18th CIC Workshop 2004, Boppard, Germany

1. Tautomers - Advanced Databases for
in-silico Screening ?
Frank Oellien, 18th CIC Workshop 2004, Boppard

2. Overview
• Motivation for tautomers in screening data sets
• Tautomer enumeration approach
• Workflow
• Examples for ‘enhanced’ Database Search
• Results for ‘enhanced’ Database Search
• Summary and Future Tasks

3. Biological Relevance of Tautomers
Targets: Bacteria, Arthropods, and Parasites
Variance for physiological pH expected – not static values !

4. Ambiguities in Proteins and Ligands
Proteins (X-Ray structures)
• Flexibility (e.g. Gln, Asn)
• Ionisation states (e.g. Glu, Asp)
• Tautomerism (e.g. His)
Ligands (Compound Libraries)
•
•
•
•
Conformations
Ionization states
Stereo centers
Tautomers

5. Software: Virtual Screening
Types of Virtual Screening Software
• High-throughput Docking of ligands into
protein X-Ray structures (Gold, FlexX)
• DB for pharmacophore search (Catalyst,
Unity)
Current VS software applications adress:
•
•
•
•
Conformations
Ionization states
Stereo centers
Tautomers

X

X (exception FlexX 2004)

6. Biological Relevance of Tautomers
Tautomeric states of ligands can be relevant
for biological interactions
• Derivates of tetrazole, triazole, thiazole,
pyrazole, iminopyrimidine, …
• Brandstetter et al., MMP-8-Inhibitors
J. Biol. Chem. 276, 2001, 17405-12.
• Pospisil et al., Ligands of herpesviral
thymidine kinases, Helvet. Chim. Acta 85,
2002, 3237-50.

7. Software: Tautomer Generation
Tautomer Generation Applications
• Agent 2.0 (ETH Zurich- Switzerland)
• QUAC PAC 1.1 (OpenEyes)
• StereoPlex (Tripos)
• no extensions by the means of user-defined rules
• no tautomer-sensitive duplicate check
Aim: Easily extensible and scriptable software that
allows the integration and automation of tautomer
generation in our existing screening workflow.
 CACTVS: Chemical data management system

8. Tautomer enumeration
• C core library, Tcl command layer
• Main command:
ens transform $eh $tlist <direction> <reactionmode> <flags>
<overlapmode> <excludelist> <maxtautomers> <timeout>
tlist: Transformation definition - SMIRKS line notation
[#1:1][O:2][C:3]#[N:4]>>[O:2]=[C:3]=[N:4][#1:1]
• preferred tautomer forms
• tautomer sensitive duplicate check
• 21 pre-defined rules (up to 1,11-H-shifts)
• user-defined tautomer sets

9. Examples
N
N
H2N
N
H1
N
H
N
H2N
N
H2
H
N
HN
N
N
H2N
N
N
N
N
H 11
N
HN
N
H2N
N
4
H
N
N
N
H 12
HN
HN
N
H
N
N
H2N
N
5
N
HN
8
N
H
HN
N
N
H 13
HN
N
14
N
H 10
N
H
OH
N
HN
N
N
OH
N
HN
N
H
OH
N
H9
N
N
N
O
OH
H2N
OH
H
N
N
H3
HN
7
O
HN
H2N
N
HN
OH
H
N
N
6
HN
N
N
N
OH
O
H2N
H
N
OH
O
OH
O
O
N
H
H
N
HN
HN
N
N
15
Only 2 transformation rules are needed (1,3 and 1,5-H-shift)

10. Database Expansion
1400000
x
3,4
1200000
1000000
x
3,5
800000
x
3,2
600000
400000
x
3,6
x
3,6
x
3,0
x
2,7
200000
0
Maybridge
no tautom ers
Specs
with tautom ers
TimTec
V itasM
A sinex
Platinum
A sinex
Gold
ChemDiv

11. Tautomer Enumeration - Benchmark
Platform: SGI Fuel R1400 / 600 MHz, 1 GB RAM
Performance depends on
• nature of the compounds
• number of tautomers
SupplierDB
Compounds/min Multiplier
Maybridge Screening
> 150
2,5
Asinex Platinum
> 250
2,9
VitasM (in-hose Stock)
> 560
3
Tripos Leadscreen
> 1400
2,2

12. Tautomeric Fingerprints
a) Asinex (Platinum Collection)
100
40
30
60
20
10
40
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 20 21
20
b) Specs (Screening Collection)
100
0
1
4
7
10
13
16
20 24 28 34
No of tautomer forms
39
Tautomer frequency
Tautomer frequency
50
80
46
80
52
69
50
115
40
30
60
20
10
40
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21
20
0
1
9
17 25 33 41 49 57 65 74 84 95 108 125 149 182 210 348 742
No of tautomer forms

13. Virtual Screening Workflow @ Intervet
2D / 3D
Structure DB
(MDL)
PreProcessing
Tautomer
Generation
Specific 3D
Databases
(Catalyst, Unity)
Tautomer-sensitive
Duplicate check
Data
Analysis
Virtual
Screening

14. Example I - MMP-8 (PDB Entry Code: 1JJ9)
Matrix Metalloproteinase Inhibitor: 8-Barbiturate
H. Brandstetter
et al., MMP-8Inhibitors
J. Biol. Chem.
276, 2001,
p 17405-12.

15. http://home.t-online.de/home/kubinyi/dd-18.pdf

16. Example I – MMP-8 Pharmacophore from X-RAY
Matrix Metalloproteinase Inhibitor: 8-Barbiturate
H-Bond Donor: green
H-Bond Acceptor: magenta
Hydrophobic aliphatic: blue
Ring Aromatic: brown

17. Example I – MMP-8 Testdatabase
993 molecules selected from NCI 2000 Database (Catalyst Version)
2D / 3D
Structure DB
(NCI 2000)
Divers Compound
Selection (Cerius 2 4.9)
3D
Database
(Catalyst,
No Tautomers)
Tautomer
Generation (CACTVS)
994 compounds including
8-Barbiturate (X-RAY) *
3D
Database
Default conditions for Catalyst
database building – exception:
max conformers 150
(Catalyst,
with tautomers)
1536 compounds including
5 8-Barbiturate Tautomers
* Knowledge of
stable tautomeric
form is needed as
prerequisite.

18. Example I – MMP-8 Search on Testdatabase
Questions:
• Covergage of the suggested tautomeric states for the 8barbiturate by our workflow ?
• Conformer generation of Catalyst resemble X-Ray ?
• Fit value significant for X-Ray and also conformers ?
• Signal to noise relationship – fit values of other hits ?
3D
Database
Exotic luxury or
useful effort
(Catalyst,
WithTautomers)
1536 compounds including
5 8-Barbiturate Tautomers
?
3D
Database
(Catalyst,
No Tautomers)
994 compounds including
8-Barbiturate (X-RAY)

19. Example I – MMP-8 Results
Best Search – no tautomers:
• 29 compounds found
• 8-barbiture acid found in database, but scores less significant
(BestFit 3.2)
• 8-barbiture tautomer form (X-ray) BestFit 7.2 (AV= 3.0 SD = 1.7)
• second best scored hit show BestFit 6.2
• only 3 hits score higher then BestFit 4
• best non X-Ray conformer scores BestFit 6.3

20. Example I – MMP-8 Results
Best Search - with tautomers:
• 30 compounds found (22 unique, 8 tautomeric duplicates)
• 8-barbiture BestFit 7.2 (AV= 2.0 SD = 1.5)
• second best scored hit show BestFit 5.5
• only 3 hits score higher then BestFit 4
• non tautomeric 8-barbiture scores BestFit 3.2
• 60 % Overlap between both search results (all top scoring hits in
common)

21. Example I – MMP-8 Summary
• Significant better fit value and hit separation in
case of a database search including tautomers
• X-ray structure closely resembled
• Number unique hits reduced
• Significant more structures have to be
converted – time consuming aspect
Critical aspect:
• Hit rate (unique hits) is lower for database
including tautomers (?)
• X-ray structure or known physiological
conditions in the protein appear to be important
for sensitive pharmacophore searches

22. Example II - CDK of Eimeria tenella
Cyclin Dependent Kinase – Homology Model based on Sequence
Analysis
C. Beyer et. al.
Oral & Poster Presentation
at the 18. Darmstädter
Molecular Modelling
Workshop 2004
J.H. Kinnaird et al.
International Journal for Parasitology 34,
2004, 683–692

23. Example II - CDK of Eimeria tenella - hiphop
Qualitative pharmacophore model derived from human CDK2 best
selective inhibitors – prefilter for docking libraries
Feature mapping
of pharmacophore
hypothesis with
CDK2 selective
molecules
H-Bond Donor: green
H-Bond Acceptor: magenta
Hydrophobic: blue
Ring Aromatic: brown

24. Example II - CDK of Eimeria tenella - database
993 molecules selected from NCI 2000 Database (Catalyst
Version) plus 123 known human CDK1/2 inhibitors. 93 ligands
show activity against CDK2.
3D
Database
3D
Database
(Catalyst,
WithTautomers)
(Catalyst,
No Tautomers)
2368 compounds including
837 CDK1/2 inhibitor tautomers *
*733 CDK2 inhibitor tautomers
1116 compounds including
123 CDK1/2 known inhibitors

25. Example II - CDK of Eimeria tenella - Results
A) Search results without tautomers:
• best hypothesis finds 55.5 % of the CDK2 known
• Inhibitors (AV 39.8 % SD 9.5 %)
• Best selective (selectivity higher 4 included)
• Number of hits 81
B) Search results with tautomers:
• best hypothesis finds 72.2. % of the CDK2 known
• Inhibitors (AV 66.4 % SD 10.0 %)
• Best selective (selectivity higher 4 included)
• Number of unique hits 61
• Overlap of best hypo search in A) with results in B) 93 %

26. Example II - CDK of Eimeria tenella - Results
A) Search results without tautomers:
• GH Score 0.54 for best pharmacophore model
B) Search results with tautomers:
• GH Score 0.77 for best pharmacophore model of A)

27. Example II - CDK of Eimeria tenella - Summary
• Number of true hits better in case of tautomers
• High overlap among top scoring ligands for both
searches
Remark: SBF models under way
Critical aspect:
• Number of unique hits is reduced by using
tautomer databases
• All kinds of tautomeric states are considered.

28. Future Tasks
• Modifications of tautomeric rules
• Automatisation of database building
workflow
• Implementation of defined Ionisations
• Further investigations with examples

29. Acknowledgements
Dr. J. Cramer, C. Bayer
Dr. J. Schröder, PD Dr. P. Selzer
Intervet Innovation GmbH
Dr. W.-D. Ihlenfeldt
Χemistry GmbH
Dr. O. Sacher
Molecular Networks GmbH
Dr. T. Hidaka
Takeda Pharmaceutical Ltd.

30. Paul Selzer
Richard Marhöfer
Andreas Rohwer
Jörg Schröder
Jörg Cramer
BIOCHEMINFORMATICS
Who we are ...
Anette Klinger
Carsten Beyer
Frank Oellien
Kristin Engels
Andreas Krasky
Hon Tran