Integrated multi-spectroscopic and molecular docking techniques to probe the interaction mechanism between maltase and 1-deoxynojirimycin, an α-glucosidase inhibitor
Introduction
Type 2 diabetes mellitus is a chronic metabolic disorder characterized by impaired carbohydrate metabolism, and ultimately leads to insulin resistance and hyperglycemia [1]. It often accompanies with several long-term complications such as nephropathy, hypertension, atherosclerosis and hyperlipidemia [2]. Despite the ongoing efforts to fight diabetes, the emerging challenge remains grim as the number of diabetics are reached to 425 million in 2017 worldwide reported by International Diabetic Foundation (IDF) [3]. Glycemic control strategy is considered as a consensus for therapy of type 2 diabetes mellitus. In humans, salivary and pancreatic α-amylase and four intestinal mucosal α-glucosidase activities are responsible for the generation of dietary glucose from starchy foods [4,5]. Therefore, inhibition of these digesting proteins, such as α-glucosidase and α-amylase, are regarded as a viable therapeutic target to control postprandial glucose [6].
α-Glucosidase is the most abundant protein of a wide variety of organisms including bacteria, yeasts, fungi, archaea, plants, and animals, which plays an important role in maintaining the normal physiological function of cells and participates in carbohydrate and energy metabolism [7]. Considering the difficult to obtain pure α-glucosidase from mammalian, α-glucosidase (maltase) from Saccharomyces cerevisiae is often selected as a model protein, not only for its high stability and ready availability in a pure form, but also for the fact that it has been the representative of many studies that used for screening α-glucosidase inhibitors and studying the interaction mechanism, such as α-glucosidase inhibitor apigenin, luteolin, kaempferol [5,6,8]. Maltase from Saccharomyces cerevisiae belongs to glycoside hydrolase family 13 and its catalytic domains are formed by (β/α)8 barrel-folds [7]. Besides, the catalytic nucleophiles are located at the C-terminal end of the fourth β-strand of the catalytic domain. 1-Deoxynojirimycin, (DNJ, structure shown in Fig. 1A), an iminosugar, is a saccharide decoy that can inhibit a wide range of enzymes, which were involved in important biological processes such as intestinal digestion, post-translational processing of glycoproteins or lysosomal catabolism of glycoconjugates, because of its structural resemblance to sugar moiety of natural substrates of glucosidases [9]. Consequently, it displays a broad spectrum of bioactivities such as antidiabetic, antitumor, and anti-HIV [10]. Thereinto, many researches were focused on the antidiabetic effect of DNJ due to its excellent property of inhibition α-glucosidase activity, improved pharmacokinetic, bioavailability and fewer side effects [11,12]. On the other hands, researchers have found that DNJ is a secondary metabolite generated by plant (such as mulberry), insect (such as silkworm larvae), microorganisms (such as Bacillus and Streptomyces). Several studies reported that DNJ can alleviate the increase of blood sugar level and complications of diabetes [13,14] and show a potent inhibition on α-glucosidase [11]. To the best of our knowledge, these studies are focused on the inhibition of DNJ against α-glucosidase and the regulatory glucose-lowering effect, lack of understanding of the inhibitory mechanism has seriously limited the development of DNJ and its derivatives. Therefore, it is very interesting and significant to reveal how DNJ inhibit the activity of α-glucosidase.
From the pharmacology point of view, investigation of interaction mechanism between drugs and proteins is of great significance, because this interaction not only reflects the ability of proteins forming bonds with drugs, but also facilitates the development of new medicines in terms of an accurate comprehending of their interactions with body proteins [15,16]. Hence, interaction studies on drugs binding to proteins have become a hotspot in drug design, chemical biology and molecular recognition mechanism. The binding of small molecules to proteins induces changes that deeply modify the binding, catalytic, thermodynamic and kinetic parameters of these macromolecules, often resulting in new and unexpected properties [17]. Therefore, study on the binding of DNJ to α-glucosidase becomes very significant. Furthermore, these binding mechanism information are necessary for pharmaceutical companies to design new and effective drugs.
In this study, the inhibitory effect of DNJ on maltase was determined by kinetic analysis. Then, the interaction mechanism between DNJ and maltase was investigated by fluorescence spectroscopy, UV–vis spectroscopy, circular dichroism (CD) spectrum, dynamic light scattering coupled with molecular docking study at the molecule level. The inhibitory type, binding properties and thermodynamic parameters were also determined. These results were expected to provide accurate and full basic data for revealing the DNJ-induced structural changes and binding mechanisms with maltase. Furthermore, it increased our understanding about DNJ that used as medicinal materials for prevention of type 2 diabetes mellitus.
Section snippets
Materials
α-Glucosidase (maltase, EC 3.2.1.20) from Saccharomyces cerevisiae was purchased from Sigma-Aldrich and dissolved in phosphate buffer (50 mM, pH 6.0). 1-Deoxynojirimycin was purified from Streptomyces lavendulae UN-8 in our laboratory and the purity was >98% by high performance liquid chromatography. Acarbose was obtained from Bayer HealthCare Company Ltd. (Beijing, China). All other reagents and solvents were of analytical reagent grade, and ultrapure water was used throughout the experiment.
Comparison of DNJ and acarbose on maltase activity
It was found that the activity of maltase was significantly inhibited by various concentrations of DNJ in a concentration dependent-manner (Fig. 1B). The concentration of DNJ and acarbose (positive control, structure shown in Fig. 1A) resulting in 50% maltase activity lost (IC50) were estimated to be (1.5 ± 0.1) × 10−3 and (1.25 ± 0.05) × 10−3 mol L−1, respectively. The results suggested that DNJ has equivalent maltase inhibitory activity compared with acarbose.
Subsequently, the reversibility of
Conclusions
In summary, multi-spectroscopy techniques, such as fluorescence spectroscopy, UV–vis spectroscopy, circular dichroism (CD) spectrum, dynamic light scattering coupled with kinetic analysis and molecular docking study have been used to provide valuable information about the interaction of DNJ and maltase. Activity of maltase was inhibited by DNJ in a concentration dependent-manner. DNJ displayed a significant inhibitory activity on maltase with IC50 of (1.5 ± 0.1) × 10−3 mol L−1. It reversibly
Acknowledgements
This work was financially supported by the Innovation Project of Guangxi Graduate Education (YCBZ2017022), National Science Foundation of China (31560448, 31760452, 21506039), the National Science Foundation of Guangxi (2015GXNSFBA139052, 2016GXNSFAA380130, 2016GXNSFAA380140).
Conflicts of interest
There are no conflicts of interest to declare.
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These authors contributed equally to this work.