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WO1994023068A1 - Procede de triage concernant des agonistes de recepteurs - Google Patents

Procede de triage concernant des agonistes de recepteurs Download PDF

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Publication number
WO1994023068A1
WO1994023068A1 PCT/US1994/003795 US9403795W WO9423068A1 WO 1994023068 A1 WO1994023068 A1 WO 1994023068A1 US 9403795 W US9403795 W US 9403795W WO 9423068 A1 WO9423068 A1 WO 9423068A1
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Prior art keywords
receptor
promoter
cell
region
agonist
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PCT/US1994/003795
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English (en)
Inventor
Ronald P. Mcdonnell
Maty Tzukerman
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Ligand Pharmaceuticals, Incorporated
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Application filed by Ligand Pharmaceuticals, Incorporated filed Critical Ligand Pharmaceuticals, Incorporated
Priority to AU65561/94A priority Critical patent/AU6556194A/en
Priority to EP94913374A priority patent/EP0694078A4/fr
Priority to JP6522489A priority patent/JPH08512197A/ja
Priority to BR9406616A priority patent/BR9406616A/pt
Publication of WO1994023068A1 publication Critical patent/WO1994023068A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention relates to methods and constructs useful for identifying or screening agonists active at cell receptors, such as at hormone receptors, e.g. , the estrogen receptor.
  • 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.
  • 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.
  • a hormone response element e.g. , a promoter
  • 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.
  • 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.
  • 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.
  • TAF1 and TAF2 vary depending upon the responsive promoter and cell type and, in some cases, both are required for full transcriptional stimulation.
  • TAF1 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.
  • functional context is meant that only a few amino acids (i.e., up to 10) , or preferably only (i.e. 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.
  • 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 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 TAFl 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.
  • mutations are introduced into the TAF2 region which make the TAF2 nonfunctional, while allowing TAFl to exhibit its functional activity.
  • 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.
  • an assay to screen for an agonist at a receptor which interacts with only one of the TAFl 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.
  • 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.
  • a modified receptor can be used to identify promoter- and cell-type specific requirements for TAFl or TAF2 activity of a receptor. Experiments can now be performed to determine such promoter- and cell-type specific differences in activi-ty. 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 TAFl 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 TAFl action. Such preparation may be recruitment of basic transcription factors, alteration of chromatin structure, or causing removal of a transcriptional repressor.
  • 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 TAFl region is unable to access the transcription apparatus until the TAF2 region has acted appropriately to prepare it for TAFl action.
  • cell specificity for TAFl or TAF2 activity may reflect the presence or absence of a TAFl or TAF2-type function in a cell that mimics the presence of TAFl or TAF2, respectively.
  • a mimetic in a cell will allow a receptor construct having a mutated and inactive TAFl 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 TAFl or TAF2 in any particular cell, dependent on the functionalities present in those promoters.
  • a cell is provided with a specific receptor construct having a selected TAFl and/or TAF2 activity, and having a suitable response element (e.g. , containing a promoter region) linked to an operative reporter gene (e.g. , encoding an enzyme activity which is readily detectable) .
  • a suitable response element e.g. , containing a promoter region
  • an operative reporter gene e.g. , 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.
  • 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.
  • 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 TAFl and a TAF2 region.
  • 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
  • the cell is chosen such that no, or minimal, transcription of the promoter occurs in the presence of a receptor having only an un utated TAF region corresponding to that mutated above (and not the other TAF region) .
  • the cell is also chosen such that transcription occurs in the presence of a receptor having the above nonmutated region alone.
  • 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.
  • 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.
  • 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.
  • 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.
  • the agonist is a human hormone agonist
  • a nuclear receptor e.g.. a human hormone receptor is encoded by the nucleic acid within the cell.
  • 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.
  • 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 TAFl region present in the cell, but there is transcriptional activity with a receptor having an operative TAFl region alone available.
  • 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.
  • 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.
  • a liver cell e.g.. HepG2
  • a complex C3 promoter together provide a useful assay for agonists active at a TAFl region only.
  • 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 TAFl region, the promoter is active. Thus, agonists active at the receptor TAFl region can be identified as those which cause expression of the reporter gene.
  • 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.
  • the invention features a method for treating or preventing an estrogen related disease or condition.
  • estrogen related disease is meant a disease that is caused or associated with an elevated or depressed level of the hormone estrogen.
  • hormone is meant a naturally occurring biochemical that will function as a receptor agonist. Synthetic hormones are more properly referred to as agonists.
  • estrogen related diseases include osteoporosis, breast cancer, uterine cancer, and endo etriosis.
  • Examples of estrogen related conditions include vasomotor abnormalities, hot flashes, depression, other psychiatric abnormalities and uterine fibroids.
  • the patient may be unable to produce estrogen in an amount required by the body.
  • 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 hormone 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) .
  • keoxifene- like transcriptional profile is meant the production of a normalized response similar to keoxifene. This profile demonstrates a relatively low TAFl 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) TAFl response than with a wild type receptor at higher concentrations (about 10 '7 M) , see Fig. 8E compared to Figs.
  • 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.
  • uterine/breast sparing activity is meant the prevention or reduction of the proliferation of tumorous cancer cells which can be measured by standard techniques.
  • 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 0-10 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.
  • 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.
  • Fig. 1 Transcription Activation by ER-wt (wild type estrogen receptor) and Truncated ER Mutants. Schematic organization of ER-wt, ERN282G and ER179C (A) .
  • CV1 (B) HepG2 (C) and HS57-8T (D) cells, 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-jS-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 "7 M 17- -estradiol (E 2 ) 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-/3-gal expression vector.
  • Carrier DNA was added to 20 ⁇ g total DNA.
  • 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.
  • a minus receptor control was included.
  • Cultures were treated with or without 10 "7 M 17-3-estradiol (E 2 ) 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-TAF1 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 pA 2 -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' 7 M jS-estradiol (E 2 ) for 36 hours and assayed for luciferase activity. Data presentation is described in Fig. 1.
  • Fig. 5 Activation of ER-TAF1 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 "7 M of 17-j8-estradiol (A) , E 2 , Tamoxifen (B) , 4-hydroxy- Tamoxifen (C) , Nafoxidine (D) , Clomiphene (E) , for 36 hours and assayed for luciferase activity.
  • Data presentation was described in Fig. 1.
  • ER represents Er-wt
  • ER" 1 represents Er-TAFl
  • TAF2 represents Er-179c
  • TAF2 m 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 4°C with 5 nM of 3 H-17-j8-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 TAFl and TAF2 as Functionally Dependent Activators of Transcription.
  • 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.
  • B the TAF2 region of the receptor interacts with a transcriptional repressor (I) , displacing it or altering its structure (C) to permit the TAFl activation sequence access to the general transcription apparatus (GTA) .
  • GTA general transcription apparatus
  • TAF2 function of the receptor can be supplied by other transcription factors, allowing TAFl 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.
  • the partial agonist activity of the triphenylethylene-derived estrogen agonists depends on the conformation induced by the ligand and the effect that this conformation has on the presentation of TAFl to the transcription apparatus.
  • Fig. 8 shows that the partial agonist activities of the triphenylethylene derived antiestrogens depends on TAFl 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-j8-gal and pGem4 as carrier DNA to a total amount of 20 ⁇ g.
  • Cultures were treated with various concentrations of 17-0-estradiol (A) , Clomiphene (B) , Nafoxidine (C) , 4-OH-Tamoxifen (D) and keoxifene (E) , for 36 hours and assayed for luciferase activity.
  • 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%.
  • TRAP + MNC tartrate acid phosphatase resistant multinucleated cells
  • Estrogen agonist activity of keoxifene (keox) on MCF-7 cell proliferation The activity of estrogen in this assay is maximum at 10 *10M , and induces proliferation to 1500% of control.
  • Fig. 11 is a diagram showing pC3-LUC.
  • 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 TAFl 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.
  • Estrogen Receptor 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) .
  • progesterone glucocorticoid
  • androgen and mineralocorticoid receptors
  • 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.
  • Estrogen Receptor can be readily used in equivalent procedures within the scope of the claims.
  • 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 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 may be 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) .
  • TAFl a sequence at the amino terminus of the receptor
  • TAF2 a sequence confined to the carboxyl 60 amino acids
  • AF domains e.g. Cavailles et al., J. of Cellular Bio.. 341, 1994
  • 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 Bfrl and SacI .
  • the DNA encoding the TAFl receptor derivative was excised from the plasmid YePERN282G using Bfrl 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 polymera ⁇ e (Boehringer Mannheim Co.) and ligated into the unique EcoRV site within the mammalian expression vector pRST7 (Berger et al., 41 J. Steroid Bioche . Mol. Biol. 733, 1992) .
  • Receptor mutations The wild type estrogen receptor cDNA was cloned into pGEM-HZf(+) (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 mutagenesi ⁇ (Kunkel et al., 154 Methods in
  • 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 Ba Hl 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.
  • pA2-LUC contains a 835 bp fragment (-821 to +14) of the Xenopus vitellogenin A2 gene promoter (Vik et al., 30
  • 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).
  • 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 (5xl0 3 cells/well) , in phenol red-free DMEM containing 10% FBS. DNA was introduced into cells using calcium phosphate e-e—precipitation. Plasmid DNA was diluted in 1 ml of 1 mM Tris, pH 7.4, 0.1 mM EDTA, 0.25 M CaCl 2 . 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 Na 2 HP0 4 ) and precipitates were allowed to form for 20 minutes.
  • 2X HBS pH 6.9 280 mM NaCl, 50 mM HEPES, 1.5 mM Na 2 HP0 4
  • 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.
  • PBS phosphate-buffered saline
  • yeast receptor proteins Expression vectors producing ER-TAFl were constructed by replacing the Bfrl-Mlul fragment of YEpElO (Pham et al., 88 Proc. Natl. Acad. Sci. USA 3125, 1991) with the corresponding fragment of pRST7ER-TAFl. 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) .
  • 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.
  • TAFl ER179C
  • TAF2G TAF2 activation sequence.
  • These constructs encode proteins structurally similar to those used previously in mammalian (Berry et al., 9 EMBO J. 2811, 1990) and yeast cells (Pham et al., 6 Mol. Endo. 1043, 1992) .
  • ERE vitellogenin estrogen response element
  • ERE-tk-LUC thymidine kinase promoter
  • 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.
  • 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 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.
  • ER179C was about 35% as active as ER-wt (Fig. 2C)
  • CV-1 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.
  • TAFl 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, 1990, and Tasset et al., 62 Cell 1177, 1990).
  • ER responsive promoter e.g. 9 EMBO J. 2811, 1990, and Tasset et al., 62 Cell 1177, 1990.
  • the activity of the TAFl activator when analyzed in the context of a full length receptor molecule was different from that when analyzed as a deletion mutant (ERN282G, Fig. 2) .
  • TAFl activity does not function independently, but rather requires additional carboxyl-terminal sequences for appropriate function.
  • 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.
  • ER TAF domains in ER function were 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 Regulation of ER-TAFl and ER179C Activity by Estrogen Receptor Agonists
  • triphenylethylene-derived estrogen receptor antagonists i.e. , tamoxifen, nafoxidine
  • TAFl or TAF2 transactivator ⁇ 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) .
  • the ER-TAFl activator functions very well on the complex C3 promoter, but less well on the other promoters examined.
  • the activity profiles of ER-TAFl and ER179C are clearly distinct, suggesting dissimilar mechanisms of action.
  • 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 TAFl is as good a transcriptional activator as ER-wt.
  • TAF2 alone does not activate transcription, it suggests that no mimetic for TAFl exists for transcription of this promoter in this particular cell line.
  • TAF2 is the dominant transcriptional activator and TAF2 is a transcriptional facilitator
  • TAF2 is a transcriptional facilitator
  • TAF2 has the effect of mimicking the function of TAF2, and that in mammalian cells TAF2 has a similar role.
  • TAFl 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 TAFl is able to interact with the transcriptional apparatus.
  • Example 5 Screening For and Use Of Compounds With
  • Keoxifene Like Transcriptional Profiles
  • the tri-phenylethylene derived anti- estrogen keoxifene exhibits bone sparing activity while having no significant effects on uterine proliferation.
  • tamoxifen a related anti-estrogen
  • tamoxifen is bone sparing but functions as a partial estrogen agonist in the uterus promoting an undesirable proliferative effect.
  • 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.
  • 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.
  • Rats are given a dorsal ovariecto y 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 ⁇ l. 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.
  • 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 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 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 maximum at 10' , ⁇ M, and induce proliferation to 1500% of control.
  • Agonists and their type can be quickly identified in the above systems.
  • the experiment described in Example 4, and illustrated in Fig. 5, is useful to identify an agonist and then define its type of activity.
  • 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.
  • desired agonist activity e.g.. agonists active only at TAFl regions which mimic the activity of estrogen and are useful for treatment of osteoporosis.
  • 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 Remington'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.
  • sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449.
  • antioxidants and suspending agents may be used. Id.
  • 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.
  • 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.g.. liposomes) may be utilized.
  • 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.
  • 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 .in vivo, ordinarily in a mammal, preferably in a human, or .in vitro.
  • 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.
  • parenterally intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms.
  • 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.
  • human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
  • 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.
  • the dosage for the products of the present invention can range broadly depending upon the desired affects and the therapeutic indication.
  • 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.

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Abstract

On décrit un procédé de triage qui concerne un agoniste de récepteur et consiste à utiliser un acide nucléique codant un récepteur doté d'une première région TAF, qui peut activer une transcription à partir d'un promoteur, et d'une deuxième région TAF mutée pour disposer du contexte fonctionnel de la deuxième région TAF mais incapable par elle-même d'activer la transcription du promoteur. Cet acide nucléique est placé dans une cellule incapable de transcription à partir du promoteur en présence de cette seule deuxième région TAF mais capable de transcription à partir du promoteur en présence de la première région TAF. Cette cellule comporte aussi un produit d'assemblage reporter contenant le promoteur, qui est transcrit quand ce promoteur est activé en présence de la première région TAF. Ce procédé consiste en outre à mettre la cellule en contact avec un agoniste potentiel dans des conditions telles que ce contact de la cellule avec un agoniste connu du récepteur entraîne la transcription à partir du promoteur et accroît le niveau du produit provenant du produit d'assemblage reporter. Ce procédé consiste enfin à mesurer le niveau d'accroissement du produit provenant de ce produit d'assemblage pour obtenir une indication de l'activité agoniste de l'agoniste potentiel.
PCT/US1994/003795 1993-04-07 1994-04-06 Procede de triage concernant des agonistes de recepteurs WO1994023068A1 (fr)

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AU65561/94A AU6556194A (en) 1993-04-07 1994-04-06 Method for screening for receptor agonists
EP94913374A EP0694078A4 (fr) 1993-04-07 1994-04-06 Procede de triage concernant des agonistes de recepteurs
JP6522489A JPH08512197A (ja) 1993-04-07 1994-04-06 受容体アゴニストのスクリーニング方法
BR9406616A BR9406616A (pt) 1993-04-07 1994-04-06 Método para triagem de agonistas receptores

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US08/179,750 1994-01-10
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996041013A1 (fr) * 1995-06-07 1996-12-19 Ligand Pharmaceuticals Incorporated Procedes de depistage d'agonistes et d'antagonistes de recepteurs
US5728534A (en) * 1996-07-19 1998-03-17 New England Medical Center Hospitals, Inc. Methods for identifying cardiovascular therapeutic agents
EP0777476A4 (fr) * 1994-08-22 1999-06-23 Lilly Co Eli Procedes enrayant le cancer de l'uterus
US5972881A (en) * 1995-09-18 1999-10-26 Ligand Pharmaceuticals Incorporated Treating NIDDM with RXR agonists
US6692928B1 (en) 1996-07-19 2004-02-17 New England Medical Center Hospitals, Inc. Methods for identifying cardiovascular agents
WO2004018041A1 (fr) 2002-08-08 2004-03-04 Laboratoires Fournier S.A. Utilisation d'un agoniste de ppar alpha pour traiter une prise de poids liee a un traitement par les agonistes de ppar gamma
US7084171B2 (en) 1997-04-11 2006-08-01 Neorx Corporation Compounds and therapies for the prevention of vascular and non-vascular pathologies
US7094550B2 (en) 1993-05-13 2006-08-22 Neorx Corporation Method to determine TGF-beta
EP1741446A2 (fr) 2000-01-21 2007-01-10 Novartis AG Combinaisons à base d'inhibiteurs de DPP-IV et d'antidiabetiques
WO2007033266A2 (fr) 2005-09-14 2007-03-22 Takeda Pharmaceutical Company Limited Administration d'inhibiteurs de dipeptidyl peptidase
US7259188B2 (en) 1998-06-12 2007-08-21 Llgand Pharmaceuticals Incorporated Methods and pharmaceutical compositions for treatment of anti-estrogen resistant breast cancer using RXR modulators
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method
US7727980B2 (en) 2001-02-23 2010-06-01 Ligand Pharmaceuticals Incorporated Tricyclic androgen receptor modulator compounds and methods
US7816372B2 (en) 2003-08-22 2010-10-19 Ligand Pharmaceuticals Incorporated 6-cycloamino-2-quinolinone derivatives as androgen receptor modulator compounds
WO2011041293A1 (fr) 2009-09-30 2011-04-07 Takeda Pharmaceutical Company Limited Dérivés pyrazolo [1, 5—a] pyrimidines comme inhibiteurs de kinase 1 régulatrice de signal d'apoptose
WO2011097079A1 (fr) 2010-02-03 2011-08-11 Takeda Pharmaceutical Company Limited Inhibiteurs de kinase 1 régulant le signal d'apoptose
WO2015059632A1 (fr) 2013-10-23 2015-04-30 Acadia Pharmaceuticals Inc. Traitement d'une maladie ou un trouble neurodégénératif
WO2020112889A2 (fr) 2018-11-26 2020-06-04 Denali Therapeutics Inc. Procédés de traitement du métabolisme lipidique dérégulé

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US5071773A (en) * 1986-10-24 1991-12-10 The Salk Institute For Biological Studies Hormone receptor-related bioassays

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EMBO Journal, Volume 11, issued 1992, P.S. DANIELIAN et al., "Identification of a Conserved Region Required for Hormone Dependent Transcriptional Activation by Steroid Hormone Receptors", pages 1025-1033, see entire document. *
Molecular and Cellular Endocrinology, Volume 34, issued 1985, J. SIMARD et al., "Keoxifene Shows Pure Antiestrogen Activity in Pituitary Gonadotrophs", pages 141-144, see entire document. *
See also references of EP0694078A4 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7094550B2 (en) 1993-05-13 2006-08-22 Neorx Corporation Method to determine TGF-beta
EP0777476A4 (fr) * 1994-08-22 1999-06-23 Lilly Co Eli Procedes enrayant le cancer de l'uterus
WO1996041013A1 (fr) * 1995-06-07 1996-12-19 Ligand Pharmaceuticals Incorporated Procedes de depistage d'agonistes et d'antagonistes de recepteurs
US6316404B1 (en) 1995-09-18 2001-11-13 Ligand Pharmaceuticals Incorporated Treating NIDDM with RXR agonists
US6028052A (en) * 1995-09-18 2000-02-22 Ligand Pharmaceuticals Incorporated Treating NIDDM with RXR agonists
US6228862B1 (en) 1995-09-18 2001-05-08 Ligand Pharmaceuticals Incorporated Treating NIDDM with RXR agonists
US5972881A (en) * 1995-09-18 1999-10-26 Ligand Pharmaceuticals Incorporated Treating NIDDM with RXR agonists
US6521633B2 (en) 1995-09-18 2003-02-18 Ligand Pharmaceuticals Incorporated Treating NIDDM with RXR agonists
US6448019B1 (en) 1996-07-19 2002-09-10 New England Medical Center Hospitals, Inc. Methods for identifying vasoprotective agents
US6692928B1 (en) 1996-07-19 2004-02-17 New England Medical Center Hospitals, Inc. Methods for identifying cardiovascular agents
US5728534A (en) * 1996-07-19 1998-03-17 New England Medical Center Hospitals, Inc. Methods for identifying cardiovascular therapeutic agents
US7511070B2 (en) 1997-04-11 2009-03-31 Poniard Pharmaceuticals, Inc. Compounds and therapies for the prevention of vascular and non-vascular pathologies
US7084171B2 (en) 1997-04-11 2006-08-01 Neorx Corporation Compounds and therapies for the prevention of vascular and non-vascular pathologies
US7259188B2 (en) 1998-06-12 2007-08-21 Llgand Pharmaceuticals Incorporated Methods and pharmaceutical compositions for treatment of anti-estrogen resistant breast cancer using RXR modulators
EP1743655A1 (fr) 2000-01-21 2007-01-17 Novartis AG Combinaisons à base d'inhibiteurs de DPP-IV et d'antidiabétiques
EP1741446A2 (fr) 2000-01-21 2007-01-10 Novartis AG Combinaisons à base d'inhibiteurs de DPP-IV et d'antidiabetiques
US7727980B2 (en) 2001-02-23 2010-06-01 Ligand Pharmaceuticals Incorporated Tricyclic androgen receptor modulator compounds and methods
US7625410B2 (en) 2001-05-02 2009-12-01 Boston Scientific Scimed, Inc. Stent device and method
WO2004018041A1 (fr) 2002-08-08 2004-03-04 Laboratoires Fournier S.A. Utilisation d'un agoniste de ppar alpha pour traiter une prise de poids liee a un traitement par les agonistes de ppar gamma
US7816372B2 (en) 2003-08-22 2010-10-19 Ligand Pharmaceuticals Incorporated 6-cycloamino-2-quinolinone derivatives as androgen receptor modulator compounds
WO2007033266A2 (fr) 2005-09-14 2007-03-22 Takeda Pharmaceutical Company Limited Administration d'inhibiteurs de dipeptidyl peptidase
WO2011041293A1 (fr) 2009-09-30 2011-04-07 Takeda Pharmaceutical Company Limited Dérivés pyrazolo [1, 5—a] pyrimidines comme inhibiteurs de kinase 1 régulatrice de signal d'apoptose
WO2011097079A1 (fr) 2010-02-03 2011-08-11 Takeda Pharmaceutical Company Limited Inhibiteurs de kinase 1 régulant le signal d'apoptose
WO2015059632A1 (fr) 2013-10-23 2015-04-30 Acadia Pharmaceuticals Inc. Traitement d'une maladie ou un trouble neurodégénératif
WO2020112889A2 (fr) 2018-11-26 2020-06-04 Denali Therapeutics Inc. Procédés de traitement du métabolisme lipidique dérégulé

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EP0694078A4 (fr) 1997-06-25
AU6556194A (en) 1994-10-24
CA2160135A1 (fr) 1994-10-13
JPH08512197A (ja) 1996-12-24
EP0694078A1 (fr) 1996-01-31

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