Novel Modulators of Ah Receptor Signaling

Sammy Khalil

Dr. Siva Kumar KolluriDepartment 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)

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

Viktor Yushchenko TCDD poisoning in 2004

AhR

AhR

ARNT

Cytosol

Nucleus

Aryl Hydrocarbon Receptor Pathway

AR

NT

AhR

Cytosol

Nucleus

Aryl Hydrocarbon Receptor Pathway

XREDNA

XRE

Aryl Hydrocarbon Receptor Pathway

Cytosol

Transcription

Nucleus

XREDNA

XRE

AR

NT

AhR

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

XRE

DNA

XRE GAL

Reporter PlasmidGALGALGAL GAL LucTranscription

Luciferase

Luciferin

Light

AR

NT

AhR

Methods and Design

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

Screening of Library NPL480 Plate 4

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

Treatment

Luc/BGal

Gal- Luc

AhR

ARNT

ARNT

Results

Screening of Library NPL480 Plate 4

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

Vehicle TCDD TCDD CGS A9 A10 B9 B10 C9 C10 D9 D10 E9 E10 F9 F10 G9 G10 H9 H10

Treatment

Luc/ BGal

Gal-luc

AhR

ARNT

ARNT

Results

Exp 1.15: Screening of Library NPL480 Plate 5

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

VehicleTCDDTCDD

CGSA3A4A5A6A7A8A9

A10A11B3B4B5B6B7B8B9

B10B11C3C4C5C6C7C8C9C10C11D3D4D5D6D7D8D9D10D11E3E4E5E6E7E8E9

E10E11F3F4 F5 F6F7 F8 F9F10F11G3G4G5G6G7G8G9G10G11

H3H4H5H6H7H8H9H10H11

Treatment

Luc/BGal

Gal Luc

AhR

ARNT

ARNT

Results

Exp 1.15B: Screening of Library NPL480 Plate 5

0

1000000

2000000

3000000

4000000

5000000

6000000

VehicleTCDD TCDD

CGS A3 A4 A5 B3 B4 B5 C3 C4 C5 D3 D4 D5 E3 E4 E5 F3 F4 F5 G3 G4 G5 H3 H4 H5

Treatment

Luc/BGal

Gal Luc

AhR

ARNT

ARNT

Results

Exp 1.16: Screening of Library NPL480 Plate 6

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

Treatment

Luc/ BGal

Gal-luc

AhR

ARNT

ARNT

Results

Screening of Library NPL480: Repeats

0

500000

1000000

1500000

2000000

2500000

3000000

VehicleTCDDTCDDCGS P1B8P2G2P3D10P4A4P5D9P2A2P2H6P3E4 P4E7P1H12P2G3P3G9P4B4P2C7P2H9 P3F5P4E8P2H3P3G10P4H3 P1A5P2C8P2C11P4H8P4H10P2D6P2D7P2D8P2G9P2H5

Treatment

Luc/BGal

Gal-Luc

AhR

ARNT

ARNT

Results

Screening of Library NPL480: Repeats

0

500000

1000000

1500000

2000000

2500000

3000000

Vehicle TCDD TCDD CGS

P4 A12 20UmP4 F2 20uMP4 F6 20uMP4 G10 20uMP4 H1 20uMP4 H8 20uMP4 A4 10uMP4 B4 10 UmP4 H9 20uMP4 H10 10uMP5 B7 20uMP5 D9 10uMP5 E10 20uMP5 G3 20uMP6 G2 20uMP6 H2 20uMP1 B8 10uMP1 H12 10uMP2 G2 10uMP3 G2 10uMP2 G3 10uMP3 D10 10uMP3 G9 20uMP1 A8 10uMP1 A12 10uMP1 F11 10uMP2 F2 10uMP4 H3 10uMP2 H3 10uMP3 G10 10uM

Sample

Luc/BGal

GalLuc

AhR

ARNT

ARNT

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 GrantHoward Hughes Medical InstituteDr. Siva Kumar KolluriDr. Kevin AhernDaniel KochEdmond O’Donnell