Re-dock of Roscovitine Against Human Cyclin-Dependent Kinase 2 with Molegro Virtual Docker

Transcription

1 Tutorial Re-dock of Roscovitine Against Human Cyclin-Dependent Kinase 2 with Molegro Virtual Docker Prof. Dr. Walter Filgueira de Azevedo Jr. walter@azevedolab.net azevedolab.net 1

2 Introduction In this tutorial, we show how to re-dock roscovitine to the ATP-binding pocket of cyclin-dependent kinase 2 (CDK2). This structure was first solved in 1997 (De Azevedo et al., 1997) using X-ray crystallography. The structure of the complex was solved at 2.4 Å resolution. Roscovitine is a competitive inhibitor with IC 50 in the nanomolar range. The figure here shows the electrostatic molecular surface of ATP-binding pocket with the structure of the inhibitor bound to it. CDK2 has been intensively studied as target for development of anticancer drugs (De Azevedo, 2016). Electrostatic surface for ATP-binding pocket of CDK2 in complex with roscovitine. Red and blue colors indicate negative and positive charges, respectively. Neutral regions are shown in white. This figure was generated using Molegro Virtual Docker (Thomsen & Christensen, 2006). PDB access code: 2A4L. 2

3 Introduction To run this tutorial you need to have the Molegro Virtual Docker (MVD) (Thomsen & Christensen, 2006) installed and running in your computer. In addition, you need have internet access. You should go to the Protein Data Bank (PDB) (Berman et al., 2000; Berman et al., 2002; Westbrook et al., 2003) ( and download the atomic coordinates for CDK2 in complex with roscovitine (PDB access code: 2A4L) (De Azevedo et al., 1997). Electrostatic surface for ATP-binding pocket of CDK2 in complex with roscovitine. Red and blue colors indicate negative and positive charges, respectively. Neutral regions are shown in white. This figure was generated using Molegro Virtual Docker (Thomsen & Christensen, 2006). PDB access code: 2A4L. 3

4 Re-docking Using Molegro Virtual Docker (Flowchart) In the flowchart below, we see the main steps to re-dock a ligand in the structure of a protein using Molegro Virtual Docker (MVD) (Thomsen & Christensen, 2006). PDB File Detect Cavities Customize Search Algorithm Analyze Docking Results Load PDB into MVD Docking Wizard Define Pose Clustering Poses and Docking Results Identify Active Ligand Select Reference Ligand Verify Errors and Warnings Select Docking View Choose Scoring Function and Define Binding Site Set up Docking Execution 4

5 Re-docking Using MVD We consider that you have MVD installed in your computer, and it is open, as shown below. We used the version 6.0, but this tutorial should work for earlier version. We used MVD for Windows, it is mostly the same for Mac OS X and Linux versions. 5

6 Re-docking Using MVD (Import Molecule) To load a PDB file, click on File-> Import Molecule We could also drag and drop the PDB file on the graphical screen. 6

7 Re-docking Using MVD (Import Molecule) Go to the directory, where you have the PDB file (2a4l). Click on the PDB file and open it. 7

8 Re-docking Using MVD (Import Molecule) You have the following pop-up window, that shows the PDB file content. Click on Preparation. 8

9 Re-docking Using MVD (Import Molecule) Change Assign All Below toalways. 9

10 Re-docking Using MVD (Import Molecule) Now click on Import. 10

11 Re-docking Using MVD (Import Molecule) You have the molecule on the graphical screen. On the left, you have the Workspace Explorer, click on the check boxes on the left of each content to turn on and off the visualization of the specific part of molecule. 11

12 Re-docking Using MVD (Import Molecule) Clicking on the + signal to expand the tree. For instance, click on + signal on the left of Water. 12

13 Re-docking Using MVD (Import Molecule) Now you have the 82 water molecules, which were found in the crystallographic structure 2A4L. Click on the - to return to the previous situation. 13

14 Re-docking Using MVD (Import Molecule) Let s check the ligands. Click on + on the left of the Ligand. 14

15 Re-docking Using MVD (Active Ligand) You have as active ligand, RRC_300[A], which is the code for roscovitine. You were lucky, MVD not always find the right active. It is better to check at the PDB site, for information about ligand. The active ligand is the one to be re-docked. 15

16 Re-docking Using MVD (Active Ligand) Keep in mind its complete information (RRC_300[A]), you need it later on to specify the reference ligand in the MVD Docking Wizard. It could happen that you have hundreds of ligands, but only one is the active ligand. 16

17 Re-docking Using MVD (Active Ligand) If you are not sure which ligand to choose to re-dock, you should get additional information about the molecular system you are about to simulate. For instance, for enzymes, most likely the active ligand is the inhibitor, with binding-affinity information. 17

18 Re-docking Using MVD (Docking View) Click on View->Docking View, as shown below. 18

19 Re-docking Using MVD (Docking View) MVD change the representation of the ligand, from Ball and Stick to Stick. Uncheck the boxes for water and protein that you will have a clear view of the ligand. 19

20 Re-docking Using MVD (Docking View) This is the crystallographic position of the ligand. Bring back protein and water molecules. 20

21 Re-docking Using MVD (Detect Cavities) Click on Preparation->Detect Cavities, as shown here. 21

22 Re-docking Using MVD (Detect Cavities) Click OK. 22

23 Re-docking Using MVD (Detect Cavities) You have potential binding pockets shown in green. 23

24 Re-docking Using MVD (Detect Cavities) The active ligand should be at least partially inserted in the predicted cavity. 24

25 Re-docking Using MVD (Docking Wizard) We are ready to re-dock. Click on Docking->Docking Wizard. 25

26 Re-docking Using MVD (Reference Ligand) In the new window, you have to choose the reference ligand (RRC_300[A]), as shown here. 26

27 Re-docking Using MVD (Reference Ligand) Click Next. 27

28 Re-docking Using MVD (Scoring Function) Now we have to choose the scoring function. MVD has four options, shown here. 28

29 Re-docking Using MVD (Scoring Function) We choose MolDock Score and click all options for ligand evaluation, except for Displaceable Water, as shown below. Then we click Next. 29

30 Re-docking Using MVD (Search Algorithm) Now we have to choose the search algorithm, we also have four options, as shown below. 30

31 Re-docking Using MVD (Search Algorithm) We choose MolDock Optimizer. 31

32 Re-docking Using MVD (Search Algorithm) We changed the number of runs to 20, the rest we leave the default values. Then we click Next. 32

33 Re-docking Using MVD (Pose Clustering) We changed the Max number of poses returned to 50 and modify the other parameters as shown below. 33

34 Re-docking Using MVD (Errors and Warnings) No Errors and Warnings. Click Next. 34

35 Re-docking Using MVD (Docking Execution) Now we have to choose where the results will be stored. Choose the same folder where you have your PDB file. Click on to choose the directory. 35

36 Re-docking Using MVD (Docking Execution) Move to the directory you want to leave the docking results and click OK. 36

37 Re-docking Using MVD (Docking Execution) Change pose format to mol2, as shown below. 37

38 Re-docking Using MVD (Docking Execution) You are ready to dock. Click Start. 38

39 Re-docking Using MVD (Docking Execution) The process should start, and you can follow the docking process. 39

40 Re-docking Using MVD (Docking Execution) Once finished, you get the message Finished. Your results are in the file DockingResults.mvdresults. You can check this file drag the Icon Results to the graphical screen. 40

41 Re-docking Using MVD (Docking Execution) Click on Close. 41

42 Re-docking Using MVD (Docking Results) MVD generated 1000 poses. We could include additional terms for analysis of the docking results clicking on Settings. 42

43 Re-docking Using MVD (Docking Results) Click on additional terms to assess pose protein interactions. 43

44 Re-docking Using MVD (Docking Results) Click on Table. 44

45 Re-docking Using MVD (Docking Results) Now we change the Sorting Criteria to any of the available scoring functions, for instance MolDock Score. 45

46 Re-docking Using MVD (Docking Results) As we can see, ranking poses with MolDock Score generated poses with RMSD < 2.0 Å. We could visualize clicking on the box on left and then clicking OK. 46

47 Re-docking Using MVD (Docking Results) We could also generate a CSV file with all scoring function values for all poses, clicking Edit-> Check All. 47

48 Re-docking Using MVD (Docking Results) Then click on Open checked poses on Molegro Data Modeller. 48

49 Re-docking Using MVD (Docking Results) You open Molegro Data Modeller (MDM) and have all poses in the spreadsheet, as shown below. 49

50 Re-docking Using MVD (Docking Results) Click on File->Export Dataset, as shown here. 50

51 Re-docking Using MVD (Docking Results) Click OK. 51

52 Re-docking Using MVD (Docking Results) Choose a folder and the file name for your results, and click on Save. MDM will save the results as a CSV file. You can now close MDM. 52

53 Re-docking Using MVD (Docking Results) Back to MVD, you can check your pose on the graphical screen. Click on Edit- >Uncheck All. 53

54 Re-docking Using MVD (Docking Results) Leave Sorting criteria as Re-rank Score. 54

55 Re-docking Using MVD (Docking Results) Click on the lowest score pose. Then click OK. 55

56 Re-docking Using MVD (Docking Results) To have only pose and ligand, uncheck all others. 56

57 Re-docking Using MVD (Docking Results) You have pose (red) and ligand (white). Use mouse middle button to zoom in and out. Use mouse left button to rotated the molecule. 57

58 Re-docking Using MVD (Docking Results) Below we have a nice view of the docking result. Click on File->Exit to finish the program. 58

59 Colophon This tutorial was produced in a DELL Inspiron notebook with 6GB of memory, a 700 GB hard disk, and an Intel Core i5-3337u 1.80 GHz running Windows 8.1. Text and layout were generated using PowerPoint 2013 and graphical figures were generated by Molegro Virtual Docker. This tutorial uses Arial font. 59

60 Author I graduated in Physics (BSc in Physics) at University of Sao Paulo (USP) in I completed a Master Degree in Applied Physics also at USP (1992), working under supervision of Prof. Yvonne P. Mascarenhas, the founder of crystallography in Brazil. My dissertation was about X-ray crystallography applied to organometallics compounds (De Azevedo Jr. et al.,1995). During my PhD I worked under supervision of Prof. Sung-Hou Kim (University of California, Berkeley. Department of Chemistry), on a split PhD program with a fellowship from Brazilian Research Council (CNPq)( ). My PhD was about the crystallographic structure of CDK2 (Cyclin-Dependent Kinase 2) (De Azevedo Jr. et al., 1996). In 1996, I returned to Brazil. In April 1997, I finished my PhD and moved to Sao Jose do Rio Preto (SP, Brazil) (UNESP) and worked there from 1997 to In 1997, I started the Laboratory of Biomolecular Systems- Department of Physics-UNESP - São Paulo State University. In 2005, I moved to Porto Alegre/RS (Brazil), where I am now. My current position is coordinator of the Laboratory of Computational Systems Biology at Pontifical Catholic University of Rio Grande do Sul (PUCRS). My research interests are focused on application of computer simulations to analyze protein-ligand interactions. I'm also interested in the development of biological inspired computing and machine learning algorithms. We apply these algorithms to molecular docking simulations, proteinligand interactions and other scientific and technological problems. I published over 160 scientific papers about protein structures and computer simulation methods applied to the study of biological systems (H-index: 36). These publications have over 4000 citations. I am editor for the following journals: 60

61 References Berman HM, Westbrook J, Feng Z et al. The Protein Data Bank. Nucleic Acids Res 2000; 28(1): Berman HM, Battistuz T, Bhat TN et al. The Protein Data Bank. Acta Crystallogr D Biol Crystallogr 2002; 58(Pt 6 No 1): De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strnad M, Kim SH. Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. Eur J Biochem. 1997; 243(1-2): De Azevedo WF. Opinion Paper: Targeting Multiple Cyclin-Dependent Kinases (CDKs): A New Strategy for Molecular Docking Studies. Curr Drug Targets. 2016;17(1):2. Thomsen, R.; Christensen, M.H. MolDock: a new technique for high-accuracy molecular docking. J. Med. Chem., 2006, 49(11), Westbrook, J.; Feng, Z.; Chen, L.; Yang, H.; Berman, H. M. The Protein Data Bank and structural genomics. Nucleic Acids Res 2003; 31(1): Last update on July 4 th