Our results demonstrate proof of concept for SGK1 inhibition as a therapeutic target for cardiac arrhythmias and identify a small molecule lead, that with further medicinal chemistry can form the basis for novel and specific SGK1 inhibitors., we sought to identify small molecule SGK1 inhibitors for use as pharmacological probes to validate SGK1 as a therapeutic target in the treatment of cardiac arrhythmias.Using a combination of structure-based and ligand-based virtual screens (SBVS and LBVS) to query a reference library for small molecule inhibitors of SGK1, we clustered compounds based on previously validated metrics of activity (using a filtered library for Lipinski properties (rule-of-5)).Consequently, treatment of SCD in high risk patients relies on the implantation of internal cardiac defibrillators (ICDs), an invasive procedure associated with significant cost and potential morbidity.Therefore, there is a clear unmet need for novel therapeutic approaches to the treatment of arrhythmias in these disease populations.
Compounds with demonstrable SGK1 inhibitory potential were re-screened using an 8-point dosing curve to obtain IC50s.9-point dose response curves on selected inhibitors that showed SGK1 inhibition at 20 μM dosage, identified several potential lead compounds (Fig. Based on a cell culture assay (see below), we focused on 5377051 and determined its IC (in cell culture) to be 2.1 μM (Fig. As a first measure of the biological effectiveness of our putative SGK1 inhibitors in living cells, we examined the phosphorylation of an SGK1 target in cultured neonatal rat ventricular myocytes (NRVMs). At the time of infection, cells were also treated with various concentrations of SGK1 inhibitors 53770136, two of the molecules identified from the second-generation CADD-generated screen and validated as SGK1 inhibitors in the in vitro kinase assay. Inhibition of SGK1 in cultured cardiomyocytes by lead compounds.Cells were infected with either an adenovirus expressing a constitutively active form of SGK1 (Ad. Cell lysates were subjected to immunoblotting with an antibody against phospho (ser38)-glycogen synthase kinase beta (p-GSK3β), a well-established SGK1 substrate 48 hours after adenoviral infection. SGK1-CA caused an increase in p-GSK3β, which was inhibited by both 53770136 at sub-micromolar concentrations, suggesting inhibition of SGK1 activity in cardiomyocytes (Fig. (A) Inhibition of SGK1 activity assessed by GSK3β phosphorylation in CMs infected by Ad. (B) Different concentrations of 5377051 were assessed at 48 hours after treatment with immunoblotting (left) and quantitated (right). Effect of SGK1 inhibitors on Akt-induced phosphorylation of GSK3-beta in CMs infected by Ad.-myr-Akt was assessed by immunoblotting.The chemotypes identified using the LVBS in our second-generation screen were filtered as detailed in the methods section to exclude chemicals that do not dock within the ATP-binding site of the SGK1 model.This 2 generation screen yielded an additional 64 compounds that were again assessed using the in vitro kinase assay to exclude chemicals with poor SGK1 inhibitory activity.Using computer-aided drug discovery coupled with in vitro kinase assays, we identified a novel class of SGK1 inhibitors.