Abstract:Nonsteroidal antiestrogens display antagonistic as well as agonistic properties when compared with estrogens. These observations and studies of their molecular interactions suggest that part of their activity is mediated through estrogen receptor (ER)-mediated pathways. However, other data, concerning mainly effects on growth and cellular proliferation, cannot be explained on this basis. Recent investigations have shown binding of these molecules to a number of other intracellular binding sites beside ER. Usin… Show more
“…Antiestrogen binding sites are cytosolic, membrane-bound protein complexes that tightly bind tamoxifen and 4-OHT but exhibit virtually no affinity for estradiol. , The structure and natural function of AEBS remain poorly understood; however, there is some evidence to suggest that AEBS overexpression plays a role in tamoxifen resistance . Additionally, AEBS are commonly expressed in hormone refractory, ER-negative breast cancer cell lines. , Therefore, a targeting strategy that utilizes a ligand, such as 4-hydroxytamoxifen, that has high affinity to both ER and AEBS could serve to deliver a cytotoxin to a broader range of breast cancer cell types. 3 Cocrystal structure of 4-OHT bound to the ligand binding domain of ERα.…”
Section: Designmentioning
confidence: 99%
“…40 Additionally, AEBS are commonly expressed in hormone refractory, ER-negative breast cancer cell lines. 41,42 Therefore, a targeting strategy that utilizes a ligand, such as 4-hydroxytamoxifen, that has high affinity to both ER and AEBS could serve to deliver a cytotoxin to a broader range of breast cancer cell types.…”
The anthracycline antitumor drug doxorubicin (DOX) has been utilized for decades as a broad-spectrum chemotherapeutic. Recent literature evidence documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. Targeting a doxorubicin-formaldehyde conjugate specifically to cancer cells may provide a more efficacious chemotherapeutic. The design and 11-step synthesis of doxorubicin-formaldehyde conjugates targeted to the estrogen receptor, which is commonly overexpressed in breast cancer cells, are reported. The formaldehyde is incorporated in a masked form as an N-Mannich linkage between doxorubicin and salicylamide. The salicylamide triggering molecule, previously developed to release the doxorubicin-formaldehyde active metabolite, is tethered via derivatized ethylene glycols to an E and Z mixture of 4-hydroxytamoxifen. The targeting group, E/Z-4-hydroxytamoxifen, was selected for its ability to tightly bind the estrogen receptor and antiestrogen binding sites. The targeted doxorubicin-formaldehyde conjugates' estrogen receptor binding and in vitro growth inhibition were evaluated as a function of tether length. The lead compound, DOX-TEG-TAM, bearing a triethylene glycol tether, binds the estrogen receptor with a binding affinity of 2.5% relative to E/Z-4-hydroxytamoxifen and inhibits the growth of four breast cancer cell lines with 4-fold up to 140-fold enhanced activity relative to doxorubicin.
“…Antiestrogen binding sites are cytosolic, membrane-bound protein complexes that tightly bind tamoxifen and 4-OHT but exhibit virtually no affinity for estradiol. , The structure and natural function of AEBS remain poorly understood; however, there is some evidence to suggest that AEBS overexpression plays a role in tamoxifen resistance . Additionally, AEBS are commonly expressed in hormone refractory, ER-negative breast cancer cell lines. , Therefore, a targeting strategy that utilizes a ligand, such as 4-hydroxytamoxifen, that has high affinity to both ER and AEBS could serve to deliver a cytotoxin to a broader range of breast cancer cell types. 3 Cocrystal structure of 4-OHT bound to the ligand binding domain of ERα.…”
Section: Designmentioning
confidence: 99%
“…40 Additionally, AEBS are commonly expressed in hormone refractory, ER-negative breast cancer cell lines. 41,42 Therefore, a targeting strategy that utilizes a ligand, such as 4-hydroxytamoxifen, that has high affinity to both ER and AEBS could serve to deliver a cytotoxin to a broader range of breast cancer cell types.…”
The anthracycline antitumor drug doxorubicin (DOX) has been utilized for decades as a broad-spectrum chemotherapeutic. Recent literature evidence documents the role of formaldehyde in the cytotoxic mechanism, and anthracycline-formaldehyde conjugates possess substantially enhanced activity in vitro and in vivo. Targeting a doxorubicin-formaldehyde conjugate specifically to cancer cells may provide a more efficacious chemotherapeutic. The design and 11-step synthesis of doxorubicin-formaldehyde conjugates targeted to the estrogen receptor, which is commonly overexpressed in breast cancer cells, are reported. The formaldehyde is incorporated in a masked form as an N-Mannich linkage between doxorubicin and salicylamide. The salicylamide triggering molecule, previously developed to release the doxorubicin-formaldehyde active metabolite, is tethered via derivatized ethylene glycols to an E and Z mixture of 4-hydroxytamoxifen. The targeting group, E/Z-4-hydroxytamoxifen, was selected for its ability to tightly bind the estrogen receptor and antiestrogen binding sites. The targeted doxorubicin-formaldehyde conjugates' estrogen receptor binding and in vitro growth inhibition were evaluated as a function of tether length. The lead compound, DOX-TEG-TAM, bearing a triethylene glycol tether, binds the estrogen receptor with a binding affinity of 2.5% relative to E/Z-4-hydroxytamoxifen and inhibits the growth of four breast cancer cell lines with 4-fold up to 140-fold enhanced activity relative to doxorubicin.
“…Given the ubiquitous presence of antiestrogen binding sites (AEBS) that specifically bind triphenylethylenic antiestrogens such as tamoxifen with high affinity ( K D = 10 -9 M), several group ( − ) have focused attention on the potential existence of a protein or proteins that are different from ER and mediate several of the biological activities of tamoxifen ( , ). By developing specific AEBS-targeted ligands based on the tamoxifen backbone but without ER affinity, we ( , ) and others ( , ) have hypothesized that AEBS mediates estrogen receptor-independent biological activities of tamoxifen. Moreover, the AEBS prototypical ligand DPPE ( N , N -diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl), which was expected to be a specific ligand of a new intracellular histamine receptor () has been shown to potentiate the chemotherapeutic index of cytotoxic drugs in vitro ( , ).…”
Our quest to identify target proteins involved in the activity of tamoxifen led to the design of photoaffinity ligand analogues of tamoxifen able to cross-link such proteins. A new tritiated photoprobe, 4-(2-morpholinoethoxy)benzophenone (MBoPE), was synthesized and used to identify proteins involved in tamoxifen binding in rat liver. MBoPE, which has structural features in common with the potential antagonist of the intracellular histamine receptor (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl: DPPE) is unable to bind the estrogen receptor although it does compete with tamoxifen for an antiestrogen binding site (AEBS). This tritiated benzophenone derivative was obtained by metal-catalyzed halogen-tritium replacement reaction. Because of its high specific activity, four target proteins could be photolabeled, three of which were identified with M(r) of 60,000, 49,500, and 14,000, while the fourth at 27,500 was in too low an amount and could not be sequenced. The 49.5 kDa protein corresponded by mass spectrometry to the microsomal epoxide hydrolase already identified with an aryl azide photoprobe [Mesange, F., et al. (1998) Biochem. J. 334, 107-112]. The 60 and 14 kDa proteins were identified as the carboxylesterase (ES10) and the liver fatty acid binding protein (L-FABP), respectively. The inhibitory effect of tamoxifen on carboxylesterase activity and the competitive efficacy of oleic acid on [(3)H]tamoxifen binding suggest that both proteins are AEBS subunits. Moreover, treatment of hepatocytes with antisense mRNA directed against ES10 or L-FABP abolished both tamoxifen and MBoPE binding. On the basis of previous pharmacological arguments, the 27.5 kDa protein might correspond to the sigma I receptor. Altogether, these results confirm that the microsomal epoxide hydrolase is a target for tamoxifen and provide evidence of two new target proteins implicated in cell lipid metabolism.
“…In addition we found that tamoxifen and N-morpholino-2- [4-(phenylmethyl) [15,16] ; it was antiproliferative on MCF-7 cells [11,[17][18][19][20] but had no effect on the proliferation of Rtx-6. We have established a positive correlation between the affinity of DPPE-based derivatives for AEBS and their antiproliferative properties on MCF-7 cells.…”
A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039–17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.
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