1. Predicting Indirect DNA Damage by Simulating Metabolic Activation of Chemicals
Ovanes Mekenyan, Milen Todorov, Ksenia Gerova
Laboratory of Mathematical Chemistry, Bulgaria
2nd McKim Workshop on Reducing Data Redundancy in Cancer Assessment
Baltimore, 8-10 May 2012

2. Outlook Goal Methods Data Predicting: Conclusions
AMES mutagenicity without metabolic activation
AMES metabolic activation chemicals negative as parents
Illustrating metabolic activation
False positives after metabolic activation
False negatives after metabolic activation
Conclusions

3. Outlook Goal Methods Data Predicting: Conclusions
AMES mutagenicity without metabolic activation
AMES metabolic activation chemicals negative as parents
Illustrating metabolic activation
False positives after metabolic activation
False negatives after metabolic activation
Conclusions

4. Goal
Predicting indirect DNA damage in the General Workflow Diagram for screening large chemicals inventories for carcinogenicity

5. General Flow Diagram for Screening Large Inventories for carcinogenicity
Inventory
Classify as
Genotoxic Bacterial Mutagen
High Carcinogenicity
Potential?
High Priority for Tumor Promotion Assays
No-Threshold
Risk Assessment Y
Direct DNA Reactive
Chemicals
Ames Positive w/o S9 Y Y N
Generate metabolites
DNA reactive
Metabolites Y Y
Ames Positive with S9
Protein Reactive
Chemicals
Intermediate Priority for Tumor Promotion Assays
Threshold Effect
Risk Assessment
CTA Assays for Nongenotoxic/
Epigenetic
Chemicals
Receptor-Based Screening
Low Carcinogenit Potential

6. General Flow Diagram for Screening Large Inventories for carcinogenicity
Inventory
Protein OASIS Y
Chrom Ab ?
MicroNucl ? N
Direct DNA reactive
Ames Positive w/o S9 Y
Bacterial Mutagen
In vivo Mammal Tests Y N
Generate metabolites Y
Indirect DNA reactive Y
Ames Positive with S9
Refine TIMES/
Structural alerts
Chrom Ab ?
MicroNucl ? N
Return for further screening
Protein Reactive
Oxidative
stress?
Receptor-Based Epigenetic Screen
Low Carcinogenit Potential

7. Outlook Goal Methods Data Predicting: Conclusions
AMES mutagenicity without metabolic activation
AMES metabolic activation chemicals negative as parents
Illustrating metabolic activation
False positives after metabolic activation
False negatives after metabolic activation
Conclusions

8. Methods: DNA binding profile by OASIS DNA binding profile by OECD
QSAR Toolbox profiles for DNA binding
DNA binding profile by OASIS
DNA binding profile by OECD

9. Illustration of the DNA binding profile of the QSAR Toolbox

11. Known DNA (covalent) binding mechanisms

12. Known DNA (covalent) binding mechanisms

13. Structural boundaries of the category

14. Structural boundaries of the category

15. Methods: DNA binding profile by OASIS DNA binding profile by OECD
QSAR Toolbox profiles for DNA binding
DNA binding profile by OASIS
DNA binding profile by OECD
TIMES Metabolic simulator for rat liver S9

16. OASIS Metabolic Simulator
Prioritized list of non-enzymatic (abiotic) and enzymatic molecular transformations;
Molecular transformations are characterized by:
Source and product fragments;
Inhibiting “masks” preventing the application of metabolic reactions if necessary;
Substructure-matching software engine applies the simulated biochemical
Reproduces the documented metabolic pathways and toxicity endpoint resulting from metabolic activation of chemicals 26

17. Illustration the OASIS Metabolic Simulators
(extract from the Rat in vivo metabolism simulator)

18. Simulator of metabolism
Substrate Principle transformations Metabolites
Simulator of metabolism
Aliphatic C-oxidation
Epoxide Hydration
Epoxidation
Aliphatic C-oxidation
Aliphatic C-oxidation
O-Glucuronidation

19. Simulator of metabolism
Substrate Principle transformations Metabolites
Aliphatic C-oxidation
P= 0.97
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

20. Substrate Principle transformations Metabolites - Isopropenylbenzene
Aliphatic C-oxidation
P= 0.97
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

21. Match? - No! Substrate Principle transformations Metabolites
- Isopropenylbenzene
Aliphatic C-oxidation
P= 0.97
Match?
- No!
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

22. Substrate Principle transformations Metabolites - Isopropenylbenzene
Aliphatic C-oxidation
P= 0.97
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

23. Match? - No! Substrate Principle transformations Metabolites
- Isopropenylbenzene
Aliphatic C-oxidation
P= 0.97
Epoxide Hydration
P= 0.96
Match?
- No!
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

24. Match? - Yes! Substrate Principle transformations Metabolites
- Isopropenylbenzene
Generated map
1.1
Epoxidation
Aliphatic C-oxidation
P= 0.97
Epoxide Hydration
P= 0.96
Epoxidation
RESULT
Match?
- Yes!
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

25. Match? - Yes! Substrate Principle transformations Metabolites
- Isopropenylbenzene
Generated map
C-oxidation
1.2
Aliphatic C-oxidation
P= 0.97
Epoxidation
1.1
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
RESULT
Match?
- Yes!
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

26. Match? - No! Substrate Principle transformations Metabolites
- Isopropenylbenzene
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
Match?
- No!
P= 0.93
O-Glucuronidation
P= 0.90

27. Match? - No! Substrate Principle transformations Metabolites
- Isopropenylbenzene
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90
Match?
- No!

28. Match? - No! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
Match?
- No!
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
Epoxide Hydration
P= 0.96
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

29. Match? - Yes! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
Epoxide Hydration
P= 0.96
RESULT
Match?
- Yes!
2.1
Hydration
Epoxidation
P= 0.95
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

30. Match? - Yes! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
RESULT
Match?
- Yes!
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
P= 0.96
Hydration
2.1
Epoxidation
P= 0.95
3.1
C-oxidation
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

31. Match? - Yes! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
RESULT
Match?
- Yes!
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
P= 0.96
C-oxidation
2.2
Hydration
2.1
Epoxidation
P= 0.95
3.1
C-oxidation
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

32. Match? - No! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
Match?
- No!
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
P= 0.96
C-oxidation
2.2
Hydration
2.1
Epoxidation
P= 0.95
3.1
C-oxidation
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

33. Match? - No! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
Match?
- No!
P= 0.96
C-oxidation
2.2
Hydration
2.1
Epoxidation
P= 0.95
3.1
C-oxidation
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

34. (Conjugated aldehyde group prevents epoxidation)
Substrate Principle transformations Metabolites
- Metabolite 2.2.
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
P= 0.96
C-oxidation
2.2
Hydration
Epoxidation
2.1
(Conjugated aldehyde group prevents epoxidation)
Match?
- Yes!
3.1
C-oxidation
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

35. Match? - No! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
P= 0.96
C-oxidation
2.2
Hydration
Epoxidation
2.1
P= 0.95
3.1
C-oxidation
Aliphatic C-oxidation
Match?
- No!
P= 0.94
Aliphatic C-oxidation
P= 0.93
O-Glucuronidation
P= 0.90

36. Match? - Yes! Substrate Principle transformations Metabolites
Generated map
Aliphatic C-oxidation
P= 0.97
Epoxidation
C-oxidation
1.1
1.2
1.1
1.2
Epoxide Hydration
P= 0.96
C-oxidation
2.2
Hydration
Epoxidation
2.1
P= 0.95
3.1
C-oxidation
3.2
C-oxidation
Aliphatic C-oxidation
P= 0.94
Aliphatic C-oxidation
RESULT
Match?
- Yes!
P= 0.93
O-Glucuronidation
P= 0.90

37. Bridging the “Parent Gap”
Metabolic Simulators
Bridging the “Parent Gap”
Library of
Biotransformations
& Abiotic Reactions
Parent
Chemicals
Algorithm for optimizing
Transformation
Probabilities
(Rate constants)
Metabolic
Simulators
Documented
Partial Maps
Metabolic
Maps and
Reactivity
Profiles
Virtual metabolism uses a heuristic substructure search engine applied to a hierarchy of possible molecular transformations

38. Simulated Metabolic Activation of 2-Acetylaminofluorene
(AMES mutagenicity in Rat liverS9)
Documented

39. The OASIS Simulators of Mammalian
Metabolism
Liver S9 metabolism
Different level of biological organisms (US EPA)
Rat liver subcellular (microsomal)
Rat liver cellular (in vitro)
Organism (in vivo)
In vivo metabolism – rat liver (in vivo MNT)
In vivo detoxification logic
In vivo bioactivation
Skin metabolism