Illuminating Drug Toxicity We offer proteomic screening using our MetAbArray for in vitro toxicity evaluation, mechanistic studies, and biomarker identification of your compounds.

Illuminating Drug Toxicity The MetAbArray is a plate-based In-cell ELISA assay that takes cells incubated with a compound: rapidly fixes the cells to block all biological actions including post - translational modifications. makes them permeable. reacts them with antibodies to a set of metabolic proteins. and then uses secondary antibodies labeled with IR dyes that have a large linear dynamic range to generate a quantitative measure of the protein signals. This assay can be done with from 2 to more than 100 well characterized antibodies, each specific to a different key metabolic enzyme/signaling protein.

Relative Compound Ranking Protein Changes Are Evaluated in Terms of % of Maximal for Each Target The method is sufficiently quantitative that data can be evaluated as a function of maximal changes that occur across many drugs. For example, across the many drugs examined to date in different cells at different conditions of energy source, phosphorylation of the S6 ribosomal protein can increase up to 7 fold or decreases by 50 fold from a DMSO control, and this is taken into account in determining the significance of the change for many proteins.

THE METABARRAY ALLOWS US TO PROVIDE YOU WITH A MECHANISTIC UNDERSTANDING OF THE WANTED AND UNWANTED EFFECTS OF YOUR COMPOUNDS ON ALL ASPECTS OF METABOLISM. IT ALSO PROVIDES AN ANTIBODY-BASED BIOMARKER PROFILE FOR USE IN IN VIVO STUDIES. HSP60 JNK PATHWAY C-JUN PKR eIF2alpha PERK Induces same genes as CHOP1 ATF4 Up reg of CHOP1 Mitochondrial UPR (yellow) ER UPR(blue) APOPTOSIS INHIBITS PROTEIN TRANSLATION p38MAPK HSP90 Up reg of Heme Oxygenase P P P P eIF2alpha ATG13 P P

Illuminating Drug Toxicity TO UNDERSTAND THE MECHANISM OF TOXICITY OF A COMPOUND, PARTICULARLY SIGNALING PATHWAYS, IT IS IMPORTANT TO CONSIDER THE TIME LINE OF EXPERIMENTS. THE METABARRAY READILY ALLOWS THIS. A CELL’S RESPONSE TO DRUG-INDUCED STRESS IS HIGHLY DYNAMIC. TIMES COURSES OF THE EFFECT OF STAUROSPORINE ON HEPG2 CELLS AND FIBROBLASTS (HDFN) IN GLUCOSE MEDIUM. important considerations in evaluating a compound’s effect.

The MetAbArray can be used to compare different growth conditions e.g. cells can be treated while in glucose media versus galactose and glutamine to simulate different substrate availabilities. DRUG TOXICITY IS A FUNCTION OF CELL STATE SUCH AS SUBSTRATE AVAILABILITY AND ENERGY DEMANDS, ASPECTS THAT DISTINGUISH PRIMARY FROM TRANSFORMED CELLS. Data for Biguanides

Illuminating Drug Toxicity Protein synthesis signaling autophagy ER stressapoptosisOx stress FA synth KrebsGlu met FAO Mito/ox phos a.a.met HEAT MAP OF PROTEIN CHANGES INDUCED BY THIAZOLIDINEDIONES Arrows show protein changes unique enough to TRO among the glitazones in HepG2 (and not seen in cardios) to be related to toxicity..

mTor sK6 4E-BP1 in S6 AKT AMPK GCN PERK eIF2a PROTEIN SYNTHESIS Pi inhibits Phospho 4E-BP1 & phospho S6 activate translation GSK3 UPR GLUCOSE & FAO METABOLISM S6 Pi 4E-BP1 Pi CUSTOM METABARRAYS CAN BE GENERATED TO FOCUS ON SPECIFIC PATHWAY STUDIES AND IDENTIFY THE MOST ROBUST BIOMARKERS FOR SUBSEQUENT ANIMAL STUDIES.

Illuminating Drug Toxicity The MetProf Methodology SUMMARY The MetAbArray platform is a high throughput, cost effective and quantitative platform for identifying both the wanted and unwanted effects of compounds on cells. It readily provides evaluation of likely tissue variation.

Illuminating Drug Toxicity AND BECAUSE IT IS ANTIBODY BASED: THE ASSAY DATA ARE IMMEDIATELY ACTIONABLE IN SUBSEQUENT ANIMAL STUDIES, AND EVEN IN PATIENT SCREENS USING IHC OR ELISA METHODS. 10

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