Design and Synthesis of 3-(β-d-Glucopyranosyl)-4-amino/4-guanidino Pyrazole Derivatives and Analysis of Their Glycogen Phosphorylase Inhibitory Potential

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C-β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion–ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1H-pyrazole (3) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-pyrazole (4) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics—generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion–ion interactions; for 4, the most stable tautomer did not have the His377 backbone C=O interaction and while ion–ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted

Synthesis, in silico and kinetics evaluation of N-(β-D-glucopyranosyl)-2-arylimidazole-4(5)-carboxamides and N-(β-D-glucopyranosyl)-4(5)-arylimidazole-2-carboxamides as glycogen phosphorylase inhibitors

Recently studied N-(β-D-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides proved to be low micromolar inhibitors of glycogen phosphorylase (GP), a validated target for the treatment of type 2 diabetes mellitus. Since in other settings, the bioisosteric replacement of the 1,2,4-triazole moiety by imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound-state DFT calculations was performed on N-(β-D-glucopyranosyl)-arylimidazole-carboxamides revealing potential for strong GP inhibition. The syntheses of the target compounds involved formation of an amide bond between per-O-acetylated β-D-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments against rabbit muscle GPb revealed low micromolar inhibitors with the best inhibition constants (Kis) of ~ 3-4 µM obtained for 1- and 2-naphthyl substituted N-(β-D-glucopyranosyl)-imidazolecarboxamides, 2b-c. The predicted protein-ligand interactions responsible for the observed potencies are discussed and will facilitate the structure-based design of other inhibitors targeting this important therapeutic target. Meanwhile, the importance of careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed

Aromatic stacking facilitated self-assembly of ultra-short ionic complementary peptide sequence: β-sheet nanofibres with remarkable gelation and interfacial properties

Understanding peptide self-assembly mechanisms and stability of the formed assemblies is crucial for the development of functional nanomaterials. Herein, we have adopted a rational design approach to demonstrate how a minimal structural modification to a nonassembling ultrashort ionic self-complementary tetrapeptide FEFK (Phe4) remarkably enhanced the stability of self-assembly into β-sheet nanofibers and induced hydrogelation. This was achieved by replacing flexible phenylalanine residue (F) by the rigid phenylglycine (Phg), resulting in a constrained analogue PhgEPhgK (Phg4), which positioned aromatic rings in an orientation favorable for aromatic stacking. Phg4 self-assembly into stable β-sheet ladders was facilitated by π-staking of aromatic side chains alongside hydrogen bonding between backbone amides along the nanofiber axis. The contribution of these noncovalent interactions in stabilizing self-assembly was predicted by in silico modeling using molecular dynamics simulations and semiempirical quantum mechanics calculations. In aqueous medium, Phg4 β-sheet nanofibers entangled at a critical gelation concentration ≥20 mg/mL forming a network of nanofibrous hydrogels. Phg4 also demonstrated a unique surface activity in the presence of immiscible oils and was superior to commercial emulsifiers in stabilizing oil-in-water (O/W) emulsions. This was attributed to interfacial adsorption of amphiphilic nanofibrils forming nanofibrilized microspheres. To our knowledge, Phg4 is the shortest ionic self-complementary peptide rationally designed to self-assemble into stable β-sheet nanofibers capable of gelation and emulsification. Our results suggest that ultrashort ionic-complementary constrained peptides or UICPs have significant potential for the development of cost-effective, sustainable, and multifunctional soft bionanomaterials

Identification of C-β-d-Glucopyranosyl Azole-Type Inhibitors of Glycogen Phosphorylase That Reduce Glycogenolysis in Hepatocytes: In Silico Design, Synthesis, in Vitro Kinetics, and ex Vivo Studies

Several C-β-d-glucopyranosyl azoles have recently been uncovered as among the most potent glycogen phosphorylase (GP) catalytic site inhibitors discovered to date. Toward further exploring their translational potential, ex vivo experiments have been performed for their effectiveness in reduction of glycogenolysis in hepatocytes. New compounds for these experiments were predicted in silico where, for the first time, effective ranking of GP catalytic site inhibitor potencies using the molecular mechanics-generalized Born surface area (MM-GBSA) method has been demonstrated. For a congeneric training set of 27 ligands, excellent statistics in terms of Pearson (RP) and Spearman (RS) correlations (both 0.98), predictive index (PI = 0.99), and area under the receiver operating characteristic curve (AU-ROC = 0.99) for predicted versus experimental binding affinities were obtained, with ligand tautomeric/ionization states additionally considered using density functional theory (DFT). Seven 2-aryl-4(5)-(β-d-glucopyranosyl)-imidazoles and 2-aryl-4-(β-d-glucopyranosyl)-thiazoles were subsequently synthesized, and kinetics experiments against rabbit muscle GPb revealed new potent inhibitors with best Ki values in the low micromolar range (5c = 1.97 μM; 13b = 4.58 μM). Ten C-β-d-glucopyranosyl azoles were then tested ex vivo in mouse primary hepatocytes. Four of these (5a–c and 9d) demonstrated significant reduction of glucagon stimulated glycogenolysis (IC50 = 30–60 μM). Structural and predicted physicochemical properties associated with their effectiveness were analyzed with permeability related parameters identified as crucial factors. The most effective ligand series 5 contained an imidazole ring, and the calculated pKa (Epik: 6.2; Jaguar 5.5) for protonated imidazole suggests that cellular permeation through the neutral state is favored, while within the cell, there is predicted more favorable binding to GP in the protonated form

In Silico and In Vitro Studies of Potential Novel Anti-Cancer Agents Targeting Angiogenin and Glycogen Phosphorylase

Glioblastoma remains the most aggressive cancer of the central nervous system, characterised by its high proliferative rate, invasiveness, and angiogenesis. Increased drug resistance and side effects have greatly reduced the overall efficacy of current treatments, and in most cases, these tumours are recurring. Novel therapeutic strategies combined with conventional treatments are therefore imperative for enhancing the overall outcome and quality of life for cancer patients. Tumour growth is not only closely associated with angiogenesis, they are able to undergo metabolic rewiring which increases their glucose uptake and consumption to ensure their survival. Inhibition of key angiogenic factors as well as inactivating this metabolic switch could be used as novel therapeutic strategies. As such, the proteins human angiogenin, an angiogenic factor, and glycogen phosphorylase, a key regulator of glucose levels, are both attractive pharmaceutical targets for the design of novel anti-cancer agents. An in silico screening of the ZINC-15 Biogenic database targeting the cell-binding and active site of human angiogenin (hAng) is presented. A docking consensus scoring approach was implemented and, using a Simple Sum rank combination of Glide-SP and -XP, GOLD, and AutoDock Vina, the compounds sorted and ranked. The protein-ligand interactions and MM-GBSA binding free energies of native ligand (4a) displaying potent anti-angiogenic activity, was studied and used as a benchmark for the selection of ten diverse compounds demonstrating dual-action inhibition. Furthermore, this thesis investigated several binding sites of glycogen phosphorylase, namely the allosteric, quercetin, catalytic, and inhibitor sites, for the discovery of potential anti-glioblastoma compounds. For the allosteric site, a benchmarking study was carried out to assess different combinations of docking programs, as well as exploiting pharmacophore models to enrich the overall accuracy of the results. The Simple Sum Rank combination using Glide-SP and -XP docking scores, and filtering using a 4-point pharmacophore model was selected determined from the superior statistical metrics obtained including the EF, EF’, and BEDROC statistical performance metrics. Following the virtual screening, 29 compounds were selected based on their superior docking scores and interactions with key binding site residues. For the GP quercetin binding site, a similarity search using the ZINC-12 database was performed, and together with the selection of several known flavonoid compounds previously identified as GP inhibitors, these compounds were screened using Glide-SP and -XP docking. Additionally, a screening of the ZINC-15 and Analyticon databases were performed and compounds filtered using the docking scores of the cognate ligand quercetin for comparison, but also considering Lipinski’s rule of five, Jorgensen’s rule of three, Veber’s rules, and E-pharmacophore/receptor cavity pose filters. Furthermore, the key interactions formed between the site residues and quercetin was used to guide the selection of the final 27 compounds. For the GP inhibitor site, the docking predictions (Glide-SP and -XP docking scores and residue interactions) of baicalein in comparison to the previously studied flavonoid, chrysin, was assessed. Towards validating predicted compounds as true inhibitors, baicalein, together with the selected allosteric (26 compounds) and quercetin (27 compound) candidates, their % inhibition against rmGPb was initially determined (using 100 μM compound concentration). Subsequently, nine compounds were taken forward for further enzyme kinetic assays revealing low to moderate activities. Additionally, all phase 2 compounds showed > 95 % inhibition against rmGPb. Through X-ray crystallographic studies, the binding sites/mode of these compounds were determined, with additionally a novel binding discovered on the surface of GP. Subsequently, five of these compounds binding at the inhibitor, quercetin and allosteric sites, along with one new allosteric and three previously studied catalytic site inhibitors were taken forward for cell viability assays. Their effects on cell viability and growth in healthy human foetal glial cells (SVG p12), and three glioblastoma cell lines (U87 MG, T98G, and U251 MG), in comparison to the known inhibitor, flavopiridol, is reported. Compounds CP-91149, baicalein, and pelargonidin showed significant decrease in cell viabilities overall, which was reflected in their low μM IC50 values. This is an important outcome towards validating GP as a novel target for glioblastoma. The future work entails combination treatment with TMZ, repeating cell viability assays in hypoxic conditions, as well as flow cytometry experiments to elucidate the effects on the cell cycle, proliferation rate and apoptosis induction. Additionally, ten potential anti-angiogenic compounds have been identified that can be further assessed at the cellular level

Design and Synthesis of 3-(β-d-Glucopyranosyl)-4-amino/4-guanidino Pyrazole Derivatives and Analysis of Their Glycogen Phosphorylase Inhibitory Potential

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C-β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion–ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1H-pyrazole (3) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-pyrazole (4) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics—generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion–ion interactions; for 4, the most stable tautomer did not have the His377 backbone C=O interaction and while ion–ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted.</jats:p

Design and Synthesis of 3-(β-<span style="font-variant: small-caps">d</span>-Glucopyranosyl)-4-amino/4-guanidino Pyrazole Derivatives and Analysis of Their Glycogen Phosphorylase Inhibitory Potential

Glycogen phosphorylase (GP) is a key regulator of glucose levels and, with that, an important target for the discovery of novel treatments against type 2 diabetes. β-d-Glucopyranosyl derivatives have provided some of the most potent GP inhibitors discovered to date. In this regard, C-β-d-glucopyranosyl azole type inhibitors proved to be particularly effective, with 2- and 4-β-d-glucopyranosyl imidazoles among the most potent designed to date. His377 backbone C=O hydrogen bonding and ion–ion interactions of the protonated imidazole with Asp283 from the 280s loop, stabilizing the inactive state, were proposed as crucial to the observed potencies. Towards further exploring these features, 4-amino-3-(β-d-glucopyranosyl)-5-phenyl-1H-pyrazole (3) and 3-(β-d-glucopyranosyl)-4-guanidino-5-phenyl-1H-pyrazole (4) were designed and synthesized with the potential to exploit similar interactions. Binding assay experiments against rabbit muscle GPb revealed 3 as a moderate inhibitor (IC50 = 565 µM), but 4 displayed no inhibition at 625 µM concentration. Towards understanding the observed inhibitions, docking and post-docking molecular mechanics—generalized Born surface area (MM-GBSA) binding free energy calculations were performed, together with Monte Carlo and density functional theory (DFT) calculations on the free unbound ligands. The computations revealed that while 3 was predicted to hydrogen bond with His377 C=O in its favoured tautomeric state, the interactions with Asp283 were not direct and there were no ion–ion interactions; for 4, the most stable tautomer did not have the His377 backbone C=O interaction and while ion–ion interactions and direct hydrogen bonding with Asp283 were predicted, the conformational strain and entropy loss of the ligand in the bound state was significant. The importance of consideration of tautomeric states and ligand strain for glucose analogues in the confined space of the catalytic site with the 280s loop in the closed position was highlighted

Multidisciplinary docking, kinetics and X-ray crystallography studies of baicalein acting as a glycogen phosphorylase inhibitor and determination of its’ potential against glioblastoma in cellular models

Glycogen phosphorylase (GP) is the rate-determining enzyme in the glycogenolysis pathway. Glioblastoma (GBM) is amongst the most aggressive cancers of the central nervous system. The role of GP and glycogen metabolism in the context of cancer cell metabolic reprogramming is recognised, so that GP inhibitors may have potential treatment benefits. Here, baicalein (5,6,7-trihydroxyflavone) is studied as a GP inhibitor, and for its effects on glycogenolysis and glioblastoma at the cellular level. The compound is revealed as a potent GP inhibitor against human brain GPa (K i  = 32.54 μM), human liver GPa (K i  = 8.77 μM) and rabbit muscle GPb (K i  = 5.66 μM) isoforms. It is also an effective inhibitor of glycogenolysis (IC 50  = 119.6 μM), measured in HepG2 cells. Most significantly, baicalein demonstrated anti-cancer potential through concentration- and time-dependent decrease in cell viability for three GBM cell-lines (U-251 MG, U-87 MG, T98-G) with IC 50 values of ∼20–55 μM (48- and 72-h). Its effectiveness against T98-G suggests potential against GBM with resistance to temozolomide (the first-line therapy) due to a positive O6-methylguanine-DNA methyltransferase (MGMT) status. The solved X-ray structure of rabbit muscle GP–baicalein complex will facilitate structure-based design of GP inhibitors. Further exploration of baicalein and other GP inhibitors with different isoform specificities against glioblastoma is suggested