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

2. 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

3. 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

4. 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

5. Aryl Hydrocarbon Receptor Pathway
AhR
Cytosol
AhR
ARNT
Nucleus

6. Aryl Hydrocarbon Receptor Pathway
Cytosol
ARNT
AhR
XRE
XRE
DNA
Nucleus

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

8. 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.

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

10. 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

11. 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

12. 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

13. Results

14. Results

15. Results

16. Results

17. Results

18. Results

19. Results

20. 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

21. 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