interaction of glucagon g-protein coupled receptor with known

Research ArticleInteraction of Glucagon G-Protein CoupledReceptor with Known Natural Antidiabetic CompoundsMultiscoring In Silico Approach

M H Baig1 K Ahmad2 Q Hasan1 M K A Khan3 N S Rao4 M A Kamal56 and I Choi1

1School of Biotechnology Yeungnam University Gyeongsan 712749 Republic of Korea2Department of Biosciences Integral University Lucknow 226026 India3Department of Bioengineering Integral University Lucknow 226026 India4School of Computational and Integrative Sciences Jawaharlal Nehru University New Delhi 110067 India5King Fahd Medical Research Center King Abdulaziz University PO Box 80216 Jeddah 21589 Saudi Arabia6Enzymoics 7 Peterlee Place Hebersham NSW 2770 Australia

Correspondence should be addressed to M H Baig mohdhassanbaiggmailcom and I Choi inhochoiynuackr

Received 24 April 2015 Accepted 15 June 2015

Academic Editor Shreesh Ojha

Copyright copy 2015 M H Baig et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Glucagon receptor (GCGR) is a secretin-like (class B) family of G-protein coupled receptors (GPCRs) in humans that plays animportant role in elevating the glucose concentration in blood and has thus become one of the promising therapeutic targets fortreatment of type 2 diabetes mellitus GCGR based inhibitors for the treatment of type 2 diabetes are either glucagon neutralizersor small molecular antagonists Management of diabetes without any side effects is still a challenge to the medical system and thesearch for a new and effective natural GCGR antagonist is an important area for the treatment of type 2 diabetes In the present studya number of natural compounds containing antidiabetic properties were selected from the literature and their binding potentialagainst GCGR was determined using molecular docking and other in silico approaches Among all selected natural compoundscurcumin was found to be the most effective compound against GCGR followed by amorfrutin 1 and 4-hydroxyderricin Thesecompounds were rescored to confirm the accuracy of binding using another scoring function (119909-score)The final conclusions weredrawn based on the results obtained from the GOLD and 119909-score Further experiments were conducted to identify the atomic levelinteractions of selected compounds with GCGR

1 Introduction

Diabetesmellitus is a groupofmetabolic diseases inwhich thehuman body is unable to utilize and store available glucosewhich results in the blood glucose level rising above thethreshold level Globally diabetes has affected 347 millionpeople to date [1] The most common symptoms of this dis-ease include weight loss polyuria polydipsia and polypha-gia [2] Whenever glucose levels decrease in the blood(such as under fasting situations) glucagon-a 29-amino acidpeptidal hormone is secreted by pancreatic 120572-cells whichenhances the blood glucose level [3] Increased glucagonin the blood leads to the promotion of glycogenolysis and

gluconeogenesis in the liver while the insulin inhibitoryeffect of these processes is attenuated ultimately enhancingthe blood glucose level [4] The combined action of insulinand glucagon is required to maintain glucose homeostasisinside the body [5 6] Therefore two strategies have beenapplied to control diabetic hyperglycemia to date reducingcirculating glucagon levels and inhibiting glucagon mediatedeffects in target body tissues and cells Several studies havedemonstrated significant blood glucose lowering effects indiabetic animalmodels through application of potent peptideantagonists [7 8] and immunoneutralization of glucagonin diabetic animals has been shown to reduce glucagon-stimulated hyperglycemia [9 10]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 497253 6 pageshttpdxdoiorg1011552015497253

2 Evidence-Based Complementary and Alternative Medicine

The glucagon receptor (GCGR) is a 62 kDa proteinactivated by glucagon that is a member of G-protein coupledreceptor (GPCR) superfamily [11] In humans the glucagonreceptor is encoded by the GCGR gene [12 13] Glucagonreceptors are primarily expressed in the liver and kidney withlesser amounts found in the heart adipose tissue spleen thy-mus adrenal glands pancreas cerebral cortex and gastroin-testinal tract By binding to GCGR glucagon sends a signalinside the cell which activates adenylyl cyclase leading tothe generation of high cAMP levels [14] In addition GCGRalso couples to an intracellular Ca2+-mediated pathway [15]GCGR activation leads to increase in metabolic processessuch as glycogenolysis and gluconeogenesis resulting inincreased glucose concentrations in hepatic cells and tissues[16 17]

Since GCGR plays an important role in elevating the glu-cose concentration in blood (glycemia) and there are manysmall-molecule inhibitors available for receptors of theGPCRfamily [18] it is a potent target for the development of small-molecule antagonistinhibitors A number of antagonistswith varying degrees of potency and structures have beenreported in recent years [19] GCGR based inhibitors for thetreatment of type 2 diabetes are either glucagon neutralizingantibodies [20 21] or small molecular antagonists [22ndash24]These compounds have been shown to effectively terminatethe GCGR action However concerns about safety toleranceand stimulation of adverse immune response when usingthese types of agents against GCGR for the treatment oftype 2 diabetes have led to investigations to identify drugsor compounds of natural origin to combat this problemIndeed GCGR antagonistinhibitors of natural origin maybe safe and favorable therapeutic agents for the treatmentof type 2 diabetes Accordingly it is important to search fornew and effective GCGR antagonists from natural sources[25]Therefore the present study was conducted to search fornatural antagonists against GCGR in silico All natural com-pounds selected in this study were collected from the avail-able literature and have been reported to have antidiabeticproperties [26ndash28] Additionally molecular docking studieshave been conducted to investigate the binding affinity ofall selected compounds The results were then reevaluatedusing two different scoring functions to confirm the accuracyof our results Overall the results presented herein enabledcalculation of the accessible surface area (ASA) of GCGR(uncomplexed) and its docked complex with the selectedcompounds for analysis of quantification of the packing ofresidues in GCGR before and after the binding of ligands

2 Methods

21 Preparation of Enzyme and Ligand for Docking The 3Dcrystal structure of human GCGR was retrieved from theRCSB protein databank (pdb ID 4L6R) (httpwwwrcsborgpdbexploreexploredostructureId=4L6R) [29] Beforeconducting the molecular docking calculations all watermolecules and other heteroatoms were removed A CharMm[30] force field was applied to the structure of GCGRfollowed by 1000-step energyminimization using the steepestdescent method The Distance-Dependent Dielectrics type

implicit solvent model was used to conduct the energyminimization step with the RMS gradient set to 01 A totalof 83 natural compounds were selected in this study Allnatural compounds used in this study were selected fromthe available literature The 3D structures of all naturalcompounds were extracted from the PubChem Compounddatabase A Cff force field [31] which is a general purposeclass II force field with good parameter coverage for manyorganicmolecules was applied to all the structures As class IIforce field it has additional cross terms in its potential energyfunction relative to other class I force fields

22 Molecular Docking Molecular docking was conductedto investigate the interaction of all the natural compoundsagainst GCGR Genetic Optimization for Ligand Docking50 (GOLD) [32] was used for docking of all the selectedcompounds against GCGR The annealing parameters wereset to 50 and 25 to evaluate van der Waals and hydrogenbonding docking respectivelyThe population size was set to100 with a selection pressure of 12The number of operationswas fixed to 100000 with 5 islands a niche size of 2migration value of 10 mutation value of 100 and crossoverof 100 The binding energies of docked molecules were alsocalculated using 119909-score [33] All molecular graphicsmaterialof docked complexes was prepared using Pymol

23 Accessible Surface Area Calculation Differences in theaccessible surface area (ASA) of the GCGR before andafter the binding of identified inhibitors were calculatedfor validation of effectiveness of these compounds usingNACCESS version 211 [34] The accessible surface area 119860of an atom is the area on the surface of a sphere of radius119877 on each point of which the center of a solvent moleculecan be placed in contact with this atom without penetratingany other atom of the molecule The radius 119877 is given by thesum of the van der Waalsrsquo radius of the atom and the selectedradius of the solvent molecule An approximation to this areais computed by this program using the following formula

Accessible surface area is

119860 = sum(

119877

radic1198772minus 1198852

119894

)sdot119863 sdot 119871119894 119863 =

Δ119885

2

+ Δ1015840119885 (1)

where 119871119894is the length of the arc drawn on a given section 119894119885

119894

is the perpendicular distance from the center of the sphere tothe section 119894Δ119885 is the spacing between the sections andΔ1015840119885is Δ1198852 or 119877 minus 119885

119894 whichever is smaller Summation is over

all of the arcs drawn for the given atom The accessibility isdefined simply as the accessible surface area divided by 41205871198772multiplied by 100

3 Results and Discussion

Glucagon G-protein coupled receptor class B GPCR hasbecome a promising therapeutic drug target for the treatmentof type 2 diabetes mellitus (T2DM) [35 36] Earlier stud-ies have reported that blockade of glucagon receptor gene(GCGR) activity is useful for the treatment of T2DM [25

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Residues involved in binding all the finally selected compounds against GCGR

Compounds Gold fitness score x-score (Kcalmol) Residues involvedHydrogen bonding Hydrophobic interaction

Curcumin 4853 minus835 Y149 I235 Y145 K187 V191 I194 D195 M231 I235 E362 F365 L386Amorfrutin 1 4218 minus837 K187 M231 Q232 I235 Y239 L307 V311 E362 V363 F3654-hydroxyderricin 3906 minus856 No H-bond Y145 K187 V191 I194 M231 I235 E362 F365 L386

35 36] However management of diabetes without any sideeffects is still a challenge to the medical system [37] This hasled to increasing demand for natural products with antidia-betic activity with fewer or no side effects Molecular dockingis considered to be an important tool for investigation of themode of interaction of ligands with the target and elucidationof the underlying binding mechanism [38 39] In this studywe determined the binding potential of several natural com-pounds with known antidiabetic properties against GCGRusing molecular docking and other in silico approaches Theprime objective of the present study was to identify thebinding potential of several natural antidiabetic compoundsagainst GCGR using the molecular docking approach Inthis regard we used an in silico approach to identify naturalcompounds with the potential for use in the treatment ofGCGR Additionally molecular docking simulation studieswere conducted to investigate possible binding modes of allselected natural compounds against GCGR Several plausiblebinding modes were detected and ranked based on their goldfitness score Moreover these compounds were rescored toconfirm the accuracy of binding using another scoring func-tion (119909-score)The final conclusions were drawn based on theresults obtained from GOLD and the 119909-score Curcumin aprincipal component of turmeric (Curcuma longa Linn) anda popular spice in Asian cuisine was found to be the mosteffective against GCGR (gold fitness score of 5353) followedby amorfrutin 1 widely available traditionalmedicine isolatedfrom licorice (Glycyrrhiza foetida) and 4-hydroxyderricinisolated from root ofA keiskei whichwere found to bindwithgold fitness scores of 4818 and 4406 respectively Rescoringof these docked results using 119909-score revealed that curcuminamorfrutin 1 and 4-hydroxyderricin bind within the activesite of GCGR with binding free energies of minus835 minus837 andminus856 kcalmol respectively Table 1 illustrates the bindingscore of the finally selected compounds against GCGR Thebinding mode of the selected inhibitors within the activesite of GCGR is shown in Figures 1ndash3 The results obtainedfrom both scoring functions were also found to be in goodagreementwith each otherThe scores obtainedusing all threefunctions are shown in the graph (Figure 4)

This study revealed that the binding of all natural com-pounds within the active site of GCGR is largely dominatedby hydrophobic interactions There were only three aminoacid residues of GCGR (K187 Y149 and I235) found toparticipate in generation of hydrogen bonds with curcuminand amorfrutin 1 V191 I194 M231 I235 E362 and F365

PHE-365GLU-362

LEU-386

TYR-145

TYR-149

ILE-235

GLY-234MET-231

LYS-187VAL-191ILE-194

Figure 1 Binding of curcumin within the active site of GCGR

LEU-386

TYR-145

PHE-365GLU-362

ILE-235LYS-187

VAL-191MET-231

ILE-194

Figure 2 Binding of 4-hydroxyderricin within the active site ofGCGR

were found to be common active site residues involved inthe proper accommodation of natural compounds withinthe active site of GCGR via hydrophobic contact The roleof these important active site residues has already beendiscussed in previous studies [25 40] Further experimentswere conducted to identify atomic level interactions of finallyselected compounds with GCGR and to quantify the packingof residues This is important to understanding of proteinstability and drug design Comparison of the accessiblesurface area for the uncomplexed protein and that complexedwith inhibitor provides amethod of assessing the goodness ofpacking of the residue in a protein structure or its importancein the binding of ligands [41] If a residue loses more than10 A2 of accessible surface area during transformation fromthe uncomplexed to the complexed state it is considered to be


PHE-365VAL-363

GLU-362

VAL-311LEU-307

TYR-239ILE-235

GLN-232

MET-231LYS-187

Figure 3 Binding of amorfrutin 1 within the active site of GCGR

0

10

20

30

40

50

60

Curcumin Amorfrutin 1

GFSx-score

minus10

minus20

4-hydroxyderricin

Figure 4 Comparison of both scoring functions used in this study

very actively involved in the interaction [42] Changes in theaccessible surface area of all residues involved in the bindingof compounds within the active site of GCGRwere calculatedusing NACCESS

Changes in accessible surface area (ΔASA) in A2 of theinteracting residues of GCGR (uncomplexed) and in complexwith curcumin 4-hydroxyderricin and amorfrutin 1 areshown in Tables 2 and 3 Changes in the total accessiblesurface area of GCGR before and after its interaction withthe selected compounds were also calculated (Table 2) Theresults revealed that the uncomplexedGCGR had a total ASAof 18930488 A2 which was reduced to 18637703 18661982and 18674038 A2 after its complex formationwith curcumin4-hydroxyderricin and amorfrutin 1 respectively This largechange in the accessible surface area of GCGR provides solidevidence of the effectiveness of these selected compoundsChanges in the accessible surface area (ASA) in responseto complex formation for each amino acid are shown in

Table 2 Total change in ASA of GCGR in uncomplexed andcomplexed form

Complexeduncomplexed Change in ASA (A2)4L6R (UC) 189304884L6R (complexed with curcumin) 186377034L6R (complexed with 4-hydroxyderricin) 186619824L6R (complexed with amorfrutin 1) 18674038

Tota

l ASA

Unc

ompl

exed

190001895018900188501880018750187001865018600185501850018450

Com

plex

ed w

itham

orfru

tin 1

Com

plex

ed w

ithcu

rcum

in

Com

plex

ed w

ith4-

hydr

oxyd

erric

in

Figure 5 Change in total ASA of GCGR (uncomplexed andcomplexed)

Table 3 and Figure 5 Many residues were found to have morethan 10 A2 of accessible surface area complex formation Forexample M231 had an ASA of 32642 A2 which decreasedto 9531 0767 and 9363 A2 after the binding of curcumin4-hydroxyderricin and amorfrutin 1 respectively Similarresults were observed in the case of other active site residues(L307 V311 E362 V363 F365 and L386) which undergo ahigh reduction inASAbefore and after binding of the selectednatural compounds This encompasses the small standardand large interface sizes as discussed by Conte et al [43]and thus represents a good sampling of the space of proteininterfaces

4 Conclusion

This study explored molecular interactions between GCGRand some well-known antidiabetic natural compoundsMolecular docking studies and their reevaluation usingthe 119909-score suggest that curcumin amorfrutin 1 and 4-hydroxyderricin have higher scores than other natural com-poundsThe large change in the accessible surface area of theamino acid residues involved in the interaction also explainsthe efficacy of the binding of these compounds Analysis ofASA further explores the important active site amino acid


Table 3 Change is ASA (A2) of important active site residues of GCGR

Complexeduncomplexed Y145 K187 V191 I194 M231 Q232 I235 L307 V311 E362 V363 F365 L3864L6R (UC) 32032 16624 14878 11591 32642 40005 25348 25793 32228 35789 45668 82247 483724L6R (complexed with curcumin) 439 2329 0 0439 9531 35199 0574 11298 12427 3194 45668 44311 59734L6R (complexed with 4-hydroxyderricin) 1771 2547 0 0006 0767 18509 0 10332 19146 745 45668 43348 159664L6R (complexed with amorfrutin 1) 32032 1797 6628 11591 9363 22045 0 1915 0657 0423 36381 41514 28608

residues Such information may also aid in future designof versatile GCGR-inhibitors Overall identification of thesenatural compounds may lead to design of a potent drug tocombat type 2 diabetes with minimal side effects

Abbreviations

GCGR Glucagon G-protein coupled receptorGPCR G-protein coupled receptorT2DM Type 2 diabetes mellitusGOLD Genetic Optimization for Ligand DockingASA Accessible surface areaCff Consistent force field

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by the National Research Founda-tion of Korea (NRF) grant funded by the Korean government(MSIP) (no 2014R1A2A2A01006324)

References

[1] G Danaei MM Finucane Y Lu et al ldquoNational regional andglobal trends in fasting plasma glucose and diabetes prevalencesince 1980 systematic analysis of health examination surveysand epidemiological studies with 370 country-years and 2sdot7million participantsrdquo The Lancet vol 378 no 9785 pp 31ndash402011

[2] D W Cooke and L Plotnick ldquoType 1 diabetes mellitus inpediatricsrdquo Pediatrics in Review vol 29 no 11 pp 374ndash3852008

[3] R Burcelin E B Katz and M J Charron ldquoMolecular andcellular aspects of the glucagon receptor role in diabetes andmetabolismrdquoDiabetes ampMetabolism vol 22 no 6 pp 373ndash3961996

[4] A Consoli ldquoRole of liver in pathophysiology of NIDDMrdquoDiabetes Care vol 15 no 3 pp 430ndash441 1992

[5] R H Unger ldquoRole of glucagon in the pathogenesis of diabetesthe status of the controversyrdquo Metabolism vol 27 no 11 pp1691ndash1709 1978

[6] W A Muller G R Faloona E Aguilar-Parada and R HUnger ldquoAbnormal alpha-cell function in diabetes response tocarbohydrate and protein ingestionrdquo The New England Journalof Medicine vol 283 no 3 pp 109ndash115 1970

[7] D G Johnson C U Goebel V J Hruby M D Bregmanand D Trivedi ldquoHyperglycemia of diabetic rats decreased by

a glucagon receptor antagonistrdquo Science vol 215 no 4536 pp1115ndash1116 1982

[8] C G Unson E M Gurzenda and R B Merrifield ldquoBiologicalactivities of des-His1[Glu9]glucagon amide a glucagon antago-nistrdquo Peptides vol 10 no 6 pp 1171ndash1177 1989

[9] C L Brand P N Jorgensen U Knigge et al ldquoRole of glucagonin maintenance of euglycemia in fed and fasted ratsrdquo AmericanJournal of Physiology Endocrinology and Metabolism vol 269no 3 part 1 pp E469ndashE477 1995

[10] C L Brand B Rolin P N Joslashrgensen I Svendsen J SKristensen and J J Holst ldquoImmunoneutralization of endoge-nous glucagon with monoclonal glucagon antibody normalizeshyperglycaemia in moderately streptozotocin-diabetic ratsrdquoDiabetologia vol 37 no 10 pp 985ndash993 1994

[11] K L Pierce R T Premont and R J Lefkowitz ldquoSeven-transmembrane receptorsrdquo Nature Reviews Molecular Cell Biol-ogy vol 3 no 9 pp 639ndash650 2002

[12] S Lok J L Kuijper L J Jelinek et al ldquoThe human glucagonreceptor encoding gene structure cDNA sequence and chro-mosomal localizationrdquo Gene vol 140 no 2 pp 203ndash209 1994

[13] S Menzel M Stoffel R Espinosa III A A Fernald M M leBeau and G I Bell ldquoLocalization of the glucagon receptor geneto human chromosome band 17q25rdquo Genomics vol 20 no 2pp 327ndash328 1994

[14] D J Drucker ldquoBiologic actions and therapeutic potential of theproglucagon-derived peptidesrdquo Nature Reviews Endocrinologyvol 1 no 1 pp 22ndash31 2005

[15] L H Hansen J Gromada P Bouchelouche et al ldquoGlucagon-mediated Ca2+ signaling in BHK cells expressing cloned humanglucagon receptorsrdquoAmerican Journal of PhysiologymdashCell Phys-iology vol 274 no 6 pp C1552ndashC1562 1998

[16] E Beale T Andreone S Koch M Granner and D GrannerldquoInsulin and glucagon regulate cytosolic phosphoenolpyruvatecarboxykinase (GTP) mRNA in rat liverrdquo Diabetes vol 33 no4 pp 328ndash332 1984

[17] J C Yoon P Puigserver G Chen et al ldquoControl of hepaticgluconeogenesis through the transcriptional coactivator PGC-1rdquo Nature vol 413 no 6852 pp 131ndash138 2001

[18] J Davey ldquoG-protein-coupled receptors new approaches tomaximise the impact of GPCRs in drug discoveryrdquo ExpertOpinion onTherapeutic Targets vol 8 no 2 pp 165ndash170 2004

[19] A Ling ldquoSmall-molecule glucagon receptor antagonistsrdquoDrugsof the Future vol 27 no 10 pp 987ndash993 2002

[20] K Tan D Tsiolakis and V Marks ldquoEffect of glucagon antibod-ies on plasma glucose insulin and somatostatin in the fastingand fed ratrdquo Diabetologia vol 28 no 7 pp 435ndash440 1985

[21] K W Sloop M D Michael and J S Moyers ldquoGlucagon as atarget for the treatment of type 2 diabetesrdquo Expert Opinion onTherapeutic Targets vol 9 no 3 pp 593ndash600 2005

[22] S A Qureshi M R Candelore D Xie et al ldquoA novelglucagon receptor antagonist inhibits glucagon-mediated bio-logical effectsrdquo Diabetes vol 53 no 12 pp 3267ndash3273 2004


[23] J Lau G Behrens U G Sidelmann et al ldquoNew beta-alaninederivatives are orally available glucagon receptor antagonistsrdquoJournal of Medicinal Chemistry vol 50 no 1 pp 113ndash128 2007

[24] R Liang L Abrardo E J Brady et al ldquoDesign and synthesisof conformationally constrained tri-substituted ureas as potentantagonists of the human glucagon receptorrdquo Bioorganic ampMedicinal Chemistry Letters vol 17 no 3 pp 587ndash592 2007

[25] S Grover J K Dhanjal S Goyal A Grover and D SundarldquoComputational identification of novel natural inhibitors ofglucagon receptor for checking type II diabetes mellitusrdquo BMCBioinformatics vol 15 supplement 16 p S13 2014

[26] C Coman O D Rugina and C Socaciu ldquoPlants and naturalcompounds with antidiabetic actionrdquo Notulae Botanicae HortiAgrobotanici Cluj-Napoca vol 40 no 1 pp 314ndash325 2012

[27] H-Y Hung K Qian S L Morris-Natschke C-S Hsu and K-H Lee ldquoRecent discovery of plant-derived anti-diabetic naturalproductsrdquo Natural Product Reports vol 29 no 5 pp 580ndash6062012

[28] K Shapiro and W C Gong ldquoNatural products used fordiabetesrdquo Journal of the American Pharmaceutical Associationvol 42 no 2 pp 217ndash226 2002

[29] F Y Siu M He C de Graaf et al ldquoStructure of the humanglucagon class B G-protein-coupled receptorrdquo Nature vol 499no 7459 pp 444ndash449 2013

[30] B R Brooks R E Bruccoleri B D Olafson D J States SSwaminathan and M Karplus ldquoCHARMM a program formacromolecular energy minimization and dynamics calcula-tionsrdquo Journal of Computational Chemistry vol 4 no 2 pp 187ndash217 1983

[31] A Warshel M Levitt and S Lifson ldquoConsistent force field forcalculation of vibrational spectra and conformations of someamides and lactam ringsrdquo Journal ofMolecular Spectroscopy vol33 no 1 pp 84ndash99 1970

[32] G Jones P Willett R C Glen A R Leach and R TaylorldquoDevelopment and validation of a genetic algorithm for flexibledockingrdquo Journal of Molecular Biology vol 267 no 3 pp 727ndash748 1997

[33] R Wang L Lai and S Wang ldquoFurther development andvalidation of empirical scoring functions for structure-basedbinding affinity predictionrdquo Journal of Computer-Aided Molec-ular Design vol 16 no 1 pp 11ndash26 2002

[34] S J Hubbard and J M Thornton NACCESS Computer Pro-gram Department of Biochemistry and Molecular BiologyUniversity College London UK 1993

[35] C M Koth J M Murray S Mukund et al ldquoMolecular basisfor negative regulation of the glucagon receptorrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 109 no 36 pp 14393ndash14398 2012

[36] J Mu S A Qureshi E J Brady et al ldquoAnti-diabetic efficacyand impact on amino acid metabolism of GRA1 a novel small-molecule glucagon receptor antagonistrdquo PLoS ONE vol 7 no11 Article ID e49572 2012

[37] A Saxena and N K Vikram ldquoRole of selected Indian plantsin management of type 2 diabetes a reviewrdquo The Journal ofAlternative amp Complementary Medicine vol 10 no 2 pp 369ndash378 2004

[38] A Hashim M S Khan M S Khan M H Baig and S AhmadldquoAntioxidant and 120572-amylase inhibitory property of Phyllanthusvirgatus L an in vitro andmolecular interaction studyrdquo BioMedResearch International vol 2013 Article ID 729393 12 pages2013

[39] M H Baig S M Rizvi S Shakil M A Kamal and S KhanldquoA neuroinformatics study describing molecular interactionof Cisplatin with Acetylcholinesterase a plausible cause foranticancer drug induced neurotoxicityrdquo CNS amp NeurologicalDisorders-Drug Targets vol 13 no 2 pp 265ndash270 2014

[40] L J Miller Q Chen P C-H Lam et al ldquoRefinement ofglucagon-like peptide 1 docking to its intact receptor usingmid-region photolabile probes andmolecularmodelingrdquoThe Journalof Biological Chemistry vol 286 no 18 pp 15895ndash15907 2011

[41] U Samanta R P Bahadur and P Chakrabarti ldquoQuantifyingthe accessible surface area of protein residues in their localenvironmentrdquo Protein Engineering vol 15 no 8 pp 659ndash6672002

[42] B K Sahoo K S Ghosh and S Dasgupta ldquoMolecularinteractions of isoxazolcurcumin with human serum albuminspectroscopic and molecular modeling studiesrdquo Biopolymersvol 91 no 2 pp 108ndash119 2009

[43] L L Conte C Chothia and J Janin ldquoThe atomic structure ofprotein-protein recognition sitesrdquo Journal of Molecular Biologyvol 285 no 5 pp 2177ndash2198 1999

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The glucagon receptor (GCGR) is a 62 kDa proteinactivated by glucagon that is a member of G-protein coupledreceptor (GPCR) superfamily [11] In humans the glucagonreceptor is encoded by the GCGR gene [12 13] Glucagonreceptors are primarily expressed in the liver and kidney withlesser amounts found in the heart adipose tissue spleen thy-mus adrenal glands pancreas cerebral cortex and gastroin-testinal tract By binding to GCGR glucagon sends a signalinside the cell which activates adenylyl cyclase leading tothe generation of high cAMP levels [14] In addition GCGRalso couples to an intracellular Ca2+-mediated pathway [15]GCGR activation leads to increase in metabolic processessuch as glycogenolysis and gluconeogenesis resulting inincreased glucose concentrations in hepatic cells and tissues[16 17]

Since GCGR plays an important role in elevating the glu-cose concentration in blood (glycemia) and there are manysmall-molecule inhibitors available for receptors of theGPCRfamily [18] it is a potent target for the development of small-molecule antagonistinhibitors A number of antagonistswith varying degrees of potency and structures have beenreported in recent years [19] GCGR based inhibitors for thetreatment of type 2 diabetes are either glucagon neutralizingantibodies [20 21] or small molecular antagonists [22ndash24]These compounds have been shown to effectively terminatethe GCGR action However concerns about safety toleranceand stimulation of adverse immune response when usingthese types of agents against GCGR for the treatment oftype 2 diabetes have led to investigations to identify drugsor compounds of natural origin to combat this problemIndeed GCGR antagonistinhibitors of natural origin maybe safe and favorable therapeutic agents for the treatmentof type 2 diabetes Accordingly it is important to search fornew and effective GCGR antagonists from natural sources[25]Therefore the present study was conducted to search fornatural antagonists against GCGR in silico All natural com-pounds selected in this study were collected from the avail-able literature and have been reported to have antidiabeticproperties [26ndash28] Additionally molecular docking studieshave been conducted to investigate the binding affinity ofall selected compounds The results were then reevaluatedusing two different scoring functions to confirm the accuracyof our results Overall the results presented herein enabledcalculation of the accessible surface area (ASA) of GCGR(uncomplexed) and its docked complex with the selectedcompounds for analysis of quantification of the packing ofresidues in GCGR before and after the binding of ligands

2 Methods

21 Preparation of Enzyme and Ligand for Docking The 3Dcrystal structure of human GCGR was retrieved from theRCSB protein databank (pdb ID 4L6R) (httpwwwrcsborgpdbexploreexploredostructureId=4L6R) [29] Beforeconducting the molecular docking calculations all watermolecules and other heteroatoms were removed A CharMm[30] force field was applied to the structure of GCGRfollowed by 1000-step energyminimization using the steepestdescent method The Distance-Dependent Dielectrics type

implicit solvent model was used to conduct the energyminimization step with the RMS gradient set to 01 A totalof 83 natural compounds were selected in this study Allnatural compounds used in this study were selected fromthe available literature The 3D structures of all naturalcompounds were extracted from the PubChem Compounddatabase A Cff force field [31] which is a general purposeclass II force field with good parameter coverage for manyorganicmolecules was applied to all the structures As class IIforce field it has additional cross terms in its potential energyfunction relative to other class I force fields

22 Molecular Docking Molecular docking was conductedto investigate the interaction of all the natural compoundsagainst GCGR Genetic Optimization for Ligand Docking50 (GOLD) [32] was used for docking of all the selectedcompounds against GCGR The annealing parameters wereset to 50 and 25 to evaluate van der Waals and hydrogenbonding docking respectivelyThe population size was set to100 with a selection pressure of 12The number of operationswas fixed to 100000 with 5 islands a niche size of 2migration value of 10 mutation value of 100 and crossoverof 100 The binding energies of docked molecules were alsocalculated using 119909-score [33] All molecular graphicsmaterialof docked complexes was prepared using Pymol

23 Accessible Surface Area Calculation Differences in theaccessible surface area (ASA) of the GCGR before andafter the binding of identified inhibitors were calculatedfor validation of effectiveness of these compounds usingNACCESS version 211 [34] The accessible surface area 119860of an atom is the area on the surface of a sphere of radius119877 on each point of which the center of a solvent moleculecan be placed in contact with this atom without penetratingany other atom of the molecule The radius 119877 is given by thesum of the van der Waalsrsquo radius of the atom and the selectedradius of the solvent molecule An approximation to this areais computed by this program using the following formula

Accessible surface area is

119860 = sum(

119877

radic1198772minus 1198852

119894

)sdot119863 sdot 119871119894 119863 =

Δ119885

2

+ Δ1015840119885 (1)

where 119871119894is the length of the arc drawn on a given section 119894119885

119894

is the perpendicular distance from the center of the sphere tothe section 119894Δ119885 is the spacing between the sections andΔ1015840119885is Δ1198852 or 119877 minus 119885

119894 whichever is smaller Summation is over

all of the arcs drawn for the given atom The accessibility isdefined simply as the accessible surface area divided by 41205871198772multiplied by 100

3 Results and Discussion

Glucagon G-protein coupled receptor class B GPCR hasbecome a promising therapeutic drug target for the treatmentof type 2 diabetes mellitus (T2DM) [35 36] Earlier stud-ies have reported that blockade of glucagon receptor gene(GCGR) activity is useful for the treatment of T2DM [25







PHE-365GLU-362

LEU-386

TYR-145

TYR-149

ILE-235

GLY-234MET-231



LEU-386

TYR-145

PHE-365GLU-362

ILE-235LYS-187

VAL-191MET-231

ILE-194




PHE-365VAL-363

GLU-362

VAL-311LEU-307

TYR-239ILE-235

GLN-232

MET-231LYS-187


0

10

20

30

40

50

60


GFSx-score

minus10

minus20

4-hydroxyderricin






Tota

l ASA

Unc

ompl

exed

190001895018900188501880018750187001865018600185501850018450

Com

plex

ed w

itham

orfru

tin 1

Com

plex

ed w

ithcu

rcum

in

Com

plex

ed w

ith4-

hydr

oxyd

erric

in



4 Conclusion






Abbreviations




Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















PHE-365GLU-362

LEU-386

TYR-145

TYR-149

ILE-235

GLY-234MET-231



LEU-386

TYR-145

PHE-365GLU-362

ILE-235LYS-187

VAL-191MET-231

ILE-194




PHE-365VAL-363

GLU-362

VAL-311LEU-307

TYR-239ILE-235

GLN-232

MET-231LYS-187


0

10

20

30

40

50

60


GFSx-score

minus10

minus20

4-hydroxyderricin






Tota

l ASA

Unc

ompl

exed

190001895018900188501880018750187001865018600185501850018450

Com

plex

ed w

itham

orfru

tin 1

Com

plex

ed w

ithcu

rcum

in

Com

plex

ed w

ith4-

hydr

oxyd

erric

in



4 Conclusion






Abbreviations




Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PHE-365VAL-363

GLU-362

VAL-311LEU-307

TYR-239ILE-235

GLN-232

MET-231LYS-187


0

10

20

30

40

50

60


GFSx-score

minus10

minus20

4-hydroxyderricin






Tota

l ASA

Unc

ompl

exed

190001895018900188501880018750187001865018600185501850018450

Com

plex

ed w

itham

orfru

tin 1

Com

plex

ed w

ithcu

rcum

in

Com

plex

ed w

ith4-

hydr

oxyd

erric

in



4 Conclusion






Abbreviations




Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Abbreviations




Acknowledgment


References



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















interaction of glucagon g-protein coupled receptor with known

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