Creating a pharmacophore from a single protein-ligand complex

Transcription

1 1 Creating a pharmacphre frm a single prtein-ligand cmplex basic minutes User Cntrls Advanced cntrls (pt.) Macrmlecule Active site Dwnlad PDB file using -letter cde Discver the crrect ligand Fcus n active site and switch t active site Check and eventually crrect ligand chemistry Create a structure-based pharmacphre Ligand bx Create pharmacphre (buttn r menu) OR: Create pharmacphre fr Catalyst/MOE/ Phase Change bnd type Change atm type Create new bnd Mve t cre Mve t envirnment Cmpund switching Descriptin: Type 1ke6 in the upper right area f the screen and press the buttn Dwnlad 1ke6 [1]. The prtein will be dwnladed and displayed (this is the macrmlecule ) [2]. Click n the yellw bx within the prtein representing the ligand - an animated zm begins ending in [3]. Since ligand structures in the PDB nly cntain incmplete infrmatin, yu shuld always check whether all bnds are typed crrectly. Bnd types can be changed by selecting a bnd (by clicking n it either in the 2D r 3D ) and by using the retype bnd buttn [] r the keys 1, 2, r 3. Once the ligand is chemically crrect, create a pharmacphre by pressing Ctrl-F9 (Cmd-F9 n OS X) []. Where t g frm here: Pharmacphre mdeling: Creating shared and merged feature pharmacphres Custmizing pharmacphre creatin preferences Virtual screening 1 2 3

2 2 Pharmacphre mdeling: Create shared & merged feature pharmacphres using chemical features frm three PDB structures medium 1 minutes User Cntrls Advanced cntrls (pt.) Macrmlecule Active site Alignment Dwnlad PDB File 1ke6 Check & crrect ligand Create pharmacphre Add ligand and pharmacphre t alignment Repeat the last steps fr 1ke7 and 1ke8 Create a shared feature pharmacphre frm the 3 pharmacphres Align the three ligands t the new pharmacphre Ligand bx Create pharmacphre (buttn r menu) Add mlecule t alignment Add pharmacphre t alignment Generate shared feature pharmacphre (by features) Generate merged feature pharmacphre Set reference element Center all structures Alignment cntext menu Descriptin: Type 1ke6 in the upper right area f the screen and press the buttn Dwnlad 1ke6. The prtein will be dwnladed and displayed in macrmlecule. Click n the yellw bx t fcus n ligand LS2201. Check the ligand fr its crrectness. Press ctrl-f9 t create a pharmacphre (cmd-f9 n OS X). Use the data exchange widget and select add mlecule t alignment [1a] and the mlecule will be added t the alignment, increasing a cunter in the crrespnding tab. Use the data exchange widget and select add pharmacphre t alignment [1b] t d the same with the pharmacphre. Repeat these steps with 1ke7, and 1ke8. Finally, yu shuld have six elements in the alignment. Click n the Alignment tab t wrk with them. Select the three pharmacphres in the alignment list using Ctrl-click r multiple selectin [2], and press the Generate shared feature pharmacphre (by features) buttn [3]. A new pharmacphre called Shared [LS2201-1, LS3201-1, LS2-1] is appended t the list. Mark this element and make it the reference element using the Set reference buttn, which will change the display style f this element t red and bld. Nw use the ctrl key (cmd n OS X) t additinally select the mlecules identified by the blue icns []. Additinally, hide the three pharmacphres derived frm 1ke6, 1ke7, and 1ke8 by clicking n the eye symbls n the right next t these alignment entries []. Nw press the align buttn. Yu will see the three ligands aligned t the shared feature pharmacphre derived frm the three PDB cmplexes []. Where t g frm here: Custmizing pharmacphre creatin preferences Virtual screening in LigandScut 1 2 3

3 3 Virtual screening and cmparing active/inactive hit retrieval rates advanced 1 minutes User Cntrls Advanced cntrls (pt.) Macrmlecule Active site Create pharmacphre fr virtual screening Initiate virtual screening Refine pharmacphre mdel Repeat virtual screening Ligand bx Create pharmacphre (buttn r menu) Initiate virtual screening Virtual screening with mitted features Descriptin: Virtual screening aims at the maximum enrichment f active cmpunds in a hit list. Therefre, such methds are usually validated by assessing the accuracy f discriminatin between actives and decys. A gd pharmacphre mdel will be able t identify a significant prtin f knwn active cmpunds and as few decys as pssible. In this tutrial, tw different types f precalculated multicnfrmatinal databases are available: A library cntaining a selectin f knwn active cmpunds and a library f decys. Type 1ke7 in the upper right area f the screen, press the buttn Dwnlad 1ke7. Create a pharmacphre f the prteinligand cmplex as described in the LigandScut tutrial sheet 1 Creating a pharmacphre frm a single prtein-ligand cmplex [1]. Perfrm virtual screening using a multi-cnfrmatinal database with knwn active cmpunds (filename cdk2- ligands.ldb ) fr screening using the Screen pharmacphre against external library item lcated in the Pharmacphre menu [2]. Virtual screening will be initiated after selecting the library file (press the butten with the three dts t pen a file dialg) and the number f mittable features [3]. Since the initial pharmacphre was derived frm all the interactins f nly ne single ligand, it is rather restrictive and retrieves nly few hits. Dwnsize the number f features by deleting less imprtant features [] r increase the tlerances f the features by selecting a feature and then chsing Pharmacphre -> Increase Selected Feature Tlerance frm the menu. Cmpare the rati f active cmpunds and decys (library filename cdk2-decys.ldb ) retrieved by yur pharmacphre in rder t estimate its predictive pwer []. Further adapt the pharmacphre in rder t imprve the rati f actives and decys btained by virtual screening: Remve nn-essential chemical features and exclusin vlume spheres in rder t increase sensitivity; adapt the size f the feature spheres by pressing ctrl-shift-plus r the crrespnding menu entry ( Pharmacphre - Decrease Selected Feature Tlerance ) r use the pharmacphre frm tutrial card 2 using the data exchange widgets [6]. Where t g frm here: Pharmacphre mdeling: Creating shared and merged feature pharmacphres Custmizing pharmacphre creatin preferences Analyzing screening results

4 Analyze LigandScut pharmacphre screening results medium 10 minutes Prerequisites: Virtual screening hit list User Cntrls Advanced cntrls (pt.) Library (spreadsheet) Hierarchy Create r imprt screening results Srt virtual screening hit list Inspect and cmpare hits Discver cntrls f the library Library (Spreadsheet) Viewer cntrls Calculatin f Gaussin Shape Similarity Scre Descriptin: Perfrm virtual screening t btain a hit list as described n tutrial card Virtual screening and cmparing active/inactive hit retrieval rates r insert external screening results frm a library file via the File - Insert menu entry. Results f virtual screening are depicted in the library [1]. Srt the hit list by a single click n the Pharm. Fit Scre table header [2]. Yu may invert the srting rder by an additinal click. Select a cmpund f interest by single click n the crrespnding table rw t be depicted in the active site tgether with the riginal ligand [3]. Multiple cmpunds are selected when hlding shift r ctrl (cmd n MAC OS X) while clicking. Keep cmpunds visible independent frm the current selectin state by clicking the eye symbl in the table left t the mlecule name in the library spreadsheet []. The visibility f the cre mlecule (i.e., the riginal ligand) and ther items in the active site is tggled with the eye symbl lcated in the hierarchy n the right side next t the mlecule name []. Select a custm clr fr the cre mlecule using the square icn next t the eye icn in the hierarchy. Calculatin f the Gaussian Shape Similarity Scre ffers further functinality fr scring. Use the Calculate Gaussin Shape Similarity Scre functin lcated in the Library menu fr calculatin. A new prperty Shape Tanimt Scre will be added in the library and yu can use the spreadsheet functinality fr srting and ranking as described abve. Where t g frm here: Pharmacphre mdeling: Creating shared and merged feature pharmacphres Custmizing pharmacphre creatin preferences 2 3 1

5 View and rescre dcking pses with pharmacphres medium 10 minutes Prerequisites: dcking pses frm external surces User Cntrls Advanced cntrls (pt.) Library (spreadsheet) Hierarchy Dwnlad PDB File Switch t active site Imprt dcking pses Analyze dcking pses Rescre dcking pses Align dcking pses t pharmacphre Discver the er cntrls f the library Library (Spreadsheet) Viewer cntrls Descriptin: Dwnlad 1ke7 and create the pharmacphre f the prtein-ligand cmplex. Imprt the crrespnding dcking pses stred in MDL SDF file frmat (tutrial filename 1ke7-dcking.sdf ) via the File - Insert menu entry [1]. Make sure that the Align Mlecules Autmatically t Shwn Pharmacphre ptin lcated in the Library menu is deactivated [2], which ensures that the crdinates f the dcking pses relative t the prtein are preserved. The cmpunds are imprted t Ligand- Scut including all prperties given in the SD file, such as the dcking scre. Srt the list f dcking pses by a single click n the table header crrespnding t the dcking scre [3]. Invert the srting rder by an additinal click if necessary. LigandScut ffers fur different ptins fr rescring dcking pses based n the 3D pharmacphre that is currently visible in LigandScut: The calculatin f the pharmacphre fit f imprted dcking pses cnsidering r ignring excluded vlume spheres, and the calculatin f the pharmacphre fit f the dcking pses after alignment t the pharmacphre cnsidering r ignring excluded vlume spheres. Alignment t the pharmacphre befre scring may be useful t ptimize dcking pses []. All fur mdes are accessed via the Library menu. Yu can mdify the pharmacphre by deleting features, adding excluded vlumes, etc. t reflect the imprtant features f the binding site that shuld be fulfilled by the highest ranked hits. Alignment f imprted dcking pses t the shwn pharmacphre is perfrmed autmatically upn selectin f a spreadsheet line (i.e. a single dcking pse) if the Align Mlecules Autmatically t Shwn Pharmacphre ptin lcated in the Library menu is activated. Pses may als be aligned t the pharmacphre using LigandScut s alignment algrithm using the Align buttn lcated in the upper right crner f the library []. Where t g frm here: Custmizing pharmacphre creatin preferences 1 2 3

6 6 Espress: Create ligand-based 3D pharmacphres intermediate 1 minutes User Cntrls Advanced cntrls (pt.) Ligand-based Mdeling Imprt ligands Cluster ligand-set in 3D Generate ligand-based pharmacphre mdel 3D Clustering Generate ligand-based pharmacphre mdel (buttn r menu) Use different settings fr cnfrmatinal mdel generatin Use different settings fr clustering Use different settings fr ligand-based pharmacphre mdel generatin Descriptin: Open the smiles file cntaining yur ligands ( cdk2.smi ) by chsing the File-Open menu. The ligands are imprted and shwn in 2D n the right side f the screen [1]. Set all candidates fr the mdel t training set by chsing Ligand-Set -> Flag Selected Mlecules as Training Set. Cluster the ligand set accrding t their 3D pharmacphre characteristics using the cluster buttn n the bttm f the 3D [2]. Keep the default setting in the fllwing dialg and start the clustering prcess by pressing the OK buttn. Espress will create a new clumn in the ligand table called Cluster ID. Srt the table using this clumn by clicking n the clumn header Cluster ID. Set all mlecules with cluster ID 1 t test set by using the table cntrls [3]. Nw click n the buttn Create ligand-based pharmacphre [] using the default values t start pharmacphre generatin. A ligand-based mdel will be created and displayed in the 3D. Feature patterns [] in the table indicate, which features are met by which ligand. A click n a clred square indicates, which feature is linked t the crrespnding square. The list n the right lwer crner f the screen shws several pharmacphre slutins: by selecting a different slutin, it is shwn in the 3D with the ligands aligned accrdingly [6]. Yu can use the data exchange cntrls n the upper right crner f the 3D t mve the currently shwn pharmacphre t ther s (e.g. screening) r save it using the File -> Save menu and selecting a file type that represents pharmacphres. Where t g frm here: Align structure-based pharmacphre mdels with ligand-based nes Virtual screening in LigandScut

7 7 Screen several databases using the Screening Perspective intermediate minutes User Cntrls Advanced cntrls (pt.) Screening Add pharmacphre mdel t screening Lad virtual database Perfrm virtual screening Lad Screening Database (Buttn) Perfrm Screening (Buttn) nne Descriptin: In cntrast t the virtual screening prcedure in the Structure-based Mdeling perspective which is designed fr pharmacphre mdel refinement with respect t ligand-binding pcket prperties, the Screening perspective prvides a fast and cnvenient way f perfrming multiple virtual screens with different screening databases against yur final pharmacphre mdel, which can be useful fr validatin. Imprt yur favrite pharmacphre mdel (e.g. the ne created frm tutrial card 1 r 6) int the Screening perspective either by using the Exchange Data Widget [1] r the Open entry in the File menu. Click n the Lad Screening Database buttn [2] t lad the screening database cdk2-ligands.ldb. Enable the laded screening database fr screening by clicking n the grey buttn left t the screening database entry [3]. Nw, select the pharmacphre mdel frm the Pharmacphres list t make sure it is shwn in the 3D. Next t the Lad Screening Database buttn, the Perfrm Screening and Delete Pharmacphre buttn will be enabled. Click n the Perfrm Screening buttn t initiate the virtual screening. View the results in the table at the bttm f the screen []. Yu can add mre databases and pharmacphres fr further virtual screening experiments. Please make sure that nly ne pharmacphre is visible befre yu start screening. Where t g frm here: Analyze screening results Advanced screening: ROC curves and mdel cmbinatin 1 2 3

8 8 Advanced screening: ROC curves and mdel cmbinatin advanced 1 minutes User Cntrls Advanced cntrls (pt.) Screening Add pharmacphre mdels t screening Lad virtual database Perfrm virtual screening Plt ROC curve Cmbine mdels by OR Lad Screening Database (Buttn) Perfrm Screening (Buttn) Plt ROC Curve (Buttn) Blean Expressin (Field) Descriptin: In cntrast t the virtual screening prcedure in the Structure-based Mdeling perspective which is designed fr pharmacphre mdel refinement with respect t ligand-binding pcket prperties, the Screening perspective prvides means fr statistical evaluatin f screening perfrmance. Imprt the structure-based pharmacphre mdels frm PDB entry 1ke7 (tutrial card 1) and the ligand-based CDK2 pharmacphre (tutrial card 2) int the Screening perspective either by using the Exchange Data Widget [1] r, alternatively, lad a saved versin using the Open entry in the File menu. Click n the Lad Screening Database buttn [2] t lad the screening database cdk2-ligands.ldb, and click the same buttn again and lad the database cdk2-decys.ldb. Click nce n the tri-state selectr left t the database names t mark cdk2-ligands.ldb as a database cntainaing actives (green database symbl), and twice n the tri-state slectr left f cdk2-decys.ldb t mark this database as decys (red database symbl) [3]. Nw, select the pharmacphre mdel 1ke7 frm the Pharmacphres list t make sure it is shwn in the 3D. Click n the Perfrm Screening buttn t initiate the virtual screening []. After screening has finished, press the Plt ROC Curve buttn [] t plt a ROC curve [6]. Nw select bth pharmacphres in the Pharmacphres table. Numbers in curly brackets ( {1} and {2} ) indicate the number f the pharmacphre. Enter 1 r 2 int the blean expressin field [7] indicating that a virtual hit in this screening run shuld either fit int the first (1) r t the secnd (2) pharmacphre. Press the screening buttn [] again t re-screen, and the ROC curve buttn [] again t cmpare the virtual screening statistics. View the virtual hits in the table at the bttm f the screen [8]. Yu can add mre databases and pharmacphres fr further virtual screening experiments. Where t g frm here: Analyze screening results

9 9 Creating a pharmacphre single prtein-ligand cmplex Creating pharmacphre mdels infrm emptya binding sites advanced basic 2 minutes minutes Macrmlecule Macrmlecule Hierarchical Active site Active site User Cntrls User Cntrls Advanced cntrls (pt.) Advanced cntrls (pt.) Open PDB file Ligand bx Perfrm virtual screening Dwnlad PDB file using Ligand bx Change bnd type Create new binding site Ligand accessible surface Create new pharmacphre -letter cde Create pharmacphre Change atm type Switch t active site prperty display mdels frmnew retrieved Discver the crrect (buttn r menu) Create bndhit Create ligand accessible Place pharmacphre ligand OR: Create pharmacph- mlecules Mve t cre re fr Catalyst/MOE/ Fcus n active site and Mvemerged t envirnment surface features n ligand accessible Generate feature Phase switch t active site Cmpund switching Analyze the surface surface mdels Check and eventually cr Create pharmacphre Mve pharmacphre rect ligand chemistry mdel Create a single-click phar- features in 3D space macphre Descriptin: Descriptin: Type 1ke6 in the upper right area f the screen, an press the buttn Dwnlad 1ke6 [1]. The prtein will be dwnladed and displayed (this isright the macrmlecule [2]. Click n the Dwnlad yellw bx1ke7 within representing the ligand Type 1ke7 in the upper area f the screen, ) and press the buttn [1]. the The prtein prtein will be dwnladed and - an animated zm begins ending in [3]. Since ligand structures in the PDB nly cntain incmplete infrmatin, yu shuld displayed in the macrmlecule. In the hierarchical, select the amin acids that are knwn t frm the binding pcket f the always check whether all bnds are typed crrectly. Bnd types can be changed by selecting a bnd (by clicking n it either in selected prtein. Define the prtein binding site using Mlecule -> Create New Active Site [2]. Click n the yellw bx within the the 2D r 3D ) and by using the retype bnd buttn [] r the keys 1, 2, r 3. Once the ligand is chemically crrect, create prtein representing the site. Create(Cmd-F9 the binding pcket ligand accessible surface using Pharmacphre -> Create Ligand a pharmacphre bybinding pressing Ctrl-F9 n OS X) []. Accessible Surface. Clr the surface accrding t HBA and HBD ptential [3]. Select the apprpriate pints n the surface and place Where t gfeatures frm here: pharmacphre at apprpriate psitins using the Pharmacphre -> Create New Feature cmmand []. Repeat the Exprting a pharmacphre t Catalyst, Phase and MOE prcedure fr psitive and negative inizable features and use the mdel fr virtual screening. Place exclusin vlume spheres n Pharmacphre mdeling: Creating shared and merged feature pharmacphres prtein atms clse t the ligand accessible surface. Custmizing pharmacphre creatin preferences Where t g frm here: Insert hits btained frm VS int the binding site Create new pharmacphre mdels frm minimized hit structures btained 1 Refine the mdels 2 3

10 10 Creating a pharmacphre frm a single prtein-ligand cmplex Dcking a mlecule int a binding site using the built-in AutDck prgram intermediate basic 1 minutes minutes Macrmlecule Macrmlecule Active site Active site Table User Cntrls User Cntrls Advanced cntrls (pt.) Advanced cntrls (pt.) Dwnlad PDB file using Ligand bx Filter pses based n Dwnlad PDB file using Ligand bx Change bnd type letter cde Insert smiles string pharmacphre features -letter cde Create pharmacphre Change atm type Minimize pses Switch t active site Dck(buttn mlecule Discver the crrect r menu) Create new bnd Re-scre pses by estimating Insertligand mlecule int the Analyze pses OR: dcking Create pharmacph Mve t cre re fr Catalyst/MOE/ Fcus free Mve t envirnment energy f binding the binding siten active site and Phase switch t active site Cmpund switching Create new binding site Perfrm the dcking Check and eventually cr Dck mlecules int new experiment rect ligand chemistry binding site Create a single-click phar- macphre Descriptin: Descriptin: Type 1ke6 in the upper right area f the screen, an press the buttn Dwnlad 1ke6 [1]. The prtein will be dwnladed and displayed (this isright the macrmlecule [2]. Click n the Dwnlad yellw bx1ke7 within representing the ligand Type 1ke7 in the upper area f the screen, ) and press the buttn [1]. the The prtein prtein will be dwnladed and - an animated zm begins ending in [3]. Since ligand structures in the PDB nly cntain incmplete infrmatin, yu shuld displayed in the macrmlecule. Click n the yellw bx within the prtein representing the ligand in rder t zm int the always check whether all bnds are typed crrectly. Bnd types can be changed by selecting a bnd (by clicking n it either in binding site. Insert the mlecules that yu want t dck int the binding site, using File -> Insert [2]. Select the mlecules that yu the 2D r 3D ) and by using the retype bnd buttn [] r the keys 1, 2, r 3. Once the ligand is chemically crrect, create want t dck frm the by table belw the 3D windw. a pharmacphre pressing Ctrl-F9 (Cmd-F9Prceed n OS with X) [].the dcking experiment by using Mlecule -> Dck Ligands (AutDck.2) [3]. After the experiment is finished, the results will be displayed in the table [], each cmpund ranked accrding t the Where t g frm here: mst favrable interactin energy. Analyze the dcked pses by creating pharmacphre mdels fr ing the interactins with the Exprting a pharmacphre t Catalyst, Phase and MOE prtein. Filter the pses based n the interactins cmmn t thse f the riginal ligand. Pharmacphre mdeling: Creating shared and merged feature pharmacphres Custmizing pharmacphre creatin preferences Where t g frm here: Minimize the dcked pses Calculate the free energy f binding and the MMFF9 estimated binding enthalpies Calculate the pharmacphre fit scre based n the mdel generated frm the riginal ligand structure Create a new binding site by selecting amin acids with the muse (hld left buttn while pressing the Shift key and mve ver the regin yu want t investigate) and repeat the dcking experiment in the new binding site

11 11 Creating a pharmacphre frm a single prtein-ligand cmplex Analyzing Mlecular Dynamics Trajectries with LigandScut advanced basic 1 minutes minutes Macrmlecule Macrmlecule Active site Active site User Cntrls User Cntrls Advanced cntrls (pt.) Advanced cntrls (pt.) Open PDB file Ligand bx Create shared feature Dwnlad PDB file using Ligand bx Change bnd type Switch t active site Mlecular dynamics mdels -letter cde Create pharmacphre Change atm type Lad mlecularthe dynamics trajectry Widget Perfrm virtual Discver crrect (buttn r menu) Create newscreening bnd trajectry Playback trajectry MD derived ligandfile OR: Create pharmacph- with Mve t cre repharmacphre fr Catalyst/MOE/ Fcus active site and Create Mve t envirnment Analyze then trajectry pharmacphre mdels Phase switch t active site Cmpund switching Create pharmacphre mdels Check and eventually crmdels Align pharmacphre rect ligand chemistry Create a single-click phar- mdels macphre Descriptin: Descriptin: Type 1ke6 in the upper right area f the screen, an press the buttn Dwnlad 1ke6 [1]. The prtein will be dwnladed and (this is thethe macrmlecule [2]. Click dynamics n the yellw bx within the prtein the ligand - an Lad thedisplayed PDB file cntaining initial frame f ) yur mlecular simulatin (1ke7_start.pdb) [1].representing Click n the yellw bx within animated zm begins ending in [3]. Since ligand structures in the PDB nly cntain incmplete infrmatin, yu shuld the prtein representing the ligand in rder t zm int the binding site. Lad the DCD file f the mlecular dynamics trajectry always check whether all bnds are typed crrectly. Bnd types can be changed by selecting a bnd (by clicking n it either in (1ke7_trajectries.dcd) [2] and select the trajectry in the hierarchical [3]. Press the play buttn in the MD trajectry widget [] fr the 2D r 3D ) and by using the retype bnd buttn [] r the keys 1, 2, r 3. Once the ligand is chemically crrect, create ing the results f the simulatins []. Pause a pharmacphre by dynamics pressing Ctrl-F9 (Cmd-F9 n OSatX)any [].time and generate a pharmacphre mdel. Mve the pharmacphre mdel int the alignment perspective. Repeat this several times. In the alignment perspective, analyze the features t g frm here: [6]. thatwhere are cmmn in all mdels Exprting a pharmacphre t Catalyst, Phase and MOE Pharmacphre mdeling: Creating shared and merged feature pharmacphres Where t g frm here: Custmizing pharmacphre creatin preferences Align pharmacphre mdels generated frm MD trajectry Create shared feature and merged feature mdels frm the different pharmacphre families Perfrm virtual screening with the different mdels 1 2 3

12 12 LigandScut Mlecule Tables: Filtering & Exprt intermediate 1 minutes User Cntrls Advanced cntrls (pt.) IDBGEN Database generatr Structure-based Table Create a multicnfrmatinal database frm a smiles file Examine the cmpunds Calculate physicchemical prperties Filter and exprt mlecular structures IDBGEN GUI Lad LDB file Calculate standard prperties Apply filter rules Exprt 2D structure files Exprt t Micrsft Excel Descriptin: Start the IDBGEN GUI mlecular database generatr [1]. Chse a smiles file f mlecules as input file and specify the utput file. Set the icn FAST ptin fr cnfrmatin generatin [2]. After cmpletin, lad the resulting ldb file int the structure-based perspective and fcus n the table [3]. Calculate sme physicchemical parameters and mlecular descriptrs using Library -> Calculate Standard Prperties. Apply filters [, ] and the dynamically generated results. Where t g frm here: Exprt the mlecular structures tgether with the calculated standard prperties t a 2D SDF file Exprt the filtered cmpund list t a Micrsft Excel file

13 13 a pharmacphre frm a single prtein-ligand cmplex In Creating Silic Fragment Screening with LigandScut advanced basic 1 minutes minutes Macrmlecular Macrmlecule Screening Active site Active site User Cntrls User Cntrls Advanced cntrls (pt.) Advanced cntrls (pt.) Create structure based Ligand bx Re-dck resulting mlecules Dwnlad PDB file using Ligand bx Change bnd type pharmacphre mdel Create pharmacphre and minimize -letter cde Create pharmacphre Change atm type Switch t screening Set features Re-scre thenew mlecules Discver the crrect (buttnptinal r menu) Create bnd by Perfrm fragment screening Initiate virtual screening estimating the free ligand OR: Create pharmacph Mve t cre energy Fcus n active site and Library re fr Catalyst/MOE/ binding Mve t envirnment Inject hits back int prtein f switch Cmpund switching binding site t active site InjectPhase hits int prtein Exprt results Check and eventually cr Jin fragments t create binding site rect ligand chemistry new mlecules Create a single-click phar- Minimize hits Merge mlecules macphre Descriptin: Descriptin: Type 1ke6 in the upper right area f the screen, an press the buttn Dwnlad 1ke6 [1]. The prtein will be dwnladed and displayed (this isright the macrmlecule n Dwnlad the yellw1ke7 bx [1], within prtein representing the ligand - an Type 1ke7 in the upper area f the screen, ) press [2]. the Click buttn clickthe n the yellw bx, and generate a animated zm begins ending in [3]. Since ligand structures in the PDB nly cntain incmplete infrmatin, yu shuld pharmacphre mdel. Mve the mdel t the Screening. Lad the tutrial fragment library (fragmentstutrial.ldb). Set the left always check whether all bnds are typed crrectly. Bnd types can be changed by selecting a bnd (by clicking n it either in three features as ptinal by selecting them (press Shift+left muse buttn and draw a rectangle arund them) and use the the 2D r 3D ) and by using the retype bnd buttn [] r the keys 1, 2, r 3. Once the ligand is chemically crrect, create Pharmacphre -> Mark asctrl-f9 Optinal cmmand a pharmacphre byfeatures pressing (Cmd-F9 n [2]. OS Perfrm X) []. screening, examine the resulting hits, and inject the mst suitable nes back int the prtein binding site [3]. Reset the three left features as mandatry using the Pharmacphre -> Unmark Features as Wherecmmand, t g frm Optinal sethere: the right tw features as ptinal and repeat the screening. Examine the resulting hits, and inject the mst Exprting a pharmacphre t Catalyst, Phase and MOE suitable nes back int the prtein binding site. In the Structure-based perspective, minimize each fragment and bserve their Pharmacphre mdeling: Creating shared and merged feature pharmacphres behavir. Using the er ptin in the hierarchical, make tw fragments visible that verlap with ne atm. Delete ne f the Custmizing pharmacphre creatin preferences verlapping atms and merge the mlecules using the Mlecule -> Merge Visible Mlecules cmmand. Create a bnd between bth fragments. Minimize the resulting new mlecule and create a pharmacphre mdel. Analyze the interactins and cmpare them t the mdel generated frm the initial ligand. Repeat this prcedure fr ther fragment cmbinatins []. 1 Where t g frm here: 2 3 Re-dck, minimize, and re-scre newly created mlecules in rder t identify the mst interesting candidates Exprt the mst favrable designs as an Excel spreadsheet, r as 3D r 2D sdf files