Research Article Screening of Mutated K-Ras Inhibitors ...downloads.hindawi.com/archive/2014/431696.pdf · g/mL suggesting that components had a synergistic e ... HHH HH H H H H Pheophorbide

Research ArticleIn Silico Screening of Mutated K-Ras Inhibitors from MalaysianTyphonium flagelliforme for Non-Small Cell Lung Cancer

Ayesha Fatima and H F Yee

Department of Pharmaceutical Technology Faculty of Pharmaceutical Sciences UCSI University Jalan Menara Gading 1Taman Connaught Cheras 56000 Kuala Lumpur Malaysia

Correspondence should be addressed to Ayesha Fatima ayeshaucsiuniversityedumy

Received 6 February 2014 Accepted 19 August 2014 Published 21 September 2014

Academic Editor Tatsuya Akutsu

Copyright copy 2014 A Fatima and H F Yee This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

K-ras is an oncogenic GTPase responsible for at least 15ndash25 of all non-small cell lung cancer cases worldwide Lung cancer ofboth types is increasing with an alarming rate due to smoking habits in Malaysia among men and women Natural products alwaysoffer alternate treatment therapies that are safe and effective Typhonium flagelliforme or Keladi Tikus is a local plant known topossess anticancer properties The whole extract is considered more potent than individual constituents Since K-ras is the keyprotein in lung cancer our aim was to identify the constituents of the plant that could target the mutated K-ras Using dockingstrategies reported potentially active compounds of Typhonium flagelliforme were docked into the allosteric surface pockets andswitch regions of the K-ras protein to identify possible inhibitors The selected ligands were found to have a high binding affinityfor the switch II and the interphase region of the ras-SOS binding surface

1 Introduction

Cancer is a major health problem in Malaysia with a totalof 2048 cases registered with National Cancer Registry(NCR) in 2006 [1] The disease is now the third leadingcause of premature deaths in our country Lung cancer isamong the top five cancers affecting both male and femalein Malaysia at 94 percent in peninsula Malaysia with 2100Malaysians diagnosed every year [2 3] Smoking relateddiseases are increasing in Malaysia especially lung cancer [34] A recent study conducted by Liam et al (2013) highlightedadenocarcinomas as themost frequent types of cancer amongMalaysian men and women smoker and nonsmokers Withan incidence rate of 1098 cases of cancer per 100000 itis imperative to find treatments that are safe and effective[5]

Lung cancer can be divided into two major classes basedon its biology therapy and prognosis namely non-small celllung cancer (NSCLC) and small cell lung cancer (SCLC)NSCLC accounts for 75 to 80 of all lung cancer incidents

while small cell lung cancer accounts for 15 to 25 of alllung cancer [6]

The ras pathway is an important signaling pathwaythat allows cell proliferation in response to stimulation ofthe epidermal growth factor receptor [7 8] These signalsaffect the production and regulation of other key proteinsinvolved in cell proliferation Studies have reported that K-rasmutation occurring in NSCLC varies between 16 and 40[8] K-ras a GTPase also known as V-Ki-ras-2 (Kirsten ratsarcoma viral oncogene) is a protein that in human is encodedby the K-Ras gene [9]The normal protein is an essential partof the ras signaling pathway acting as a molecular switchIn the ldquooffrdquo state it is bound to the guanine diphosphatenucleotide (GDP) It is turned on via the growth factorstimuli The guanine nucleotide exchange factor also knownas the son of sevenless (SOS) protein and the growth factorreceptor-bound protein 2 (Grb-2) together enable the K-rasto become activated by exchanging the GDPmolecule for themore active guanine triphosphate nucleotide (GTP) Onceturned ldquoonrdquo it recruits and activates downstream proteins

Hindawi Publishing CorporationAdvances in BioinformaticsVolume 2014 Article ID 431696 7 pageshttpdxdoiorg1011552014431696

2 Advances in Bioinformatics

necessary for the propagation of growth factor and otherreceptor signals It possesses an intrinsic weak enzymaticactivity which is enhanced by interaction with the GTPaseactivating protein (GAP) leading to cleavage of the terminalphosphate of the nucleotide guanine triphosphate (GTP)converting it into the guanine diphosphate (GDP) Uponconversion of GTP to GDP K-ras is switched ldquooffrdquo Despitebeing a weak GTPase K-ras possesses two very importantfeatures involved in its switching ldquoonrdquo and ldquooffrdquo Calledswitches because of their ability to change the conformationof the protein in the active and inactive state the regionsare involved in interacting with the nucleotides The guaninenucleotide pocket of theK-ras is highly conserved and is linedwith residues 11ndash16 [10 11] Because of the specific interactionsof amino acid residues of this region with the GTP mutationsat the 12 and 13 amino acid positions in the enzyme lead topermanent cell proliferation because it cannot be hydrolysedand hence the ras signaling function is unable to be turnedldquooffrdquo [12ndash14] The mutated K-ras (mut-Kras) is an interestingdrug target of several studies [15ndash18]Themajor reason beingthat it provides fast resistance to the available drug therapySeveral EGFR MEK inhibitors have been tried in singleand combination However drug resistance develops quickly[17 19ndash21]

Medicinal plants with anti-cancer effects are commonlyused as alternative medicine because of their safety andtoxicity profiles Several herbal medicines have been studiedfor finding effective treatment of lung cancer Only fewstudies on the use of Malaysian medicinal plants as treat-ment options have been reported [4 21 22] Typhoniumflagelliforme (Keladi tikus) is one such plant that is foundlocally in Malaysia that has been studied for its inhibitionof proliferation in human lung cancer cell line Its activeingredients including phytol and its derivative hexadecanoicacid 1-hexadecene and pheophorbide related compound-shave shown some promising results as anticancer whenwhole extracts have been used Lai et al reported that theycould not find single constituents as effective when comparedto the extract [22 23]

Computer studies have recently provided insights into themechanics of K-ras protein [24ndash26] Researchers have offeredin depth study of the various mutations and the effect theyhave on the ldquoon-offrdquo states of the protein With sophisticatedsoftware being available to researchers they have recentlyreported direct inhibition of the protein as therapeutic targetMaurer et al (2012) have carried out an in-depth study of theallosteric binding pockets on the protein that maybe targetedin the ldquooffrdquo state of the mut-K-ras [24]

In this study we explored the inhibitory effects of thesome of the reported potent constituents of T flagelliformeon the lung cancer cell lines using docking studies withAutodock Vina [27] We used the reported structures of theactive ingredients and docked them into reported allostericbinding sites [25 28] on the mut-K-ras to determine theprobable binding sites of the constituents We also tried torelate the experimental results obtained by Lai et al [22 23] toour computational observations to gain meaningful insightsinto the use of the proposed plant constituents as probableinhibitors

2 Materials and Methods

The three dimensional structures of the G12D mutated K-rasstructure inhibitor bound to SOS pocket (PDB ID 4DST)and two G12C mutated K-ras structure bound to allostericsites (PDB ID 4LUC and 4LYF) Autodock Vina 42 [27]was used to dock all ligands to the K-ras protein Beforethat we used Autodock tools downloaded from The ScrippsResearch Institute to prepare the ligand and protein file [2930] All water molecules were removed and Kollman chargesadded as described in the Autodock Vina 42 manual [31 32]The grid box dimensions were obtained from the grid boxwidget by keeping the bound ligand sites as box centersControl studies were performed with all ligand bound in thecrystal structures before docking with test ligands from Tflagelliforme Pheophorbide a and two related epimers weredrawn inChemSketch [33] based on reported structure by Laiet al [22] while hexadecene and hexadecanoic acid structurewere obtained from PubchemThe 2D structures of the activeconstituents are tabulated in Table 1

3 Results and Discussions

The results of highest binding affinity of the five activeconstituents for the GTP bound K-ras as well as the mutatedprotein structures are tabulated in Table 2

T flagelliforme is a local Malaysian plant with anticanceractivity when taken as a fresh juice prepared from freshlycrushed plant Researchers have reported its activity in lungcancer as a whole extract in dichloromethane [22 23] Laiet al (2010) reported that the extract contained at least 11chemical compounds of hydrophobic character [22] Themost predominant compounds were pherophorbide a pheo-phorbide a1015840 pyropheophorbide a methyl pyropheophorbidea hexadecanoic acid oleic acid linoleic acid linolenic acidcampesterol stigmasterol and 120573-sitosterol The in vitro stud-ies conducted by the group on the NCI-H23 lung cancer cellline had concluded that individual compound isolates hadfailed to show a significant anti-cancer behavior Howeverthe whole extracts were found to have an IC

50of 27 120583gmL

suggesting that components had a synergistic effect onantiproliferation of cancerous cells They also suggested thatthe constituents showed increased activity upon exposureto light The authors had also earlier reported an activewhole extract of the plant comprising of hexadecanoic acid1-hexadecene phytol and a phytol derivative with an IC

50of

75 120583gmL against NCI-H23 cell lines [23]Mohan et al (2010) conducted their study on the leukemic

cells and showed the selectivity of the dichloromethanefractions for the cancerous cells However they did notindicate the effect of any particular constituent on the cancercells [34]

In our study we focused on determining whether theproposed constituents by Lai et al (2008 2010) were able totarget the K-ras protein either directly or allosterically Fordirect inhibition we docked the selected ligands at or nearthe SOS binding pocket using the PDB structure 4DST [24]The target binding site proposed by the authors is near theras switches I and II and binding to this area was indicated

Advances in Bioinformatics 3

Table 1 Two dimensional structures of the constituents used in the study

Name Structure

Hexadecanoic acidH

O

O

H H H H H H H H

H H H H H H HH

Hexadecene

H

H H H H H H H H

H H H H H H H H

H

Pheophorbide a epimer

H2C

H3C

CH3

NH

H

HO

H

HN

O

CH3

N

N

H3C

H

HO

OCH3

O

CH3

Pheophorbide a1015840 epimer

H2C

H3C

CH3

NH

H

HO

H

HN

O

CH3

N

N

H3C

HH

OO

CH3

O

CH3

4 Advances in Bioinformatics

Table 1 Continued

Name Structure

Pyropheophorbide epimer

H2C

H3C

CH3

NH

H

HO

H

O

CH3

N

N

H3C

H

H O

CH3

N

H

Table 2 Binding affinity values obtained for the control and test ligands

Protein Binding affinity KcalmolControl Pheophorbide A Pheophorbide A1015840 Pyropheophorbide A 1-Hexadecene Hexadecanoic acid

G12D mutatedK-ras GDP SOSbinding pocket(4DST)

minus54 minus72 minus75 minus71 minus40 minus41

G12C mutatedK-ras GDPallosteric bindingpocket (4LUC)

minus80 minus70 minus68 minus73 minus48 minus46

G12C mutatedK-ras GDPallosteric bindingpocket (4LYF)

minus67 minus67 minus66 minus65 minus45 minus50

as interfering with ras-SOS binding surface [24]This findingis important because the Ras-SOS complex is essential foractivation of the K-ras since SOS initiates the GTP exchangeprocess to the protein According to the resolved structureof the ras-SOS complex (PDB ID 1BKD) [35] the CDC25binding region is tightly bound to the Switch II of ras andcauses the disruption of theGDPbound structure [35] Tyr 64of the ras appears to be the anchoring residue for theRas-SOScomplex Hence the inhibitors should be designed to targetthe switch regions or the binding surface between the protein-protein complex Where small molecules can modulate theswitch regions of the ras due to space confinement largermolecules can target the accessible surface areas between theprotein-protein complex

Our semiflexibledockingexperiment on theK-rasmoleculewith control ligand 46-dichloro-2-methyl-3-aminoethyl-indole (4DST) [24] had a binding affinity of minus54 KcalmolAmong the test ligands the highest binding affinities wereshown by the pheophorbide epimers The observed docked

GTP

Hexadecanoic acid (red) occupies the same binding pocket as the reported inhibitor

The pheophorbide epimers (yellow blue and magenta) and 1-hexadecene (green) in alternate position on the surface of the protein

Figure 1 Docked poses of flagelliforme constituents to GTP boundK-ras

poses given in Figure 1 showed that the epimers were notnear the binding site of the control ligand

The test ligand that showed affinity for the same bindingsite as the control molecule was hexadecanoic acid with

Advances in Bioinformatics 5

binding affinity minus41 Kcalmol It appeared to overlap thecontrol molecule at the same positionThe authors suggestedthat the binding pocket involves residues Lys5 Leu6 Val7Ile55 Leu56 and Thr74 [24] Our results showed mostly thesame binding site except that Thr 74 was placed a bit furtherfrom the ligands but within 5 A Our results showed thebinding pocket residues to be Lys5 Leu6 Val7 Glu37 Ser39Arg40 Asp54 Ile55 Leu56 Gln70 Tyr71 Met72 Thr74 andGly75 They further elaborated that the binding of the indolederivative expanded the pocket to accommodate the ligandArecent report by Grant et al (2011) also established this regionas one of the allosteric binding pockets important in findinginhibitors for K-ras [25] Hexadecanoic acid is a long chainedhydrocarbon that folds into a U-shape when docking intothe pocket This folding makes the molecule snugly fit intothe pocket From this result we can assume that when thecancer cells were incubated with the T flagelliforme extract[23] the hexadecanoic acid could possibly target the K-ras atthis surface pocket

The other ligands presented another interesting positionThe pheophorbide epimers docked strongly into a depressionon the surface of the K-ras that is also the surface forinteraction with the SOS protein [35] The residues involvedthat formed the binding pocket for the epimers and 1-hexadecene were Arg73 Thr74 Gly75 Glu76 and Gly77 Thestrong binding affinity between the protein and the epimerswas the result of the hydrogen bonding between the ligandsand Arg73 and Gly75

For another docking experiment we used two of the PDBstructures 4LUC and 4LYF reported by Ostrem et al (2013)since it presented two different ligands a sulphonamide anda vinyl sulphonamide that caused changes in the switchII region to accommodate the ligand [28] The researchersfocused onfinding inhibitors that could bind tomutatedGDPbound K-ras and change its structure such that it would notbe able to exchange the GDP molecule for the GTP to beactivated

The authors showed that their test ligands targeted theswitch II region that falls in the loop region between thecentral 120573-sheet 1205722 and 1205723 helices of the ras The pockethad been earlier reported by Taveras et al [36] We used thesame binding pocket lined with residues Val7 Val9 Gly10Ala11 Thr58 Ala59 Gly60 Gln61 Glu62 Glu63 Arg68Tyr71 andMet72 Our results revealed that the pheophorbideepimers had the strongest affinity However the docked posespresented an entirely different picture

In case of the structure PDB ID 4LUC pheophorbidea could insert its side chain (ndashCH

2ndashCH2ndashCOOH) into the

pocket (Figure 2)The same side chain of pheophorbide a1015840 didnot penetrate much Pyropheophorbide a occupied positionlined by residues Phe90 Glu91 His94 His95 Gln129 Asp132Leu133 and Ser136 Being bulky molecules they could notpenetrate inside the pocket however could make a hydrogenbond with Glu63 His95 and Tyr96 that contributed tothe binding affinity of minus70 for pheophorbide a 68 forpheophorbide a1015840 and minus73 Kcalmol for pyropheophorbide aHexadecanoic acid and hexadecene could easily slide intothe pocket and make polar contact with Arg68 This wasperhaps due to the long hydrophobic chain despite the low

Figure 2 Docked poses of the flagelliforme constituents to thebinding pocket of K-ras (PDB ID 4LUC) The ligands are hexade-canoic acid (red) pheophorbide a (yellow) phephorbide a1015840 (bluepyropheophorbide a and magenta) and 1-hexadecene (green))

binding affinity The pheophorbide a and a1015840 showed that theyoccupied the binding region that included residues 61ndash64 and68 on K-ras with pheophorbide a interacting with Glu63As already pointed out [35] these residue are involved inthe contact surface between the Ras-SOS proteins Henceif pheophorbide is able to bind to these residues it couldprevent the interaction of the two proteins in a pronouncedmanner

The other K-ras structure (PDB ID 4LYF) has a vinyl-sulphonamide covalently bound to the K-ras in the samepocket area The authors indicated that when this compoundbinds to the pocket it modifies the switch II position andcompletely disorders switch I [25]

Our docking experiment given in Figure 3 on thisexpanded pocket allowed the 1-hexadecene and hexadecanoicacid to go deep into the pocket and occupy the same bindingpocket as the inhibitor and formed hydrophobic interactionsinside their binding pocket As had been pointed out bythe authors the disorderliness of the switch regions causedby the ligands could lead to SOS protein not being able tobind efficiently to the ras protein leading to inhibition ofthe exchange of nucleotides and ultimate inactivity of theprotein [25] However pheophorbides targeted the bindingregion comprised of Lys5 Arg73Thr74 Gly75 Glu76 Val103Lys104 Asp105 and Ser106 which according to Boriack-Sjodin et al (1998) is part of the site of interaction betweenras-SOS [35] Pheophorbide a exhibited the highest affinityof minus67 Kcalmol while pyropheophorbide a molecule withbinding affinity 65 Kcalmol formed a hydrogen bond withArg73 Because of their big size and span of the ring we canhypothise that these compounds cover the area of interactionbetween K-ras and SOS and hence may be important directinhibitors of the K-ras at the ras-SOS interacting region

Taking these results together we can say that K-ras couldbe a good target for the pheophorbides of the flagelliformeThey have two hotspots on the protein One is the regionof surface contact between the SOS protein and the K-ras and the other could be the switch two region Wecarried out experiments in both the activated state andinactivated state and in both states Our results showedthat pheophorbides naturally targeted the SOS interactingresidues preferable in the activated (4DST) and inactivated

6 Advances in Bioinformatics

(a) (b)

Figure 3 Docked poses of the flagelliforme constituents to the binding pocket K-ras PDB ID 4LYF (a) Control ligand vinylsulphonamidedocked in the switch II region (b) Test ligands pheophorbide a (yellow) pheophorbide a1015840 (blue) and pyropheophorbide a (magenta) boundat the SOS-interaction site while 1-hexadecene (green) and hexadecanoic acid (red) are bound in the switch II region

state (4LUC 4LYF)The hexadecanoic acid and 1-hexadecenelooked for hydrophobic pockets that it could slide into andtheir preferable hotspot was switch II

4 Conclusion

Hence we can conclude that T flagelliforme constituentscould target several allosteric sites on the K-ras Since thisprotein is the most important signaling molecule in cancercells inhibiting this protein would induce apoptosis of lungcancer cells Combining our results with experimental evi-dence from Lai et al (2008 2010) where whole extracts aremore potent than individual constituents we can hypothesizethat K-ras could have been the probable target of pheophor-bides and other constituents Pheophorbides bind to the SOSbinding spot on K-ras and could possibly prevent a stronginteraction between the nucleotide exchange protein SOS andK-ras while 1-hexadecane and hexadecanoic acid bind toswitch II region of the K-ras Both events combined wouldeventually inhibit the growth signals in the cancerous cellsFurther studies are required to conclusively indicate K-ras atthe target for the pheophorbides and other constituents

Conflict of Interests

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

References

[1] Z A Omar Z M Ali and S I Tamin Nor Eds MalaysianCancer StatisticsmdashData and Figure Penisular Malysia NationalCancer Registry Ministry of Health Malaysia 2006

[2] National Cancer Society Malaysia httpcancerorgmylearn-about-cancerabout-cancer

[3] G C C Lim ldquoOverview of cancer inMalaysiardquo Japanese Journalof Clinical Oncology vol 32 pp S37ndashS42 2002

[4] S Mohan S I Abdelwahab S-C Cheah et al ldquoApoptosis effectof girinimbine isolated from Murraya koenigii on lung cancercells in vitrordquo Evidence-based Complementary and AlternativeMedicine vol 2013 Article ID 689865 12 pages 2013

[5] C-K Liam M I A Wahid P Rajadurai Y-K Cheah and TS-Y Ng ldquoEpidermal growth factor receptor mutations in lungadenocarcinoma in Malaysian patientsrdquo Journal of ThoracicOncology vol 8 no 6 pp 766ndash772 2013

[6] TheAmericanCancer Society ldquoLearn about cancerrdquo httpwwwcancerorgcancerlungcancer-non-smallcelldetailedguidenon-small-cell-lung-cancer-what-is-non-small-cell-lung-cancer

[7] A Fernandez-Medarde and E Santos ldquoRas in cancer anddevelopmental diseasesrdquo Genes and Cancer vol 2 no 3 pp344ndash358 2011

[8] P M K Westcott and M D To ldquoThe genetics and biology ofKRAS in lung cancerrdquo Chinese Journal of Cancer vol 32 no 2pp 63ndash70 2013

[9] MVMilburn L TongAMDevos et al ldquoMolecular switch forsignal transduction structural differences between active andinactive forms of protooncogenic ras proteinsrdquo Science vol 247no 4945 pp 939ndash945 1990

[10] D A Eberhard B E Johnson L C Amler et al ldquoMutationsin the epidermal growth factor receptor and in KRAS arepredictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and incombination with erlotinibrdquo Journal of Clinical Oncology vol23 no 25 pp 5900ndash5909 2005

[11] A T Brunger M V Milburn L Tong et al ldquoCrystal structureof an active form of RAS protein a complex of a GTP analogand theHRAS p21 catalytic domainrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 87 no12 pp 4849ndash4853 1990

[12] EMassarelli M Varella-Garcia X Tang et al ldquoKRASmutationis an important predictor of resistance to therapy with epider-mal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancerrdquo Clinical Cancer Research vol 13 no 10pp 2890ndash2896 2007

[13] Y-S Chang K-T Yeh N C Hsu S-H Lin T-J Chang andJ-G Chang ldquoDetection of N- H- and KRAS codons 12 13 and

Advances in Bioinformatics 7

61 mutations with universal RAS primermultiplex PCR andN-H- andKRAS-specific primer extensionrdquoClinical Biochemistryvol 43 no 3 pp 296ndash301 2010

[14] M-J Yang C-K Hsu H-J Chang et al ldquoThe KRAS mutationis highly correlated with EGFR alterations in patients with non-small cell lung cancerrdquo Fooyin Journal of Health Sciences vol 1no 2 pp 65ndash71 2009

[15] G J Riely J Marks andW Pao ldquoKRASmutations in non-smallcell lung cancerrdquo Proceedings of the American Thoracic Societyvol 6 no 2 pp 201ndash205 2009

[16] S Jancık J Drabek D Radzioch and M Hajduch ldquoClinicalrelevance of KRAS in human cancersrdquo Journal of Biomedicineand Biotechnology vol 2010 Article ID 150960 13 pages 2010

[17] R Dienstmann S De Dosso E Felip and J Tabernero ldquoDrugdevelopment to overcome resistance to EGFR inhibitors in lungand colorectal cancerrdquoMolecular Oncology vol 6 no 1 pp 15ndash26 2012

[18] J C Soria T S Mok F Cappuzzo and P A Janne ldquoEGFR-mutated oncogene-addicted non-small cell lung cancer currenttrends and future prospectsrdquoCancer Treatment Reviews vol 38no 5 pp 416ndash430 2012

[19] P Ulivi D Calistri W Zoli and D Amadori ldquoPredictivemolecular markers for EGFR-TKI in non-small cell lung cancerpatients new insights and critical aspectsrdquo Journal of NucleicAcids Investigation vol 1 no 1 pp 47ndash54 2010

[20] G M Stella M Luisetti S Inghilleri et al ldquoTargeting EGFRin non-small-cell lung cancer lessons experiences strategiesrdquoRespiratory Medicine vol 106 no 2 pp 173ndash183 2012

[21] H-F Hsu K-H Huang K-J Lu et al ldquoTyphonium blumeiextract inhibits proliferation of human lung adenocarcinomaA549 cells via induction of cell cycle arrest and apoptosisrdquoJournal of Ethnopharmacology vol 135 no 2 pp 492ndash500 2011

[22] C S Lai R H M H Mas N K Nair S M Mansorand V Navaratnam ldquoChemical constituents and in vitro anti-cancer activity of Typhonium flagelliforme (Araceae)rdquo Journal ofEthnopharmacology vol 127 no 2 pp 486ndash494 2010

[23] C-S Lai R H M H Mas N K Nair M I A MajidS M Mansor and V Navaratnam ldquoTyphonium flagelliformeinhibits cancer cell growth in vitro and induces apoptosisan evaluation by the bioactivity guided approachrdquo Journal ofEthnopharmacology vol 118 no 1 pp 14ndash20 2008

[24] T Maurer L S Garrenton A Oh et al ldquoSmall-moleculeligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activityrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 109 no 14 pp 5299ndash5304 2012

[25] B J Grant S Lukman H J Hocker et al ldquoNovel allosteric siteson ras for lead generationrdquo PLoS ONE vol 6 no 10 Article IDe25711 2011

[26] F Shima Y Yoshikawa M Ye et al ldquoIn silico discovery ofsmall-molecule Ras inhibitors that display antitumor activityby blocking the Ras-effector interactionrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 110 no 20 pp 8182ndash8187 2013

[27] O Trott and A J Olson ldquoSoftware news and update AutoDockVina improving the speed and accuracy of docking with a newscoring function efficient optimization and multithreadingrdquoJournal of Computational Chemistry vol 31 no 2 pp 455ndash4612010

[28] J M Ostrem U Peters M L Sos J A Wells and K M ShokatldquoK-Ras(G12C) inhibitors allosterically control GTP affinity and

effector interactionsrdquo Nature vol 503 no 7477 pp 548ndash5512013

[29] G M Morris D S Goodsell R S Halliday et al ldquoAutomateddocking using a Lamarckian genetic algorithm and an empiricalbinding free energy functionrdquo Journal of Computational Chem-istry vol 19 no 14 pp 1639ndash1662 1998

[30] G Morris and R Huey ldquoAutoDock4 and AutoDockTools4automated docking with selective receptor flexibilityrdquo Journalof Computational Chemistry vol 30 pp 2785ndash2791 2009

[31] R Huey G M Morris and S ForliUsing AutoDock 4 and Vinawith AutoDockTools A Tutorial 2011

[32] D Seeliger and B L de Groot ldquoLigand docking and bindingsite analysis with PyMOL and AutodockVinardquo Journal ofComputer-Aided Molecular Design vol 24 no 5 pp 417ndash4222010

[33] ACDChemSketch Version 12 Advanced Chemistry Develop-ment ACDChemSketch Toronto Canada 2014 httpwwwacdlabscom

[34] S Mohan A B Abdul S I Abdelwahab et al ldquoTyphoniumflagelliforme induces apoptosis in CEMss cells via activationof caspase-9 PARP cleavage and cytochrome c release itsactivation coupled with G0G1 phase cell cycle arrestrdquo Journalof Ethnopharmacology vol 131 no 3 pp 592ndash600 2010

[35] P A Boriack-Sjodin SMMargarit D Bar-Sagi and J KuriyanldquoThe structural basis of the activation of Ras by SosrdquoNature vol394 no 6691 pp 337ndash343 1998

[36] A G Taveras S W Remiszewski R J Doll et al ldquoRasoncoprotein inhibitors the discovery of potent ras nucleotideexchange inhibitors and the structural determination of a drug-protein complexrdquo Bioorganic and Medicinal Chemistry vol 5no 1 pp 125ndash133 1997

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