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    • The fact that GSK recognition of its substrate

    2021-11-26

    The fact that GSK-3 recognition of its substrate involves pre-phosphorylation supports the rational for using synthetic phosphorylated peptides as substrate competitive inhibitors [57]. Phosphorylated peptides derived from the N-terminal pseudosubstrate sequence of GSK-3β were very weak inhibitors of GSK-3 (IC50s in the range of mM, unpublished results from our laboratory and [56], [58]). On contrast, a peptide derived from heat shock factor-1 (HSF-1), 1KEAPPAPPQS(p)P11 (termed L803), was found to be a potent inhibitor in an in vitro kinase assay [57]. A cell-permeable version of this peptide with myristic Auranofin attached to its N-terminus (L803-mts) was generated [57]. L803-mts is highly selective toward GSK-3, water soluble, and stable in serum, and shows low toxicity as determined by histopathology and single-dose maximal-tolerated dose (MTD) analyses in mice [57], [59]. L803-mts has biological activity in neuronal cells and in vivo systems. It provides neuroprotection in cultured neuronal cells exposed to the Parkinson ‘inducer’ 6-hydroxydopamine and to trisialoganglioside [60], [61]. Mice treated with L803-mts show that the compound has anti-depressive-like activity in the forced swimming tests [18] and after traumatic brain injury (TBI) [62]. Recently, L803-mts was shown to ameliorate intra-neuronal amyloid beta peptide loads and improve cognitive deficits in an Alzheimer's mouse model [35]. L803-mts has lower toxicity in neurons than other GSK-3 inhibitors [63]. The results obtained with L803-mts were encouraging enough to support further development. A structure-based design approach was undertaken combining mutagenesis and computational docking analyses. These studies suggested that substrates make important contacts with four residues within GSK-3β: Phe 67, Gln 89, Phe 93, and Asn 95 [49], [64] (Fig. 1A). Phe 67 resides in the P-loop and is a conserved site within the protein kinase family. As expected, mutation at this site completely impairs the enzyme activity [49]. Residues Gln 89, Phe 93, and Asn 95 reside in the “89–95 loop” that is highly conserved in GSK-3s of vertebrates [64], and which, together with the P-loop, delimits a promiscuous binding cavity for GSK-3 substrates (Fig. 1A). While Gln 89 and Asn 95 are located at the bottom of the cavity, Phe 93 is highly exposed and located opposite the phosphate binding pocket [64]. Recognition of substrate thus combines the promiscuity of the 89–95 binding loop, which allows interaction with a broad selection of substrates, with the strict demand for primed phosphorylation. These features of the protein together define substrate specificity. The studies with L803-mts indicated that the inhibitor has similar but non-overlapping interactions with GSK-3β as compared to a natural substrate [63] (Fig. 1B). Like the substrate, L803-mts docks into the phosphate binding pocket via the phosphorylated serine moiety (position 10), but unlike the substrate, L803-mts does not interact with Gln 89 or Asn 95. L803-mts forms a tight contact with Phe 93 within the 89-95 loop and also interacts with a “hydrophobic patch” (Val 214, Ile 217, and Tyr 216) located in the C-terminal lobe of the kinase, facing the ATP binding site [64] (Fig. 1B). We concluded that substrates and substrate competitive inhibitors interact with different geometries in the substrate binding trough of the kinase. The different binding modes likely enhance the inhibitory properties of the substrate competitive inhibitors. For example, in aqueous surroundings, hydrophobic interactions are energetically more favorable than polar and charged interactions, and therefore binding of the inhibitor to the hydrophobic patch might hamper its dissociation.
    Refinement of a substrate competitive inhibitor The finding that the binding geometry of L803-mts with GSK-3β is directed by hydrophobic interactions led us to predict that increasing the peptide's hydrophobicity would enhance inhibition. Indeed, replacement of the polar amino acid glutamine (at position 9) in L803-mts with alanine or proline improved inhibition by 4 and 10 fold, respectively [64]. In an attempt to further understand the binding mode of L803, we mapped preferred binding sites of amino acid side chains on the surface of GSK-3, using the ANCHORSmap procedure [65]. The low free energy anchoring spots in the substrate binding region are shown in Fig. 1A. This analysis indicated that the positive cavity strongly prefers a negative anchor (Glu), whereas the cavity near loop 89–95 is promiscuous and binds a variety of amino acids (Arg, Lys, His, Gln, Leu, Met, Phe, Trp, and Tyr). The computational study also suggested that GSK-3β residue Phe 93 would provide an interaction site for an additional residue of the inhibitor (Fig. 1A, C). Experimentally, addition of a phenylalanine residue to the C-terminus of L803-mts (L803F-mts) improved inhibition two fold [64].