A Novel Potent and Selective Inhibitor of Glycogen Synthase Kinase-3 (GSK-3)

Glycogen synthase kinase 3 (GSK‐3) is a multi‐tasking serine/threonine protein kinase that is constitutively active and has over 50 known substrates. There are two isoforms of GSK‐3, GSK‐3α and GSK‐3β, which share nearly identical sequences in their catalytic domains. GSK‐3 has been shown to regulate critical cellular functions and has been implicated in several human diseases including pathological inflammation, diabetes, and Alzheimer's. Elevated expression/activity of GSK‐3 can result in dysfunctional inflammatory cytokine production which can be a key component of pathological processes. While GSK‐3 inhibitors have been identified and there are ongoing clinical trials involving GSK‐3 inhibitors (e.g. Tideglusib), given the challenge of finding compounds that selectively inhibit a specified kinase and the plethora of known substrates for GSK‐3, there is a critical need for the identification of novel, specific inhibitors of GSK‐3. Our group has identified a small organic compound, COB‐187, that exhibits potent inhibition of GSK‐3 activity in a non‐cell‐based molecular assay. An extensive screen utilizing 414 kinase assays (representing 404 unique kinases) revealed that COB‐187 is highly specific for GSK‐3 and inhibits the activity of both GSK‐3 isoforms. COB‐187 inhibited the activity of GSK‐3 with greater potency than Tideglusib and was significantly more selective. This result led us to hypothesize that COB‐187 inhibits cellular GSK‐3 activity. Thus, PMA differentiated THP‐1 human macrophages (ATCC, Manassas, VA) were used to investigate the effect of COB‐187 on cellular GSK‐3 function. GSK‐3 is known to phosphorylate β‐catenin at Ser33/37/Thr41 which targets this protein for degradation. Thus, a hallmark of GSK‐3 inhibition is a decrease in Ser33/37/Thr41 phosphorylation and an increase in β‐catenin levels. In addition, there is an increase in β‐catenin phosphorylation at Ser675. Western blot analysis of protein isolated from COB‐187 treated (5 hr.) THP‐1 cells compared to carrier control (DMSO) treated cells, revealed a dose‐dependent reduction in β‐catenin phosphorylation at Ser33/37/Thr41 and consequent increase in β‐catenin levels as well as an increase in phosphorylation at Ser675. Immunocytochemistry showed increased levels of β‐catenin and localization to the nucleus in COB‐187 treated cells. The human cytokine proteome profiler array kit from R&D systems (Minneapolis, MN) revealed marked a reduction in the expression of inflammatory cytokines (e.g. IL‐1β, TNF‐α, IFN‐γ, CXCL10) in LPS stimulated THP‐1 macrophages treated with COB‐187. Based on these results, COB‐187 appears to be a specific and potent GSK‐3 inhibitor and might represent an avenue for the development of novel small molecule therapeutics for diseases caused by aberrant GSK‐3 activity. Support or Funding Information NIH R15GM110602 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .