- Email: [email protected]
318 A rational approach to the design of anti-estrogens : The case of hydroxylated triphenylethylenes (TPE)
106s
317
DEFECTIVE ACTIVATION OF THE ESTROGEN RECEPTOR BY TRIPHENYLETHYLENEANTIESTROGENS. J.L. Borgna, E. Evans, J. Fauque and H. Rochefort - U 148 INSERM, 60 Rue de Navacelles, 34100 MONTPELLIER FRANCE. Triphenylethylene antiestrogens such as tamoxifen inhibit estrogen action by preventing its binding to the estrogen receptor (RE). In an attempt to explain the low and dissociated agonist activity of tamoxifen, we have compared in vitro the properties of the RE bound to estradiol (E2) or to 4-hydroxytamoxifen (OHT), a high affinity metabolite of tamoxifen. The kinetics and equilibrium parameters of the RE/ligand interaction are very similar for OHT and E . However using molybdate as a probe to inhibit the in vitro activation of RE, we f2 ound 3 series of alterations of the RE activation triggered by OHT which concerned : the hormone binding site ; the DNA binding domain and the degree of interaction with a monoclonal antibody (836) able to discriminate the activated and stabilized RE. We suggest that the conformations of RE activated by E2 or OHT are different and that this difference may account for the defective agonistic activity of antiestrogens.
318
AR4lICWLAETRXQiTOZ-IEDESIGNOFAh'IT-ESIKGEN S :MEC'&OFHYDFXXUIZ)~(?pE) M.Pons~, F.Mlcheli, A.Crastes de Paulet*, J.Gl.lbertx , J.F.MfquzlX , G.Pr&igouxt, M.lb+tal~ T.Ojaso3+and J.P.Raynaud+. *INSEWU58, 34030HmtPelLier; 'CEXXWXS, 94320lhiais;' Iab.Cristallographie et Physique CristalYne,Uli~.Bordeaw,33405'I$lence;+Rarssel_UcLaf,75007~s_Rance. It has been hypthedd that the activity of available IPE anti-estrogens in ho&eperdenf humn breast cancer thzrapy may be due to a nechanisn vhereby the estrogen receptor (ER) concentrates the conpamd in the acts as a cytotoxin. zhe physiological significance of thebinding cell and the alky~ncethoxy side-&sin
ofthese~dstosn * anti-estrogen binding site(AEBS)m Is unknown. In a firsta stepto rationalize theurders'stardingof them&anismof actimof these ccnpcunds, %e haw synthesized tcalve hmlogcustriphenylacrylonitrile derivatives vitha p-OHor m groupon ooe or mre of thephenylrings(seefig.). Noneof theseccnpcu& competed significantly for b&ingtotheAEZBSin atkidrey supernatant. lhefrr&in ratandrmLSeuteruscytosol at 0" and 25'C.Iheuntive binding affinities (FUW)forERuzre d substituted skeletonhadcnREAof60.1(%= la). An map in Rl or % en&z&red veryluaaffirdty whereasanO_arp inRgaveac~dwithanKBAequivalenttothatofE2,erpha sizil?gthe +ortance of this positionininteractionwifzhER.~tith bettercwpetitorsthanE . an additional OH-grcup in R1 or waresignificantly 3 the ? No furtherincraa.seinRBA~ ~r,ad for thetrihydroxyderlvative. Ihe effect0 inttitionof ahydrophobicQ+ra~P decreasedaffinityas expectedin R, tut side-chain alsoinpitim R (in the sag positionas the allcymmthoxy d essaseccnd OR-grcupuaspresent in F$. lhe confonmticns of t.anntien) of thesederivatiws arecouparedtothcse of-estrogen l&a& and theirrelative Partial agorrist/antagonistactitity is discllssed.
319
PROGESTINS, ANTIPROGESTINS AND PROGESTERONE RECEPTORS IN BREAST CANCER: AND BIOLOGICAL ACTIONS. Kathryn B. Horwitz, Departments of Medicine and Biochemistry, University Health Sciences Center, Denver, CO 80262 USA.
l!ECHANISMS of
Colorado
T47Dco are estrogen receptor (ER) negative, progesterone receptor (PR) positive, and antiestrogen resistant, human breast cancer cells. They are an excellent model to study direct progestin effects without the interference of added estrogens. We now report that in cultured T47D co, progestins are growth inhibitors. At physiological concentrations, the synthetic progestins, R5020 and medroxyprogesterone acetate (MPA), inhibit cell growth more than 50% after 10 days of treatment. Progesterone is also antiproliferative, but it requires IO-fold higher concentrations because it is rapidly (tf = 2 hrs) metabolized in culture. Other classes of steroids have no effect. RU38 486, a new synthetic antiprogestin, has very high affinity for PR, but weaker biological activity than the progestins, and partially blocks the R5020 effect of growth. Unlike progesterone, the synthetic progestins exchange poorly from PR. Al though they translocate PR to nuclei very effectively, synthetic progestins profoundly inhibit subsequent cytoplasmic PR replenishment. in calls treated with progesterone Thus, for 1 hour, nuclear translocation and processing is followed by cytoplasmic PR replenishment 18-24 hours later. In contrast, a 1 hour pulse of R5020, MPA, or RU38 486, blocks replenishment completely for more than 4 days. in situ photoaffinity labeling studies of nuclear PR subunits using [3H]R5020 and [3H]RU38 486 will be described.
317
DEFECTIVE ACTIVATION OF THE ESTROGEN RECEPTOR BY TRIPHENYLETHYLENEANTIESTROGENS. J.L. Borgna, E. Evans, J. Fauque and H. Rochefort - U 148 INSERM, 60 Rue de Navacelles, 34100 MONTPELLIER FRANCE. Triphenylethylene antiestrogens such as tamoxifen inhibit estrogen action by preventing its binding to the estrogen receptor (RE). In an attempt to explain the low and dissociated agonist activity of tamoxifen, we have compared in vitro the properties of the RE bound to estradiol (E2) or to 4-hydroxytamoxifen (OHT), a high affinity metabolite of tamoxifen. The kinetics and equilibrium parameters of the RE/ligand interaction are very similar for OHT and E . However using molybdate as a probe to inhibit the in vitro activation of RE, we f2 ound 3 series of alterations of the RE activation triggered by OHT which concerned : the hormone binding site ; the DNA binding domain and the degree of interaction with a monoclonal antibody (836) able to discriminate the activated and stabilized RE. We suggest that the conformations of RE activated by E2 or OHT are different and that this difference may account for the defective agonistic activity of antiestrogens.
318
AR4lICWLAETRXQiTOZ-IEDESIGNOFAh'IT-ESIKGEN S :MEC'&OFHYDFXXUIZ)~(?pE) M.Pons~, F.Mlcheli, A.Crastes de Paulet*, J.Gl.lbertx , J.F.MfquzlX , G.Pr&igouxt, M.lb+tal~ T.Ojaso3+and J.P.Raynaud+. *INSEWU58, 34030HmtPelLier; 'CEXXWXS, 94320lhiais;' Iab.Cristallographie et Physique CristalYne,Uli~.Bordeaw,33405'I$lence;+Rarssel_UcLaf,75007~s_Rance. It has been hypthedd that the activity of available IPE anti-estrogens in ho&eperdenf humn breast cancer thzrapy may be due to a nechanisn vhereby the estrogen receptor (ER) concentrates the conpamd in the acts as a cytotoxin. zhe physiological significance of thebinding cell and the alky~ncethoxy side-&sin
ofthese~dstosn * anti-estrogen binding site(AEBS)m Is unknown. In a firsta stepto rationalize theurders'stardingof them&anismof actimof these ccnpcunds, %e haw synthesized tcalve hmlogcustriphenylacrylonitrile derivatives vitha p-OHor m groupon ooe or mre of thephenylrings(seefig.). Noneof theseccnpcu& competed significantly for b&ingtotheAEZBSin atkidrey supernatant. lhefrr&in ratandrmLSeuteruscytosol at 0" and 25'C.Iheuntive binding affinities (FUW)forERuzre d substituted skeletonhadcnREAof60.1(%= la). An map in Rl or % en&z&red veryluaaffirdty whereasanO_arp inRgaveac~dwithanKBAequivalenttothatofE2,erpha sizil?gthe +ortance of this positionininteractionwifzhER.~tith bettercwpetitorsthanE . an additional OH-grcup in R1 or waresignificantly 3 the ? No furtherincraa.seinRBA~ ~r,ad for thetrihydroxyderlvative. Ihe effect0 inttitionof ahydrophobicQ+ra~P decreasedaffinityas expectedin R, tut side-chain alsoinpitim R (in the sag positionas the allcymmthoxy d essaseccnd OR-grcupuaspresent in F$. lhe confonmticns of t.anntien) of thesederivatiws arecouparedtothcse of-estrogen l&a& and theirrelative Partial agorrist/antagonistactitity is discllssed.
319
PROGESTINS, ANTIPROGESTINS AND PROGESTERONE RECEPTORS IN BREAST CANCER: AND BIOLOGICAL ACTIONS. Kathryn B. Horwitz, Departments of Medicine and Biochemistry, University Health Sciences Center, Denver, CO 80262 USA.
l!ECHANISMS of
Colorado
T47Dco are estrogen receptor (ER) negative, progesterone receptor (PR) positive, and antiestrogen resistant, human breast cancer cells. They are an excellent model to study direct progestin effects without the interference of added estrogens. We now report that in cultured T47D co, progestins are growth inhibitors. At physiological concentrations, the synthetic progestins, R5020 and medroxyprogesterone acetate (MPA), inhibit cell growth more than 50% after 10 days of treatment. Progesterone is also antiproliferative, but it requires IO-fold higher concentrations because it is rapidly (tf = 2 hrs) metabolized in culture. Other classes of steroids have no effect. RU38 486, a new synthetic antiprogestin, has very high affinity for PR, but weaker biological activity than the progestins, and partially blocks the R5020 effect of growth. Unlike progesterone, the synthetic progestins exchange poorly from PR. Al though they translocate PR to nuclei very effectively, synthetic progestins profoundly inhibit subsequent cytoplasmic PR replenishment. in calls treated with progesterone Thus, for 1 hour, nuclear translocation and processing is followed by cytoplasmic PR replenishment 18-24 hours later. In contrast, a 1 hour pulse of R5020, MPA, or RU38 486, blocks replenishment completely for more than 4 days. in situ photoaffinity labeling studies of nuclear PR subunits using [3H]R5020 and [3H]RU38 486 will be described.