Defining Chemical Target and Pathway Toxicity Mechanisms with Primary Human Cell Systems

Defining Chemical Target and Pathway Toxicity Mechanisms with Primary Human Cell Systems

Ellen L. Berg, PhD

BioSeek, Inc.

eChemInfo, Philadelphia PAOctober 2009

BioSeek

BioSeek

Presentation Overview

• Why is predictive toxicology so hard?

Biological complexity

• A role for cell-based assays in predictive toxicology

BioMAP primary human cell systems

Examples & challenges

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BioSeek

Biological Systems Are Complex

Scale (meters) (Time)

molecules pathways cells tissues humans

10-9 M 10-8 M 10-7 M 10-6 M 10-5 M 10-4 M 10-3 M 10-2 M 10-1 M 1 M

Human exposureMolecular targets

10-6 sec 102 sec 104 sec 105 sec 108 sec

Modular design / Networked architecture

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Biological Systems have a Modular Design

• Great diversity from few components

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• The ability to compartmentalize

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Scale (meters) (Time)

molecules pathways cells tissues humans

10-9 M 10-8 M 10-7 M 10-6 M 10-5 M 10-4 M 10-3 M 10-2 M 10-1 M 1 M10-6 sec 102 sec 104 sec 105 sec 108 sec

components

interactions

BioSeek

Biological Systems have a Networked Architecture

components

interactions

• Rapid responses to environment

Efficient information flow

• Framework for control systems

Feedback mechanisms, etc.

• Many potential outcomes

System “wiring” determines outcome

Scale (meters) (Time)

molecules pathways cells tissues humans

10-9 M 10-8 M 10-7 M 10-6 M 10-5 M 10-4 M 10-3 M 10-2 M 10-1 M 1 M10-6 sec 102 sec 104 sec 105 sec 108 sec

BioSeek

Biological Systems are Dominated by Control Systems

• Dominate in networks with demanding

performance requirements

• Important for providing tolerance to

perturbations (robustness)

• “Hidden nature”

Unexpected fragility

Scale (meters) (Time)

molecules pathways cells tissues humans

10-9 M 10-8 M 10-7 M 10-6 M 10-5 M 10-4 M 10-3 M 10-2 M 10-1 M 1 M10-6 sec 102 sec 104 sec 105 sec 108 sec

components

interactions

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Modular, Networked ArchitectureConsequences for Drug Discovery & Predictive Toxicology

• Drug targets function in multiple biological processes

Unexpected side effects are often only discovered in clinical testing

• Drugs can have effects far downstream of the target

Hard to predict outcomes

• Problems become amplified since most drugs have multiple

targets

Targets may interact in unexpected ways

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BioMAP® Technology Bridging the Gap

Scale (meters) (Time)

molecules pathways cells tissues humans

10-9 M 10-8 M 10-7 M 10-6 M 10-5 M 10-4 M 10-3 M 10-2 M 10-1 M 1 M

Human exposureMolecular targets

10-6 sec 102 sec 104 sec 105 sec 108 sec

BioMAP Technology

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BioMAP® Technology Platform

Assays

Human primary cells Disease-like culture conditions

LPS

BF4T

SM3C

Profile Database Informatics

Biological responses to drugs and stored in the database

Specialized informatics tools are used to mine and analyze biological data

BioSeek

• BioMAP Systems are cell-based assays that BioMAP Systems are cell-based assays that model complex human disease biologymodel complex human disease biology

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BioMAP® Technology Platform

Assays

LPS

BF4T

SM3C

Human primary cells Disease-like culture conditions

>30 BioMAP Systems

BioSeek

• Assay endpoints include human clinical Assay endpoints include human clinical biomarkers and risk factors (proteins)biomarkers and risk factors (proteins)

Cytokines, chemokines, mediators

Cell surface receptors

Proteases, enzymes (MMPs, plasminogen activators)

Others (hemostatic factors, matrix components)

Clinically relevant, extracellular biomarkers

BioMAP® Technology Platform

Assays

LPS

BF4T

SM3C

Human primary cells Disease-like culture conditions

>30 BioMAP Systems

BioSeek

AssaysAssays

Human primary cells Disease-like culture conditions

LPS

BF4T

SM3C

Profile DatabaseProfile Database

Biological responses to drugs and stored in the database

BioMAP® Technology Platform

• > 2000 agents> 2000 agents

• Approved drugsApproved drugs

• Clinical stage & Clinical stage &

failed drugsfailed drugs

• Experimental Experimental

compoundscompounds

• BiologicsBiologics

• ToxicantsToxicants

BioSeek

BioMAP Systems are Validated Corticosteroids (Prednisolone) Are Active in Inflammation Systems

BioMAP Systems

Readout Parameters (Biomarkers)Cytotoxicity Readouts

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Control (no drug)

99% significance envelope

DoseResponse

Profiles retain shape over multiple concentrationsProfiles retain shape over multiple concentrations

BioSeek

E-selectin

TNF-α

IL-8

BioMAP Systems are Validated Activities of Corticosteroids Match Clinical Effects

PGE2

IL-8MCP-1

MCP-1, IL-8, E-sel. decreaseLeukocyte recruitment

Many, e.g. Jilma et al., 2000

PGE2 decreasePain, swellingSebaldt et al., 1990

Readouts in BioMAP show the same pattern as has been reported for patients receiving steroid therapy

Readouts in BioMAP show the same pattern as has been reported for patients receiving steroid therapy

Collagen I & III

Collagen I, III decreaseSkin atrophyAutio et al., 1994

MMP-1

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PAI-1SAA

PAI-1, SAA increaseCV complications

Sartori et al., 1999Fyfe et al., 1997

PAI-1

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Predictive Toxicology

• Characterizing chemicals for mode of action

• Defining toxicity mechanisms

Pathway mechanisms

Unknown targets or target-independent mechanisms

• Chemical prioritization

ToxCastTM Phase I Proof-of-concept study

BioSeek

MicrotubuleStabilizers

Mitochondrial ET chain

Retinoids

Hsp90

CDK

NFκB

MEK

DNAsynthesis

JNK

Proteinsynthesis

MicrotubuleDestabilizers

Estrogen R

PI-3K

Ca++

Mobilization

Classification of Drugs By Mechanism Pairwise Correlation of BioMAP Reveals Functional Similarities

mTOR

PKC Activation

p38 MAPK

HMG-CoAreductase

Calcineurin

Transcription

Mechanism of Action(On-Target)

Pathway

Relationships

BioSeek

Defining Toxicity Mechanisms

Sodium Azide Chlorambucil

Cycloheximide A23187 Thapsigargin

Oxidative /Nitrosative Stress

ER Stress Unfolded Protein Response

Rotenone

BioSeek

ToxCast ChemicalsDiversity of Mechanisms

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Houck, 2009

BioSeek

BioMAP Profiles of Compounds in Example Cluster

• Pyraclostrobin and Tryfloxystrobin are strobilium herbicides

• BioMAP profiles are highly similar to one another

Tryfloxystrobin

Pyraclostrobin

Pyraclostrobin, 13.33 µM

Pyraclostrobin, 4.44 µM

Pyraclostrobin, 1.48 µM

Tryfloxystrobin, 40 µM

Tryfloxystrobin, 13.33 µM

Tryfloxystrobin, 4.44 µM

BioSeek

Trifluoxystrobin

Reference Database Search Identifies MOA

Tryfloxystrobin

Search results:

• Search reference database for compounds with similar profiles Correlation (Pearson-based) metrics

BioSeek

Trifluoxystrobin

Search results:

• Optimized search algorithms use combination of metrics

• Confirms mechanism of action

• Of >3000 reference profiles searched, only 7

compounds are significantly similar

• All are known inhibitors of mitochondrial

function

Tryfloxystrobin

Reference Database Search Identifies MOA

BioSeek22

cAMP Elevation

DNA Alkylation

NFκB

Tubulin Inhibition

Mitochondrial Dysfunction

ToxCast ChemicalsDiversity of Mechanisms

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Challenges for Predictive Toxicology Applications

• Data require specialized analysis approaches

Compound cytotoxicities complicate data interpretation

• Assay readouts reflect activities of multiple targets

Concentration effects are complicated

• Diversity of assay endpoint ranges and dynamics

• Assay readouts can be independent

• Can only predict human toxicity, not animal toxicities

Species differences are amplified in complex systems

• Limited data available for validation

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Cytotoxicity Effects

• Cell type selective toxicity

• Activation state-dependent toxicity

Toxicity

Cell Type

Endothelial Cells Epithelial Cells SMC Fibroblasts EC

Activation State

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Lo

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Readout Parameters (Biomarkers)

BioMAP Systems

Readout Parameters (Biomarkers)

BioMAP Systems

Rapamycin Genistein

Concentration Effects

• Diversity of concentration effects

BioSeek

Concentration Effects

• EC50, Slope, Magnitude of effect

• Plateau?

• Cytotoxicity

Rapamycin Genistein

No effect

MaxInhibition

Increasing Concentration Increasing Concentration

Cytotoxicity

HLA

-DR

Lev

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Plateau

EC50Magnitude

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Concentration Effects

• Bimodal distribution - Reverse dosing

No effect

MaxInhibition

Increasing Concentration

Rea

dout

Lev

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Summary

• Biological complexity is a challenge to predictive toxicology

• Cell-based assays have a role in defining mechanisms of action

Complex assays are effective

Target and pathway mechanisms can be identified

• Specialized approaches to data analysis are required

Data are heterogeneous and multiparameter

Analysis requires managing concentration and cytotoxicity effects

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Acknowledgements

• BioSeek Alison O’Mahony

Jennifer Melrose

Elen Rosler

Sylvie Privat

Dat Nguyen

Mark Polokoff

Stephanie Tong

Jian Yang

Antal Berenyi

• Stanford Eugene Butcher

Rob Tibshirani

Trevor Hastie

• EPA David Dix

Keith Houck

Richard Judson

Bob Kavlock

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