Search Results (22)

Background: Lafora disease (LD) is an ultra-rare and fatal neurodegenerative disorder with limited therapeutic options. Current treatments primarily address symptoms, with modest efficacy in halting disease progression, thus highlighting the urgent need for novel therapeutic approaches. Gene therapy, antisense oligonucleotides, and recombinant enzymes have recently been, and still are, under investigation. Drug repurposing may offer a promising approach to identify new, possibly effective, therapies. Methods: This study aims to investigate the conditions for repurposing empagliflozin, an SGLT2 (sodium/glucose cotransporter-2) inhibitor, as a potential treatment for LD and to establish a clinical protocol. Clinical phase: This 12-month prospective observational study will assess the safety and clinical efficacy of empagliflozin in two patients with early to intermediate LD stage. The primary endpoints will include changes in the severity of epilepsy and cognitive function, while the secondary endpoints will assess motor function, global function, and autonomy. Multiple clinical and instrumental evaluations (including MRI and PET with ¹⁸F-fluorodeoxyglucose) will be performed before and during treatment. Safety monitoring will include regular clinical assessments and reports of adverse events. Preclinical phase: In silico studies (using both molecular docking calculations and reverse ligand-based screening) and in vitro cell-based assays will allow us to investigate the effects of empagliflozin (and other gliflozins) on some key targets likely implicated in LD pathogenesis, such as GLUT1, GLUT3, glycogen synthase (hGYS), and glycogen phosphorylase (GP), as suggested in the literature and digital platforms for in silico target fishing. Results: The expected outcome of this study is twofold, i.e., (i) assessing the safety and tolerability of empagliflozin in LD patients and (ii) gathering preliminary data on its potential efficacy in improving clinical and neurologic features. Additionally, the in silico and in vitro studies may provide new insights into the mechanisms through which empagliflozin may exert its therapeutic effects in LD. Conclusion: The findings of this study are expected to provide evidence in support of the repurposing of empagliflozin for the treatment of LD. Full article

Compound 1 was previously identified by our team as a glycogen phosphorylase (GP) inhibitor with glucose-lowering activity and demonstrated to have protective effects against myocardial and cerebral ischemia. However, its impact on muscle function has not been clarified. This study is the first to evaluate the long-term effects of GP inhibitors on muscle function and metabolism. After a 28-day administration of Compound 1, we performed assays to assess muscle function and biochemical parameters in rats. We observed reductions in peak holding force, duration, tetanic contraction force, single-contraction force, and electromyographic signals under 20 s and 10 min contraction stimuli. The metabolic analysis showed no significant effects on muscle glycogen, ATP, lactic acid, and uric acid levels at low doses. In contrast, medium to high doses resulted in increased glycogen, decreased ATP, and reduced lactic acid (only at high doses), without affecting uric acid. These findings suggest that Compound 1 may adversely affect muscle function in rats, potentially due to the glycogen inhibition effects of GP inhibitors. This study provides crucial safety data and insights into the long-term effects of GP inhibitors on rat muscles, which will guide future developments and applications. Full article

Recently studied N-(β-d-glucopyranosyl)-3-aryl-1,2,4-triazole-5-carboxamides have proven 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 with imidazole resulted in significantly more efficient GP inhibitors, in silico calculations using Glide molecular docking along with unbound state DFT calculations were performed on N-(β-d-glucopyranosyl)-arylimidazole-carboxamides, revealing their potential for strong GP inhibition. The syntheses of the target compounds involved the formation of an amide bond between per-O-acetylated β-d-glucopyranosylamine and the corresponding arylimidazole-carboxylic acids. Kinetics experiments on rabbit muscle GPb revealed low micromolar inhibitors, with the best inhibition constants (K_is) 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 the careful consideration of ligand tautomeric states in binding calculations is highlighted, with the usefulness of DFT calculations in this regard proposed. Full article

Tetrazole heterocycle is a promising scaffold in drug design, and it is incorporated into active pharmaceutical ingredients of medications of various actions: hypotensives, diuretics, antihistamines, antibiotics, analgesics, and others. This heterocyclic system is metabolically stable and easily participates in various intermolecular interactions with different biological targets through hydrogen bonding, conjugation, or van der Waals forces. In the present review, a systematic analysis of the activity of tetrazole derivatives against type 2 diabetes mellitus (T2DM) has been performed. As it was shown, the tetrazolyl moiety is a key fragment of many antidiabetic agents with different activities, including the following: peroxisome proliferator-activated receptors (PPARs) agonists, protein tyrosine phosphatase 1B (PTP1B) inhibitors, aldose reductase (AR) inhibitors, dipeptidyl peptidase-4 (DPP-4) inhibitors and glucagon-like peptide 1 (GLP-1) agonists, G protein-coupled receptor (GPCRs) agonists, glycogen phosphorylases (GP) Inhibitors, α-glycosidase (AG) Inhibitors, sodium glucose co-transporter (SGLT) inhibitors, fructose-1,6-bisphosphatase (FBPase) inhibitors, IkB kinase ε (IKKε) and TANK binding kinase 1 (TBK1) inhibitors, and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In many cases, the tetrazole-containing leader compounds markedly exceed the activity of medications already known and used in T2DM therapy, and some of them are undergoing clinical trials. In addition, tetrazole derivatives are very often used to act on diabetes-related targets or to treat post-diabetic disorders. Full article

The high conservation of the three subtypes of glycogen phosphorylase (GP) presents significant challenges for specific inhibitor studies targeting GP. Our prior screening revealed that compound 1 exhibited unequal inhibitory activity against the three GP subtypes, with a noticeable effect against brain GP (PYGB). The commercially available ingliforib demonstrated potent inhibitory activity specifically against liver GP (PYGL). To guide the further design and screening of high-specificity inhibitors, the possible reasons for the differential inhibitory activity of two compounds against different GP subtypes were analyzed, with ingliforib as a reference, through molecular docking and molecular dynamics simulations. Initially, the study predicted the binding modes of ligands with the three GP receptor subtypes using molecular docking. Subsequently, this was validated by molecular dynamics experiments, and possible amino acid residues that had important interactions were explored. The strong correlation between the calculated interaction free energies and experimental inhibitory activity implied the reasonable binding conformations of the compounds. These findings offer insight into the different inhibitory activity of compound 1 and ingliforib against all three GP subtypes and provide guidance for the design of specific target molecules that regulate subtype selectivity. Full article

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. Full article

Brain-type glycogen phosphorylase (PYGB) inhibitors are recognized as prospective drugs for treating ischemic brain injury. We previously reported compound 1 as a novel glycogen phosphorylase inhibitor with brain-protective properties. In this study, we validated whether PYGB could be used as the therapeutic target for hypoxic-ischemic diseases and investigated whether compound 1 exerts a protective effect against astrocyte hypoxia/reoxygenation (H/R) injury by targeting PYGB. A gene-silencing strategy was initially applied to downregulate PYGB proteins in mouse astrocytes, which was followed by a series of cellular experiments with compound 1. Next, we compared relevant indicators that could prove the protective effect of compound 1 on brain injury, finding that after PYGB knockdown, compound 1 could not obviously alleviate astrocytes H/R injury, as evidenced by cell viability, which was not significantly improved, and lactate dehydrogenase (LDH) leakage rate, intracellular glucose content, and post-ischemic reactive oxygen species (ROS) level, which were not remarkably reduced. At the same time, cellular energy metabolism did not improve, and the degree of extracellular acidification was not downregulated after administration of compound 1 after PYGB knockdown. In addition, it could neither significantly increase the level of mitochondrial aerobic energy metabolism nor inhibit the expression of apoptosis-associated proteins. The above results indicate that compound 1 could target PYGB to exert its protective effect against cellular H/R injury in mouse astrocytes. Simultaneously, we further demonstrated that PYGB could be an efficient therapeutic target for ischemic-hypoxic diseases. This study provides a new reference for further in-depth study of the action mechanism of the efficacy of compound 1. Full article

Glycosylidene-spiro-morpholin(on)es are scarcely described skeletons in the literature. In this work, we have systematically explored the synthetic routes towards such morpholinones based on the reactions of O-peracylated hept-2-ulopyranosonamide derivatives of D-gluco and D-galacto configuration. Koenigs–Knorr type glycosylation of 2-chloroethanol, allylic and propargylic alcohols by (glyculosylbromide)onamides furnished the expected glycosides. The 2-chloroethyl glycosides were ring closed to the corresponding spiro-morpholinones by treatment with K₂CO₃. The (allyl glyculosid)onamides gave diastereomeric mixtures of spiro-5-hydroxymorpholinones by ozonolysis and 5-iodomethylmorpholinones under iodonium ion mediated conditions. The ozonolytic method has not yet been known for the construction of morpholine rings, therefore, it was also extended to O-allyl mandelamide. The 5-hydroxymorpholinones were subjected to oxidation and acid catalyzed elimination reactions to give the corresponding morpholine-3,5-dions and 5,6-didehydro-morpholin-3-ones, respectively. Base induced elimination of the 5-iodomethylmorpholinones gave 5-methyl-2H-1,4-oxazin-3(4H)-ones. O-Acyl protecting groups of all of the above compounds were removed under Zemplén conditions. Some of the D-gluco configured unprotected compounds were tested as inhibitors of glycogen phosphorylase, but showed no significant effect. Full article

Brain-type glycogen phosphorylase inhibitors are potential new drugs for treating ischemic brain injury. In our previous study, we reported compound 1 as a novel brain-type glycogen phosphorylase inhibitor with cardioprotective properties. We also found that compound 1 has high blood–brain barrier permeability through the ADMET prediction website. In this study, we deeply analyzed the protective effect of compound 1 on hypoxic-ischemic brain injury, finding that compound 1 could alleviate the hypoxia/reoxygenation (H/R) injury of astrocytes by improving cell viability and reducing LDH leakage rate, intracellular glucose content, and post-ischemic ROS level. At the same time, compound 1 could reduce the level of ATP in brain cells after ischemia, improve cellular energy metabolism, downregulate the degree of extracellular acidification, and improve metabolic acidosis. It could also increase the level of mitochondrial aerobic energy metabolism during brain cell reperfusion, reduce anaerobic glycolysis, and inhibit apoptosis and the expression of apoptosis-related proteins. The above results indicated that compound 1 is involved in the regulation of glucose metabolism, can control cell apoptosis, and has protective and potential therapeutic effects on cerebral ischemia-reperfusion injury, which provides a new reference and possibility for the development of novel drugs for the treatment of ischemic brain injury. Full article

Hordatines are a characteristic class of secondary metabolites found in barley which have been reported to be present in barley malt, beer and, recently, brewer´s spent grain (BSG). However, little is known about their biological activities such as antioxidative effects in beer or antifungal activity as their main task within the plants. We conducted an in vitro investigation of the activity of hordatines isolated from BSG towards enzymes of glucose metabolism. Hordatine-rich fractions from BSG were prepared by solid-liquid extraction (SLE) with 60% acetone followed by purification and fractionation. The fractions were characterised and investigated for their in vitro inhibitory potential on α-glucosidase and glycogen phosphorylase α (GPα). Both enzymes are relevant within the human glucose metabolism regarding the digestion of carbohydrates as well as the liberation of glucose from the liver. In total, 10 hordatine-rich fractions varying in the composition of different hordatines were separated and analysed by mass spectrometry. Hordatine A, B and C, as well as hydroxylated aglycons and many glycosides, were detected in the fractions. The total hordatine content was analysed by HPLC-DAD using a semi-quantitative approach and ranged from 60.7 ± 3.1 to 259.6 ± 6.1 µg p-coumaric acid equivalents/mg fraction. Regarding the biological activity of fractions, no inhibitory effect on GPα was observed, whereas an inhibitory effect on α-glucosidase was detected (IC₅₀ values: 77.5 ± 6.5–194.1 ± 2.6 µg/mL). Overall, the results confirmed that hordatines are present in BSG in relatively high amounts and provided evidence that they are potent inhibitors of α-glucosidase. Further research is needed to confirm these results and identify the active hordatine structure. Full article

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway. GP inhibitors are currently under investigation as a new liver-targeted approach to managing type 2 diabetes mellitus (DM). The aim of the present study was to evaluate the inhibitory activity of a panel of 52 structurally related chromone derivatives; namely, flavonoids, 2-styrylchromones, 2-styrylchromone-related derivatives [2-(4-arylbuta-1,3-dien-1-yl)chromones], and 4- and 5-styrylpyrazoles against GP, using in silico and in vitro microanalysis screening systems. Several of the tested compounds showed a potent inhibitory effect. The structure–activity relationship study indicated that for 2-styrylchromones and 2-styrylchromone-related derivatives, the hydroxylations at the A and B rings, and in the flavonoid family, as well as the hydroxylation of the A ring, were determinants for the inhibitory activity. To support the in vitro experimental findings, molecular docking studies were performed, revealing clear hydrogen bonding patterns that favored the inhibitory effects of flavonoids, 2-styrylchromones, and 2-styrylchromone-related derivatives. Interestingly, the potency of the most active compounds increased almost four-fold when the concentration of glucose increased, presenting an IC₅₀ < 10 µM. This effect may reduce the risk of hypoglycemia, a commonly reported side effect of antidiabetic agents. This work contributes with important considerations and provides a better understanding of potential scaffolds for the study of novel GP inhibitors. Full article

With a yearly production of about 39 million tons, brewer’s spent grain (BSG) is the most abundant brewing industry byproduct. Because it is rich in fiber and protein, it is commonly used as cattle feed but could also be used within the human diet. Additionally, it contains many bioactive substances such as hydroxycinnamic acids that are known to be antioxidants and potent inhibitors of enzymes of glucose metabolism. Therefore, our study aim was to prepare different extracts—A1-A7 (solid-liquid extraction with 60% acetone); HE1-HE6 (alkaline hydrolysis followed by ethyl acetate extraction) and HA1-HA3 (60% acetone extraction of alkaline residue)—from various BSGs which were characterized for their total phenolic (TPC) and total flavonoid (TFC) contents, before conducting in vitro studies on their effects on the glucose metabolism enzymes α-amylase, α-glucosidase, dipeptidyl peptidase IV (DPP IV), and glycogen phosphorylase α (GPα). Depending on the extraction procedures, TPCs ranged from 20–350 µg gallic acid equivalents/mg extract and TFCs were as high as 94 µg catechin equivalents/mg extract. Strong inhibition of glucose metabolism enzymes was also observed: the IC₅₀ values for α-glucosidase inhibition ranged from 67.4 ± 8.1 µg/mL to 268.1 ± 29.4 µg/mL, for DPP IV inhibition they ranged from 290.6 ± 97.4 to 778.4 ± 95.5 µg/mL and for GPα enzyme inhibition from 12.6 ± 1.1 to 261 ± 6 µg/mL. However, the extracts did not strongly inhibit α-amylase. In general, the A extracts from solid-liquid extraction with 60% acetone showed stronger inhibitory potential towards a-glucosidase and GPα than other extracts whereby no correlation with TPC or TFC were observed. Additionally, DPP IV was mainly inhibited by HE extracts but the effect was not of biological relevance. Our results show that BSG is a potent source of α-glucosidase and GPα inhibitors, but further research is needed to identify these bioactive compounds within BSG extracts focusing on extracts from solid-liquid extraction with 60% acetone. Full article

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC₅₀ value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols. Full article

Oximes have been studied for decades because of their significant roles as acetylcholinesterase reactivators. Over the last twenty years, a large number of oximes have been reported with useful pharmaceutical properties, including compounds with antibacterial, anticancer, anti-arthritis, and anti-stroke activities. Many oximes are kinase inhibitors and have been shown to inhibit over 40 different kinases, including AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K), cyclin-dependent kinase (CDK), serine/threonine kinases glycogen synthase kinase 3 α/β (GSK-3α/β), Aurora A, B-Raf, Chk1, death-associated protein-kinase-related 2 (DRAK2), phosphorylase kinase (PhK), serum and glucocorticoid-regulated kinase (SGK), Janus tyrosine kinase (JAK), and multiple receptor and non-receptor tyrosine kinases. Some oximes are inhibitors of lipoxygenase 5, human neutrophil elastase, and proteinase 3. The oxime group contains two H-bond acceptors (nitrogen and oxygen atoms) and one H-bond donor (OH group), versus only one H-bond acceptor present in carbonyl groups. This feature, together with the high polarity of oxime groups, may lead to a significantly different mode of interaction with receptor binding sites compared to corresponding carbonyl compounds, despite small changes in the total size and shape of the compound. In addition, oximes can generate nitric oxide. This review is focused on oximes as kinase inhibitors with anticancer and anti-inflammatory activities. Oximes with non-kinase targets or mechanisms of anti-inflammatory activity are also discussed. Full article

A current trend in the quest for new therapies for complex, multifactorial diseases, such as diabetes mellitus (DM), is to find dual or even multi-target inhibitors. In DM, the sodium dependent glucose cotransporter 2 (SGLT2) in the kidneys and the glycogen phosphorylase (GP) in the liver are validated targets. Several (β-D-glucopyranosylaryl)methyl (het)arene type compounds, called gliflozins, are marketed drugs that target SGLT2. For GP, low nanomolar glucose analogue inhibitors exist. The purpose of this study was to identify dual acting compounds which inhibit both SGLTs and GP. To this end, we have extended the structure-activity relationships of SGLT2 and GP inhibitors to scarcely known (C-β-D-glucopyranosylhetaryl)methyl arene type compounds and studied several (C-β-D-glucopyranosylhetaryl)arene type GP inhibitors against SGLT. New compounds, such as 5-arylmethyl-3-(β-D-glucopyranosyl)-1,2,4-oxadiazoles, 5-arylmethyl-2-(β-D-glucopyranosyl)-1,3,4-oxadiazoles, 4-arylmethyl-2-(β-D-glucopyranosyl)pyrimidines and 4(5)-benzyl-2-(β-D-glucopyranosyl)imidazole were prepared by adapting our previous synthetic methods. None of the studied compounds exhibited cytotoxicity and all of them were assayed for their SGLT1 and 2 inhibitory potentials in a SGLT-overexpressing TSA201 cell system. GP inhibition was also determined by known methods. Several newly synthesized (C-β-D-glucopyranosylhetaryl)methyl arene derivatives had low micromolar SGLT2 inhibitory activity; however, none of these compounds inhibited GP. On the other hand, several (C-β-D-glucopyranosylhetaryl)arene type GP inhibitor compounds with low micromolar efficacy against SGLT2 were identified. The best dual inhibitor, 2-(β-D-glucopyranosyl)-4(5)-(2-naphthyl)-imidazole, had a K_i of 31 nM for GP and IC₅₀ of 3.5 μM for SGLT2. This first example of an SGLT-GP dual inhibitor can prospectively be developed into even more efficient dual-target compounds with potential applications in future antidiabetic therapy. Full article