Two different genes encode GSK isoforms and that have
Two different genes encode GSK-3 isoforms α and β that have very similar catalytic domains but significantly differ in their N- and C-termini. The α and β isoforms split from a common precursor approximately at the emergence of vertebrates, suggesting that at least one of the isoforms took on a new function tied to the emergent vertebrate system (SP, unpublished data). Recent studies have addressed the possible differences between the two GSK-3 isoforms , , , , , yet their precise roles in different tissues are not fully understood. At present it seems that most GSK-3 inhibitors do not discriminate between the two isoforms. Development of isoforms-specific inhibitors is desired but will be a challenging task.
GSK-3 substrate competitive peptide inhibitor L803-mts Most of the protein kinase inhibitors developed so far are ATP competitors. This type of molecule competes for the ATP binding site of the kinase and often shows off-target effects due to serious limitations in their specificity. The low specificity of these inhibitors is due to the fact that the ATP binding site is highly conserved among diverse protein kinases. Non-ATP competitive inhibitors, on the other hand, such as substrate competitive inhibitors, are expected to be more specific as the substrate octanoic acid have a certain degree of variability among the various protein kinases. Substrate competitive inhibitors usually make a weak binding interaction with the enzyme (as does the substrate); this may be considered a disadvantage. It may also explain why this type of inhibitors has been overlooked in high-throughput screening efforts to select protein kinase inhibitors. Yet, chemical modifications can improve the specific binding affinity and the in vivo efficacy of substrate competitive inhibitors . Another problem concerns the in vitro assays used to test and evaluate given protein kinase inhibitor candidates. As the substrate's concentration in these assays is far above that of its physiological concentration, in vitro kinase assays do not reflect the true potency of substrate competitive inhibitors. Thus, in many cases, substrate competitive inhibitors will show better efficacy in cells than in cell-free conditions and we recommend that searches for this type of inhibitor be undertaken in cellular conditions. Overall, despite the difficulties in discovery and development, the search for substrate competitive inhibitors is worthwhile. In the case of GSK-3, the use of substrate competitive inhibitors may be particularly advantageous. GSK-3 is essential for the well-being of the cell and drastic inhibition of GSK-3 causes damage. This is demonstrated by the fact that GSK-3-knockout mice die late in gestation . In addition, ‘pathological’ GSK-3 activity does not exceed 2- to 3-fold over ‘normal’ levels. Thus, a moderate-to-weak inhibition of the enzyme (about 50%) is actually desired for treating conditions associated with elevated levels of GSK-3 activity. Understanding the mode of interaction of GSK-3 with its substrates is a central theme in the design and development of substrate competitive inhibitors. It has been known for many years that GSK-3 substrate recognition is unique as, unlike many other protein kinases, it requires prephosphorylation , . GSK-3 substrates have recognition motifs of S1XXXS2(p), where S1 is the site phosphorylated by GSK-3 and S2(p) is the ‘priming site’ prephosphorylated by a different kinase , . Apparently, the GSK-3 requirement for prephosphorylation is very strict. Lack of prephosphorylation and/or replacement of S2(p) with a phosphor-tyrosine residue or glutamic acid diminishes substrate phospho-tyrosine by GSK-3 . The three-dimensional structure of GSK-3β in crystals provided new insights into this phenomenon. The catalytic domain possesses a positive binding pocket (Arg 96, Arg 180, and Lys 205) that interacts with negatively charged groups such as phosphate and sulfate ions , . Mutation of Arg 96, Arg 180, and Lys 205 prevented GSK-3 phosphorylation of its substrates , .