Structure based inhibitor design targeting glycogen phosphorylase b. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-β-d-glucopyranosylamines

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 β- D-glucopyranose-NH-CO-R 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

(E)-2-(4-Arylbut-1-en-3-yn-1-yl)chromones as synthons for the synthesis of xanthone-1,2,3-triazole dyads

Xanthone-1,2,3-triazole dyads have been synthesized by two different approaches, both starting from novel (E)-2-(4-arylbut-1-en-3-yn-1-yl)chromones, prepared through a base-catalyzed aldol reaction of 2-methylchromone and arylpropargyl aldehydes. In the first method, the xanthone moiety is built by Diels-Alder reaction of the referred unsaturated chromones with N-methylmaleimide under microwave irradiation, followed by oxidation of the obtained adducts with DDQ, whereas the 1,2,3-triazole ring results from the cycloaddition reaction of the acetylene moiety with sodium azide. The second strategy first involves the cycloaddition reaction with sodium azide to provide the 1,2,3-triazole ring, followed by methylation of the triazole NH group prior to Diels-Alder reaction with N-methylmaleimide. The last step in this synthesis of novel xanthone-1,2,3-triazole dyads entails oxidation of the cycloadducts with DDQ

Auf dem Weg zu einem strukturbasierten Wirkstoffdesign fuer den Typ-2-Diabetes

Diabetes type 2 is a complex disease characterized by altered glucose metabolism and insulin resistance. Almost half of all people with diabetes type 2 are not aware they have this life threatening condition, as they can show symptoms years after the onset of the disease. Glycogen phosphorylase, an allosteric enzyme, plays a pivotal role in controlling the metabolism of glycogen. It catalyzes the first step in the degradation of glycogen by releasing glucose-1-phosphate from a long chain of glucose residues. As an allosteric enzyme, the inactive T state is switched to the active R state by a change in conformation controlled by phosphorylation of a single residue (Ser14) by phosphorylase kinase. Phosphorylation leads to transition between the two forms of the enzyme, called phosphorylase a and b. Glycogen phosphorylase is a dimer composed of two identical subunits. Each subunit has a molecular weight of 97.444 Da, consists of 842 amino acids and an essential co-factor, pyridoxal-5´-phosphate (PLP). Various binding sites on the enzyme are known, notably the catalytic, allosteric, new allosteric and inhibitor sites. By means of kinetic in vitro experiments and X-ray crystallography experiments, these binding sites were targeted by studying a large number of glycogen phosphorylase inhibitors as potential hyperglycaemic drugs. The essential inhibitory and binding properties of specific compounds were analyzed in an effort to provide rationalizations for the affinities of these compounds and to exploit the molecular interactions with the goal to design new better inhibitors. Most of the inhibitors studied were glucose analogues and were found to bind at the catalytic site of the enzyme but interesting results were also found for more binding sites. A novel binding site was also discovered and mapped out. These studies have given new insights into fundamental structural aspects of the enzyme enhancing our understanding of how the enzyme recognizes and specifically binds ligands, which could be of potential therapeutic value in the treatment of diabetes type 2.Typ-2-Diabetes ist eine komplexe Stoffwechselkrankheit, die sich durch einen veränderten Glucosestoffwechsel sowie Insulinresistenz auszeichnet. Fast die Hälfte aller Menschen mit Typ-2-Diabetes sind sich nicht bewußt, daß sie an dieser lebensgefährlichen Krankheit leiden, da sie oft schleichend beginnnt und sich die ersten Symptome erst nach Jahren zeigen. Die Glycogenphosphorylase (PYG), ein allosterisches aktiviertes Enzym, ist das Schlüsselenzym der Glycogenolyse, die den Glycosestoffwechsel steuert. Es katalysiert den ersten Schritt des Glycosestoffwechsels, bei dem freies Phosphat am C-Atom 1 der Glycose angebunden wird, wobei die glykosidische Bindung zwischen den Glycose-Molekülen aufgespalten und Glucose-1-phosphat entsteht. PYG ist die Ursache der Konformationsänderung von der inaktiven T-Form zur aktiven R-Form in der Phosphorylierung eines Serin-Restes (Ser14) durch die Phosphorylase-Kinase. Die Phosphorylierung führt zum Übergang zwischen den zwei Enzymformen, die Phosphorylase a und b genannt werden. Glykogenphosphorylase ist ein Dimer, das aus zwei identischen Monomeren besteht. Jedes Monomer besitzt eine molekulare Masse von 97.444 Da und besteht aus 842 Aminosäuren, sowie dem Kofactor Pyridoxal-5´-Phosphat (PLP). Mehrere Bindungstellen für Inhibitoren an diesem Enzym sind bekannt, insbesondere die sogenannte katalytische, die allosterische, die neue allosterische und die Inhibitor Bindungsstelle. In dieser Arbeit wurden mithilfe von kinetischen in vitro Experimenten und kristallographischen Roentgenstruktur-Untersuchungen die Bindungs- and Inhibitor-Eigenschaften der verschiedenen Bindungsstellen des Enzyms gegenüber einer großen Anzahl ausgewählter Inhibitoren ausführlich untersucht. Die Analyse der Ergebnisse dieser Untersuchungen ermöglichen ein besseres Verständnis der molekularen Wechselwirkungen, mit Hinblick auf die gezielte Entwicklung neuer hyperglykämischer Wirkstoffe mit besserem Wirkungsgrad. Die meisten der untersuchten Inhibitoren waren Glykose-Derivate, und die Bindung fand in der Regel an der katalytischen Bindungsstelle des Enzyms statt. Aber auch für die anderen Bindungsstellen wurden interessante Ergebnisse gefunden. Es wurde auch eine neue Bindungstelle im Enzym entdeckt und charakterisiert. Die Ergebnisse dieser Arbeit vermitteln neue Erkenntnisse über die Struktur des Enzyms, sowie über die Mechanismem der Erkennung spezifischer Liganden und ihrer selektiven Bindung, und sind daher von potentiellen therapeuthischem Wert für die Behandlung der Typ 2 Diabetes- Krankheit

FR258900, a potential anti-hyperglycemic drug, binds at the allosteric site of glycogen phosphorylase

FR258900 has been discovered as a novel inhibitor of human liver glycogen phosphorylase a and proved to suppress hepatic glycogen breakdown and reduce plasma glucose concentrations in diabetic mice models. To elucidate the mechanism of inhibition, we have determined the crystal structure of the cocrystallized rabbit muscle glycogen phosphorylase b-FR258900 complex and refined it to 2.2 angstrom resolution. The structure demonstrates that the inhibitor binds at the allosteric activator site, where the physiological activator AMP binds. The contacts from FR258900 to glycogen phosphorylase are dominated by nonpolar van der Waals interactions with Gln71, Gln72, Phe196, and Val45' (from the symmetry-related subunit), and also by ionic interactions from the carboxylate groups to the three arginine residues (Arg242, Arg309, and Arg310) that form the allosteric phosphate-recognition subsite. The binding of FR258900 to the protein promotes conformational changes that stabilize an inactive T-state quaternary conformation of the enzyme. The ligand-binding mode is different from those of the potent phenoxy- phthalate and acyl urea inhibitors, previously described, illustrating the broad specificity of the allosteric site