Novel pyrazolopyrimidine derivatives as GSK-3 inhibitors

A series of [1-aryl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]arylhydrazones were discovered as novel inhibitors glycogen synthase kinase-3 (GSK-3). Based on initial modeling a detailed SAR was constructed. Modification of the interior binding aryl ring (Ar(1)) determined this to be a tight binding region with little room for modification. As predicted from the model, a large variety of modifications could be incorporated into the hydrazone aryl ring. This work led to GSK-3 inhibitors in the low nano-molar range.     

Novel GSK-3 inhibitors with improved cellular activity

A novel series of [1-(1H-benzimidazol-7-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl] arylhydrazones was synthesized and shown to potently inhibit glycogen synthase kinase-3 (GSK-3). In light of detailed structure-activity relationships and structural knowledge of the GSK-3 binding pocket, a benzimidazole substituent was incorporated onto the pyrazolopyrimidine core resulting in improved potency over previous analogs. More importantly, these derivatives show low nanomolar efficacy for stimulating glycogen synthesis in vitro and therefore may be useful in the treatment of type 2 diabetes mellitus.     

Novel human mPGES-1 inhibitors identified through structure-based virtual screening

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase after exposure to pro-inflammatory stimuli and, therefore, represents a novel target for therapeutic treatment of acute and chronic inflammatory disorders. It is essential to identify mPGES-1 inhibitors with novel scaffolds as new leads or hits for the purpose of drug design and discovery that aim to develop the next-generation anti-inflammatory drugs. Herein we report novel mPGES-1 inhibitors identified through a combination of large-scale structure-based virtual screening, flexible docking, molecular dynamics simulations, binding free energy calculations, and in vitro assays on the actual inhibitory activity of the computationally selected compounds. The computational studies are based on our recently developed three-dimensional (3D) structural model of mPGES-1 in its open state. The combined computational and experimental studies have led to identification of new mPGES-1 inhibitors with new scaffolds. In particular, (Z)-5-benzylidene-2-iminothiazolidin-4-one is a promising novel scaffold for the further rational design and discovery of new mPGES-1 inhibitors. To our best knowledge, this is the first time a 3D structural model of the open state mPGES-1 is used in structure-based virtual screening of a large library of available compounds for the mPGES-1 inhibitor identification. The positive experimental results suggest that our recently modeled trimeric structure of mPGES-1 in its open state is ready for the structure-based drug design and discovery.     

GSK-3beta inhibitors reduce protein degradation in muscles from septic rats and in dexamethasone-treated myotubes

Sepsis is associated with muscle wasting, mainly reflecting increased muscle proteolysis. Recent studies suggest that inhibition of GSK-3beta activity may counteract catabolic stimuli in skeletal muscle. We tested the hypothesis that treatment of muscles from septic rats with the GSK-3beta inhibitors LiCl and TDZD-8 would reduce sepsis-induced muscle proteolysis. Because muscle wasting during sepsis is, at least in part, mediated by glucocorticoids, we also tested the effects of GSK-3beta inhibitors on protein degradation in dexamethasone-treated cultured myotubes. Treatment of incubated extensor digitorum longus muscles with LiCl or TDZD-8 reduced basal and sepsis-induced protein breakdown rates. When cultured myotubes were treated with LiCl or one of the GSK-3beta inhibitors SB216763 or SB415286, protein degradation was reduced. Treatment of incubated muscles or cultured myotubes with LiCl, but not the other GSK-3beta inhibitors, resulted in increased phosphorylation of GSK-3beta at Ser9, consistent with inactivation of the kinase and suggesting that the other inhibitors used in the present experiments inhibit GSK-3beta by phosphorylation-independent mechanisms. The present results suggest that GSK-3beta inhibitors may be used to prevent or treat sepsis-induced, glucocorticoid-regulated muscle proteolysis.     

GSK-3 inhibitors induce chromosome instability

Background

Several mechanisms operate during mitosis to ensure accurate chromosome segregation. However, during tumour evolution these mechanisms go awry resulting in chromosome instability. While several lines of evidence suggest that mutations in adenomatous polyposis coli (APC) may promote chromosome instability, at least in colon cancer, the underlying mechanisms remain unclear. Here, we turn our attention to GSK-3 – a protein kinase, which in concert with APC, targets β-catenin for proteolysis – and ask whether GSK-3 is required for accurate chromosome segregation.

Results

To probe the role of GSK-3 in mitosis, we inhibited GSK-3 kinase activity in cells using a panel of small molecule inhibitors, including SB-415286, AR-A014418, 1-Azakenpaullone and CHIR99021. Analysis of synchronised HeLa cells shows that GSK-3 inhibitors do not prevent G1/S progression or cell division. They do, however, significantly delay mitotic exit, largely because inhibitor-treated cells have difficulty aligning all their chromosomes. Although bipolar spindles form and the majority of chromosomes biorient, one or more chromosomes often remain mono-oriented near the spindle poles. Despite a prolonged mitotic delay, anaphase frequently initiates without the last chromosome aligning, resulting in chromosome non-disjunction. To rule out the possibility of "off-target" effects, we also used RNA interference to selectively repress GSK-3β. Cells deficient for GSK-3β exhibit a similar chromosome alignment defect, with chromosomes clustered near the spindle poles. GSK-3β repression also results in cells accumulating micronuclei, a hallmark of chromosome missegregation.

Conclusion

Thus, not only do our observations indicate a role for GSK-3 in accurate chromosome segregation, but they also raise the possibility that, if used as therapeutic agents, GSK-3 inhibitors may induce unwanted side effects by inducing chromosome instability.     

Novel and selective DNA methyltransferase inhibitors: Docking-based virtual screening and experimental evaluation

The DNA methyltransferase (DNMT) enzyme family consists of four members with diverse functions and represents one of the most promising targets for the development of novel anticancer drugs. However, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects that have been developed several decades ago. In this work, we conducted a virtual screening of more than 65,000 lead-like compounds selected from the National Cancer Institute collection using a multistep docking approach with a previously validated homology model of the catalytic domain of human DNMT1. Experimental evaluation of top-ranked molecules led to the discovery of novel small molecule DNMT1 inhibitors. Virtual screening hits were further evaluated for DNMT3B inhibition revealing several compounds with selectivity towards DNMT1. These are the first small molecules reported with biochemical selectivity towards an individual DNMT enzyme capable of binding in the same pocket as the native substrate cytosine, and are promising candidates for further rational optimization and development as anticancer drugs. The availability of enzyme-selective inhibitors will also be of great significance for understanding the role of individual DNMT enzymes in epigenetic regulation.     

Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors

GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.     

Non-ATP competitive glycogen synthase kinase 3beta (GSK-3beta) inhibitors: study of structural requirements for thiadiazolidinone derivatives

The 2,4-disubstituted thiadiazolidinones (TDZD) were described as the first non-ATP competitive GSK-3beta inhibitors. New modifications in this heterocyclic ring are here reported to study the influence on the biological activity. The basic skeleton of 1,2,4-thiadiazole and also one of the carbonyl groups are kept, while different modifications are introduced in positions 3 and 5, respectively. The GSK-3beta activity of the new thiadiazole derivatives here synthesized showed IC(50) values for some of the compounds in the micromolar range. Additionally, ATP competition studies have been carried out, showing that as well as the first generation of TDZD, these new compounds act in a non-competitive manner. With this study, additional requirements for the biological activity of the TDZD family have been delineated.     

Structure based virtual screening of GSK-3β: Importance of protein flexibility and induced fit

GSK-3β, one of the vital enzymes responsible for various phosphorylation catalysis. Induced fit mechanism and the presence of conserved water molecule(s) in the active site poses complexity during the process of virtual screening. The present investigation reveals the practical strategy to handle the induced fit mechanism of GSK-3β though flexible docking protocol. This protocol provides an enrichment of 70% in top 1% of the dataset with a rank correlation of >0.9 and found better in comparison to earlier reported protocols.     

GSK-3beta negatively regulates skeletal myotube hypertrophy

Todetermine whether changes in glycogen synthase kinase-3 (GSK-3)phosphorylation contribute to muscle hypertrophy, we delineated theeffects of GSK-3 activity on C₂C₁₂ myotubesize. We also examined possible insulin-like growth factor I (IGF-I) signaling of NFAT (nuclear factors of activated T cells)-inducible geneactivity and possible modulation of NFAT activation by GSK-3. Application of IGF-I (250 ng/ml) or LiCl (10 mM) alone (i.e., bothinhibit GSK-3 activity) increased the area ofC₂C₁₂ myotubes by 80 and 85%, respectively.The application of IGF-I (250 ng/ml) elevated GSK-3 phosphorylationand reduced GSK-3 kinase activity by ~800% and ~25%,respectively. LY-294002 (100 µM) and wortmannin (150 µM), specificinhibitors of phosphatidylinositol 3'-kinase, attenuated IGF-I-inducedGSK-3 phosphorylation by 67 and 92%, respectively. IGF-I suppressedthe kinase activity of GSK-3. IGF-I (250 ng/ml), but not LiCl (10 mM), induced an increase in NFAT-activated luciferase reporteractivity. Cotransfection of a constitutively active GSK-3(cGSK-3) inhibited the induction by IGF-I of NFAT-inducible reporteractivity. LiCl, which inhibits GSK-3, removed the block by cGSK-3on IGF-I-inducible NFAT-responsive reporter gene activity. These datasuggest that the IGF-I-induced increase in skeletal myotube size issignaled, in part, through the inhibition of GSK-3.

     

Discovery of dual binding site acetylcholinesterase inhibitors identified by pharmacophore modeling and sequential virtual screening techniques

Dual binding site acetylcholinesterase (AChE) inhibitors are promising for the treatment of Alzheimer’s disease (AD). They alleviate the cognitive deficits and AD-modifying agents, by inhibiting the β-amyloid (Aβ) peptide aggregation, through binding to both the catalytic and peripheral anionic sites, the so called dual binding site of the AChE enzyme. In this Letter, chemical features based 3D-pharmacophore models were developed based on the eight potent and structurally diverse AChE inhibitors (I-VIII) obtained from high-throughput in vitro screening technique. The best 3D-pharmacophore model, Hypo1, consists of two hydrogen-bond acceptor lipid, one hydrophobe, and two hydrophobic aliphatic features obtained by Catalyst/HIPHOP algorithm adopted in Discovery studio program. Hypo1 was used as a 3D query in sequential virtual screening study to filter three small compound databases. Further, a total of nine compounds were selected and followed on in vitro analysis. Finally, we identified two leads—Specs1 (IC₅₀ = 3.279 μM) and Spec2 (IC₅₀ = 5.986 μM) dual binding site compounds from Specs database, having good AChE enzyme inhibitory activity.     

A-kinase anchoring protein AKAP220 binds to glycogen synthase kinase-3beta (GSK-3beta ) and mediates protein kinase A-dependent inhibition of GSK-3beta

Glycogen synthase kinase-3 (GSK-3) is regulated by various extracellular ligands and phosphorylates many substrates, thereby regulating cellular functions. Using yeast two-hybrid screening, we found that GSK-3beta binds to AKAP220, which is known to act as an A-kinase anchoring protein. GSK-3beta formed a complex with AKAP220 in intact cells at the endogenous level. Cyclic AMP-dependent protein kinase (PKA) and type 1 protein phosphatase (PP1) were also detected in this complex, suggesting that AKAP220, GSK-3beta, PKA, and PP1 form a quaternary complex. It has been reported that PKA phosphorylates GSK-3beta, thereby decreasing its activity. When COS cells were treated with dibutyryl cyclic AMP to activate PKA, the activity of GSK-3beta bound to AKAP220 decreased more markedly than the total GSK-3beta activity. Calyculin A, a protein phosphatase inhibitor, also inhibited the activity of GSK-3beta bound to AKAP220 more strongly than the total GSK-3beta activity. These results suggest that PKA and PP1 regulate the activity of GSK-3beta efficiently by forming a complex with AKAP220.     

Structure-based virtual screening of Src kinase inhibitors

Src is an important target in multiple processes associated with tumor growth and development, including proliferation, neovascularization, and metastasis. In this study, hit identification was performed by virtual screening of commercial and in-house compound libraries. Docking studies for the hits were performed, and scoring functions were used to evaluate the docking results and to rank ligand-binding affinities. Subsequently, hit optimization for potent and selective candidate Src inhibitors was performed through focused library design and docking analyses. Consequently, we report that a novel compound ‘43’ with an IC₅₀ value of 89 nM, representing (S)-N-(4-(5-chlorobenzo[d][1,3]dioxol-4-ylamino)-7-(2-methoxyethoxy)quinazolin-6-yl)pyrrolidine-2-carboxamide, is highly selective for Src in comparison to EGFR (IC₅₀ ratio > 80-fold) and VEGFR-2 (IC₅₀ ratio > 110-fold). Compound 43 exerted anti-proliferative effects on Src-expressing PC3 human prostate cancer and A431 human epidermoid carcinoma cells, with calculated IC₅₀ values of 1.52 and 0.78 μM, respectively. Moreover, compound 43 (0.1 μM) suppressed the phosphorylation of extracellular signal-regulated kinases and p90 ribosomal S6 kinase, downstream molecules of Src, in a time-dependent manner, in both PC3 and A431 cell lines. The docking structure of compound 43 with Src disclosed that the chlorobenzodioxole moiety and pyrrolidine ring of C-6 quinazoline appeared to fit tightly into the hydrophobic pocket of Src. Additionally, the pyrrolidine NH forms a hydrogen bond with the carboxyl group of Asp348. These results confirm the successful application of virtual screening studies in the lead discovery process, and suggest that our novel compound 43 can be an effective Src inhibitor candidate for further lead optimization.     

Selective immunoproteasome inhibitors with non-peptide scaffolds identified from structure-based virtual screening

As a major component of the crucial nonlysosomal protein degradation pathway in the cells, the proteasome has been implicated in many diseases such as Alzheimer’s disease, Huntington’s disease, inflammatory bowel diseases, autoimmune diseases, multiple myeloma (MM) and other cancers. There are two main proteasome subtypes: the constitutive proteasome which is expressed in all eukaryotic cells and the immunoproteasome which is expressed in immune cells and can be induced in other cell types. Majority of currently available proteasome inhibitors are peptide backbone-based, having short half-lives in the body. It is highly desirable to identify novel, immunoproteasome-selective inhibitors with non-peptide scaffolds for development of novel therapeutics. Through combined virtual screening and experimental studies targeting the immunoproteasome, we have identified a set of novel immunoproteasome inhibitors with diverse non-peptide scaffolds. Some of the identified inhibitors have significant selectivity for the immunoproteasome over the constitutive proteasome. Unlike most of the currently available proteasome inhibitors, these new inhibitors lacking electrophilic pharmacophores are not expected to form a covalent bond with proteasome after the binding. These non-peptide scaffolds may provide a new platform for future rational drug design and discovery targeting the immunoproteasome.     

Identification of potential glycogen kinase-3 inhibitors by structure based virtual screening

Glycogen synthase kinase-3 (GSK3) is a serine/threonine kinase that has attracted much drug discovery attention in recent years. Structural crystallography of the kinase has produced several high resolution inhibitor-GSK3 complexes and this is offering valuable information about the important pharmacophoric features present in the inhibitor, the protein target and the bioactive conformation. The availability of several GSK3-inhibitor co-crystals was successfully exploited to derive a pharmacophore query which retains the all important inhibitor-GSK3 interaction chemistry. A hypothesis containing three features: two hydrogen bond donors and one hydrogen acceptor was found to explain much of the inhibitor-GSK3 interaction. Subsequently, the query has been submitted to three databases for electronic screening. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of 21 novel potential leads were proposed after thorough examination by a combination of methods: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta binding site, (ii) detailed analysis of their FlexX, G_Score, PMF_Score, ChemScore and D_Score values, (iii) comparative investigation of the docking scores of the hits with that of the thus far reported inhibitors (iv) determination of the binding mode and examination of how the hits retain interactions with the important amino acid residues of the kinase binding site. The hydrophobic heterocycles identified in this investigation are expected to be important additions to the armamentarium of GSK3 hyperactivity antagonism. Further more, the present work may further our current knowledge of the molecular basis of activation, inhibition and regulation of this pharmaceutically important kinase.     

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.     

Discovery of novel PRL-3 inhibitors based on the structure-based virtual screening

The inhibitors of phosphatase of regenerating liver-3 (PRL-3) have been shown to be useful as therapeutics for the treatment of cancer. We have been able to identify 12 novel PRL-3 inhibitors by means of the virtual screening with docking simulations under the consideration of the effects of ligand solvation in the scoring function. Because the newly identified inhibitors are structurally diverse and reveal a significant potency with IC(50) values ranging from 10 to 50muM, all of them can be considered for further development by structure-activity relationship or de novo design methods. Structural features relevant to the interactions of the newly identified inhibitors with the amino acid residues in the active site and the peripheral binding site of PRL-3 are discussed in detail.     

Involvement of GSK-3beta in TWEAK-mediated NF-kappaB activation

Glycogen synthase kinase-3beta (GSK-3beta) is a key component of several signaling pathways. We found that a short variant of 'TNF-like weak inducer of apoptosis' (shortTWEAK) formed a complex with GSK-3beta in a yeast two-hybrid system. We demonstrate that shortTWEAK and GSK-3beta colocalize in the nucleus of human neuroblastoma cells. We also show that TWEAK is internalized in different cell lines and that it translocates to the nucleus. This event causes the degradation of IkappaBalpha, the nuclear translocation of both GSK-3beta and p65, and the induction of NF-kappaB-driven gene expression. We demonstrate that the induction of IL-8 expression by TWEAK can be counteracted by LiCl. Taken together, these data suggest that GSK-3beta plays an important role in the signal transduction pathway between TWEAK and NF-kappaB.     

Novel inhibitors of As(III) S-adenosylmethionine methyltransferase (AS3MT) identified by virtual screening

Due to increased interest in As(III) S-adenosylmethionine methyltransferase (AS3MT), a search for chemical probes that can help elucidate function was initiated. A homology model was built based on related enzymes, and virtual screening produced 426 potential hits. Evaluation of these compounds in a functional enzymatic assay revealed several modest inhibitors including an O-substituted 2-amino-3-cyano indole scaffold. Two iterations of near neighbor searches revealed compound 5 as a potent inhibitor of AS3MT with good selectivity over representative methyltransferases DOT1L and NSD2 as well as a representative set of diverse receptors. Compound 5 should prove to be a useful tool to investigate the role of AS3MT and a potential starting point for further optimization.