Methods for treatment of diseases where GSK 3-beta is desired, and methods to identify compounds usefule for that

#This invention relates generally to the field of medicinal chemistry, and specifically to methods of inhibiting the activity of glycogen synthase kinase 3 (“GSK3”) in non-neoplastic conditions, and to methods of identifying compounds that inhibit GSK3 via increasing protein kinase G (“PKG”) activity and inhibiting cGMP phosphodiesterase (“PDE”).

#The cellular actions of insulin include increased glucose transport, glycogen synthesis, and lipogenesis and decreased gluconeogenesis, glycogen and fat break down. The result is reduced hepatic glucose output and increased peripheral glucose utilization. In type II diabetes, most of the intracellular actions of insulin are reduced or absent. To find ways to combat insulin resistance in type II diabetes, much research has been focused on the insulin-regulated signaling pathways that normally mediate glucose production and/or glucose utilization.

#GSK3 was originally identified by its ability to phosphorylate and inactivate glycogen synthase (GS), the rate-limiting enzyme in converting glucose into glycogen. GSK3 is regulated by insulin through insulin receptor/PI3K/PKB signal cascade and directly by PKB phosphorylation. Insulin binding to insulin receptor causes the trans-phosphorylation and activation of the receptors intracellular tyrosine kinases and thus results in the activation of PI3K. Active PI3K phosphorylates and activates PKB. PKB phosphorylates Ser21 of GSK3□ and Ser9 of GSK3□ to inactivate GSK3. The phosphorylation of GS by GSK3 inactivates GS activity. Hence, when GSK 3 is inhibited, it can not phosphorylate GS. Therefore, GS remains active and synthesizes glycogen to keep the blood glucose level low.

#FIG. 1 is is a Western blot of SW480 cell lysates from drug-treated cell lysates in the absence of added cGMP, where cells were treated in culture for 48 hours with DMSO (0.03%, lanes 1 and 2), exsulind (200, 400 and 600 μM; lanes 3, 4, 5) and E4021 (0.1, 1 and 10 μM, lanes 6, 7, 8).

#FIG. 2 is a Western blot of SW480 cell lysates from drug-treated cell lysates in the presence of added cGMP, where cells were treated in culture for 48 hours with DMSO (0.03%, lanes 1 and 2), exsulind (200, 400 and 600uM; lanes 3, 4, 5) and E4021 (0. 1, 1 and 10 μM, lanes 6, 7, 8).

#FIG. 3 is a Western blot of SW480 cell lysates from drug-treated cell lysates where cell were treated for 48 hours with various concentrations of exisulind, compounds 38 and compound A as indicated. PKG1β indicates protein level of PKG1β and GSK3β-P-ser9 indicates the level of inactivation of GSK31β by phosphorylation.

#This invention is a method to inhibit GSK3 in a non-neoplastic cell in a way that also causes the degradation and phosphorylation of β-catenin. In brief, this invention involves a method of inhibiting GSK3 by activating PKG in a sustained manner in a non-neoplastic cell. If accomplished in the manner we teach, this can be done without inhibiting COX I or II enzymes.

#As we unexpectedly discovered, activated PKG inhibits GSK3. At the same time, activated PKG causes β-catenin phosphorylation, which as discussed above leads to β-catenin degradation by the ubiquitin-proteasome pathway. Thus, we have a method of inhibiting GSK3 that avoids the β-catenin accumulation associated with carcinogenesis.

#One manner of activating PKG is directly. Another method is indirect, e.g., by increasing the level of cGMP in a cell. cGMP can be increased in a cell by inhibiting the cGMP phosphodiesterase(s) (“PDE”) in the cell. cGMP PDEs include PDE1, PDE2 and PDE5.