The replacement of a phenol group by an aniline or acetanilide group enhances the cytotoxicity of 2-ferrocenyl-1,1-diphenyl-but-1-ene compounds against breast cancer cells

International audienceWe have previously shown that conjugated ferrocenyl p-phenols show strong cytotoxic effects against both the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell lines, possibly via oxidative quinone methide formation. We now present a series of analogous amine and acetamide compounds: 2-ferrocenyl-1-(4-aminophenyl)-1-phenyl-but-1-ene (Z+E-2), 2-ferrocenyl-1-(4-N-acetylaminophenyl)-1-phenyl-but-1-ene (Z-3), and their corresponding organic molecules 1-(4-aminophenyl)-1,2-bis-phenyl-but-1-ene (Z+E-4) and 1-(4-N-acetamidophenyl)-1,2-bis-phenyl-but-1-ene (Z+E-5). All of the compounds have adequate relative binding affinity values for the estrogen receptor; between 2.8% and 5.7% for ERα, and between 0.18% and 15.5% for ERβ, as well as exothermic ligand binding in in silico ER docking experiments. Compounds 2 and 3 show dual estrogenic/cytotoxic activity on the MCF-7 cell line; they are proliferative at low concentrations (0.1 μM) and antiproliferative at high concentrations (10 μM). On the MDA-MB-231 cell line, the ferrocenyl complexes 2 and 3 are antiproliferative with IC50 values of 0.8 μM for 2 and 0.65 μM for 3, while the purely organic molecules 4 and 5 show no effect. Electrochemical experiments suggest that both 2 and 3 can be transformed to oxidized quinoid-type species, analogous to what had previously been observed for the ferrocene phenols

The replacement of a phenol group by an aniline or acetanilide group enhances the cytotoxicity of 2-ferrocenyl-1,1-diphenyl-but-1-ene compounds against breast cancer cells

International audienceWe have previously shown that conjugated ferrocenyl p-phenols show strong cytotoxic effects against both the hormone-dependent MCF-7 and hormone-independent MDA-MB-231 breast cancer cell lines, possibly via oxidative quinone methide formation. We now present a series of analogous amine and acetamide compounds: 2-ferrocenyl-1-(4-aminophenyl)-1-phenyl-but-1-ene (Z+E-2), 2-ferrocenyl-1-(4-N-acetylaminophenyl)-1-phenyl-but-1-ene (Z-3), and their corresponding organic molecules 1-(4-aminophenyl)-1,2-bis-phenyl-but-1-ene (Z+E-4) and 1-(4-N-acetamidophenyl)-1,2-bis-phenyl-but-1-ene (Z+E-5). All of the compounds have adequate relative binding affinity values for the estrogen receptor; between 2.8% and 5.7% for ERα, and between 0.18% and 15.5% for ERβ, as well as exothermic ligand binding in in silico ER docking experiments. Compounds 2 and 3 show dual estrogenic/cytotoxic activity on the MCF-7 cell line; they are proliferative at low concentrations (0.1 μM) and antiproliferative at high concentrations (10 μM). On the MDA-MB-231 cell line, the ferrocenyl complexes 2 and 3 are antiproliferative with IC50 values of 0.8 μM for 2 and 0.65 μM for 3, while the purely organic molecules 4 and 5 show no effect. Electrochemical experiments suggest that both 2 and 3 can be transformed to oxidized quinoid-type species, analogous to what had previously been observed for the ferrocene phenols

N and P-K status of herbage : Use for diagnosis of grasslands

National audienc

Selective estrogen receptor modulators in the ruthenocene series. Synthesis and biological behavior

A series of ruthenocene derivatives, 1-[4-(O(CH2)nN(CH3)2)phenyl]-1-(4-hydroxyphenyl)-2-ruthenocenylbut-1-ene, with n = 2-5, based on the structure of the breast cancer drug tamoxifen has been prepared. These compounds were obtained, via a McMurry cross-coupling reaction, as a mixture of Z and E isomers that could not be separated by HPLC. The relative binding affinity values for estrogen receptor α (ERα) for n = 2 and 3 were very high (85 and 53%) and surpassed even that of hydroxytamoxifen (38.5%), the active metabolite of tamoxifen. Ruthenocene derivatives act as anti-estrogens as effective (n = 2) or slightly more effective (n = 3-5) than hydroxytamoxifen on ERα-positive breast cancer cell lines but, unlike ferrocifens, do not show antiproliferative effects on ERα-negative breast cancer cell lines. Electrochemical studies showed that the ruthenocifen radical cations are unstable, which may account for this behavior. Some of these compounds could be useful as radiopharmaceuticals for ERα-positive breast cancer tumors

Synthesis, Biochemical Properties and Molecular Modelling Studies of Organometallic Specific Estrogen Receptor Modulators (SERMs), the Ferrocifens and Hydroxyferrocifens: Evidence for an Antiproliferative Effect of Hydroxyferrocifens on both Hormone-Dependent and Hormone-Independent Breast Cancer Cell Lines

SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Structuring detergents for extracting and stabilizing functional membrane proteins.

BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field

BmrA purification with C4Cn and detergent exchange.

<p>(<b>A</b>) SDS-PAGE of the sequential extraction of BmrA. As detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>, the membrane fraction (lane T) was incubated with C4C3 and then centrifuged to give the supernatant S and the pellet P. The latter, enriched in BmrA, was suspended in the presence of C4C7 and then centrifuged to give the corresponding supernatant S and pellet P. Arrows indicate the position of BmrA, C4C3 and C4C7. The C4C7 supernatant was then subjected to DLS (B), Ni-affinity chromatography (<b>C</b>) and gel filtration carried out with FC12 (<b>D</b>) from which respective pools indicated by stars were loaded onto SDS-PAGE.</p

Extraction of membrane proteins by C4Cn derivatives.

<p>Extraction of ABC transporters by C4Cn, prokaryotic BmrA (A) and YheI/YheH (B) expressed in <i>E. coli</i>, human ABCG2 expressed in <i>Sf</i>9 (C) or HEK293 (D) cells, together with AcrB expressed in <i>E. coli</i> (E) and the SR-Ca²⁺-ATPase (F), was carried out on the corresponding membrane fractions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. After solubilisation, extracted and non-extracted materials were separated by high-speed centrifugation, generating a supernatant S and a pellet P which were loaded on a 10% SDS-PAGE, and after migration either stained with Coomassie blue or submitted to a Western blot for ABCG2 (upper lanes in panels in C and D). Arrows indicate the position of each monomer. Positive control experiments were carried out with DDM, FC12 and C12E8, negative controls were carried out without detergent (<i>No det</i>). The red dotted line indicates the threshold of extraction.</p

Crystallization of BmrA extracted with C4C10.

<p>BmrA was extracted and purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g005" target="_blank">Figure 5</a>, using C4C10 instead of C4C7 and exchanging it with FC12. (A) The protein, concentrated to 10 mg/ml, and mixed with 1 mM doxorubicin, crystallized after 10 days in 0.2 M KSCN, 20% PEG 3350, and was (B) analyzed at the ESRF beamline ID23EH-2.</p

C4Cn preserve a functional state of BmrA.

<p>(<b>A</b>) Binding of daunorubicin to BmrA monitored by intrinsic (Tryptophan, Trp) fluorescence quenching. BmrA was extracted and purified either with C4C10 (circles) or FC12 (triangles) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4</a>, the former being subsequently exchanged by FC12 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4D</a>. The purified protein was then incubated with increasing concentrations of daunorubicin, the binding of the drug being probed by the variation of intrinsic fluorescence of BmrA, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>. (<b>B</b>) VO₄-sensitive ATPase activity of BmrA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>) in different fractions: BmrA-enriched membrane fraction (“-“ bar) corresponding to 0.5 µmol/min.mg and taken as 100%; BmrA-enriched membrane fraction solubilized with 1% SDS, FC12, DDM, or C4C3+C4C7 (“Extraction/C4Cn” bar, carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Fig. 4</a>); BmrA extracted with FC12 and then purified by metal affinity and gel filtration with FC12 (“Purification/FC12” bar); BmrA extracted with C4C3+C4C7 and then purified by metal affinity with C4C7 followed by detergent exchange with FC12 using gel filtration as carried out in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4</a> (“Purification/C4Cn, FC12 exchange” bar). (<b>C</b>) Intrinsic fluorescence quenching monitoring of C4C10 binding on BmrA. BmrA was extracted either with C4C10 or FC12 and then purified with FC12 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#pone-0018036-g004" target="_blank">Figure 4</a> generating two populations on which C4C10 binding was monitored by probing the quenching of intrinsic fluorescence of 1 µM BmrA as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018036#s2" target="_blank">Methods</a>.</p