Selectivity of binding to estrogen receptors α and β as determined by fluorescence polarization
results and conclusions: ER α
compounds tested for
estrogenicity
120
50
0
0
1
2
3
50
0
0
• 50 = 54.2 nM
• Ki = 47.8 nM
IC
Cl
O
Br
O
O
O
Permethrin
O
O
• All three compounds used in agriculture
and ranching applications
• Permethrin also found in household products like tick repellent and pet shampoos
flourescence polarization
assay
-4
-2
0
2
Fenvalerate
180
160
140
140
140
120
120
120
100
100
80
60
-6
-4
-2
0
2
4
100
80
60
-6
-4
-2
0
2
4
80
60
-6
-4
-2
• No binding
0
2
4
log[Fenvalerate] (uM)
log [Deltamethrin] (uM)
Log[Permethrin] (uM)
• No binding
• No binding
esults and conclusions: ER β
PPT
DPN
PPt
Bisphenol A
BPA
dPn
300
300
300
250
250
250
200
200
200
150
150
150
100
100
100
50
0
0
1
2
3
Polarization (mP)
Polarization (mP)
Fluorescence polarization (FP) measures
the amount of polarized light emitted
by a fluorescent molecule when excited
by polarized light. The larger the mol• 50 = 54.2 nM
• No binding
• 50 = 54.2 nM
ecule, the slower is rotates in solution
• Ki = 47.8 nM
• Ki = 47.8 nM
and the more polarized light (the higher
Permethrin
Deltamethrin
Fenvalerate
polarization) it will have. A small molecule rotates more quickly and will have a lower polarization
value. The ability of the
compounds shown above to
bind to the ER and displace
a fluormone (a fluorescent
ER ligand) from ER α or
•
•N
•
ER β was measured using FP. If the compound did not bind to
For all assays, the polarization of the ER fluormone complex is the high polarization standard
the ER, the fluormone stayed bound to the ER and the polarizashown in red, while the polarization of the displaced fluormone is the low standard shown in
tion of the large complex was high. If the competitor did bind to
the ER, it displaced the fluormoe and the polarization of the fluormone alone was low. These assays were conducted in 96 and
384 well plates. A variety of concentrations of the competitors
were run with the ER fluormone complex. The polarization was
graphed versus the concentration to get a binding curve.
Polarization (mP)
50
0
0
1
2
3
50
0
-4
-2
0
2
4
Log[BPA] (uM)
log[DPN] (nM)
IC
IC
Log[PPT] (nM)
300
300
250
250
200
200
150
150
240
220
200
180
160
140
100
80
60
-4
-3
-2
-1
0
1
2
3
4
Polarization (mP)
120
Polarization (mP)
Polarization (mP)
100
50
-4
-2
0
2
Log[Deltamethrin] (uM)
N
Log[Permethrin] (uM)
N
Various synthetic compounds ubiquitous in the environment have been implicated as xenoestrogens, or chemicals that can bind to the Er thus mimicking
estrogen. they have been linked to many health problems including various
forms of cancer, sexual and developmental defects, and reproductive abnormalities. Because the Er has a large binding pocket, a diverse range of chemicals
can act as xenoestrogens. though these chemicals are not as potent in binding
as natural estrogen, the total xenoestrogen burden due to their ubiquity is cause
for concern. this study also examines the binding of bisphenol A and three
pyrethroid pesticides, Permethrin, fenvalerate and deltamethrin, with both Er
α and ER β.
-6
E R a lp h a F e n va le ra te 6 h o u rs
160
260
Binding modes of SERMs
within the ligand binding pocket of ER α (blue) and ER β
(green): The effect of the different shape pocket on ER selectivity is shown.
40
• 50 = 31.7 uM
• Ki = 14.07 uM
Deltamethrin
160
CN
60
Log [BPA] (uM)
Polarization (mP)
O
O
3
E R a lp h a a n d D e lta m e th rin 6 h o u rs
Er alpha Permethrin 6 hours
Polarization (mP)
Br
2
• No binding
180
OH
1
80
log[DPN] (nM)
Log[PPT] (nM)
• Found in plastics and epoxy coatings used in food containers
Polarization (mP)
• ER β selective SERM
100
Polarization (mP)
CN
HO
OH
100
100
Polarization (mP)
Small molecules have been identified which bind each receptor with differing
binding affinities. These selective estrogen receptor modulators (SERMs) hold
the potential to be pharmacologically effective in treating diseases specific to
one type of estrogen receptor while not affecting the other. For example, ER α
and ER β are both present in breast tissue, and the ratio of ER β to ER α is being examined as one indicator in determining the likelihood of successful treatment of breast cancer by certain drugs. Here we use changes in the fluorescence
polarization to calculate binding affinities for DPN and PPT, two SERMs, to
ER α and ER β.
140
200
N
Cl
Cl
160
250
200
Pyrethroid Pesticides: Permethrin, Deltamethrin,
and Fenvalerate
CN
180
300
150
HO
O
200
150
HO
Ligand Binding Domain of ER
α and ER β: The residues that
differ in the binding pocket
between the two receptors are
shown in black. These differences are largely responsible
for ER ligand selectivity.
350
250
OH
Bisphenol Bisphenol
A A
dPn
IC
N
DPN
OH
DPN
PPt
300
Polarization (mP)
Estrogen’s role in cell growth and proliferation has long been appreciated both
in the normal development of secondary sexual characteristics and in diseased
states in cancers of the breast, ovaries and uterus. We are beginning to appreci- PPT
ate estrogen’s expanded role in maintaining such diverse functions as the skin’s
elasticity, the health of the central nervous system, bone density and cardiac
health. Estrogen plays out its roles in varied tissues by binding to two major li• ER α selective SERM
gand activated nuclear receptors, estrogen receptor alpha (ER α) and estrogen
Bisphenol A
receptor beta (ER β). Though the ligand binding cavities of the two receptors
differ by only two amino acids, the overall degree of homology between ER α
and ER β is low. The body uses the receptor selectivity to its advantage by dispersing the receptors in varying ratios to different tissues.
PPT
R
introduction
4
6
100
50
-4
-3
-2
-1
0
1
Log[Fenvalerate] (uM)
2
3
4
Selectivity of Binding to Estrogen Receptors Α and Β as Determined by Fluorescence Polarization
of 1
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