Transcript Novel Modulators of Ah Receptor Signaling

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 2,3,7,8tetrachlorodibenzo-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
Aryl Hydrocarbon Receptor Pathway
AhR
Cytosol
AhR
Nucleus
Aryl Hydrocarbon Receptor Pathway
Cytosol
AhR
DNA
XRE
Nucleus
XRE
Aryl Hydrocarbon Receptor Pathway
Cytosol
AhR
DNA
XRE
Nucleus
XRE
Transcription
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 GALcoupled reporter gene (luciferase)
 Luciferase abundance can be measured by the light it emits when it is
coupled with its substrate, luciferin
Light
AhR
Luciferin
Luciferase
XRE
DNA
XRE
GAL
GAL
GAL
Reporter Plasmid
GAL
GAL
Luc
Transcription
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
Results
Screening of Library NPL480 Plate 4
4500000
4000000
3500000
Gal-luc
2500000
AhR
ARNT
2000000
ARNT
1500000
1000000
500000
Treatment
H
10
H
9
10
G
9
G
F1
0
F9
E1
0
E9
D
10
D
9
C
10
C
9
B1
0
B9
A1
0
A9
S
C
G
TC
D
D
TC
D
D
ic
le
0
Ve
h
Luc/ BGal
3000000
V
eh
TCicl
e
TCD D
D
C D
G
S
A
3
A
4
A
5
A
6
A
7
A
8
A
A 9
10
A
11
B
3
B
4
B
5
B
6
B
7
B
8
B
B 9
1
B 0
11
C
3
C
4
C
5
C
6
C
7
C
8
C
C 9
1
C 0
11
D
3
D
4
D
5
D
6
D
7
D
8
D
D 9
10
D
11
E3
E4
E5
E6
E7
E8
E
E1 9
E10
1
F3
F4
F5
F6
F7
F8
F9
F1
F10
1
G
3
G
4
G
5
G
6
G
7
G
8
G
G 9
1
G 0
11
H
3
H
4
H
5
H
6
H
7
H
8
H
H9
10
H
11
Luc/BGal
Results
Exp 1.15: Screening of Library NPL480 Plate 5
4500000
4000000
3500000
3000000
2500000
Gal Luc
AhR
2000000
ARNT
ARNT
1500000
1000000
500000
0
Treatment
Results
Exp 1.15B: Screening of Library NPL480 Plate 5
6000000
4000000
Gal Luc
AhR
3000000
ARNT
ARNT
2000000
1000000
Treatment
H5
H4
H3
G5
G4
G3
F5
F4
F3
E5
E4
E3
D5
D4
D3
C5
C4
C3
B5
B4
B3
A5
A4
A3
CG
S
hic
le
TC
DD
TC
DD
0
Ve
Luc/BGal
5000000
Results
le
TC
D
D
TC
D
D
CG
S
P1
B8
P2
G
2
P3
D1
0
P4
A4
P5
D9
P2
A2
P2
H6
P3
E4
P4
E7
P1
H1
2
P2
G
3
P3
G
9
P4
B4
P2
C7
P2
H9
P3
F5
P4
E8
P2
H3
P3
G
10
P4
H3
P1
A5
P2
C8
P2
C1
1
P4
H8
P4
H1
0
P2
D6
P2
D7
P2
D8
P2
G
9
P2
H5
Ve
hi
c
Luc/BGal
Results
Screening of Library NPL480: Repeats
3000000
2500000
2000000
Gal-Luc
1500000
AhR
ARNT
ARNT
1000000
500000
0
Treatment
C
G
A1 S
2
20
U
P4
m
F2
20
P4
uM
F6
20
P4
uM
G
10
20
P4
uM
H1
20
P4
uM
H8
20
P4
u
A4 M
10
P4
uM
B4
10
P4
U
H9 m
20
P4
uM
H1
0
10
P5
uM
B7
20
P5
uM
D9
10
P5
uM
E1
0
20
P5
uM
G
3
20
P6
uM
G
2
20
P6
uM
H2
20
P1
uM
B8
10
P1
uM
H1
2
P2 10u
M
G
2
10
P3
uM
G
2
10
P2
uM
G
3
10
P3
uM
D1
0
10
P3
uM
G
9
20
P1
uM
A8
10
P1
uM
A1
2
10
P1
uM
F1
1
10
P2
uM
F2
10
P4
u
H3 M
10
P2
uM
H3
10
P3
uM
G
10
10
uM
P4
Ve
hi
cl
e
TC
D
D
TC
D
D
Luc/BGal
Results
Screening of Library NPL480: Repeats
3000000
2500000
2000000
1500000
GalLuc
AhR
ARNT
ARNT
1000000
500000
0
Sample
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