A Novel 5-Chloro-N-phenyl-1H-indole-2-carboxamide Derivative as Brain-Type Glycogen Phosphorylase Inhibitor: Validation of Target PYGB.

Brain-type glycogen phosphorylase (PYGB) inhibitors are recognized as prospective drugs for treating ischemic brain injury. We previously reported compound 1 as a novel glycogen phosphorylase inhibitor with brain-protective properties. In this study, we validated whether PYGB could be used as the therapeutic target for hypoxic-ischemic diseases and investigated whether compound 1 exerts a protective effect against astrocyte hypoxia/reoxygenation (H/R) injury by targeting PYGB. A gene-silencing strategy was initially applied to downregulate PYGB proteins in mouse astrocytes, which was followed by a series of cellular experiments with compound 1. Next, we compared relevant indicators that could prove the protective effect of compound 1 on brain injury, finding that after PYGB knockdown, compound 1 could not obviously alleviate astrocytes H/R injury, as evidenced by cell viability, which was not significantly improved, and lactate dehydrogenase (LDH) leakage rate, intracellular glucose content, and post-ischemic reactive oxygen species (ROS) level, which were not remarkably reduced. At the same time, cellular energy metabolism did not improve, and the degree of extracellular acidification was not downregulated after administration of compound 1 after PYGB knockdown. In addition, it could neither significantly increase the level of mitochondrial aerobic energy metabolism nor inhibit the expression of apoptosis-associated proteins. The above results indicate that compound 1 could target PYGB to exert its protective effect against cellular H/R injury in mouse astrocytes. Simultaneously, we further demonstrated that PYGB could be an efficient therapeutic target for ischemic-hypoxic diseases. This study provides a new reference for further in-depth study of the action mechanism of the efficacy of compound 1.

Glycopyranosylidene-Spiro-Morpholinones: Evaluation of the Synthetic Possibilities Based on Glyculosonamide Derivatives and a New Method for the Construction of the Morpholine Ring.

Glycosylidene-spiro-morpholin(on)es are scarcely described skeletons in the literature. In this work, we have systematically explored the synthetic routes towards such morpholinones based on the reactions of O-peracylated hept-2-ulopyranosonamide derivatives of D-gluco and D-galacto configuration. Koenigs-Knorr type glycosylation of 2-chloroethanol, allylic and propargylic alcohols by (glyculosylbromide)onamides furnished the expected glycosides. The 2-chloroethyl glycosides were ring closed to the corresponding spiro-morpholinones by treatment with K2CO3. The (allyl glyculosid)onamides gave diastereomeric mixtures of spiro-5-hydroxymorpholinones by ozonolysis and 5-iodomethylmorpholinones under iodonium ion mediated conditions. The ozonolytic method has not yet been known for the construction of morpholine rings, therefore, it was also extended to O-allyl mandelamide. The 5-hydroxymorpholinones were subjected to oxidation and acid catalyzed elimination reactions to give the corresponding morpholine-3,5-dions and 5,6-didehydro-morpholin-3-ones, respectively. Base induced elimination of the 5-iodomethylmorpholinones gave 5-methyl-2H-1,4-oxazin-3(4H)-ones. O-Acyl protecting groups of all of the above compounds were removed under Zemplén conditions. Some of the D-gluco configured unprotected compounds were tested as inhibitors of glycogen phosphorylase, but showed no significant effect.

A Novel 5-Chloro-N-phenyl-1H-indole-2-carboxamide Derivative as Brain-Type Glycogen Phosphorylase Inhibitor: Potential Therapeutic Effect on Cerebral Ischemia.

Brain-type glycogen phosphorylase inhibitors are potential new drugs for treating ischemic brain injury. In our previous study, we reported compound 1 as a novel brain-type glycogen phosphorylase inhibitor with cardioprotective properties. We also found that compound 1 has high blood-brain barrier permeability through the ADMET prediction website. In this study, we deeply analyzed the protective effect of compound 1 on hypoxic-ischemic brain injury, finding that compound 1 could alleviate the hypoxia/reoxygenation (H/R) injury of astrocytes by improving cell viability and reducing LDH leakage rate, intracellular glucose content, and post-ischemic ROS level. At the same time, compound 1 could reduce the level of ATP in brain cells after ischemia, improve cellular energy metabolism, downregulate the degree of extracellular acidification, and improve metabolic acidosis. It could also increase the level of mitochondrial aerobic energy metabolism during brain cell reperfusion, reduce anaerobic glycolysis, and inhibit apoptosis and the expression of apoptosis-related proteins. The above results indicated that compound 1 is involved in the regulation of glucose metabolism, can control cell apoptosis, and has protective and potential therapeutic effects on cerebral ischemia-reperfusion injury, which provides a new reference and possibility for the development of novel drugs for the treatment of ischemic brain injury.

Long-term pharmacokinetic and pharmacological evaluations of a novel indole-benzazepinone derivative on obese Type 2 diabetes mellitus.

Background: Owing to the chronic nature of Type 2 diabetes mellitus, antidiabetic drugs must have long-lasting efficacy. Compound 1 has a good inhibitory effect on acute hyperglycemia, but its long-term hypoglycemic effect has not been evaluated. Results: Preliminary prediction and in vitro experimental pharmacokinetic results support the use of compound 1 for long-term in vivo experiments. Long-term experiments demonstrated that compound 1 significantly reduces blood glucose, improves the oral glucose tolerance of obese mice and has a positive effect on body weight, free fatty acid, hepatocyte steatosis and inflammatory cell infiltration. Conclusion: These findings lay a good foundation for the further exploration and development of novel glycogen phosphorylase inhibitors.

Discovery of novel heterocyclic derivatives as potential glycogen phosphorylase inhibitors with a cardioprotective effect.

The purpose of this study was to evaluate the effect of GP inhibitor as a potential pharmaceutical target on MI/R injury. Four different structural types of novel compounds (I, II, III, and IV) were designed and synthesized, obtaining 31 novel GP inhibitors. SAR studies revealed that the conjugates of 5-chloroindole with benzo six-membered heterocyclic were found to elevate the activity. In particular, compound IIIh (IC₅₀ = 0.21 ± 0.03 µM) emerged as a potent derivative against RMGPa, being approximately 2-fold less potent than that of PSN-357. In order to screen out a compound for in vivo activity test, we further conducted an experiment of inhibition against three different subtypes of GPa (HLGPa, HMGPa and HBGPa) and the corresponding affinity experiment. As a result, compound IIIh showed strong inhibitory activity against the above three subtypes of GP, especially on HBGPa (IC₅₀ = 0.09 ± 0.002 µM), which was relatively close to that of positive control ingliforib (IC₅₀ = 0.16 ± 0.02 µM). The affinity of compound IIIh to HBGPa was 4.3 times higher than that of HLGPa, and 1.1 times higher than that of HMGPa. This fact further proved that compound IIIh has a higher inhibitory effect on HMGPa than the other two subtypes. Besides, in vivo activity evaluation demonstrated that compound IIIh exhibited obviously cardioprotective effect on MI/R injury mice. The discovery of compound IIIh provides a new strategy for developing novel GP inhibitors with myocardial ischemia protection.

Vitamin C deficiency induces hypoglycemia and cognitive disorder through S-nitrosylation-mediated activation of glycogen synthase kinase 3ß.

Vitamin C (VC, l-ascorbic acid) is an essential nutrient that plays a key role in metabolism and functions as a potent antioxidant in regulating the S-nitrosylation and denitrosylation of target proteins. The precise function of VC deprivation in glucose homeostasis is still unknown. In the absence of L-gulono-1,4-lactone oxidoreductase, an essential enzyme for the last step of VC synthesis, VC deprivation resulted in persistent hypoglycemia and subsequent impairment of cognitive functions in female but not male mouse pups. The cognitive disorders caused by VC deprivation were largely reversed when these female pups were given glucose. VC deprivation-induced S-nitrosylation of glycogen synthase kinase 3ß (GSK3ß) at Cys14, which activated GSK3ß and inactivated glycogen synthase to decrease glycogen synthesis and storage under the feeding condition, while VC deprivation inactivated glycogen phosphorylase to decrease glycogenolysis under the fasting condition, ultimately leading to hypoglycemia and cognitive disorders. Treatment with Nω-Nitro-l-arginine methyl ester (l-NAME), a specific inhibitor of nitric oxide synthase, on the other hand, effectively prevented S-nitrosylation and activation of GSK3ß in female pups in response to the VC deprivation and reversed hypoglycemia and cognitive disorders. Overall, this research identifies S-nitrosylation of GSK3ß and subsequent GSK3ß activation as a previously unknown mechanism controlling glucose homeostasis in female pups in response to VC deprivation, implying that VC supplementation in the prevention of hypoglycemia and cognitive disorders should be considered in the certain groups of people, particularly young females.

Metabolic alterations and mitochondrial dysfunction underlie hepatocellular carcinoma cell death induced by a glycogen metabolic inhibitor.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. There is an urgent need for new targets to treat HCC due to limited treatment options and drug resistance. Many cancer cells are known to have high amount of glycogen than their tissue of origin and inhibition of glycogen catabolism induces cancer cell death by apoptosis. To further understand the role of glycogen in HCC and target it for pharmacotherapy, we studied metabolic adaptations and mitochondrial function in HepG2 cells after pharmacological inhibition of glycogen phosphorylase (GP) by CP-91149 (CP). GP inhibition increased the glycogen levels in HepG2 cells without affecting overall glucose uptake. Glycolytic capacity and importantly glycolytic reserve decreased significantly. Electron microscopy revealed that CP treatment altered mitochondrial morphology leading to mitochondrial swelling with less defined cristae. A concomitant decrease in mitochondrial oxygen consumption and mitochondria-linked ATP generation was observed. Metabolomics and enzyme activity / expression studies showed a decrease in the pentose phosphate pathway. In addition, CP treatment decreased the growth of HepG2 3D tumor spheroids in a dose- and time-dependent manner. Taken together, our study provides insights into metabolic alterations and mitochondrial dysfunction accompanying apoptosis in HepG2 cells upon GP inhibition. Our study can aid in the understanding of the mechanism and development of metabolic inhibitors to treat HCC.

A Proteomic Platform Unveils the Brain Glycogen Phosphorylase as a Potential Therapeutic Target for Glioblastoma Multiforme.

In the last few years, several efforts have been made to identify original strategies against glioblastoma multiforme (GBM): this requires a more detailed investigation of the molecular mechanism of GBM so that novel targets can be identified for new possible therapeutic agents. Here, using a combined biochemical and proteomic approach, we evaluated the ability of a blood-brain barrier-permeable 2,3-benzodiazepin-4-one, called 1g, to interfere with the activity and the expression of brain glycogen phosphorylase (PYGB) on U87MG cell line in parallel with the capability of this compound to inhibit the cell growth and cycle. Thus, our results highlighted PYGB as a potential therapeutic target in GBM prompting 1g as a capable anticancer drug thanks to its ability to negatively modulate the uptake and metabolism of glucose, the so-called "Warburg effect", whose increase is considered a common feature of cancer cells in respect of their normal counterparts.

Photoelectrochemical sandwich immunoassay of brain glycogen phosphorylase based on methyl orange-sensitized TiO2 nanorods.

The photoelectrochemical immunoassay of glycogen phosphorylase BB (GPBB) was studied. A methyl orange/TiO2 nanorod heterojunction was constructed on a fluorine-doped tin oxide electrode by hydrothermal synthesis, calcination, and chemical adsorption. A sandwich immune structure consisting of GPBB as the first antibody, GPBB, and a CdS@mesoporous silica-ascorbic acid (AA)-GPBB as secondary antibody composite was constructed on each of the selected well surfaces of a 96-well microplate. By adding mercaptoethylamine to structurally destroy the secondary antibody composite and release the electron donor AA, the amplification of photocurrent, and thus the "off-on" photoelectrochemical biosensing of GPBB were realized. The use of the 96-well microplate provides good reproducibility of the assembled immune structures and eliminates the possible effect of the photogenerated hole-induced protein oxidation on the photocurrent. The relevant electrodes and materials were characterized by electrochemistry, UV-vis diffuse reflectance spectra, Fourier transform infrared spectroscopy, X-ray diffractometer, scanning electron microscopy/energy dispersive spectroscopy, transmission electron microscopy and BET method. Under the optimal conditions, the photocurrent was linear with the logarithm of GPBB concentration from 0.005 to 200 ng mL-1 and with a limit of detection of 1.7 pg mL-1 (S/N = 3). Satisfactory results were obtained in the analysis of real serum samples. A sandwich immune structure consisting of GPBB first antibody, GPBB, and a CdS@mesoporous silica-ascorbic acid (AA)-GPBB secondary antibody composite was constructed on each of the selected well surfaces of a 96-well microplate. By adding mercaptoethylamine to structurally destroy the secondary antibody composite and release the electron donor AA, the amplification of photocurrent, and thus the "off-on" photoelectrochemical biosensing of GPBB were realized.

Insights into Phosphorylation-Induced Protein Allostery and Conformational Dynamics of Glycogen Phosphorylase via Integrative Structural Mass Spectrometry and In Silico Modeling.

Allosteric regulation plays a fundamental role in innumerable biological processes. Understanding its dynamic mechanism and impact at the molecular level is of great importance in disease diagnosis and drug discovery. Glycogen phosphorylase (GP) is a phosphoprotein responding to allosteric regulation and has significant biological importance to glycogen metabolism. Although the atomic structures of GP have been previously solved, the conformational dynamics of GP related to allostery regulation remain largely elusive due to its macromolecular size (∼196 kDa). Here, we integrated native top-down mass spectrometry (nTD-MS), hydrogen-deuterium exchange MS (HDX-MS), protection factor (PF) analysis, molecular dynamics (MD) simulations, and allostery signaling analysis to examine the structural basis and dynamics for the allosteric regulation of GP by phosphorylation. nTD-MS reveals differences in structural stability as well as oligomeric state between the unphosphorylated (GPb) and phosphorylated (GPa) forms. HDX-MS, PF analysis, and MD simulations further pinpoint the structural differences between GPb and GPa involving the binding interfaces (the N-terminal and tower-tower helices), catalytic site, and PLP-binding region. More importantly, it also allowed us to complete the missing link of the long-range communication process from the N-terminal tail to the catalytic site caused by phosphorylation. This integrative MS and in silico-based platform is highly complementary to biophysical approaches and yields valuable insights into protein structures and dynamic regulation.

Glycogen phosphorylase isoform regulation of glucose and energy sensor expression in male versus female rat hypothalamic astrocyte primary cultures.

Astrocyte glycogen constitutes the primary energy fuel reserve in the brain. Current research investigated the novel premise that glycogen turnover governs astrocyte responsiveness to critical metabolic and neurotransmitter (norepinephrine) regulatory signals in a sex-dimorphic manner. Here, rat hypothalamic astrocyte glycogen phosphorylase (GP) gene expression was silenced by short-interfering RNA (siRNA) to investigate how glycogen metabolism controlled by GP-brain type (GPbb) or GP-muscle type (GPmm) activity affects glucose [glucose transporter-2 (GLUT2)] and energy [5'-AMP-activated protein kinase (AMPK)] sensor and adrenergic receptor (AR) proteins in each sex. Results show that in the presence of glucose, glycogen turnover is regulated by GPbb in the male or by GPmm in the female, yet in the absence of glucose, glycogen breakdown is controlled by GPbb in each sex. GLUT2 expression is governed by GPmm-mediated glycogen breakdown in glucose-supplied astrocytes of each sex, but glycogenolysis controls glucoprivic GLUT2 up-regulation in male only. GPbb-mediated glycogen disassembly causes divergent changes in total AMPK versus phosphoAMPK profiles in male. During glucoprivation, glycogenolysis up-regulates AMPK content in male astrocytes by GPbb- and GPmm-dependent mechanisms, whereas GPbb-mediated glycogen breakdown inhibits phosphoAMPK expression in female. GPbb and GPmm activity governs alpha2-AR and beta1-AR protein levels in male, but has no effect on these profiles in the female. Outcomes provide novel evidence for sex-specific glycogen regulation of glucose- and energy-sensory protein expression in hypothalamic astrocytes, and identify GP isoforms that mediate such control in each sex. Results also show that glycogen regulation of hypothalamic astrocyte receptivity to norepinephrine is male-specific. Further studies are needed to characterize the molecular mechanisms that underlie sex differences in glycogen control of astrocyte protein expression.

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation.

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.

Effect of Betaine and Arginine on Interaction of αB-Crystallin with Glycogen Phosphorylase b.

Protein-protein interactions (PPIs) play an important role in many biological processes in a living cell. Among them chaperone-client interactions are the most important. In this work PPIs of αB-crystallin and glycogen phosphorylase b (Phb) in the presence of betaine (Bet) and arginine (Arg) at 48 °C and ionic strength of 0.15 M were studied using methods of dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation. It was shown that Bet enhanced, while Arg reduced both the stability of αB-crystallin and its adsorption capacity (AC0) to the target protein at the stage of aggregate growth. Thus, the anti-aggregation activity of αB-crystallin increased in the presence of Bet and decreased under the influence of Arg, which resulted in inhibition or acceleration of Phb aggregation, respectively. Our data show that chemical chaperones can influence the tertiary and quaternary structure of both the target protein and the protein chaperone. The presence of the substrate protein also affects the quaternary structure of αB-crystallin, causing its disassembly. This is inextricably linked to the anti-aggregation activity of αB-crystallin, which in turn affects its PPI with the target protein. Thus, our studies contribute to understanding the mechanism of interaction between chaperones and proteins.

Mechanochemical Promoted Heterocycles: A Solvent-free Route to Triazole Carbohydrates as Glycogen Phosphorylase Inhibitors.

AIMS: This study aimed to recommend a novel way for the preparation of carbohydrates containing triazole derivatives. BACKGROUND: Triazoles containing derivatives have numerous biological activities. Ball milling is a fast, modest, green process with massive potential. One of the greatest interesting applications of this technique is in the arena of heterocycles. OBJECTIVE: Solvent-free click reactions are facilitated via the activation of copper powder using a ball milling mechanochemical procedure. An optimization study of parameters affecting the reaction rate, such as reaction time, size, and milling ball number, has been conducted. Different substrates have been tested using this adopted procedure considering in all cases, in high yields and purity, the corresponding chiral optically pure five-membered glycoconjugates containing 1,2,3-triazole. METHODS: Three milling balls of 10 mm in diameter were placed in the milling jar (50 mL; stainless steel). 1 mmol of alkyne, 2 mmol of azide, and 1 mmol of Cu powder (63 mg) were added, respectively, in the presented order. Milling was assured for 25 min at 650 rpm deprived of solvent. RESULTS: The cycloaddition results and the deprotection of the cycloadducts were affected by the selection of the protective groups. Cleavage of the acetyl protecting groups provided water-soluble triazoles. The four 1,4-di-substituted 1,2,3-triazoles synthesized via deacetylation were tested against glycogen phosphorylase. The best inhibitor of rabbit muscle glycogen phosphorylase was 2-Amino-3-{2-[1-(3,4,5,6-tetrahydroxytetrahydro- pyran-2-ylmethyl)-1H-[1,2,3]triazol-4-yl]-ethylsulfanyl}-propionic acid b (Ki = 40.8 ± 3.2 µM). This novel procedure affords an eco-friendly reaction profile (catalyst-free) affording high yields and short reaction times. CONCLUSION: In this work, acetyl protective groups were used to the corresponding deprotected watersoluble triazole analogous to recognizing glycogen phosphorylase inhibitors. Triazole 6a was the most effective inhibitor of RMGP b with a Ki value of 40.8 µM.

Diagnostic accuracy of glycogen phosphorylase BB for myocardial infarction: A systematic review and meta-analysis.

PURPOSE: We tried to investigate the diagnostic accuracy of glycogen phosphorylase BB as a cardiac marker for myocardial infarction. METHODS: We searched through different electronic databases (PubMed, Google-scholar, Embase, and Cochrane Library) to locate relevant articles. Studies, with sufficient data to reconstruct a 2 × 2 contingency table, met our inclusion criteria were included. Three reviewers independently screened the articles. Discrepancies were resolved by other reviewers. Unpublished data were requested from the authors of the study via email. Subsequently, data extraction was done using a standardized form and quality assessment of studies using the QUADAS-2 tool. Meta-analysis was done using a bivariate model using R software. RESULTS: Fourteen studies were selected for the final evaluation, which yielded the summary points: pooled sensitivity 87.77% (77.52%-93.72%, I2  = 86%), pooled specificity 88.45% (75.59%-94.99%, I2  = 88%), pooled DOR 49.37(14.53-167.72, I2  = 89%), and AUC of SROC was 0.923. The lambda value of the HSROC curve was 3.670. The Fagan plot showed that GPBB increases the pretest probability of myocardial infarction from 46% to 81% when positive, and it lowers the same probability to 12% when negative. CONCLUSION: With these results, we can conclude that GPBB has modest accuracy in screening myocardial infarction, but the limitations of the study warrant further high-quality studies to confirm its usefulness in predicting myocardial infarction (MI).

Formation and physicochemical properties of glycogen phosphorylase in complex with a cationic polyelectrolyte.

The accumulation of rabbit muscle glycogen phosphorylase b (RMGPb) in electrostatic complexes with the cationic polyelectrolyte poly 2-(dimethylamino) ethyl methacrylate in its quenched form (QPDMAEMA) was studied in two buffer solutions. In the N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, large complexes of RMGPb-QPDMAEMA were formed which adopted smaller sizes as QPDMAEMA concentration increased. However, in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer, the hydrodynamic radius of the formed complexes gradually increased as the polymer concentration increased. Zeta potential measurements (ζp) showed that RMGPb significantly changed the ζp of the QPDMAEMA aggregates. Fluorescence studies showed that the interaction between RMGPb and QPDMAEAMA was enhanced as polymer concentration increased. Specifically, 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence indicated that in the BES buffer the aggregates became denser as more QPDMAEMA was added, while in the HEPES buffer the density of the formed structures decreased. RMGPb's secondary structure was examined by Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) and Circular Dichroism (CD) showing that QPDMAEMA interaction with RMGPb does not induce any changes to the secondary structure of the enzyme. These observations suggest that cationic polyelectrolytes may be utilized for the formulation of RMGPb in multifunctional nanostructures and be further exploited in innovative biotechnology applications and bioinspired materials development.

A glucose-based molecular rotor inhibitor of glycogen phosphorylase as a probe of cellular enzymatic function.

Molecular rotors belong to a family of fluorescent compounds characterized as molecular switches, where a fluorescence on/off signal signifies a change in the molecule's microenvironment. Herein, the successful synthesis and detailed study of (E)-2-cyano-3-(p-(dimethylamino)phenyl)-N-(ß-D-glucopyranosyl)acrylamide (RotA), is reported. RotA was found to be a strong inhibitor of rabbit muscle glycogen phosphorylase (RMGPb), that binds at the catalytic site of the enzyme. RotA's interactions with the residues lining the catalytic site of RMGPb were determined by X-ray crystallography. Spectroscopic studies coupled with theoretical calculations proved that RotA is a molecular rotor. When bound in the catalytic channel of RMGPb, it behaved as a light switch, generating a strong fluorescence signal, allowing utilization of RotA as a probe that locates glycogen phosphorylase (GP). RotA, mono-, di- and per-acetylated derivatives, as well as nanoparticles with RotA encapsulated in polyethylene glycol-poly-L-histidine, were used in live cell fluorescence microscopy imaging to test the delivery of RotA through the plasma membrane of HepG2 and A431 cells, with the nanoparticles providing the best results. Once in the intracellular milieu, RotA exhibits remarkable colocalization with GP and significant biological effects, both in cell growth and inhibition of GP.

Outliers in SAR and QSAR: 4. effects of allosteric protein-ligand interactions on the classical quantitative structure-activity relationships.

Effects of allosteric interactions on the classical structure-activity relationship (SAR) and quantitative SAR (QSAR) have been investigated. Apprehending the outliers in SAR and QSAR studies can improve the quality, predictability, and use of QSAR in designing unknown compounds in drug discovery research. We explored allosteric protein-ligand interactions as a possible source of outliers in SAR/QSAR. We used glycogen phosphorylase as an example of a protein that has an allosteric site. Examination of the ligand-bound x-ray crystal structures of glycogen phosphorylase revealed that many inhibitors bound at more than one binding site. The results of QSAR analyses of the inhibitors included a QSAR that recognized an outlier bound at a distinctive allosteric binding site. The case provided an example of constructive use of QSAR identifying outliers with alternative binding modes. Other allosteric QSARs that captured our attention were the inverted parabola/bilinear QSARs. The x-ray crystal structures and the QSAR analyses indicated that the inverted parabola QSARs could be associated with the conformational changes in the allosteric interactions. Our results showed that the normal parabola, as well as the inverted parabola QSARs, can describe the allosteric interactions. Examination of the ligand-bound X-ray crystal structures of glycogen phosphorylase revealed that many inhibitors bound at more than one binding site. The results of QSAR analyses of the inhibitors included a QSAR that recognized an outlier bound at a distinctive allosteric binding site.

An In Silico and an In Vitro Inhibition Analysis of Glycogen Phosphorylase by Flavonoids, Styrylchromones, and Pyrazoles.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway. GP inhibitors are currently under investigation as a new liver-targeted approach to managing type 2 diabetes mellitus (DM). The aim of the present study was to evaluate the inhibitory activity of a panel of 52 structurally related chromone derivatives; namely, flavonoids, 2-styrylchromones, 2-styrylchromone-related derivatives [2-(4-arylbuta-1,3-dien-1-yl)chromones], and 4- and 5-styrylpyrazoles against GP, using in silico and in vitro microanalysis screening systems. Several of the tested compounds showed a potent inhibitory effect. The structure-activity relationship study indicated that for 2-styrylchromones and 2-styrylchromone-related derivatives, the hydroxylations at the A and B rings, and in the flavonoid family, as well as the hydroxylation of the A ring, were determinants for the inhibitory activity. To support the in vitro experimental findings, molecular docking studies were performed, revealing clear hydrogen bonding patterns that favored the inhibitory effects of flavonoids, 2-styrylchromones, and 2-styrylchromone-related derivatives. Interestingly, the potency of the most active compounds increased almost four-fold when the concentration of glucose increased, presenting an IC50 < 10 µM. This effect may reduce the risk of hypoglycemia, a commonly reported side effect of antidiabetic agents. This work contributes with important considerations and provides a better understanding of potential scaffolds for the study of novel GP inhibitors.

Circulating cardiomyocyte-derived extracellular vesicles reflect cardiac injury during systemic inflammatory response syndrome in mice.

The release of extracellular vesicles (EVs) is increased under cellular stress and cardiomyocyte damaging conditions. However, whether the cardiomyocyte-derived EVs eventually reach the systemic circulation and whether their number in the bloodstream reflects cardiac injury, remains unknown. Wild type C57B/6 and conditional transgenic mice expressing green fluorescent protein (GFP) by cardiomyocytes were studied in lipopolysaccharide (LPS)-induced systemic inflammatory response syndrome (SIRS). EVs were separated both from platelet-free plasma and from the conditioned medium of isolated cardiomyocytes of the left ventricular wall. Size distribution and concentration of the released particles were determined by Nanoparticle Tracking Analysis. The presence of GFP + cardiomyocyte-derived circulating EVs was monitored by flow cytometry and cardiac function was assessed by echocardiography. In LPS-treated mice, systemic inflammation and the consequent cardiomyopathy were verified by elevated plasma levels of TNFα, GDF-15, and cardiac troponin I, and by a decrease in the ejection fraction. Furthermore, we demonstrated elevated levels of circulating small- and medium-sized EVs in the LPS-injected mice. Importantly, we detected GFP+ cardiomyocyte-derived EVs in the circulation of control mice, and the number of these circulating GFP+ vesicles increased significantly upon intraperitoneal LPS administration (P = 0.029). The cardiomyocyte-derived GFP+ EVs were also positive for intravesicular troponin I (cTnI) and muscle-associated glycogen phosphorylase (PYGM). This is the first direct demonstration that cardiomyocyte-derived EVs are present in the circulation and that the increased number of cardiac-derived EVs in the blood reflects cardiac injury in LPS-induced systemic inflammation (SIRS).