The absolute requirement for substrate prephosphorylation ra

The absolute requirement for substrate prephosphorylation raised the possibility that short phosphorylated peptides might serve as selective substrate competitive inhibitors. A set of phosphorylated peptides patterned after known GSK-3 substrates was generated and shown to inhibit GSK-3 in vitro in the micromolar range (100–300 μM) [37]. Peptide L803 was found to be the best inhibitory sequence. L803 is derived from heat shock factor-1 (HSF-1). L803 has the sequence KEAPPAPPQS(p)P) and contains two modified residues; the GSK-3 phosphorylation site and the glutamic PPDA synthesis upstream to the P1 site were both replaced by alanine [37]. In an advanced version of L803, a myristic acid was attached to the N-terminus of the peptide, now termed L803-mts, to enhance cell permeability [37]. L803-mts was very specific for GSK-3 and did not inhibit a wide repertoire of protein kinases [37] (HE-F, unpublished results). It was stable in serum and showed low toxicity as determined by histopathology analysis and single-dose, maximal-tolerated dose (MTD) tests (HE-F, unpublished results). The ability of L803-mts to reduce phosphorylation of physiological targets of GSK-3 such as glycogen synthase, IRS-1, and β-catenin further provided a proof of concept [37] (HE-F, unpublished results). The in vivo impact of L803-mts has been tested in diabetes and CNS models. Insulin resistant and obese ob/ob mice were injected intraperitoneally (i.p.) daily with L803-mts for 3 weeks. L803-mts reduced plasma glucose levels and improved glucose tolerance; it did not change body weight or food consumption compared to controls [38]. Detailed investigation of the insulin-sensitive tissues indicated that L803-mts suppresses hepatic gluconeogenesis, increases glucose transporter-4 (GLUT4) in the skeletal muscle and increases glycogen content in both tissue [38]. Similar results were reported in a different model of high-fat diet-induced diabetes [39]. Together, the studies suggested that inhibition of GSK-3 by L803-mts reduces hyperglycemia and reverses the diabetic phenotype. GSK-3 was implicated in various aspects of the nervous system, including cell survival, cell polarity, and synaptic plasticity [9], [40], [41], [42]. The finding that the mood stabilizer lithium is an effective GSK-3 inhibitor further implicated GSK-3 in cognition and behavior [43]. We evaluated the impact of L803-mts on depressive behavior using the forced swim test (FST), a common protocol used to assess activity of antidepressants [44]. In this test, the animal is subjected to a swimming trial and the immobility time, namely the time it spends without moving, is measured. The immobility time reflects the depressive degree of the animal [44]. Administration of L803-mts significantly reduced the mouse-immobility time in the FST; in addition, L803-mts increased β-catenin levels in the mouse hippocampus [45]. These results indicated that inhibition of GSK-3 can promote antidepressive like activity; they further provided proof of concept for the in vivo inhibitory capacity of L803-mts in the brain. The important role of GSK-3 in behavior was also demonstrated using heterozygote GSK3β+/− mice and using wild-type mice treated with the GSK-3 inhibitor AR-A014418 [46], [47]. In another model system of traumatic brain injury (TBI), mice developed depressive behavior after the insult and this was abolished by pretreatment with L803-mts [48]. L803-mts has also been tested in models of Parkinson's disease, axon growth, and schizophrenia with positive results [49], [50], [51].
Design of substrate competitive inhibitors is mainly based on our understanding of the kinase/substrate binding mode. The prerequisite of pre-phosphorylation of GSK-3′s substrates suggests that phosphorylation is an important element in substrate recognition. Still, it is likely that the GSK-3 catalytic core makes specific interactions with other regions of the substrate. The availability of the 3D structures of GSK-3β and a phosphorylated peptide derived from its substrate CREB (pCREB) enabled computational attack of this problem. Protein–protein docking analysis of pCREB with GSK-3β identified new docking sites, Phe 67, Gln 89, and Asn 95, which are spatially close to the active site and which interact with the substrate's core (i.e., S1XXXS2(p)). Specifically, Arg 135 of pCREB forms hydrogen bonds with Gln 89 and Asn95 of GSK-3β and Tyr 134 of pCREB interacts with Phe 67 of GSK-3β [33]. In addition, and as expected, the priming site, Ser 133, interacted with the phosphate binding pocket of GSK-3β (i.e., Arg 96, Arg 180, Lys 205). Mutations of Gln 89, Asn 95, or Phe 67 reduced the ability of GSK-3β to phosphorylate its substrates [33]. Mutation of both Gln 89 and Asn 95 severely impaired GSK-3 phosphorylation of peptide substrates derived from CREB, IRS-1, and glycogen synthase (Fig. 2A). The polar natures of Gln 89 and Asn 95 allow interaction with a wide variety of polar/charged groups. This may explain the broad specificity of GSK-3. Indeed, we could identify a polar or charged amino acid located at position +2 downstream to the phosphorylated priming site (S2(p)) in a number of substrates (Fig. 2B). We suggest that like CREB/Arg 135, these residues interact with Gln 89 and Asn 95. The preferential conservation of Gln 89 and Asn 95 across the GSK-3 family [33] also implies a special role for these residues in substrate recognition. Phe 67, on the other hand, is located in the conserved P-loop, and is more generally conserved across protein kinases. Mutation of Phe 67, however, did not impair the catalytic activity of GSK-3 per se[33], suggesting that this residue is indeed important for substrate recognition.