Browsing by Subject "Glycogen synthase"

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    Discovery, Characterization, and Development of Small Molecule Inhibitors of Glycogen Synthase
    (2020-06) Tang, Buyun; Hurley, Thomas D.; Roach, Peter J.; Georgiadis, Millie M.; Johnson, Steven M.; Elmendorf, Jeffrey S.
    The over-accumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Glycogen synthase (GS) is the rate-limiting enzyme for glycogen synthesis. Recent evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these diseases. Herein, we describe the discovery, characterization, and development of small molecule inhibitors of GS through a multicomponent study including biochemical, biophysical, and cellular assays. Adopting an affinity-based fluorescence polarization assay, we identified a substituted imidazole molecule (H23), as a first-in-class inhibitor of yeast glycogen synthase 2 (yGsy2) from the 50,000 ChemBridge DIVERSet library. Structural data derived from X-ray crystallography at 2.85 Å, and enzyme kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2. Medicinal chemistry efforts examining over 500 H23 analogs produced structure-activity relationship (SAR) profiles that led to the identification of potent pyrazole and isoflavone compounds with low micromolar potency against human glycogen synthase 1 (hGYS1). Notably, several of the isoflavones demonstrated cellular efficacy toward suppressing glycogen accumulation. In an alternative effort to screen inhibitors directly against human GS, an activity-based assay was designed using a two-step colorimetric approach. This assay led to the identification of compounds with submicromolar potency to hGYS1 from a chemical library comprised of 10,000 compounds. One of the hit molecules, hexachlorophene, was crystallized bound to the active site of yGsy2. The structure was determined to 3.15 Å. Additional kinetic, mutagenic, and SAR studies validated the binding of hexachlorophene in the catalytic pocket and its non-competitive mode of inhibition. In summary, these two novel assays provided feasible biochemical platforms for large-scale screening of small molecule modulators of GS. The newly-developed, potent analogs possess diverse promising scaffolds for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.
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    Glycogen metabolism in Lafora disease
    (2018-02) Contreras, Christopher J.; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.; Herring, B. Paul
    Glycogen, a branched polymer of glucose, serves as an osmotically neutral means of storing glucose. Covalent phosphate is a trace component of mammalian glycogen and has been a point of interest with respect to Lafora disease, a fatal form of juvenile myoclonus epilepsy. Mutations in either the EPM2A or EPM2B genes, which encode laforin and malin respectively, account for ~90% of disease cases. A characteristic of Lafora disease is the formation of Lafora bodies, which are mainly composed of an excess amount of abnormal glycogen that is poorly branched and insoluble. Laforin-/- and malin-/- knockout mice share several characteristics of the human disease, formation of Lafora bodies in various tissues, increased glycogen phosphorylation and development of neurological symptoms. The source of phosphate in glycogen has been an area of interest and here we provide evidence that glycogen synthase is capable of incorporating phosphate into glycogen. Mice lacking the glycogen targeting subunit PTG of the PP1 protein phosphatase have decreased glycogen stores in a number of tissues. When crossed with mice lacking either laforin or malin, the double knockout mice no longer over-accumulate glycogen, Lafora body formation is almost absent and the neurological disorders are normalized. Another question has been whether the abnormal glycogen in the Lafora disease mouse models can be metabolized. Using exercise to provoke glycogen degradation, we show that in laforin-/- and malin-/- mice the insoluble, abnormal glycogen appears to be metabolically inactive. These studies suggest that a therapeutic approach to Lafora disease may be to reduce the overall glycogen levels in cells so that insoluble, metabolically inert pools of the polysaccharide do not accumulate.
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    A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase
    (Elsevier, 2017-01) Maile, C.A.; Hingst, J. R.; Mahalingan, K. K.; O'Reilly, A. O.; Cleasby, M. E.; Mickelson, J. R.; McCue, M. E.; Anderson, S. M.; Hurley, T. D.; Wojtaszewski, J. F. P.; Piercy, R. J.; Biochemistry and Molecular Biology, School of Medicine
    BACKGROUND: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS: Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy.
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    Incorporation of phosphate into glycogen by glycogen synthase
    (Elsevier, 2016-05-01) Contreras, Christopher J.; Segvich, Dyann M.; Mahalingan, Krishna; Chikwana, Vimbai M.; Kirley, Terence L.; Hurley, Thomas D.; DePaoli-Roach, Anna A.; Roach, Peter J.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.
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    Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation
    (ACS Publications, 2017-01-10) Mahalingan, Krishna K.; Baskaran, Sulochanadevi; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D.; Biochemistry and Molecular Biology, School of Medicine
    Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G6P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G6P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. Based on these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme, but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.
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    Structural basis for regulated inhibition and substrate selection in yeast glycogen synthase
    (2017-02) Mahalingan, Krishna Kishore; Hurley, Thomas D.; Elmendorf, Jeffrey; Georgiadis, Millie M.; Roach, Peter J.
    Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain.