Structure activity relationship studies on C2 side chain substituted 1,1-bis(4-methoxyphenyl)-2-phenylalkenes and 1,1,2-tris(4-methoxyphenyl)alkenes

1,1-bis(4-Methoxyphenyl)-2-phenylalkenes (1a-9a) and 1,1,2-tris(4-methoxyphenyl)alkenes (1b-9b) with various C2-substituents (H (1a, 1b), methyl (2a, 2b), ethyl (3a, 3b), propyl (4a, 4b), butyl (5a, 5b), 2-cyanoethyl (6a, 6b), 3-cyanopropyl (7a, 7b), 3-aminopropyl (8a, 8b), 3-carboxypropyl (9a, 9b)) were tested for cytotoxic effects on hormone dependent MCF-7 cells. The effects were correlated with agonistic and antagonist properties determined on the MCF-7-2a cell line stably transfected with the plasmid ERE(wtc)luc. We demonstrated that the antiproliferative effects did not result from an interaction with the estrogen receptor (ER). The most cytotoxic compounds 5,5-bis(4-methoxyphenyl)-4-phenylpent-4-enylamine (8a) and 4,5,5-tris(4-methoxyphenyl)pent-4-enyl (8b) showed cytocidal effects without having significant agonistic and antagonistic properties.     

Biological evaluation of novel estrogen-platinum(II) hybrid molecules on uterine and ovarian cancers-molecular modeling studies

We have recently reported the synthesis of a series of original 17beta-estradiol-linked platinum(II) hybrid molecules. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER(+) and ER(-)) human uterine and ovarian cancers. The hybrid molecules present higher affinity than that of 17beta-estradiol for the estrogen receptor alpha (ERalpha). The cytotoxicity and the affinity of the hybrid molecules are explained using molecular modeling analysis. This study further confirms that the derivatives made of a 2-(2'-aminoethyl)pyridine ligand displayed superior activity against the cell lines particularly when the connecting arm is 8-10 carbon atoms long. Molecular modeling shows that a long side chain can facilitate the access of the platinum(II) moiety to DNA. The novel compounds also prove to be moderately cytotoxic against platinum resistant endometrial and ovarian cancer cell lines.     

Maximizing production of estrogen receptor beta with the baculovirus expression system

Steroid hormone/nuclear receptor expression in cultured insect cell lines is routinely driven by a baculovirus vector. An advantage of the baculovirus production of these receptors is that large amounts of functional receptors are obtained for subsequent in vitro studies. Most laboratories produce nuclear receptors in Spodoptera frugiperda (Sf)9 cells. However, no one has determined whether this cell line is optimal for the production of any nuclear receptor. We compared the time course and level of estrogen receptor beta (ER beta) production from a baculovirus in two S. frugiperda cell lines, IPLB-SF21AE (Sf21) and Sf9, and two Trichloplusia ni cell lines, Tn368 and BTI-TN5b1-4 (High Five). Cells were harvested at various times (0.5-5 days) after infection. ER beta expression and activity was determined by specific [3H]estradiol (E2) binding, Western blot analysis, and estrogen response element (ERE) binding in vitro. The highest functional, bioactive ER beta expression both at the earliest time after infection and in the amount of ER beta produced/cell was with the Sf21 cell line. Baculovirus expressed ER beta-bound EREs with high affinity in a DNA sequence-dependent manner. We conclude that Sf21 cells are the best-suited cells for ER beta production.     

Synthesis of 17beta-estradiol-linked platinum(II) complexes and their cytocidal activity on estrogen-dependent and -independent breast tumor cells

The synthesis of two new highly potent 17beta-estradiol-linked platinum(II) complexes is described. The new molecules are linked at position 16 of the steroid nucleus with an alkyl chain. They are made from estrone in nine chemical steps with an overall yield exceeding 10%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER(+) and ER(-)) human breast tumor cell lines: MCF-7 and MDA-MB-231. The novel compounds prove to be highly cytotoxic against breast cancer cell lines. The most cytotoxic derivative shows high affinity for the estrogen receptor alpha.     

Synthesis of unique 17beta-estradiol homo-dimers, estrogen receptors binding affinity evaluation and cytocidal activity on breast, intestinal and skin cancer cell lines

A rapid and efficient synthesis of a series of C2-symmetric 17beta-estradiol homo-dimers is described. The new molecules are linked at position 17alpha of the steroid nucleus with either an alkyl chain or a polyethylene glycol chain. They are made from estrone in only five chemical steps with an overall yield exceeding 30%. The biological activity of these compounds was evaluated in vitro on estrogen dependent and independent (ER+ and ER-) human breast tumor cell lines: MCF-7 and MDA-MB-231. Some of the dimers present selective cytotoxic activity against the ER+ cell line. However, they are not very cytotoxic when compared to the antiestrogen tamoxifen. Unfortunately, they show only weak affinity for the estrogen receptor alpha (ERalpha) and no affinity for the estrogen receptor beta (ERbeta). The new compounds were also tested on human intestinal (HT-29) cancer and on murine skin cancer (B16-F10) cell lines for further biological assessment. Interestingly, the dimers were found to be cytotoxic to the murine skin cancer cell line but were inactive towards the intestinal cancer cell line.     

The immunomodulating properties and antitumor activity of block copolymers of ethylene oxide and propylene oxide (proxanols)

Influence of block copolymers of ethylene and propylene oxides (proxanols) on the human natural killer activity was studied. Proxanols had no effect on the lymphocytes viability and increased their cytotoxic activity against target cells K 562 in vitro. Certain antitumor properties of proxanols were demonstrated in vivo (especially for ascitic tumors). The proxanols activity was chiefly determined by the membranotropic properties of its main polymer chain.     

Synthesis, pharmacological evaluation, and structure-activity relationships of benzopyran derivatives with potent SERM activity

The synthesis, binding affinity for estrogen receptor subtypes (ER alpha and ER beta) and pharmacological activity on rat uterus of a new class of potent ligands, characterized by a 3-phenylbenzopyran scaffold with a basic side chain in position 4, are reported. Some of these compounds, endowed with very high receptor affinity, showed potent inhibition of agonist-stimulated uterine growth, with no or limited proliferative effect. Binding affinity mostly depended on the nature and position of substituents at the 3-phenyl ring, while the uterine activity seems to be affected by basic chain length. Compound 9c (CHF4227) showed excellent binding affinity and antagonist activity on the uterus. The docking of benzopyran derivatives explained the structure-affinity relationships observed for 3-phenyl substitution: a small, hydrophobic 4'-substituent could interact with a small accessory binding cavity, while di-substitution at 4' and 3' led to some ER alpha selectivity. This selectivity can be ascribed to differences in amino acid composition and side chain conformation in the region accommodating the 3-phenyl ring at human ER alpha and ER beta ligand-binding domain.     

Endoplasmic reticulum aminopeptidase associated with antigen processing regulates quality of processed peptides presented by MHC class I molecules

Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13(a) histocompatibility gene and presented by D(b) MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-D(b) complex is inhibited when other peptides compete for binding D(b). Conversely, improving the binding of SVL9 peptide to D(b) suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.     

Conformational correlation and coupled motion between residue A21 and B25 side chain observed in crystal structures of insulin mutants at position A21

The C-terminal residue of the insulin A chain is invariant and kept as asparagine in all known insulin molecules from hagfish through birds to mammals. To get information on the role of this conserved residue, which is still unclear, the three-dimensional structures of four human insulin mutants, A21 Asn-->Gly, A21 Asn-->Asp, A21 Asn-->Ala, and A21 Asn-->Gln DesB30, were determined by X-ray crystallography. The four mutants crystallize separately into two kinds (rhombohedral and cubic) of crystals. In the refined structures, conformational correlation and coupled motion between the A chain C-terminal residue A21 and the B25 side chain was observed, in contrast to the nearly unchanged general structures as compared with the native insulin structures in their respective crystals. A detailed analysis suggests that residue A21 can affect insulin receptor binding by interaction with the B25 side chain and the B chain C-terminal segment to assist the B25 side chain rearranging into the 'active' conformation.     

Biosynthetic and functional properties of an Arg-Gly-Asp-directed receptor involved in human melanoma cell attachment to vitronectin, fibrinogen, and von Willebrand factor

M21 human melanoma cells express an Arg-Gly-Asp-directed adhesion receptor composed of noncovalently associated alpha and beta chains. To establish the structural and functional properties of this receptor on M21 human melanoma cells, stable variant cell lines were selected that express altered alpha chain levels. One of these variants, M21-L, fails to synthesize alpha chain protein or its mRNA, yet does produce normal levels of the beta chain. In these cells the beta chain does not reach the cell surface but rather accumulates within the cell. M21-L cells lacking the alpha chain are incapable of attaching to vitronectin, von Willebrand factor, fibrinogen, or an Arg-Gly-Asp-containing heptapeptide yet attach normally to fibronectin, whereas the unselected M21 cells attach to all of these adhesive proteins. In addition, a monoclonal antibody, LM609 generated to a functional site on the intact receptor, is capable of preventing M21 cell attachment to vitronectin, von Willebrand factor, fibrinogen, and the Arg-Gly-Asp peptide but not to fibronectin. Following a 2-min biosynthetic pulse-label, the newly synthesized alpha chain remains in free form for 5 min and then associates with previously synthesized beta chain present in an intracellular pool. Once oligomerization takes place, the receptor gains the capacity to recognize Arg-Gly-Asp, and at this time the epitope recognized by monoclonal antibody LM609 is formed.     

Tmp21, a novel MHC-I interacting protein, preferentially binds to Β2-microglobulin-free MHC-I heavy chains

MHC-I molecules play a critical role in immune surveillance against viruses by presenting peptides to cytotoxic T lymphocytes. Although the mechanisms by which MHC-I molecules assemble and acquire peptides in the ER are well characterized, how MHC-I molecules traffic to the cell surface remains poorly understood. To identify novel proteins that regulate the intracellular transport of MHC-I molecules, MHC-I-interacting proteins were isolated by affinity purification, and their identity was determined by mass spectrometry. Among the identified MHC-I-associated proteins was Tmp21, the human ortholog of yeast Emp24p, which mediates the ER-Golgi trafficking of a subset of proteins. Here, we show that Tmp21 binds to human classical and non-classical MHC-I molecules. The Tmp21-MHC-I complex lacks Β(2)-microglobulin, and the number of the complexes is increased when free MHC-I heavy chains are more abundant. Taken together, these results suggest that Tmp21 is a novel protein that preferentially binds to Β(2)-microglobulin-free MHC-I heavy chains.     

Subcellular localization and membrane topology of the melon ethylene receptor CmERS1

Ethylene receptors are multispanning membrane proteins that negatively regulate ethylene responses via the formation of a signaling complex with downstream elements. To better understand their biochemical functions, we investigated the membrane topology and subcellular localization of CmERS1, a melon (Cucumis melo) ethylene receptor that has three putative transmembrane domains at the N terminus. Analyses using membrane fractionation and green fluorescent protein imaging approaches indicate that CmERS1 is predominantly associated with the endoplasmic reticulum (ER) membrane. Detergent treatments of melon microsomes showed that the receptor protein is integrally bound to the ER membrane. A protease protection assay and N-glycosylation analysis were used to determine membrane topology. The results indicate that CmERS1 spans the membrane three times, with its N terminus facing the luminal space and the large C-terminal portion lying on the cytosolic side of the ER membrane. This orientation provides a platform for interaction with the cytosolic signaling elements. The three N-terminal transmembrane segments were found to function as topogenic sequences to determine the final topology. High conservation of these topogenic sequences in all ethylene receptor homologs identified thus far suggests that these proteins may share the same membrane topology.     

Design, synthesis, cytocidal activity and estrogen receptor α affinity of doxorubicin conjugates at 16α-position of estrogen for site-specific treatment of estrogen receptor positive breast cancer

Doxorubicin (DOX) is an important medicine for the treatment of breast cancer, which is the most frequently diagnosed and the most lethal cancer in women worldwide. However, the clinical use of DOX is impeded by serious toxic effects such as cardiomyopathy and congestive heart failure. Covalently linking DOX to estrogen to selectively deliver the drug to estrogen receptor-positive (ER(+)) cancer tissues is one of the strategies under investigation for improving the efficacy and decreasing the cardiac toxicity of DOX. However, conjugation of drug performed until now was at 3- or 17-position of estrogen, which is not ideal since the hydroxyl groups at this position are important for receptor binding affinity. In this study, we designed, prepared and evaluated in vitro the first estrogen-doxorubicin conjugates at 16α-position of estradiol termed E-DOXs (8a-d). DOX was conjugated using a 3-9 carbon atoms alkylamide linking arm. E-DOXs were prepared from estrone using a seven-step procedure to afford the desired conjugates in low to moderate yields. The antiproliferative activities of the E-DOX 8a conjugate through a 3-carbon spacer chain on ER(+) MCF7 and HT-29 are in the micromolar range while inactive on M21 and the ER(-) MDA-MB-231 cells (>50μM). Compound 8a exhibits a selectivity ratio (ER(+)/ER(-) cell lines) of >3.5. Compounds 8b-8d bearing alkylamide linking arms ranging from 5 to 9 carbon atoms were inactive at the concentrations tested (>50μM). Interestingly, compounds 8a-8c exhibited affinity for the estrogen receptor α (ERα) in the nanomolar range (72-100nM) whereas compound 8d exhibited no affinity at concentrations up to 215nM. These results indicate that a short alkylamide spacer is required to maintain both antiproliferative activity toward ER(+) MCF7 and affinity for the ERα of the E-DOX conjugates. Compound 8a is potentially a promising conjugate to target ER(+) breast cancer and might be useful also for the design of more potent E-DOX conjugates.