Glucopyranosylidene-spiro-iminothiazolidinone, a new bicyclic ring system: synthesis, derivatization, and evaluation for inhibition of glycogen phosphorylase by enzyme kinetic and crystallographic methods.

The reaction of thiourea with O-perbenzoylated C-(1-bromo-1-deoxy-ß-D-glucopyranosyl)formamide gave the new anomeric spirocycle 1R-1,5-anhydro-D-glucitol-spiro-[1,5]-2-imino-1,3-thiazolidin-4-one. Acylation and sulfonylation with the corresponding acyl chlorides (RCOCl or RSO2Cl where R=tBu, Ph, 4-Me-C6H4, 1- and 2-naphthyl) produced the corresponding 2-acylimino- and 2-sulfonylimino-thiazolidinones, respectively. Alkylation by MeI, allyl-bromide and BnBr produced mixtures of the respective N-alkylimino- and N,N'-dialkyl-imino-thiazolidinones, while reactions with 1,2-dibromoethane and 1,3-dibromopropane furnished spirocyclic 5,6-dihydro-imidazo[2,1-b]thiazolidin-3-one and 6,7-dihydro-5H-thiazolidino[3,2-a]pyrimidin-3-one, respectively. Removal of the O-benzoyl protecting groups by the Zemplén protocol led to test compounds most of which proved micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). Best inhibitors were the 2-benzoylimino- (Ki=9µM) and the 2-naphthoylimino-thiazolidinones (Ki=10 µM). Crystallographic studies of the unsubstituted spiro-thiazolidinone and the above most efficient inhibitors in complex with RMGPb confirmed the preference and inhibitory effect that aromatic (and especially 2-naphthyl) derivatives show for the catalytic site promoting the inactive conformation of the enzyme.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods.

N-(4-Substituted-benzoyl)-N'-(ß-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated ß-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3µM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the ß-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.

Halogen-substituted (C-ß-D-glucopyranosyl)-hydroquinone regioisomers: synthesis, enzymatic evaluation and their binding to glycogen phosphorylase.

Electrophilic halogenation of C-(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl) 1,4-dimethoxybenzene (1) afforded regioselectively products halogenated at the para position to the D-glucosyl moiety (8, 9) that were deacetylated to 3 (chloride) and 16 (bromide). For preparing meta regioisomers, 1 was efficiently oxidized with CAN to afford C-(2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosyl) 1,4-benzoquinone 2 which, in either MeOH or H(2)O-THF containing few equivalents of AcCl, added hydrochloric acid to produce predominantly meta (with respect to the sugar moiety) chlorinated hydroquinone derivatives 5 and 18, this latter being deacetylated to 4. The deacetylated meta (4, 5) or para (3, 16) halohydroquinones were evaluated as inhibitors of glycogen phosphorylase (GP, a molecular target for inhibition of hepatic glycogenolysis under high glucose concentrations) by kinetics and X-ray crystallography. These compounds are competitive inhibitors of GPb with respect to α-D-glucose-1-phosphate. The measured IC(50) values (µM) [169.9±10.0 (3), 95 (4), 39.8±0.3 (5) 136.4±4.9 (16)] showed that the meta halogenated inhibitors (4, 5) are more potent than their para analogs (3, 16). The crystal structures of GPb in complex with these compounds at high resolution (1.97-2.05 Å) revealed that the inhibitors are accommodated at the catalytic site and stabilize the T conformation of the enzyme. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions with protein residues of the different substituents on the aromatic part of the inhibitors.

The binding of ß-d-glucopyranosyl-thiosemicarbazone derivatives to glycogen phosphorylase: A new class of inhibitors.

Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(ß-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC(50) values ranging from 5.7 to 524.3µM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95-2.23Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO(2), F, Cl, Br, OH, OMe, CF(3), or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the ß-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the ß-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.

Synthesis of new glycosyl biuret and urea derivatives as potential glycoenzyme inhibitors.

O-peracetylated 1-(beta-D-glucopyranosyl)-5-phenylbiuret was prepared in the reaction of O-peracetylated beta-D-glucopyranosylisocyanate and phenylurea. The reaction of O-peracetylated N-beta-D-glucopyranosylurea with phenylisocyanate furnished the corresponding 1-(beta-D-glucopyranosyl)-3,5-diphenyl- as well as 3-(beta-D-glucopyranosyl)-1,5-diphenyl biurets besides 1-(beta-D-glucopyranosyl)-3-phenylurea. O-Peracetylated 1-(beta-D-glucopyranosyl)-5-(beta-D-glycopyranosyl)biurets were obtained in one-pot reactions of O-peracetylated beta-D-glucopyranosylamine with OCNCOCl followed by a second glycopyranosylamine of beta-D-gluco, beta-D-galacto and beta-D-xylo configurations. O-Acyl protected 1-(beta-D-glucopyranosyl)-3-(beta-D-glycopyranosylcarbonyl)ureas were obtained from the reaction of beta-D-glucopyranosylisocyanate with C-(glycopyranosyl)formamides of beta-D-gluco and beta-D-galacto configurations. The O-acyl protecting groups were removed under acid- or base-catalyzed transesterification conditions, except for the N-acylurea derivatives where the cleavage of the N-acyl groups was faster than deprotection. Some of the new compounds exhibited moderate inhibition against rabbit muscle glycogen phosphorylase b and human salivary alpha-amylase.

Glucose-based spiro-isoxazolines: a new family of potent glycogen phosphorylase inhibitors.

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 92.5 microM. 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.

Naturally occurring pentacyclic triterpenes as inhibitors of glycogen phosphorylase: synthesis, structure-activity relationships, and X-ray crystallographic studies.

Twenty-five naturally occurring pentacyclic triterpenes, 15 of which were synthesized in this study, were biologically evaluated as inhibitors of rabbit muscle glycogen phosphorylase a (GPa). From SAR studies, the presence of a sugar moiety in triterpene saponins resulted in a markedly decreased activity ( 7, 18- 20) or no activity ( 21, 22). These saponins, however, might find their value as potential natural prodrugs which are much more water-soluble than their corresponding aglycones. To elucidate the mechanism of GP inhibition, we have determined the crystal structures of the GPb-asiatic acid and GPb-maslinic acid complexes. The X-ray analysis indicates that the inhibitors bind at the allosteric activator site, where the physiological activator AMP binds. Pentacyclic triterpenes represent a promising class of multiple-target antidiabetic agents that exert hypoglycemic effects, at least in part, through GP inhibition.

Crystallographic and computational studies on 4-phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide, an inhibitor of glycogen phosphorylase: comparison with alpha-D-glucose, N-acetyl-beta-D-glucopyranosylamine and N-benzoyl-N'-beta-D-glucopyranosyl urea binding.

4-Phenyl-N-(beta-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide (glucosyltriazolylacetamide) has been studied in kinetic and crystallographic experiments with glycogen phosphorylase b (GPb), in an effort to utilize its potential as a lead for the design of potent antihyperglycaemic agents. Docking and molecular dynamics (MD) calculations have been used to monitor more closely the binding modes in operation and compare the results with experiment. Kinetic experiments in the direction of glycogen synthesis showed that glucosyltriazolylacetamide is a better inhibitor (K(i) = 0.18 mM) than the parent compound alpha-D-glucose (K(i) = 1.7 mM) or beta-D-glucose (K(i) = 7.4 mM) but less potent inhibitor than the lead compound N-acetyl-beta-D-glucopyranosylamine (K(i) = 32 microM). To elucidate the molecular basis underlying the inhibition of the newly identified compound, we determined the structure of GPb in complex with glucosyltriazolylacetamide at 100 K to 1.88 A resolution, and the structure of the compound in the free form. Glucosyltriazolylacetamide is accommodated in the catalytic site of the enzyme and the glucopyranose interacts in a manner similar to that observed in the GPb-alpha-D-glucose complex, while the substituent group in the beta-position of the C1 atom makes additional hydrogen bonding and van der Waals interactions to the protein. A bifurcated donor type hydrogen bonding involving O3H, N3, and N4 is seen as an important structural motif strengthening the binding of glucosyltriazolylacetamide with GP which necessitated change in the torsion about C8-N2 bond by about 62 degrees going from its free to the complex form with GPb. On binding to GP, glucosyltriazolylacetamide induces significant conformational changes in the vicinity of this site. Specifically, the 280s loop (residues 282-288) shifts 0.7 to 3.1 A (CA atoms) to accommodate glucosyltriazolylacetamide. These conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding compared with the lead compound. In the molecular modeling calculations, the GOLD docking runs with and without the crystallographic ordered cavity waters using the GoldScore scoring function, and without cavity waters using the ChemScore scoring function successfully reproduced the crystallographic binding conformation. However, the GLIDE docking calculations both with (GLIDE XP) and without (GLIDE SP and XP) the cavity water molecules were, impressively, further able to accurately reproduce the finer details of the GPb-glucosyltriazolylacetamide complex structure. The importance of cavity waters in flexible receptor MD calculations compared to "rigid" (docking) is analyzed and highlighted, while in the MD itself very little conformational flexibility of the glucosyltriazolylacetamide ligand was observed over the time scale of the simulations.

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 A 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.