Synthetic flavonoid derivatives targeting the glycogen phosphorylase inhibitor site: QM/MM-PBSA motivated synthesis of substituted 5,7-dihydroxyflavones, crystallography, in vitro kinetics and ex-vivo cellular experiments reveal novel potent inhibitors

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 (Ki 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 (IC50 = 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

A multidisciplinary study of 3-(β-d-glucopyranosyl)-5-substituted-1,2,4-triazole derivatives as glycogen phosphorylase inhibitors: Computation, synthesis, crystallography and kinetics reveal new potent inhibitors

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 '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 copyright: Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Glycogen phosphorylase revisited: extending the resolution of the R- and T-state structures of the free enzyme and in complex with allosteric activators

The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7–17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed between helices α4 and α16 (residues 104–115 and 497–508, respectively), while in the R-state it is packed against helix α1 (residues 22′–38′) and towards the loop connecting helices α4′ and α5′ of the neighbouring subunit. The allosteric binding site where AMP and IMP bind is formed by the ordering of a loop (residues 313–326) which is disordered in the free structure, and adopts a conformation dictated mainly by the type of nucleotide that binds at this site

van der Waals interactions govern C-[beta]-D-glucopyranosyl triazoles' nM inhibitory potency in human liver glycogen phosphorylase

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High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-<i>N</i>-acyl-β-<span style="font-variant: small-caps">d</span>-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors

Structure-based design and synthesis of two biphenyl-N-acyl-&#946;-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data

Probing the [beta]-pocket of the active site of human liver glycogen phosphorylase with 3-(C-[beta]-D-glucopyranosyl)-5-(4-substituted-phenyl)-1, 2, 4-triazole inhibitors

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Nanomolar Inhibitors of Glycogen Phosphorylase Based on β-d-Glucosaminyl Heterocycles: A Combined Synthetic, Enzyme Kinetic, and Protein Crystallography Study

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Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X-ray Crystallography

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Natural flavonoids as antidiabetic agents. The binding of gallic and ellagic acids to glycogen phosphorylase b

We present a study on the binding of gallic acid and its dimer ellagic acid to glycogen phosphorylase (GP). Ellagic acid is a potent inhibitor with K(i)s of 13.4 and 7.5 mu M, in contrast to gallic acid which displays K(i)s of 1.7 and 3.9 mM for GPb and GPa, respectively. Both compounds are competitive inhibitors with respect to the substrate, glucose-1-phoshate, and non-competitive to the allosteric activator, AMP. However, only ellagic acid functions with glucose in a strongly synergistic mode. The crystal structures of the GPb-gallic acid and GPb-ellagic acid complexes were determined at high resolution, revealing that both ligands bind to the inhibitor binding site of the enzyme and highlight the structural basis for the significant difference in their inhibitory potency. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved