Sylvie Y. Blond

Highly Potent and Specific GSK-3β Inhibitors That Block Tau Phosphorylation and Decrease α-Synuclein Protein Expression in a Cellular Model of Parkinson's Disease

Research by Klein and co‐workers suggests that the inhibition of GSK‐3β by small molecules may offer an important strategy in the treatment of a number of central nervous system (CNS) disorders including Alzheimers disease, Parkinsons disease, and bipolar disorders. Based on results from kinase‐screening assays that identified a staurosporine analogue as a modest inhibitor of GSK‐3β, a series of 3‐indolyl‐4‐indazolylmaleimides was prepared for study in both enzymatic and cell‐based assays. Most strikingly, whereas we identified ligands having poor to high potency for GSK‐3β inhibition, only ligands with a Ki value of less than 8 nM, namely maleimides 18 and 22, were found to inhibit Tau phosphorylation at a GSK‐3β‐specific site (Ser 396/404). Accordingly, maleimides 18 and 22 may protect neuronal cells against cell death by decreasing the level of α‐Syn protein expression. We conclude that the GSK‐3β inhibitors described herein offer promise in defending cells against MPP+‐induced neurotoxicity and that such compounds will be valuable to explore in animal models of Parkinsons disease as well as in other Tau‐related neurodegenerative disease states.

From a Natural Product Lead to the Identification of Potent and Selective Benzofuran-3-yl-(indol-3-yl)maleimides as Glycogen Synthase Kinase 3β Inhibitors that Suppress Proliferation and Survival of Pancreatic Cancer Cells

Recent studies have demonstrated that glycogen synthase kinase 3beta (GSK-3beta) is overexpressed in human colon and pancreatic carcinomas, contributing to cancer cell proliferation and survival. Here, we report the design, synthesis, and biological evaluation of benzofuran-3-yl-(indol-3-yl)maleimides, potent GSK-3beta inhibitors. Some of these compounds show picomolar inhibitory activity toward GSK-3beta and an enhanced selectivity against cyclin-dependent kinase 2 (CDK-2). Selected GSK-3beta inhibitors were tested in the pancreatic cancer cell lines MiaPaCa-2, BXPC-3, and HupT3. We determined that some of these compounds, namely compounds 5, 6, 11, 20, and 26, demonstrate antiproliferative activity against some or all of the pancreatic cancer cells at low micromolar to nanomolar concentrations. We found that the treatment of pancreatic cancer cells with GSK-3beta inhibitors 5 and 26 resulted in suppression of GSK-3beta activity and a distinct decrease of the X-linked inhibitor of apoptosis (XIAP) expression, leading to significant apoptosis. The present data suggest a possible role for GSK-3beta inhibitors in cancer therapy, in addition to their more prominent applications in CNS disorders.

Design, Synthesis, Docking, and Biological Evaluation of Novel Diazide-containing Isoxazole- and Pyrazole-based Histone Deacetylase Probes

The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.

Endoplasmic Reticulum Chaperone Protein GRP-78 Mediates Endocytosis of Dentin Matrix Protein 1

Dentin matrix protein 1 (DMP1), a phosphorylated protein present in the mineral phase of both vertebrates and invertebrates, is a key regulatory protein during biogenic formation of mineral deposits. Previously we showed that DMP1 is localized in the nuclear compartment of preosteoblasts and preodontoblasts. In the nucleus DMP1 might play an important role in the regulation of genes that control osteoblast or odontoblast differentiation. Here, we show that cellular uptake of DMP1 occurs through endocytosis. Interestingly, this process is initiated by DMP1 binding to the glucose-regulated protein-78 (GRP-78) localized on the plasma membrane of preodontoblast cells. Binding of DMP1 to GRP-78 receptor was determined to be specific and saturable with a binding dissociation constant KD = 85 nm. We further depict a road map for the endocytosed DMP1 and demonstrate that the internalization is mediated primarily by caveolae and that the vesicles containing DMP1 are routed to the nucleus along microtubules. Immunohistochemical analysis and binding studies performed with biotin-labeled DMP1 confirm spatial co-localization of DMP1 and GRP-78 in the preodontoblasts of a developing mouse molar. Co-localization of DMP1 with GRP-78 was also observed in T4-4 preodontoblast cells, dental pulp stem cells, and primary preodontoblasts. By small interfering RNA techniques, we demonstrate that the receptor for DMP1 is GRP-78. Therefore, binding of DMP1 with GRP-78 receptor might be an important mechanism by which DMP1 is internalized and transported to the nucleus during bone and tooth development.

The Affinity of a Major Ca2+ Binding Site on GRP78 Is Differentially Enhanced by ADP and ATP

GRP78 is a major protein regulated by the mammalian endoplasmic reticulum stress response, and up-regulation has been shown to be important in protecting cells from challenge with cytotoxic agents. GRP78 has ATPase activity, acts as a chaperone, and interacts specifically with other proteins, such as caspases, as part of a mechanism regulating apoptosis. GRP78 is also reported to have a possible role as a Ca2+ storage protein. In order to understand the potential biological effects of Ca2+ and ATP/ADP binding on the biology of GRP78, we have determined its ligand binding properties. We show here for the first time that GRP78 can bind Ca2+, ATP, and ADP, each with a 1:1 stoichiometry, and that the binding of cation and nucleotide is cooperative. These observations do not support the hypothesis that GRP78 is a dynamic Ca2+ storage protein. Furthermore, we demonstrate that whereas Mg2+ enhances GRP78 binding to ADP and ATP to the same extent, Ca2+ shows a differential enhancement. In the presence of Ca2+, the KD for ATP is lowered ∼11-fold, and the KD for ADP is lowered around 930-fold. The KD for Ca2+ is lowered ∼40-fold in the presence of ATP and around 880-fold with ADP. These findings may explain the biological requirement for a nucleotide exchange factor to remove ADP from GRP78. Taken together, our data suggest that the Ca2+-binding property of GRP78 may be part of a signal transduction pathway that modulates complex interactions between GRP78, ATP/ADP, secretory proteins, and caspases, and this ultimately has important consequences for cell viability.

Substrate Binding Induces Depolymerization of the C-terminal Peptide Binding Domain of Murine GRP78/BiP

To investigate the role of each domain in BiP/GRP78 function, we have used a full-length recombinant BiP engineered to contain two enterokinase sites; one site is located after an N-terminal FLAG epitope, and a second site has been inserted at the junction between the N- and C-terminal domains (FLAG-BiP.ent). FLAG-BiP.ent oligomerizes into multiple species that interconvert with each other in a slow, concentration- and temperature-dependent equilibrium. Binding of ATP or AMP-PNP (adenosine 5′-(β,γ-imino)triphosphate), but not ADP, or of a peptidic substrate induces depolymerization of FLAG-BiP.ent and stabilization of monomeric species. Enterokinase cleavage of monomeric, nucleotide-free BiP.ent results in the physical dissociation of the 44-kDa N-terminal ATPase fragment (N44.ent) from the 30-kDa C-terminal substrate binding domain (C30.ent). Upon dissociation, the freed C-terminal substrate binding domain readily undergoes self-association while N44.ent remains monomeric. Enterokinase cleavage performed in the presence of a synthetic peptide prevents oligomerization of the freed C30.ent domain. Addition of ATP during enterokinase cleavage has no effect on C30.ent oligomerization. Our data clearly indicate that binding of a specific peptide onto the C-terminal domain, or ATP onto the N-terminal domain, induces internal conformational change(s) within the C30 domain that result(s) in BiP depolymerization.

Purification and properties of BiP.

Publisher Summary The chapter presents a study related to purification and properties BiP, a member of the heat shock protein 70 (HSP70) family localized in the endoplasmic reticulum of eukaryotic cells. The chapter focuses on different approaches that can be used to biochemically characterize mammalian BiP. Several of the methods can be adapted to any particular protein, multidomain proteins, and secreted proteins, to the applications and limitations of histidine and epitope tagging in the purification of recombinant proteins; and of course to the characterization of HSP70-related proteins. The chapter includes a detailed protocol for the characterization of peptide specificity by using affinity panning of phage display libraries that includes biotinylation of BiP and the use of the avidin-biotin system to immobilize protein on enzyme-linked immunosorbent assay (ELISA) microtitration plates. This technique is used to identify epitopes recognized by monoclonal antibodies and used the information to tag recombinant proteins. The method presented can be adapted to any protein that exhibits a peptidic substrate recognition activity (molecular chaperones, antibodies, and other peptide acceptors, peptidic hormone receptors, enzyme inhibitors,) and to other applications of phage display libraries.

Stress Chaperone GRP-78 Functions in Mineralized Matrix Formation

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78.

Use of molecular modeling, docking, and 3D-QSAR studies for the determination of the binding mode of benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3β inhibitors

Molecular modeling and docking studies along with three-dimensional quantitative structure relationships (3D-QSAR) studies have been used to determine the correct binding mode of glycogen synthase kinase 3β (GSK-3β) inhibitors. The approaches of comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) are used for the 3D-QSAR of 51 substituted benzofuran-3-yl-(indol-3-yl)maleimides as GSK-3β inhibitors. Two binding modes of the inhibitors to the binding site of GSK-3β are investigated. The binding mode 1 yielded better 3D-QSAR correlations using both CoMFA and CoMSIA methodologies. The three-component CoMFA model from the steric and electrostatic fields for the experimentally determined pIC50 values has the following statistics: R2(cv) = 0.386 nd SE(cv) = 0.854 for the cross-validation, and R2 = 0.811 and SE = 0.474 for the fitted correlation. F (3,47) = 67.034, and probability of R2 = 0 (3,47) = 0.000. The binding mode suggested by the results of this study is consistent with the preliminary results of X-ray crystal structures of inhibitor-bound GSK-3β. The 3D-QSAR models were used for the estimation of the inhibitory potency of two additional compounds.

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

Human glutathione transferase π (GST π) has been crystallized as a homodimer, with a subunit molecular mass of ∼23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST π, resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in Km for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the KmGSH, whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in Vmax. The GST π retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.