Novel Modulators of Ah Receptor Signaling Sammy Khalil Dr. Siva Kumar Kolluri Department of Environmental and Molecular Toxicology

Aryl Hydrocarbon Receptor The Aryl Hydrocarbon Receptor (AhR) is a ligand activated cytosolic transcription factor AhR is particularly known to mediate toxic effects of many aryl hydrocarbons including 2,3,7,8-tetrachlorodibenzo-p-dioxin (also known as TCDD or Dioxin) It well known that it has been around for millions of years due to evidence in evolutionary studies (ie fish have it too), but we don’t fully understand what its originally there for just that it is and it works

AhR Mechanism Upon ligand binding, AhR heterodimerizes with its partner protein aryl hydrocarbon nuclear translocator (ARNT) This complex translocates to the nucleus and binds to sites in the DNA upstream of genes including many that encode for xenobiotic metabolizing enzymes, such as Xenobiotic Response Element (XRE) promoter sequence

AhR and TCDD TCDD binds with high affinity to AhR Upon binding with AhR, dioxin elicits various AhR-dependent events Hepatoxicity Immune suppression Modulation of cell proliferation TCDD is highly resistant to metabolic degradation Does not kill cells, but is carcinogenic (ASK if I spelled hepatoxicity right) Viktor Yushchenko TCDD poisoning in 2004

Aryl Hydrocarbon Receptor Pathway AhR Cytosol AhR ARNT Nucleus

Aryl Hydrocarbon Receptor Pathway Cytosol ARNT AhR XRE XRE DNA Nucleus

Aryl Hydrocarbon Receptor Pathway Cytosol ARNT AhR XRE XRE Transcription DNA Nucleus

Objective Some of AhR biological effects are beneficial Immune suppression for treating hyper immune disorders like rheumatoid arthritis. Inhibition of cell proliferation is desirable for cancer treatment. The purpose of our research is to identify compounds that activate AhR transcription. Some of these compounds may have beneficial effects functioning through AhR without the unwanted toxic effects of dioxins.

Hypothesis Different AhR modulators elicit selective responses through AhR, and AhR signaling can be exploited for beneficial effects.

Methods and Design Transfect mammalian cells using: pGal4-Luciferase Reporter plasmid Gal4-DNA binding sequences driving a luciferase reporter gene Gal4-DNA binding domain linked to AhR Gal4-transactivation domain linked to ARNT Assay is designed to measure level of response to ligand candidates by the AhR-ARNT Complex

Methods and Design The reporter plasmid holds Gal4-DNA binding sequences followed by luciferase coding gene sequence When AhR is bound with a ligand, AhR-ARNT complex activates GAL-coupled reporter gene (luciferase) Luciferase abundance can be measured by the light it emits when it is coupled with its substrate, luciferin Light ARNT AhR Change to what dan explained, gal not xre Luciferin Luciferase XRE DNA GAL GAL GAL GAL GAL Transcription Luc Reporter Plasmid

Methods and Design 96-well plate format Treat cells with compounds from a library 96-well plate format Measure luciferase activity in the cells Positive hits display measurable luciferase activity Negative hits display no luciferase activity NT TCDD

Results

Results

Results

Results

Results

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Results

Overall Plan Identify novel modulators of AhR by screening Test whether the compounds will enhance endogenous genes regulated by AhR Confirm that outcomes are AhR dependent Explore potential as possible therapeutic agents

Acknowledgements Dept. of Environmental and Molecular Toxicology EMT Minority Student Training Grant Howard Hughes Medical Institute Dr. Siva Kumar Kolluri Dr. Kevin Ahern Daniel Koch Edmond O’Donnell