Batya Plotkin

Lithium-mediated phosphorylation of glycogen synthase kinase-3β involves PI3 kinase-dependent activation of protein kinase C-α

Lithium, a known mood-stabilizer frequently used in treatment of bipolar disorders, is an effective glycogen synthase kinase-3β (GSK-3β) inhibitor. This led to the idea that GSK-3β is an in vivo target directly inhibited by lithium. As lithium is a weak in vitro inhibitor of GSK-3β (IC50=2 mM), however, we speculated that it inhibits GSK-3β via an indirect, yet unknown, mechanism. The present studies show that lithium increased the phosphorylation of a key inhibitory site of GSK-3β, serine-9 (Ser-9), in HEK293 cells and in PC12 cells. This phosphorylation was significantly reduced by protein kinase C (PKC) inhibitors GF109203X and Ro31-8425, as well as GÖ6976, and effective inhibitor toward conventional PKC isoforms (cPKC). Consistent with these results, lithium increased PKC-α activity approximately twofold in both cell lines. Because PI3 kinase is a potential upstream regulator of cPKC, its inhibition by wortmannin or LY294002 also abolished the lithium-induced serine phosphorylation of GSK-3β in HEK293 and PC12 cells. Moreover, lithium did not activate PKB, and in addition, its activity was not dependent on the presence of medium inositol nor did it affect the autophosphorylation activity of GSK-3β. Finally, intracerebroventricular injection of lithium increased GSK-3β Ser-9 phosphorylation and enhanced PKC-α activity 1.8-fold in mouse hippocampus, confirming this lithium response in vivo. Our studies propose a new mechanism by which lithium indirectly inhibits GSK-3β via phosphatidylinositol 3 kinase-dependent activation of PKC-α.

Identification of Glycogen Synthase Kinase-3 Inhibitors with a Selective Sting for Glycogen Synthase Kinase-3α

The glycogen synthase kinase-3 (GSK-3) has been linked to the pathogenesis of colorectal cancer, diabetes, cardiovascular disease, acute myeloid leukemia (AML), and Alzheimers disease (AD). The debate on the respective contributions of GSK-3α and GSK-3β to AD pathology and AML is ongoing. Thus, the identification of potent GSK-3α-selective inhibitors, endowed with favorable pharmacokinetic properties, may elucidate the effect of GSK-3α inhibition in AD and AML models. The analysis of all available crystallized GSK-3 structures provided a simplified scheme of the relevant hot spots responsible for ligand binding and potency. This resulted in the identification of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest GSK-3α selectivity reported so far. In addition, compound 14d did not display significant inhibition of 48 out of 50 kinases in the test panel. The GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay.

The role of histidines 26 and 33 in the structural stabilization of cytochrome c

Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro-44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule. These changes resulted in a decrease in the global stability of the protein. Further mutation of Asn-52 to Ile led to a total recovery of the wild-type stability of the sulfur-iron bond, and a partial restoration of the global stability of the protein. Substitution of Phe for His-33 did not alter the sulfur-iron bond but caused a pronounced increase in the global stability of the protein. It is suggested that this effect results from hydrophobic interaction of the Phe-33 side chain with the lower loop on the right side of the protein. Such an interaction also explains the observation that the same mutation reversed the loss of global stability caused by substitution of Val to His-26, but did not restore the strength of the sulfur-iron bond that this mutation had brought about.

Structure-based optimization of oxadiazole-based GSK-3 inhibitors

Inhibition of glycogen synthase kinase-3 (GSK-3) induces neuroprotective effects, e.g. decreases β-amyloid production and reduces tau hyperphosphorylation, which are both associated with Alzheimers disease (AD). The two isoforms of GSK-3 in mammalians are GSK-3α and β, which share 98% homology in their catalytic domains. We investigated GSK-3 inhibitors based on 2 different scaffolds in order to elucidate the demands of the ATP-binding pocket [1]. Particularly, the oxadiazole scaffold provided potent and selective GSK-3 inhibitors. For example, the most potent inhibitor of the present series, the acetamide 26d, is characterized by an IC50 of 2 nM for GSK-3α and 17 nM for GSK-3β. In addition, the benzodioxane 8g showed up to 27-fold selectivity for GSK-3α over GSK-3β, with an IC50 of 35 nM for GSK-3α. Two GSK-3 inhibitors were further profiled for efficacy and toxicity in the wild-type (wt) zebrafish embryo assay to evaluate simultaneously permeability and safety.

The reactivity of cytochrome c with soft ligands

The spectral changes caused by binding soft ligands to the cytochrome c iron and their correlation to ligand affinities support the hypothesis that the iron—methionine sulfur bond of this heme protein is enhanced by delocalization of the metal l2, electrons into the empty 3d orbitals of the ligand atom. These findings also explain the unique spectrum of cytochrome c in the far red.

A unique type of GSK-3 inhibitor brings new opportunities to the clinic

A substrate peptide that the kinase GSK-3 converts into its own inhibitor improves symptoms and cognitive function in an Alzheimer’s disease model. GSK-3 makes its own inhibitor One of the main challenges in developing kinase inhibitors has been achieving specificity. Licht-Murava et al. discovered a substrate peptide derivative, L807, that the kinase GSK-3 converted into an inhibitor within the catalytic site of the enzyme. This peptide was highly selective for GSK-3 when tested against a panel of 139 kinases, and a membrane-permeable form of it inhibited GSK-3 activity in cells and animals and, more importantly, improved cellular symptoms, cognitive function, and social behaviors in a mouse model of Alzheimer’s disease. Thus, this new mechanism of inhibition may finally enable effective and selective GSK-3 inhibitors to reach the clinic. Development of protein kinase inhibitors is a focus of many drug discovery programs. A major problem, however, is the limited specificity of the commonly used adenosine triphosphate–competitive inhibitors and the weak inhibition of the more selective substrate-competitive inhibitors. Glycogen synthase kinase–3 (GSK-3) is a promising drug target for treating neurodegenerative disorders, including Alzheimer’s disease (AD), but most GSK-3 inhibitors have not reached the clinic. We describe a new type of GSK-3 inhibitor, L807mts, that acts through a substrate-to-inhibitor conversion mechanism that occurs within the catalytic site of the enzyme. We determined that L807mts was a potent and highly selective GSK-3 inhibitor with reasonable pharmacological and safety properties when tested in rodents. Treatment with L807mts enhanced the clearance of β-amyloid loads, reduced inflammation, enhanced autophagic flux, and improved cognitive and social skills in the 5XFAD AD mouse model. This new modality of GSK-3 inhibition may be therapeutic in patients with AD or other central nervous system disorders associated with dysregulated GSK-3.

Design, Synthesis, and Biological Evaluation of 1-Phenylpyrazolo[3,4-e]pyrrolo[3,4-g]indolizine-4,6(1H,5H)-diones as New Glycogen Synthase Kinase-3β Inhibitors

Compound 5 was selected from our in-house library as a suitable starting point for the rational design of new GSK-3β inhibitors. MC/FEP calculations of 5 led to the identification of a structural class of new GSK-3β inhibitors. Compound 18 inhibited GSK-3β with an IC50 of 0.24 μM and inhibited tau phosphorylation in a cell-based assay. It proved to be a selective inhibitor of GSK-3 against a panel of 17 kinases and showed >10-fold selectivity against CDK2. Calculated physicochemical properties and Volsurf predictions suggested that compound 18 has the potential to diffuse passively across the blood-brain barrier.

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 Alzheimers 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.

Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium Cinctum ☆

Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V. This cytochrome was active in the respiratory chain of beef heart Keilin-Hartree particles. Cytochrome c-553 had a molecular mass of 13,200 Da, pI 4.9, and Em of 384 m V, and was active in light induced electron transport of Euglena gracilis chloroplast fragments. Cytochrome c-554 had a molecular mass of 13,500 Da, pI 4.4, and Em of 326 m V. This cytochrome was inactive in light induced electron transport but competed with cytochrome c-552 of Euglena in the assay. The acidic cytochrome c-557 was present in very small quantities. The properties of the soluble c-type cytochromes of P. cinctum are compatible with the classification of dinoflagellates as primitive eucaryotes.