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удК616.98:578.825.11-085(045)=111 Оригинальная статья original article teGUMeNt BASeD IN-SILICO DRUG tARGetING OF HeRPeS SIMPLex VIRUS-1 Ashish Runthala – India, Pilani, Birla Institute of Technology & Science, Biological Sciences Department, Lecturer, M.Sc., M.E., Ph.D; Amit Kumar Singh – India, Gurgaon, ILS Bioservices, Trainee Application Specialist, B.Tech.

аСПекты медикаментоЗного лечения вПг-тиПа 1 Ашиш Рунтала – Индия, Пилани, Бирла, Научно-технологический Институт, Кафедра Биологических Наук, Магистр Естественных Наук, Доктор Философии; Амит Кумар Синг – Индия, Гургаон, Специалист-стажер, Бакалавр Технологических Наук.

дата поступления – 10.04.10 г. дата принятия в печать – 15.06.2010 г.

Ashish Runthala, Amit Kumar Singh. Tegument based in-silico Drug Targeting of Herpes Simplex Virus-1. Saratov Journal of Medical Scientific Research, 2010, vol. 6, № 2, p. 353-357.

alpha trans inducing factor (-tif) is a herpes simplex virus type 1 (hSV-1) virion tegument protein present in the tegument layer between the capsid and the envelope, in association with cellular proteins and trans-activated viral activities. -tif stimulates the transcription of hSV-1 immediate early genes during lytic virus replication. the presence or absence of functional -tif protein in vivo is always associated with viral activities. its role behind latent hSV-1 infection is ambiguous. no drug is designed for hSV till now based on this protein and thus its conformational details can be very important for drug designing purpose. Methods. docking studies on this protein becomes logical.

four different ligand molecules viz. adenosine-3’-5’-diphosphate, p1-(5’-adenosyl) p4-(5’-(2’-deoxy-thymidyl)) tetraphosphate, 9-hydroxymethylguanine, 9-(4-hydroxybutyl)-n2-phenylguanine were screened for the study. Results.

p1-(5’-adenosyl) p4-(5’-(2’-deoxy-thymidyl) tetraphophate was found to be the best ligand as it showed the lowest docking energy of – 9.238 kcal/mol. Conclusions. the best ligand was found to bind different sites of the -tif protein and hence can be utilized to combat hSV-1 successfully, with a synergistic effect of multiplicity of drug molecules on the target protein.

Keywords: -tif, tegument, hSV-1, Modeller, deep viewer-Swiss pdb viewer, procheck, Rasmol, docking, arguslab, chimera.

Ашиш Рунтала, Амит Кумар Синг. Аспекты медикаментозного лечения ВПГ-типа 1 Саратовский научно.

медицинский журнал, 2010, том 6, № 2, с. 353-357.

Альфа-транспорт-индуцирующий фактор является вирионом вируса простого герпеса типа 1, покрытого протеином между капсидом и оболочкой, связанным с клеточными протеинами и обеспечивающий транспортную функцию. Альфа-транспорт-индуцирующий фактор стимулирует быструю расшифровку генетического аппарата ВПГ типа 1 в течение вирусной репликации. Наличие или отсутствие функционального альфа транспорт индуцирующего протеина в условиях эксперимента всегда связано с вирусной активностью. его роль после перенесенной латентной вирусной инфекции ВПГ типа 1 – значительна. до настоящего времени не было разработано лекарственных препаратов на основе протеина для лечения ВПГ типа 1. Поэтому его структурные особенности важны для создания нового препарата. Сравнительный анализ белка заключался в его поэтапном изучении. четыре различные молекулярные лиганды, т.е. Аденозин -3'-5'-дифосфат, P1-(5’-Аденозил) P4-(5’(2’-ди-окситимидил)) Тетрафосфат, 9-Гидроксиметилгуанин, 9-(4-Гидроксибутил)-N2-Фенилгуанин – были взяты для изучения. В результате исследования P1-(5’-Аденозил) P4-(5’-(2’-диокси-Тимидил) Тетрафосфат – оказался лучшей лигандой, т.к. показал наименьшую энергию – 9.238 ккал/моль. доказано, что лучшая лиганда связывает различные части транспорт индуцирующего протеина и таким образом может быть выделен для успешной борьбы с ВПГ типа 1, с подобным эффектом множества лекарственных молекул в основе протеина.

Introduction. proteins are the basis of functional name herpes comes from the latin term herpes which, in machinery in a cell. proteins are the building blocks of turn, comes from the greek word herpein which means to an organism as they are involved in regulating the vari- creep [3]. this reflects the creeping or spreading nature ous activities [1]. Structural representations are the most of the skin lesions caused by many herpes virus types.

useful parameters for understanding protein function. So, once a patient has become infected by herpes virus, the the Structure prediction aims at deriving detailed struc- infection remains for entire life. the initial infection may tural information of high resolution for understanding bio- be followed by latency with subsequent reactivation.

logical function qualitatively [2]. understanding structure herpes viruses infect most of the human population and has potential applications in the various genome proj- persons living past middle age usually have antibodies ects being undertaken, such as mapping the functions of to most of the herpes viruses except human herpes Viproteins in metabolic pathways for whole genomes and rus type 8 (hhV-8).

deducing significant evolutionary relationships. herpes viruses are enveloped viruses. they bud herpes viruses are a leading cause of human viral from inner nuclear membrane, modified by the inserdisease, second only to influenza and cold viruses. they tion of herpes glycol-proteins. in the mature virus, these are capable of causing overt disease or remaining silent glycoproteins determine the cell to be infected because for many years only to be reactivated, like shingles. the of the availability of the appropriate receptors. the viral membrane is quite fragile and a virus with a damaged envelope is not infectious. this essentially means that Corresponding author – ashish Runthala e-mail: ashish.runthala@gmail.com the virus readily falls apart and so the virus can only be Saratov Journal of Medical Scientific Research. 2010. Vol. 6. № 2.

354 инфекционные БолеЗни obtained by direct contact with mucosal surfaces or se- sence of functional -tif protein in vivo with respect to cretions of an infected person. besides drying, the virus viral replication and latency. a hypothesis for the mechais also sensitive to acids, detergents and organic solvents nism of latent hSV-1 infection that includes a role for this as might be expected for a virus with a lipid envelope. protein is also proposed.

these viruses have a doughnut shaped capsomere Methods. homology modeling was carried out, as of about 100-200 nm in diameter with an icosahedral we found a high homology between target sequence of nucleocapsid, containing 162 capsomeres. the space -tif and the known structures. this process is concepbetween the envelope and capsid is called tegument. it tually very simple. the target sequence is aligned with contains virally-encoded proteins and enzymes involved known structures in protein data bank (pdb) database in the initiation of replication. -tif, a herpes simplex vi- through basic local alignment Search tool in the pdb rus type 1 (hSV-1) tegument protein, in association with dataset (pdb-blaSt) tool using position-Specific iteratcellular proteins, transactivates viral immediate early ed blaSt algorithm (pSi-blaSt) [6, 7] available on nagenes. in order to examine the role of -tif during acute tional centre for biotechnology information (ncbi) web and latent infection in vivo, a mutant virus containing a server with the already modeled sequences, for which the 12-base-pair insertion in the -tif gene, lacking trans- structure has already been determined by experimental activating function of -tif was examined in mice. fol- methods. faSt alignment (faSta) format of this -tif lowing corneal inoculation, mutant parental virus (17+) target protein can be accessed from genoMe bank and the revertant (1814R) strains replicated effectively (genbank) with accession number aaa45862.1.

in eyes and trigeminal ganglia with 30 - 60% mortality pSi-blaSt on the target sequence resulted in Virion rates. protein 16 (Vp16) as the known template structure to neither equal plaque forming units (pfu) nor par- model -tif protein sequence, as it showed highest 86% ticle numbers replicated in trigeminal ganglia and there- sequence similarity, with 2e-180 e-value, which is an fore don’t kill any mice. even low inoculated amounts are excellent score for a template structure. this template is sufficient to establish latent infection in some animals. a transcriptional Regulatory protein [9]. its chain a is the Since no infection is detected at all in mouse trigeminal template for the target sequence, being the conserved ganglia, it poses a target regarding the commencement core of the herpes Simplex Virus transcriptional of latency soon after primary infection. latency with the Regulatory protein. its conformational details were taken infection of reverent strain 1814 is detected right after from protein data bank (pdb) databank [10].

the presence of virus in the sensory ganglia, during 24 Results.

to 48 hour tenure after infection. thus, though -tif may Modeling by Modeller9vbe required for lytic infection in vivo, it is dispensable Modeller9v7 was used to generate 100 structural for the establishment of reactivation from latent infection. model decoys which were then checked for their dope these details support the hypothesis that the latent and (discrete optimized potential energy) scores along with lytic pathways of hSV-1 are distinct and that latency is the z-score for the statistical accuracy of the prediction.

established soon after infection without any requirement through energy graph displayed in figure 1, model for viral replication. however, -tif levels reaching neu- was found to have the reasonably low dope score of ronal nuclei may be a critical determinant of lytic or latent -47037.96484with the lowest z score of 0.18014 and was infection. -tif is a hSV-1 virion protein present in the thus found to be the most stable model conformation.

tegument layer between the capsid and the envelope. Assessment -tif stimulates the transcription of hSV-1 immediate the model was obtained by homology modeling. but, early genes during lysis and virus replication. this study there is a need to access this resultant model for the examines the consequences of the presence and ab- errors. the resultant structure was assessed by various Figure1: normalized dope or z score of analysis for selecting the best model decoy among generated models, shows that model 91 has the reasonably low dope score of-47037.96484 forming the best set with the lowest z score of 0.18014 and was thus selected for later analysis Саратовский научно-медицинский журнал. 2010. Том 6. № 2.

INFeCtIOUS DISeASeS Table -TIF protein conformation prediction was found to be the native state considering the following Ramachandran analytical statistics number of percentage residues Most favoured regions 356 85.additional allowed regions 42 generously allowed regions 9 2.disallowed regions 11 2.non-glycine and non-proline 418 residues end residues ( excluding glycine & proline) glycine residues proline residues Figure 2: Ramachandran plot for the best model highlights the total number of residues structural details, showing the native fold for the protein tools, described below. this was all needed for the structural validation of the resultant structure of the modeling process.

RMSD (Root Mean Square Deviation): first it is logical to confirm that the designed -tif conformation is not digressing from the template and the possible native state [18]. it can be easily studied by determining the RMSd of -tif conformation against Vp16 template structure in most of the protein structural visualization programs like deepView, Swiss pdb Viewer etc. the RMSd value, considering the alpha carbons, was determined to be 1.17a° and 1.16a° when backbone atoms and side chain atoms were considered.

Whatcheck: through protein Motif (promotif) then, it was revealed that this structure has a variety of structural details [12]. the -tif has 1 chain with 3 strands Figure 3: -tif protein of hSV-1. its secondary structure is having the number and percentage of alpha helices and highlighted, helices – Red 3, 10 helices respectively equal to 10, 34.7% and 2, 1%.

Strands-green, turns – cyan. figure clearly displays high the 3 strands of this structure lies in the locations of 255proportion of turns as compared to strands. it also depicts that helix forms the hydrophobic core of the protein -tif 258, 277-280 and 283-284 as displayed in figure 2.

Procheck: protein Structure checks (pRocheck) showed that the structure having 490 residues has 82.5% residues lying in the core, 10% in allowed region, 2.2 % in generously allowed region and 2.6% in disallowed region [15]. overall Ramachandran plot showed that 85.2% residues lie in the most favorable region. no main chain or side chain parameters were found worse in the depicted structure. all residues showed a maximum deviation of 4.0 a° with 12 bad contacts throughout the resultant conformation. bond lengths and bond angles were also found to be 96.7 % and 87.5% in the structurally stable region.

the procheck.prm file was set according to our desired parameters. procheck v. 3.3 was used for the analysis. this tool resulted in 10 different plots. these were the Ramachandran plot, glycine & proline Ramachandran plot, side chain torsion angles chi1-chi2 plots, Main-chain parameters, Side-chain parameters, Residue properties, Main-chain bond length distributions, Mainchain bond angle distributions, Root Mean Square distances from planarity, distorted geometry plots; overall g-value is less than -0.5 which is considered as a good Figure 4: Structural details of different ligands screened for value. procheck result is summed up below (table 1 and docking -tif protein. A. adenosine-3’-5’-diphosphate, B.

table 2).

p1-(5’-adenosyl) p4-(5’-(2’-deoxy-thymidyl)) tetra phosphate, C 9-hydroxymethylguanine, D 9-(4-hydroxybutyl)-n2- Model Visualization: Structural models were visualphenylguanine ized by university of california San-francisco (ucSf) Saratov Journal of Medical Scientific Research. 2010. Vol. 6. № 2.

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