Joseph M. Hayes

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.

New inhibitors of glycogen phosphorylase as potential antidiabetic agents.

The protein glycogen phosphorylase has been linked to type 2 diabetes, indicating the importance of this target to human health. Hence, the search for potent and selective inhibitors of this enzyme, which may lead to antihyperglycaemic drugs, has received particular attention. Glycogen phosphorylase is a typical allosteric protein with five different ligand binding sites, thus offering multiple opportunities for modulation of enzyme activity. The present survey is focused on recent new molecules, potential inhibitors of the enzyme. The biological activity can be modified by these molecules through direct binding, allosteric effects or other structural changes. Progress in our understanding of the mechanism of action of these inhibitors has been made by the determination of high-resolution enzyme inhibitor structures (both muscle and liver). The knowledge of the three-dimensional structures of protein-ligand complexes allows analysis of how the ligands interact with the target and has the potential to facilitate structure-based drug design. In this review, the synthesis, structure determination and computational studies of the most recent inhibitors of glycogen phosphorylase at the different binding sites are presented and analyzed.

Kinetics, in silico docking, molecular dynamics, and MM‐GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP‐binding site inhibitors: The role of water molecules examined

With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC50 > 50 μM), indirubin‐3′‐oxime (IC50 = 144 nM), KT5720 (Ki = 18.4 nM) and staurosporine (Ki = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP‐binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MM‐GBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc‐staurosporine complex has the greatest number of receptor‐ligand hydrogen bonds, while for the indirubin‐3′‐oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor‐enzyme contacts. The MM‐GBSA results revealed the source of staurosporines low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1‐water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schrödinger induced‐fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor‐ligand contacts. Proteins 2011.

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.

Antihypertensive drug valsartan in solution and at the AT1 receptor: conformational analysis, dynamic NMR spectroscopy, in silico docking, and molecular dynamics simulations.

The conformational properties of AT1 antagonist valsartan have been analyzed both in solution and at the binding site of the receptor. Low energy conformations of valsartan in solution were explored by NMR spectroscopy and molecular modeling studies. The NMR results showed the existence of two distinct and almost isoenergetic conformations for valsartan (cis:trans ratio around the amide bond approximately 40:60) that coalesce at the temperature range of 55-60 degrees C in agreement with previous in solution conformational analysis study (Fang et al. Magn. Reson. Chem. 2007, 45, 929-936). Quantum mechanics and ONIOM calculations revealed that the bulky valsartan substituents actually contribute to stabilization of the transition state for interconversion. In silico docking and Molecular Dynamic studies were applied to study binding of valsartan at the AT1 receptor site models, explicitly solvated and embedded in lipid bilayers and solvent molecules. These studies revealed that the majority of docked poses adopted a trans (major) conformation. Of paramount and maybe biological importance are the MD simulations results which showed that the two acidic groups of valsartan are bridged through LYS199 enabling it for multiple hydrogen bond interactions. In a lipid bilayer environment these interactions are enhanced, designating the important role of lipid bilayers for the better binding of valsartan and its stabilization at the active site.

The σ-hole phenomenon of halogen atoms forms the structural basis of the strong inhibitory potency of C5 halogen substituted glucopyranosyl nucleosides towards glycogen phosphorylase b.

C5 halogen substituted glucopyranosyl nucleosides (1‐(β‐D‐glucopyranosyl)‐5‐X‐uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective Ki values of 1.02, 3.27, and 1.94 μM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ‐hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1‐(β‐D‐glucopyranosyl)uracil (Ki=12.39 μM), as revealed by X‐ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM‐PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell.

Computation as a tool for glycogen phosphorylase inhibitor design.

Glycogen phosphorylase is an important therapeutic target for the potential treatment of type 2 diabetes. The importance of computation in the search for potent, selective and drug-like glycogen phosphorylase inhibitors which may eventually lead to antihyperglycemic drugs is now firmly established. Acting solo or more effectively in combination with experiment in a multidisciplinary approach to structure based drug design, current day modeling methods are an effective means of reducing the time and money spent on costly experimental procedures. Glycogen phosphorylase is an allosteric protein with five different ligand binding sites, hence offering multiple opportunities for modulation of enzyme activity. However, the binding sites have their own individual characteristics, so that different modeling approaches may be more effective for each. This review is focused on advances in the modelling and design of new inhibitors of the enzyme aimed towards providing the reader with some useful hints towards more successful computer-aided inhibitor (drug) design targeting glycogen phosphorylase.

Natural products and their derivatives as inhibitors of glycogen phosphorylase: potential treatment for type 2 diabetes

Glycogen phosphorylase (GP) (EC 2.4.1.1) is an important therapeutic target for the potential treatment of type 2 diabetes. The search for potent, selective and drug-like GP inhibitors which may eventually lead to hypoglycaemic agents has to date uncovered a number of natural product inhibitors with both pharmaceutical and nutraceutical potential. GP is an allosteric protein with at least six different ligand binding sites that modulate its enzymatic activity. Hence, inhibitors with considerable structural diversity can be designed. This review is focused on advances in the discovery of natural products and their derivatives as GP inhibitors.

MM-GB(PB)SA Calculations of Protein-Ligand Binding Free Energies

The importance of computational chemistry in modern scientific research is well established. Continuous improvement in software and algorithms for the modeling of chemical interactions has transformed molecular modeling into a powerful tool for many current day research projects. From a medical perspective, one of the ultimate goals in computer-aided drug design (CADD) is the accurate prediction of ligand-binding affinities to a macromolecular target, which can facilitate and speed the routine identification of new candidates in early stage drug discovery projects (Gilson & Zhou, 2007; Hayes & Leonidas, 2010). In particular, structure-based modeling provides an efficient pathway towards exploiting known three-dimensional structural data in the design and proposal of new molecules for experimental evaluation. Docking calculations are now widely used in highthroughput virtual screening of structurally diverse molecules from available compound libraries/databases against specific targets. Once initial “hits” or “lead” molecules are identified (normally low μM inhibitors), modification of their chemical features in the “lead optimization” phase can improve their binding affinities and fine-tune other desirable druglike properties. However, docking calculations currently have limited success beyond the lead identification stage, where more accurate lower-throughput computational methods are needed. In this regard, the Molecular Mechanics/Generalized Born Surface Area (MMGBSA) and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methods calculate binding free energies using molecular mechanics (forcefields) and continuum (implicit) solvation models (Kollman et al., 2000).They have been successfully applied across a range of targets and are implemented in software programs such as Amber (Case et al., 2005), Delphi (Rocchia et al., 2001) and Schrodinger (Du et al., 2011). With a target readership from beginner to expert, the current chapter provides an extensive and critical overview of MM-GB(PB)SA methods and their applications. The theoretical foundation of the MM-GB(PB)SA method is first described. We then discuss key aspects which improve the accuracy of results, and highlight potential caveats due to the approximations inherent in the methods. The chapter concludes with a review of recent representative applications, which illustrate both successes and limitations. The emphasis of this chapter is on structure-

Crystallographic and computational studies on 4-phenyl-N-(β-D-glucopyranosyl)-1H-1,2,3-triazole-1-acetamide, an inhibitor of glycogen phosphorylase: Comparison with α-D-glucose, N-acetyl-β-D-glucopyranosylamine and N-benzoyl-N′-β-D-glucopyranosyl urea binding

4‐Phenyl‐N‐(β‐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 (Ki = 0.18 mM) than the parent compound α‐D‐glucose (Ki = 1.7 mM) or β‐D‐glucose (Ki = 7.4 mM) but less potent inhibitor than the lead compound N‐acetyl‐β‐D‐glucopyranosylamine (Ki = 32 μM). 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 Å 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‐α‐D‐glucose complex, while the substituent group in the β‐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° 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 Å (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. Proteins 2008.