[go: up one dir, main page]

WO1998037053A1 - Identification d'un recepteur unique specifique d'un retinoide particulier induisant l'apoptose cellulaire - Google Patents

Identification d'un recepteur unique specifique d'un retinoide particulier induisant l'apoptose cellulaire Download PDF

Info

Publication number
WO1998037053A1
WO1998037053A1 PCT/US1998/002911 US9802911W WO9837053A1 WO 1998037053 A1 WO1998037053 A1 WO 1998037053A1 US 9802911 W US9802911 W US 9802911W WO 9837053 A1 WO9837053 A1 WO 9837053A1
Authority
WO
WIPO (PCT)
Prior art keywords
receptor
adamantyl
apoptosis
binding
ahpn
Prior art date
Application number
PCT/US1998/002911
Other languages
English (en)
Inventor
Joseph A. Fontana
Braham Shroot
Philippe Nedoncelle
Original Assignee
Galderma Research & Development, S.N.C.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Galderma Research & Development, S.N.C. filed Critical Galderma Research & Development, S.N.C.
Priority to JP53675298A priority Critical patent/JP2001517211A/ja
Priority to AU66561/98A priority patent/AU746452B2/en
Priority to EP98908556A priority patent/EP0986533A4/fr
Priority to CA002281523A priority patent/CA2281523A1/fr
Publication of WO1998037053A1 publication Critical patent/WO1998037053A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/105—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic
    • C07C65/11—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic with carboxyl groups on a condensed ring system containing two rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings

Definitions

  • This invention relates to a novel receptor, found in both nuclear and cytoplasmic cell extracts which binds to a retinoid that induces apoptosis.
  • This receptor is believed to be involved in apoptosis. Therefore, it should be useful as a screening agent for identifying other ligands which induce apoptosis.
  • Retinoids are defined as substances that can elicit specific biological responses by binding to and activating a specific receptor or set of receptors. Retinoids are known to play a fundamental role in normal cell growth and differentiation. (Roberts, A.B. et al, in "The Retinoids,” ed. by M.B. Sporn, A.B. Roberts and D. S. Goodman, Vol. 2, pp. 209-256, Academic Press, Oi ndo, Fla., (1984); Sporn, M.B. et al, J. Amer. Acad. Dermatol, 15:756- 764 (1986)).
  • Retinoic acid receptors mediate gene transcription through a variety of mechanisms. These nuclear receptors can bind to specific DNA consensus sequences termed retinoid receptor response elements (RAREs or RXREs) which are located in the regulatory regions of the retinoid target genes (Gudas, L.J., Cell Growth Differ., 3:655-662 (1992); Lohnes et al, Cell Scl, 16 (Suppl.):69-76 (1992)). Nuclear receptor binding to these response elements preferably occurs through heterodimer formation between the RAR and RXR, although homodimer binding and subsequent gene activation has also been found (Hermann et al, Mol.
  • RAREs retinoid receptor response elements
  • the RXRs can mediate gene transcription via heterodimer formation with the RARs, with the vitamin D, thyroid hormone (Yu et al, Cell, 67:1251-1266
  • the retinoid receptor response elements usually consist of direct repeats (DRs) in which the half-sites are separated by a number of base pair spacers. Selectivity for binding appears to be determined by the number of base pairs utilized as spacers, as well as by the sequence of the response element itself (Kim et al, Mol. Endocrinol, 6:1489-1501 (1992); Mader et al, J. Biol. Chem., 268:591-600 (1993)). RAR and RXR inhibition of AP-1 -mediated gene transcription that does not require RAR or RXR binding to DNA has also been observed (Pfahl, Endocrin.
  • DRs direct repeats
  • RAR homologous recombination studies have suggested that RAR functional redundancy exists among the different RARs (Li et al, Proc. Natl. Acad. Scl, USA, 90:1590-1594 (1993); Lohnes et al, Cell, 73: 643-658 (1993); Lufkin et al, Proc. Natl. Acad. Scl, USA, 90:7225-7229 (1993)).
  • the various receptor subtypes possess distinct functions and may indeed modulate the activity of distinct genes (Nagpal et al, Cell, 70:1007-1019 (1992); Boylan et al, Mol. Cell BioL, 15:843-851 (1995)).
  • RARs bind both RA and its isomer 9-cis-RA, while the RXRs only bind 9-cis-RA (Allenby et al, J. Biol. Chem., 269: 16689-16695 (1995), and references cited therein).
  • conformationally restricted retinoids have been synthesized that selectively bind to and enhance transcriptional activation by selective RAR and RXR subtypes (Graupner et al, Biochem. Biophys. Res.
  • retinoids that selectively transactivate RAR ⁇ (Bernard et al, Bio- chem. Biophys. Res. Comm., 186(2):977-983 (1992)). Because of the ability of retinoids to affect cell growth and differentiation, these compounds have been disclosed to be useful for the treatment or prevention of diseases and conditions involving abnormal cell proliferation and differentiation. For example, the usage of retinoids as efficient therapeu- tics for the treatment of various skin diseases and neoplasms has been reported (Roberts, A. B.
  • retinoid therapy has typically been achiev- ed with a regimen which combines retinoid administration with the administration of other differentiation or cytotoxic agents.
  • isotretoin 13-cis-retinoic acid
  • etretinate have been used, as well as 9-cis retinoic acid and N-(9-hydroxyphenyl)retinoid.
  • retinoids have been reported to be useful for the treatment of a variety of dermatoses including psoriasis, cystic acne, cutaneous disorders of keratinazation, among others.
  • retinoids have important potential as anti-cancer agents.
  • retinoid compounds have been disclosed to have potential for the prevention of skin cancer, for the treatment of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) for the treatment of other hematopoietic malignancies such as myelodysplastic syndrome, juvenile chronic myelogenous leukemia, Sezary syndrome, squa- mous cell carcinomas of the upper aerodigestive tract, non-small lung cancer, and human head and neck carcinomas.
  • AML acute myeloid leukemia
  • APL acute promyelocytic leukemia
  • other hematopoietic malignancies such as myelodysplastic syndrome, juvenile chronic myelogenous leukemia, Sezary syndrome, squa- mous cell carcinomas of the upper aerodigestive tract, non-small lung cancer, and human head and neck carcinomas.
  • AHPN a specific adamantyl retinoid derivative 6-(3-(l-adamantyl)-4-hydroxyphenyl)-2-naphthoic acid
  • retinoids have been reported to have potential as anticancer agents, and specifically for inducing apoptosis
  • the identification of retinoids having improved therapeutic properties which are suitable for the treatment or prevention of cancer would be highly beneficial.
  • the identification of retinoids which induce apoptosis would be highly beneficial.
  • the identification of receptor(s) involved in the induction of apoptosis e.g., the receptor bound by AHPN, would be highly useful as a screening agent for selecting other apoptosis inducing compounds.
  • novel receptor it is another object of the invention to use such novel receptor as a screening agent, e.g., in competition binding assays, to select other receptors that induce apoptosis. It is another object of the invention to provide a nucleic acid sequence encoding such novel receptor.
  • the present invention is directed to a novel receptor, which is not of the RAR or RXR type that is believed to be involved in apoptosis that binds to AHPN.
  • This receptor is apparently expressed in both the nuclear and cytoplasmic fractions of cells.
  • This receptor has an apparent molecular weight of about 90 kDa. Binding studies, and this apparent molecular weight, the results of which are discussed in detail infra, strongly suggest that this receptor is not of the RAR or RXR type, and is of a highly unique nature.
  • This receptor specifically binds to AHPN, an adaman- tyl retinoid compound shown to induce apoptosis in a variety of cell types, many of which are resistant to the proliferative and/or differentiating effects of retinoids.
  • the binding of AHPN to this receptor is only minimally displaced by all-trans retinoic acid in a competitive binding assay.
  • a receptor that specifically binds AHPN is intended to refer to a receptor that binds AHPN, wherein the binding of
  • AHPN to such receptor is substantially unaffected by compounds that do not specifically bind to such receptor.
  • “Substantially unaffected” preferably means that at least 75% of the bound AHPN compound remains bound in the presence of an excess of a compound that does not specifically bind such receptor, more preferably at least 90% remains bound and most preferably in excess of 95% remains bound.
  • a compound which only "minimally displaces" the binding of a compound to a receptor e.g., the minimal displacement of AHPN to its receptor by the addition of an excess of all-trans retinoic acid means that at most about 25% of bound compound, e.g., AHPN is displaced, more preferably at most about 10% is displaced and most preferably 5% or less of the bound compound, e.g., AHPN, is displaced from its receptor in a competitive binding assay.
  • the AHPN compound also binds to the RAR ⁇ receptor. Consequent- ly, this receptor should be well suited as a screening agent to select other ligands that induce apoptosis. Also, the corresponding DNA may be used to identify related receptors. These receptors will also provide useful screening agents for selecting ligands that induce apoptosis.
  • the present invention is directed to the use of AHPN and radioisotopically labeled forms thereof as a screening agent to assess qualita- tively and/or quantitatively the expression of the subject novel receptor by selected cells, e.g., tumor cell lines or other cells for which the modulation of apoptosis is therapeutically desirable.
  • Screen agents will be useful in both in vitro and in vivo assays for selecting target cells susceptible to apoptosis.
  • the present invention is directed to a novel means of synthesizing radioisotopically labeled derivatives of AHPN as well as the novel intermediates and radioisotopically labeled derivatives produced by such methods. This method will, in general, comprise the following steps:
  • the present invention embraces to carbon 13 or carbon 14 containing AHPN derivatives, and the use thereof as screening agents to identify other ligands that induce apoptosis.
  • These derivatives can be pro- prised by replacing the benzene ring or the carboxylic acid substituent of AHPN with a C 13 or C 14 labeled carbon dioxide or benzene.
  • these radioisotopically labeled forms are also useful in metabolism studies in in vitro and in vivo systems.
  • FIG. 1 Scatchard analysis of [ 3 H] 6-[3-(l-adamantyl)-4-methoxy phenyl]-2- naphthalenecarboxylic acid binding to MDA-MB-468 nuclear extracts. K D value is the mean of three separate experiments. Binding was performed as described in methodology section infra.
  • Fig. 2 Competitive binding between tRA (20 ⁇ M) or 6-[3-(l-adamantyl)-4- methoxy phenyl]-2-naphthalenecarboxylic acid (20 ⁇ M) for the [ 3 H] 6-
  • RARs and RXRs nuclear receptors termed RARs and RXRs.
  • RAREs, RXREs specific consensus sequences located in the regulatory regions of genes modulating their expression.
  • a retinoid compound 6-[3-( 1 - adamantyl)-4-methoxy phenyl] -2-naphthalenecarboxylic acid (AHPN) which induces apoptosis in a variety of cell types, many of which display resistance to the antiproliferative and/or differentiating effects of retinoids.
  • AHPN retinoid compound 6-[3-( 1 - adamantyl)-4-methoxy phenyl] -2-naphthalenecarboxylic acid
  • the present invention relates to the discovery that this retinoid binds to a unique receptor which has been found in both the nuclear and cytoplasmic fractions of cells.
  • the 6-[3- (l-adamantyl)-4-methoxy phenyl]-2-naphthalenecarboxylic acid (AHPN) receptor differs from the RARs and RXRs in its ligand specificity. Binding of AHPN to its receptor, while minimally displaced by all trans retinoic acid
  • the AHPN compound was initially synthesized as a conformationally restricted retinoid which displayed selective binding to the RAR ⁇ receptor.
  • AHPN was observed to display a 3-fold higher kD for the receptor RAR ⁇ than all trans retinoic acid (tRA) and further displayed a 9-fold and 70-fold higher kD than tRA for the RAR ⁇ and RAR ⁇ receptors respectively.
  • tRA trans retinoic acid
  • AHPN was found to be a potent inducer of G, cell cycle arrest and apoptosis in breast carcinoma cells both resistant and sensitive to the antiproliferative action of tRA, (Shao, Z-M., Dawson, M.I.. Li, X.-S., Rishi, A.K., Sheikh, M.S., Han, Q.-X., Ordonex, J.V., Shroot, B., and Fontana, J.A. Oncogene, 11, 493-504 (1995)).
  • 6-[3-( 1 -adamantyl)-4-methoxy phenyl] -2-naphthalenecarboxylic acid is significantly less potent than tRA in its ability to transactivate endoge- nous retinoid receptors on transfected ⁇ 2 RARE and (TRE) 3 _-TK reporter constructs in breast carcinoma cells both sensitive and resistant to the antiproliferative effects of tRA but in which 6-[3-(l-adamantyl)-4-methoxy phenyl]-2-naphthalenecarboxylic acid induces apoptosis (Shao, Z-M., Dawson, M.I..
  • MDA-MB-468 human breast carcinoma cells like the majority of estrogen receptor negative breast carcinoma cells, are refractory to the antiproliferative effects of tRA; do not possess RAR ⁇ ; express low levels of RAR ⁇ ; and only express moderate levels of RAR ⁇ . Also, they areakily sensitive to AHPN-mediated apoptosis.
  • the inventors thereupon examined [ 3 H]-6-[3-(l- adamantyl)-4-methoxy phenyl]-2-naphthalenecarboxylic acid binding to RAR ⁇ as well as RXR ⁇ and RXR ⁇ in vitro utilizing recombinant receptors. No binding of [ 3 H]-6-[3-(l-adamantyl)-4-methoxy phenyl]-2- naphthalenecarboxylic acid to the RXRs was detected while RAR ⁇ displayed a kD of 440nM (results not shown).
  • the RARs and RXRs possess molecular weights of 50kDa and 54kDa respectively.
  • Petkovich, M., Brand, N.J., Krust, A. and Chambon, P. Nature, 330, 444-450 (1987); Mangelsdorf, D.J., Ong. E.S., Dyck, J.A. and Evans, R.M. Nature, 345, 224-229 (1990) We further characterized the AHPN receptor as to its mass.
  • AHPN AHPN triggers apoptosis in a number of cell lines.
  • AHPN and isotopically labeled forms thereof should provide useful screening agents for identifying other receptors involved in apoptosis. Further, these compounds should be useful for the affinity purification of such receptors and for assaying the expression of such receptors by selected cell types (both quantitatively and qualitatively).
  • isotopically-labeled AHPN derivatives will include, in particular, AHPN substituted with radiolabels including deuterium, tritium, carbon 13 or carbon 14, iodine 125, indium 131 , yttrium 90, and other well known radiolabels used for diagnostic and/or therapeutic purposes.
  • radiolabels including deuterium, tritium, carbon 13 or carbon 14, iodine 125, indium 131 , yttrium 90, and other well known radiolabels used for diagnostic and/or therapeutic purposes.
  • the radiolabel will comprise deuterium or tritium, and most preferably tritium.
  • the AHPN can be mono-, di- or poly-isotopically-labeled. Preferably, it is di-isotopically-labeled.
  • the 6 (3-(l-Adamantyl)-4-hydroxyphenyl)-2-naphthoic acid com- pound of the present invention has the following formula:
  • AHPN AHPN
  • CD437 3-(l- adamantyl)-4-hydroxyphenyl)-2-naphthoic acid
  • the isotopically-labeled 6-[3-(l-adamantyl)-4-methoxy phenyl]-2- naphthalenecarboxylic acid used for screening will preferably comprise [ 3 H]- 6-[3-(l-adamantyl)-4-hydroxy-5-[ 3 H]-phenyl]-2-naphthalenecarboxylic acid having the following formula:
  • This isotopically-labeled AHPN derivative is referred to interchangeably in this application as [ 3 H] CD437 or [ 3 H]AHPN, or [ ⁇ ]-6-[3-(l-adamantyl)-4- hydroxy-5-[ 3 H]-phenyl]-2-naphthalenecarboxylic acid.
  • the radioisotopically labeled AHPN or CD437 derivatives defined above preferably will have a specific activity which is greater than 20Ci/mmole and more preferably at least equal to 50 Ci/mmole or approximately 1850 GBq/mmole.
  • the present invention also provides a novel method for the preparation of the above-identified isotopically-labeled compounds. This synthesis is depicted schematically in Figure 4. This method is principally characterized by the fact that a mono-, di- or poly- halogenated alkyl ester of 6-[3-(l-adamantyl)-4-methoxy phenyl]-2- naphthalenecarboxylic acid is prepared (compound II).
  • the alkyl ester of 6-[3-(l-adamantyl)-4-methoxy phenyl]-2- naphthalenecarboxylic acid used in such reaction will preferably comprise the methyl ester.
  • This compound may be prepared by known methods, preferably as described in Example 9 of US 4,717,720.
  • the halogenated derivative used as the starting material will preferably comprise a brominated derivative. However, other halogen derivatives should be suitable also.
  • the present invention also relates to the intermediate compounds of the process as described above.
  • the reduction of the halogenated derivative is preferably carried out at room temperature and under ambient pressure, for example, at 15-30°C under a pressure of 1 bar.
  • ambient pressure for example, at 15-30°C under a pressure of 1 bar.
  • these methods can be varied using known procedures.
  • the present invention also embraces the production of C 13 or C 14 isotopically labeled derivatives of AHPN.
  • Isotopically-labeled AHPN substituted with carbon 13 or carbon 14 can be obtained by replacing the benzene ring or the carboxylic acid. The replacement of such functional groups, through the use of reagents such as isotopically-labeled carbon dioxide or benzene, can be effected by well known methods.
  • the isotopically labeled compounds are useful screening agents. For example, they may be used to assess qualitatively and/or quantitatively the expression of the subject novel receptor (or related receptors) and/or RAR ⁇ on specific types of cells. Also, these labeled compounds may be used to identify related receptors. This is highly useful as such receptors potentially also will be involved in apoptosis.
  • these screening agents may be used in affinity assays for the affinitiy purification of the subject receptor, and in competitive binding assays to identify other ligands that induce apoptosis.
  • affinity assays for the affinitiy purification of the subject receptor
  • competitive binding assays to identify other ligands that induce apoptosis.
  • the isotopically-labeled 6-[3-( 1 -adamantyl)-4-methoxy phenyl]-2-naphthalenecarboxylic acid can be used in a competition binding assay against a ligand potentially to be tested for apoptosis activity.
  • the activity of the ligand on the new receptor and/or RAR ⁇ can be determined. This should correlate to the ability of such ligand to induce apoptosis.
  • one method of conducting such an assay comprises initially incubating the isotopically-labeled AHPN in a medium containing the subject receptor and/or RAR ⁇ until the isotopically- labeled AHPN becomes bound to the subject receptor and/or RAR ⁇ to the point of saturation. Thereafter, the bound receptor of the present invention and/or RAR ⁇ is washed and incubated with an excess (e.g., 200 times) of the ligand to be tested.
  • an excess e.g. 200 times
  • the relative selective activity of the ligand on the subject receptor and/or RAR ⁇ can be determined.
  • the saturation curve of the isotopically-labeled 6- [3-( 1 -adamantyl)-4-methoxy phenyl]-2-naphthalenecarboxylic acid to the subject receptor and/or RAR ⁇ has been previously determined, then the isotopically-labeled 6-[3-(l-adamantyl)-4-methoxy phenyl]-2- naphthalenecarboxylic acid and ligand can be incubated with the subject receptor and/or RAR ⁇ at the same time, and the degree of exclusion of the binding of the isotopically-labeled AHPN to the subject receptor and/or
  • RAR ⁇ used to measure the relative selective activity of the ligand on the new receptor and/or RAR ⁇ , if any.
  • the subject receptor and/or RAR ⁇ proteins used in the competition binding assays of the methods of the present invention will generally be recovered from cell lysates of an appropriate cell or cell culture, e.g., one which has been transfected with a recombinant plasmid capable of expressing the subject novel receptor and/or RAR ⁇ proteins.
  • any appropriate means of producing a sufficient quantity of the subject receptor and/or RAR ⁇ receptor to allow for the conducting of the identification methods of the present invention can be employed.
  • the specific binding of the isotopically-labeled AHPN derivative be determined by titrating the bound isotopically labeled AHPN against an excess quantity of the same compound in a non-labeled form.
  • the isotopically-labeled AHPN is first incubated with the subject receptor and/or RAR ⁇ to the point of saturated binding. An appropriate binding curve is then generated which shows total binding, both specific binding to the subject receptor and/or RAR ⁇ pocket, and nonspecific binding to other associated structures (e.g. lipids).
  • the subject receptor and/or RAR ⁇ bound with the isotopically-labeled AHPN is incubated with a large excess of the non-labeled version of the isotopically-labeled AHPN (e.g., 200 times or greater concentration).
  • a large excess of the non-labeled version of the isotopically-labeled AHPN e.g. 200 times or greater concentration.
  • Any isotopically-labeled AHPN that remains bound represents nonspecifically bound isotopically-labeled AHPN.
  • a binding curve can be generated which shows the total specific binding of the isotopically- labeled AHPN to the subject novel receptor and/or RAR ⁇ .
  • other ligands that preferably induce apoptosis comparably or even better than AHPN may be selected.
  • Another use of the isotopically-labeled AHPN derivatives provided herein are in metabolism studies. This may be effected using a mass spectrometer to locate metabolites of both the isotopically-labeled and non-labeled ligands.
  • a 1:1 mixture of a 13 CD 3 isotopically-labeled AHPN and a non-labeled AHPN are administered to an in vitro system (e.g., an appropriate cell line) or an in vivo system (e.g., an animal model, such as laboratory mice), followed by extraction of metabolites from the system (e.g., whole cell lysates or animal bodily fluids).
  • an in vitro system e.g., an appropriate cell line
  • an in vivo system e.g., an animal model, such as laboratory mice
  • metabolites e.g., whole cell lysates or animal bodily fluids.
  • the metabolites can then be identified using known methods.
  • the novel receptor which is disclosed herein, based on the fact it binds AHPN, a compound known to induce apoptosis, is believed to be involved in cell apoptosis. Therefore, this receptor should be useful for the identification of other ligands that induce apoptosis. As explained above, the identification of other ligands that induce apoptosis may be identified in competition binding assays such as are known in the art and described above. In general, these assays will evaluate whether a particular ligand significantly competes with AHPN or an isotopic labeled form thereof in binding the novel receptor disclosed herein.
  • a source of the subject receptor of sufficient purity and quantity to conduct the assay is required.
  • the purification of a suitable receptor containing extract is disclosed in the example which follows. It is expected that other mammalian cells, e.g., others for which
  • AHPN induces apoptosis, will also express this receptor.
  • Cells which express this receptor or similar receptors can be identified using radioisotopically labeled derivatives of AHPN as a radiomarker. These cells can then be utilized as an alternative source of receptor.
  • the subject receptor may be sequenced by known methods, and the sequence utilized to construct hybridization probe(s) in order to isolate the compounding nucleic acid sequence from a DNA library. Methods of purifying and sequencing receptors and constructing probes based on such sequences are well within the level of ordinary skill in the art. As discussed above, the subject receptor is believed to be unique relative to other known nuclear receptors, e.g., it is distinct from RAR and RXR receptors.
  • the DNA encoding such receptor should be useful as a hybridization probe, i.e, to identify DNAs encoding related receptors, which also may be involved in apoptosis. These hybridization assays will be conducted by known methods.
  • DNAs will exhibit a high level of sequence identity with the subject DNA, i.e., at least 50% of the residues will likely be conserved and preferably 80 - 90% of the residues, or more.
  • DNAs which hybridize under conditions of high stringency to a DNA encoding the subject receptor or a fragment thereof will be selected. These DNAs will be sequenc- ed using known methods and expressed by recombinant methods. The resultant expression products will also be useful in assays for selecting other ligands which induce apoptosis.
  • the compounds screened for apoptosis inducing activity with the subject receptor can be selected randomly, e.g., using random compound libraries.
  • the compounds screened for apoptosis activity will include retinoid compounds as numerous retinoid compounds have been synthesized and have been reported in the literature.
  • One particular class of retinoid compounds that desirably will be screened for putative apoptosis activity comprises adamantyl containing retinoid compounds.
  • the present Applicant has developed numerous novel adamantyl retinoid derivatives, some of which have already been shown to induce apoptosis. The selection of other putative retinoids having similar apoptosis inducing activity will be determined based on their binding to the novel receptor provided herein.
  • a preferred means of identifying such ligands will comprise a competitive binding assay using the radioisotopically labeled forms of AHPN provided herein. Also, once other ligands are identified, these ligands or radiolabeled forms thereof may also be used in competitive binding assays to identify other apoptosis inducing ligands.
  • Ligands that bind the subject receptor potentially should induce apoptosis. Therefore, they are potentially useful as therapeutics in methods wherein induction of apoptosis is therapeutically desirable.
  • these ligands may be used to selectively kill desired target cells, e.g., cancer cells.
  • desired target cells e.g., cancer cells.
  • a particular advantage is that these ligands can be used to selectively kill cells which are resistant to the antiproliferative and/or differentiating effects of other retinoids.
  • Such ligands will be administered by known methods, e.g., orally, topically, intravenously, intramuscularly, rectally, intranasally, transdermally, etc. These ligands will be provided in a pharmaceutically acceptable form, e.g., by the addition of pharmaceutically acceptable excipients and carriers.
  • a pharmaceutically acceptable form e.g., by the addition of pharmaceutically acceptable excipients and carriers.
  • Extract Nuclear and cytoplasmic extracts were prepared as previously describ- ed. (Ausbel, F.M.,. Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.G., and Struhl, K., (Eds.), Current Protocols in Molecular Biology, Volume 2, pp. 12.1.1-12.1.7, 1990)). In brief, cells are harvested, resuspend- ed in a hypotonic buffer of lOmM HEPES, pH 7.9, 1.5mM MgCl 2 , lOmM KCl, 0.2 mM PMSF, 0.5 mM DDT.
  • hypotonic buffer of lOmM HEPES, pH 7.9, 1.5mM MgCl 2 , lOmM KCl, 0.2 mM PMSF, 0.5 mM DDT.
  • the cells are then allowed to swell, are disrupted by homogenization and the nuclei harvested therefrom by differential centrifugation.
  • the resultant nuclear extract is then resuspended in a small volume of low-salt buffer (20mM HEPES pH 7.9, 25 % glycerol, 1.5 mM DTT).
  • the salt concentration is then increased in a drop wise fashion to 300 mM and the extracted nuclei dialyzed against 20mM HEPES, pH 7.9. 20 % glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF and 0.5 mM DTT.
  • the tritiated compound obtained in (c) (140 mCi) was then dissolved in 2 ml of DMF. 2 mg of sodium methanethiolate was added and the mixture heated at 100° C for 12 hours. This reaction did not proceed to completion. Accordingly, an additional 2 mg of sodium methanethiolate were added and the mixture reheated to 100°C for 12 hours. DMF was evaporated, and IN hydrochlorhydric acid was added thereto to produce an acidic reaction medium having a pH of 2. The reaction medium was then extracted with ethyl acetate and the organic phase separated therefrom. This organic phase was then dried over magnesium sulfate, and evaporated.
  • the obtained residue was chromatographed on silica by elution with methanol and dichloromethane.
  • the product was rechromatographed on a reverse phase silica by elution with acetonitrile-water-acetic acid.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention se rapporte à un nouveau récepteur qui n'est pas du type RAR, qui se lie à AHPN et qui semble être impliqué dans le phénomène d'apoptose. Ce récepteur peut servir à identifier des ligands qui induisent l'apoptose.
PCT/US1998/002911 1997-02-25 1998-02-25 Identification d'un recepteur unique specifique d'un retinoide particulier induisant l'apoptose cellulaire WO1998037053A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP53675298A JP2001517211A (ja) 1997-02-25 1998-02-25 細胞アポトーシスを誘導する特有なレチノイドに特異的な独創的レセプターの同定
AU66561/98A AU746452B2 (en) 1997-02-25 1998-02-25 Identification of a unique receptor specific for a particular retinoid inducing cellular apoptosis
EP98908556A EP0986533A4 (fr) 1997-02-25 1998-02-25 Identification d'un recepteur unique specifique d'un retinoide particulier induisant l'apoptose cellulaire
CA002281523A CA2281523A1 (fr) 1997-02-25 1998-02-25 Identification d'un recepteur unique specifique d'un retinoide particulier induisant l'apoptose cellulaire

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US3900297P 1997-02-25 1997-02-25
US60/039,002 1997-02-25

Publications (1)

Publication Number Publication Date
WO1998037053A1 true WO1998037053A1 (fr) 1998-08-27

Family

ID=21903128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/002911 WO1998037053A1 (fr) 1997-02-25 1998-02-25 Identification d'un recepteur unique specifique d'un retinoide particulier induisant l'apoptose cellulaire

Country Status (5)

Country Link
EP (1) EP0986533A4 (fr)
JP (1) JP2001517211A (fr)
AU (1) AU746452B2 (fr)
CA (1) CA2281523A1 (fr)
WO (1) WO1998037053A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003048101A1 (fr) * 2001-11-30 2003-06-12 The Burnham Institute Induction de l'apoptose dans les cellules cancereuses

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5073361A (en) * 1989-07-20 1991-12-17 Centre International De Recherches Dermatologiques Galderma (Cird Galderma Compound marked with tritium, its preparation and its use in the location of nuclear receptors of retinoids
US5149631A (en) * 1989-07-20 1992-09-22 Centre International De Recherches Dermatologiques Galderma (Cird Galderma) Compound marked with tritium, its preparation and its use in the location of nuclear receptors of retinoids

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5073361A (en) * 1989-07-20 1991-12-17 Centre International De Recherches Dermatologiques Galderma (Cird Galderma Compound marked with tritium, its preparation and its use in the location of nuclear receptors of retinoids
US5149631A (en) * 1989-07-20 1992-09-22 Centre International De Recherches Dermatologiques Galderma (Cird Galderma) Compound marked with tritium, its preparation and its use in the location of nuclear receptors of retinoids

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP0986533A4 *
SHAO Z.-M., ET AL.: "P53 INDEPENDENT G0/G1 ARREST AND APOPTOSIS INDUCED BY A NOVEL RETINOID IN HUMAN BREAST CANCER CELLS.", ONCOGENE, NATURE PUBLISHING GROUP, GB, vol. 11., 1 January 1995 (1995-01-01), GB, pages 493 - 504., XP002910714, ISSN: 0950-9232 *

Also Published As

Publication number Publication date
AU6656198A (en) 1998-09-09
CA2281523A1 (fr) 1998-08-27
JP2001517211A (ja) 2001-10-02
EP0986533A1 (fr) 2000-03-22
AU746452B2 (en) 2002-05-02
EP0986533A4 (fr) 2001-05-23

Similar Documents

Publication Publication Date Title
Sun et al. Identification of receptor-selective retinoids that are potent inhibitors of the growth of human head and neck squamous cell carcinoma cells
Wu et al. Inhibition of trans-retinoic acid-resistant human breast cancer cell growth by retinoid X receptor-selective retinoids
Schüle et al. Retinoic acid is a negative regulator of AP-1-responsive genes.
Lehmann et al. RAR72 expression is regulated through a retinoic acid response element embedded in Sp1 sites
Duester et al. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis
EP0807624B1 (fr) Dérivé d'acide retinoique
Wang et al. Endogenous bile acids are ligands for the nuclear receptor FXR/BAR
Lehmann et al. Identification of retinoids with nuclear receptor subtype-selective activities
Jones et al. Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle
Hashimoto Retinobenzoic acids and nuclear retinoic acid receptors
JP2002504809A (ja) 核ホルモン受容体によって仲介される過程の修飾に有用な化合物、このような化合物の同定方法および使用
JPH09504111A (ja) Ｇａｌ４−受容体構築体の新規な使用
Beard et al. Synthesis and structure-activity relationships of stilbene retinoid analogs substituted with heteroaromatic carboxylic acids
US5707800A (en) Retinoic acid response elements and assays employing same
US5824484A (en) RXR homodimer formation
Harding et al. Retinoic acid activation and thyroid hormone repression of the human alcohol dehydrogenase gene ADH3.
Pignatello et al. Multiple factors contribute to the toxicity of the aromatic retinoid, TTNPB (Ro 13-7410): binding affinities and disposition
Sass et al. Metabolism of oral 9-cis-retinoic acid in the human. Identification of 9-cis-retinoyl-beta-glucuronide and 9-cis-4-oxo-retinoyl-beta-glucuronide as urinary metabolites.
AU746452B2 (en) Identification of a unique receptor specific for a particular retinoid inducing cellular apoptosis
Hofmann Retinoids—“Differentiation agents” for cancer treatment and prevention
DE69513689T2 (de) Trizyclische retinoidverbindungen verfahren zu ihrer herstellung und verwendung
US5196577A (en) Compound marked with tritium, its preparation and its use in particular in the determination of the affinity of retinoids for their nuclear receptors and their cytosolic binding protein
AU4582202A (en) Identification of a unique receptor specific for a particular retinoid inducing cellular apoptosis
Alam et al. Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity
WO1995033745A2 (fr) Nouveaux composes utiles pour moduler l'expression de genes sensibles aux retinoides et/ou presentant une activite anti ap-1

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2281523

Country of ref document: CA

Kind code of ref document: A

Ref document number: 2281523

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 1998 536752

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1998908556

Country of ref document: EP

Ref document number: 66561/98

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 09367978

Country of ref document: US

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1998908556

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 66561/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1998908556

Country of ref document: EP