In silico paper

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  • In Silico Research: Inhibiting binding GTP on Methyltransferase ofDengue VirusJuan E Maldonado Weng Primary ArticleGustavo A MartnezAbstract: The objective of this paper is to present the process of identifying a drug that will easily fight against theDengue Virus (DNV). The process was begun using a model of the methyltransferase enzyme with a GuanosineTriphosphate (GTP) of the virus within an active spot. Methyltransferase is an enzyme that transfers a methylgroup to the DNA of a cell. GTP is the molecule that produces the energy in order for that enzyme to transfer themethyl group properly. The purpose of developing this drug is to find a compound that will inhibit the transfer ofmethyl to the virus DNA. On this project, the Pymol program was used to generate three benzene rings werepositioned in the pocket where the GTP normally connects to the enzyme. These rings are then to be used to createthe pharmacophore model, which will show the hydrophobic regions. The hydrophobic regions are what will showwhich the compound will fit properly within the pocket in order to inhibit the GTP. That compound will besearched for in the ZINC Pharmer database. After processing the model through the database, over sixty thousandhits were found compatible in that region of the methyltransferase. These were filtered and separated into fivedifferent groups. From these groups, the twenty-five compounds with the highest affinity were chosen, and ofthose, the top three with the highest affinity were placed into the methyltransferase pocket in order to compare withthe benzene rings and the GTP. The compound with the highest affinity (-10.4) was DENV-M2_1.IntroductionDengue is a serious life affectingdisease infecting thousands of people whilemillions of others are at risk. The diseasecan be classified as three types of disease:Dengue Fever, Hemorrhagic Fever, orShock Syndrome. Antiviral drug discoveryis becoming increasingly important due tothe global threat of viral disease pandemics.Many members of the genus Flavivirus aresignificant human pathogens, among whichdengue virus (DENV) alone poses a publichealth threat to 2.5 billion worldwide,leading to 50100 million human infectionseach year. Neither vaccine nor effectivetherapeutics is currently available forDENV. Development of a DENV vaccinehas been challenging, because of the need tosimultaneously immunize and induce a long-lasting protection against all four serotypesof DENV This statement was from areview paper by Christian G. Noble and histeam. These diseases are very severe, andbased on CDC reports from 2013; manycommon symptoms include joint and muscleCartogram 1- InfectionCartogram of the annualnumber of infections for allages as a proportion ofNational or subnational (China)geographical areaObtained from Bahtt et al.Review Paper 2013
  • pain, elevated corporal temperatures, andsevere headaches. These diseases in severecases can cause death. According to CDC in2010, there were 12,580 confirmed cases ofpeople diagnosed with dengue in PuertoRico. The tropical regions are under themost danger because the numbers ofmosquitoes that transmit the disease preferthese areas.The Aedes mosquito is the commoncarrier of the Dengue virus. The Denguevirus is a member of the family Flaviviridaeand the genus Flavivirus. The Dengue virushas very similar structural properties to othermembers of the family like the West NileVirus. The viral transmission poses a realthreat to people and, with no cure available;the amount of cases will increase by theincoming years.Through research by computersimulation, the search for a cure is easier.Utilizing many advance software andprogramming, the structure of importantproteins can be ideally researched. Withthese structures, the main components, suchas amino acids with their spacingarrangement and chemical properties, couldbe studied then inhibited. Throughinhibition of certain useful structures, theviral infection could be stopped.In the case of the Dengue Virus, themethyltransferase is an important protein forthe spread and development of the virus.Methyltransferases (MTases) play key rolesin normal physiology and human diseasesthrough methylating DNA, RNA, andproteins. Almost all MTases use S-adenosyl-L-methionine (SAM) as a methyl donor andgenerate S-adenosyl-Lhomocysteine (SAH)as a by-product. Pharmacologicalmodulation of MTases by small moleculesrepresents a novel approach to therapeuticintervention in cancer and other diseases.(Siew Pheng Lim et al, 2011) Themethyltransferase transfers a methyl groupfrom a donator towards an acceptor whichcould be a strand of DNA or RNA. Thismethyl group will help mark the genesneeded to be expressed. This protein isneeded for the virus to develop properly.The mechanism that the protein utilizes tohave the appropriate energy is to bind with aGTP molecule. This molecule is the energysource that enables the methyltransferase tocomplete the transfers.Through In Sillico research, the GTP,used for the Methyltransferase of DENV-2,can be inhibited utilizing a higher affinityImage 1 Image 2
  • compound. Finding a compound that couldhave a stronger force of attraction towardsthe site where GTP would be. If an alternatecompound would take GTPs place, thenthere is a possibility the protein would failcausing a cease in the virus effect on theorganism.Materials and MethodThe first step, which was the mostimportant, was to identify a biologicalproblem and to undergo research tounderstand the virus. The methyltransferase3D molecule structure was downloadedfrom the online Protein Database (pbd.org).This database provided much neededinformation and was very accessible. Themolecule can be seen in image 1 as it wouldin the PyMol program. From the angle ofthe first image, there is a gap or pocketwhere a molecule could be placed. To see ifit were true, the molecule went through avigorous process to locate the hotspotsand to observe where the GTP would bind.From there, the model in the PyMol programhad the GTP properly placed into the pocketand the amino acids that interact well aroundit, as seen in Image 2.A grid was constructed in order toseclude the pocket and the region where theGTP connects with the protein. Within thegrid, several benzene rings were found, andwith these, the binding site of the GTP.Benzenes are important structures which aremade up of carbon rings. These structureshelp identify the methyltransferase pocket.These three benzene rings were thenused to create a pharmacophore model withthe Ligand-Scout program. This modeldepicts the hydrophobic regions, thephysical arrangement, and other chemicalproperties of the compound that will be usedto filter out the search. From the millions ofcompounds in the database, thepharmacophore model will help find theones that fit the pocket. The search engineutilized was ZINC Pharmer, which foundmany matches. The processes werecompleted by the use of a cluster of serversworking together.From the results, a long list ofcompounds appeared. Organization was themost important asset from this step onward.Excel was utilized to have all thecompounds in tables. From there, the tablewas assorted from highest to lowest affinity,and, finally, thecompound with thehighest affinity waschosen. Thiscompound, along withthe other two highestcompounds, weremodeled in PyMol andtested to see if there isa visual match withthe benzenes. Aftervisualizing theinteractions, the nextideal step would beBio Assay to test thesecompounds in a realworld scenario.ResultsThe structureof the pharmacophoremodel was runthrough the ZINCTable 1: Highest AffinityCompoundsRank Compound Affinity1 DENV-M2_1 -10.42 DENV-M2_2 -10.33 DENV-M2_3 -10.24 DENV-M2_4 -10.25 DENV-M2_5 -10.26 DENV-M2_6 -10.27 DENV-M2_7 -10.18 DENV-M2_8 -10.19 DENV-M2_9 -10.110 DENV-M2_10 -10.011 DENV-M2_11 -10.012 DENV-M2_12 -10.013 DENV-M2_13 -10.014 DENV-M2_14 -10.015 DENV-M2_15 -10.016 DENV-M2_16 -9.917 DENV-M2_17 -9.918 DENV-M2_18 -9.919 DENV-M2_19 -9.920 DENV-M2_20 -9.921 DENV-M2_21 -9.922 DENV-M2_22 -9.923 DENV-M2_23 -9.924 DENV-M2_24 -9.825 DENV-M2_25 -9.8
  • Pharmer. (The image of the pharmacophoremodel can be seen as image 3; the yellowspheres represent the benzenes and the grayspheres the amino acids and othermolecules.) This enabled access tothousands of different compounds that couldpossibly fit within the pocket of themethyltransferase and inhibit its interactionwith the GTP. These compounds werefiltered and divided into groups dependingon their size or molecular weight.These compounds had varyingaffinities, which means that they will attachto the pocket of the protein at differentstrengths or intensities. In a chart showingthem from highest affinities, twenty-fivecompounds were separated, and the threewith the highest affinities were chosen to beplaced within the methyltransferase in orderto see their interaction with the protein.These three proteins were denominated:DENV-M2_1, with an affinity of -10.4;DENV-M2_2, with an affinity of -10.3; andDENV-M2_3, with an affinity of -10.2. Theother twenty two compounds are shown intable 1. The affinity on the table varies from-10.4 to -9.8. From all the compoundscollected, there were a total of a hundredand sixty-two (162) compounds, varyingfrom -10.4 to -9.5, but there were over25,000 compounds which had lesser affinity.These three compounds were thenplaced into the pocket of themethyltransferase, and compared with theplacement of the benzene rings and the GTPmolecule. The compound that showed tooccupy the most space and position itself ina similar way to the benzene rings wasDENV-M2_1. These factors demonstrate thecompounds affinity of -10.4. The placementof the DENV-M2_1 also shows that therewould not be any possibility for the GTPmolecule to make its way into the pocket.(The