In Silico Research: Inhibiting binding GTP on Methyltransferase of Dengue VirusJuan E Maldonado Weng Primary Article

Gustavo A Martínez

Abstract: The objective of this paper is to present the process of identifying a drug that will easily fight against the Dengue Virus (DNV). The process was begun using a model of the methyltransferase enzyme with a Guanosine Triphosphate (GTP) of the virus within an active spot. Methyltransferase is an enzyme that transfers a methyl group to the DNA of a cell. GTP is the molecule that produces the energy in order for that enzyme to transfer the methyl group properly. The purpose of developing this drug is to find a compound that will inhibit the transfer of methyl to the virus’ DNA. On this project, the Pymol program was used to generate three benzene rings were positioned in the pocket where the GTP normally connects to the enzyme. These rings are then to be used to create the pharmacophore model, which will show the hydrophobic regions. The hydrophobic regions are what will show which the compound will fit properly within the pocket in order to inhibit the GTP. That compound will be searched for in the ZINC Pharmer database. After processing the model through the database, over sixty thousand hits were found compatible in that region of the methyltransferase. These were filtered and separated into five different groups. From these groups, the twenty-five compounds with the highest affinity were chosen, and of those, the top three with the highest affinity were placed into the methyltransferase pocket in order to compare with the benzene rings and the GTP. The compound with the highest affinity (-10.4) was DENV-M2_1.

Introduction

Dengue is a serious life affecting disease infecting thousands of people while millions of others are at risk. The disease can be classified as three types of disease: Dengue Fever, Hemorrhagic Fever, or Shock Syndrome. “Antiviral drug discovery is becoming increasingly important due to the global threat of viral disease pandemics. Many members of the genus Flavivirus are significant human pathogens, among which dengue virus (DENV) alone poses a public health threat to 2.5 billion worldwide,

leading to 50–100 million human infections each year. Neither vaccine nor effective therapeutics is currently available for DENV. Development of a DENV vaccine has been challenging, because of the need to simultaneously immunize and induce a long-lasting protection against all four serotypes of DENV…” This statement was from a review paper by Christian G. Noble and his team. These diseases are very severe, and based on CDC reports from 2013; many common symptoms include joint and muscle pain, elevated corporal temperatures, and

Cartogram 1- Infection

“Cartogram of the annual number of infections for all ages as a proportion ofNational or subnational (China) geographical area”

Obtained from Bahtt et al. Review Paper 2013

Image 1 Image 2

severe headaches. These diseases in severe cases can cause death. According to CDC in 2010, there were 12,580 confirmed cases of people diagnosed with dengue in Puerto Rico. The tropical regions are under the most danger because the numbers of mosquitoes that transmit the disease prefer these areas.

The Aedes mosquito is the common carrier of the Dengue virus. The Dengue virus is a member of the family Flaviviridae and the genus Flavivirus. The Dengue virus has very similar structural properties to other members of the family like the West Nile Virus. The viral transmission poses a real threat to people and, with no cure available; the amount of cases will increase by the incoming years.

Through research by computer simulation, the search for a cure is easier. Utilizing many advance software and programming, the structure of important proteins can be ideally researched. With these structures, the main components, such as amino acids with their spacing arrangement and chemical properties, could be studied then inhibited. Through inhibition of certain useful structures, the viral infection could be stopped.

In the case of the Dengue Virus, the methyltransferase is an important protein for

the spread and development of the virus. “Methyltransferases (MTases) play key roles in normal physiology and human diseases through methylating DNA, RNA, and proteins. Almost all MTases use S-adenosyl-L-methionine (SAM) as a methyl donor and generate S-adenosyl-Lhomocysteine (SAH) as a by-product. Pharmacological modulation of MTases by small molecules represents a novel approach to therapeutic intervention in cancer and other diseases.” (Siew Pheng Lim et al, 2011) The methyltransferase transfers a methyl group from a donator towards an acceptor which could be a strand of DNA or RNA. This methyl group will help mark the genes needed to be expressed. This protein is needed for the virus to develop properly. The mechanism that the protein utilizes to have the appropriate energy is to bind with a GTP molecule. This molecule is the energy source that enables the methyltransferase to complete the transfers.

Through In Sillico research, the GTP, used for the Methyltransferase of DENV-2, can be inhibited utilizing a higher affinity compound. Finding a compound that could have a stronger force of attraction towards the site where GTP would be. If an alternate compound would take GTP’s place, then there is a possibility the protein would fail causing a cease in the virus’ effect on the organism.

Materials and Method

The first step, which was the most important, was to identify a biological problem and to undergo research to understand the virus. The methyltransferase 3D molecule structure was downloaded from the online Protein Database (pbd.org). This database provided much needed information and was very accessible. The molecule can be seen in image 1 as it would in the PyMol program. From the angle of the first image, there is a gap or pocket where a molecule could be placed. To see if it were true, the molecule went through a vigorous process to locate the “hotspots” and to observe where the GTP would bind. From there, the model in the PyMol program had the GTP properly placed into the pocket and the amino acids that interact well around it, as seen in Image 2.

A grid was constructed in order to seclude the pocket and the region where the GTP connects with the protein. Within the grid, several benzene rings were found, and with these, the binding site of the GTP. Benzenes are important structures which are made up of carbon rings. These structures help identify the methyltransferase’ pocket.

These three benzene rings were then used to create a pharmacophore model with the Ligand-Scout program. This model depicts the hydrophobic regions, the physical arrangement, and other chemical properties of the compound that will be used to filter out the search. From the millions of compounds in the database, the pharmacophore model will help find the ones that fit the pocket. The search engine utilized was ZINC Pharmer, which found

many matches. The processes were completed by the use of a cluster of servers working together.

From the results, a long list of compounds appeared. Organization was the most important asset from this step onward. Excel was utilized to have all the compounds in tables. From there, the table was assorted from highest to lowest affinity, and, finally, the compound with the highest affinity was chosen. This compound, along with the other two highest compounds, were modeled in PyMol and tested to see if there is a visual match with the benzenes. After visualizing the interactions, the next ideal step would be Bio Assay to test these compounds in a real world scenario.

Results

The structure of the pharmacophore model was run through the ZINC Pharmer. (The image of the pharmacophore model can be seen as image 3; the yellow spheres represent the benzenes and the gray spheres the amino acids and other molecules.) This enabled access to thousands of different compounds

Table 1: Highest Affinity Compounds

Rank 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

Image 3 Image 4

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that could possibly fit within the pocket of the methyltransferase and inhibit its interaction with the GTP. These compounds were filtered and divided into groups depending on their size or molecular weight.

These compounds had varying affinities, which means that they will attach to the pocket of the protein at different strengths or intensities. In a chart showing them from highest affinities, twenty-five compounds were separated, and the three with the highest affinities were chosen to be placed within the methyltransferase in order to 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; and DENV-M2_3, with an affinity of -10.2. The other twenty two compounds are shown in table 1. The affinity on the table varies from -10.4 to -9.8. From all the compounds collected, there were a total of a hundred and sixty-two (162) compounds, varying from -10.4 to -9.5, but there were over 25,000 compounds which had lesser affinity.

These three compounds were then placed into the pocket of the methyltransferase, and compared with the placement of the benzene rings and the GTP molecule. The compound that showed to occupy the most space and position itself in a similar way to the benzene rings was DENV-M2_1. These factors demonstrate the compound’s affinity of -10.4. The placement of the DENV-M2_1 also shows that there would not be any possibility for the GTP molecule to make its way into the pocket. (The image of the DENV-M2_1 molecule interacting at the target can be seen in image 4.) This proves the hypothesis stated that a high affinity compound can be found for methyltransferase.

Discussion

After many hours in front of a computer, many compounds have been explored but the Dengue Virus’ methyltransferase has finally found a compound deemed worthy as a GTP inhibitor. Thus, the hypothesis was proven.

This work has, theoretically, shown there is an existing compound that is capable of inhibiting the transfer of methyl to the DNA of the Dengue Virus. Further work with this project will be performing a Bioassay. This means that the compound and the protein will be tested physically and biologically, not just within a computer program. The compound, DENV-M2_1, could be proven as a possible treatment for the Dengue Virus. The virus continuously spreads by the mosquito, but if prevented, this possible new drug could save many lives that are at risk.

The possible advantages for this drug would be that it could help for the research of other viruses including the West Nile Virus. If the viruses have a similar structure, then it is possible for compound to effectively interact the same way. More research needs to be conducted with this compound for further implications.

Research via computer simulation is also an impressive technique because it helps being more precise and accurate with results. It would be more cost effective if a team studies three to five compounds rather than with every compound trying to find a match. This could help improve the way the scientific community confronts biological problems. This is a big improvement than rather taking stabs at the dark. The community can use the opportunity to expand its knowledge faster and more cost effective.

Acknowledgements

This research could not have been completed without the guidance of Dr. Héctor Maldonado and his In Sillico Research team. We are grateful for the opportunity to join them during the short time. Thanks to them, we had a great time learning their methods and using their tools. We hope to see the team tackle new problems and make new discoveries.

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