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Office of Research and DevelopmentNational Center for Computational Toxicology
August 25, 2011
Tox21 & ToxCast Chemical Landscapes: Laying the Foundation for 21st Century Toxicology
5th Meeting on U.S. Government Chemical Databases & Open Chemistry, Frederick, MD
Ann RichardNational Center for Computational Toxicology
Office of Research and DevelopmentNational Center for Computational Toxicology 2
NCCT’s ToxCast Program
StructuresHTS
In Vitro
In VivoQSAR
Office of Research and DevelopmentNational Center for Computational Toxicology
Generic Chemicals
Test Substances
NCCT Public Data http://www.epa.gov/ncct/
ToxRefDBToxCast
ACToR DSSTox
Tox21
Office of Research and DevelopmentNational Center for Computational Toxicology
NCCT Public Data http://www.epa.gov/ncct/
ToxRefDBToxCast
Chemical Structure AnnotationChemical Management
Chemical QC
DSSToxACToR
Tox21
Office of Research and DevelopmentNational Center for Computational Toxicology
NCCT Public Data http://www.epa.gov/ncct/
ToxRefDB
Tox21
ToxCast
DSSTox
Data IntegrationChemical – Assay Linkage
In vivo data LinkageAnalysis tools
ACToR
Office of Research and DevelopmentNational Center for Computational Toxicology
2007 2009
2008 2010
ToxCast ProjectTo
xCas
t con
cept
pap
er To
xSci
EPA NCCT launches ToxCast
ToxC
ast P
hase
I co
ntra
cts
issue
d
ACToR launchedNAS Report released
Phas
e I t
estin
g be
gins
Phase I data results
ToxC
ast D
ata A
nalys
is
Sum
mit
ToxRefDB web access w/in ACToR
Full Phase I data publication/release
Phase I (309) Phase II: (700)
Office of Research and DevelopmentNational Center for Computational Toxicology
Tox21 Project
2005 2007 2009
10K
5.6K
1.4K
Tox21 Chemical Library2.8K
2006 2008 20102004
2011
EPA Tox21 Plate A
NTP HTS Plate A
ToxCast Phase I NCGC Drug
Plates A,B
Tox21 EPA Plates A’,B,C
NTP Tox21Plates A’,B,C
NCGC DrugPlates A’,B’,C
ToxCast Phase II
Office of Research and DevelopmentNational Center for Computational Toxicology 8
Tox21 Chemical x Assay Landscape
# Chemicals>500
~1000 ~10,000
# Assays100
ToxCast Phase I & II
960 Chem x >500 assays
Tox21
~10,000 Chem x 50-100 assays
1408 Chem x >100 assays
NTP HTSPlate A
Drugs
NCCT/EPA NIEHS/NTP NIH/NCGCEnvironmentalIndustrialPesticidesFood UseDrugsToxicology
1536 well microplate format(1408 cmpds/plate) x 9 plates
FDACFSAN/CDER
Office of Research and DevelopmentNational Center for Computational Toxicology
e1k 820 unique(1000 samples)
To be tested in endocrine-related subset of ToxCast assaysToxCast_
960
Phase I & II(a,b)
111 (+24 PhIIc) failed drugs, CFSAN, pesticides, EPA high interest compounds tested in >500 HTS assays
100
IIc
Tox21: EPA Chemical Inventories
TOX21_EPA_3726
Office of Research and DevelopmentNational Center for Computational Toxicology
Tox21: EPA ToxCast Chemicals ToxCast Phase I (293 unique)
EPA pesticidal actives w/ rich in vivo data PFOAs, BPA, metabolite/parent pairs
ToxCast Phase II a,b (776) +c (100) EPA pesticides, high interest EPA and stakeholder inventories, data rich
chemicals (EDSP, OPPT, Antimicrobials, Inerts, …) 135 Pharma failed drugs w/ pre-clinical & clinical tox data FDA CFSAN data rich, NCTR LTKB Priority 1 drugs Toxicity reference chemicals, data-rich chemicals, NTP immunotox
Broaden selections from ToxCast Phase II nomination lists to include in Tox21 EPA set
Office of Research and DevelopmentNational Center for Computational Toxicology
211 volatile/ insufficient sample
COA/MSDS review
Unable to procureToo expensive
4371 compounds Able to purchase
3726 unique substances
EPA Tox21 Inventory
e.g., EPA Tox21 Chemical Procurement
~7,000 compounds Candidates for procurement Complex mixturesIll-defined substancesNo structureInsoluble (est. LogP)Volatiles (est. VP)InorganicsExplosiveReactivePolymers …
435 Insoluble in DMSO (approx 10%)
Solubilize in DMSO
HPV, MPV SubstancesAntimicrobials
Pesticide actives Pesticide inertsWater contaminants
Industrial chemicals
NCCT Programs EU, FDAIn vivo data availability (EPA, NTP, FDA)
Endocrine disruptors
~19,000 compounds EPA Tox21/ToxCast Phase II Chemical Nominations (over 100 lists)
Green Chemistry
11
Office of Research and DevelopmentNational Center for Computational Toxicology
Tox21 Chemicals: EPA Selection Strategy
Nominations & tracking of inventory overlaps by CAS Prioritize for procurement Cost & availability Filter by MW, physchem, volatility, MSDS cautions DMSO solubility
Office of Research and DevelopmentNational Center for Computational Toxicology
13
ToxCast PhI&PhII 960:# Compounds per Inventory (960 x 16)
PesticideInerts
Water
Consumer
Antimicrobials
Green Chemistry
HPV
MPV
TRI
IRIS
EDSP
GRAS
AIR
PesticideIn-erts; Series1;
243
Water; Se-ries1; 217
Consumer; Series1; 188
Antimi-crobials; Series1;
91
Green Chem-istry; Series1;
75
HPV; Se-ries1; 232
MPV; Se-ries1; 83
TRI; Series1; 216
IRIS; Se-ries1; 240
EDSP; Se-ries1; 120
GRAS; Se-ries1; 26
AIR; Series1; 90
SUM
580
94
202
SUM; Se-ries1; 0
580; Se-ries1; 0
94; Se-ries1; 0
202; Se-ries1; 0
Series1; 0
SUM; Se-
ries2; 580
580; Se-
ries2; 94
94; Se-
ries2; 202
202; Se-
ries2; 111
Se-ries2; 329
960 Total chemicals 2740 total overlaps across 16 diverse inventories Excellent coverage of multiple high-interest inventories Broad diversity of chemical-use and types Large overlap with data-rich inventories
Office of Research and DevelopmentNational Center for Computational Toxicology
14
ToxCast_960 Pharmaceuticals: Multipurposing (255)
111 Failed pharmaceuticals – no overlaps 144 additional cmpds classified as drugs; on other lists with other uses 137 of these appear on 2 or more lists Caffeine appears on 18 lists
HPVCSI_1081CPDBAS_677EPAFHM_276
NTP_ToxCarcLong_SubstanceNTP_ReproTox
NTP_Phthalates_IURNTP_ImmunoTox
NTP_DevToxOPPIN_DER
Nongenotoxic_hepatocarcinogensFDA_CFSAN_repro_dev
FDA_CFSAN_chronic_subchronicFDA_CFSAN_carc
FDA_GRASfragrance
PFCsOECD
NTP_ImmunotoxLTKB_Priority1,2_PhaseII
ECHA_organic_CMR_2010FAR_EDSP_173
EPA_Fooduse_InertsEPA_PCCL
EPA_OW_Priority2EPA_DNT
EPA_AntimicrobialsEPA_OPPT-RAD_All
OPPT_Categoriesv-Liver_22+14ref
v-EmbryoSuperfund_PPRTV
IRIS+NominExposure_Summit
EPA_DfEGreen Plasticizers_Nisha
phthalate_metabolitePhthalates
0 20 40 60 80 100 120 140 160
Office of Research and DevelopmentNational Center for Computational Toxicology 15
Chemical Sample Registration Workflow
Place chemical orders Procure 2x100mg
compound in tared, barcoded vials
Store neat
Solubilize in DMSO
+
Partition to Matrix
racks
Solution Plates
Insoluble
Review COA
QC structure
Register in DSSTox Inventory
Register in Sample Tracking
Database
X
Bottle IDs
Remove from DSSTox
Update
Solution IDs
Update
AssayProviders
ACToR/PubChem
NCGC
Plate Address
Plate ID
Assay Results
Analytical QC
Analytical QC
Office of Research and DevelopmentNational Center for Computational Toxicology
Publish QC summary results in association with assay data 16
Tox21 Analytical QC
LC-MS
GC-MS
PASS = Confirm parent ion peak and >90% purity
Fail, inconclusive or analytical method inappropriate
Retest at later time point under assay conditions for stability
A copy of each parent Tox21 assay plate (352 cmpds/plate) will be subjected to analytical QC for assessing purity, identity, stability
Office of Research and DevelopmentNational Center for Computational Toxicology
Analytical QC Summary PDF results will be available for each sample
NCGC Analytical Chemist (W. Leister) will review all preliminary LC results and QC “FAIL” compounds, and supervise follow-up testing
Prime objective of QC is to inform analysis & interpretation of assay results:
high confidence low confidence fail
Office of Research and DevelopmentNational Center for Computational Toxicology
Structure-based Data MiningPubChem ChemSpider EPA ACToR
Analog SearchingAIM DSSTox
External Resources
S t r u c
t u r
e
Chemical structure - CIDSubstance details - SIDProject inventory record - RID
Cl
Cl
OO
OH
OH
Cl
NH2NH
N
N
N
PNHO
S OO
NN N
Cl
N
OH
OO
Cl
F
FFN+
O
O-
NO
O
NHS
DSSTox
Test Sample
AssayResults
Structures
ToxCast/Tox21 Chemical Registry
SAR Modeling
CID Table
RID Table
SID Table
Tox21ToxCast
ToxRefDB
7
Office of Research and DevelopmentNational Center for Computational ToxicologyTox21 Sample Tracking Database
DSSTox RID Bottle ID ( COA ID) Solution ID ( QC ID)
Chemical structure - CIDSubstance details - SIDProject inventory record - RID
Cl
Cl
OO
OH
OH
Cl
NH2NH
N
N
N
PNHO
S OO
NN N
Cl
N
OH
OO
Cl
F
FFN+
O
O-
NO
O
NHS
DSSTox
Test Sample
AssayResults
Structures
ToxCast/Tox21 Chemical Registry
Bottle_ID
Soln_IDQC_ID
DSSTox_RID
COA_ID
8
Office of Research and DevelopmentNational Center for Computational Toxicology
ACToR/ToxMiner
Solution ID Plate ID Plate Address ID
DSSTox RID Assay nameAssay detailsAssay outcome
AssayResults
DSSTox RID Bottle ID ( COA ID) Solution ID ( QC ID)
Tox21 Sample Tracking Database
Chemical structure - CIDSubstance details - SIDProject inventory record - RID
Cl
Cl
OO
OH
OH
Cl
NH2NH
N
N
N
PNHO
S OO
NN N
Cl
N
OH
OO
Cl
F
FFN+
O
O-
NO
O
NHS
DSSTox
Test Sample
Structures
ToxCast/Tox21 Chemical Registry
9
PubChem_CID
S t r u c t u
r e
PubChem_SID
Tox21_ID
Chemical Information & Sample QC
“Whoever is careless with the truth in small matters cannot be trusted with important matters.”
– Albert Einstein
Gene-expression
Chemical SupplierLot/Batch
Certificate of AnalysisSolubility
Purity & Stability
Chemical Sample Details
Chemical Name Toxicology literature Public databases
Cheminformatics resourcesCAS SMILES
InChI / StructureSubstance details
ChemicalAnnotation
LayerCl
Cl
O
O
OH
Name is misspelled or incorrect
CAS is invalid or retired CAS and name do not
agree Name and structure do
not agree Name is insufficient for
structure assignment Insufficient description
of substance
Chemical Annotation of Public resources
Generic Chemical of Toxicological
Interest
Chemical Information & Sample QC
Sample Annotation & QC
Not same as generic chemical
Salt, isomer, … Name does
not match info on COA
hydrate, stereo Wrong MW COA expired
Purity <90% Active impurities Sample degrades Rxn with solvent Hydrolysis
Procure from
ChemicalSupplier
Name, CAS, purity (COA)
MW, dose
IC 50
No CAS or wrong Name wrong Structure wrong
Office of Research and DevelopmentNational Center for Computational Toxicology 24
Tox21 Cheminformatics
Plate IDs, Plate Addresses
Solution IDs
Bottle IDs
Assay Results
Analytical QC
Tox21/ToxCast
FeaturesClassesDescriptorsProperties
Mixture IDs Chemical Substance: SID
Structure: CID
Parent: PID
2D, 3DConformersTautomers
SaltsComplexes
(Metabolites?)
CAS/Name
Groups: MapID
Analogs, classifiers, stereo family, etc.
Creating SAR-ready files: How to process salts, complexes, charged species How to aggregate results for resulting structure “duplicates” How to deal with enriched features & “families” of structures within dataset
Office of Research and DevelopmentNational Center for Computational Toxicology 25
DSSTox TOX21E1_3619
NCGC PharmaceuticalsNTP compoundsFDA compoundsEPA compounds
13 macromolecule155 mixture or formulation1 unspecified or multiple forms45 inorganics100 organometallics173 complexes219 salts12 no structures
91 “parent” groupings (2-3)
MW ranges from 30 (formalin) to 1700 (tannic acid)
AlkylbenzenesChlorobenzenesMethyl phenolsNitrobenzenesAromatic aminesPhthalatesPerfluorinates…
Office of Research and DevelopmentNational Center for Computational Toxicology
Tox21 Structural Library
Feature enrichment
Feature diversity
Office of Research and DevelopmentNational Center for Computational Toxicology
Tox21 Reaction Features: Commonalities
NTP Tox21
NCGC Tox21
EPA Tox21
DrugsFood additivesAntimicrobialsWater contaminantsHPVsToxicants…
Metabogen reaction features generated using “MOSES” software by Molecular Networks
Office of Research and DevelopmentNational Center for Computational Toxicology
Histogram
Column 1
0 200 400 600 800 1000 1200
Cou
nt
0
100
200
300
400
500
600
700
Count Histogram
Column 1
0 200 400 600 800 1000 1200
Cou
nt
0
10
20
30
40
50
Count
Histogram
Col um n 1
0 200 400 600 800 10 00 120 0
Cou
nt
0
10
20
30
40
50
Coun t
Tox21 NTP & EPA
Tox21 NCGC
# M
olec
ules
Molecular Weight
NTP & EPA collections enriched with lower MW compounds compared to NCGC drug collection
Mean = 231
Mean = 304
Tox21: Molecular Weight Distributions
Office of Research and DevelopmentNational Center for Computational Toxicology
-5
0
5
1015
LOGP
0
1
2
3
TPSA
1
2
3
4
CO
MP
LEX
ITY
ToxCast_PhaseI
ToxCast/Tox21 property distributions
Chemical properties computed using “Adrianna” software by Molecular Networks.
LOG P = Octanol/Water partition coefficient
TPSA = log (Total Polar Surface Area)
Complexity = log (complexity based on paths, branching, atoms)
Office of Research and DevelopmentNational Center for Computational Toxicology
-5
0
5
1015
LOGP
0
1
2
3
TPSA
1
2
3
4
CO
MP
LEX
ITY
ToxCast/Tox21 property distributions
Chemical properties computed using “Adrianna” software by Molecular Networks.
LOG P = Octanol/Water partition coefficient
TPSA = log (Total Polar Surface Area)
Complexity = log (complexity based on paths, branching, atoms)
ToxCast_PhaseIToxCast_PhaseII
Office of Research and DevelopmentNational Center for Computational Toxicology
-5
0
5
1015
LOGP
0
1
2
3
TPSA
1
2
3
4
CO
MP
LEX
ITY
ToxCast/Tox21 property distributions
Chemical properties computed using “Adrianna” software by Molecular Networks.
LOG P = Octanol/Water partition coefficient
TPSA = log (Total Polar Surface Area)
Complexity = log (complexity based on paths, branching, atoms)
111 failed drugs
ToxCast_PhaseIIToxCast_PhaseI
Office of Research and DevelopmentNational Center for Computational Toxicology
-5
0
5
1015
LOGP
0
1
2
3
TPSA
1
2
3
4
CO
MP
LEX
ITY
ToxCast/Tox21 property distributions
Chemical properties computed using “Adrianna” software by Molecular Networks.
LOG P = Octanol/Water partition coefficient
TPSA = log (Total Polar Surface Area)
Complexity = log (complexity based on paths, branching, atoms)
Tox21111 failed drugs
ToxCast_PhaseIIToxCast_PhaseI
Office of Research and DevelopmentNational Center for Computational Toxicology
Expanding Applicability
QSAR In vitro/HTS
In Vivo
Defined organicsVirtual chemicalsVolatilesDMSO insolublesReactives
MixturesFormulations
Proprietary substancesOrganometallics
MetalsSalts
metabolismADME
Defined structure + Biological profile
Office of Research and DevelopmentNational Center for Computational Toxicology
Acknowledgements:
EPA NCCT ToxCast Team:Robert Kavlock - DirectorDavid Dix Keith Houck Matt Martin (ToxRefDB)Richard Judson (ACToR)
EPA NCCT DSSTox:Maritja Wolf– Lockheed Martin, Contractor to the EPAIndira Thillainadarajah – EPA:SEEPatra Volarath – EPA Post Doc
External Collaborators: Chihae Yang, FDA/CFSANChris Austin & colleagues, NCGC/NIHRay Tice & colleagues, NTP/NIEHS
This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy.