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Glycogen synthase kinase-3 (GSK-3) isoforms α and β have diverse roles within cell biology, and have been linked with multiple diseases that include prominent CNS conditions such as Alzheimer's disease and several psychiatric disorders. In this study, motivated by computation, we aimed to identify novel ATP-binding site inhibitors of GSK-3 with CNS-active potential. A ligand screening (docking) protocol against GSK-3β was first optimized, employing an active/decoy benchmarking set, with the final protocol selected based on statistical performance analysis. The optimized protocol involved pre-filtering of ligands using a three-point 3D-pharmacophore, followed by Glide-SP docking applying hinge region hydrogen bonding constraints. Using this approach, the Biogenic subset of the ZINC15 compound database was screened, focused on compounds with potential for CNS-activity. Twelve compounds (generation I) were selected for experimental validation using in vitro GSK-3β binding assays. Two hit compounds, 1 and 2, with 6-amino-7H-benzo[e]perimidin-7-one and 1-(phenylamino)-3H-naphtho[1,2,3-de]quinoline-2,7-dione type scaffolds were identified with IC50 values of 1.63 µM and 20.55 µM, respectively. Ten analogues of 2 (generation II) were selected for structure activity relationship (SAR) analysis and revealed four low micromolar inhibitors (<10 µM), with 19 (IC50 = 4.1 µM)~five times more potent than initial hit compound 2. Selectivity screening of low micromolar inhibitors 14 and 19 (comparing aryl- and alkyl-substituents) against 10 homologous kinases revealed unique selectivity profiles, with both compounds more potent against the GSK-3α isoform (IC50s~2 µM) and, additionally, inhibitors of PKBβ (IC50s < 25 µM). Compound 14 also inhibited ERK2 and 19, PKCγ, but generally good selectivity for GSK-3 isoforms over the other kinases was observed. The compounds had excellent predicted oral bioavailability and CNS-activity profiles, presenting promising candidates for future testing in cellular models of disease. [ABSTRACT FROM AUTHOR]

A 3D model visualization and basic molecular modeling laboratory suitable for first-year undergraduates studying introductory medicinal chemistry is presented. The 2 h practical is embedded within a series of lectures on drug design, target--drug interactions, enzymes, receptors, nucleic acids, and basic pharmacokinetics. Serving as a teaching aid to the lecture material, 3D models of biological macromolecules exploiting Schrödinger software and the Maestro graphical user interface (GUI) is explored to enhance student learning. A considerably positive response was received firom the participants. Background and details of the laboratory are outlined, while the student handout with answers is included as Supporting Information. [ABSTRACT FROM AUTHOR]

Abstract: Flavonoids have been discovered as novel inhibitors of glycogen phosphorylase (GP), a target to control hyperglycemia in type 2 diabetes. To elucidate the mechanism of inhibition, we have determined the crystal structure of the GPb-chrysin complex at 1.9Å resolution. Chrysin is accommodated at the inhibitor site intercalating between the aromatic side chains of Phe285 and Tyr613 through π-stacking interactions. Chrysin binds to GPb ∼15 times weaker (K i =19.01μM) than flavopiridol (K i =1.24μM), exclusively at the inhibitor site, and both inhibitors display similar behavior with respect to AMP. To identify the source of flavopiridols’ stronger affinity, molecular docking with Glide and postdocking binding free energy calculations using QM/MM-PBSA have been performed and compared. Whereas docking failed to correctly rank inhibitor binding conformations, the QM/MM-PBSA method employing M06-2X/6-31+G* to model the π-stacking interactions correctly reproduced the experimental results. Flavopiridols’ greater binding affinity is sourced to favorable interactions of the cationic 4-hydroxypiperidin-1-yl substituent with GPb, with desolvation effects limited by the substituent conformation adopted in the crystallographic complex. Further successful predictions using QM/MM-PBSA for the flavonoid quercetagetin (which binds at the allosteric site) leads us to propose the methodology as a useful and inexpensive tool to predict flavonoid binding. [Copyright &y& Elsevier]

Abstract: A series of glucopyranosylidene-spiro-isoxazolines was prepared through regio- and stereoselective [3+2]-cycloaddition between the methylene acetylated exo-glucal and aromatic nitrile oxides. The deprotected cycloadducts were evaluated as inhibitors of muscle glycogen phosphorylase b. The carbohydrate-based family of five inhibitors displays K i values ranging from 0.63 to 92.5μM. The X-ray structures of the enzyme–ligand complexes show that the inhibitors bind preferentially at the catalytic site of the enzyme retaining the less active T-state conformation. Docking calculations with GLIDE in extra-precision (XP) mode yielded excellent agreement with experiment, as judged by comparison of the predicted binding modes of the five ligands with the crystallographic conformations and the good correlation between the docking scores and the experimental free binding energies. Use of docking constraints on the well-defined positions of the glucopyranose moiety in the catalytic site and redocking of GLIDE-XP poses using electrostatic potential fit-determined ligand partial charges in quantum polarized ligand docking (QPLD) produced the best results in this regard. [Copyright &y& Elsevier]

Recently studied N-(β-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(β-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated β-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (Kis) of ~3–4 µM obtained for 1- and 2-naphthyl-substituted N-(β-d-glucopyranosyl)-imidazolecarboxamides, 2b–c. The predicted protein–ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed. [ABSTRACT FROM AUTHOR]

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C-β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion–ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1H-pyrazole (3) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-pyrazole (4) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics—generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion–ion interactions; for 4, the most stable tautomer did not have the His377 backbone C=O interaction and while ion–ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted. [ABSTRACT FROM AUTHOR]

Abstract: Bovine seminal ribonuclease (BS-RNase) is a 27kDa homodimeric enzyme and a member of the pancreatic RNase A superfamily. It is the only RNase with a quaternary structure and it is a mixture of two dimeric forms. In the most abundant form the active site is formed by the swapping of the N-terminal segments. BS-RNase is a potent antitumor agent with severe side effects such as aspermatogenicity, and immunosuppression. As a first step towards the design of potent inhibitors of this enzyme we mapped its active site through the study of the binding of uridine 2′-phosphate (U2′p), uridine 3′-phosphate (U3′p), uridine 5′-diphosphate (UDP), cytidine 3′-phosphate (C3′p), and cytidine 5-phosphate (C5′p), by kinetics, and X-ray crystallography. These phosphonucleotides are potent inhibitors with C3′p being the most potent with a K i value of 22μM. Absorption, distribution, metabolism, and excretion pharmacokinetic property predictions reveal U2′p, U3′p, and C5′p as the most promising with respect to oral bioavailability. In vivo studies on the aspermatogenic effect have shown that C3′p and C5′p inhibit significantly this biological action of BS-RNase. [Copyright &y& Elsevier]

A series of gas-phase nucleophilic substitution reactions at sulfur of methanesulfinyl derivatives by small anions (chloride, cyanide, hydroxide, methoxide, amide, and phosphide, identical to the leaving group in each case) were examined by Hartree-Fock, MP2, and DFT computations. In most cases, substitution was found to follow an addition-elimination mechanism, resulting in a triple-well potential energy surface with small barriers of activation on either side of the central, tetracoordinate-sulfur minimum. The geometries of the central minima, as in the analogous methanesulfenyl cases, are unsymmetrical trigonal bipyramidal, with the nucleophiles and leaving groups occupying apical positions and the sulfur lone pair an equatorial position. The apparent exception, cyanide, may undergo an SN2 displacement. [ABSTRACT FROM AUTHOR]

3-(β- d -Glucopyranosyl)-5-substituted-1,2,4-triazoles have been revealed as an effective scaffold for the development of potent glycogen phosphorylase (GP) inhibitors but with the potency very sensitive to the nature of the alkyl/aryl 5-substituent (Kun et al., Eur. J. Med. Chem. 2014, 76, 567). For a training set of these ligands, quantum mechanics-polarized ligand docking (QM-PLD) demonstrated good potential to identify larger differences in potencies (predictive index PI = 0.82) and potent inhibitors with K i 's < 10 μM (AU-ROC = 0.86). Accordingly, in silico screening of 2335 new analogues exploiting the ZINC docking database was performed and nine predicted candidates selected for synthesis. The compounds were prepared in O -perbenzoylated forms by either ring transformation of 5-β- d -glucopyranosyl tetrazole by N -benzyl-arenecarboximidoyl chlorides, ring closure of C -(β- d -glucopyranosyl)formamidrazone with aroyl chlorides, or that of N -(β- d -glucopyranosylcarbonyl)arenethiocarboxamides by hydrazine, followed by deprotections. Kinetics experiments against rabbit muscle GPb (rmGPb) and human liver GPa (hlGPa) revealed five compounds as potent low μM inhibitors with three of these on the submicromolar range for rmGPa. X-ray crystallographic analysis sourced the potency to a combination of favorable interactions from the 1,2,4-triazole and suitable aryl substituents in the GP catalytic site. The compounds also revealed promising calculated pharmacokinetic profiles. [ABSTRACT FROM AUTHOR]

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and GBM at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (K i = 32.54 μM), human liver GPa (K i = 8.77 μM) and rabbit muscle GPb (K i = 5.66 μM) isoforms. It is also an effective inhibitor of glycogenolysis (IC 50 = 119.6 μM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC 50 values of ∼20–55 μM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O6-methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP–baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against GBM is suggested. • Baicalein revealed as a potent inhibitor of all three glycogen phosphorylase (GP) isforms: liver, muscle and brain. • Its binding at GP was predicted using docking and validated using X-ray crystallography. • The compound binds at the GP inhibitor site, intercalated between Phe285 and Tyr613. • Baicalein revealed as an effective inhibitor of glycogenolysis (IC50 = 119.6 μM), measured in HepG2 cells. • It demonstrated promising anti-cancer potential against three glioblastoma cell-lines. [ABSTRACT FROM AUTHOR]

Glucocorticoids are steroid hormones widely used to control many inflammatory conditions. These effects are primarily attributed to glucocorticoid receptor transrepressional activities but with concomitant receptor transactivation associated with considerable side effects. Accordingly, there is an immediate need for selective glucocorticoid receptor agonists able to dissociate transactivation from transrepression. Triterpenoids have structural similarities with glucocorticoids and exhibit anti-inflammatory and apoptotic activities via mechanisms that are not well-defined. In this study, we examined whether echinocystic acid and its 3-O-glucoside derivative act, at least in part, through the regulation of glucocorticoid receptor and whether they can constitute selective receptor activators. We showed that echinocystic acid and its glucoside induced glucocorticoid receptor nuclear translocation by 75% and 55%. They suppressed the nuclear factor-kappa beta transcriptional activity by 20% and 70%, respectively, whereas they have no glucocorticoid receptor transactivation capability and stimulatory effect on the expression of the phosphoenolopyruvate carboxykinase target gene in HeLa cells. Interestingly, their suppressive effect is diminished in glucocorticoid receptor low level COS-7 cells, verifying the receptor involvement in this process. Induced fit docking calculations predicted favorable binding in the ligand binding domain and structural characteristics which can be considered consistent with the experimental observations. Further, glucocorticoids exert apoptotic activities; we have demonstrated here that the echinocystic acids in combination with the synthetic glucocorticoid, dexamethasone, induce apoptosis. Taken together, our results indicate that echinocystic acids are potent glucocorticoid receptor regulators with selective transrepressional activities (dissociated from transactivation), highlighting the potential of echinocystic acid derivatives as more promising treatments for inflammatory conditions. [ABSTRACT FROM AUTHOR]

Phosphorylase kinase (PhK) has been linked with a number of conditions such as glycogen storage diseases, psoriasis, type 2 diabetes and more recently, cancer (Camus et al., 2012 [6] ). However, with few reported structural studies on PhK inhibitors, this hinders a structure based drug design approach. In this study, the inhibitory potential of 38 indirubin analogues have been investigated. 11 of these ligands had IC 50 values in the range 0.170–0.360 μM, with indirubin-3′-acetoxime ( 1c ) the most potent. 7-Bromoindirubin-3′-oxime ( 13b ), an antitumor compound which induces caspase-independent cell-death (Ribas et al., 2006 [20] ) is revealed as a specific inhibitor of PhK (IC 50 = 1.8 μM). Binding assay experiments performed using both PhK-holo and PhK-γtrnc confirmed the inhibitory effects to arise from binding at the kinase domain (γ subunit). High level computations using QM/MM-PBSA binding free energy calculations were in good agreement with experimental binding data, as determined using statistical analysis, and support binding at the ATP-binding site. The value of a QM description for the binding of halogenated ligands exhibiting σ-hole effects is highlighted. A new statistical metric, the ‘sum of the modified logarithm of ranks’ (SMLR), has been defined which measures performance of a model for both the “early recognition” (ranking earlier/higher) of active compounds and their relative ordering by potency. Through a detailed structure activity relationship analysis considering other kinases (CDK2, CDK5 and GSK-3α/β), 6′(Z) and 7(L) indirubin substitutions have been identified to achieve selective PhK inhibition. The key PhK binding site residues involved can also be targeted using other ligand scaffolds in future work. [ABSTRACT FROM AUTHOR]

Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 N -acyl-β- d -glucopyranosylamines putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a ‘consensus scoring’ approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants’ values, in vitro, ranged from 5 to 377 μM while two of them were effective at causing inactivation of GP in rat hepatocytes at low μM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: All possible isomers of N-β-d-glucopyranosyl aryl-substituted oxadiazolecarboxamides were synthesised. O-Peracetylated N-cyanocarbonyl-β-d-glucopyranosylamine was transformed into the corresponding N-glucosyl tetrazole-5-carboxamide, which upon acylation gave N-glucosyl 5-aryl-1,3,4-oxadiazole-2-carboxamides. The nitrile group of the N-cyanocarbonyl derivative was converted to amidoxime which was ring closed by acylation to N-glucosyl 5-aryl-1,2,4-oxadiazole-3-carboxamides. A one-pot reaction of protected β-d-glucopyranosylamine with oxalyl chloride and then with arenecarboxamidoximes furnished N-glucosyl 3-aryl-1,2,4-oxadiazole-5-carboxamides. Removal of the O-acetyl protecting groups by the Zemplén method produced test compounds which were evaluated as inhibitors of glycogen phosphorylase. Best inhibitors of these series were N-(β-d-glucopyranosyl) 5-(naphth-1-yl)-1,2,4-oxadiazol-3-carboxamide (K i =30μM), N-(β-d-glucopyranosyl) 5-(naphth-2-yl)-1,3,4-oxadiazol-2-carboxamide (K i =33μM), and N-(β-d-glucopyranosyl) 3-phenyl-1,2,4-oxadiazol-5-carboxamide (K i =104μM). ADMET property predictions revealed these compounds to have promising oral drug-like properties without any toxicity. [Copyright &y& Elsevier]

Glycogen phosphorylase is a molecular target for the design of potential hypoglycemic agents. Structure-based design pinpointed that the 3′-position of glucopyranose equipped with a suitable group has the potential to form interactions with enzyme's cofactor, pyridoxal 5′-phosphate (PLP), thus enhancing the inhibitory potency. Hence, we have investigated the binding of two ligands, 1-( β- d-glucopyranosyl)5-fluorouracil (GlcFU) and its 3′-CH2OH glucopyranose derivative. Both ligands were found to be low micromolar inhibitors with Ki values of 7.9 and 27.1 μ m, respectively. X-ray crystallography revealed that the 3′-CH2OH glucopyranose substituent is indeed involved in additional molecular interactions with the PLP γ-phosphate compared with GlcFU. However, it is 3.4 times less potent. To elucidate this discovery, docking followed by postdocking Quantum Mechanics/Molecular Mechanics - Poisson-Boltzmann Surface Area (QM/MM-PBSA) binding affinity calculations were performed. While the docking predictions failed to reflect the kinetic results, the QM/MM-PBSA revealed that the desolvation energy cost for binding of the 3′-CH2OH-substituted glucopyranose derivative out-weigh the enthalpy gains from the extra contacts formed. The benefits of performing postdocking calculations employing a more accurate solvation model and the QM/MM-PBSA methodology in lead optimization are therefore highlighted, specifically when the role of a highly polar/charged binding interface is significant. [ABSTRACT FROM AUTHOR]

Abstract: Design of inhibitors of glycogen phosphorylase (GP) with pharmaceutical applications in improving glycaemic control in type 2 diabetes is a promising therapeutic strategy. The catalytic site of muscle glycogen phosphorylase b (GPb) has been probed with five deoxy-fluro-glucose derivatives. These inhibitors had fluorine instead of hydroxyl at the 3′ position of the glucose moiety and a variety of pyrimidine derivatives at the 1′ position. The best of this carbohydrate-based family of five inhibitors displays a K i value of 46μM. To elucidate the mechanism of inhibition for these compounds, the crystal structures of GPb in complex with each ligand were determined and refined to high resolution. The structures demonstrated that the inhibitors bind preferentially at the catalytic site and promote the less active T state conformation of the enzyme by making several favorable contacts with residues of the 280s loop. Fluorine is engaged in hydrogen bond interactions but does not improve glucose potency. The pyrimidine groups are located between residues 284–286 of the 280s loop, Ala383 of the 380s loop, and His341 of the β-pocket. These interactions appear important in stabilizing the inactive quaternary T state of the enzyme. As a follow up to recent computations performed on β-d-glucose pyrimidine derivatives, tautomeric forms of ligands 1–5 were considered as potential binding states. Using Glide-XP docking and QM/MM calculations, the ligands 2 and 5 are predicted to bind in different tautomeric states in their respective GPb complexes. Also, using α-d-glucose as a benchmark model, a series of substitutions for glucose –OH at the 3′ (equatorial) position were investigated for their potential to improve the binding affinity of glucose-based GPb catalytic site inhibitors. Glide-XP and quantum mechanics polarized ligand (QPLD-SP/XP) docking calculations revealed favorable binding at this position to be dominated by hydrogen bond contributions; none of the substitutions (including fluorine) out-performed the native –OH substituent which can act both as hydrogen bond donor and acceptor. The structural analyses of these compounds can be exploited towards the development of better inhibitors. [Copyright &y& Elsevier]

Inhibition of the human O-linked β-N-acetylglucosaminidase (hOGA, GH84) enzyme is pharmacologically relevant in several diseases such as neurodegenerative and cardiovascular disorders, type 2 diabetes, and cancer. Human lysosomal hexosaminidases (hHexA and hHexB, GH20) are mechanistically related enzymes; therefore, selective inhibition of these enzymes is crucial in terms of potential applications. In order to extend the structure–activity relationships of OGA inhibitors, a series of 2-acetamido-2-deoxy-d-glucono-1,5-lactone sulfonylhydrazones was prepared from d-glucosamine. The synthetic sequence involved condensation of N-acetyl-3,4,6-tri-O-acetyl-d-glucosamine with arenesulfonylhydrazines, followed by MnO2 oxidation to the corresponding glucono-1,5-lactone sulfonylhydrazones. Removal of the O-acetyl protecting groups by NH3/MeOH furnished the test compounds. Evaluation of these compounds by enzyme kinetic methods against hOGA and hHexB revealed potent nanomolar competitive inhibition of both enzymes, with no significant selectivity towards either. The most efficient inhibitor of hOGA was 2-acetamido-2-deoxy-d-glucono-1,5-lactone 1-naphthalenesulfonylhydrazone (5f, Ki = 27 nM). This compound had a Ki of 6.8 nM towards hHexB. To assess the binding mode of these inhibitors to hOGA, computational studies (Prime protein–ligand refinement and QM/MM optimizations) were performed, which suggested the binding preference of the glucono-1,5-lactone sulfonylhydrazones in an s-cis conformation for all test compounds. [ABSTRACT FROM AUTHOR]

O -GlcNAcylation is a dynamic post-translational modification mediated by O -linked β- N -acetylglucosamine transferase (OGT) and O -GlcNAc hydrolase (OGA), that adds or removes a single β- N -acetylglucosamine (GlcNAc) moiety to or from serine/threonine residues of nucleocytosolic and mitochondrial proteins, respectively. The perturbed homeostasis of O -GlcNAc cycling results in several pathological conditions. Human OGA is a promising therapeutic target in diseases where aberrantly low levels of O -GlcNAc are experienced, such as tauopathy in Alzheimer's disease. A new class of potent OGA inhibitors, 2-acetamido-2-deoxy- d -glucono-1,5-lactone (thio)semicarbazones, have been identified. Eight inhibitors were designed and synthesized in five steps starting from d -glucosamine and with 15–55% overall yields. A heterologous OGA expression protocol with strain selection and isolation has been optimized that resulted in stable, active and full length human OGA (hOGA) isomorph. Thermal denaturation kinetics of hOGA revealed environmental factors affecting hOGA stability. From kinetics experiments, the synthesized compounds proved to be efficient competitive inhibitors of hOGA with K i -s in the range of ∼30–250 nM and moderate selectivity with respect to lysosomal β-hexosaminidases. In silico studies consisting of Prime protein-ligand refinements, QM/MM optimizations and QM/MM-PBSA binding free energy calculations revealed the factors governing the observed potencies, and led to design of the most potent analogue 2-acetamido-2-deoxy- d -glucono-1,5-lactone 4-(2-naphthyl)-semicarbazone 6g (K i = 36 nM). The protocol employed has applications in future structure based inhibitor design targeting OGA. [Display omitted] • Synthesis of 2-acetamido-2-deoxy-D-glucono-1,5-lactone semicarbazones. • New, low nanomolar, competitive inhibitors of human OGA. • Expression and stability studies on human OGA. • In silico studies on the binding mode of the new inhibitors. [ABSTRACT FROM AUTHOR]

• Synthetic flavonoids synthesized based on QM/MM-PBSA binding free energy predictions. • Three compounds revealed as more potent than any natural flavonoid GP inhibitor. • Binding is synergistic with glucose and could be regulated by blood glucose levels. • Compound 43 an effective inhibitor of glycogenolysis in hepatocytes (IC 50 = 70 µM). • X-ray crystallography studied the interactions responsible for the observed potencies. Glycogen phosphorylase (GP) is an important target for the development of new anti-hyperglycaemic agents. Flavonoids are novel inhibitors of GP, but their mode of action is unspecific in terms of the GP binding sites involved. Towards design of synthetic flavonoid analogues acting specifically at the inhibitor site and to exploit the site's hydrophobic pocket, chrysin has been employed as a lead compound for the in silico screening of 1169 new analogues with different B ring substitutions. QM/MM-PBSA binding free energy calculations guided the final selection of eight compounds, subsequently synthesised using a Baker-Venkataraman rearrangement-cyclisation approach. Kinetics experiments against rabbit muscle GPa and GPb together with human liver GPa, revealed three of these compounds (11 , 20 and 43) among the most potent that bind at the site (K i s < 4 µM for all three isoforms), and more potent than previously reported natural flavonoid inhibitors. Multiple inhibition studies revealed binding exclusively at the inhibitor site. The binding is synergistic with glucose suggesting that inhibition could be regulated by blood glucose levels and would decrease as normoglycaemia is achieved. Compound 43 was an effective inhibitor of glycogenolysis in hepatocytes (IC 50 = 70 µM), further promoting these compounds for optimization of their drug-like potential. X-ray crystallography studies revealed the B-ring interactions responsible for the observed potencies. [ABSTRACT FROM AUTHOR]

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist. [ABSTRACT FROM AUTHOR]