Glycogen synthase kinase-3 inhibitors

Details Glycogen synthase kinase-3 inhibitors Glycogen synthase kinase-3 inhibitors

2001-09-14

2004-08-24

Russel, Jeffrey Edwin (Department: 1654)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S015000, C514S007600, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000

Reexamination Certificate

active

06780625

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to novel peptide inhibitors of glycogen synthase kinase-3 (GSK-3) and the use thereof for regulating biological conditions mediated by GSK-3 activity. The invention particularly relates to the use of such inhibitors to potentiate insulin signaling in type-2 diabetics, to treat neurodegenerative disorders as well as affective disorders, and to reduce neuronal cell death resulting from ischemic insult. The invention further relates to a computer-assisted method of structure based drug design of GSK-3 inhibitors based on the three-dimensional structure of a peptide substrate of GSK-3.
2. Description of the Related Art
Protein kinases, the enzymes that phosphorylate protein substrates, are key players in the signalling of extracellular events to the cytoplasm and the nucleus, and take part in practically any event relating to the life and death of cells, including mitosis, differentiation and apoptosis. As such, protein kinases have long been favorable drug targets. However, they have presented a problem in that the inhibition of many kinases could lead to cell death, because their activity is crucial to the well-being of the cell. Although this is a desirable effect for anticancer drugs, it is a major drawback for most other therapeutics. As a member of the family of protein kinases, glycogen synthase kinase-3 (GSK-3) is a cytoplasmic serine-threonine kinase that is involved in insulin signaling and metabolic regulation, as well as in Wnt signaling and the scheme of cell fate during embryonic development. Two similar isoforms of the enzyme, termed GSK-3&agr; and GSK-3&bgr;, have been identified. The idea that GSK-3 is a favorable drug target among the protein kinase family is based on the fact that unlike other protein kinases, which are typically activated by signaling pathways, GSK-3 is normally activated in resting cells, and its activity is attenuated by the activation of certain signaling pathways such as after the binding of insulin to its cell-surface receptor. Activation of the insulin receptor leads to the activation of protein kinase B (PKB, also called Akt), which in turn phosphorylates GSK-3, thereby inactivating it. The inhibition of GSK-3 presumably leads to the activation of glycogen synthesis. The intricate insulin-signaling pathway is further complicated by negative-feedback regulation of insulin signaling by GSK-3 itself, which phosphorylates insulin-receptor substrate-1 on serine residues (Eldar-Finkelman et al., 1997).
Therefore, synthetic GSK-3 inhibitors might mimic the action of certain hormones and growth factors, such as insulin, which use the GSK-3 pathway. In certain pathological situations, this scheme might permit the bypassing of a defective receptor, or another faulty component of the signaling machinery, so that the biological signal will take effect even when some upstream players of the signaling cascade are at fault, such as in non-insulin-independent type 2 diabetes.
The regulation of glycogen catabolism in cells is a critical biological function that involves a complex array of signaling elements, including the hormone insulin. Through a variety of mediators, insulin exerts it regulatory effect by increasing the synthesis of glycogen by glycogen synthase (GS). A key event in insulin action is the phosphorylation of insulin receptor substrates (IRS-1, IRS-2) on multiple-tyrosine residues, which results in simultaneous activation of several signaling components, including PI3 kinase (Myers et al, 1992)). Similarly, the activity of glycogen synthase is suppressed by its phosphorylation.
One of the earliest changes associated with the onset of type 2 (non-insulin dependent) diabetes is insulin resistance. Insulin resistance is characterized by hyperinsulemia and hyperglycemia. Although the precise molecular mechanism underlying insulin resistance is unknown, defects in downstream components of the insulin signaling pathway may be the cause. Among the downstream components of insulin signaling is glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase that has recently been recognized as an important signaling molecule in a variety of cellular processes. High activity of GSK-3 impairs insulin action in intact cells (Eldar-Finkelman et al, 1997). This impairment results from the phosphorylation of insulin receptor substrate-1 (IRS-1) serine residues by GSK-3. Likewise, increased GSK-3 activity expressed in cells results in suppression of glycogen synthase activity (Eldar-Finkelman et al, 1996). The laboratory of the present inventor found that GSK-3 activity is significantly increased in epididymal fat tissue of diabetic mice (Eldar-Finkelman et al, 1999). Subsequently, increased GSK-3 activity was detected in skeletal muscle of type 2 diabetes patients (Nickoulina et al, 2000). Thus, the inhibition of GSK-3 activity may represent a way to increase insulin activity in vivo.
GSK-3 is also considered to be an important player in the pathogenesis of Alzheimer's disease. GSK-3 was identified as one of the kinases that phosphorylates tau, a microtubule-associated protein, that is responsible for formation of paired helical filaments (PHF), an early characteristics of Alzheimer's disease. Apparently, abnormal hyperphosphorylation of tau is the cause for destabilization of microtubules and PHF formation. Despite the fact that several protein kinases were shown to promote phosphorylation of tau, only GSK-3 phosphorylation directly affected tau ability to promote microtubule self-assembly (Hanger et al., 1992; Mandelkow et al., 1992; Mulot et al., 1994; Mulot et al., 1995). Further evidence came from studies of cells overexpressing GSK-3 and from transgenic mice that specifically expressed GSK-3 in brain. In both cases GSK-3 led to generation of the PHF like epitope tau (Lucas et al., 2001).
Another mechanism that can link GSK-3 with Alzheimer's disease is its role in cell apoptosis. The fact that insulin is a survival factor of neurons (Barber et al., 2001) and initiates its anti-apoptotic action through activation of PI3 kinase and PKB (Barber et al., 2001), suggested that GSK-3, which is negatively regulated by these signaling components, promotes neuronal apoptosis. Studies have indeed confirmed this view, and showed that GSK-3 is critically important in life and death decision. Furthermore, its apoptotic function was shown to be independent of PI3 kinase. Overexpression of GSK-3 in PC12 cells caused apoptosis (Pap et al., 1998). Activation of GSK-3 in cerebellar granule neurons mediated migration and cell death (Tong et al., 2001). In human neuroblastoma SH-SY5Y cells, overexpression of GSK-3 facilitated stauroaporine-induced cell apoptosis (Bijur et al., 2000). Additional studies also indicated that inhibition of GSK-3 rescued cell death (as expected). These studies showed that expression of Frat1, a GSK-3 &bgr; inhibitor, was sufficient to rescue neurons from death induced by inhibition of PI3 kinase (Crowder et al., 2000). Recent work described the development of small molecules SB-216763 and SB-415286 (Glaxo SmithKline Pharmaceutical) that specifically inhibited GSK-3. Treatment of primary neurons with these compounds protected neuronal death induced by reduction in PI3 kinase activity (Cross et al., 2001).
Another implication of GSK-3 was detected in the context of affective disorders, i.e., bipolar disorder and manic depression. This linkage was based on the findings that lithium, a primary mood stabilizer frequently used in bipolar disease is a strong and specific inhibitor of GSK-3 at the therapeutic concentration range used in clinics (Klein et al., 1996; Stambolic et al., 1996; Phiel et al., 2001). The discovery has led to a series of studies that were undertaken to determine if lithium can mimic loss of GSK-3 activity in cellular processes. Indeed, lithium was shown to cause activation of glycogen synthesis (Cheng et al., 1983), stabilization and accumulation of &bgr;-catenine (Stambolic et al., 1996), induction of axis duplication in

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