Predicting Indirect DNA Damage by Simulating Metabolic Activation of Chemicals

Predicting Indirect DNA Damage by Simulating Metabolic Activation of Chemicals

1

2nd McKim Workshop on Reducing Data Redundancy in Cancer AssessmentBaltimore, 8-10 May 2012

Ovanes Mekenyan, Milen Todorov, Ksenia Gerova

Laboratory of Mathematical Chemistry, Bulgaria

2

Outlook

• Goal• Methods• Data• Predicting:

• 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



• Conclusions

4

• Predicting indirect DNA damage in the General Workflow Diagram for screening large chemicals inventories for carcinogenicity

Goal

5

General Flow Diagram for Screening Large Inventories for carcinogenicity

Inventory

Direct DNA Reactive

Chemicals

DNA reactiveMetabolites

Ames Positive with S9

Ames Positive w/o S9

Classify asGenotoxic Bacterial Mutagen

High Carcinogenicity

Potential?

Generate metabolites

YY

Receptor-Based Screening

Low Carcinogenit

Potential

Y

N

Y Y

Protein Reactive

Chemicals

High Priority for Tumor

Promotion Assays

No-ThresholdRisk Assessment

CTA Assays for Nongenotoxic/

EpigeneticChemicals

Intermediate Priority for

Tumor Promotion

AssaysThreshold EffectRisk Assessment

6

General Flow Diagram for Screening Large Inventories for carcinogenicity

Inventory

Direct DNA reactive

Indirect DNA reactive

Ames Positive with S9

Ames Positive w/o S9

Bacterial Mutagen

Chrom Ab ?MicroNucl ?

Protein OASIS

Generate metabolites

N

Y

Y

Receptor-Based Epigenetic

Screen

Low Carcinogenit

Potential

Y

Chrom Ab ?MicroNucl ?

Refine TIMES/Structural alerts

N

YY

N

Oxidative stress?

In vivo Mammal Tests

Protein Reactive

Return for further screening

7

Outlook



• Conclusions

DNA binding profile by OASIS

DNA binding profile by OECD

8

Methods:

• QSAR Toolbox profiles for DNA binding

Illustration of the DNA binding profile of the QSAR Toolbox

9

Known DNA (covalent) binding mechanisms

Known DNA (covalent) binding mechanisms

Structural boundaries of the category

Structural boundaries of the category

DNA binding profile by OASIS

DNA binding profile by OECD

25

Methods:

• QSAR Toolbox profiles for DNA binding

• TIMES Metabolic simulator for rat liver S9

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

Illustration the OASIS Metabolic Simulators

(extract from the Rat in vivo metabolism simulator)

27

28

Substrate Principle transformations MetabolitesSimulator of metabolismSimulator of metabolism

Aliphatic C-oxidation

C CH3 C CH2OH

Epoxidation

C C C C

O


C CH2OH C C

O

H

Epoxide Hydration

C C

O

C C OHHO


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

29

Substrate Principle transformations MetabolitesSimulator of metabolismSimulator of metabolism


C CH3 C CH2OH

Epoxidation

C C C C

O


C CH2OH C C

O

H

Epoxide Hydration

C C

O

C C OHHO


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.90

P= 0.93

P= 0.94

P= 0.95

P= 0.96

P= 0.97

30

Substrate Principle transformations Metabolites


C CH3 C CH2OH

Epoxidation

C C C C

O


C CH2OH C C

O

H

Epoxide Hydration

C C

O

C C OHHO


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.94

P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

CH2C CH3

- Isopropenylbenzene

31

Match? - No! C CH2OH C CO

H



C CH3 C CH2OH

Epoxidation

C C C C

O

C C

O

C C OHHO


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.94

P= 0.93

P= 0.90

P= 0.95

P= 0.96

Aliphatic C-oxidationCH2C CH3

P= 0.97

Epoxide Hydration


32

C CH2OH C C

O

H


P= 0.97


C CH3 C CH2OH

Epoxidation

C C C C

O

C C

O

C C OHHO


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.94

P= 0.93

P= 0.90

P= 0.95

CH2C CH3

P= 0.96


Epoxide Hydration


33

Match? - No! C C

O

HO C C OH


C CH2OH C C

O

H


C CH3 C CH2OH

Epoxidation

C C C C

O


C C

O

HC C

O

OH

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.94

P= 0.95

CH2C CH3

P= 0.93

P= 0.90

O-Glucuronidation

P= 0.96


Epoxide Hydration

P= 0.97


34


C C

O

C C

Epoxidation

RESULTMatch? - Yes!C

H2CCH3

OC

H2C CH3

P= 0.95

C CH2OH C C

O

H


C C

O

C C OHHOP= 0.96

Epoxide Hydration


C CH3 C CH2OH


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.94

P= 0.93

P= 0.90

P= 0.97

Generated map

1.1

Epoxidation


35



RESULTMatch? - Yes!

CH2C CH3

C CH3 C CH2OH

CC CH2OHH2

P= 0.94

C CH2OH C C

O

H


P= 0.96

Epoxide Hydration

C C

O

C C OHHO

Epoxidation

C C C C

O

P= 0.95


C C

O

HC C

O

OH

C OH C O

O

HOH H

OH

H

OHH

COOH

H

P= 0.93

P= 0.90

O-Glucuronidation

P= 0.97


1.1

C-oxidation

1.2

Generated map

Epoxidation

36


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

C CH2OH C C

O

H

Epoxide Hydration



C C

O

C C OHHO


C C

O

C C

Epoxidation

Match?

CH2C CH3

C CH3 C CH2OH

- No!

P= 0.94

P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

Generated map

Epoxidation C-oxidation


1.1 1.2

37


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

C CH2OH C C

O

H

Epoxide Hydration



C C

O

C C OHHO


C C

O

C C

Epoxidation

Match?

CH2C CH3

- No!

C CH3 C CH2OHP= 0.94

P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

Generated map



1.1 1.2

38


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

C CH2OH C C

O

H

Epoxide Hydration



C C

O

C C OHHO


C C

O

C C

Epoxidation

Match? - No!


P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

Generated map


1.1 1.2

CH2C

OCH3

- Metabolite 1.1

39


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

C CH2OH C C

O

H

Epoxide Hydration




C C

O

C C

Epoxidation

Match?- Yes!


P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

CH2C

OCH3

C C

O

HO C C OH RESULT

C OH

OH

CH2

1.1 1.2

2.1

Hydration

C-oxidationEpoxidation

Generated map- Metabolite 1.1

40


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




C C

O

C C

Epoxidation

Match?- Yes!


P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

1.1 1.2

RESULTC OH

OH

CH2

- Metabolite 2.1

C C

O

C C OHHO

C CH2OH C CO

H

C OH

O

CH2

1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

41


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




C C

O

C C

Epoxidation

Match?- Yes!


P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

1.1 1.2

RESULT

C C

O

C C OHHO

C CH2OH C CO

H 1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

- Metabolite 1.2.

CC CH2OHH2 O

C

HCCH2

C-oxidation2.2

42


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




C C

O

C C

Epoxidation

Match?- No!


P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

1.1 1.2

C C

O

C C OHHO

1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

- Metabolite 2.2.

OC

HCCH2

C-oxidation2.2

C CH2OH C C

O

H

43


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




C C

O

C C

Epoxidation

Match?- No!


P= 0.93

P= 0.90

P= 0.95

P= 0.96

P= 0.97

1.1 1.2

C C

O

C C OHHO

1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

OC

HCCH2

C-oxidation2.2

C CH2OH C C

O

H

- Metabolite 2.2.

44


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




Epoxidation

Match?- Yes!


P= 0.93

P= 0.90

P= 0.96

P= 0.97

1.1 1.2

C C

O

C C OHHO

1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

OC

HCCH2

C-oxidation2.2

C CH2OH C C

O

H

C C

O

C C

(Conjugated aldehyde group prevents epoxidation)

- Metabolite 2.2.

45


C C

O

HC C

O

OH

O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




Epoxidation

Match?- No! C CH3 C CH2OHP= 0.94

P= 0.93

P= 0.90

P= 0.96

P= 0.97

1.1 1.2

C C

O

C C OHHO

1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

OC

HCCH2

C-oxidation2.2

C CH2OH C C

O

H

C C

O

C CP= 0.95

- Metabolite 2.2.

46


O-Glucuronidation

C OH C O

O

HOH H

OH

H

OHH

COOH

H

Epoxide Hydration




Epoxidation

Match?- Yes!


P= 0.93

P= 0.90

P= 0.96

P= 0.97

1.1 1.2

C C

O

C C OHHO

1.1 1.2

2.1

Generated map


3.1

C-oxidation

Hydration

OC

HCCH2

C-oxidation2.2

C CH2OH C C

O

H

C C

O

C CP= 0.95

C C

O

H

C C

O

OH

RESULT

OC

CCH2

OH

3.2

C-oxidation

- Metabolite 2.2.

Metabolic SimulatorsBridging the “Parent Gap”

Virtual metabolism uses a heuristic substructure search engine applied to a hierarchy of possible molecular transformations

Library ofBiotransformations& Abiotic Reactions

Documented Partial Maps

Algorithm for optimizingTransformationProbabilities

(Rate constants)

MetabolicMaps and ReactivityProfiles

Metabolic Simulators

ParentChemicals

Simulated Metabolic Activation of 2-AcetylaminofluoreneSimulated Metabolic Activation of 2-Acetylaminofluorene(AMES mutagenicity in Rat liverS9)(AMES mutagenicity in Rat liverS9)

NH

O

NH

O

OH

NH

O

O

NH2

O

HO

O

NHOH

O

N+HO

NH

OHO

NH

O

O

NH

O

O

NH

OHO

NH

OHO

OHNH

OHO

OH

NH

OHO

O

NH

OHO

O

N+H

HO

ON+H

OH

O

. . . . . .

NHX

OO

X = H, OH,

O

Activated metabolites

Documented

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 logicIn vivo bioactivation

•Skin metabolism

Documents

Predicting Indirect DNA Damage by Simulating Metabolic Activation of Chemicals