THINK can be started from the Windows menu START > All Programs > THINK or by double clicking on think.exe in the Windows Explorer. When THINK starts the console and explorer windows open. THINK uses a console window to enter commands or open dialogs. This is the main window used by the program. Additional windows will be created when necessary to display molecules, data or perform searches.

Items on the dialogs may appear as:

• Push buttons
• Radio buttons - sets of circular buttons denoting mutually exclusive options (eg display style)
• Check boxes - square boxes for items that may be independently toggled on or off. The box is checked when the when toggled on
• Choosers - drop-down sub-menus indicated by an arrow to the right of some text enclosed in a box
• Keyboard entry items
• Lists - either individual or multiple selections are supported depending on the context. Double-picking a name within the list is equivalent to a single selection and pushing the associated button.

Bubble help is available for dialog items, tools etc.

The THINK software can be used under Windows and Linux with the KNIME workflow environment. The THINK plugins are downloaded from http://www.treweren.com and extracted into the plugins folder under the KNIME directory tree. It is also necessary to create the environment variables THINK_EXEC as the path to the THINK software and THINK_WORKING as a path to a folder in which temporary files can be created.

Molecular modelling or informatics procedures are created, configured and executed by users as a visual workflow in KNIME. THINK using KNIME allows integration with in-house developed and other commercial tools such as those available from TRIPOS and Schroedinger. The main capabilities are illustrated by the following videos (with sound commentaries):
2D functionality Structure-based Virtual Screening Pharmacophores and map volume constraints Focused subset 3D De Novo

As the THINK nodes are designed for use with native KNIME nodes and those provided by other third party developers, applications might include using other nodes to create a table of molecules (which have connection tables in a SMILES or SDF column) and the outputs might be connected to other nodes for statistical analysis or further computation. KNIME allows much higher productivity than writing command mode scripts and using a workflow enables visualisation of the computational steps in the modelling procedure.

Each facility or utility in THINK has its own command, which may take one or more keywords to supply additional options. A keyword may take a value (eg to supply a filename). Commands are case independent and may be specified in upper, lower or mixed case.

The syntax for a THINK command is:

Command keyword[=value[,value]] [ keyword[=value[,value]] ...]

where items in [] brackets may be optional. A few commands (eg EXIT) do not take any keywords. Spaces are required to separate the command from any subsequent keywords, and between keywords. Any values required by a keyword must be specified using an "=" between the keyword and the value, with no spaces, ie keyword=value. If the keyword may take a list of values, these should be separated by commas, again with no spaces, ie keyword=value1,value2.

It is also possible to omit the keyword= for manditory keywords provided they are specified in the same order as the help file. Advanced users may find this practice saves typing but it should not be used in script files.

The HELP command will list all the commands. A full list of keywords and values available for any command may be obtained by issuing the command HELP followed by the name of the command for which information is required or a the command followed by a question mark.

THINK allows multiple commands to be specified on a single command line separated by a semi-colon (;).

Errors encountered while running KNIME workflows can be obscure and nodes developed by different third parties are likely to have different style error reporting.

If THINK detects an error, this is normally reported to the console window with an error number and some explanatory text. This information is also written to the log file (see section 1.3).

When THINK detects an error it will display a pop-up error message containing the error number, some explanatory text and three buttons: Continue, Cancel and More. Picking Cancel will cancel the operation immediately; picking Continue will allow the calculation to continue if possible. Picking the More button will display a second pop-up error window giving more information about the error. This information may not be very helpful to the user, but can be useful to the THINK developers when determining the cause of the error. The second pop-up window contains Continue and Cancel buttons; these have the same effect as their equivalents on the first window.

All messages from the first pop-up error window are echoed to the THINK console window and written to the log file (see section 1.3).

When THINK detects an error it will write the error message containing the error number and some explanatory text.

All messages from the first pop-up error window are echoed to the THINK console window and written to the log file (see section 1.3).

All commands issued during the execution of a THINK node are written to a file named output.log creating in the working directory except for the Display node for which the log file is named output2.log. The log file is overwritten when the next node is executed.

All commands issued during a THINK session are recorded in a log file. This is named "think[n].log" where n is a counter and is created in the current working directory. The log file contains the commands issued by the dialogs. A log file may sometimes be subsequently replayed as a script by issuing the command "CALL xxx" where xxx is the name of the log file. However, in many circumstances it will need to be edited first using a text editor. Note that THINK will automatically create a new log file incrementing n in the name "think[n].log" each time the program is started.

All commands issued during a THINK session are recorded in a log file. This is named "think[n].log" where n is a counter and is created in the current working directory. A log file may sometimes be subsequently replayed as a script by issuing the command "CALL xxx" where xxx is the name of the log file. However, in many circumstances it will need to be edited first using a text editor. Note that THINK will automatically create a new log file incrementing n in the name "think[n].log" each time the program is started.

Conventionally molecules are assigned names or identifiers and these are usually the unique column identifiers in KNIME data tables. In addition, within molecules atoms have atom types, serial numbers and sometimes different group numbers. It is occasionally necessary to understand the molecule and atom specifications used by THINK.

Some dialogs require the user to identify a molecule. Occasionally a dialog requires atoms to be identified by their atom specifications (eg when using the ROTATE dialog rather than the mouse for rotation). To do this successfully as well as understand some output from THINK requires some understanding of the molecule and atom specifications used by THINK.

Many commands require the user to identify a molecule. Occasionally the user is required to identify atoms by typing their atom specifications (eg when using the ROTATE command). To do this successfully requires some understanding of the molecule and atom specifications used by THINK.

Symbols (see section 1.5.1) may be used to identify atoms or residues. This is a convenient method of identifying a group of atoms in a single operation (eg the active site of a protein). The symbol must be an array comprising one or more array elements, and each element contains a separate atom or residue specification. Each array element is taken in turn when the symbol is processed. The symbol name must contain more than four characters to avoid confusion with atom or residue names (eg SITEA would be interpreted as a symbol but SITE would be treated as a residue name).

The array is terminated by a blank or undefined element. When a symbol is used for the first time, this will occur automatically. However, if an array symbol is reused, and the new array contains fewer elements, it is important that a blank element is specified after the last valid element to ensure that THINK will determine the array length correctly. For instance, if the new array only contains three elements, then the fourth element should be set to a blank value.

If the symbol is to be used in place of an atom specification (eg when defining the bond for bond rotation) the array element may contain any portions of the full atom specification. For instance, CA_TYR(33) would refer to the alpha carbon in residue TYR(33); and (23:29) would refer to all atoms with serial numbers in the range 23-29. However, if the symbol is to be used in place of a residue specification, the array element must contain only the residue components of the full atom specification, ie residue(sequence)chain. The leading underscore "_" character must be supplied before the symbol name.

In the examples below the symbol names have been highlighted to distinguish them from the rest of the commands. See section 1.5.1 for full details on creating symbols.

1.5   Command Scripts and Symbols

The THINK command set includes simple control commands such as LET, WHILE and IF to enable the user to create command scripts. Apart from LET commands, these must be saved in files (they cannot be entered directly into the console window) and are played by issuing the command "CALL xxx" where xxx is the name of the script file. The "@" character may be used in place of CALL. Commands within scripts may be in upper, lower or mixed case. LET commands may be issued from command scripts or typed directly into the console window.

Command scripts may be nested up to 10 levels deep. A nested script is invoked by the command "CALL xxx", where xxx is the name of the script. A nested script ends, and control is returned to the calling script, when a RETURN command is encountered. All nested command scripts should finish with a RETURN command.

If a command script is interrupted by typing <CTRL-C> or picking the Cancel button, all script files are closed and control is returned to the THINK console window.

1.5.1 Symbols and Operators

Command scripts support local and global symbols. Local symbols only exist within the command script being executed (and any nested scripts below the current level) and are deleted at the end of the script. Global symbols persist after the script has terminated. Both types of symbol are set via the LET command: local symbols use the form "LET a = b" whereas global symbols use the form "LET a := b", replacing the "=" with ":=". Note that spaces are optional around operators and the "=" or ":=". Text strings should be enclosed within double quotes ""; names of symbols may be enclosed within single quotes '' or left unquoted. However, use of single quotes around symbols and spaces around operators is recommended to avoid ambiguities. Symbol names may use any alphanumeric characters (A-Z, 0-9) but must not conflict with the THINK commands (issuing the command HELP will generate a full list of commands). The symbols P1 to P9 have a special meaning within command scripts (see section 1.5.2) and should be avoided for user-defined symbols.

A symbol (local or global) may contain a single scalar value or a one-dimensional array. Each member of an array is known as an array element and is identified by its position within the array (eg ATOMS(3) would be the third element in the array ATOMS). Each element within an array is set by a separate LET command (unlike some other scripting languages, there is no way to set the contents of the whole array through a single command).

THINK can extract (but not set) substrings of any symbol or array element for use in another operation. A substring is specified as the name of the symbol or array element, followed by the range of characters required. If the first value in the range is omitted or replaced with a "*", THINK will start at the beginning of the string; if the second value is omitted or replaced with "*", THINK will finish at the end of the string. For instance if the symbol ALPHA contained the alphabet in a single text string, ALPHA(5:8) would return the string "EFGH"; ALPHA(:3) would return "ABC" and ALPHA(23:*) would return "WXYZ".

The following arithmetic, string and bit operators are supported in LET statements:

The arithmetic operators are processed in the order they are listed above, with exponentiation having the highest priority. The string substitution operator is very rarely used in user-written command scripts, but is used extensively in the scripts that generate the THINK dialogs. THINK also supports relational operators - these are described in section 1.5.3. The LET command may also be omitted where this does not cause any ambiguity.

1.5.2 Script Arguments

Up to nine values may be passed as arguments to a command script. This allows a single script to be re-used (eg on different molecules) without having to change the script file before each repetition. The arguments are specified after the name of the command script in the CALL command, and are separated by spaces. Any text strings that include spaces must be enclosed in double quotes "" to ensure the whole string is treated as a single argument.

Within the command script, the arguments are identified by the special local symbols P1 to P9. Unlike other local symbols, P1-P9 only exist within the current command script and are not inherited by any nested command scripts below the current level.

1.5.3 Control Commands and Relational Operators

Normally the statements in a command script are executed in the order in which they appear in the file. This order may be changed through the use of:

• GOTO commands to jump to a label somewhere else in the file
• IF-[ELSEIF-][ELSE-]ENDIF commands to execute statements if a condition is true (the portions in brackets [] may be omitted)
• WHILE-END commands repeatedly perform a set of statements while a condition is true

The label used by a "GOTO xxx" command is identified by the statement "LABEL xxx". The IF-ENDIF commands have the form:

IF condition_1
  block_1
ELSEIF condition_2
  block_2
ELSE
  block_3
ENDIF

where the statements in block_1 are executed if the condition condition_1 is true, otherwise the statements in block_2 are executed if condition_2 is true. If neither condition is true, the statements in block_3 will be executed. Either or both of the ELSEIF-block_2 and ELSE-block_3 sections may be omitted; only the IF-block_1 and ENDIF sections are mandatory.

The WHILE-END commands have the form:

WHILE condition
  block_a
END

where the statements in block_a are repeatedly executed while condition is true. Note that block_a must include a statement that makes condition false, otherwise the loop will never terminate and THINK will not respond to any further commands.

The conditions used by the IF-ENDIF and WHILE-END commands compare the values of two symbols or constants using one of the following relational operators:

When the logical operations give a non zero result the condition is considered to be TRUE.

Care must be taken when comparing text strings (they are case-sensitive) and when comparing real numbers using an "equal to" test. It is recommended that real numbers are compared using the "greater than" or "less than" relational operators.

1.5.4 Error Handling

If an error is encountered during a command script, THINK will execute an implicit GOTO command and jump to an error location. This may be defined in one of three ways:

• a label called ON_ERROR, ie "LABEL ON_ERROR"
• a label in the current command script whose name is stored in the symbol ON_ERROR. For instance "LET ON_ERROR = ERROR1" would be used with "LABEL ERROR1"
• storing the value "CONTINUE" in the ON_ERROR symbol, ie "LET ON_ERROR = CONTINUE"

Use of the ON_ERROR symbol allows the destination of the error jump to be changed whilst the script is executing simply by changing the value of the symbol. The special value "CONTINUE" indicates that the error should be ignored and the THINK should execute the next line in the command script.

For more advanced error control it is possible to have a different labels for different errors. Each error message written by THINK has a unique number and this can be used in an array of symbols such as setting ON_ERROR(101) to catch no molecules present (error message number 101).

1.5.5 Input and Output

Text strings can be written to an external file, the THINK log file or the console window through the WRITE command: "WRITE file text" where file is the name of the file to receive the data and text is one of the following:

• a single text string with no spaces
• a text string (which may contain spaces) enclosed in double quotes ""
• the name of a symbol containing the data to be written

File may be specified as LOGFILE or CONSOLE to write data to the THINK log file or console window respectively. If an error occurs whilst the data is being written out, THINK will execute an implicit GOTO command and will jump to the FILE_ERROR location. This is analogous to the ON_ERROR error location (see section 1.5.4) and may be an explicit label ("LABEL FILE_END") or a symbol containing the name of a label or the value "CONTINUE".

Data may be read from a file into a symbol using the corresponding "READ file symbol" command, where file is the name of the file containing the data. Attempting to read past the end of the file will cause THINK to execute an implicit GOTO and jump to the FILE_END location. This may take the same range of values as the FILE_ERROR and ON_ERROR locations.

1.5.6 Intrinsic Functions

THINK includes a variety of numerical, string and system instrinsic functions which are prefixed by "$". Values returned by intrinsic functions can be used like symbols. Each function takes a number of arguments and returns a single integer, real or string value that may be assigned to a variable.

In the table below, ival indicates an integer argument, rval a real argument and cval a character argument.

Molecules are usually read using the File > Open dialog, or the file explorer (see below). Under Windows files may also be dragged from the Windows Explorer and dropped in the Console window. The dialog does not include functionality to read a subset of molecules from a file. The option to disable automatic hydrogen addition can be important when reading fragments for substructure searching and is found on the File Explorer dialog.

The file explorer contains a list of all SMILES, PDB and SD files that have been opened by THINK displayed in lower case. It maintains a hierarchy of these files so that the output from searches are located under the file which was searched with a default name derived from the query. If the user wishes to read a new file using the explorer, it must first be added to the list of recognised files by using the Add button. Once the file is visible in the list it can be opened by double-picking the file, or by picking Open from the pop-up menu displayed by the right mouse button.

A file may be "closed" when all the molecules that have been read from that file automatically deleted from THINK (but not from the file on disk), by picking Close from the right mouse button pop-up menu.

Commands		Dialogs
OPEN FILE=dopamine.smi		File > Open
OPEN FILE=dopamine.smi		Select filename from File Explorer then Open
CLOSE FILE=dopamine		Select filename from File Explorer then Close

Note: In THINK v1.25 the file path and extension is omitted from the close command.

Molecules are read through the OPEN command. This command provides the maximum flexibility - the other routes read all the molecules in the file in a single operation.

When using the OPEN command to read SMILES, PDB or SD files, the user has the option to read selected molecules from the file through the MOLECULE keyword, using the molecule names or positions within the file to identify the desired molecules. The molecule position is specified through the keyword construct "MOLECULE=#n" where n is 1 for the first molecule, 2 for the second, etc. If the file contains molecule names then either the name ("MOLECULE=name") or the position may be used to identify the molecule. Several molecules may be read in a single operation if name includes wildcard characters.

The capability to suppress hydrogen addition is important when reading queries for substructure searching. The OPEN command allows the user to override the default setting through the "OPTIONS=NOHYDROGENS" and "OPTIONS=HYDROGENS" keywords respectively. When automatic hydrogen addition is suppressed, only hydrogen atoms that are explicitly included in the file will appear in the molecule. Any hydrogens that would normally be added as a result of THINK interpreting elements of the form [CH] or [CHn] in SMILES files or interpreting the hydrogen-count field in SD files are omitted.

Commands		Dialogs
OPEN FILE=dopamine.smi		File > Open
OPEN FILE=capsaicin.smi MOLECULE=#5		Selective read not supported
OPEN FILE=dopamine.sdf MOLECULE=DOPAMINE(1)		Selective read not supported
OPEN FILE=dopamine.sdf MOLECULE=DOPAMINE(1%)		Selective read not supported
OPEN MOLECULE=dopamine.smi OPTIONS=NOHYDROGENS		File explorer option

File names that include spaces may be used, providing they are enclosed in double quotes "".

Molecules may be used using the File Save As dialog which saves all molecules loaded within THINK. It is possible to save a subset of molecules using the popup menu in a spreadsheet or tile display.

Commands		Dialogs
SAVE FILE=dopamine.sdf		File > Save
SAVE FILE=dopamines.sdf MOLECULES=@SELECTED		Use popup menu in spreadsheet or tile display

Molecules may be saved via the SAVE command. The "MOLECULE=name" keyword allows specified molecules to be saved using wildcards and/or a comma separated list of molecule names. By default any field information associated with the molecules will be written to the SMILES or SD file; this may be suppressed through the "OPTION=NOFIELDS" keyword.

If the user wishes to generate and save conformers the "FORMAT=CONFORMERS" keyword must be specified.

THINK provides the option to reduce the number of molecules saved to a file using the "OPTION=FILTER" keyword to omit those that contain undesirable substructures or property values. This option would normally be used when saving the enumerated molecules from a combinatorial chemistry library (see Chapter 15), but may be used when saving any set of molecules. The substructure and property value filters are taken from a learn file created by an earlier data analysis calculation (see Chapter 11). The name of the learn file is automatically taken from the name of the field that contains activity data (set through the CUSTOMISE command using the ACTIVITY keyword). If the activity field is not set then the file "default.lrn" in the THINK_EXEC directory will be used.

Commands		Dialogs
SAVE FILE=dopamine.sdf		File > Save
SAVE FORMAT=CONFORMERS FILE=dopamine.sdf		Conformer save not supported
SAVE FILE=dopamines.sdf MOLECULES=S1,S2,S6,S7*		Specific save not supported
SAVE FILE=dopamines.sdf MOLECULES=@SELECTED		Use popup menu in spreadsheet or tile display
CUSTOMISE ACTIVITY=LOGK SAVE FILE=LIB1.SMI OPTIONS=FILTER,NOFIELDS		Options not supported

Molecules are normally read into THINK from an external file (see section 2.1). THINK does not provide a KNIME node for creating new molecules.

Although molecules are normally read into THINK from an external file (see section 2.1), there are occasions when it may be more convenient to create a simple molecule by typing the appropriate SMILES string instead of creating a file and then reading the file into THINK. Molecules may be entered by typing the SMILES string into the Edit > Create dialog and then picking the Create button.

Commands		Dialogs
OPEN FILE=TTY MOLECULE=c1cncc1		Edit > Create

Although molecules are normally read into THINK from an external file (see section 2.1), there are occasions when it may be more convenient to create a simple molecule by typing the appropriate SMILES string instead of creating a file and then reading the file into THINK. Molecules may be entered by using the OPEN command and supplying "TTY" as the name of the input file. The SMILES string is specified using the MOLECULE keyword. Note that the filename is case-sensitive (to support the LINUX operating system) and "TTY" must be supplied in uppercase.

Commands		Dialogs
OPEN FILE=TTY MOLECULE=c1cncc1		Edit > Create

The display node includes functionality to edit a molecule in 2D using the mouse, the drawing tools and the right mouse button popup menu. The drawing tools provide a subset of the functionality found on the menu and are described in the following table.

The 2D molecule display includes functionality to edit a molecule using the mouse, the drawing tools and the right mouse button popup menu. The drawing tools provide a subset of the functionality found on the menu and are described in the following table.

Tool	Menu item	Action
	Element ..	Selects the element for new or existing picked atoms (includes a periodic table) This is also used to specify R-groups for combinatorial chemistry reaction schemes and other wildcard queries.
	Insert atom	Insert atom at picked coordinates
	Insert bond	Insert bond between picked atoms
	Bond order ..	Changes the order of the picked bond
	Sprout atom	Insert atom connected to picked atom
	Delete atom	Delete picked atom
	Delete bond	Delete bond between picked atoms or bond
	Rings > 6 atoms	Insert 6-membered saturated ring at picked coordinates, atom or bond
	Rings > 5 atoms	Insert 5-membered saturated ring at picked coordinates, atom or bond
	Rings > 4 atoms	Insert 4-membered saturated ring at picked coordinates, atom or bond
	Rings > 3 atoms	Insert 3-membered saturated ring at picked coordinates, atom or bond
	Rings > Aromatic 6	Insert 6-membered aromatic ring at picked coordinates, atom or bond
	Rings > Aromatic 5	Insert 5-membered aromatic ring at picked coordinates, atom or bond
	Undo	Undo previous action (maximum 10)
	Flip/Rotate	Flips molecule about X or Y axes and rotates about Z axes
	Tidy	Regenerates 2D coordinates
	Save	Saves molecule

Notes

• When a bond is picked with the Insert Bond tool the bond order cycles through the series single > double > triple.
• When an atom is picked with a ring insertion tool, the atom is included in the ring.
• When a bond is picked with a ring insertion tool, the bond is included in the ring. This can be used to build fused rings. The order of the existing bond will be changed to aromatic when an aromatic ring is being inserted.
• Hydrogens are not normally displayed and need not be drawn - they are added automatically when using other functionality in the program.
• The delete tool can be combined with the shift key to delete a fragment by clicking an atom or a chain by clicking a bond.
• A reaction drawing mode can be enable/disabled using the right mouse button popup menu.

The text command mode provides some functionality to modify or delete existing atoms and bonds but does not offer functionality to create new atoms.

Bonds may be made or broken within the MODIFY command using the MAKE-BOND and BREAK-BOND keywords respectively. The bond is defined by the two atoms and the bond order:

The MAKE-BOND keyword may be used to change the bond order of an existing bond.

The atom type, name, serial number or group number of any atom or collection of atoms may be changed using the MODIFY command. The atom(s) to be altered are identified with the CHANGE keyword, and the new data is supplied with the TYPE, NAME, SERIAL and GROUP keywords. If a set of atoms is to be changed, it may be specified as a range of serial numbers, such as (3:7), or via a symbol (see sections 1.4 and 1.5.1). All the atoms in the set will be given the new name, etc, which may lead to multiple atoms sharing the same identification, so this option must be used with caution.

If the keyword construct "SERIAL=#" is used, the serial number of each atom in the set will be altered to reflect the position of the atom within the molecule. Thus, the first atom will be given serial number 1, the second 2, etc. When applied to all the atoms in a molecule, this is a quick method of assigning unique serial numbers.

Atoms may be deleted using the "DELETE=atoms" keyword. After deleting atoms or changing the bonds or atom types within a molecule, it is recommended that the molecule is rebuilt to update the coordinates and connectivity. THINK will regenerate the 2D or 3D coordinates or just the connectivity depending upon the REBUILD option used (2D, 3D or CONNECTIONS respectively). The molecule to be rebuilt is identified with the MOLECULE keyword.

The molecule name may be altered. The molecule is identified with the MOLECULE keyword and the new name is supplied with the TO keyword. If desired, the conformation number may be set or altered as part of the same command.

THINK does not include a node to delete a molecule. However, the standard KNIME filter node allows rows (molecules) to be eliminated. Some THINK nodes have the option to process just the first molecule which often eliminates the need to use the filter node.

The Edit > Delete dialog is used to delete molecules. An individual molecule may be deleted by selecting its name from the list, or all the molecules in the list may be cleared in a single operation. Selective lists of molecules may be deleted by changing the filter at the top of the dialog (the default filter is "*", which lists all molecules) and then deleting all the molecules in the resulting list.

Molecules may be deleted using the DELETE command. The molecule(s) are specified with the MOLECULE keyword, and may include wildcards. The keyword construct "MOLECULE=*" may be used to delete all molecules.

Individual atoms or sets of atoms may be deleted using the DELETE keyword within the MODIFY command.

Although there are other third party nodes which can be used to display molecules, most of these do not provide the functionality required to view a series of super-imposed molecules for instance docked into a protein.

In THINK 1.42, the display node is only supported under Windows.

Click on the box to display the corresponding dialog.

Within THINK both the molecules and their data may be visualised in a variety of different ways. The term visualisation incorporates simple data listings (eg lists of torsion angles) as well as molecular properties (eg lipophilicity).

The input table is a set of molecules with a SDF column with 2D or 3D coordinates or a SMILES column. Implicit inputs are associated with previously executed Search, Similarity or Docking nodes. THINK will automatically generate 2D or 3D coordinates if they are required for the display mode and absent from the input table.

Before any molecule can be visualised it must be read into THINK for instance from a SMILES, PDB or SD file. If the file contains several molecules, of which only a subset is required, these may be selectively loaded (see section 2.1). Alternatively, all the molecules in the file may be read and the desired molecules specified as part of the visualisation.

Commands		Dialogs
OPEN FILE=capsaicin.smi		File > Open
OPEN FILE=m2.sdf MOLECULE=TC1*		Selective read not supported
OPEN FILE=m2.sdf		File > Open

When a molecule is read from a SMILES or SD file, any hydrogens specified as explicit atoms in the file will be automatically included in the picture. Hydrogens that are specified implicitly via a hydrogen-count (ie as [CHn] in a SMILES file or through the hydrogen-count flag in a SD file) are excluded from the picture, as are any hydrogen atoms that are added automatically to complete the valencies.

THINK will automatically generate the 2D or 3D coordinates in order to display the molecules if the necessary data was not read from the SMILES or SD file.

Molecules may be displayed in various 3D styles, using a 2D representation with one molecule per screen, or with a tiled display showing many molecules on a single screen.

Molecules may be displayed in various 3D styles, using a 2D representation with one molecule per screen, or with a tiled display showing many molecules on a single screen.

Pictures may be displayed on the screen or printed on the default printer using the DISPLAY and PLOT commands respectively. To avoid producing large quantities of output on the printer, it is recommended that the conformer display is avoided and that molecules are printed selectively when more than one or two are present (unless otherwise specified, all molecules will be displayed or printed).

The Edit > Rotate dialog is used to manipulate molecules without displaying them. A global rotation takes all the atoms and applies a rotation about the X, Y or Z axis through the coordinate origin. Note that in the dialog, the rotation takes place as soon as the angle is entered into X, Y or Z box.

Bond rotations may also be performed through the Edit > Rotate dialog. The two connected atoms are specified as Atom 1 and Atom 2, and the angle of rotation is supplied in degrees. All the atoms connected to Atom 1 (except through Atom 2) will be moved.

The simplest keyboard manipulation is a global rotation, which takes all the atoms and applies a rotation about the X, Y or Z axis through the coordinate origin. This is achieved with the ROTATE command using the ABOUT and ANGLE keywords to define the axis and angle (in degrees) respectively.

Bond rotations are achieved using the "ABOUT=atom1-atom2" keyword construct, when all the atoms connected to atom1 (except throught Atom 2) will be rotate by the number of degrees specified with the ANGLE keyword.

The ROTATE command may also be used to orientate a molecule along the line between two atoms (such as a bond); on to a plane defined by 3 or more atoms; to superimpose one molecule on another by 3 or more pairs of atoms; and to orientate maps to their best plane projections.

Any 2D or 3D display may be annotated by labelling the atoms within the molecule, although this is not recommended with the tiled display because it produces a cluttered picture. THINK provides options to label the molecules with their:

• Atom type
• Serial number
• Group number
• Atom name
• Residue name
• Sequence number, insertion code and chain id

3D pictures can be annotated to show the CPK or VdW inter-atomic contacts, using dashed lines to connect each pair of atoms that are in contact. The type of contacts shown (CPK or VdW) is controlled though the CUSTOMISE CONTACTS setting.

3D pictures can be annotated to show the CPK or VdW inter-atomic contacts, using dashed lines to connect each pair of atoms that are in contact.

For each molecule loaded, THINK will automatically calculate a functional group key, or fingerprint, indicating the presence or absence of various functional groups within that molecule. The keys can then be displayed as a 2D key plot: each molecule is represented by a row on the plot and each functional group by a column. If a particular functional group is present in a molecule, a point is placed the intersection of the appropriate row and column. There is insufficient room on the plot to show the molecules explicitly, but picking any point on the plot will pop-up a 2D representation of the associated molecule. The functional group associated with any column on the plot can be displayed in a pop-up window by picking the box on the horizontal bar across the bottom of the plot.

The rows on the 2D key plot may be colour-coded to show the activity of each molecule. The activity data is taken from a field that was loaded when the molecules were read from the SMILES or SD file; the user must specify the name of this field.

Commands		Dialogs	Output
DISPLAY MODE=KEYS ACTIVITY=EC50		View > Keys
PLOT MODE=KEYS		View > Keys

An alternative way of visualising the functional group keys is to display them as a histogram showing the number of molecules that contain each functional group. The histogram may also be used to show the number of times each functional group occurs in highly active or inactive molecules (ie to ignore molecules that lie in the middle of the activity range). To achieve this, the user needs to supply the name of the field containing the activity data, and a significance value in the range 0-0.5. Molecules whose activities lie within this fraction of the top or bottom of the activity range are included in the histogram. If a significance value of 0.5 is specified then all molecules will be included. The activity-coded histogram is drawn with the numbers of active molecules above the origin line and inactive molecules below the line, using the appropriate colours for active and inactive molecules.

Commands		Dialogs	Output
CUSTOMISE ACTIVE=RED INACTIVE=BLUE		File > Preferences > Active:RED; Inactive:BLUE
DISPLAY MODE=HISTOGRAM		View > Keys
PLOT MODE=HISTOGRAM ACTIVITY=EC50 SIGNIFICANCE=.25		View > Keys

For each molecule loaded, THINK can calculate a functional group key, or fingerprint, indicating the presence or absence of various functional groups within that molecule.

Commands		Dialogs
KEY MOLECULE=*		Automatically calculated

THINK will evaluate or calculate a set of 2D and 3D molecular properties for the current molecules. These values, along with the contents of any data fields loaded from the SMILES or SD file (external data fields) may be viewed in a spreadsheet. Normally, all molecules within THINK would be included in the spreadsheet, but a subset of molecules may be used.

THINK will evaluate or calculate a set of 2D and 3D molecular properties for the current molecules. These values, along with the contents of any data fields loaded from the SMILES or SD file (external data fields) may be listed or exported.

Once calculated, the rows in the spreadsheet may be reordered by sorting the contents of a column into ascending or descending order, or rows may be deleted. Columns may be deleted or reinserted (inserting a column that is already present merely changes its position in the spreadsheet). All these operations are available through the pop-up menu displayed when a column is picked with the right mouse button. As an alternative to using the pop-up menu, a column may be sorted by picking the column title once or twice (the second pick reverses the sorting order) and may be moved to a new location in the spreadsheet by dragging the column title.

Picking the name of a molecule from the first column will pop-up a 2D representation of that molecule and will also update the picture window (if present) to show the same molecule. If a 3D display window is open, the contents of the window are changed to the molecule(s) that have been selected. Multiple molecules can be selected by holding down the SHIFT key when picking to select a range. Holding down the CONTROL key allows molecules to be toggled in and out of the selection. The pop-up menu has functionality to

• Save the selected molecules to a named file
• Invert the selection
• Select similar molecules to the most recently selected molecule
• Select molecules in the same family (with the molecule name ending -n where n is a number)

Two of the columns in the spreadsheet may be plotted against each other, using a third column to colour-code the points representing the molecules. This is done by setting two columns as the X- and Y-axes of the plot and the third as the "activity" column (even if it does not really contain activity data) through the right mouse button pop-up menu. The plot will be drawn as soon as the X- and Y-axes have been defined. Molecules may also be selected off this plot to be displayed as with the worksheet including use of the SHIFT and CONTROL keys. In addition, a sphere select centred on a molecule can be used by moving the mouse while holding the left button down to select molecules in the selected range. Alternatively a rectangle select can be used when there is no molecule at the coordinates when the left button is pushed down. Rectange and sphere selections can also be modified using the SHIFT and CONTROL keys. The same pop-up menu for manipulating selections is available in the plot and the spreadsheet.

If a learn file created by an earlier data analysis calculation (see Chapter 11) is available, the properties of the current molecules may be compared with those contained within the file. In this case, the spreadsheet will only contain those properties that occur in the learn file. Values that lie outside the property ranges taken from the file are highlighted. The filename for the learn file is derived from the activity field by adding the suffix ".lrn" and must be present in the working folder.

When reviewing 3D/SITE search results, it can be useful to compare a known ligand and the docked conformers of that ligand. If the known ligand is specified as the query then field containing the RMS deviations for all atoms and non-H atoms mapped by serial number are included. A further field RMS-Match contains the RMS deviations of just those atoms which matched the original 3D/SITE search query. If the protein is designated as the query then the contributions to the score (G-TOT) are computed and included in the table.

Commands		Dialogs	Output
LIST INFO=PROPERTIES OUTPUT=WINDOW		View > Table
LIST INFO=PROPERTIES OUTPUT=WINDOW ACTIVITY=ec50		View > Table

4.5   View Diversity

The property diversity of the molecules can be shown in a panel plot or a 3D cube plot. In the former, the plot is divided into 25 separate tiles or individual plots, each representing a different number of centres. Within each tile, volume is plotted along one axis and lipophilicity along the other. In a 3D cube plot, volume, lipophilicity and the number of centres are plotted along the three axes of a cube that can be rotated.

If the molecules have associated activity data (read from a field in the SMILES or SD file), they may be colour-coded in the diversity plot to show their activity. The user must supply the name of the field containing the activity data.

See Chapter 11 for more information on the calculation of property diversity

Commands		Dialogs	Output
PLOT MODE=PANEL MOLECULE=TC1*		View > Diversity
DISPLAY MODE=CUBE ACTIVITY=EC50		View > Diversity

4.6   View Atom Data

THINK can list various types of atomic data to the THINK console window or to a printer. This data currently includes:

The listing will be displayed in the THINK console window, or it may be sent to a printer if "OUTPUT=PRINTER" is specified. At the end of the molecular property listing, THINK lists the range of each property in the form min:max. Note that the molecular properties will be listed to the spreadsheet if "OUTPUT=WINDOW" is specified.

Commands		Dialogs
LIST INFO=TYPES		View > Atom Data > Data:Types; List
LIST INFO=FLEXIBILITY OUTPUT=PRINTER		View > Atom Data > Data:Properties; Print

	Conformers may be generated for a molecule and exported from the node as a table.
	The configuration options dialog is used to specify the conformer generation mode and when this is not systematic (ie regular or random sampling) the number of conformers required can be specified. This dialog includes several less frequently used options which are described below.
	The configuration bond rotations dialog is used to set the rotational increments about the bonds.

It is not necessary to generate conformers prior to performing a 3D search, docking or generating pharmacophores. THINK will generate them automatically without storing in order to conserve memory and improve performance. This chapter serves to describe the use of the conformational generation settings. The conformers generated may be used by some other software.

Conformer generation is sometimes described as conformer search because it is searching through the conformational space of the molecule.

Conformers may be generated for a molecule and displayed or saved to a file. The torsion angles about the rotatable bonds for all acceptable conformers may be listed to the console window.

It is not necessary to generate conformers prior to performing a 3D search, docking or generating pharmacophores. THINK will generate them automatically without storing them. This chapter serves to describe the use of the conformational generation settings. These settings are independent of the molecules being processed and the operation being performed (display, listing, 3D searching, etc). They are global settings defined through the customise utility.

Conformer generation is sometimes described as conformer search because it is searching through the conformational space of the molecule.

The input table is a set of molecules with a SDF column with 2D or 3D coordinates or a SMILES column. THINK will automatically generate 2D or 3D coordinates if they are required for the display mode and absent from the input table.

By default, only the first molecule (row) in the table is processed. The option to process all rows is found on the THINK setup tab.

The molecule or molecules whose conformers are required must be read into THINK from SMILES or SD files before they can be processed. Molecules may also be read from PDB files. However, these are normally used to store proteins or peptides which are frequently too large or too flexible for conformer generation.

The molecule or molecules whose conformers are required must be read into THINK from SMILES or SD files before they can be processed. If the file contains additional molecules, the desired subset may be read selectively. Alternatively, the molecules to be processed may be identified during the calculation. Molecules may also be read from PDB files. However, these are normally used to store proteins or peptides which are frequently too large or too flexible for conformer generation.

The generated conformers are written to the output table in SDF format. Each conformer is given a unique name of the form molecule(n) where molecule is the name of the original molecule and n is an integer number used to distinguish the conformers.

Conformers are automatically generated as they are displayed, listed or saved to an SD file. They are also generated implicitly during 3D searches.

Within the SD file, the conformers are saved as separate 3D molecules. Each conformer is given a unique name of the form molecule(n) where molecule is the name of the original molecule and n is an integer number used to distinguish the conformers. There is no option to store conformers in a SMILES file because the SMILES format does not include coordinate information, and without coordinates there is no way to re-create the conformers when the file is read.

The conformers are transient - they have no separate existence within THINK and are discarded as soon as they have been displayed, listed or saved - so the user is left with the original molecule at the end of the calculation. If the conformers are required for further analysis, they should be saved to an SD file and then read back from the file as new molecules.

Commands		Dialogs
DISPLAY MODE=CONFORMERS ...		View > Conformers
LIST INFO=CONFORMERS		View > Atom Data > Data:Conformers
SAVE FILE=dconf.sdf FORMAT=CONFORMERS		Option not supported

A pharmacophore is a 3D representation of the features in a molecule. It provides a mechanism by which the important features of the molecule (ie those used in drug-receptor interactions) can be represented without the "clutter" of the other atoms in the molecule. This makes it easier to compare the features across a set of molecules. The term centres is often used to refer to the important features, and will be used in this chapter.

THINK uses 3-point (triangular) and 4-point (tetrahedral) pharmacophores. If a molecule contains more than 3 or 4 important features then a set of pharmacophores is obtained by taking all possible groups of 3 or 4 features from the molecule. The distances between the points in the pharmacophore are calculated exactly, but then allocated to distance bins. This allows the infinite range of distances available to be represented by a finite set of values: each distance is represented by the bin into whose range it falls.

THINK can also generate 2-centre pharmacophores. However, these are only supported within site searches (see Chapter 7), so are not described in this section.

The input table is a set of molecules with a SDF column with 2D or 3D coordinates or a SMILES column. THINK will automatically generate 3D coordinates if they are absent from the input table. It is advisable to convert salts to their parent forms and normal practice to eliminate molecules which are not drug-like using the Parent node.

The molecules must be read into THINK before their pharmacophores can be generated. They may be read from any type of file. However, the user should be extremely cautious about generating pharmacophores for a protein or peptide, since these molecules are very flexible and can contain large numbers of centres, and therefore may generate very large numbers of pharmacophores.

If a subset of molecules from a file is required, these may either be read selectively from the file, or the whole file may be read and the desired molecules specified as part of the pharmacophore calculation.

Commands		Dialogs
OPEN FILE=capsaicin.smi		File > Open
OPEN FILE=m2.sdf MOLECULE=TC1*		Selective read not supported

Most users favour 4-point pharmacophores, except for series of molecules where some molecules have only a few centres when 3-point pharmacophores are more appropriate. The implementation uses fuzzy pharmacophores which are designed to handle the tolerances on the exact positions of the centres (atoms) that define a pharmacophore, which affects the distances between the atoms. When calculating a pharmacophore, THINK allows a tolerance of ±x on each distance (see THINK theory manual for more information).

Most users favour 4-point pharmacophores, except for series of molecules where some molecules have only a few centres when 3-point pharmacophores are more appropriate. There is also an option to control whether THINK requires all hydrogen donors to have an attached hydrogen atom before they are treated as centres. The implementation uses fuzzy pharmacophores which are designed to handle the tolerances on the exact positions of the centres (atoms) that define a pharmacophore, which affects the distances between the atoms. When calculating a pharmacophore, THINK allows a tolerance of ±x on each distance (see THINK theory manual for more information).

The definition of a set of pharmacophores consists of three main items:

• The number of points (centres) in a pharmacophore (3 or 4)
• The distance bins used
• The feature types used

All these options can be altered through the CUSTOMISE command.

Most users favour 4-point pharmacophores, except for series of molecules where some molecules have only a few centres when 3-point pharmacophores are more appropriate. There is also an option to control whether THINK requires all hydrogen donors to have an attached hydrogen atom before they are treated as centres. The implementation uses fuzzy pharmacophores which are designed to handle the tolerances on the exact positions of the centres (atoms) that define a pharmacophore, which affects the distances between the atoms. When calculating a pharmacophore, THINK allows a tolerance of ±x on each distance (see THINK theory manual for more information).

The distance bins are used to transform the distances within each pharmacophore (3 distances in a 3-point pharmacophore, 6 in a 4-point pharmacophore) into a set of integers that give a more compact representation of the pharmacophore. The upper limit of each distance bin should be specified (in ascending order) with the BINS keyword, separated by commas with no intervening spaces ie BINS=a,b,c,d,e where a,b,c,d,e are the bin limits.

By default, THINK will recognise all 12 supported feature types (HDON, HACC, POS, NEG, AROM, LIP, ACID, BASE, MET, LPD, USR1, and USR4) in the molecule. THINK can be tailored to ignore one or more of these atom types by specifying the list of acceptable types with the TYPES keyword. The types should be supplied as a list of 1-letter codes (D, A, P, etc) or centre names (HDON, HACC, POS, etc) separated by commas and with no intervening spaces (eg TYPES=HDON,HACC,AROM,LIP).

In most circumstances the Boltzman population pharmacophore profiles are preferred which weight each pharmacophore count by the energy dependent Boltzman population and express the result as a percentage. There are options to use a simple count per conformer, a count per molecule and a percentage population.

The output from the pharmacophore node consists of one of the following:

• Rows of 3D coordinates for each pharmacophores (SD format); pharmacophore identifier and population for use by the Search node;
• Pharmacophore profile consisting of the pharmacophore identifiers and their populations for use by the FocusedSet and DiverseSet nodes;
• A table of molecules as rows and pharmacophore populations as columns for use with general statistic nodes.

There are several options to reduce the size of the output table.

• The number of pharmacophores output with 3D coordinates can be usefully limited to the most common 'n' pharmacophores.
• In most cases, pharmacophores with low populations are of little interest and can be safely omitted.
• The average population for a pharmacophore in a series of molecules is not always a reliable indicator that all molecules exhibit a pharmacophore because the population for individual molecules may vary. Consequently, when creating maps for pharmacophores intended to represent the union of volume occupied by all the active molecules when exhibiting that pharmacophore, it can be useful to validate that most of the series of active molecules exhibit the pharmacophore.

The pharmacophores are automatically calculated when the user creates a pharmacophore file or (less commonly) lists them to the console window. If the user wishes to list or save the pharmacophores for selected molecules then their names should be specified as part of the SAVE or LIST command.

The pharmacophore file is written using a compressed format that is described in the THINK Theory Manual.

Commands		Dialogs
SAVE FILE=asp.phm		File > Save
SAVE FILE=M2.PHM MOLECULE=TC*		Selective calculation and save not supported
LIST INFO=PHARMS MOLECULE=TYR		View > Atom Data > Data:Pharms; Filter=TYR; List

If the molecules have not been previously filtered by drug-like or other desirable properties, this can be performed by adding OPTIONS=FILTER to the SAVE command. Elimination of salts (ie conversion to parent form) must be performed as a prior step.

This functionality is not implemented for Windows dialogs or command mode.

7.1   Molecule and Pharmacophore Inputs

The input table of molecules must contain a SDF or SMILES column. If 3D coordinates are not provided they will be generated automatically by THINK. The implementation uses fuzzy pharmacophores which are designed to handle the tolerances on the exact positions of the centres (atoms) that define a pharmacophore, which affects the distances between the atoms. When calculating a pharmacophore, THINK allows a tolerance of ±x on each distance (see THINK theory manual for more information).

The pharmacophore profile is an output of the Pharmacophore node. This should have the same number of centres and distance tolerance.

7.2   Pharmacophore Options

The essential differences between the DiverseSet and FocusedSet nodes, is the criterion for selecting molecules. To be included in the DiverseSet output, a molecule must exhibit a minimum number of additional pharmacophores whereas in the FocusedSet node, molecules are selected if they exhibit more pharmacophores which are in the starting profile than a default threshold.

Using 4-centre pharmacophores gives a larger number of possible pharmacophores and therefore a larger measure of diversity. However, some molecules with little functionality may have too few centres to exhibit 4-centre pharmacophores. The distance tolerance

The bond rotation and conformational generation options would normally be set the same as in the Pharmacophore node for generating the starting profile.

7.3   Subset Calculation

Molecule selection based on pharmacophores tends to be slower than Searching or Docking because all conformations have to be processed. As a consequence DiverseSet and FocusedSet nodes are normally not used for hundreds of thousands of molecules. Progress reports are provided in the normal way and the priority of the THINK process doing the calculation is lowered to ensure that the PC remains responsive.

The input table contains the query or queries in SMILES or SD format. Where multiple rows are present only the first row is processed by default. However, occasionally for substructure searches it can be useful to process multiple queries. When a substructure or 3D search is to be performed, THINK will not add hydrogens to complete missing valencies. For a pharmacophore search, the input is normally a selected row from the output of the Pharmacophore node.

A second optional input is available for the table with a SDF or SMILES column of molecules to be searched. For relatively small numbers of molecules this offers an alternative to reading them from a file.

The molecule to be used as the query during the search is read from a SMILES, PDB or SD file. It need not be the only molecule in the file - the user may either read the entire file and then select the desired molecule. If a substructure or 3D search is to be performed, it is recommended that the NOHYDROGENS option is used to prevent THINK automatically adding hydrogens or interpreting the hydrogen-count field in SD files to complete the molecule.

For 3D searches, the relative positions of the atoms within the query molecule are important and consequently these must be read from a PDB or SD file since these contain 3D coordinates.

THINK does not support the MDL query file format.

The molecules in an SD or SMILES format file may be searched directly without either reading them into a KNIME table or a third party database by selecting the file to be searched. If the molecules to be searched already exist in a KNIME table with a SDF or SMILES column, then this can be used for the second (optional) input.

The filter option reduces the number of molecules examined during a search by eliminating those that are not "drug-like" because containing undesirable substructures or property values.

Unlike many other software packages that require molecules to be converted to a proprietary format before they can be searched, THINK searches molecules directly from a SMILES or SD file. This eliminates the need to perform time-consuming data conversions.

Commands		Dialogs
SEARCH ... FILE=capsaicin.smi ...		Search > 2D or 3D or Similarity > Browse

In command mode, the MODE keyword determines the type of search while the GUI provides several alternative dialogs.

Commands		Dialogs
SEARCH MODE=SUBSTRUCTURE ... SEARCH MODE=EXACT ...		Search > 2D
SEARCH MODE=SIMILARITY ...		Search > Similarity
SEARCH MODE=3D ...		Search > 3D

In all types of 2D and 3D searches, the user has the option of matching atoms according to their element symbol or atom type, with atom type matching being the more restrictive search. In addition, the user can choose whether bond orders should be checked during the search.

The filter option reduces the number of molecules examined during a search by eliminating those that are not "drug-like" or containing undesirable substructures or property values. This is done by applying the filters contained in a learn file generated during an earlier data analysis (see Chapter 10). The name of the learn file is taken from the name of the field containing activity data (set via the CUSTOMISE command); if the field is not set then the file "default.lrn" in the THINK_EXEC directory will be used.

The output consists of 3 tables:

• A table of hits (molecules which match the search criteria) with the SD and SMILES columns.
• Summary information including the number of hits for each query.
• Information about the failures including a brief summary of why they didn't match the query

The results of a search are stored in an output file that can have one of the following formats:

• SMILES - usually used for 2D searches
• SDfile - used for 3D and site search results
• Listing - a simple list of the names of the molecules that match the query

There are no restrictions on the format that may be used with each type of search. The format of the input file (the file being searched) does not affect the format of the results file (eg the results may be stored in an SD file even though input file uses the SMILES format). The file format is deduced automatically from the file name.

Commands		Dialogs
SEARCH ... OUTPUT=dopamine-s1.smi ...		Search > 2D or 3D or Similarity > Search

The time taken by a search is dependent on the numbers of molecules being searched and for 3D/Pharmacophore searches the flexibility of these molecules. During a search the priority of the THINK process is lowered so that other activities on the computer may be more responsive. The progress of the search is reported in the usual way.

When using the GUI, to perform an Exact match search the Exact option needs to be selected on the 2D search dialog. Similarly on the 3D search dialog the Site option needs to be selected to perform a Site search.

If insufficient information is specified for the search, the input file does not exist or the output file already exists etc, then an error message is generated. For all types of searches, each molecule is read from the input file in turn and compared with the query. If necessary, the molecule will be converted to 3D before being compared.

For a 2D or normal 3D search, all the atoms in the query must be matched within the molecule for it to be accepted as a hit and saved in the results file. In a site search, only the atoms within one pharmacophore must be matched. This means that several hits may be accepted from a single conformer of a ligand, if it can be matched with more than one of the query pharmacophores.

Commands		Dialogs
SEARCH MODE=SUBSTRUCTURE QUERY=CAPQUERY#1 FILE=capsaicin.smi OUTPUT=CAPHITS.SDF OPTIONS=ORDER		Search > 2D
SEARCH MODE=SIMILARITY QUERY=CAPQUERY#1 FILE=capsaicin.smi CUTOFF=0.75		Search > Similarity

The output tables can be viewed or analysed and the structures visualised by a range of KNIME nodes. The THINK display node is designed for visualising 3D hits with a pharmacophore query together with an optional volume map. It can also be used to visualise other search results in 3D or 2D.

If the search results are stored in a SMILES or SD file, they can be viewed, listed and analysed in exactly the same way as any other set of molecules. The query may be superimposed as the molecules are displayed, and the mapping between the atoms in the query and the corresponding atoms in each hit molecule can be shown using dashed lines. This requires both the query molecule and the hits to be present in THINK (the query is not stored as part of the results file). In THINK v1.25 the mapping can only be shown on 3D pictures.

Commands		Dialogs	Output
OPEN FILE=dopamine-s1.smi		File > Open
DISPLAY MODE=TILE QUERY=dopamine		View > Molecule > Query:dopamine
OPEN FILE=1ir3-s1.sdf		File > Open
DISPLAY MODE=3D STYLE=STICK OPTION=INTERACTION QUERY=1IR3		View > Molecule > 3D Stick; Query:1IR3; Lines Show: Interactions

When using THINK from KNIME, the map calculation and display are integral parts of the Display and Pharmacophore nodes when a volume constraint can be associated with each pharmacophore. These maps can also be used as site contraints for Docking and De Novo derivative generation when the pharmacophore is used as the search query. There is no separate Maps Node or KNIME data type to support maps.

THINK provides additional capabilities including the use of a volume map to constraint the results of a SITE search or a de novo derivative generation.

9.1   Read Molecules

The molecules are normally read from an SD file which were the results of a SITE search or less commonly a 3D search or a 3D de novo derivative generation. In these cases the molecules are already aligned and the alignment step is skipped.

9.2   Align Molecules

The molecule alignment is the most critical step of the procedure and is dependent on the selection of conformers and identifying features to overlap. It can be tempting and erroneous to align molecules based on the reactivity of their functional groups rather than the potential interactions with a protein receptor. The pharmacophore technology used in THINK is designed for pragmatically aligning molecules see Ligand Query Maps 9.5. For molecules with little or no flexibility it is conceivable to generate appropriate conformations interactively and align the molecules as described in section 4.2.

9.3   Calculate Map

A volume map may be constructed for a single molecule such as the most active or a Ligand map for series of molecules such as those that exhibit submicromolar activity.

When selecting a Ligand map for active molecules it is necessary to configure the data field to be used, the type of activity and the partitioning into actives and inactives. The Activity tab on the Preferences dialog or the CUSTOMISE command provides these options. When partitioning molecules into actives and inactives, either absolute activity measures can be used or a factor (0.1-1.0) can be specified when the activity range is divided into 3 regions: most active; intermediate and least active. Thus 0.4 would class the top 40% by value as active and the lowest 40% as inactive leaving the mid range 20% as indeterminant.

Commands		Dialogs	Output
CALCULATE MAP=MOL1_VDW TYPE=VDW MOLECULE=MOL1 GRID=0.5		Calculate > Map
CALCULATE MAP=LIGAND TYPE=LIGAND BEST=10 MOLECULE=@actives GRID=0.5		Calculate > Map
DISPLAY MAP=LIGAND MOLECULE=* MODE=3D STYLE=STICK		View > Molecule

9.4   Display Map and Molecules

It is sometimes apparent that molecules use different binding modes in the protein receptor site. Under these circumstances some active molecules might within the volume map of the most active molecule while others may not. This may be further complicated by molecules which can bind in more than one mode - a default SITE search would only save one conformer per molecule. It may be possible to conclude that a binding mode is associated with activity by comparing how a series of molecules bind and their activities. It is also possible to adjust the SITE query so that results for only one binding mode are saved.

9.5   Ligand Query Maps

The Ligand Query procedure considers multiple overlap modes for a series of molecules and identifies the pharmacophore and ligand volume map which best accomodates the set of active molecules. The approach does not require knowledge of the protein receptor site although it relies on the active molecules binding in precisely the same way. This can be validated by searching training sets and subsequently used to identify molecules which should exhibit activity.

The identification of potential binding sites and the docking of molecules into a binding site are two distinct steps in THINK provided by the FindSites and Docking nodes respectively. THINK uses pharmacophore technology for docking which means that docked molecules will have well defined interactions within the binding site. For the best results, the conformations of both the small molecule and the protein are refined to give the best interaction score. One might expect a correlation between score, binding free energy and inhibition but because of various assumptions and approximations quantitative predictions are not normally reliable. Typically, up to 10-30% of the molecules with the best scores exhibit µMolar activity.

	The FindSites node reads in a PDB file and creates site queries based on docked ligands, PDB binding site records and/or scanning the protein to locate binding pockets.
	The output consists of The Site Queries for use with the Docking node. The Interactions with bound ligands which can be used as residue constraints in the Advanced Options. A table of bound ligands which can be used to validate Site Queries with the Docking node.
	The configuration Options Dialog is used to specify the PDB file and select which creation modes are used etc.
	The configuration Advanced Dialog is used to specify residues with which interactions are required.
:
	The Docking node attempts to dock molecules into the binding pocket defined by the site query.
	The output is described below.
	The configuration Options Dialog is used to set the main docking options.
	The configuration Bond Rotation Dialog is used to change the bond rotational increments.
	The configuration Advanced Dialog is used when some more advanced options need are changed.
:
	The promiscuity node performs multiple searches (normally in parallel) using a set of queries stored concatenated in a single PDB format file. Normally the set of site queries are associated with selectivity or potential promiscuity of the ligands. Each site query can have different configuration options such as the number of centres, warp speed etc which are stored in the file. See find-a-drug.pdb for example.

Docking small molecules into protein binding sites and scoring how well the molecule interacts might indicate the potential of that molecule to inhibit. (Note: Binding and inhibition are not necessarily the same). THINK includes capabilities to create site queries for binding pockets, generate conformations for molecules and dock them using pharmacophore technology to the binding target where the conformations of both small molecule and protein can be refined to give an optimum score.

A crystal structure of ligand-protein complex provides valuable information about one binding mode. However, THINK's capability to search for all sites might identify additional residues in the same binding pocket as the bound ligand which might interact with other ligands. In some cases, the PDB site records are of little use, for instance when they define catalytic residues rather than a binding site.

When the precision of the crystal structure is low or the flexibility is high it can be adviseable to increase the distance tolerance although this will inevitably give more hits in subsequent searches.

Residue constraints reduce the number of hits and without reducing the number of relevant hits if there is also evidence that interaction with specific residues is associated with desirable biological activity. In most cases, it is appropriate to review the Interaction table output from this node and view the binding site using the Display node in order to be satisfied that requiring interactions with specific residues is appropriate.

As the Docking node has no functionality to select a site query from Sites output table, it is common to use a generic KNIME node to filter this output.

The part of the protein or binding site in which molecules are docked is identified by:

• Using a known bound ligand
• The SITE record in a PDB file
• THINK functionality to scan a protein and identify binding pockets.

A default query for a site search may be created within THINK and stored as a PDB file but it may be necessary to edit this file to shift the coordinates of centres, remove centres, indicate required centres, adjust tolerances etc. Further information can be found in the THINK theory manual. The query and the active site of the protein can be saved to the same file.

Commands		Dialogs
MODIFY INTERACT=^1B7Y LIGAND=PAA5P SAVE FILE=1B7Y-Q1.PDB MOLECULES=INTERACT,1B7Y		Edit > Site > Ligand:PAA5P; Create; Save

The extended active site is often used to provide supplementary data to the query, for instance to ensure that ligands found during the search do not occupy the same space as the protein itself. The protein, or the subset of the protein around the receptor site, should be read before the search is initiated.

The Site query created by the FindSites node is a required input which defines both the interactions and the residues in the binding site. If the set of molecules being searched for potential ligands is relatively small these can be supplied as a table with a SDF or SMILES column as the second input. Otherwise a potential ligand file may be configured in the Options dialog.

The site query is read from a PDB file created by the command sequence describe earlier. The file of potential ligands to be searched is specified on the Search Dialog or using the FILE keyword of the SEARCH command.

Several of the more advanced controls for docking, especially those that effect pharmacophores in general, use the CUSTOM command or the PREFERENCES dialog, The 3D search dialog with the SITE option is used to perform docking.

The output consists of 3 tables:

• A table of hits (molecules which match the search criteria) with the SD and SMILES columns.
• Summary information including the number of hits for each query.
• Information about the failures including a brief summary of why they didn't match the query

The results of a search are normally stored in a 3D SDF output file. This uses serial numbers to map the atoms to the corresponding atoms in the site query and the protein. The conformational numbers ae used to distinguish different conformers without implying a separation in conformational space.

The output tables can be viewed or analysed and the structures visualised by a range of KNIME nodes. The THINK display node is designed for visualising 3D hits with or without the site query and binding site of the protein. The query and/or the protein may be superimposed as the molecules are displayed, and the mapping between the atoms in the site query or the protein and the corresponding atoms in each hit molecule can be shown using dashed lines.

If the search results are stored in a SMILES or SD file, they can be viewed, listed and analysed in exactly the same way as any other set of molecules. The query may be superimposed as the molecules are displayed, and the mapping between the atoms in the query and the corresponding atoms in each hit molecule can be shown using dashed lines. This requires both the query molecule and the hits to be present in THINK (the query is not stored as part of the results file). In THINK the mapping can only be shown on 3D pictures.

Commands		Dialogs	Output
OPEN FILE=1ir3-s1.sdf		File > Open
DISPLAY MODE=3D STYLE=STICK OPTION=INTERACTION QUERY=1IR3		View > Molecule > 3D Stick; Query:1IR3; Lines Show: Interactions

The THINK functionality to visualise property diversity and to select subsets based on property values is not accessible from KNIME.

The property diversity examines the volume, lipophilicity and number of centres in a set of molecules and determines the diversity of the set from these factors.

There are two routes by which the diversity of a set of molecules may be calculated. One route takes molecules directly from a file, and allows the user to save the set of selected (diverse) molecules in a results file. This route provides more flexibility in changing the calculation options. The other route uses molecules currently loaded within THINK and is designed to show the diversity of the current molecules, rather than to select a diverse subset.

In the following sections, the number in parentheses in each title indicates the route to which the section refers.

11.1   Identify File to be Processed (1)

In route 1, the set of molecules to be analysed is read directly from a SMILES or SD file - there is no need for the molecules to be read into THINK before the calculation is performed. The file format is deduced automatically from the file extension supplied as part of the file name. The molecules could be read from a PDB file, but this format is normally used only for peptides and proteins, which are not suitable for property diversity calculations.

Commands		Dialogs
SELECT FILE=capsacin.smi ...		Calculate > Selection > File or Browse

11.2   Read Molecules to be Processed (2)

If route 2 is being used to calculate diversity, the molecules must be read into THINK before the calculation is initiated. If a subset of molecules from a file is required, these may either be read selectively from the file, or the whole file may be read and the desired molecules specified as part of the diversity calculation.

Commands		Dialogs
OPEN FILE=capsacin.smi		File > Open
OPEN FILE=m2.sdf MOLECULE=TC1*		Selective read not supported

11.3   Set Calculation Options (1)

The diversity calculation uses a three-dimensional property space plotting molecular volume, lipophilicity and the number of centres (0-24) along the three axes. Each axis is divided into 25 cells; thus using 25³ cells to cover the entire property space. During the calculation each molecule is allocated to a cell unless that cell is already fully occupied, in which case the molecule is rejected. The user can limit the maximum number of molecules allowed in a cell.

Normally, the diversity calculation would start from an empty property space. However, the user may choose to append the current calculation to the results of a previous calculation, in which case the cell occupancies from the previous diversity calculation are retained and used as the initial values for the current calculation.

Commands		Dialogs
SELECT ... LIMIT=3 ...		Calculate > Selection > Limit=3
SELECT ... MODE=ADD LIMIT=5 ...		All options not available

11.4   Supply Results File (1)

A simple list of the molecules selected by the diversity calculation can be stored in a results file. Alternatively, the selected molecules themselves may be stored in a SMILES or SD file. The file format required is deduced automatically from the file name.

Commands		Dialogs
SELECT ... OUTPUT=m3div.set		Calculate > Selection > Select

11.5   Select Plot Style (1,2)

The diversity of the selected molecules can be shown in a panel plot or a 3D cube plot. In the former, the plot is divided into 25 separate tiles or individual plots, each representing a different number of centres. Within each tile, volume is plotted along one axis and lipophilicity along the other.

In a 3D cube plot, volume, lipophilicity and the number of centres are plotted along the three axes of a cube that can be rotated.

Commands		Dialogs
SELECT ... DISPLAY=PANEL ...		Calculate > Selection > Panel
SELECT ... DISPLAY=CUBE ...		Calculate > Selection > Cube
DISPLAY ... MODE=PANEL ...		View > Diversity > Panel
DISPLAY ... MODE=CUBE ...		View > Diversity > Cube

11.6   Define Activity-Based Colour-Coding (2)

When route 2 is used, the user may opt to colour-code the molecules in the diversity plot according to their activity. This can only be achieved if activity data was loaded with the molecules. The user must supply the name of the field containing the data.

Commands		Dialogs
DISPLAY ... ACTIVITY=EC50		View > Diversity > Field:

11.7   Calculate Diversity (1,2)

In a route 1 diversity calculation, each molecule is read in turn from the file. The volume, lipophilicity and number of centres are calculated, and the molecule is then allocated to a cell in the property space. If the cell is already full the molecule is rejected, otherwise it is accepted and added to the list of selected molecules. Thus, the order of the molecules in the input file may affect the final selection.

The method used in a route 2 calculation is similar, except that the molecules do not need to be read from an input file. There is no cell limit in a route 2 calculation, so all molecules are retained and placed in the diversity plot. At the end of the calculation the molecule represented by any point on the plot may be viewed by picking that point.

Commands		Dialogs	Output
SELECT FILE=capsaicin.smi LIMIT=5 DISPLAY=PANEL OUTPUT=div.lis		Calculate > Selection
DISPLAY MODE=CUBE MOLECULE=TC1* ACTIVITY=EC50		View > Diversity

The THINK functionality for Data Analysis is not accessible from KNIME.

The data analysis examines the properties and/or keys of a set of molecules and attempts to identify features that may be responsible for undesirable characteristics. The results of the analysis are stored in a learn file that can be used in subsequent THINK calculations to highlight or eliminate molecules whose properties lie outside the acceptable ranges or which contain unacceptable functional groups.

12.1   Read Molecules

THINK will analyse all molecules that have been loaded into the program. They may be read from a SMILES or SD file. Any external property data required for the analysis must be included in the file in data fields - these fields will be loaded automatically as the molecules are read. The file must contain a field of activity data.

Commands		Dialogs
OPEN FILE=capsaicin.smi		File > Open

12.2   Select Activity Field

The field containing the activity data must be identified.

Commands		Dialogs
LEARN ... ACTIVITY=EC50 ...		Calculate > Analysis > Field:EC50

12.3   Set Activity Options

By default, the activity values will be taken directly from the activity field, with the highest values indicating the most active molecules. Alternatively, the interpretation of activity may be reversed, so that molecules with low values are considered the most active. There is also an option to convert the activities into their logarithmic values, for instance if binding constants are used as the activity data.

The molecules need to be divided into active and inactive molecules for the analysis. This is done through a user-supplied significance value that must lie within the range 0-0.5. Molecules that lie within this fraction of the top or bottom of the activity range are considered active or inactive; molecules that lie in the middle are ignored during the analysis. If a significance value of 0.5 is specified then all molecules will be included in the calculation.

Commands		Dialogs
LEARN ... SIGNIFICANCE=0.3 ...		Calculate > Analysis > Significance=0.3
LEARN ... OPTIONS=LOW,LOG ...		Calculate > Analysis

12.4   Select Data to be Analysed

THINK can include some or all of the functional group keys, 2D properties and/or external data fields in the data analysis. The fields, 2D properties and keys are identified by keywords:

See section 4.4 for a full list of the 2D properties.

Commands		Dialogs
LEARN ... PROPERTIES=KEYS ...		Calculate > Analysis > Keys
LEARN ... PROPERTIES=KEY#5,KEY#12,LOGP,DIPOLE,...		Full control is not supported

12.5   Analyse Data

During the analysis calculation, THINK first calculates any 2D properties and functional group keys that are required before attempting to extract discriminating features that could identify the inactive molecules. The results of an analysis calculation are saved in a learn file whose filename is taken from the name of the activity field and uses the file extension ".lrn". Thus, if the activities are taken from the field called EC50, the results will be stored in a file called "ec50.lrn". Amongst other information, the learn file contains the acceptable range of values for each discriminating property (2D property or external data field) and the most significant unacceptable functional groups.

Commands		Dialogs
LEARN ACTIVITY=EC50 SIGNIFICANCE=0.5		Calculate > Analysis > .. Analyse
LEARN ACTIVITY=EC50 SIGNIFICANCE=0.3 PROPERTIES=FIELDS		Calculate > Analysis > .. Analyse

12.6   Apply Learn File

The learn file generated by data analysis may be used to provide additional rejection criteria for de novo structure generation. It may also be used in the property spreadsheet to highlight values that lie outside the acceptable ranges.

Click on the box to display the corresponding dialog.

A hierarchical clustering algorithm has been implemented in THINK which is appropriate to cluster a few thousand molecules. This clustering capability is useful in order to find representative molecules from a larger set such as the results of a search. The functional group keys are used for the same similarity measure that is used for similarity searching.

The input table is a set of molecules with a SDF column with 2D or 3D coordinates or a SMILES column.

THINK will analyse all molecules that have been loaded into the program. They may be read from a SMILES or SD file.

Commands		Dialogs
OPEN FILE=capsaicin.smi		File > Open

The output tables contain the molecules with columns for SD file, SMILES and the cluster identifier in a column named ClusterID. The first table contains all the molecules while the second table only contains a representative for each cluster.

A numerical identifier for each cluster is stored in a field named Cluster-ID and representive for each cluster is placed in the Selected set of molecules.

This capability is not available from KNIME.

A dendogram is produced showing the hierarchy of clusters. The molecule corresponding to the Y-coordinate on the dendogram is displayed by clicking. The up and down arrow keys can then be used to display other molecules in that cluster.

If the spreadsheet or tile display is used, the selected molecules are the representives of each cluster. It can sometimes be useful to sort the spreadsheet eg on Cluster-ID so that the members of a cluster can be selected and saved.

This capability is not available in command mode.

	The De Novo node generates derivatives of a starting molecule or molecule(s).
	The Options dialog is used to set the mode and control the number of molecules generated. In addition, this dialog allows some fine control of the drug-like filters.
	The output consists of a table of molecules with 2D or 3D SD column and a SMILES column. In addition to various molecular properties, an index to the last transformation is included.

A genetic algorithm is used to generate the new molecules which in 2D mode uses a set of transformations which can substitute groups, increase or decrease chain lengths etc. In 3D mode, the transformations are limited to functional group substitutions via a single attachment atom. Drug-like substructure and property filters are always applied to the molecules generated.

Click on the box to display the corresponding dialog.

De novo molecule generation uses a genetic algorithm to generate derivatives of a starting molecule using a set of user-defined rules to substitute functional groups, increase or decrease chain lengths, etc. The new molecules are checked for undesirable substructures or properties before passing though an annealing process which constrains the generated molecules to be close derivatives.

The input table is a set of molecules with a SDF column with 2D or 3D coordinates or a SMILES column. Normally only the first molecule in the table is used.

De novo generation uses an existing molecule as the starting template. This molecule, often referred to as a query, is usually read from a SMILES or SD file. It need not be the only molecule in the file - the user may either read the entire file and then select the desired molecule, or may selectively read just the query molecule from the file (see section 2.1).

Commands		Dialogs
OPEN FILE=capsaicin.smi		File > Open

The parts of the molecule being modified can also be restricted by atom group number. These can be set by the user with the Display Node although for 3D docked starting molecules (and other 3D hits) the atoms not matched to the query are set to a group number of 999 and changes are automatically restricted to the unmatched atoms.

The parts of the molecule being modified can also be restricted by atom group number. These can be set by the user with the Display Node although for 3D docked starting molecules (and other 3D hits) the atoms not matched to the query are set to a group number of 999.

Commands		Dialogs
SUGGEST MOLECULE=CAPSAICIN#1 MAXIMUM=250 ACTIVITY=EC50 OPTIONS=LOW,LOG SIGNIFICANCE=.5 DISPLAY=PANEL		Search > De Novo

A diversity plot is displayed as the molecules are generated.

Combinatorial chemistry consists of two distinct steps:

• Performing one or more R-group searches to convert reagents into R-groups (requires the 2D module)
• Enumerating the molecules by "plugging together" R-groups from two or more files to form a library

The input table defines a reaction query with a SDF column with 2D coordinates or a SMILES column. This can be created using the Display node or third party software such as the Marvin sketcher from ChemAxon and its R-group designations. A second optional input is available for the table of reagent molecules with a SDF or SMILES column. For relatively small numbers of reagents this offers an alternative to reading them from a file.

It is also possible to use just the corresponding reactant of the generic reaction query as the input instead, but in most cases this offers no advantages.

The molecule to be used as the query during the R-group search is read from a SMILES or RD file. It need not be the only molecule in the file - the user may either read the entire file and then select the desired molecule, or may selectively read just the query molecule from the file (see section 2.1).

The SMILES string used to search the reagents is known as an R-group query and consists of a substructure and the connection points. The substructure is the portion that is common to all reagents accepted by the search; this portion will be discarded when the reagent is converted into an R-group. The connection points are indicated by numeric atom types or wildcard atom types:

• [0] for a generic connection atom
• [n] for an explicit connection atom
• [wildcard] for a generic connection atom

The [0] and [n] connections in the query will be matched to any atom type except hydrogen in the reagent. The [wildcard] connection will only be matched to selected atom types - see the THINK Theory Manual for a full list of supported wildcards. The [0] connection is not usually used in reactions but is a useful means of generating R-groups which can be re-used with in a variety of libraries.

It is also possible to store the R-group queries in separate molecules. For example, to build a coreless amide library of the form [R1]C(=O)N[R2] from acid chlorides and amines would require two R-group queries: one to look for acid chlorides and convert them into suitable R-groups, and the other to perform the same operation on amines. The reagents to be used, and the R-group query required to select them are listed in the table below:

If the query contains multiple connection points, THINK will use explicit connection atoms in the resulting R-groups, even though the the query may have contained generic connection points. Since the order in which explicit connections are allocated to generic connection points is undefined, it is recommended that explicit connections ([n]) are used in these queries.

Commands		Dialogs
OPEN FILE=rx-amide.smi		File > Open

Note: Reactions and R-group queries should never have missing hydrogens as these cause serious problems.

It is necessary to specify which R-group is to be created by each Rgroup node.

The set of molecules for the R-group search can either be stored in a file, when its file name needs to be configured or connected to the second optional input. This input must have a SDF or SMILES column.

Many files of reagents store their molecules in the salt form and this will cause spurious results from R-group searches. The THINK R-group search incorporates the ability to convert the molecules into their parent forms. This conversion takes place before the reagent is compared with the query.

If the drug-like filter option is used, the upper limit is applied to the resulting R-group (not to the original molecule), and the lower limit is ignored in order to avoid eliminating solutions that could be satisfied in enumerated molecules by other R-groups.

In addition to selecting the molecule which contains the R-group for reaction queries it is necessary to specify which R-group to use. The SITE keyword is used to specify any atom in the R-group (usually the connection atom by atom name ie without the square brackets).

Commands		Dialogs
SEARCH ... QUERY=rx-amide#1 SITE=2 ...		Search > 2D > R-group; Site=2

Unlike many other software packages that require molecules to be converted to a proprietary format before they can be searched, THINK searches molecules directly from a SMILES or SD file. This eliminates the need to perform time-consuming data conversions.

Commands		Dialogs
SEARCH ... FILE=aldrich.smi ...		Search > 2D > Browse

An R-group search is a specialised form of 2D search (see Chapter 7) that checks connectivity and automatically converts matching molecules into R-groups. The user can choose whether atoms should be matched according to their element symbol or atom type and bond orders should normally be checked; these settings may be controlled through the OPTIONS=ORDER and OPTIONS=TYPE keywords respectively.

Many files of reagents store their molecules in the salt form and this will cause spurious results from R-group searches. The THINK R-group search incorporates the ability to convert the molecules into their parent forms through the OPTIONS=PARENT keyword. This conversion takes place before the reagent is compared with the query.

Commands		Dialogs
SEARCH MODE=R-GROUP ... OPTIONS=ORDER,PARENT		Search > 2D > R-group; Order; Parent

If the FILTER option is used, the upper limit is applied to the resulting R-group (not to the original molecule), and the lower limit is ignored in order to avoid eliminating solutions that could be satisfied in enumerated molecules by other R-groups.

The output consists of 3 tables:

• A table of R-groups in the SD and SMILES columns.
• Summary information including the number of hits for each query.
• Information about the failures including a brief summary of why they didn't match the query.

The R-groups resulting from an R-group search are stored in an output file using the SMILES or SD format, depending upon the file extension supplied for the output file. Normally the SMILES format would be used because this is more compact that the SD format.

Commands		Dialogs
SEARCH ... OUTPUT=gp2.smi ...		Search > 2D > Search

The time taken by a search is dependent on the numbers of molecules being searched During a search the priority of the THINK process is lowered so that other activities on the computer may be more responsive. The progress of the search is reported in the usual way.

Once the query molecule, reagent and R-group files have been supplied, the user can initiate the search. Each reagent molecule is read from the file in turn and compared with the query; if it matches then it is converted into an R-group and saved to the R-group file. THINK automatically eliminates duplicate R-groups - this situation would occur if the reagent file contained several copies of the same molecule, for instance in different salt forms. THINK will also automatically ignore all molecules with multiple reaction sites since it would be impossible to predict which reaction site is used when physically making the library.

Commands		Dialogs
SEARCH MODE=R-GROUP QUERY=RQUERY#1 FILE=aldrich.smi OUTPUT=gp2.smi OPTIONS=ORDER,PARENT		Search > 2D > R-group; ... Search

The enumeration inputs are the Reaction Query (where the core of the library is the product) and at least one R-group input. The remaining R-group inputs are optional. It is important that the number of connected R-groups match those needed for the library and that these R-group tables are connected to the correct inputs. The R-groups generated by the Rgroup node are generic and can be connected in any substituent position.

One some occasions it may be desirable to read R-groups from files in which the results of previous R-group searches have been saved using the Open node and connect these to the Enumeration inputs.

For Fragment Based Drug Design, the simplest core in the bound fragment (with the 3D coordinates to interact with the protein). Alternatively, a different core group can be used together with an optional ligand (with 3D coordinates to interact with the protein) to which the core is fitted. In both cases it is essential that the core is modified to indicate which atoms are the group connections for which there is an optional dialog.

Once the R-groups for a desired library have been created, they can be used to create the complete molecules. To achieve this, the R-groups must first be read into THINK. The core group (if any) is treated as an R-group, and therefore must also be read into THINK. The generic reaction (which contains the core group as a product) may be used as the reactants will be ignored. Since there may be a large number of R-groups, it is recommended that all other molecules are first deleted to reduce the amount of memory required.

For Fragment Based Drug Design, the simplest core in the bound fragment (with the 3D coordinates to interact with the protein). Alternatively, a different core group can be used together with an optional query substructure (with 3D coordinates to interact with the protein but typically without hydrogens) to which the core is fitted. In both cases it is essential that the core is modified to indicate which atoms are the group connections for which the 2D Edit functionality or keyboard commands may be used.

The R-group files may be read in any order. THINK will use all the R-groups loaded from each file to enumerate the library. If a subset of R-groups is required from a file, then either the required R-groups should be read selectively (see section 2.1), or the entire file should be read and the undesired R-groups deleted.

Note that all the R-groups to be used at any single position in the library must be read from the same file. This is because THINK uses the file name to identify the R-groups for each position in the enumerated molecules. The R-groups from a file may be used at more than one position in the library (eg when building peptides), providing exactly the same set of R-groups is used in each position.

Commands		Dialogs
OPEN FILE=rx-amide.smi OPEN FILE=g1.smi		File > Open
DELETE MOLECULE=PRO@G1		Edit > Delete
OPEN FILE=g2.smi MOLECULE=A*		Selective read not supported

Before a library can be enumerated, THINK needs to know which R-groups are to be attached to each position. The R-groups are identified by the name of the file from which they were read (when any molecule is read, THINK maintains a record of the file from which it came). Only the file names are required (not the file extensions).

If the R-groups contain generic connection points ([0]) then it is important that the file names are supplied in the correct order, with the core file (if any) first. If all the R-groups contain explicit connection points ([1], [2], etc) then the files can be supplied in any order. If the same set of R-groups are to be used at several positions, for instance when building a peptide, then the file name must be supplied once for each position (as in the second example below).

Commands		Dialogs
SAVE ... R-GROUPS=amide-rx,gp1,gp2		File > Enumerate
SAVE ... R-GROUPS=ncap,amino,amino,amino,amino,cap

It is essential to configure the Enumeration node so that the correct number of inputs is processed by selected the number of R-groups in the library core.

An enumerated library may contain many thousands of molecules, many of which are of no further interest because they have undesirable properties. Many of these can be filtered out by using the Drug-Like filter option.

If the protein site is connected, then it will be used as a volume constraint for the enumeration and the conformation of the enumerated molecule randomly sampled. In addition, if any residue restraints are specified then a merit score (see section 14.2) will be calculated and the conformation with the highest merit score will be retained.

An enumerated library may contain many thousands (or millions) of molecules, many of which are of no further interest because they have undesirable properties. These molecules may be filtered out from the library by applying the criteria from a learn file. This file, created during an earlier data analysis calculation (see Chapter 11), contains a list of undesirable substructures and of desirable property values. When applied during library enumeration, molecules which contain any of the undesirable substructures or whose properties lie outside any of the desirable ranges are automatically discarded instead of being saved to the file. Note that these filters are applied to the enumerated molecules, not the constituent R-groups. The learn file is assumed to have the same name as the field that contains activity data (set through the ACTIVITY keyword in the CUSTOMISE command). If this field is not set then the learn file "default.lrn" in the THINK_EXEC directory will be used.

Commands		Dialogs
CUSTOMISE ACTIVITY=LOGK		Not supported
SAVE ... OPTIONS=FILTER		File > Enumerate > Filter

If a query is specified, then it will be used to orientate the core group unless the reserved name of CORE is specified, in which case the initial core orientation is preserved. Any protein will then be used as a volume constraint for the enumeration and the conformation of the enumerated molecule randomly sampled. In addition, if any residue restraints are specified then a merit score (see section 14.2) will be calculated and the conformation with the highest merit score will be retained.

The molecules within a library are enumerated by permuting the R-groups within the sets. Thus the total number of molecules generated is the product of the sizes of the sets, although this may be reduced by applying filters (see above). The enumerated molecules are saved to a file for subsequent use within THINK or other software packages. Because of the sheer number of molecules that may be created, it is recommended that the SMILES format is used since this is the most compact representation of the molecules.

THINK automatically constructs a temporary copy of each enumerated molecule, compares it with the filter criteria (if set) and saves it to the file before discarding it. The molecule is constructed by plugging together the R-groups according to a set of rules:

• All explicit connection atoms (atoms with atom types >0) are paired - an explicit connection atom can only be joined to a connection atom with the same value
• All generic connection atoms (atoms with atom type 0) are converted into explicit connection atoms and paired
  
• Each pair of connection atoms is removed and the remaining bonds joined together

Commands		Dialogs
SAVE FILE=lib1.smi R-GROUPS=L1,G1,G2 OPTIONS=FILTER		File > Enumerate > ... Save
SAVE FILE=lib1.smi R-GROUPS=LIGAND2,G1 QUERY=CORE		Not supported

See the THINK Theory Manual for more information on the rules governing the conversion of generic connection atoms into explicit connection atoms.