Molecular mechanism of interaction between human sweet taste receptors and antidiabetic agents of Gymnema sylvestre through docking studies
Sivakumar S1*, Bharathy G
1,2
1Department of Biochemistry, Sri Sankara Arts and Science College, Enathur Kanchipuram- 631561, Tamil Nadu,
India 2Department of Chemistry, Sri Ramanujar Engineering College, Kolapakkam, Vandalur, Chennai-600048, Tamil Na-
du, India
ABSTRACT
Diabetes mellitus is a group of metabolic disorder characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. It is classified into two types, type1 and type 2. In this study, sequence analysis of taste receptors and docking studies with antidiabetic agents of Gymnema sylvestre had been carried out. The ac-tive components gurmarin and gymnemic acid of Gymnema sylvestre inhibit the sweet taste response which are going to act as antidiabetic agents. The results showed that the gurmarin and gymnemic acid showed antidiabetic activity. By using bioinformatics tools such as ExPASy translate tool, tertiary structure prediction and docking, the interaction of these active components had been carried out. The results of the present study, first time identified the binding sites of gurmarin and gymnemic acid in both Taste type 1 Receptor 2 and Taste type 1 Receptor 3 (T1R2/T1R3) receptors.
Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. It is classi-fied into two types, type 1 and type 2. Type 1 Diabetes in which insulin is not secreted by pancreas, it is also called juvenile onset diabetes. Type 2 diabetes not caused by low levels of insulin, instead the body cells do not recognize insulin in the blood stream. The Com-plications of diabetes mellitus are heart disease, Neph-ropathy, Retinopathy and Neuropathy. Thus, it needs proper treatment agents.
The senses of vision, hearing, touch, olfaction, and taste have the critical roles of providing an organism with faithful representation of the external world. In its simplest form, taste perception is responsible for basic food and bestows the organism with valuable discrimi-natory power. For example, sweet receptors allow rec-ognition of high-caloric food sources, while signaling through bitter receptors may stimulate behavioral aversion to noxious substances. Mammals are believed to have five basic taste modalities: sweet, bitter, sour, salty, and umami (the taste monosodium glutamate). Extensive psychophysical studies humans have re-
ported that different regions of the tongue display dif-ferent gustatory preferences, and numerous physiolog-ical studies in animals have shown that taste receptor cells may selectively respond to different tastants. In mammals, taste receptor cells are assembled into taste buds that are distributed into different papillae in the tongue epithelium. Circumvallate papillae are found at the very back of the tongue, contain hundreds (mice) to thousands (human) of taste buds, and are particular-ly sensitive to bitter substances. Foliate papillae local-ize to the posterior lateral edge of the tongue, contain dozens to hundreds of taste buds, and are particularly sensitive to sour and bitter. Fungiform papillae contain a single or a few taste buds, are at the front of the ton-gue, and are thought to mediate much of the sweet taste modality (Hoon et al., 1999).
Gymnema sylvestre R. Br. (Asclepiadaceae) is an herb distributed throughout the world. The leaves of the plant are widely used for the treatment of diabetes and as a diuretic in Indian proprietary medicines. Gym-nemic acid is the main active chemical constituent iso-lated from the Gymnema sylvestre plant. The plant is commonly known as Periploca of the woods (English); Gurmar (Hindi); Meshashringi, madhunashini (San-skrit); Kavali, kalikardori (Marathi); Dhuleti, marda-shingi (Gujrathi); Adigam, cherukurinja (Tamil); Poda-patri (Telgu) and Sannagerasehambu (Kannada). The word “Gymnema” is derived from a Hindu word “Gur-mar” meaning “destroyer of sugar” and it is believed that it might neutralize the excess of sugar present in the body in Diabetes mellitus (Saneja et al., 2010). From the leaves of an Indian plant, Gymnema sylvestre,
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ISSN: 2231-010X Research Article
* Corresponding Author Email: [email protected] Contact: +91-9865866168 Received on: 29-10-2012 Revised on: 27-11-2012 Accepted on: 03-12-2012
Sivakumar and Bharathy G (2012) Int. J. Res. Phytochem. Pharmacol., 2(4), 164-170
Imoto and associates isolated a novel peptide which suppresses sweet taste responses of the gustatory fi-bers in the rat and mouse (Imoto et al., 1991). This peptide, given the name of gurmarin, comprises 35 amino acid residues. Incidentally the leaves of this plant have long been known to contain sweet taste suppressing glycosides, gymnemic acids (Yoshie et al., 1994).
Table 1: Energy values of docked complex with the
help of Swiss PDB Viewer
S. No. Docked Complex E (KJ/Mol)
1. Gurt1m2 complex -15682.338
2. Gymt1m2 complex -15682.338
3. Gurt1m3 complex -16442.779
4. Gymt1m3 complex -16442.779
A wide variety of chemically diverse compounds taste sweet including natural sugars, such as glucose, fruc-tose, sucrose, and sugar alcohols. Non-carbohydrate
sweet-tasting substances include several chemically distinct groups of small molecule artificial sweeteners, such as sucralose, saccharin, cyclamate, and acesul-fame K, as well as dipeptide sweeteners, such as aspar-tame and neotame. Sweet-tasting proteins have been found as naturally occurring molecules from plants (or rarely from animals), including thaumatin, monellin, mabinlin, brazzein, egg white lysozyme, and curcu-lin/neoculin (Assadi-Porter et al., 2010). In addition, the glycoprotein riboflavin-binding protein from chick-en egg, a pure sweet antagonist protein, has been re-ported to inhibit specifically the taste of sweet protein but not carbohydrates or artificial sweeteners (Maeha-shi et al., 2007). Interestingly, sweet compounds have a wide variety of structural features as evidenced by the vastly different chemical structures of sugars, ar-tificial sweetener, sweet proteins, and so on. Previous studies have indicated that the sweet taste receptor has multiple ligand binding sites. The Taste type 1 Re-ceptors available in three isofroms namely Taste type 1 Receptor 1 (T1R1), Taste type 1 Receptor 2 (T1R2) and
Taste type 1 Receptor 3 (T1R3) (Nelson et al., 2001). For example, aspartame is recognized at the extracellu-lar domain of human T1R2 (hT1R2), while cyclamate is received at the transmembrane domain of human T1R3 (hT1R3). Sweet proteins such as monellin, brazze-in, and neoculin have been shown to interact with the extracellular domain of hT1R3 (Imada et al., 2010). Psychophysically, sweet and bitter have long been con-sidered separate taste qualities, evident already to the newborn human. The identification of different recep-tors for sweet and bitter located on separate cells of the taste buds substantiated this separation (Wang et al., 2009).
The objectives of the present study were to do the se-quence analysis of taste receptors and docking studies with antidiabetic agents, gurmarin and gymnemic acid, of Gymnema sylvestre. The major bioactive constitu-ents of Gymnema sylvestre are a group of oleanane type triterpenoid saponins known as gymnemic acids. Gymnena sylvestre therapy not only produced blood
glucose homesostasis but also increased the activities of the enzyme affording the utilization of glucose by insulin dependent pathways. The sweet taste response is mediated by taste cell surface receptors that utilize the adenylate cyclase system as secondary messenger system. Gymnema sylvestre acts on two sites: First, the taste buds in the oral cavity; second, the absorptive surface to the intestines. The structure of those taste buds, which detect sugar in the mouth, is similar to the structure of the tissue that absorbs sugar in the intes-tine. The important active ingredient in Gymnema syl-vestre is an organic acid called “Gymnemic acid”. The gymnemic acid is made up of molecules whose atom arrangement is similar to that of glucose molecules. Those molecules fill the receptor location on the taste buds for a period of one to two hours, thereby pre-venting the buds from being activated by any sugar molecules present in the food. Similarly, the glucose-like molecules in the gymnemic acid fill the receptor locations in the absorptive external layers of the intes-tine, thereby preventing the intestine from absorbing
Table 2: Interacting aminoacids of taste recepotrs with ligands
S. No.
Docked Complex
Interacting Amino acid residues No. of Amino acid residues
the sugar molecules. Gymnema leaf extract, notably the peptide ‘Gurmarin’, has been found to interfere with the ability of the taste buds on the tongue to taste sweet and bitter. G. Sylvestre helps to promote weight loss possibly through its ability to reduce cravings for sweets and control blood sugar levels. Gurmarin is a polypeptide isolated from the leaves that consists of 35 amino acid residues, including three intramolecular disulfide bonds (Kamei, 1992). It has been reported that the gurmarin peptide block the ability to taste sweet or bitter flavors and thus reduces sweet cravings (Saneja et al., 2010).
Figure 1: Three dimensional structure of gurmarin and
gymnemic acid
Figure 2: dimensional structure of taste receptors
T1R2 and T1R3
In mammals, sweet taste perception is mediated by the heterodimeric G-protein-coupled receptor, T1R2/T1R3. An interesting characteristic of this sweet taste recep-tor is that it has multiple ligand binding sites (Imada et al., 2010). Although there have been several studies on agonists of sweet taste receptors, little is known about antagonists of these receptors. Thus present study designed to investigate interaction domains of T1R2/T1R3 with its antagonists such as gurmarin and gymnemic acid.
Generally, the oral hypoglycemic agents are antagon-ists of taste receptors (T1R2/T1R3) and work by reduc-ing the sugar intake, thus promoting glucose control in peripheral tissues and have no effect on insulin secre-tion. Thus, these two receptors are chosen for the present study as the targets to develop dual antagonis-tic ligands through molecular docking. In this study, tertiary structure of the T1R2 and T1R3 predicted using EsyPred Homology modeling server. In addition, inte-raction sites for gurmarin and gymnemic acid to T1R2 and T1R3 were also identified with the help of Pacth-Dock docking tool.
Figure 3: Docking structure of taste receptors T1R2
and T1R3 with the phytocompounds
METHODOLOGY
Bioinformatics tools
In-silico translation was carried out using ExPASy Trans-late tool. Tertiary structure of taste receptors were predicted by EsyPred homology server. PatchDock server was used as docking tool. Swiss PDB Viewer tool was used to predict energy of docked complexes. DS ViewerLite 4.1 tool was used for ligand interaction site prediction.
Medicinal compounds
Sivakumar and Bharathy G (2012) Int. J. Res. Phytochem. Pharmacol., 2(4), 164-170
Gurmarin (PDB ID: 1GUR) and gymnemic acid (Pub-Chem ID: 11953919) are selected as antagonist for taste receptors (T1R2/T1R3). Three dimensional struc-tures of these ligands were retrieved from PDB and PubChem databases respectively.
Figure 4: Binding pockets of taste receptors T1R2 and
T1R3 with its phytocompounds
Human Taste receptors (T1R2/T1R3)
The nucleotide sequences of taste receptors type 1 member 2 and taste receptor type 1 member 3 were obtained from the Genbank database of NCBI with ID NM_152232.1 and NM_152228.1 respectively. ExPASy translate tool was a tool, which allows the translation of a nucleotide (DNA/RNA) sequence to a protein se-quence. It was used to translate nucleotide sequence to protein sequence. The tool used for docking was PatchDock. Energy Minimization studies were carried out using Swiss PDB Viewer tool. Interaction region between docked receptor and ligands was determined using ViewerLite 4.1 tool.
RESULTS
The tertiary structure of phytocompounds namely gurmarin and gymnemic acid were depicted in figure 1. The protein structure of the two receptors namely T1R2 and T1R3 were predicted using EsyPred Server and are shown in figure 2. The taste receptors T1R2 and T1R3 belong to the same class of G protein–coupled receptors (GPCRs) as metabotropic glutamate receptors (MGR1) and are expected to share their 3D structure. While docking, both the two phytocom-pounds were made interactions with taste receptors (T1R2/T1R3). The docking interaction between the taste receptors (T1R2/T1R3) with the compounds namely gurmarin and gymnemic acid are presented in figure 3. The two compounds were docked with taste receptors (T1R2/T1R3) in the same pocket irrespective of the receptors but gurmarin and gymnemic acid docked to different pockets on a same receptor. In these results, the aminoacids were displayed in sec-ondary structure form. The ligands are represented in CPK model. The details of docking interaction between
compounds and the receptor are presented in figure 4 and the energy values were shown in Table-1. The lists of interacting amino acid residues present in the bind-ing pocket prediction were listed in the Table-2.
DISCUSSION
The broad scope of anti-diabetic therapy is to restrict blood glucose control, by controlling fasting glucose levels and by controlling elevations in postprandial blood glucose
in which it adapts some definite mechan-
ism. Traditional medicine plays an important role in the health care of human population where 80% of the world population depends on herbal medication. Hence, it is more essential to develop more targeted and effective ligands against diabetes. In-silico drug screening is an effective alternative for identification of lead compounds. Lead compounds could be identified and tested using molecular docking for their effective-ness against major molecules of interest for diabetes diseases. A variety of computational factors are used to identify novel compounds (Pitchai et al., 2012).
The retrieved nucleotide sequences of T1R2 and T1R3 taste receptors were translated to amino acid se-quence using ExPASy translate tool. The translated amino acid residues present in T1R2 are 805 residues and in T1R3 are 763 residues. The translated se-quences were submitted to EsyPred Server for tertiary structure prediction. The predicted structure of T1R2 contain amino acid residues from 25
th residue to 556
th
residue whereas in case of T1R3 1st
residue to 479th
residue. The predicted structures were depicted in fig-ure 2. The predicted structures have mainly two do-mains of taste receptors namely amino terminal do-main (ATD) and Cystein rich domain (CRD). The only portion of the receptor that was modeled in the case of T1R3 is the extracellular ligand binding domain (LB1 and LB2) in Walters’ (2002) study. The homology model of hT1R2/hT1R3 VFTM (closed-open/Active form) was constructed with the Modeller program (Liu et al., 2011). Homology models of the T1R2 VFT domain were constructed with the program Homology (Accelrys) by using available structures from the Protein Data Bank: 1EWK, 1EWT, 1EWV, and 3KS9 of the metabotropic glutamate receptors (mGluR1); 2E4U, 2E4V, 2E4W, 2E4X, and 2E4Y of mGluR3; and 2E4Z of mGluR7 (Zhang et al., 2010). The ATD, CRD and transmembrane helix domain (TMD) of T1R2 and T1R3 have been modeled based on the crystal structures of metabotropic gluta-mate receptor type 1, tumor necrosis factor receptor, and bovine rhodopsin, respectively. The CRD is a ~70 amino acids domain with nine highly conserved cyste-ines, which bridges the ATD and TMD of the mGluR (Cui et al., 2006).
Taste transduction for a variety of sweet stimuli de-pends upon the presence of the T1r3 receptor protein, which combines with a related protein in the T1r family of receptors (T1r2) to form a functional sweet taste receptor (Brasser et al., 2010). An automatic molecular
Sivakumar and Bharathy G (2012) Int. J. Res. Phytochem. Pharmacol., 2(4), 164-170
docking program, AutoDock was used for the docking studies of T1R2 and T1R3 receptors with their ligands (Liu et al., 2011). Ligands were introduced into the T1R2 and T1R3 receptor models by using the program Biodock (BioPredict) (Zhang et al., 2010). In the present study, tertiary structure of heterodimeric G-protein-coupled receptors, T1R2/T1R3 receptors were success-fully modeled using EsyPred server and they were uti-lized for docking studies with gurmarin and gymnemic acid. Several compounds such as lactisole and gym-nemic acid and ions such as Zn
2+ are known to repress
the intensity of sweet taste according to human sen-sory evaluation (Imada et al., 2010). However, there is currently no support for such a complex interaction studies. The data presented here provide useful con-straints for future modeling and prediction of sweet protein–sweet receptor interactions.
Calculated binding energies and ligand-bound com-plexes for the ligands were examined carefully. In the present study the novel gurmarin and gymnemic acid, are interacted with T1R3 receptor with low docking energy, and high energy score with T1R2 receptor as shown in table 1. Lactisole inhibits the hT1R2/hT1R3 human sweet taste receptor by binding to the TMD of hT1R3 (Jiang et al., 2005). Both the phytocompounds found to interact to the ATD domain. Lactisole, a broad-acting sweet antagonist, suppresses the sweet taste of sugars, protein sweeteners, and artificial swee-teners. This shows that these compounds must possess an inhibiting effect on with T1R3 receptor. Thus these 2 compounds could also be considered as taste recep-tor ligands and could be developed in to drugs for the treatment of type-2 diabetes. Taste qualities, such as sweet, sour, salty, and bitter, have been used as de-scriptors of taste since ancient times. Gymnemic acids are a group of related triterpenes that suppress or ab-olish the sweetness of all known sweeteners. In hu-mans gymnemic acids suppress sweet in every tastant (Hellekant et al., 1998). Another interesting issue that has not yet received an explanation is the mechanism of action of substances that can suppress sweet taste, in particular gymnemic acid (Temussi, 2007). Thus, the present docking study provides answer for the role of gymnemic acid for suppression of sweet taste.
Substances that selectively modify specific physiologi-cal functions have proven useful for exploring the neural representation of sensory information. For taste, lingual application of gurmarin, a protein isolated from the plant Gymnema sylvestre, significantly atte-nuates integrated chorda tympani (CT) nerve res-ponses to sweet-tasting substances in rats and mice. Additionally, palatal gurmarin treatment suppresses integrated responses to sugars, sodium saccharin, and sweet-tasting amino acids recorded from the greater superficial petrosal (GSP) nerve in rats. Advances in molecular biology suggest the existence of a single mammalian receptor for sweets, T1R2/T1R3, as this candidate responded to all sweet taste stimuli tested
(Lemon et al., 2003). The docking interaction of the phytocompounds namely novel gurmarin and gym-nemic acid with both taste receptors indicates that these two compounds could posses an efficacy to inhi-bit the sugar consumption. This confirms that these two compounds could be considered for drug design and development for the treatment of both type-1 and type-2 diabetes after in- vitro, in- vivo and clinical stu-dies.
Molecular docking of tastants to binding sites other than the assumed active (orthosteric) site allows the identification of putative allosteric sites. For T1R2 and T1R3, Li et al., refered as ‘‘active’’ or ‘‘orthosteric’’ to the site corresponding to where glutamate is found to be bound in the crystallographic structure of the close-ly relatedmGR1. Aspartame binds preferentially and with strong affinity to the active site in human and mouse T1R3. For human T1R2, the allosteric site is pre-ferable in the active-open conformation, whereas the active site is better for binding in the active close con-formation with K60-E63-W341-P348-R352-Q355 ami-noacid residues. There are 22 residues directly involved in binding in the active site of hT1R2 model. These are: Y103, D142, N143, S144, S165, A166, I167, S168, Y215, R270, V272, V274, F275, S301, E302, S303, A305, T326, R378, L379, S380, and R383 (Li et al., 2011).
Predicted binding site for gurmarin and gymnemic acid with taste receptors are represented in the Table 2. Docking studies of Walters (2002) show that proposed active conformations of the high-potency sweeteners neotame, superaspartame, and SC-45647 could inte-ract favorably in the binding site of T1R3 receptor, forming ion pairs or ionic hydrogen bonds with His-163, Glu-318, and His-407, in addition to hydrophobic interactions with numerous nonpolar side-chains. Gurmarin acts as sweet response inhibitor by binding to taste receptors (Ninomiya et al., 1997; Harada and Kasahara, 2000). The T1R taste receptors, like other type 3 G-protein-coupled receptors (GPCRs), have a large amino terminal extracellular domain. Type 3 GPCRs typically function as dimers, but each monomer can independently bind ligand. Brazzein binds to T1R2 with binding pocket comprised off E61, E63, D278 and D307 (Jiang et al., 2005a). Each subunit has a large ATD or Venus fly trap (VFT) domain linked by an extracellu-lar cysteine-rich domain (CRD) to a seven transmem-brane helical domain (TMD). hT1R2–hT1R3 has mul-tiple ligand binding sites for these various sweeteners. For example, the ATD of hT1R2 is responsible for bind-ing to aspartame and sugar derivatives. Neoculin binds the ATD of hT1R3. In contrast, cyclamate and neohes-peridin dihydrochalcone (NHDC) bind the TMD of hT1R3 as agonists, whereas this region also serves as the allosteric binding site for saccharin and lactisole as antagonists. Several ligand-binding sites were pro-posed by a molecular modeling–based docking simula-tion for the sweet taste receptor. ATD or VFT domain of hT1R2 contains ten amino acid residues namely
Sivakumar and Bharathy G (2012) Int. J. Res. Phytochem. Pharmacol., 2(4), 164-170
D142, D307, E302, Y103, P277, D278, S144, S165, E382, R383. This pocket preferably bound by peptide deriva-tives (Masuda et al., 2012). D535 of hT1R3 of CRD is essential for binding to brazzein protein (Assadi-Porter et al., 2010a).
A537 and F540 within CRD region of hT1R3 were shown to be critical for responsiveness to brazzein. H641A, F778A, Q637E, S640V, A733V and V779A resi-dues in the TM domain of hT1R3 are responsible for lactisole’s inhibitory effect. It is clear that lactisole shares the same pocket with cyclamate and interacts with H641 and R723 through salt bridges and hydrogen bond interactions, while F778, L782, and L644 produce hydrophobic interactions (Cui et al., 2006). R217 of T1R2 and R220 of T1R3 receptors are essential for its interaction with natural sweeteners like brazzein and monelin etc., (Assadi-Porter et al., 2010). A careful re-view of current scientific literature clearly demonstrat-ed that none of the studies were carried out with re-spect to the interaction of gurmarin and gymnemic acid to taste receptors. The present study, supporting the view that gurmarin blocks the sweet taste sensa-tion at the level of reception (Yoshie et al., 1994), may hopefully be first to visualize the binding site of the sweet taste receptor to gurmarin.
Predicted binding site for gurmarin and gymnemic acid with taste receptors are represented in the Table 2. There are 28 and 24 amino acid residues were partici-pated in the interaction of gurmarin and gymnemic acid with taste receptor T1R2 respectively. Similarly there are 37 and 19 amino acid residues participated in the interaction of gurmarin and gymnemic acid with taste receptor T1R3 respectively. Thus new binding pockets were identified in the amino terminal domain (ATD).
Positive allosteric modulators of the human sweet taste receptor have been developed as a new way of reducing dietary sugar intake (Zhang et al., 2010). Both phytocompounds gurmarin and gymnemic acidare docked with taste receptors and hypothesized that they could be able inhibit the taste signaling pathway. Hence it is clear that these two compounds could inhi-bit taste receptors there by reducing sugar intake and in turn reduces blood glucose level. Hence it is con-cluded that, these two compounds namely gurmarin and gymnemic acid, could be developed into the po-tent oral drugs for the treatment of diabetes.
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