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.     

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.     

E-pharmacophore-based virtual screening to identify GSK-3β inhibitors

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase which has attracted significant attention during recent years in drug design studies. The deregulation of GSK-3β increased the loss of hippocampal neurons by triggering apoptosis-mediating production of neurofibrillary tangles and alleviates memory deficits in Alzheimer’s disease (AD). Given its role in the formation of neurofibrillary tangles leading to AD, it has been a major therapeutic target for intervention in AD, hence was targeted in the present study. Twenty crystal structures were refined to generate pharmacophore models based on energy involvement in binding co-crystal ligands. Four common e-pharmacophore models were optimized from the 20 pharmacophore models. Shape-based screening of four e-pharmacophore models against nine established small molecule databases using Phase v3.9 had resulted in 1800 compounds having similar pharmacophore features. Rigid receptor docking (RRD) was performed for 1800 compounds and 20 co-crystal ligands with GSK-3β to generate dock complexes. Interactions of the best scoring lead obtained through RRD were further studied with quantum polarized ligand docking (QPLD), induced fit docking (IFD) and molecular mechanics/generalized Born surface area. Comparing the obtained leads to 20 co-crystal ligands resulted in 18 leads among them, lead1 had the lowest docking score, lower binding free energy and better binding orientation toward GSK-3β. The 50?ns MD simulations run confirmed the stable nature of GSK-3β-lead1 docking complex. The results from RRD, QPLD, IFD and MD simulations confirmed that lead1 might be used as a potent antagonist for GSK-3β.     

Docking,molecular dynamics,binding energy-MM-PBSA studies of naphthofuran derivatives to identify potential dual inhibitors against BACE-1 and GSK-3β

BACE-1 and GSK-3β both are potential therapeutic drug targets for Alzheimer’s disease. Recently, both these targets received attention for designing dual inhibitors. Till now only two scaffolds (triazinone and curcumin) derivatives have been reported as BACE-1 and GSK-3β dual inhibitors. In our previous work, we have reported first in class dual inhibitor for BACE-1 and GSK-3β. In this study, we have explored other naphthofuran derivatives for their potential to inhibit BACE-1 and GSK-3β through docking, molecular dynamics, binding energy (MM-PBSA). These computational methods were performed to estimate the binding affinity of naphthofuran derivatives towards the BACE-1 and GSK-3β. In the docking results, two derivatives (NS7 and NS9) showed better binding affinity as compared to previously reported inhibitors. Hydrogen bond occupancy of NS7 and NS9 generated from MD trajectories showed good interaction with the flap residues Gln73, Thr72 of BACE-1 and Arg141, Thr138 residues of GSK-3β. MM-PBSA and energy decomposition per residue revealed different components of binding energy and relative importance of amino acid involved in binding. The results showed that the binding of inhibitors was majorly governed by the hydrophobic interactions and suggesting that hydrophobic interactions might be the key to design dual inhibitors for BACE1-1 and GSK-3β. Distance between important pair of amino acid residues indicated that BACE-1 and GSK-3β adopt closed conformation and become inactive after ligand binding. The results suggested that naphthofuran derivatives might act as dual inhibitor against BACE-1 and GSK-3β.     

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.     

A common feature-based 3D-pharmacophore model generation and virtual screening: identification of potential PfDHFR inhibitors

A four-feature 3D-pharmacophore model was built from a set of 24 compounds whose activities were reported against the V1/S strain of the Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme. This is an enzyme harboring Asn51Ile + Cys59Arg + Ser108Asn + Ile164Leu mutations. The HipHop module of the Catalyst program was used to generate the model. Selection of the best model among the 10 hypotheses generated by HipHop was carried out based on rank and best-fit values or alignments of the training set compounds onto a particular hypothesis. The best model (hypo1) consisted of two H-bond donors, one hydrophobic aromatic, and one hydrophobic aliphatic features. Hypo1 was used as a query to virtually screen Maybridge2004 and NCI2000 databases. The hits obtained from the search were subsequently subjected to FlexX and Glide docking studies. Based on the binding scores and interactions in the active site of quadruple-mutant PfDHFR, a set of nine hits were identified as potential inhibitors.     

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF’s tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein’s flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC₅₀ values lower than 10 μM (three hits with IC₅₀ lower than 1 μM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC₅₀ = 300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF’s tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.     

Design of Benzene-1,2-diamines as selective inducible nitric oxide synthase inhibitors: a combined de novo design and docking analysis

Selective inhibition of inducible nitric oxide synthases (iNOS) has been a challenging problem for researchers pursuing work in finding methods to treat inflammatory disorders, shock, etc. Though many inhibitors have been studied to date, all are associated with selectivity or potency problems. Additionally, most of the reported compounds have several similarities and fewer number of novel structures are being tried. There is an increasing need to design novel molecules for this target. In this work, de novo design using LUDI, combined with docking analysis using FlexX has been employed in an attempt to identify novel scaffolds. Benzene-1,2-diamines were identified which could mimic the interactions of the substrate analogs and other inhibitors. Comparative docking scores in each of the isoforms of nitric oxide synthase were employed to recognize hits for iNOS selectivity. Figure Figure shows the docked poses of the ligand M226 along with that of the reference GW274150. (FlexX analysis)     

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.     

Toward the virtual screening of Cdc25A phosphatase inhibitors with the homology modeled protein structure

Cdc25 phosphatases have been considered as attractive drug targets for anticancer therapy due to the correlation of their overexpression with a wide variety of cancers. As a method for the discovery of novel inhibitors of Cdc25 phosphatases, we have evaluated the computer-aided drug design protocol involving the homology modeling of Cdc25A and virtual screening with the two docking tools: FlexX and the modified AutoDock program implementing the effects of ligand solvation in the scoring function. The homology modeling with the X-ray crystal structure of Cdc25B as a template provides a high-quality structure of Cdc25A that enables the structure-based inhibitor design. Of the two docking programs under consideration, AutoDock is found to be more accurate than FlexX in terms of scoring putative ligands. A detailed binding mode analysis of the known inhibitors shows that they can be stabilized in the active site of Cdc25A through the simultaneous establishment of the multiple hydrogen bonds and the hydrophobic interactions. The present study demonstrates the usefulness of the modified AutoDock program as a docking tool for virtual screening of new Cdc25 phosphatase inhibitors as well as for binding mode analysis to elucidate the activities of known inhibitors. Figure Structures and available IC50 values (in μM) of the twenty Cdc25 phosphatase inhibitors seeded in docking library     

Novel GSK-3beta inhibitors from sequential virtual screening

Glycogen synthase kinase-3 (GSK-3beta) has been emerging as a key therapeutic target for type-2 diabetics, Alzheimer's disease, cancer, and chronic inflammation. For the purpose of finding biologically active and novel compounds and providing new idea for drug-design, we performed virtual screening using commercially available database. Three-dimensional common feature pharmacophore model was developed by using HipHop program provided in Catalyst software and it was used as a query for screening database. Recursive partitioning (RP) model was developed as a filtering system, which was able to classify active and inactive compounds. Eventually, a sequential virtual screening procedure (SQSP) was conducted by applying the common feature pharmacophore and RP model in succession to discover novel potent GSK-3beta inhibitors. The final 56 hit compounds were carefully selected considering predicted docking mode in crystal structures. Subsequent enzyme assay for human GSK-3beta protein confirmed that three compounds of these hit compounds exhibit micromolar inhibitory activity. Here, we report novel hit compounds and their binding mode in the active site of GSK-3beta crystal structure.     

3D-QSAR pharmacophore-based virtual screening,molecular docking and molecular dynamics simulation toward identifying lead compounds for NS2B–NS3 protease inhibitors

NS2B–NS3 protease has been identified to serve as lead drug design target due to its significant role in West Nile viral (WNV) and dengue virus (DENV) reproduction and replication. There are currently no approved chemotherapeutic drugs and effective vaccines to inhibit DENV and WNV infections. In this work, 3D-QSAR pharmacophore model has been developed to discover potential inhibitory candidates. Validation through Fischer’s model and decoy test indicate that the developed 3D pharmacophore model is highly predictive for DENV inhibitors, which was then employed to screen ZINC chemical library to obtain reasonable hits. Following ADMET filtering, 15 hits were subjected to further filter through molecular docking and CoMFA modeling. Finally, top three hits were identified as lead compounds or potential inhibitory candidates with IC₅₀ values of ～0.4637?µM and fitness of ～57.73. It is implied from CoMFA modeling that substituents at the side site of benzotriazole such as a p-nitro group (e.g. biphenyl head) and a carbonyl (e.g. carboxylate function) at the side site of furan or amino group may improve bioactivity of ZINC85645245, respectively. Molecular dynamics simulations (MDS) were performed to discover new interactions and reinforce the binding modes from docking for the hits also. The QSAR and MDS results obtained from this work should be useful in determining structural requirements for inhibitor development as well as in designing more potential inhibitors for NS2B–NS3 protease.     

Molecular dynamics simulation studies of GSK-3β ATP competitive inhibitors: understanding the factors contributing to selectivity

Glycogen synthase kinase-3 is a constitutively acting, multifunctional serine threonine kinase, the role of which has been implicated in several physiological pathways and has emerged as a promising target for the treatment of type-II diabetes and Alzheimer’s disease. In order to provide a detailed understanding of the origin of selectivity determinants of ATP competitive inhibitors, molecular dynamics simulations in combination with MM-PBSA binding energy calculations were performed using crystal structures of GSK-3β and CDK-2 in complex with 12 ATP competitive inhibitors. Analysis of energy contributions indicate that electrostatic interaction energy dictates the selectivity of ATP competitive inhibitors against CDK-2. Key interactions as well as residues that potentially make a major contribution to the binding free energy were identified at the ATP binding site. This analysis stresses the need for the inhibitors to interact with Lys85, Thr138, and Arg141 in the binding site of GSK-3β to show selectivity. The residue-wise energy decomposition analysis further suggested the additional role of Gln185 in determining the selectivity of maleimides. The results obtained in this study can be utilized to design new selective GSK-3 ATP competitive inhibitors.     

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.     

Identification of selective MMP-9 inhibitors through multiple e-pharmacophore,ligand-based pharmacophore,molecular docking,and density functional theory approaches

Matrix metalloproteinase-9 (MMP-9) is a significant target for the development of drugs for the treatment of arthritis, CNS disorders, and cancer metastasis. The structure-based and ligand-based methods were used for the virtual screening (VS) of database compounds to obtain potent and selective MMP-9 inhibitors. Experimentally known MMP-9 inhibitors were used to grow up ligand-based three pharmacophore models utilizing Schrodinger suite. The X-ray crystallographic structures of MMP-9 with different inhibitors were used to develop five energy-optimized structure-based (e-pharmacophore) models. All developed pharmacophores were validated and applied to screen the Zinc database. Pharmacophore matched compounds were subjected to molecular docking to retrieve hits with novel scaffolds. The molecules with diverse structures, high docking scores and low binding energies for various crystal structures of MMP-9, were selected as final hits. The Induced fit docking (IFD) analysis provided significant information about the driving of inhibitor to approve a suitable bioactive conformational position in the active site of protein. Since charge transfer reaction occurs during receptor–ligand interaction, therefore, electronic features of hits (ligands) are interesting parameters to explain the binding interactions. Density functional theory (DFT) at B3LYP/6-31G* level was utilized to explore electronic features of hits. The docking study of hits using AutoDock was helpful to establish the binding interactions. The study illustrates that the combined pharmacophore approach is advantageous to identify diverse hits which have better binding affinity to the active site of the enzyme for all possible bioactive conformations. The approach used in the study is worthy to design drugs for other targets.     

Identification and evaluation of bioactive natural products as potential inhibitors of human microtubule affinity-regulating kinase 4 (MARK4)

Microtubule affinity-regulating kinase 4 (MARK4) has recently been identified as a potential drug target for several complex diseases including cancer, diabetes and neurodegenerative disorders. Inhibition of MARK4 activity is an appealing therapeutic option to treat such diseases. Here, we have performed structure-based virtual high-throughput screening of 100,000 naturally occurring compounds from ZINC database against MARK4 to find its potential inhibitors. The resulted hits were selected, based on the binding affinities, docking scores and selectivity. Further, binding energy calculation, Lipinski filtration and ADMET prediction were carried out to find safe and better hits against MARK4. Best 10 compounds bearing high specificity and binding efficiency were selected, and their binding pattern to MARK4 was analyzed in detail. Finally, 100 ns molecular dynamics simulation was performed to evaluate; the dynamics stability of MARK4-compound complex. In conclusion, these selected natural compounds from ZINC database might be potential leads against MARK4, and can further be exploited in drug design and development for associated diseases.     

Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.     

GSK-3beta directly phosphorylates and activates MARK2/PAR-1

In Alzheimer disease (AD), the microtubule-associated protein tau is found hyperphosphorylated in paired helical filaments. Among many phosphorylated sites in tau, Ser-262 is the major site for abnormal phosphorylation of tau in AD brain. The kinase known to phosphorylate this particular site is MARK2, whose activation mechanism is yet to be studied. Our first finding that treatment of cells with LiCl, a selective inhibitor of another major tau kinase, glycogen synthase kinase-3beta (GSK-3beta), inhibits phosphorylation of Ser-262 of tau led us to investigate the possible involvement of GSK-3beta in MARK2 activation. In vitro kinase reaction revealed that recombinant GSK-3beta indeed phosphorylates MARK2, whereas it failed to phosphorylate Ser-262 of tau. Our further findings led us to conclude that GSK-3beta phosphorylates MARK2 on Ser-212, one of the two reported phosphorylation sites (Thr-208 and Ser-212) found in the activation loop of MARK2. Down-regulation of either GSK-3beta or MARK2 by small interfering RNAs suppressed the level of phosphorylation on Ser-262. These results, respectively, indicated that GSK-3beta is responsible for phosphorylating Ser-262 of tau through phosphorylation and activation of MARK2 and that the phosphorylation of tau at this particular site is predominantly mediated by a GSK-3beta-MARK2 pathway. These findings are of interest in the context of the pathogenesis of AD.