Identification of mangiferin as a potential Glucokinase activator by structure-based virtual ligand screening

The natural product mangiferin (compound 7) has been identified as a potential glucokinase activator by structure-based virtual ligand screening. It was proved by enzyme activation experiment and cell-based assays in vitro, with potency in micromolar range. Meanwhile, this compound showed good antihyperglycemic activity in db/db mice without obvious side effects such as excessive hypoglycaemia.

The natural product mangiferin (compound 7) has been identified as a potential glucokinase activator by structure-based virtual ligand screening. It was proved by enzyme activation experiment and cellbased assays in vitro, with potency in micromolar range. Meanwhile, this compound showed good antihyperglycemic activity in db/db mice without obvious side effects such as excessive hypoglycaemia.
Diabetes mellitus (DM), especially type 2 diabetes mellitus (T2DM), is referred to a metabolic disorder of multiple etiologies in which chronic hyperglycemia results from absent or inadequate pancreatic insulin secretion, with or without concurrent impairment of insulin action 1 . This often leads to carbohydrates, lipids and proteins metabolism disorders, along with serious complications that results in significant disability and mortality 2 .
Glucokinase (GK, EC 2.7.1.2), a glucose phosphorylating enzyme, represents a promising molecular target for development of T2DM drugs. It is an enzyme that facilitates phosphorylation of glucose to glucose-6-phosphate 3 . GK has a pivotal position in the regulation of glucose homeostasis in vivo, which acts as a glucose sensor in pancreatic β -cell and a rate controlling enzyme for hepatic glucose metabolism and glycogen synthesis 4 . Studies showed that fifty percent of diabetics had lower liver glucokinase activity than that of the controls 5 . On the other hand, the GK activity can be elevated by glucokinase activators (GKAs), which bind to the allosteric site of GK and contribute to govern blood glucose by enhancing glucose uptake in the liver and potentiating insulin secretion in a glucose-dependent manner [6][7][8][9][10][11][12][13][14] . Until now, more than 150 patents for GKAs have been recorded 15,16 . However, all of them, even the highly promising LY2608204, failed to generate a clinically effective antidiabetic medicine owing to their notable side effect or drug tolerance 17 .
Structure-based virtual ligand screening has been an essential tool in assisting the fast and cost-efficient discovery of lead compounds 18,19 . On the other hand, natural products are important sources for drug discovery. They have been an invaluable pool of molecular scaffolds to discover biologically active lead compounds or even new therapeutic agent. Most of currently marketed drugs have been derived directly or indirectly from plant constituent.
In this study, we found that the natural product mangiferin (compound 7, zinc04098535), a C-glycosyl xanthone widely distributed in many plant species, was a potential glucokinase activator by structure-based virtual ligand screening. Previous studies showed that mangiferin could exert effective antidiabetic activities on T2DM animals [20][21][22] . However, the mechanism of this compound hasn't been really confirmed. Based on our further study on docking analysis of GK-mangiferin complex, this investigation employed in vitro and in vivo method to evaluate mangiferin's antidiabetic potential. All data reported here provide evidence that mangiferin can effectively control postprandial blood glucose levels by moderately activating glucokinase, without the occurrence of adverse effects.
The effect of screening hits on GK enzyme activation. In order to further investigate the activation effect of the twelve compounds on GK enzymatic activity, the recombinant GK protein had been successfully obtained by genetic engineering method 4 . Evaluation of the twelve hits on enzymatic activity was assessed spectrometrically by a coupled reaction with glucose-6-phosphate dehydrogenase. Data analysis displayed that both compound 7 and 12 had a positive effect on GK activation (Fig. 2). Furthermore, compound 7 and 12 showed activated effects on GK with EC 50 values of ca. 156 μ M and more than 500 μ M, respectively (Table S2). Binding affinity assay by microscale thermophoresis (MST). The binding interaction between GK and compound 7 was assayed by MST, which is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale. Results revealed that the binding affinity of compound 7 to GK was 472 ± 20.5 μ M, better than that of the known activator of GK, LY2608204 (600 ± 36.1 μ M ), a potent compound but failed in the phase II clinical trial (Table 1 and Fig. S1).

Molecular docking analysis of GK-compound 7 complex.
To further elucidate the binding mode of compound 7 with GK, we performed molecular docking. The best scoring binding conformation of compound 7 is shown as Fig. 3 In the generated docking model, compound 7 was adopted an extended conformation, which occupied the two major angle of the tri-star shaped allosteric site. Hydrogen bonds were predicted between 4′ -OH of sugar ring and Ser69, 6-OH and carbonyl oxygen of Cys220. Moreover, the π -π stacking interaction formed by the pyranone ring of 7 and Tyr214 further strengthens the binding (Fig. 3). The activation percentage was the GK enzyme activity in each group compared to that of the blank control group. Each compound was assayed triply. The value presents in a column as the mean ± SD (n = 3). *P < 0.05, **P < 0.01, compared to the blank control group.  Glucose consumption in HepG2 and C2C12 myoblast cell. HepG2 cell and mouse C2C12 myoblast cell are widely used as the cell models to study hepatocyte and myocyte functions, respectively. In present study, we employed this two cell models to evaluate the effect of compound 7 on cellular glucose consumption. Changes of glucose concentration in culture medium were measured by glucose assay kit, after cells were incubated with various concentrations of compound 7 for 24 hours. As shown in Fig. 4 a remarkable dose-dependent enhancement of glucose consumption of HepG2 cells was observed ( Fig. 4A), Meanwhile, compound 7 had a relative slight enhancement on muscle cell glucose consumption (Fig. 4B). This findings suggested that compound 7 could improve glucose metabolism level in HepG2 and muscle cells, especially in the HepG2 cell line. Therefore, compound 7 has a promising potential to govern blood glucose.

In vivo anti-diabetic activities of compound 7 in db/db mice. After administered with compound 7
(200 mg/kg) for 8 weeks, the two-hour postprandial blood glucose level of 7-treated group significantly declined, compared with the diabetic group (Fig. 5A). Oral glucose tolerance test (OGTT) was performed at the eighth week. Compared to the diabetic group, both 7-treated group and metformin-treated group showed palpable hypoglycemia and steady declines (P < 0.05) from 1st to 2nd hour (Fig. 6A). Comparing the area under the curve (AUC) among the groups, metformin and 7 treated groups showed significant reductions, with the degree of 56.55 and 23 41%, respectively (Fig. 6B). The Results revealed that compound 7 improved glucose tolerance in mice. The serum insulin level was determined according to the Mouse Insulin ELISA kit instructions. It showed that the serum insulin level of 7-treated group had significantly (P < 0.05) increases than that of the diabetic group (Fig. 5B). Meanwhile, the serum lipid level in 7-treated group was slightly increased (Table S3). Results showed that the TG level and HDL-C level of 7-treated group were obviously lower than that of the diabetic group (P < 0.05). The LDL-C level of 7-treated group was decreasd, but there was no noticeable difference comparing with the diabetic group statistically. The TC level of the 7-treated group was higher than that of the diabetic group, but there was no significant difference statistically.
Histopathological examination of tissues. The liver lipid deposition has obvious amelioration in compound 7 and metformin treated groups compared to the diabetic group. Histological morphology examination clearly showed adipose accumulation and fat vacuoles in the diabetic group. The abnormal changes were remarkably ameliorated after treatment with metformin and compound 7 (Fig. S2A). Morphology analysis revealed a marked hyperplasia of abdominal white adipose tissue (ABAT) in db/db mice. Compared with the diabetic group, the adipose tissue content and the size of ABAT adipocytes were significantly reduced in the 7-treated group (Fig. S2B). However, decreased weight gain was unapparent in the 7-treated group. (Fig. 5C). These findings were consistent with previous results that compound 7 gave rise to the redistribution of adipose depots. Furthermore, the 7-treated group and metformin-treated group had larger islets with few signs of degeneration, while the diabetic group had smaller islets with grossly disrupted architecture (Fig. 7A). Meanwhile, both two groups had returned to normal expression levels of insulin and glucagen compared to that of the diabetic group (Fig. 7B). This findings suggested that compound 7 might also play a role in the protection and repairation of islet tissues.
In this study, we found the natural product mangiferin (compound 7, zinc04098535) is a naturally potential agonists of GK by structure-based virtual ligand screening. In vitro and in vivo experiments have proved that it could effectively control diabetes by moderately activating GK without causing excessive hypoglycemia. All along, people have been searching for the agonist of the GK enzyme. However, none of the drugs has been approved by FDA. The reason is that the activity of GKAs founded currently were too strong to use as a antidiabetic drug. Mangiferin is a common, inexpensive and readily available chemical composition in nature. It could activate the GK enzyme moderately without causing considerable side effect, making this compound as a promising entity for GKAs 27 . And also, more synthetic efforts are required to generate more candidates for in vitro and in vivo pharmacodynamic evaluation.    (pH 7.5), 10 mM glucose, 1 mM ATP, 4 U/ml G6PDH (Sigma-Aldrich Co. LLC., St. Louis, MO, USA), 1 mM NADP, 2.5 mM MgCl, 50 mM KCl, 2 mM DTT, 1 mM recombinant human glucokinase and test compounds. The whole reaction mixtures were kept under 37 °C for 20 minutes. The increase in the rate of absorbance of NADPH generated in the reactions was monitored kinetically at 340 nm. The values were calculated by comparing with untreated GK. This term is called the efficacy of stimulatory concentration (SC 50 ). Then, the EC 50 of promising candidates were determined in the presence of various fixed concentrations of compound 7 (2-300 μ M). Values were calculated by fitting enzymatic rates to the Hill equation by using GraphPad Prism 5.01.
Microscale thermophoresis assay (MST). The MST assay was performed according to the supplied practice protocol. The recombinant GK protein was labeled with Monolith NTTM Protein Labeling Kit RED (Cat#L001) according to the kit instructions 28,29 . Labelled GK concentration was adjusted to 200 nM. A serial of dilution solution of compound 7 (5-10000 μ M) in the same buffer (20 mM Hepes, pH 7.5) was prepared and mixed with the above labelled GK protein with the volume ratio of 1:1. After fifteen minutes incubation, all the samples were loaded into the standard glass capillaries. They were immediately measured by MST with a LED power of 100% and a MST power of 40%. The dissociation constrant Kd values were fitted by using NT Analysis software (NanoTemper Technologies, München, Germany). Molecular docking. The X-ray co-crystal structure of glucokinase with the allosteric activator (PDB code: 3S41) was used as the docking model. The docking was performed by using ICM 3.8.2 modeling software on an Intel i7 4960 processor (MolSoft LLC, San Diego, CA). Ligand binding pocket residues were selected by using graphical tools in the ICM software to create the boundaries of the docking search. In the docking calculation, potential energy maps of the receptor were calculated using default parameters. Compounds were imported into ICM and an index file was created. Conformational sampling was based on the Monte Carlo procedure 30 , and finally the lowest-energy and the most favorable orientation of the ligand were selected.
Cell glucose consumption assay. HepG2 and C2C12 myotubes were incubated with compound under various concentrations (0-1 mM) for 24 hours, respectively. The media were collected and glucose surplus was measured at 505 nm with Glu Assay Kit (Shanghai Mind Bioengineering Co. Ltd, Shanghai, China) by Synergy Multi-Mode Microplate Reader (BioTek, USA). Glucose consumption rate was the glucose consumption in each group compared to that of the control group. Cell counting kit-8 (Dojindo, Japan) assay was performed to adjust the value of glucose consumption by calculating the ratio of glucose consumption and CCK-8 (glucose consumption/CCK-8) 32 .
Animal experimental design. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Huazhong University of Science and Technology and approved by the Ethics Committee. The experimental period was eight weeks. Six week-old male db/db mice (B6.BKS(D)-Leprdb/Nju) and lean wild type littermates were purchased from the Model Animal Research Center of Nanjing University. Animals were housed at the animal care facility of Tongji Medical college under standard conditions at constant room temperature of 25 °C, humidity of 60 ± 5%, and on a 12 hours dark/light cycle. Mice had free access to water and food throughout the study period. After 10 days acclimation, diabetic mice were randomly divided into there groups (n = 6). In the experiment, a total of 24 mice (6 normal mice, 18 diabetic mice) were used. Group 1: the normal group, normal mice were orally administered of distilled water (Vehicle); Group 2: the diabetic group, diabetic mice were orally administered of distilled water (Vehicle); Group 3: the metformin-treated group, diabetic mice were orally administered of positive drug metformin (200 mg/kg BW/D) in the same vehicle; Group 4: the 7-treated group, diabetic mice were orally administered of compound 7 (200 mg/kg BW/D) in the same vehicle.
Glucose and weight measurements. Weight and two-hour postprandial blood glucose level 33 of mice were monitored weekly. Glucose measurements were performed on blood drawn from the tail vein using a Bayer Contour Glucose Meter (Bayer, Germany).
Oral glucose tolerance test (OGTT). When the db/db mice and normal mice were intragastricly administered for 8 weeks, the 12 h fasted mice in all groups were intra gastricly given glucose (2.5 g/kg). Blood samples were collected from the tail vein at 0, 0.5, 1, 1.5, 2 h after glucose loading, and the blood glucose level of all samples were immediately measured by using a Bayer Contour Glucose Meter.
Fasting serum insulin levels and lipids levels in mice. The mice were sacrificed after treatment for 8 weeks. Blood samples and tissues for bioassay were obtained from 12 h fasting mice. Blood samples were centrifuged (4000 rpm, 15 min) and then stored at − 20 °C for further study. Fasting serum insulin level were measured by the Mouse insulin ELISA kit (TSZ, USA). Total cholesterol (TC), total triglyceride (TG), high-density Scientific RepoRts | 7:44681 | DOI: 10.1038/srep44681 lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were respectively analyzed according to the kit instructions, all assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China).
Histopathological examination. The liver, pancreas, abdominal white adipose tissue were removed after the mice were sacrificed. All tissue samples were divided into two halfs, one of which was stored at − 80 °C for further study, and the other one were stored in 10% formalin after washing with PBS buffer. The tissues embedded in paraffin and sectioned, and then stained with hematoxylin eosin for histopathological assessment through microscopic observation (Olympus, Tokyo, Japan).