRT534. In addition, the DNA of Cx26 was modeled by AlphaFold3 to examine the effect on the monomeric connexin channel. Afterwards, the modeled structure of Cx26 was used to identify putative binding events via Diff-Dock-L. In HeLa cells expressing wild-type or mutated Cx26 (Cx26L90P, Cx26F161S, Cx26R184P), VRT534 restored the channel function only of Cx26L90P and Cx26R184P. The results show that VRT534 was able to bind with higher affinity at mutated forms of human Cx26 than at the wild-type. This approach is a novel way to identify how VRT534 interacts with mutated Cx26 and can be used to screen for compounds for the restoration of Cx26-associated hearing loss.