Drug screening of α-amylase inhibitors as candidates for treating diabetes.

In the present study, the identification of potential α-amylase inhibitors is explored as a potential strategy for treating type-2 diabetes mellitus. A computationally driven approach using molecular docking was employed to search for new α-amylase inhibitors. The interactions of potential drugs with the enzyme's active site were investigated and compared with the contacts established by acarbose (a reference drug for α-amylase inhibition) in the crystallographic structure 1B2Y. For this active site characterization, both molecular docking and molecular dynamics simulations were performed, and the residues involved in the α-amylase-acarbose complex were considered to analyse the potential drug's interaction with the enzyme. Two potential α-amylase inhibitors (AN-153I105594 and AN-153I104845) have been selected following this computational strategy. Both compounds established a large number of interactions with key binding site α-amylase amino acids and obtained a comparable docking score concerning the reference drug (acarbose). Aiming to further analyse candidates' properties, their ADME (absorption, distribution, metabolism, excretion) parameters, druglikeness, organ toxicity, toxicological endpoints and median lethal dose (LD50 ) were estimated. Overall estimations are promising for both candidates, and in silico toxicity predictions suggest that a low toxicity should be expected.

Identifying the alpha-glucosidase inhibitory potential of dietary phytochemicals against diabetes mellitus type 2 via molecular interactions and dynamics simulation.

The research aims to identify the inhibitory potential of natural dietary phytochemicals against non-insulinotropic target protein alpha-glucosidase and its possible implications to diabetes mellitus type 2. A data set of sixteen plant-derived dietary molecules viz., 4,5-dimethyl-3-hydroxy-2(5H)-furanone, apigenin, bromelain, caffeic acid, cholecalciferol, dihydrokaempferol 7-o-glucopyranoside, galactomannan, genkwanin, isoimperatorin, luteolin, luteolin 7-o-glucoside, neohesperidin, oleanoic acid, pelargonidin-3-rutinoside, quercetin, and quinic acid were taken to accomplish molecular docking succeeded by their comparison with known inhibitors including acarbose, miglitol, voglibose, emiglitate, and 1-deoxynojirimycin. Among all phyto-compounds, bromelain (ΔG: -9.54 kcal/mol), cholecalciferol (-8.47 kcal/mol), luteolin (-9.02 kcal/mol), and neohesperidin (-8.53 kcal/mol) demonstrated better binding interactions with alpha-glucosidase in comparison to the best-known inhibitor, acarbose (ΔG: -7.93 kcal/mol). Molecular dynamics simulation of 10 ns duration, CYP450 site of metabolism identification, and prediction of activity spectra for substances depicted the bromelain as the most stable inhibitor compared to luteolin and acarbose. Findings of molecular interactions, molecular dynamics study, metabolism, and biological activity prediction proved bromelain as a potential alpha-glucosidase inhibitor. Thus, bromelain might be helpful as an insulin-independent therapeutic molecule towards controlling and managing diabetes mellitus type 2.

Isolated compounds from Dracaena angustifolia Roxb and acarbose synergistically/additively inhibit α-glucosidase and α-amylase: an in vitro study.

BACKGROUND: As a traditional herbal medicine, Dracaena angustifolia Roxb has been used as an anti-inflammatory agent by the Li people in Hainan, China. In preliminary phytochemical studies conducted in our lab, its fractions were found to inhibit α-glucosidase in vitro, indicating a potential for alleviating glucose dysregulation. METHODS: Through in vitro enzymatic assays, the abilities of the separated components to affect α-glucosidase and α-amylase were evaluated. By establishing concentration gradients and generating Lineweaver-Burk plots, the corresponding inhibition modes together with kinetic parameters were assessed. Following the evaluation of the outcomes of their combination with acarbose, computational docking and molecular dynamic simulations were carried out to analyse the interaction mechanisms and perform virtual screening against human enzymes. RESULTS: Compared with acarbose, 7 compounds, including flavonoid derivatives, amides and aromatic derivatives, with higher α-glucosidase inhibitory efficiencies were confirmed. It was found that those competitive/mixed candidates and acarbose interacted synergistically or additively on α-glucosidase. Moreover, 3 of them were able to inhibit α-amylase in mixed mode, and additive effects were observed in combination with acarbose. Through in silico docking, it was found that the active site residues as well as adjacent residues were involved in α-glucosidase and α-amylase binding, which were mainly achieved through hydrogen bonding. Among those dual-function flavonoids, Compound 9 was predicted to be a considerable inhibitor of human enzymes, as the formation of ligand-enzyme complexes was mediated by the residues responsible for substrate recognition and catalysis, the stabilities of which were reiterated by molecular dynamics simulations. CONCLUSION: Despite their mild effects on α-amylase, considerable α-glucosidase inhibitory efficiencies and potential synergy with acarbose were exhibited by these natural candidates. Furthermore, a stable ligand, human α-glucosidase, was predicted by the performed simulations, which provided useful information for the application of Dracaena angustifolia Roxb in diabetes treatment.

Personal Glucose Meter for α-Glucosidase Inhibitor Screening Based on the Hydrolysis of Maltose.

As a key enzyme regulating postprandial blood glucose, α-Glucosidase is considered to be an effective target for the treatment of diabetes mellitus. In this study, a simple, rapid, and effective method for enzyme inhibitors screening assay was established based on α-glucosidase catalyzes reactions in a personal glucose meter (PGM). α-glucosidase catalyzes the hydrolysis of maltose to produce glucose, which triggers the reduction of ferricyanide (K3[Fe(CN)6]) to ferrocyanide (K4[Fe(CN)6]) and generates the PGM detectable signals. When the α-glucosidase inhibitor (such as acarbose) is added, the yield of glucose and the readout of PGM decreased accordingly. This method can achieve the direct determination of α-glucosidase activity by the PGM as simple as the blood glucose tests. Under the optimal experimental conditions, the developed method was applied to evaluate the inhibitory activity of thirty-four small-molecule compounds and eighteen medicinal plants extracts on α-glucosidase. The results exhibit that lithospermic acid (52.5 ± 3.0%) and protocatechualdehyde (36.8 ± 2.8%) have higher inhibitory activity than that of positive control acarbose (31.5 ± 2.5%) at the same final concentration of 5.0 mM. Besides, the lemon extract has a good inhibitory effect on α-glucosidase with a percentage of inhibition of 43.3 ± 3.5%. Finally, the binding sites and modes of four active small-molecule compounds to α-glucosidase were investigated by molecular docking analysis. These results indicate that the PGM method is feasible to screening inhibitors from natural products with simple and rapid operations.

Alpha-amylase as molecular target for treatment of diabetes mellitus: A comprehensive review.

The alpha (α)-amylase is a calcium metalloenzyme that aids digestion by breaking down polysaccharide molecules into smaller ones such as glucose and maltose. In addition, the enzyme causes postprandial hyperglycaemia and blood glucose levels to rise. α-Amylase is a well-known therapeutic target for the treatment and maintenance of postprandial blood glucose elevations. Various enzymatic inhibitors, such as acarbose, miglitol and voglibose, have been found to be effective in targeting this enzyme, prompting researchers to express an interest in developing potent alpha-amylase inhibitor molecules. The review mainly focused on designing different derivatives of drug molecules such as benzofuran hydrazone, indole hydrazone, spiroindolone, benzotriazoles, 1,3-diaryl-3-(arylamino) propan-1-one, oxadiazole and flavonoids along with their target-receptor interactions, IC50 values and other biological activities.

Inhibitory mechanism of morin on α-glucosidase and its anti-glycation properties.

It is important to investigate the inhibition of α-glucosidase due to its correlation with type 2 diabetes. Morin was found to exert significant inhibition activity on α-glucosidase in a reversible mixed-type manner with an IC50 value of (4.48 ± 0.04) µM. Analyses of fluorescence and circular dichroism spectra indicated that the formation of the morin-α-glucosidase complex was driven mainly by hydrophobic forces and hydrogen bonding, and caused the conformational changes of α-glucosidase. The phase diagrams of fluorescence showed that the conformational change process was monophasic without intermediates. Molecular docking indicated that morin mainly interacted with amino acid residues located close to the active site of α-glucosidase, which may move to cover the active pocket to reduce the binding of the substrate and then inhibit the catalytic activity. Morin was also found to exhibit inhibition in the generation of advanced glycation end products which was related to the long term complications of diabetes.

Important Aspects of Post-Prandial Antidiabetic Drug, Acarbose.

Acarbose, a well known and efficacious α-amylase and α-glucosidase inhibitor, is a postprandial acting antidiabetic drug. DNS-based α-amylase inhibitory assays showed that use of acarbose at concentrations above 125 µg/ml resulted in release of reducing sugar in the reaction, an unexpected observation. Objective of the present study was to design experimental strategies to address this unusual finding. Acarbose was found to be susceptible to thermo-lysis. Further, besides being an inhibitor, it could also be hydrolyzed by porcine pancreatic α-amylase, but had weaker affinity for α - amylase compared to starch. GRIP docking was done for the mechanistic analysis of the active site in the enzyme for substrate, inhibitor and, inhibitor's metabolite (K2). Interaction between acarbose and α-amylase involved significant hydrogen binding compared to that of starch, producing a stronger enzyme-inhibitor complex. Further, docking analysis led us to predict the site on α-amylase where the inhibitor (acarbose) bound more tightly, which possibly affected the binding and hydrolysis of starch exerting its effective anti-diabetic function.

α-Glucosidase and α-amylase inhibitors from Myrcia spp.: a stronger alternative to acarbose?

"Pedra hume caá" is the common name of five species of Myrcia genus used as traditional medicine for the treatment of diabetes mellitus. In this study, different extracts from Myrcia salicifolia, Myrcia sphaerocarpa, and Myrcia speciosa were investigated for the first time, to identify their phenolic compounds (being 3-O-rhamnoside derivatives of myricetin and quercetin the major ones) and in vitro inhibitory potential against α-glucosidase and α-amylase. These extracts inhibited 90-500 times more α-glucosidase (IC50=0.7 to 4.1 µg ml(-1)) than acarbose and displayed a mild inhibition against α-amylase (IC50=6.1 to 29 µg mL(-1)).

Screening alpha-glucosidase and alpha-amylase inhibitors from natural compounds by molecular docking in silico.

The alpha-glucosidase inhibitor is a common oral anti-diabetic drug used for controlling carbohydrates normally converted into simple sugars and absorbed by the intestines. However, some adverse clinical effects have been observed. The present study seeks an alternative drug that can regulate the hyperglycemia by down-regulating alpha-glucosidase and alpha-amylase activity by molecular docking approach to screen the hyperglycemia antagonist against alpha-glucosidase and alpha-amylase activities from the 47 natural compounds. The docking data showed that Curcumin, 16-hydroxy-cleroda-3,13-dine-16,15-olide (16-H), Docosanol, Tetracosanol, Antroquinonol, Berberine, Catechin, Quercetin, Actinodaphnine, and Rutin from 47 natural compounds had binding ability towards alpha-amylase and alpha-glucosidase as well. Curcumin had a better biding ability of alpha-amylase than the other natural compounds. Analyzed alpha-glucosidase activity reveals natural compound inhibitors (below 0.5 mM) are Curcumin, Actinodaphnine, 16-H, Quercetin, Berberine, and Catechin when compared to the commercial drug Acarbose (3 mM). A natural compound with alpha-amylase inhibitors (below 0.5 mM) includes Curcumin, Berberine, Docosanol, 16-H, Actinodaphnine/Tetracosanol, Catechin, and Quercetin when compared to Acarbose (1 mM). When taken together, the implication is that molecular docking is a fast and effective way to screen alpha-glucosidase and alpha-amylase inhibitors as lead compounds of natural sources isolated from medicinal plants.

Combined effects of green tea extracts, green tea polyphenols or epigallocatechin gallate with acarbose on inhibition against α-amylase and α-glucosidase in vitro.

Green tea, green tea polyphenols and epigallocatechin gallate (EGCG) are confirmed to have beneficial effects in the treatment of diabetes mellitus, and a possible mechanism can be ascribed to their inhibitory effect against α-amylase and α-glucosidase in the digestive tract. In this paper, we first investigated the combined inhibitory effect of green tea extracts, green tea polyphenols or EGCG with acarbose on α-amylase and α-glucosidase in vitro. Our results indicated that the interaction between green tea extracts (green tea polyphenols or EGCG) and acarbose was complicated. The combination of green tea extracts, green tea polyphenols or EGCG with acarbose had a synergistic effect on α-amylase and α-glucosidase at low concentrations and the combined effect turned out to be antagonistic at high concentrations according to the Combination Index (CI) values. These findings not only provided some significant quantitative values, but also provide some valuable implications for the combined use of acarbose and GTE (GTP or EGCG) in the treatment of diabetes mellitus.

Novel inhibitory effect of the antidiabetic drug voglibose on melanogenesis.

Overproduction of melanin can lead to medical disorders such as postinflammatory melanoderma and melasma. Therefore, developing antimelanogenic agents is important for both medical and cosmetic purposes. In this report, we demonstrated for the first time that the antidiabetic drug voglibose is a potent antimelanogenic agent. Voglibose is a representative antidiabetic drug possessing inhibitory activity towards human α-glucosidase; it blocked the proper N-glycan modification of tyrosinase, resulting in a dramatic reduction of the tyrosinase protein level by altering its stability and subsequently decreasing melanin production. Acarbose, another antihyperglycaemic drug that has a lower inhibitory effect on human intracellular α-glucosidase compared with voglibose, did not cause any changes in either the N-glycan modification of tyrosinase or the tyrosinase protein level, indicating that voglibose was the most efficient antimelanogenic agent among the widely used antihyperglycaemic agents. Considering that voglibose was originally selected from the valiolamine derivatives in a screen for an oral antidiabetic drug with a strong inhibitory activity towards intestinal α-glucosidase and low cell permeability, we propose an alternative strategy for screening compounds from valiolamine derivatives that show high inhibitory activity towards human intracellular α-glucosidases and high cell permeability, with the goal of obtaining antimelanogenic agents that are effective inside the cells.

Molecular basis for the recognition of long-chain substrates by plant α-glucosidases.

Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher kcat/Km values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (ß/α)8 barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (ß/α)8 barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.

Maplexins, new α-glucosidase inhibitors from red maple (Acer rubrum) stems.

Thirteen gallic acid derivatives including five new gallotannins, named maplexins A-E, were isolated from red maple (Acer rubrum) stems. The compounds were identified by spectral analyses. The maplexins varied in number and location of galloyl groups attached to 1,5-anhydro-d-glucitol. The isolates were evaluated for α-glucosidase inhibitory and antioxidant activities. Maplexin E, the first compound identified with three galloyl groups linked to three different positions of 1,5-anhydro-d-glucitol, was 20 fold more potent than the α-glucosidase inhibitory drug, Acarbose (IC(50)=8 vs 160 µM). Structure-activity related studies suggested that both number and position of galloyls attached to 1,5-anhydro-d-glucitol were important for α-glucosidase inhibition.

Yeast α-glucosidase inhibition by isoflavones from plants of Leguminosae as an in vitro alternative to acarbose.

In the course of searching for new classes of α-glucosidase inhibitors originated from natural resources, 11 kinds of isoflavones, i.e., medicarpin (1), formononetin (2), mucronulatol (3), (3R)-calussequinone (5), (3R)-5'-methoxyvestitol (6), tectorigenin (7), biochanin A (8), tuberosin (9), calycosin (10), daidzein (11), and genistein (12), as well as a flavone, liquritigenin (4), were isolated as active principles responsible for the yeast α-glucosidase inhibitory activity from two leguminous plant extracts, i.e., the heartwood extract of Dalbergia odorifera and the roots extract of Pueraria thunbergiana. Each components (1-12) demonstrated a significantly potent inhibition on yeast α-glucosidase in a dose dependent manner when the p-nitrophenyl-α-D-glucopyranoside was used as a substrate in vitro. The concentration required for 50% enzyme inhibition (IC50) were calculated as 2.93 mM (1), 0.51 mM (2), 3.52 mM (7) 0.35 mM (8), 3.52 mM (9), 0.85 mM (11), and 0.15 mM (12) when that of reference drug acarbose was evaluated as 9.11 mM, in vitro. However, isoflavone glycosides, i.e., puerarin (13), daidzin (14), formononetin-7-O-ß-glucopyranoside (15), and genistin (16), exhibited a relatively poor inhibitory activity on yeast α-glucosidase as compared with the corresponding isoflavone (2, 11, 12), respectively.

New glucosidase inhibitors from an ayurvedic herbal treatment for type 2 diabetes: structures and inhibition of human intestinal maltase-glucoamylase with compounds from Salacia reticulata.

An approach to controlling blood glucose levels in individuals with type 2 diabetes is to target alpha-amylases and intestinal glucosidases using alpha-glucosidase inhibitors acarbose and miglitol. One of the intestinal glucosidases targeted is the N-terminal catalytic domain of maltase-glucoamylase (ntMGAM), one of the four intestinal glycoside hydrolase 31 enzyme activities responsible for the hydrolysis of terminal starch products into glucose. Here we present the X-ray crystallographic studies of ntMGAM in complex with a new class of alpha-glucosidase inhibitors derived from natural extracts of Salacia reticulata, a plant used traditionally in Ayuverdic medicine for the treatment of type 2 diabetes. Included in these extracts are the active compounds salacinol, kotalanol, and de-O-sulfonated kotalanol. This study reveals that de-O-sulfonated kotalanol is the most potent ntMGAM inhibitor reported to date (K(i) = 0.03 microM), some 2000-fold better than the compounds currently used in the clinic, and highlights the potential of the salacinol class of inhibitors as future drug candidates.

Discovery and biological evaluation of novel alpha-glucosidase inhibitors with in vivo antidiabetic effect.

Discovery of alpha-glucosidase inhibitors has been actively pursued with the aim to develop therapeutics for the treatment of diabetes and the other carbohydrate-mediated diseases. We have identified four novel alpha-glucosidase inhibitors by means of a drug design protocol involving the structure-based virtual screening under consideration of the effects of ligand solvation in the scoring function and in vitro enzyme assay. Because the newly identified inhibitors reveal in vivo antidiabetic activity as well as a significant potency with more than 70% inhibition of the catalytic activity of alpha-glucosidase at 50 microM, all of them seem to deserve further development to discover new drugs for diabetes. Structural features relevant to the interactions of the newly identified inhibitors with the active site residues of alpha-glucosidase are discussed in detail.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity.

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.