Potent Glycogen Synthase Kinase-3 Inhibitors Research Articles

GSK-3: An "Ace" Among Kinases.

Background: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase known to be participating in the regulation of β-catenin signaling (Wnt signaling). They help in the establishment of a multicomponent destruction complex that stimulates phosphorylation, leading to the destruction of β-catenin. Evidence about the role of increasingly active β-catenin signaling is involved in many forms of human cancer. The understanding of GSK-3 remains elusive as recent research aims to focus on developing potent GSK-3 inhibitors to target this kinase. Objective: This short review aims to highlight the regulation of GSK-3 with emphasis on Wnt signaling while highlighting its interaction with miRNAs corresponding to pluripotency and epithelial mesenchymal transition substantiating this kinase as an "Ace" among kinases in regulation of cellular processes. Result: Significant findings of miRNA regulation by GSK-3 exemplify the underpinnings of kinase-mediated transcriptional regulation in cancers. Conclusion: The review provides evidence on the role of GSK-3 as a possible master regulator of proteins and noncoding RNA, thereby implicating the fate of a cell.

Discovery of 2-(Anilino)pyrimidine-4-carboxamides as Highly Potent, Selective, and Orally Active Glycogen Synthase Kinase-3 (GSK-3) Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that serves as an important regulator of a broad range of cellular functions. It has been linked to Alzheimer's disease as well as various other diseases, including mood disorders, type 2 diabetes, and cancer. There is considerable evidence indicating that GSK-3β in the central nervous system plays a role in the production of abnormal, hyperphosphorylated, microtubule-associated tau protein found in neurofibrillary tangles associated with Alzheimer's disease. A series of analogues containing a pyrimidine-based hinge-binding heterocycle was synthesized and evaluated, leading to the identification of highly potent GSK-3 inhibitors with excellent kinase selectivity. Further evaluation of 34 and 40 in vivo demonstrated that these compounds are orally bioavailable, brain-penetrant GSK-3 inhibitors that lowered levels of phosphorylated tau in a triple-transgenic mouse Alzheimer's disease model.

The novel potent GSK3 inhibitor AF3581 reverts fragile X syndrome phenotype.

Glycogen-synthase kinase 3 (GSK3) is a kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK3 has been linked to several disease conditions such as fragile X syndrome (FXS). Recent evidences demonstrating an increased activity of GSK3 in murine models of FXS, suggest that dysregulation/hyperactivation of the GSK3 path should contribute to FXS development. A likely possibility could be that in FXS there is a functional impairment of the upstream inhibitory input over GSK3 thus making overactive the kinase. Since GSK3 signaling is a central regulatory node for critical neurodevelopmental pathways, understanding the contribution of GSK3 dysregulation to FXS, may provide novel targets for therapeutic interventions for this disease. In this study we used AF3581, a potent GSK3 inhibitor that we recently discovered, in an in vivo FXS mouse model to elucidate the crucial role of GSK3 in specific behavioral patterns (locomotor activity, sensorimotor gating and social behavior) associated with this disease. All the behavioral alterations manifested by Fmr1 knockout mice were reverted after a chronic treatment with our GSK3 inhibitor, confirming the importance of this pathway as a therapeutic target.

Tideglusib inhibition of GSK3 promotes the oxidative muscle phenotype and reduces serum creatine kinase in D2 mdx mice

Introduction Duchenne muscular dystrophy (DMD) is an X-linked disorder caused by an absence of dystrophin that compromises membrane integrity, ultimately resulting in muscle weakness, wasting, and premature death. The fast glycolytic muscle fibres are known to be most susceptible to dystrophic pathology, while slow oxidative fibres are less affected. Thus, promoting the slow oxidative phenotype has become a viable therapeutic strategy. Recent work from our lab has shown that inhibiting the enzyme glycogen synthase kinase 3 (GSK3) can promote the slow oxidative phenotype leading to enhancements in fatigue resistance. Furthermore, we have shown that inhibiting GSK3 augments muscle specific force production and myoblast fusion. Therefore, inhibiting GSK3 may aid in alleviating dystrophic pathology. Tideglusib is a potent GSK3 inhibitor currently undergoing clinical trials for myotonic dystrophy, another form of muscular dystrophy. Here, we tested whether treating the DMD preclinical mdx mouse with tideglusib would alter muscle oxidative phenotype, improve muscle function, and reduce serum creatine kinase (CK) levels, a marker of cellular damage. Methods Male DBA/2J wild type (WT) and mdx mice were ordered from Jackson Laboratories at 5-6 weeks of age. Three groups were included in this study; 1) WT healthy control, 2) mdx tideglusib (10 mg/kg/day, via oral gavage), and 3) mdx vehicle (26% peg400, 15% Chremaphor EL and water). The mdx-tideglusib and vehicle mice underwent their respective treatments for 2 weeks with ad libitum access to food and water. Subsequently, all mice were subjected to a hangwire test to assess muscle function. Mice were then euthanized and their serum extracted for CK activity analyses using a commercially available kit that was fitted onto a 96-well plate. Extensor digitorum longus (EDL) muscles were collected and homogenized for Western blotting to investigate phosphorylated (serine 9) and total GSK3b content along with myosin heavy chain (MHC) I and IIa levels. Results The hangwire test results showed that mdx mice treated with tideglusib were able to sustain hanging on the wire for a significantly longer period of time compared with the mdx vehicle group (p = 0.03). Serum CK analyses revealed that while the mdx vehicle group had significantly higher levels of activity compared with WT (p = 0.009), there was a 30% reduction in the mdx tideglusib mice that was no longer significantly different from WT. Western blotting revealed that though phosphorylated GSK3b levels were unaltered, tideglusib treatment led to a significant reduction in GSK3b content compared with vehicle (p = 0.04). Additionally, there was a significant increase in the oxidative MHC isoforms (I and IIa, p = 0.02) in the mdx tideglusib group compared with vehicle. Conclusions Our results demonstrate the potential use of tideglusib for DMD. We show that tideglusib treatment inhibited GSK3 in mdx mice through a reduction in total GSK3 content. In turn, we also found a significant increase in oxidative MHC isoforms (I and IIa), which was associated with enhanced muscle performance and reduced muscle damage.

Protective properties of glycogen synthase kinase-3 inhibition against doxorubicin-induced oxidative damage to mouse ovarian reserve

Chemotherapy induces ovarian failure in female children and young female cancer survivors. It has been shown that doxorubicin (DOX), an antitumor drug of the anthracycline group, causes gonadotoxicity via the stimulation of oxidative stress. The inhibition of glycogen synthase kinase-3 (GSK-3) was reported to be able to regulate oxidative stress. The present study assessed whether GSK-3 inhibition confers protection to the ovary against DOX-induced oxidative stress damage. An intraperitoneal injection of DOX was used to induce oxidative damage in the mouse ovary, while the inhibition of GSK-3 was achieved by the administration of SB216763, a small and potent GSK-3 inhibitor. DOX increased the mRNA expression levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the antioxidant genes heme-oxygenase-1 (HO-1) and catalase (CAT), while reducing the levels of glutathione peroxidase (GSH-Px) and superoxide dismutase-1 (SOD-1) compared with those of the control. When the GSK-3 inhibitor was combined with DOX increased more expression levels of Nrf2 mRNA and restored levels of GSH-Px and SOD-1 mRNA similar to those in the control group. DOX remarkably decreased the Nrf2 protein expression compared to that in the control, which was significantly associated with increased MDA levels. Interestingly, the SB216763 and DOX coadministration restored the Nrf2 protein expression and MDA levels to levels that were similar to those in the control. DOX significantly decreased the number of primordial, primary, preantral and antral follicles while increasing the number of atretic follicles compared to those in the control. SB216763 caused the drastic recovery of these follicles from the DOX effects. SB216763 and DOX coadministration significantly reduced the apoptotic index compared with that with DOX treatment. DOX decreased the serum AMH and E2 levels and increased the FSH levels compared to those in the controls. However, SB216763 and DOX coadministration restored AMH and E2 while decreasing the FSH levels compared to those in the DOX-treated group. In addition, SB216763 and DOX coadministration reduced the mature oocyte abnormalities that resulted from DOX-induced ovarian damage. Given these results, we suggest that GSK-3/Nrf2 is a promising protective pathway against doxorubicin-induced oxidative damage to the ovaries of females at reproductive ages. Thus, GSK-3 could be an attractive target for the research and development of new drugs for preserving ovarian function during chemotherapy.

Pharmacophore feature-based virtual screening for finding potent GSK-3 inhibitors using molecular docking and dynamics simulations.

Glycogen synthase kinase-3 (GSK-3) is a multitasking serine/threonine protein kinase, which is associated with the pathophysiology of several diseases such as diabetes, cancer, psychiatric and neurodegenerative diseases. Tideglusib is a potent, selective, and irreversible GSK-3 inhibitor that has been investigated in phase II clinical trials for the treatment of progressive supranuclear palsy and Alzheimer's disease. In the present study, we performed pharmacophore feature-based virtual screening for identifying potent targetspecific GSK-3 inhibitors. We found 64 compounds that show better GSK-3 binding potentials compared with those of Tideglusib. We further validated the obtained binding potentials by performing 20-ns molecular dynamics simulations for GSK-3 complexed with Tideglusib and with the best compound found via virtual screening in this study. Several interesting molecular-level interactions were identified, including a covalent interaction with Cys199 residue at the entrance of the GSK-3 active site. These findings are expected to play a crucial role in the binding of target-specific GSK-3 inhibitors.

Inhibitors of Glycogen Synthase Kinase 3 with Exquisite Kinome-Wide Selectivity and Their Functional Effects.

The mood stabilizer lithium, the first-line treatment for bipolar disorder, is hypothesized to exert its effects through direct inhibition of glycogen synthase kinase 3 (GSK3) and indirectly by increasing GSK3's inhibitory serine phosphorylation. GSK3 comprises two highly similar paralogs, GSK3α and GSK3β, which are key regulatory kinases in the canonical Wnt pathway. GSK3 stands as a nodal target within this pathway and is an attractive therapeutic target for multiple indications. Despite being an active field of research for the past 20 years, many GSK3 inhibitors demonstrate either poor to moderate selectivity versus the broader human kinome or physicochemical properties unsuitable for use in in vitro systems or in vivo models. A nonconventional analysis of data from a GSK3β inhibitor high-throughput screening campaign, which excluded known GSK3 inhibitor chemotypes, led to the discovery of a novel pyrazolo-tetrahydroquinolinone scaffold with unparalleled kinome-wide selectivity for the GSK3 kinases. Taking advantage of an uncommon tridentate interaction with the hinge region of GSK3, we developed highly selective and potent GSK3 inhibitors, BRD1652 and BRD0209, which demonstrated in vivo efficacy in a dopaminergic signaling paradigm modeling mood-related disorders. These new chemical probes open the way for exclusive analyses of the function of GSK3 kinases in multiple signaling pathways involved in many prevalent disorders.

Glycogen Synthase Kinase-3 (GSK-3)-Targeted Therapy and Imaging.

Glycogen synthase kinase-3 (GSK-3) is associated with various key biological processes, including glucose regulation, apoptosis, protein synthesis, cell signaling, cellular transport, gene transcription, proliferation, and intracellular communication. Accordingly, GSK-3 has been implicated in a wide variety of diseases and specifically targeted for both therapeutic and imaging applications by a large number of academic laboratories and pharmaceutical companies. Here, we review the structure, function, expression levels, and ligand-binding properties of GSK-3 and its connection to various diseases. A selected list of highly potent GSK-3 inhibitors, with IC50 <20 nM for adenosine triphosphate (ATP)-competitive inhibitors and IC50 <5 μM for non-ATP-competitive inhibitors, were analyzed for structure activity relationships. Furthermore, ubiquitous expression of GSK-3 and its possible impact on therapy and imaging are also highlighted. Finally, a rational perspective and possible route to selective and effective GSK-3 inhibitors is discussed.

Glycogen Synthase Kinase-3 (GSK3) Inhibition with Lithium in Combination with Tretinoin Induces Differentiation in Non-Promyelocytic Acute Myeloid Leukemia (AML)

Lithium (Li) is a GSK3 inhibitor that induces differentiation and anti-proliferation of AML cells in vitro. Pre-clinical studies demonstrated GSK3 inhibition leads to increased expression of the receptor of tretinoin, the retinoic acid receptor. We conducted a phase I trial of Li in combination with tretinoin for non-promyelocytic AML based on pre-clinical studies demonstrating synergistic AML cell differentiation using the two-drug combination. We report laboratory correlative data demonstrating AML cell differentiation using the combination of Li and tretinoin.Methods. 12 relapsed or refractory AML patients were enrolled. Li was administered at 300mg three times daily (twice daily in patients age ≥60) continuously and adjusted to meet the target Li plasma concentration of 0.6-1mEq/L; tretinoin doses were 22.5 mg/m2/day (dose level 1) and 45 mg/m2/day (dose level 2) administered on days 1-7 and 15-21 of a 28-day cycle. Li was given for a 3-day lead-in period prior to cycle 1. 7 patients were evaluable for laboratory correlative studies. Blood and/or bone marrow samples were collected at baseline, day 1 and day 8 of cycles 1 and 2 and at the end of the study period. Specimens were processed only if ≥5% leukemic cells were available for analysis. Immunophenotyping was performed by multi-parameter flow cytometry using fluorochrome-labeled antibodies to CD11b, CD14 and CD15 as markers for monocytic and/or granulocytic differentiation. Cells were concurrently stained for CD34, CD117 and CD38 for identification of the leukemic blast and stem cell population (CD34+CD38-). After fixation and permeabilization using a formaldehyde-based solution, intracellular staining was performed for phosphorylated Serine-9 GSK3β (pGSK3β) as a marker for GSK3 inhibition. Li directly binds GSK3 and causes reversible enzyme inhibition by Ser9 phosphorylation.Results. The target serum concentration of Li was achieved in all patients and found to inhibit GSK3 in leukemic cells. pGSK3β measured by mean fluorescence intensity (MFI) increased by a median of 1067 (168-1738) by day 30 in 4 patients with samples available for analysis; the earliest increase in pGSK3β MFI occurred at day 1 after the Li lead-in period. Six subjects (86%) demonstrated differentiation as defined by ≥10% increase in a cell population expressing at least one of the surface markers associated with monocytic or granulocytic differentiation, with median increase 27.4% (range 10.0-59.2%). Maximum increase occurred at median day 30 (range 8-60) of the study period. Three patients (50%) exhibited differentiation in both the CD34+ or CD117+ blast population and in non-blast cells. Two subjects (33%) had increased differentiation in the non-blast population only, and 1 (17%) had increased differentiation in the blast population only. Three patients had clinically stable disease (defined as no increase in blasts for ≥4 weeks from study start) and showed a median increase in differentiated cells of 27.4% (17.2-37.6%) compared to a 14.8% (10.0-19.6%) increase in the 3 patients with progressive disease (p=0.44). Four patients had day 60 bone marrow available for analysis; in 3 patients, there was a median of 29.3% (range 12-32%) increase in cells expressing at least one differentiation surface marker in the CD34+ or CD117+ blast population. In 6 subjects with evaluable sample, a median relative reduction of 67% (37-98%) of the AML stem cell population (CD34+ CD38-) was seen in the blood at day 8 to 60.Conclusions. The combination of lithium and tretinoin induces differentiation in leukemic cells. Unlike most cytotoxic AML therapies which target rapidly dividing cells, this strategy may be effective in targeting the AML stem cell population. Response to differentiation therapy was variable, suggestive of the heterogeneity of the disease. Li induced a modest effect on GSK3 inhibition at clinically achievable plasma concentrations. Though this study demonstrated the ability to inhibit GSK3 in leukemic blasts and induce differentiation, higher levels of GSK3 inhibition are likely necessary for greater clinical efficacy. As Li concentrations cannot be increased significantly due to drug toxicity, future studies investigating the use of more potent GSK3 inhibitors in AML are needed to further investigate this strategy. DisclosuresWald:Invenio Therapeutics: Equity Ownership.

Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging.GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28days exposure in rats. After 7days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH1–34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT.GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption.

Anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3

Glycogen synthase kinase 3 (GSK-3) plays a central role in cellular energy metabolism, and dysregulation of GSK-3 activity is implicated in a variety of metabolic disorders, including obesity, type 2 diabetes, and cancer. Hence, GSK-3 has emerged as an attractive target molecule for the treatment of metabolic disorders. Therefore, this research focused on identification and characterization of a novel small-molecule GSK-3 inhibitor. Compound 1a, a structure based on 3-hydroxychromone bearing isothiazolidine-1,1-dione, was identified from chemical library as a highly potent GSK-3 inhibitor. An in vitro kinase assay utilizing a panel of kinases demonstrated that compound 1a strongly inhibits GSK-3β. The potential effects of compound 1a on the inactivation of GSK-3 were confirmed in human liver HepG2 and human embryonic kidney HEK293 cells. Stabilization of glycogen synthase and β-catenin, which are direct targets of GSK-3, by compound 1a was assessed in comparison with two other GSK-3 inhibitors: LiCl and SB-415286. In mouse 3T3-L1 preadipocytes, compound 1a markedly blocked adipocyte differentiation. Consistently, intraperitoneal administration of compound 1a to diet-induced obese mice significantly ameliorated their key symptoms such as body weight gain, increased adiposity, dyslipidemia, and hepatic steatosis due to the marked reduction of whole-body lipid level. In vitro and in vivo effects were accompanied by upregulation of β-catenin stability and downregulation of the expression of several critical genes related to lipid metabolism. From these results, it can be concluded that compound 1a, a novel small-molecule inhibitor of GSK-3, has potential as a new class of therapeutic agent for obesity treatment.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Synthesis and biological evaluation of glycogen synthase kinase 3 (GSK-3) inhibitors: An fast and atom efficient access to 1-aryl-3-benzylureas

The glycogen synthase kinase 3 (GSK-3) is implicated in multiple cellular processes and has been linked to the pathogenesis of Alzheimer's disease (AD). In the course of our research topic we synthesized a library of potent GSK-3 inhibitors. We utilized the urea scaffold present in the potent and highly selective GSK-3 inhibitor AR-A014418 (AstraZeneca). This moiety suits both (a) a convergent approach utilizing readily accessible building blocks and (b) a divergent approach based on a microwave heating assisted Suzuki coupling. We established a chromatography-free purification method to generate products with sufficient purity for the biological assays. The structure-activity relationship of the library provided the rationale for the synthesis of the benzothiazolylurea 66 (IC(50)=140 nM) and the pyridylurea 62 (IC(50)=98 nM), which displayed two to threefold enhanced activity versus the reference compound 18 (AR-A014418: IC(50)=330 nM) in our assays.

Rational design of 4-amino-5,6-diaryl-furo[2,3-d]pyrimidines as potent glycogen synthase kinase-3 inhibitors

4-Amino-5,6-diaryl-furo[2,3-d]pyrimidines have been identified as inhibitors of glycogen synthase kinase-3beta (GSK-3beta). One representative derivative, 4-amino-3-(4-(benzenesulfonylamino)-phenyl)-2-(3-pyridyl)-furo[2,3-d]pyrimidine (12) exhibited potent GSK-3beta inhibitory activity in low nanomolar level of IC(50). The binding mode was proposed from a docking study.

Inhibition of GSK3 Promotes Replication and Survival of Pancreatic Beta Cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Biosynthesis of New Indigoid Inhibitors of Protein Kinases Using Recombinant Cytochrome P450 2A6

Glycogen synthase kinase-3 (GSK-3) is a potential drug target for a number of human diseases. Some indigoids have been found to be potent inhibitors of GSK-3, and individual compounds with better activity, specificity, and solubility are desired. In this work, a new disubstituted indigoid generation system was developed with a tryptophanase-deficient Escherichia coli strain as a host to express the human cytochrome P450 2A6 mutant L240C/N297Q, which catalyzes the oxidation of indole to isatin and indoxyl, which in turn react to generate indigoids. Forty-five substituted 1H-indoles from commercial sources were used as substrates in the system, and indigoid mixtures were tested as potential inhibitors of GSK-3. After preliminary screening, cell extracts with high inhibitory activity towards GSK-3alpha/beta were fractionated, and the IC50 values of twelve individual indigoids were measured for GSK-3alpha/beta as well as the protein kinases CDK1/cyclinB and CDK5/p25. Several indigoids, including an indigo, showed stronger inhibition than found in previous work. The most potent towards GSK-3alpha/beta, dimethyl indirubin 5,5'-dicarboxylate (IC50 of 51 nM), was modified by chemical reactions. One product, indirubin 5,5'-dicarboxylic acid 5-methyl ester, inhibited GSK-3alpha/beta with an IC50 of 14 nM and selectivity nearly 40-fold over CDK1 and CDK5. Indirubin-5-5'-dicarbonitrile was also modified to the corresponding 3'-oxime, which had low specificity but showed very high inhibition of all three kinases with IC50 values of 5, 13, and 10 nM towards GSK-3alpha/beta, CDK1, and CDK5, respectively. Thus, this system has the potential to generate new indigoids with therapeutic potential.