Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus
Abstract
Background
Diabetes mellitus is a chronic metabolic disorder caused due toinsulin deficiency or insulin resistance. In Type 1 diabetesautoimmune destruction of the beta cells of pancreas leads toinsulin deficiency. Type 2 diabetes involves insulin resistance ordecreased insulin secretion. Insulin is essential for maintainingblood glucose and regulating carbohydrate metabolism. Insulinreceptor (IR) is a tetrameric protein consisting of twoextracellular alpha subunits and two transmembrane betasubunits [1].The binding of insulin to alpha subunit of IR causesconformational changes in the receptor leading to the activationof tyrosine kinase beta subunit. The activated IR has the abilityto autophosphorylate and phosphorylate intracellularsubstrates that are essential for initiating other cellularresponses of insulin [2–4].These events lead to the activation ofdownstream signaling molecules that participate in the insulinsignaling pathway [5]. Insulin signaling, including activation ofIR tyrosine kinase activity, is impaired in most patients withdiabetes mellitus. This resistance to insulin then leads tohyperglycemia and other metabolic abnormalities of the disease[6,7]. Hence, compounds that augment insulin receptortyrosine kinase activity would be useful in the treatment ofdiabetes mellitus.
Bio-flavonoids are well-known for their multi-directionalbiological activities including anti-diabetic efficacy. Theflavonoids, ubiquitous in plants, are the largest class ofpolyphenols, with a common structure of diphenylpropanes(C6-C3-C6), consisting of two aromatic rings linked throughthree carbons. The six major subclasses of flavonoids includethe flavones (e.g. apigenin, luteolin), flavonols (e.g. quercetin,myricetin), flavanones (e.g. naringenin and hesperitin),flavanols (or catechins) (e.g. epicatechin and gallocatechin),anthocyanidins (e.g. cyanidin and pelargonidin) andisoflavones (e.g. genistein and daidezin) [8]. It has beendemonstrated that flavonoids can act per se as insulinsecretagogues or insulin mimetics, by influencing thepleiotropic mechanisms [9].
Musa sapientum commonly known as ‘banana’ is widely used inIndian folk medicine for the treatment of diabetes mellitus [10].The available literature confirms that flavonoids are present inbanana flowers [11].The chloroform, water and ethanol extractof Musa sapientum flowers were found to exhibit hypoglycaemicactivity in alloxan diabetic rat [12]. Intraperitonealadministration of prunin (naringenin 7-O-β-D-glucoside)produces a significant hypoglycemic effect in diabetic rats. [13].Chronic treatment with hesperitin and naringenin was found tolower the blood glucose level of db/db mice [14]. Bananaflower extracts are able to promote glucose uptake into the cells,which could be beneficial in diabetes mellitus. It can behypothesized that consumption of nutraceutical-rich extract ofbanana flower could replace some amount of insulin beingtaken for diabetes mellitus [15]. Although insulin has becomeone of the most important therapeutic agents known tomedicine, there is a continuing effort to find insulin substitutes,secretagogues, or sensitizers from synthetic or plant sources forthe treatment of diabetes mellitus [16]. In the present study wehave attempted to dock the banana flower flavonoids withinsulin receptor tyrosine kinase to understand the interactions.This insilico approach can be further investigated to generatemore effective and potential insulin receptor tyrosine kinaseactivators through ligand based drug designing approaches.
Methodology
Preparation of Protein and ligands:
The three dimensional structure of Insulin Receptor ofHomosapiens was obtained from Protein Database (PDB: ID1IR3) [17] .The list of Flavonoids and other compounds presentin banana flower were obtained from USDA database [18]. TheCID files of the ligands were obtained from NCBI Pubchem[19]. The list of the ligands present in the banana flower initiallyused are Leucocyaniin (CID155206), Cyanidin (CID128861),Malvidin (CID159287), Pelargonidin(CID440832),Peonidin(CID441773), Hesperetin (CID 72281), Naringenin (CID932), Hesperetin Triacetate (CID 457809), HesperetinDihydrochalcone ( CID 147608 ), Naringenin Pelargonidin ( CID439246), Naringenin Flavanone (CID 25244584), Hesperetin(Anion) (CID 49859576). The energy minimization of theprepared ligand was carried out with Swiss-PDB Viewer V.4.02.[20] Molinspiration [21] an online tool, was used to performQSAR studies in order to identify important molecularproperties (logP, polar surface area, number of hydrogen bonddonors and acceptors and others), as well as prediction ofbioactivity score for the most important drug targets. Variousproperties of ligands such as logP, molecular weight, H bonddonors, H bond acceptors, number of atoms were obtained.Lipinski’s Rule of Five was then applied to select probableligands. Those compounds that had more than one violation (i.eLeucocyanidin) were eliminated. Active site analysis of theInsulin Receptor was carried out using Swiss PDB Viewer(SPDBV) V.4.02 and from the PDB ligand Explorer.
Molecular Docking Studies
Binding mode and selectivity of Insulin receptor tyrosinekinase with individual flavonoids, was studied by dockingsoftwares like Autodock 4.0 [22], Autodock VINA [23].Autodock 4.0 uses Monte Carlo simulated annealing andLamarckian genetic algorithm (LGA) to create a set of possibleconformations. LGA is used as a global optimizer and energyminimization as a local search method. Possible orientations areevaluated with AMBER force field model in conjunction withfree energy scoring functions and a large set of protein-Ligandcomplexes with known protein-Ligand constants. The newestversion 4 contains side chain flexibility. Hydrogen atoms,Kollman charges were added. The grid was centered in theactive site region which involves all functional amino acidresidues. Grid maps were generated using the AutogridProgram. Docking was performed using the Lamarckian geneticalgorithm. In the present study docking was performed bycreating an initial population of 150 individuals, 5 randomtorsions to each of the 150 individuals, Lamarckian GeneticAlgorithm (LGA), was implemented with a maximum of2500000 energy evaluations. AutoDock Vina is a new opensourceprogram for drug discovery, molecular docking andvirtual screening, offering multi-core capability, highperformance and enhanced accuracy and ease of use. AutoDockVina significantly improves the average accuracy of the bindingmode predictions compared to AutoDock 4.0.
Discussion
We analyzed the interactions of the banana flower flavonoidswith insulin receptor tyrosine kinase and observed that thesecompounds can activate the kinase domain since thephosphorylated tyrosines were bound to the residues located inthe activated loop of the protein.
Structural Aspects and Interactions of IR Tyrosine kinase:
Insulin receptor is a tyrosine protein kinase that undergoesautophosphorylation upon insulin binding. The activatedreceptor phosphorylates substrate proteins on multiple Tyrresidues to propagate insulin signal transduction and insulinaction [24]. Three important structural regions are identified inthe intracellular part of the beta subunit. They arejuxtamembrane (JM) region, the kinase region, and thecarboxyl-terminal (CT) region. The JM region (Arg940-Leu987)contains one autophosphorylation site; the CT region (Leu1245-Ser1343) contains two autophosphorylation sites. Theintracellular portion of the insulin receptor, contain functionalgroups that bind to the effector molecules. Insulin receptorsubstrates (IRS-1, -2, -3 and -4) are the targets for insulinreceptor kinase. The JM region serves as a binding site for IRS-1.Phosphorylation of the juxtamembrane of the insulin receptormediates insulin's metabolic functions. The catalytic loop isformed by residues 1130-1137 and the activation loop comprisesof residues 1150-1171 [25]. Extensive studies on activated andinactive IR tyrosine kinases indicate that in the activated statethe activation loop is tris-phosphorylated and moves out of theactive site. The phosphorylated tyrosines become hydrogenbondedto residues in the activation loop [26].
The molecular properties of ligands such as logP, molecularweight, H bond donors, H bond acceptors, number of atomswere obtained using Molinspiration. This tool also gives theanalysis with respect to number of violations ie deviation fromLipinski's rule. Lipinski's Rule of Five was then applied toselect probable ligands. Those compounds that had more thanone violation (i.e Leucocyanidin) were eliminated Table 1 (seesupplementary material). Active site analysis of the InsulinReceptor was carried out using Swiss PDB Viewer (SPDBV)V.4.02 and from the PDB ligand Explorer. The active siteconsists of residues: SER 1006, LYS 1030, GLU 1077, ASP 1083,ASN 1137, and ASP 1150, MET 1079. Docking results indicatedthat majority of the compounds bind to insulin receptortyrosine kinase Table 2 (see supplementary material). Thebinding modes and interactions of each ligand with thefunctional residues of insulin receptor tyrosine kinase wereanalyzed in detail by visually inspecting the docked complexesusing PMV 1.5.2.From (Figure 1) it is evident that all the ligandsinteract with most of the residues in the binding pocket.Cyanidin binding energy value from Autodock 4.0 was foundto be -5.18 kcal/mol, Autodock Vina it was found to be -7.9kcal/mol. This compound was found to interact with MET1139, ASN 1137, ARG 1136, ASP 1083, ASP 1150, LYS 1030, SER1006 and PHE 1007. Hesperetin Dihydrochalcone bindingenergy value from Autodock 4.0 was found to be -4.78kcal/mol, Autodock Vina it was found to be -7.0 kcal/mol. Thiscompound was found to interact with ARG 1136, ASN 1137,ASP 1150 and SER 1006. Pelargonidin binding energy fromAutodock 4.0 was -4.93 kcal/mol, Autodock Vina it was foundto be -7.7 kcal/mol. This compound was found to interact withASN 1137, LYS 1030 and SER 1006. Peonidin binding energyfrom Autodock 4.0 was found to be -4.80 kcal/mol, AutodockVina it was found to be -7.7 kcal/mol. This compound wasfound to interact with ASN 1137, LYS 1030 and ASP 1150.
Hesperetin triacetate binding energy value from Autodock 4.0was found to be -6.08 kcal/mol, Autodock Vina it was found tobe -8.2 kcal/mol. This compound was found to interact withSER 1006, ASN 1137, and LYS 1030. Malvidin binding energyfrom Autodock 4.0 was found to be -5.26 kcal/mol, AutodockVina it was found to be -7.3 kcal/mol. This compound wasfound to interact with ASP 1150, LYS 1030 and SER 1006.Naringenin binding energy value from Autodock 4.0 was foundto be -6.02 kcal/mol, Autodock Vina it was found to be -8.1kcal/mol. This compound interacted with ASP 1150, ASP 1083,and LYS 1030. Naringenin pelargonidin binding energy valuefrom Autodock 4.0 was found to be -6.02 kcal/mol, AutodockVina it was found to be -8.1 kcal/mol. This compound wasfound to interact with ASP 1150 and LYS 1030 .Naringeninflavanone binding energy value from Autodock 4.0 was foundto be -5.82 kcal/mol, Autodock Vina it was found to be -8.0kcal/mol. This compound was found to interact with ASN1137, ASP 1150, LYS 1030 and SER 1006. Hesperetin bindingenergy value from Autodock 4.0 was found to be -5.78kcal/mol, Autodock Vina it was found to be -8.4 kcal/mol. Thiscompound was found to interact with ASN 1137, ASP 1150 andLYS 1030 and SER 1006.
Conclusions
The docked conformations of all the flavonoids indicate thatthe phosphorylated tyrosines namely PTR -1158, PTR -1162and PTR – 1163 remain in the activation loop .PTR -1158 wasbound to ASP 1156, PTR-1162 was hydrogen bonded to THR1160 ,ARG 1164 and PTR-1163 was bound to GLY 1166 all ofwhich are a part of the activation loop. This structuralorientation clearly suggests that insulin receptor tyrosinekinase in its active state. Hence it can be concluded thattheses flavonoids can be considered as activators of InsulinReceptor Tyrosine kinase. Based on Autodock bindingenergies it can be hypothesized that Hesperitintriacetate,Naringenin,Naringenin pelargonidin andnaringinen flavonone are potent activators of IR tyrosinekinase.
Supplementary material
Footnotes
Citation:Ganugapati et al, Bioinformation 8(5): 216-220 (2012)
Acknowledgments
We wish to thank the management of Sreenidhi Institute ofScience and Technology for providing the necessary facilitiesto carryout this work.
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