Tautomers - Advanced Databases for in-silico Screening ?

Frank Oellien, 18th CIC Workshop 2004, Boppard

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

Biological Relevance of Tautomers

Targets: Bacteria, Arthropods, and ParasitesVariance for physiological pH expected – not static values !

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

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 (exception FlexX 2004)

X

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-InhibitorsJ. Biol. Chem. 276, 2001, 17405-12.

• Pospisil et al., Ligands of herpesviral thymidine kinases, Helvet. Chim. Acta 85, 2002, 3237-50.

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 thatallows the integration and automation of tautomergeneration in our existing screening workflow.

CACTVS: Chemical data management system

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

Examples

Only 2 transformation rules are needed (1,3 and 1,5-H-shift)

N

NH

NH

N

NH2

O

NH

N N

NH

NH2

O

NH

NH

N

NH

NH

O

N

NH

N

NH

NH2

O

N

N N

NH

NH2

OH

N

NH

N

NH

NH

OH

N

NH

N

N

NH2

OH

NH

N N

N

NH2

OH

NH

NH

N

N

NH

OH

NH

N NH

N

NH2

O

NH

NH

NH

N

NH

O

NH

N NH

N

NH

OH

N

N NH

N

NH2

OH

N

NH

NH

N

NH

OH

NH

N N

NH

NH

OH

1 2 3 4 5

6 7 8 9 10

11 12 13 14 15

0

200000

400000

600000

800000

1000000

1200000

1400000

Maybridge Specs TimTec VitasM AsinexPlatinum

AsinexGold

ChemDiv

no tautom ers with tautom ers

Database Expansion

x2,7

x3,5

x3,2

x3,6

x3,0

x3,6

x3,4

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 MultiplierMaybridge Screening > 150 2,5Asinex Platinum > 250 2,9VitasM (in-hose Stock) > 560 3Tripos Leadscreen > 1400 2,2

Tautomeric Fingerprintsa) Asinex (Platinum Collection)

0

20

40

60

80

100

1 4 7 10 13 16 20 24 28 34 39 46 52 69 115No of tautomer forms

Taut

omer

freq

uenc

y

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 21

b) Specs (Screening Collection)

0

20

40

60

80

100

1 9 17 25 33 41 49 57 65 74 84 95 108 125 149 182 210 348 742No of tautomer forms

Taut

omer

freq

uenc

y

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Virtual Screening Workflow @ Intervet

2D / 3DStructure DB

(MDL)

Specific 3DDatabases

(Catalyst, Unity)

TautomerGeneration

Pre-Processing

Tautomer-sensitiveDuplicate check

Data Analysis Virtual

Screening

Example I - MMP-8 (PDB Entry Code: 1JJ9)

Matrix Metalloproteinase Inhibitor: 8-Barbiturate

H. Brandstetter et al., MMP-8-InhibitorsJ. Biol. Chem.276, 2001, p 17405-12.

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

Example I – MMP-8 Pharmacophore from X-RAY

Matrix Metalloproteinase Inhibitor: 8-Barbiturate

H-Bond Donor: greenH-Bond Acceptor: magentaHydrophobic aliphatic: blueRing Aromatic: brown

Example I – MMP-8 Testdatabase

993 molecules selected from NCI 2000 Database (Catalyst Version)

2D / 3DStructure DB

(NCI 2000)

Divers CompoundSelection (Cerius 2 4.9)

TautomerGeneration (CACTVS)

3DDatabase(Catalyst,

No Tautomers)

3DDatabase(Catalyst,

with tautomers)

1536 compounds including5 8-Barbiturate Tautomers

994 compounds including8-Barbiturate (X-RAY) *

* Knowledge of stable tautomeric form is needed as prerequisite.

Default conditions for Catalyst database building – exception: max conformers 150

Example I – MMP-8 Search on Testdatabase

Questions:• Covergage of the suggested tautomeric states for the 8-

barbiturate 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 ?

3DDatabase(Catalyst,

No Tautomers)

3DDatabase(Catalyst,

WithTautomers)

1536 compounds including5 8-Barbiturate Tautomers

994 compounds including8-Barbiturate (X-RAY)

Exotic luxury or useful effort

?

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

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 incommon)

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 aspectCritical 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

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

Qualitative pharmacophore model derived from human CDK2 best selective inhibitors – prefilter for docking libraries

H-Bond Donor: greenH-Bond Acceptor: magentaHydrophobic: blueRing Aromatic: brown

Feature mappingof pharmacophorehypothesis with CDK2 selective molecules

Example II - CDK of Eimeria tenella - hiphop

993 molecules selected from NCI 2000 Database (Catalyst Version) plus 123 known human CDK1/2 inhibitors. 93 ligands show activity against CDK2.

3DDatabase(Catalyst,

No Tautomers)

3DDatabase(Catalyst,

WithTautomers)

2368 compounds including837 CDK1/2 inhibitor tautomers *

*733 CDK2 inhibitor tautomers

1116 compounds including123 CDK1/2 known inhibitors

Example II - CDK of Eimeria tenella - database

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 %

Example II - CDK of Eimeria tenella - Results

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)

• Number of true hits better in case of tautomers • High overlap among top scoring ligands for both searchesRemark: SBF models under way

Critical aspect:• Number of unique hits is reduced by using tautomer databases

• All kinds of tautomeric states are considered.

Example II - CDK of Eimeria tenella - Summary

• Modifications of tautomeric rules• Automatisation of database building workflow

• Implementation of defined Ionisations• Further investigations with examples

Future Tasks

AcknowledgementsDr. J. Cramer, C. Bayer Dr. J. Schröder, PD Dr. P. SelzerIntervet Innovation GmbH

Dr. W.-D. IhlenfeldtΧemistry GmbH

Dr. O. SacherMolecular Networks GmbH

Dr. T. HidakaTakeda Pharmaceutical Ltd.

Who we are ...

Paul SelzerRichard Marhöfer

Jörg SchröderJörg Cramer

Andreas Rohwer

BIOCHEMINFORMATICS

Carsten Beyer

Andreas Krasky

Anette Klinger

Frank Oellien

Kristin Engels Hon Tran