Abstract
Introduction: DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Gain of function mutations in the KCNJ11 gene, encoding the KIR6.2 subunit of the IKATP potassium channel, stand at the basis of most forms of DEND syndrome. In a previous search for existing drugs with the potential of targeting Cantú Syndrome, also resulting from increased IKATP, we found a set of candidate drugs that may also possess the potential to target DEND syndrome. In the current work, we combined Molecular Modelling including Molecular Dynamics simulations, with single cell patch clamp electrophysiology, in order to test the effect of selected drug candidates on the KIR6.2 WT and DEND mutant channels. Methods: Molecular dynamics simulations were performed to investigate potential drug binding sites. To conduct in vitro studies, KIR6.2 Q52R and L164P mutants were constructed. Inside/out patch clamp electrophysiology on transiently transfected HEK293T cells was performed for establishing drug-channel inhibition relationships. Results: Molecular Dynamics simulations provided insight in potential channel interaction and shed light on possible mechanisms of action of the tested drug candidates. Effective IKIR6.2/SUR2a inhibition was obtained with the pore-blocker betaxolol (IC50 values 27–37 μM). Levobetaxolol effectively inhibited WT and L164P (IC50 values 22 μM) and Q52R (IC50 55 μM) channels. Of the SUR binding prostaglandin series, travoprost was found to be the best blocker of WT and L164P channels (IC50 2–3 μM), while Q52R inhibition was 15–20% at 10 μM. Conclusion: Our combination of MD and inside-out electrophysiology provides the rationale for drug mediated IKATP inhibition, and will be the basis for 1) screening of additional existing drugs for repurposing to address DEND syndrome, and 2) rationalized medicinal chemistry to improve IKATP inhibitor efficacy and specificity.
Highlights
DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes
The sulfonylurea drug glibenclamide potently inhibited inward and outward currents of the WT channels (IC50 0.9 ± 0.3 μM, Hill coefficient −0.41 and 0.6 ± 0.2 μM, Hill coefficient −0.40 for inward and outward currents respectively), whereas Q52R channels were insensitive for glibenclamide mediated block (
We expanded our previous findings, in which we demonstrated that betaxolol and Travoprost were able to inhibit IKIR6.2/SUR2a outward currents
Summary
DEND syndrome is a rare channelopathy characterized by a combination of developmental delay, epilepsy and severe neonatal diabetes. Whereas KIR6.2 channels harboring mild gain of function mutations result in transient or permanent ND, strong gain of function mutant KIR6.2 channels on the other hand associate with a complex phenotype of developmental delay, muscle weakness, dysmorphic features, epilepsy and neonatal diabetes, known as the DEND syndrome (Hattersley and Ashcroft, 2005). These strong gain of function mutations are generally of the indirect class
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