Note: Descriptions are shown in the official language in which they were submitted.
~ ~94/~068 21 6 01 3 5 PCT~S94/03795
METHOD FOR SCREENING FOR RE~OK AGONISTS
BACRGROUND OF THE lNV~.~ION
This invention relates to methods and
constructs useful for identifying or screening agonists
active at cell receptors, such as at hormone receptors,
e.q., the estrogen receptor.
The following is a discussion of relevant art,
none of which is admitted to be prior art to the claims.
Evans et al., U.S. Patent 5,071,773, (hereby
incorporated by reference herein) describes an assay by
which hormone receptors, ligands for such receptors, and
proteins having transcription activating properties of a
hormone receptor, can be detected. Generally, the assay
involves use of a cell which contains both DNA encoding
a hormone response element (e.g., a promoter) linked to
an operative reporter gene, and DNA encoding a receptor
protein. When a suitable hormone or ligand is provided
to the cell, a hormone receptor - hormone complex is
formed and delivered to an appropriate DNA-binding
region to thereby activate the hormone response element
and cause expression of the reporter gene. Activation
of the reporter gene is detected by standard procedures
used for detecting the product of the reporter gene.
Pierre Chambon and his group have described
many properties of the estrogen receptor, and its
alleged cell-type and promoter-context dependent
activity. These experiments were generally performed by
use of one or more truncated estrogen-receptor-encoding
genes which express receptors lacking all or a portion
of two domains termed TAFl and TAF2. These domains are
thought to be regulated by estrogen and then cause
promoter activation. A third domain is located between
these two, and is thought to bind to DNA near a promoter
activated by the receptor-hormone complex (Kumar et al.,
51 Cell 941 (1987)).
W094/~068 PCT~S94/0379~
~6o~3S
Specifically, Webster et al., 54 Cell 199
(1988), use chimeric receptors to localize regions
responsible for transcription activation function. The
authors propose that a hormone is responsible for
allowing a receptor to recognize a DNA response element,
and that the hormone induces a transcription activation
function in the hormone-binding domain.
Tora et al., 59 Cell 477 (1989), analyzed
truncated mutants of human estrogen receptor, and
described TAFl and TAF2 as two transcriptional
activation functions in the receptor. These activators
are said to exhibit cell-type specificity and promoter-
context dependency. The authors indicate that TAF2 acts
synergistically with upstream elements.
Meyer et al., 57 Cell 433 (1989), describe
inhibition of transcription stimulation by the
progesterone receptor by co-expression of the estrogen
receptor. The authors propose that the observations
reflect competition by the receptors for a limiting
transcription factor.
Berry et al., 9 EMBO Journal 2811 (1990),
describe so-called promoter- and cell-specific effects
of an agonist on estrogen-responsive genes. Truncated
and chimeric estrogen receptors were used which
contained TAFl and/or TAF2 regions from the same or
different sources.
Tassett et al., 62 Cell 177 (1990), describe
interaction of TAF1 and TAF2 regions, and competition
(squelching) for limiting factors, by comparing relative
activities of TAF regions and competitor constructs.
Fawell et al., 60 Cell 953 (1990), describe
estrogen-receptor dimerization and its alteration by
mutations in the molecule.
Metzger et al., 20 Nucleic Acids Research 2813
(1992), describe alleged promoter- and cell-specificity
of TAFl and TAF2 regions in the yeast Saccharomyces
~!~ 94/~068 PCT~S94/03795
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cerevisiae. Truncated receptors, or receptors having
regions deleted from them, were used in the analyses.
Danielian et al., 11 EMBO Journal 1025 (1992),
describe conserved regions in the estrogen receptor and
state that:
"Activities of TAFl and TAF2 vary
depending upon the responsive
promoter and cell type and, in some
cases, both are required for full
transcriptional stimulation."
The authors identify amino acids near the C-terminus of
the mouse estrogen and glucocorticoid receptors which
are said to be essential for hormone dependent
stimulation of transcription. Point mutations were
introduced either into the full-length receptor, or into
an internal deletion mutant which lacked the TAF1
region, to allow the authors to determine the effects of
mutations upon TAF2 activity in the absence or presence
of TAFl.
20 SUMMARY OF THE lNv~:~.. ION
Applicant has discovered that the activity of
TAFl and TAF2 regions in a receptor are interdependent.
That is, the activity of one region is dependent on
having the functional context of the other region
available. (By "functional context" is meant that only
a few amino acids (i.e., up to 10), or preferably only
(i.c. 1-3~ amino acids are altered in one region so that
the interaction of a hormone or transcription factor
with the region is altered to a minimum extent
(preferably, the interaction is unaltered). Such
interaction will allow full expression of the activity
of the unaltered or non-mutated region. Thus, the
functional context of one TAF region contains the
functional activities of the other TAF region with
respect to agonist binding, dimerization, and heat shock
protein interaction, but not with respect to the ability
W094/23068 PCT~S94/03795
` ~6~ ~ d
to activate transcription.) Applicant's discovery
indicates that appropriate assays for detection of
agonists of a chosen receptor require receptor
constructs in which a minimum number of mutations are
introduced into either the TAF1 or TAF2 regions to
inactivate that region, i.e., to make it nonfunctional,
without having significant effect on the activity of the
other, non-mutated, region. Thus, in one example,
mutations are introduced into the TAF2 region which make
the TAF2 nonfunctional, while allowing TAFl to exhibit
its functional activity.
Unlike prior studies which used forms of TAF1
or TAF2 having deleted regions, assays of the present
invention provide reproducible results which can be
readily interpreted. Prior constructs failed to provide
the information required to determine the agonist or
antagonist activity of any chosen molecule at a selected
receptor.
Specifically, applicant has discovered that it
is possible to provide an assay to screen for an agonist
at a receptor which interacts with only one of the TAF1
or TAF2 regions of the receptor. Such a specific assay
for an agonist allows rapid screening of large numbers
of agonists for those having specific and desired
properties. For example, an agonist at the estrogen
receptor can be readily identified, e.g., as one which
has activity similar to estrogen or tamoxifen or other
known agonists. That is, not only can an agonist be
specifically identified, but the type of agonist can be
determined in such an assay.
More specifically, applicant has identified
methods by which a modified receptor can be used to
identify promoter- and cell-type specific requirements
for TAF1 or TAF2 activity of a receptor. Experiments
can now be performed to determine such promoter- and
cell-type specific differences in activi-ty.
~'~94/23068 PCT~S94/03795
Without being bound to any specific theory,
applicant proposes that such promoter- and cell-type
specificity may be explained by a model in which the
TAF1 region acts as a dominant transcriptional
activator, and the TAF2 region as a transcriptional
facilitator. That is, the TAF2 region acts to prepare
the transcription apparatus for TAF1 action. Such
preparation may be recruitment of basic transcription
factors, alteration of chromatin structure, or causing
removal of a transcriptional repressor. Alternatively,
the TAF2 region may prepare a transcription apparatus
for other transcriptional activators, and alone have
little inherent transcription activation activity. In
such a model, the TAF1 region is unable to access the
transcription apparatus until the TAF2 region has acted
appropriately to prepare it for TAF1 action.
Applicant proposes that cell specificity for
TAF1 or TAF2 activity may reflect the presence or
absence of a TAF1 or TAF2-type function in a cell that
mimics the presence of TAF1 or TAF2, respectively. Such
a mimetic in a cell will allow a receptor construct
having a mutated and inactive TAF1 or TAF2 region, to be
active. That is, the inactive portion of the receptor
can be complemented by the active functionality present
in the cell. A similar model may exist for promoter
specificity, i.e., only selected promoters will be
activated by TAF1 or TAF2 in any particular cell,
dependent on the functionalities present in those
promoters. In this model, the difference in agonist
activity of various agonists is dependent on the effect
of that agonist on the TAF1 region or TAF2 region, and
interaction of the resulting TAF1 or TAF2 region with a
selected promoter or general transcription apparatus.
Applicant has taken advantage of these
findings to develop an assay by which agonists of
receptors can be readily identified. In-this method, a
W094/23068 PCT~S94/03795
3~ ~
cell is provided with a specific receptor construct
having a selected TAFl and/or TAF2 activity, and having
a suitable response element (e.q., containing a promoter
region) linked to an operative reporter gene (e.a.,
encoding an enzyme activity which is readily
detectable). The response element is selected in
conjunction with a specific cell so that activity of an
agonist is observed only under selected conditions.
Thus, in one example, the receptor may have an active
TAFl region, and a mutated (inactive) TAF2 region which
provides the functional context of TAF2, and the cell is
chosen such that it has a component which mimics or
replaces the TAF2 region function of the receptor on the
chosen promoter. The promoter in turn provides an
appropriate binding context to allow the component to
manifest the desired TAF functions. In this way, an
agonist which acts at the TAF1 region can be readily
identified by its ability to cause expression of the
reporter gene, despite the lack of an active TAF2 region
on the receptor.
Thus, in a first aspect, the invention
features a method for screening or assaying for an
agonist at a chosen receptor. The method includes
providing a cell containing nucleic acid encoding a
receptor having a TAF1 and a TAF2 region. One of these
TAFl and TAF2 regions is able to activate transcription
from a selected promoter, and the other region is
mutated so that, while it provides the functional
context of that region, it is not able to activate
transcription of the promoter independent of the other
TAF region.
The cell is chosen such that no, or minimal,
transcription of the promoter occurs in the presence of
a receptor having only an unmutated TAF region
corresponding to that mutated above (and not the other
TAF region). The cell is also chosen such that
~'~94/23068 ~ PCT~S94/03795
60~3
S
transcription occurs in the presence of a receptor
having the above nonmutated region alone. For example,
in a receptor construct having an operative TAF1 region
and a mutated, inoperative TAF2 region, transcription of
the promoter will not occur (i.e., no significant level
of transcription is detectable, usually less than 5-10%
of normal levels) in the presence of a receptor having
an operative TAF2 region only, but will occur in the
presence of a receptor having an operative TAFl region
only. As discussed above, applicant postulates that
such a cell contains a factor which mimics the TAF2
function of the receptor.
The cell further includes a reporter construct
which has a promoter region which is activated to cause
transcription of a reporter gene in the presence of a
receptor having an active TAF region corresponding to
that which is not mutated above. The promoter is not
activated by the presence of a receptor containing only
the TAF region corresponding to that mutated above.
The method further includes the step of
contacting the cell with a potential agonist under
conditions in which contact of the cell with a normal
agonist (e.g., estrogen for an estrogen receptor) will
cause transcription from the promoter, and thereby
increase the level of the reporter gene product. The
- method may involve transcribing the reporter construct
at a basal (low or minimal) level in the cell before the
agonist is applied. Alternatively, the method may
involve applying the agonist first, and then
transcribing the reporter construct. The receptor may
contain two TAF regions. Non-limiting examples of
receptors that may be used in the present invention
include estrogen receptors, progesterone receptors,
androgen receptors, or mutated versions of the above
receptors.
W094/23068 PCT~S94/03795
,a~6~3~
Finally, the method involves the step of
measuring the level of increase of the reporter gene
product, as an indication of the agonist activity of the
potential agonist.
In preferred embodiments, the agonist is a
human hormone agonist, and a nuclear receptor, e.q., a
human hormone receptor is encoded by the nucleic acid
within the cell. In one example, the receptor has a
mutated TAF2 region, and the cell and promoter are
chosen to exhibit no, or minimal, response to the
presence of TAF2. One example of such a cell is a liver
cell (specifically, a HepG2 cell) in which a receptor
with an operative TAF2 region has no activity. That is,
there is no inherent transcriptional activity with a
receptor having just TAF2 and no TAF1 region present in
the cell, but there is transcriptional activity with a
receptor having an operative TAFl region alone
available. Most preferably, the promoter is chosen such
that it does not require a receptor with a TAF2 function
to be provided within the chosen cell, so that any
agonist which acts in conjunction with a functional TAFl
in the receptor construct is able to show its agonist
activity.
In another aspect, the invention features a
method for detection of agonist activity by provision of
a cell having a nonmutated receptor having functional
TAFl and TAF2 regions. The cell is chosen to lack a
mimicking TAFl or TAF2 activity (i.e., a receptor having
either an active TAFl or TAF2 region, alone does not
cause activation of transcription in the cell). The
promoter is chosen so that activation is achieved in
this cell from this promoter in the presence of an
agonist for the receptor which acts only through one TAF
region and not both. For example, a liver cell (e.q.,
HepG2) and a complex C3 promoter together provide a
useful assay for agonists active at a TAF1 region only.
~94123068 ~CT~S94/03795
The liver cell and C3 promoter have a TAF2 activity, but
the promoter is not activated in the presence of a
receptor having an active TAF2 region alone. But, in
the presence of an active TAF1 region, the promoter is
active. Thus, agonists active at the receptor TAF1
region can be identified as those which cause expression
of the reporter gene.
In a preferred embodiment, the method concerns
use of a receptor in which the TAF2 region is mutated,
and provided within the selected cell and promoter
context. This cell provides a useful screening test to
determine the type of agonist tested. The level of
transcription observed is related to the agonist type as
exemplified below. The above two methods (with mutated
and nonmutated receptor constructs) may be used in
combination to detect, and grade or type agonists at a
selected receptor.
In another aspect, the invention features a
method for treating or preventing an estrogen related
disease or condition. By "estrogen related disease" is
meant a disease that is caused or associated with an
elevated or depressed level of the hormone estrogen. By
"hormone" is meant a naturally occurring biochemical
that will function as a receptor agonist. Synthetic
hormones are more properly referred to as agonists.
Examples of estrogen related diseases include
osteoporosis, breast cancer, uterine cancer, and
endometriosis. Examples of estrogen related conditions
include vasomotor abnormalities, hot flashes,
depression, other psychiatric abnormalities and uterine
fibroids. In some diseases the patient may be unable to
produce estrogen in an amount required by the body. In
other diseases, estrogen may be overproduced. The
treatment may have the effect of preventing new tumors
from developing and/or of shrinking the size of existing
tumors. The method includes forms of ho-rmone
W094/23068 PCT~S94/03795
6~35
replacement therapy. In this method, the patient is
preferably first identified as suffering from such a
disease or condition by standard techniques, and then
treated as described below.
The method involves administering a chemical
compound other than keoxifene, having a keoxifene-like
transcriptional profile to a patient (or causing
production in vivo of such a compound). By "keoxifene-
like transcriptional profile" is meant the production of
a normalized response similar to keoxifene. This
profile demonstrates a relatively low TAF1 response at
low concentrations of the compound but relatively high
response at higher concentrations of the compound. In
addition, little or no TAF2 response should be present
at all concentrations. In addition, it should have a
greater (about twice or more) TAF1 response than with a
wild type receptor at higher concentrations (about
10-7M), see Fig. 8E compared to Figs. 8A-D). The
administration of these chemical compounds with
keoxifene-like transcriptional profiles is expected to
exhibit bone protecting activity and uterine/breast
sparing activity. By "bone protecting activity" is
meant the ability to prevent bone resorption which can
be measured by standard techniques. Bone resorption is
typically associated with a loss of estrogen. Bone
resorption is typically associated with osteoporosis and
is manifest by bone dissolution due to a loss of
calcium. By uterine/breast sparing activity" is meant
the prevention or reduction of the proliferation of
tumorous cancer cells which can be measured by standard
techniques.
Examples of compounds that can be screened to
determine whether or not they have a keoxifene-like
transcriptional profile are provided in Jones, U.S.
Patent No. 4,418,068, issued November 29, 1983 and
incorporated herein by reference. As th-e Jones patent
~'~94t~068 g PCT~S94/03795
,~16013S
makes clear, other compounds that could be screened
include dihydronaphthalenes and benzothiophenes.
Other compounds that can be screened include
compounds with a similar chemical structure to keoxifene
or keoxifene-like analogs. Some of these compounds
could be produced by making substitutions of 1-10 carbon
long alkyl, alkenyl or similar-type chains in the
nitrogen-containing ring of keoxifene. Other
alterations could include altering the length or
saturation characteristic of the alkyl chain (e.g., from
O-lO carbon atoms) that links the nitrogen-containing
ring to the rest of the keoxifene compound. Other
compounds that can be screened for a keoxifene-like
profile include compounds with a chemical structure
similar to tamoxifine or tamoxifine analogs. Those
skilled in the art will readily recognize other
modifications and substitutions that can be made to
compounds that can be screened for a keoxifene-like
profile.
The present invention also includes
pharmaceutically acceptable compositions prepared for
storage and subsequent administration which include a
pharmaceutically effective amount of an above-described
product in a pharmaceutically acceptable carrier or
diluent.
Other features and advantages of the invention
will be apparent from the following description of the
preferred embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The drawings will first briefly be described.
Drawinqs
Fig. 1. Transcription Activation by ER-wt
(wild type estrogen receptor) and Truncated ER Mutants.
Schematic organization of ER-wt, ERN282G and
35 ER179C (A). CVl (B), HepG2 (C) and HS57-8T (D) cells,
W094/~068 ~ PCT~S94/03795
2~`60~35
were transiently co-transfected with increasing
concentrations of the different receptor expression
vectors as indicated, together with 9.5 ~g/ml of ERE-tk-
LUC reporter plasmid, and 5 ~g/ml of pRSV-~-gal
expression vector as an internal control for
transfection efficiency. Carrier DNA (pGEM4) was added
to adjust the total amount of DNA to 20 ~g/ml (see
below). Cultures were treated with or without 10-7M
17-~-estradiol (E2) as indicated for 36 hours and assayed
for ~-galactosidase and luciferase activity (LUC
activity is normalized for ~-gal activity). The
relative luciferase activity is calculated by dividing
the normalized luciferase value at a given point by that
obtained in the absence of transfected receptor or
ligand. A single experiment representative of four
independent experiments is detailed above. Data shown
indicate the mean + SE(m) of triplicate estimations.
Fig. 2. Transcription Activation by Mutant
ER Defective in TAF2 Activity.
Schematic organization of ER-wt and mutant ERs
used in this experiment (A). CV1 (B), HepG2 (C) and
HS578T (D) cells were transiently co-transfected with
increasing concentrations of different receptor
expression vectors as indicated, together with 9.5 ~g/ml
of ERE-tk-LUC reporter plasmid, 5 ~g of pRSV-~-gal
expression vector. Carrier DNA was added to 20 ~g total
DNA. Cultures were treated with or without 10-7M 17-
~-estradiol (E2) for 36 hours, and assayed for luciferase
and ~-gal activity. The relative luciferase activity is
calculated by dividing the normalized luciferase value
at a given point by that obtained in the absence of
transfected receptor or ligand. A single experiment
representative of four independent experiments is
detailed above. Data shown indicates the mean + SE(m)
of triplicate estimations.
~94/~068 ~CT~S94/0379~
~ ~0
~S
13
Fig. 3. Activity of TAF1 and TAF2 on the
Human C3 Gene Promoter.
CVl (A), HepG2 (B) and HS578T (C) cells were
transiently co-transfected with 0.5 ~g of the indicated
receptor expression vector, 9.5 ~g of C3-LUC reporter
plasmid, 5 ~g pRSV-~-gal and carrier DNA to a total
amount of 20 ~g DNA. In addition, a minus receptor
control was included. Cultures were treated with or
without 10-7M 17-~-estradiol (E2) for 36 hours, and
assayed for luciferase activity. The data shown are
representative curves of experiments that have been
repeated 6 times with similar results. The curves
represent averages of quadruplicate data points averaged
and normalized for transfection efficiency by
simultaneous estimation of pRSV-~-gal transcriptional
activity.
Fig. 4. Activity of ER-TAFl and ER179C on
Different Promoter Constructs.
CVl (A) and HepG2 (B) cells were transiently
co-transfected with 0.5 ~g of the indicated receptor
expression vector, 9.5 ~g of pA2-LUC reporter plasmid,
5 ~g pRSV-~-gal and carrier DNA to a total amount of
20 ~g. CVl (C) and HepG2 (D) cells were co-transfected
as described above, using the pEREMLT-LUC reporter.
Cultures were treated with or without 10-7M ~-estradiol
(E2) for 36 hours and assayed for luciferase activity.
Data presentation is described in Fig. 1.
Fig. 5. Activation of ER-TAFl and ER179C by
Triphenylethylene-derived Estrogen Partial Agonists.
HepG2 cells were co-transfected with 0.5 ~g of
the indicated receptor expression vectors, 9.5 ~g of C3-
LUC reporter, 5 ~g of pRSV-~-gal and carrier DNA to a
total amount of 20 ~g. Cultures were treated with 10-7M
of 17-~-estradiol (A), E2, Tamoxifen (B), 4-hydroxy-
Tamoxifen (C), Nafoxidine (D), Clomiphene (E), for 36
hours and assayed for luciferase activit-y. Data
W O 94/23068 i 2 ~ 6 ~ PCTrUS94/03795
14
presentation was described in Fig. 1. In Fig. 5, ER
represents Er-wt, ERn` represents Er-TAF1, TAF2
represents Er-179c, and TAF2"` represents Er-Null.
Fig. 6. Displacement of Estradiol Binding to
ER-wt and ER-TAF1 Proteins by Estrogen Agonists.
Yeast cytosols prepared from cell expressing
ER-wt or ER-TAF1 were incubated overnight at 4C with
5 nM of 3H-17-~-estradiol alone or in the presence of the
indicated concentrations of the different estrogen
agonists. Ligand binding was determined by
scintillation counting following separation of bound and
free ligand using hydroxylapatite.
Fig. 7. Model for TAF1 and TAF2 as
Functionally Dependent Activators of Transcription.
lS This schematic outlines a hypothesis for the
promoter and cell specificity of the individual
transactivators of the estrogen receptor. Interaction
of the receptor with ligand initiates a cascade of
events which exposes the receptor DNA binding region
(DBD) and promotes association of ER with DNA. Only
"estrogenic compounds" are capable of functionally
activating TAF2 region of the receptor. Upon activation
(B), the TAF2 region of the receptor interacts with a
transcriptional repressor (I), displacing it or altering
its structure (C) to permit the TAF1 activation sequence
access to the general transcription apparatus (GTA). In
certain cells and on certain promoters, TAF2 function of
the receptor can be supplied by other transcription
factors, allowing TAF1 region of the receptor to
function independently of TAF2. Therefore, binding of
the receptor to DNA is synonymous with transactivation
and can be accomplished by both estrogen agonists, as
well as antagonists that permit delivery of the receptor
to DNA. In this model, the partial agonist activity of
the triphenylethylene-derived estrogen agonists depends
on the conformation induced by the ligand and the effect
~~ 94/~068 o~3~ PCT~S94/03795
that this conformation has on the presentation of TAF1
to the transcription apparatus.
Fig. 8 shows that the partial agonist
activities of the triphenylethylene derived
antiestrogens depends on TAF1 function. HepG2 cells
were cotransfected with 0.5 ~g of the indicated receptor
expression vectors, 9.5 ~g of C3-LUC reporter, 5 ~g of
pRSV-~-gal and pGem4 as carrier DNA to a total amount
of 20 ~g. Cultures were treated with various
concentrations of 17-~-estradiol (A), Clomiphene (B),
Nafoxidine (C), 4-OH-Tamoxifen (D) and keoxifene (E),
for 36 hours and assayed for luciferase activity. The
data for panel E was obtained relative to a different
estradiol control than the other panels. Thus, the peak
in panel E appears approximately five times higher than
it would if the data had been obtained relative to the
same estradiol control that was used in panel A. The
relative luciferase activity was calculated as described
for Fig. 1. A single experiment representative of 6
independent experiments is detailed. The data shown
indicate the mean + SE(m) of triplicate estimations.
Fig. 9. Bone marrow from sham (control), OVX
(ovariectomized rats), OVX plus estrogen, and OVX plus
keoxifene treated rats were evaluated for osteoclastic
potential in the coculture assay. Bone marrow was
- combined with primary osteoblasts in the presence of
1,25-dihydroxyvitamin D3 and parathyroid hormone for 8
days and scored for the number of tartrate acid
phosphatase resistant multinucleated cells (TRAP + MNC).
The number of TRAP + MNC in the sham operated animals
was set at 100%.
Fig 10. Estrogen agonist activity of
keoxifene (keox) on MCF-7 cell proliferation. The
activity of estrogen in this assay is maximum at 10-1M,
and induces proliferation to 1500% of control.
Fig. 11 is a diagram showing pC3-LUC.
W094/23068 PCT~S94/03795
~6013~
i .
16
Methods
The methods discussed briefly above are useful
for identifying agonists of various receptors. For
example, an estrogen agonist can be identified which is
useful for treatment of osteoporosis. In the disease
state, it appears as though TAFl activity alone is
sufficient for prevention of bone loss. Thus, agonists
having activity only at the TAF1 region and not at the
TAF2 region of the receptor are useful for disease
treatment. The methods described herein allow rapid
screening of potential agonists, unlike prior methods in
which laborious procedures were involved to detect
useful agonists. Other useful receptors for which this
procedure can be used includes progesterone,
glucocorticoid, androgen and mineralocorticoid
receptors, not only in the human derived cells, but also
in other eucaryotic cell lines, such as chicken and
yeast (which are useful organisms for screens of the
present invention).
The following are specific examples of methods
of this invention. These examples make use of the
estrogen receptor, but are not limiting in the
invention. Those in the art will recognize that other
equivalent receptors, cells and promoters can be readily
used in equivalent procedures within the scope of the
claims.
Estroqen Receptor
The estrogen receptor (ER) is a member of the
nuclear receptor super-family, a class of transcription
factors whose functions are regulated by steroids,
vitamins or thyroid hormone (Beato, 56 Cell 335, 1989).
This family of regulatory proteins share common
mechanistic features in that they are transcriptionally
inactive within the cell until exposed to hormone.
Occupancy by hormone results in transformation of the
receptor to an activated state, thus allowing it to
"~94/~068 PCT~S94/03795
,.
17 3
productively interact with specific DNA sequences in the
regulatory regions of target genes. The resultant
positive or negative effects of the bound receptor on
specific gene transcription are cell-type and promoter-
context dependent. Nonetheless, the relative effect maybe measured in any particular cell/promoter construct.
Thus, the desired effect may be observed in a wide
variety of constructs.
The cDNA for ER has been cloned and used to
reconstitute estrogen responsive transcription units in
heterologous mammalian cells (Kumar et al., 5 EMBO J.
2231, 1986, and Green et al., 231 Science 1150, 1986).
This has enabled a detailed examination of the
functional domains within the protein (Kumar et al., 51
Cell 941, 1987). A functional examination of the
domains of ER in several systems has revealed the likely
structural features within the receptor which may
interface with critical cellular components to generate
a variety of hormone responsive endpoints (Danielian et
al., 11 EMBO J. 1025, 1992). In particular, two
distinct transactivation domains have been defined, a
sequence at the amino terminus of the receptor, termed
TAFl, and a sequence confined to the carboxyl 60 amino
acids, termed TAF2 (Danielian et al., supra; Berry et
al., 9 EMBO J. 2811, 1990; and Tasset et al., 62 Cell
1177, 1990), all hereby incorporated by reference
herein. Recently, investigators involved in
intracellular receptor research have favored referring
to the TAF domains, as AF domains (e.g. Cavailles et
al., J. of Cellular Bio., 341, 1994) to avoid confusion
with the discovery and cloning of TATA-binding protein
associated factors (Dynlacht et al., 66 Cell, 563,
1991) -
It appears that alterations in charged
residues of the amino terminal portion of the hormonebinding domain can result in increases or decreases in
W094/~068 PCT~S94/03795
6013S
ER transcriptional activity with no change in receptor
affinity of cognate hormone. Therefore, the regions
around residue 530 (Danielian et al., supra,) and the
region around cysteine 381 (Pakdel et al., 7 Mol
Endocrinol, 1408, 1993) may in themselves consititute AF
subdomains within TAF-2. It follows then, that changes
in such domains, such as the region around cysteine 381,
could result in mutant receptors which could
discriminate between estrogen and antiestrogen ligands,
paralleling the results obtained as detailed herein.
Futhermore the analogous situation could exist for
discreet residues in the TAF-l region of ER.
The cellular targets of ER-TAFl and ER-TAF2
have not yet been identified. A rigorous examination of
ER-TAFl and ER-TAF2 function in mammalian cells has not
been yet accomplished.
ExamPles
The following experiments characterize the
dependence of ER-TAFl and ER-TAF2 activities on cell-and
promoter-context in mammalian cells, and the role of
ligand (agonist) in manifestation of these differences.
Some of these experiments are described in Tzukerman et
al., 8 Mol. Endocrin 21, 1994, hereby incorporated by
reference herein.
The following materials and methods were used
in the example:
Receptor exPression vectors
cDNA sequences encoding the ER-wt and a TAFl -
deleted receptor derivative were excised from the
plasmids YEpwtER and YEpER179C respectively, using BfrI
and SacI. The DNA encoding the TAFl receptor derivative
was excised from the plasmid YePERN282G using BfrI and
KpnI. Construction of the vectors YEpwtER, YEpER179C
and YEpERN282G, have been described previously (Pham et
al., 6 Mol. Endo. 1043, 1992). The excised DNA was
treated with T4 DNA polymerase (Boehring-er Mannheim Co.)
-'094/~068 ~ PCT~S94/03795
60~
3S
19
and ligated into the unique EcoRV site within the
~ mammalian expression vector pRST7 (Berger et al., 41 J.
Steroid Biochem. Mol. Biol. 733, 1992).
RecePtor mutations
The wild type estrogen receptor cDNA was
cloned into pGEM-llZf(+) (Promega, Wisconsin). Specific
mutations were introduced into the hormone binding
domain of the receptor by substituting alanine for amino
acids located at positions 538, 542, and 545, using site
directed mutagenesis (Kunkel et al., 154 Methods in
Enzymoloqy 367, 1987), creating the plasmid pGERm. The
mutated hormone binding domains were introduced into ER-
wt and ER179C by exchanging the BqlII-SacI C-terminal
fragment of this vector with the analogous mutated
fragment from pGERm.
RePorter Plasmids
The reporter ERE-tk-LUC contains a single copy
of the vitellogenin ERE upstream of the herpes simplex
thymidine kinase promoter sequences linked to luciferase
(LUC). The C3-LUC reporter which contains 1.8 kb of the
human C3 gene promoter (-1807 to +58) (Vik et al., 30
Biochemistry 1080, 1991). Unique restriction sites Xhol
and BamH1 were introduced into the C3 promoter, the DNA
was then cloned into the cognate sites of the vector pl-
LUC (Berger et al., 41 J. Steroid Biochem. Mol. Biol.733, 1992), where a polyclonal site has been inserted
into the MMTV-LUC vector (see Fig. 11). Those in the
art can readily construct equivalent vectors. pA2-LUC
contains a 835 bp fragment (-821 to +14) of the xenoPus
vitellogenin A2 gene promoter (Vik et al., 30
Biochemistry 1080, 1991). pEREMLT-LUC contains a single
ERE upstream the adenovirus major late promoter
sequences (-44 to +11) (Hu and Manly, 78 Proc. Natl.
Acad. Sci. USA 820, 1981).
W094t~068 PCT~S94/03795
~lcol35
Cell culture
CVl and HS578T cells were routinely maintained
in Dulbecco's modified Eagle's medium - DMEM
(Biowittaker, Maryland) supplemented with 10% fetal
bovine serum (FBS) (Hyclone Laboratories, Utah). HepG2
cells were maintained in Minimal Essential Medium
Eagle's - MEM (Biowittaker, Maryland) containing 10%
FCS.
Transient transfection assaY
Cells were seeded 24 hours prior to
transfection in flat-bottom 96-well tissue culture
plates (5x103 cells/well), in phenol red-free DMEM
containing 10% FBS. DNA was introduced into cells using
calcium phosphate co precipitation. Plasmid DNA was
diluted in 1 ml of 1 mM Tris, pH 7.4, 0.1 mM EDTA,
0.25 M CaCl2. DNA solution was added dropwise with
vortexing into an equal volume of 2X HBS pH 6.9 (280 mM
NaCl, 50 mM HEPES, 1.5 mM Na2HPO4) and precipitates were
allowed to form for 20 minutes. Transfections (11 ~l of
DNA mix/well) were performed on a Biomek 1000 Automated
Laboratory Workstation (Beckman, California). Cells
were transfected for 6 hours and then washed with
phosphate-buffered saline (PBS) to remove the
precipitate. Cells were incubated for an additional 36
hours in phenol red-free medium containing 10% charcoal-
treated FBS, with or without hormones as indicated in
the text. Cell extracts were prepared as described by
Berger et al., 41 J. Steroid Biochem. Mol. Biol. 733,
1992, and assayed for luciferase and ~-galactosidase
activities. All determinations were performed in
triplicate in at least two independent experiments, and
were normalized for transfection efficiency by using
expression of ~-galactosidase as an internal control.
Preparation of yeast recePtor ~roteins
Expression vectors producing ER-TAF1 were
constructed by replacing the Bfrl-Mlul fragment of
` ~94/~068 ~ PCT~S94/03795
.~60~
YEpE10 (Pham et al., 88 Proc. Natl. Acad. Sci. USA 3125,
- 1991) with the corresponding fragment of pRST7ER-TAF1.
This vector and a vector producing wild type receptor
(YEPE10) were transformed into the yeast strain BJ2168
(previously described by McDonnell et al., 39 J. Steroid
Biochem. Molec. Biol. 291, 1991). Individual
transformants were picked and grown to an OD~=1.
Cultures were then induced with 100 ~M CUS04 ~ and 2 mM
chloroquine for 16 hours at 30C. Cells were then
pelleted and washed with cold water. Cells were
resuspended in 2-5X pellet volume of 10 mM Tris, 0.4 M
KCl, 2 mM EDTA, 0.5 mM PMSF, 1 ~g/ml aprotinin, 2 mM
DTT, pH 7.6, and lysed by vortexing with 0.45-0.5 mm
glass beads, intermittently with cooling on ice, until
at least 90% of the cells were observed to be open.
Extracts were centrifuged at 13,000xg and the
supernatants were recovered. Protein concentrations
were determined by Bio-Rad Protein Assay (Bio-Rad,
Richmond, CA).
~-Estradiol bindinq competition assay
All methods were performed using a Biomek 1000
automated workstation (Beckman Instrument, Fullerton,
CA). Ten-fold serial dilutions of the compounds to be
tested were made in 10 mM Tris, 0.3 M KCl, 5 mM DTT, pH
7.6, and transferred to polystyrene tubes containing
100 ~l at final concentrations of 104 M to 10-ll M
diluted compounds, 5 mM 3H-~-estradiol (Amersham, UK),
and 22 ~g total protein derived from strains producing
ER or ER-TAF1. Following an overnight incubation at 4 C,
100 ~l of a 6% hydroxylapatite slurry in 10 mM Tris,
5 mM DTT, pH 7.6 was added. The tubes were incubated
for an additional 30 minutes at 4 C, mixing after the
first 15 minutes. Hydroxylapatite pellets were washed
4X with 1 ml 1% Triton X-100 in 10 mM Tris, 5 mM DTT,
pH 7.6. Finally, the hydroxylapatite pellets were
resuspended in 800 ~l of Ecoscint A scintillation fluid
W094/~068 PCT~S94/0379~
(National Diagnostics, Manville, NJ). Activity in each
sample was measured using a LS6000IC scintillation
counter (Beckman Instruments, Fullerton, CA).
ExamPle 1: Transcriptional Activation by TAF1 and TAF2
Truncated RecePtors
Referring to Fig. 2, truncated forms of the
human estrogen receptor were prepared which lacked
either the TAFl (ER179C, see Fig. 2A) or the TAF2
(ERN282G, see Fig. 2A) activation sequence. These
constructs encode proteins structurally similar to those
used previously in mammalian (Berry et al., 9 EMB0 J.
2811, 1990) and yeast cells (Pham et al~, 6 Mol. Endo.
1043, 1992). The transcriptional activities of these ER
derivatives were assessed using a reporter plasmid
containing one copy of the vitellogenin estrogen
response element (ERE) (Klein-Hitpass et al., 76 Cell
1053, 1986) inserted upstream of the thymidine kinase
promoter (ERE-tk-LUC). The reporter plasmid and
increasing concentrations of ER or mutant ER expression
vectors were transiently transfected into the ER
negative cell lines CV-1 (monkey kidney fibroblasts),
HepG2 (human hepatocellular carcinoma) and HS578T (human
breast cancer cells), and activity assessed as
documented in Fig. 2B. All transfections were performed
in the absence or in the presence of 17-~-estradiol at
concentrations ranging from 10-5M to 10-lIM. However, due
to the number of data points obtained in this way
(>2,500) only the activities obtained using 10-7M 17-~-
estradiol are presented since this is a concentration
that elicited maximal transcriptional response in all
cell lines examined.
The ER-wt was active in all cell lines. Using
this protocol, we were unable to detect significant
TAFl- mediated transcriptional activity in either CV-1,
HepG2, HS578T (Figs. 2B, C, D) or HeLa or U20S cells
(data not shown) when assayed in the context of the
~94/~068 2~ PCT~S94/03795
3S
23
ERN282G deletion. In contrast, however, the TAF2
activation function (ER179C) exhibited substantial
activity in these cells (Figs. 2B, C & D). The
magnitude of the TAF2 transcriptional activity by ER179C
was cell-type dependent. This isolated activator
exhibited a lower efficacy relative to wild type
receptor, even at DNA concentrations that produced
saturating receptor levels. In HepG2 cells ER179C was
about 35% as active as ER-wt (Fig. 2C), whereas in CV-l
and HS578T, the ER179C demonstrated 70% and 65% of ER-wt
activity respectively (Figs. 2B & D). Transfection
efficiency and recombinant expression levels were
similar as estimated by indirect fluorescence microscopy
and flow cytometric analysis (data not shown).
The results obtained in this analysis are
consistent with the hypothesis that the TAFl and TAF2
sequences represent functionally distinct
transcriptional activators. A wild type receptor
activity requires either both activator regions or an
intact receptor context for an individual activator to
exhibit maximal transcriptional activity.
In addition to the partial activities observed
by the above ER-mutants, increasing concentrations of
transfected ER-wt in CV-l and HS578T cells led to a
progressive decrease in hormone dependent
transcriptional activity (Figs. 2B & D). This
phenomenon has been observed by others and likely
results from sequestration of limited transcription
factors or targets by the over-expressed, hormone-
activated receptor, such that activated receptorfunction is compromised (Tasset et al., 62 Cell 1177,
l99O). This "squelching" or "transcriptional
interference" supports the idea that ER requires
additional, limiting cellular transcription factors for
appropriate function. The failure of the ER-wt to
"squelch" in the HepG2 cell line (Fig. 2C) suggests
W094/~068 ~ 6~ PCT~S94/03795
24
either an increased abundance of a required co-factor,
or the involvement of an additional cell specific
component in this process.
Example 2: Transcription Activation bY a Mutant ER
Defective in TAF2 ActivitY
Previously, TAF1 and TAF2 functions were
defined as individual domains within the estrogen
receptor that were capable of supporting transcription
of an ER responsive promoter (Berry et al., 9 EMBO J.
2811, l99O, and Tasset et al., 62 Cell 1177, 1990). In
the mammalian cells tested here, we were unable to show
a distinct activity of the TAFl sequence when analyzed
in the context of the ERN282G deletion. We considered,
therefore, whether analysis of this transactivator
outside the context of the full-length receptor may not
reflect its true biological activity. Previously,
Danielian et al., supra, demonstrated that it was
possible to change three amino acids between residues
535 and 550 in the carboxyl terminus of the mouse
estrogen receptor which comprises transcriptional
activity of TAF2, but nevertheless results in a receptor
capable of binding both specific DNA and cognate ligand
with wild type affinity, indicating that these changes
did not lead to gross structural abnormalities in the
protein. Therefore, using site-directed mutagenesis we
created similar amino acid changes in the carboxyl
terminus of human ER at residues 538, 542 and 549
(Danielian et al., su~ra). (see the ER-TAF1 construct
of Fig. 1) This triple mutation was also introduced
into ER179C creating a null estrogen receptor. (See the
ER-Null construct of Figure 1) This latter construct
allowed a specific determination of the effect of these
mutations on TAF2 function. Thus, ER-Null and ER-TAFl
both serve as excellent controls because they are
inactivated for the functions being studied. One of
these constructs was used in every experiment documented
` ~94/~068 PCT~S94/03795
~ ' ' 2~60~
S
herein. Mutation of these three amino acids provides
- but one example by which the context of a TAF region can
be maintained while inactivating that region. Those in
the art will recognize that equivalent mutations in the
same or other amino acids can be readily made by
standard techniques.
The transcriptional activities of these mutant
ERs were assessed by transient transfection into CV-1
cells together with the ERE-tk-LUC reporter.
Introduction of the triple mutation into ER179C totally
abolished TAF2 activity (Fig. lB). Thus, we believed
that introduction of this mutation into the wild-type
ER, would allow an examination of TAF1 activity in the
full-length receptor context without interference from
TAF2 activity. The ability to specifically mutate the
TAF2 activator within the human estrogen receptor in
this manner is consistent with the results previously
reported for the mouse ER, and indicates that equivalent
mutations can be made in the other receptor TAF regions.
The full-length receptor containing the triple
mutation (ER-TAF1) was subsequently used for analysis of
TAF1 function in the context of the intact receptor.
Constructs encoding wild type receptor, ER-TAF1, ER179C
or the null estrogen receptor were transfected into CV-
1, HepG2 or HS578T cells, together with the ERE-tk-LUC
reporter. The results are shown in Fig. 1. In all cell
lines, the ER179C was transcriptionally active, as
observed earlier (Figs. lB, C & D), whereas the null
receptor was inactive. Interestingly, in CV-1 cells, in
the absence of a functional TAF2 activation sequence,
the ER-TAF1 protein exhibited a significant
transcriptional activity (Figs. lB, C & D). Thus, the
activity of the TAF1 activator when analyzed in the
context of a full length receptor molecule, as observed
here, was different from that when analyzed as a
deletion mutant (ERN282G, Fig. 2). This-suggests that
W094/~068 PCT~S94/03795
~6~13~
TAFl activity does not function independently, but
rather requires additional carboxyl-terminal sequences
for appropriate function. Interestingly, increasing the
concentration of transfected ER-TAFl DNA did not result
in a receptor dependent "squelching" of transcriptional
activity. This observation implies that both TAFl and
TAF2 activators and possibly the context of the full-
length receptor are required for this squelching
function.
A comparison was made of the expression level
of each of these receptors by transfecting the
expression vectors into CV-1 cells and analyzing the
hormone binding activities in the resulting cytosolic
extracts (data not shown). The Kds of the ER-wt, ER-TAFl
and ER179C were the same. The ER-wt and ER-TAFl were
synthesized in comparable levels as measured by hormone
binding activity, whereas the amino-terminally deleted
ER179C and the null receptor were expressed at about 25%
of ER-wt level. Since, all the transcriptional
responses we detected with each receptor were maximal
responses achievable, it is unlikely that receptor
expression levels are a significant factor in the
outcome of our experiments.
Example 3: ER-TAFl and ER179C ActivitY is Promoter
Specific
- The above results using the ERE-tk-LUC
reporter indicated that the TAFl activator of the
estrogen receptor functions, albeit weakly, in the
absence of an intact TAF2 function. In addition, TAFl
activity appeared to be cell-type dependent.
We extended our studies to examine the
efficacy of the individual activator functions on other
estrogen responsive promoters. To this end, we chose
the estrogen responsive C3 promoter in which a strong
ERE has recently been identified (Zawaz, 2 Gene Exp.,
39, 1992) (Vik et al. 30 Biochemistr~ 080, 1991). The
~- 941~068 ~3S PCT~S94/03795
activities of the of ER-wt, ER-TAFl and ER179C
activators were evaluated on C3 promoter-directed
transcription as depicted in Fig. 3. In HS578T cells,
the C3 promoter can be activated equally well by either
ER-wt, ER-TAFl or ER179C (Fig. 3C). In contrast however,
in HepG2 cells, the ER-TAFl activator was as active in
transcription as wild type ER, but the ER179C activator
was silent (Fig. 3B). These data suggest that, with
respect to the C3 promoter, there is a strong cell-type
bias in ER transactivator functions. In CV-l cells it
appears that the combination of the activation sequences
is required for maximal activity (Fig. 3A).
Cumulatively, these data suggest that the TAFl and TAF2
activators within ER demonstrate a dependence upon cell-
type and promoter, and furthermore, the dominantactivator of ER-mediated regulation of C3 expression is
TAFl.
The analysis of the relative contribution of
the individual ER TAF domains in ER function was
extended to include two additional promoters, namely the
adenovirus major late promoter, containing an estrogen
response element, and the vitellogenin promoter (Fig.
4). Both of these promoters were responsive to estrogen
in the presence of ER-wt. However, unlike the C3
promoter the individual activation domains of ER were
- minimally active in both cell lines examined. This
highlights further the promoter specificity of the
estrogen receptor activation domains. Similar tests to
those described above can be used to quickly identify
useful promoter and cell combinations for use in assays
for agonists discussed above (see also, Example 4,
below).
Example 4: Requlation of ER-TAFl and ER179C Activity by
Estrogen Receptor Aqonists
Certain triphenylethylene-derived estrogen
receptor antagonists (i.e., tamoxifen, nafoxidine) are
W094/~0~ 2 16 013 ~ PCT~S94/0379~
28
reported to exhibit partial agonist activities. We
therefore tested whether these compounds preferentially
activate either TAF1 or TAF2 transactivators. A series
of these compounds was evaluated in HepG2 cells using
the ER-TAF-specific receptor derivatives and the C3
promoter. On this promoter, tamoxifen, 4-hydroxy-
tamoxifen, nafoxidine and clomiphene were all potent
activators of ER-wt mediated C3 gene transcription
(Figs. 5B-E). However, none of these compounds were as
effective as estrogen in this regard (Fig. 5A). In
addition, estrogen was an efficient activator of the ER-
TAF1, whereas the partial estrogen agonists were not as
effective. In this cellular and promoter context,
ER179C was not activated by either estradiol or the
partial estrogen agonists. These data imply that even
though TAF1 activity is necessary, it alone does not
activate this promoter by triphenylethylene-derived
antihormones, suggesting that their mode of action may
be mechanistically different from that of estrogen
(Figs. 5A-F). The differences in hormonal
responsiveness of these receptor derivatives do not
relate to alterations in the affinity of the proteins
for ligands. As shown in Figs. 6A and B, the affinity
and specificity for ligands of the ER-wt and ER-TAF1
were indistinguishable. It is notable that not all the
- anti-hormones tested in this system have an identical
transcriptional profile (Figs. SB-E). The absolute
efficacy for each of the anti-hormones is different, as
is their ability to differentially activate TAF1,
suggesting subtle mechanistic differences in the
agonistic properties of these ligands.
These examples demonstrate that introduction
of specific point mutations into the human estrogen
receptor, affects ER-transcription activation function.
Mutation of TAF2 in this manner is still compatible with
wild-type binding affinities for estrogen, tamoxifen and
~-`94/~068 ~ PCT~S94/03795
6~3~
29
4-hydroxy-tamoxifen. Surprisingly, TAF1 retained
considerable transcriptional activity despite TAF2
mutation. When we deleted the entire TAF2 sequence
(ERN282G) we were unable to observe residual
transcriptional activity of TAFl in any cell line
examined. This suggests that either TAF2 or the context
of the full length receptor is required for full
manifestation of TAF1 activity. In contrast, Berry et
al. observed that a construct identical to ERN282G was
constitutively active in avian CEF cells (Berry et al.,
9 EMBO J. 2811, l99O). This may suggest a difference in
estrogen receptor function in mammalian and avian cells,
and may not reflect basic differences between the two
sets of results.
Using the modified receptors we were able to
identify cell and promoter specific differences in the
activity of ER-TAFl and ER179C. In studies which were
controlled for expression level and transfection
efficiency we saw that both activators displayed
promoter and cell type specific differences in their
activity. The most striking example of this is the
inability of ER179C to function well on any promoter in
HepG2 cells. The ER-TAF1 activator, on the other hand,
functions very well on the complex C3 promoter, but less
well on the other promoters examined. The activity
profiles of ER-TAF1 and ER179C are clearly distinct,
suggesting dissimilar mechanisms of action. On the
complex C3 promoter there is no apparent synergism
between TAF1 and TAF2, whereas it clearly exists on
other promoters. This suggests that on this promoter
the ER activation domains may interact with differing
transcription factors. The data obtained using the C3
promoter in HepG2 cells indicate that there is a
transcription factor in these cells that can
functionally replace TAF2, as TAF1 is as good a
transcriptional activator as ER-wt. However, since TAF2
W094/23068 PCT~S94/0379~
.. ~ . ~.
` "216013~
alone does not activate transcription, it suggests that
no mimetic for TAFl exists for transcription of this
promoter in this particular cell line.
The dissimilar mechanism of action and the
promoter and cell type specificity can be explained by a
model in which TAF1 is the dominant transcriptional
activator and TAF2 is a transcriptional facilitator (see
model Fig. 7). We suggest that the function of TAF2 is
to "prepare" the transcription apparatus for TAF1
function. This "preparation" function could be
recruitment of basic transcription factors, alteration
of chromatin structure or overcoming the effects of a
transcriptional repressor. On the other hand, TAF2
could "prepare" the transcription apparatus for another
transcriptional activator, but on its own would have
little inherent transcriptional activity. In support of
this hypothesis, the TAF2 activator is poorly active on
minimal promoters.
This dependence on promoter complexity is also
observed for ER-TAFl activity. Additional evidence in
support of the facilitator role of TAF2 is that in yeast
the TAFl activator is inactive on a minimal promoter.
However, a mutation of the SSN6 locus (a cellular
repressor of transcription), Keleher et al., 68 Cell,
709, 1992, results in a 100-fold increase in ER-TAFl
- activity (to a level comparable to ER-wt), whereas
ER179C activity is not effected (McDonnell et al., 89
Proc. Natl. Acad. Sci. USA 10563, 1992). We suggest
that this cellular mutation has the effect of mimicking
the function of TAF2, and that in mammalian cells TAF2
has a similar role. In this system TAF1 is unable to
access the transcription apparatus as a result of
stearic hindrance by an inhibitor. Where TAF2 is
available, then the inhibitor is displaced and TAF1 is
able to interact with the transcriptional apparatus.
~-94/~068 ~ PCT~S94/03795
`- ~60~
Example 5: Screening For and Use of Compounds With
Keoxifene Like TranscriPtional Profiles
In humans the tri-phenylethylene derived anti-
- estrogen keoxifene, exhibits bone sparing activity while
having no significant effects on uterine proliferation.
In contrast, tamoxifen, a related anti-estrogen, is bone
sparing but functions as a partial estrogen agonist in
the uterus promoting an undesirable proliferative
effect. In order to determine whether the differences
in the in vivo biological activity of tamoxifen and
keoxifene could be reconciled by their differential
ability to transcriptionally activate TAF1, these
compounds were studied in HepG2 cells using the ER-TAF
specific receptor derivatives on the C3 promoter. The
results are shown in Figure 8D and 8E.
Keoxifene had a unique transcription profile
in this promoter and cellular context. In particular,
low concentrations of keoxifene stimulated ER
transcriptional activity. At higher concentrations,
keoxifene inhibited the basal transcriptional activity
of ER and did not cause further transcriptional
activation (See Fig. 8E). On the ER-TAFl construct,
keoxifene demonstrated significant partial agonist
activity inducing C3 promoter transcription 5-fold over
background. The mechanism by which keoxifene manifests
- a transcriptional profile distinct from the related
molecule tamoxifen is unclear. However, it is likely
that these compounds induce subtle alterations in
receptor structure which facilitate distinct
interactions of the ER-TAFs with the general
transcription apparatus.
The unique profile exhibited by keoxifene in
this in vitro assay suggested that additional compounds
displaying similar transcriptional profiles may also
exhibit favorable bone protective/uterine sparing
activities. To this end we have studied-several
W094/23068 ` 2 ¦ ~ O 13 5 PCT~S94/03795
compounds derived from tamoxifen using this assay system
and have been able to split these compounds into three
distinct groups based on their ability to modulate TAF1
activity. The first category contains compounds that
resemble the activity of estrogen, the second group
resembles the activity of tamoxifen and the third group
profile similar to keoxifene.
When the keoxifene-like compounds are assayed
in the ovariectomized rat model they are expected to be
bone protective and to demonstrate no uterotrophic
activity. Rats are given a dorsal ovariectomy as
follows. The animals are anesthetized with
Ketamine:Xylazine and surgery is performed. Shave the
central back of the anesthetized rat. Make a
longitudinal incision in the skin parallel to the spine
about 1 inch long. Spread the connective tissue away
from the muscle layer with the tips of scissors. About
1 inch from the spinal column at the base of the rib
cage, make a small cut (1/4") of the muscle with
scissors. With small forceps, pull out ovarian fat.
Ovary will be visible as a cluster-like structure,
attached to the end of the uterine horn. Cut the
connective tissue that holds them together. Staunch any
bleeding. Replace fat into body cavity. Repeat on
opposite side. Clip skin together and use betadine on
- the incision to retard infection and reduce clip
removal. The next day injections are begun. Injections
are done subcutaneously in the hip daily (usually in the
morning). Vehicle is 10% ethanol and all injection
volumes are 300 ~1. After 28 days of injections, the
animals are sacrificed under anesthetic by cervical
dislocation and the body and wet uterine weights are
determined. The hindlimbs are taken for histology and
histomorphometry.
The transcriptional profile exhibited by
keoxifene in vitro is predictive of agents demonstrating
~94/~068 ~ PCT~S94/03795
,60~3
33
bone selective estrogenic activity. Thus, the use of
these ER-TAF constructs examined on this promoter and
cellular context provides a useful screen for compounds
useful for the treatment of osteoporosis.
In in vivo studies, rats are subjected to sham
or authentic ovariectomy and allowed to recover for 5
days. Rats (4-6 per group) are then injected
subcutaneously with vehicle or vehicle containing
estrogen, keoxifene, or a test compound daily for
periods up to 28 days. Animals are sacrificed, weighed,
- and evaluated for uterine wet weight, total serum
cholesterol, and bone mineral density. Established
methods are utilized with the exception that bone
mineral density of the distal femural metaphysis is
determined utilizing an Hologic mineral densitometer.
Bone marrow from test animals is evaluated for
osteoclastic potential in the coculture assay with
primary osteoblasts. Bone marrow is combined with
primary osteoblasts in the presence of 1,25-
dihydroxyvitamin D3 and parathyroid hormone for 8 daysand scored for the number of tartrate acid phosphatase
resistant multinucleated cells (TRAP + MNC). The number
of TRAP + MNC in the sham operated animals is set at
100%. Tartrate resistant acid phosphatase positive,
multinucleated cells are scored by standard methods as
nascent osteoclasts.
The in vitro effects of compounds on MCF-7
breast cell proliferation can also be studied. The
partial agonist activities of estrogen, keoxifene, and
the test compound on MCF-7 human breast carcinoma cells,
is assessed by treating the cells for 7 days in the
absence or presence of increasing concentrations of
compound. Cells are treated at day 0 and day 4 with
compound. Triplicate wells are evaluated for cell
number at termination of the experiment-on day 7. The
activity of estrogen in this assay is expected to be
W094l~068 PCT~S94/03795
~ ~`6~`i3~ ~
34
maximum at 10-l~, and induce proliferation to 1500% of
control.
The in vitro profiles of keoxifene and the
test compounds could then be determined. The activities
of increasing concentrations of compound on ER, ER-
TAF1, and ER-null receptor transactivation of the C3
promoter in HepG2 cells is determined by standard
methods as described in this document. Thus, compounds
that exhibit both bone protecting and uterine/breast
sparing activity at a given concentration, can be
identified. Higher potency compounds than keoxifene are
preferred. By "potency" is meant the amount of a
compound required to produce the desired effect. Thus,
high potency compounds will bind to the receptor with
greater affinity than keoxifene. High potency compounds
produce maximal effect at minimal dosages. As
illustrated in Fig. 8, compounds with high potency have
peaks further to the right than do compounds of lower
potency.
Use
Agonists and their type can be quickly
identified in the above systems. Specifically, the
experiment described in Example 4, and illustrated in
Fig. 5, is useful to identify an agonist and then define
its type of activity. For example, use of a wild type
receptor (ER-wt) in this assay will indicate whether the
test compound is an agonist, i.e., has activity in the
assay. The use of a mutated receptor with full
functional context (ERm), in the assay will indicate the
type of agonist, i.e., what level of activity is
observed. Examples of the range of results expected
with various test compounds are shown in Fig. 5, and
discussed in Example 4. Using such assays, one can
readily screen for desired agonist activity, e.a.,
agonists active only at TAF1 regions which mimic the
~r- ~ 94/23068 60~3~ PCT~S94/03795
activity of estrogen and are useful for treatment of
osteoporosis.
Pharmaceutical Compositions
The present invention also encompasses
pharmaceutical compositions prepared for storage and
subsequent administration, which have a pharmaceutically
effective amount of the products disclosed above in a
pharmaceutically acceptable carrier or diluent.
Acceptable carriers or diluents for therapeutic use are
well known in the pharmaceutical art, and are described,
for example, in Reminqton's Pharmaceutical Sciences,
Mack Publishing Co. (A.R. Gennaro edit. 1985).
Preservatives, stabilizers, dyes and even flavoring
agents may be provided in the pharmaceutical
composition. For example, sodium benzoate, sorbic acid
and esters of p-hydroxybenzoic acid may be added as
preservatives. Id. at 1449. In addition, antioxidants
and suspending agents may be used. Id.
The compositions of the present invention may
be formulated and used as tablets, capsules or elixirs
for oral administration; suppositories for rectal
administration; sterile solutions, suspensions for
injectable administration; and the like. Injectables
can be prepared in conventional forms, either as liquid
solutions or suspensions, solid forms suitable for
- solution or suspension in liquid prior to injection, or
as emulsions. Suitable excipients are, for example,
water, saline, dextrose, mannitol, lactose, lecithin,
albumin, sodium glutamate, cysteine hydrochloride, and
the like. In addition, if desired, the injectable
pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents,
pH buffering agents, and the like. If desired,
absorption enhancing preparations (e.a., liposomes) may
be utilized.
W094/23068 216 0 13 ~ PCT~S94/0379~
The pharmaceutically effective amount of the
composition required as a dose will depend on the route
of administration, the type of animal being treated, and
the physical characteristics of the specific animal
under consideration. The dose can be tailored to
achieve optimal efficacy but will depend on such factors
as weight, diet, concurrent medication and other factors
which those skilled in the medical arts will recognize.
In practicing the methods of the invention,
the products or compositions can be used alone or in
combination with one another, or in combination with
other therapeutic or diagnostic agents. These products
can be utilized ln vivo, ordinarily in a mammal,
preferably in a human, or ln vitro. In employing them
in vivo, the products or compositions can be
administered to the mammal in a variety of ways,
including parenterally, intravenously, subcutaneously,
intramuscularly, colonically, rectally, nasally or
intraperitoneally, employing a variety of dosage forms.
As will be readily apparent to one skilled in
the art, the useful in vivo dosage to be administered
and the particular mode of administration will vary
depending upon the age, weight and mammalian species
treated, the particular compounds employed, and the
specific use for which these compounds are employed.
The determination of effective dosage levels, that is
the dosage levels necessary to achieve the desired
result, will be within the ambit of one skilled in the
art. Typically, human clinical applications of products
are commenced at lower dosage levels, with dosage level
being increased until the desired effect is achieved.
In non-human animal studies, applications of products
are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved
or adverse side effects disappear.
` ~94t23068 ~ PCT~S94/03795
~3
37
The dosage for the products of the present
- invention can range broadly depending upon the desired
affects and the therapeutic indication. Typically,
dosages may be between about 10 ~g/kg and 100 mg/kg body
weight, preferably between about 100 ~g/kg and 10 mg/kg
body weight. Administration is preferably oral on a
daily basis.
Other embodiments are within the following
claims.
