Synthesis and characterization of fluorescent 4-hydroxytamoxifen conjugates with unique antiestrogenic properties.

Membrane receptors for steroid hormones are currently a subject of considerable debate. One approach to selectively target these putative receptors has been to couple ligands to substances that restrict cell permeability. Using this approach, an analogue of the estrogen receptor ligand 4-hydroxytamoxifen was attached to fluorescent dyes with differing degrees of predicted cell permeability. The conjugates bound to estrogen receptor in vitro, but all three conjugates, including one predicted to be cell-impermeable, inhibited estradiol-induced transcriptional activation. Fluorescence microscopy revealed cytoplasmic localization for all three conjugates. We further characterized a 4-hydroxytamoxifen analogue conjugated to a BODIPY fluorophore in breast cancer cell lines. Those experiments suggested a similar, but not identical, mode of action to 4-hydroxytamoxifen, as the fluorescent conjugate was equally effective at inhibiting proliferation of both tamoxifen-sensitive and tamoxifen-resistant breast cancer cell lines. While these findings point to significant complicating factors in designing steroid hormone mimics targeted to the plasma membrane, the results also reveal a possible new direction for designing estrogen receptor modulators.

SIRT1 in Astrocytes Regulates Glucose Metabolism and Reproductive Function.

Sirtuin 1 (Sirt1) is an NAD-dependent class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, evidence suggests that SIRT1 in neurons plays a role in the central regulation of energy balance and reproduction, but no studies have addressed the contribution of astrocytes. We show here that overexpression of SIRT1 in astrocytes causes markedly increased food intake, body weight gain, and glucose intolerance, but expression of a deacetylase-deficient SIRT1 mutant decreases food intake and body weight and improves glucose tolerance, particularly in female mice. Paradoxically, the effect of these SIRT1 mutants on insulin tolerance was reversed, with overexpression showing greater insulin sensitivity. The mice overexpressing SIRT1 were more active, generated more heat, and had elevated oxygen consumption, possibly in compensation for the increased food intake. The female overexpressing mice were also more sensitive to diet-induced obesity. Reproductively, the mice expressing the deacetylase-deficient SIRT1 mutant had impaired estrous cycles, decreased LH surges, and fewer corpora lutea, indicating decreased ovulation. The GnRH neurons were responsive to kisspeptin stimulation, but hypothalamic expression of Kiss1 was reduced in the mutant mice. Our results showed that SIRT1 signaling in astrocytes can contribute to metabolic and reproductive regulation independent of SIRT1 effects in neurons.

Neuronal SIRT1 Regulates Metabolic and Reproductive Function and the Response to Caloric Restriction.

Sirt1 is an NAD-dependent, class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. In this study, we generated mice expressing an enzymatically inactive form (N-MUT) or wild-type (WT) SIRT1 (N-OX) in mature neurons. N-OX male and female mice had impaired glucose tolerance, and N-MUT female, but not male, mice had improved glucose tolerance compared with that of WT littermates. Furthermore, glucose tolerance was improved in all mice with caloric restriction (CR) but was greater in the N-OX mice, who had better glucose tolerance than their littermates. At the reproductive level, N-OX females had impaired estrous cycles, with increased cycle length and more time in estrus. LH and progesterone surges were absent on the evening of proestrus in the N-OX mice, suggesting a defect in spontaneous ovulation, which was confirmed by the ovarian histology revealing fewer corpora lutea. Despite this defect, the mice were still fertile when mated to WT mice on the day of proestrus, indicating that the mice could respond to normal pheromonal or environmental cues. When subjected to CR, the N-OX mice went into diestrus arrest earlier than their littermates. Together, these results suggested that the overexpression of SIRT1 rendered the mice more sensitive to the metabolic improvements and suppression of reproductive cycles by CR, which was independent of circadian rhythms.

Synthesis and evaluation of 3- and 7-substituted geranylgeranyl pyrophosphate analogs.

Protein prenyl transferases have been a focus of anti-cancer drug discovery in recent years due to their roles in post-translational modification of small GTP binding proteins. Attention is now turning to the development of GGTase I inhibitors. Here, we present the synthesis and biological evaluation of four GGPP analogs versus mammalian GGTase I and the discovery that 7-allyl GGPP is a surprisingly efficient GGTase I substrate.

Obese Neuronal PPARγ Knockout Mice Are Leptin Sensitive but Show Impaired Glucose Tolerance and Fertility.

The peroxisome-proliferator activated receptor γ (PPARγ) is expressed in the hypothalamus in areas involved in energy homeostasis and glucose metabolism. In this study, we created a deletion of PPARγ brain-knockout (BKO) in mature neurons in female mice to investigate its involvement in metabolism and reproduction. We observed that there was no difference in age at puberty onset between female BKOs and littermate controls, but the BKOs gave smaller litters when mated and fewer oocytes when ovulated. The female BKO mice had regular cycles but showed an increase in the number of cycles with prolonged estrus. The mice also had increased luteinizing hormone (LH) levels during the LH surge and histological examination showed hemorrhagic corpora lutea. The mice were challenged with a 60% high-fat diet (HFD). Metabolically, the female BKO mice showed normal body weight, glucose and insulin tolerance, and leptin levels but were protected from obesity-induced leptin resistance. The neuronal knockout also prevented the reduction in estrous cycles due to the HFD. Examination of ovarian histology showed a decrease in the number of primary and secondary follicles in both genotypes due to the HFD, but the BKO ovaries showed an increase in the number of hemorrhagic follicles. In summary, our results show that neuronal PPARγ is required for optimal female fertility but is also involved in the adverse effects of diet-induced obesity by creating leptin resistance potentially through induction of the repressor Socs3.

Astrocyte-Specific Deletion of Peroxisome-Proliferator Activated Receptor-γ Impairs Glucose Metabolism and Estrous Cycling in Female Mice.

Mice lacking peroxisome-proliferator activated receptor-γ (PPARγ) in neurons do not become leptin resistant when placed on a high-fat diet (HFD). In male mice, this results in decreased food intake and increased energy expenditure, causing reduced body weight, but this difference in body weight is not observed in female mice. In addition, estrous cycles are disturbed and the ovaries present with hemorrhagic follicles. We observed that PPARγ was more highly expressed in astrocytes than neurons, so we created an inducible, conditional knockout of PPARγ in astrocytes (AKO). The AKO mice had impaired glucose tolerance and hepatic steatosis that did not worsen with HFD. Expression of gluconeogenic genes was elevated in the mouse livers, as was expression of several genes involved in lipogenesis, lipid transport, and storage. The AKO mice also had a reproductive phenotype with fewer estrous cycles, elevated plasma testosterone levels, reduced corpora lutea formation, and alterations in hypothalamic and ovarian gene expression. Thus, the phenotypes of the AKO mice were very different from those seen in the neuronal knockout mice, suggesting distinct roles for PPARγ in these two cell types.

Dissecting rapid estrogen signaling with conjugates.

Hypothesizing that rapid estrogen signaling could be modulated from different estrogen receptors with unique localization patterns, a number of groups have attempted to design drug conjugates that target or restrict compounds to specific subcellular compartments. This article will briefly discuss the history of using conjugates to dissect rapid estrogen signaling and different strategies to attempt to target estrogens and antiestrogens to different locations. It will also detail some of the potential issues that can arise with different types of conjugates, using examples drawn from the authors' own work.

Characterization of molecular and structural determinants of selective estrogen receptor downregulators.

Antiestrogens used for breast cancer therapy can be categorized into two classes that differ in their effect on estrogen receptor (ER) alpha stability. The selective estrogen receptor modulators (SERMs) stabilize ER alpha and the selective estrogen receptor downregulators (SERDs) cause a decrease in cellular ER alpha levels. A clinically relevant antiestrogen, GW7604, appears to work through a SERD-like mechanism, despite sharing the same molecular scaffold as 4-hydroxytamoxifen, a SERM. In order to investigate potential structural features of GW7604 responsible for SERD activity, GW7604 and two analogs were synthesized using a new, improved synthetic route and tested for their effects on ER alpha function and cell proliferation. The two analogs, which have an acrylamide or a methyl vinyl ketone replacing the acrylic acid group of GW7604, display lower binding affinity for ER alpha than GW7604, but show similar antagonism of estradiol-induced activation of ER alpha-mediated transcription as GW7604 and inhibit estradiol-induced proliferation of the MCF-7 cell line with a similar potency as GW7604. Unlike GW7604, neither analog has a significant effect on cellular ER alpha levels, suggesting that the carboxylate is a key determinant in GW7604 action and, for the first time, showing that this group is responsible for inducing ER alpha degradation in breast cancer cells.

Synthesis and characterization of bioactive tamoxifen-conjugated polymers.

Macromolecular conjugates of tamoxifen could perhaps be used to circumvent some of the limitations of the extensively used breast cancer drug. To test the feasibility of these conjugates, a 4-hydroxytamoxifen analogue was conjugated to a diaminoalkyl linker and then conjugated to activated esters of a poly(methacrylic acid) polymer synthesized by atom transfer radical polymerization. A polymer conjugated to the 4-hydroxytamoxifen analogue with a six-carbon linker showed high affinity for both estrogen receptor alpha and estrogen receptor beta and potent antagonism of the estrogen receptor in cell-based transcriptional reporter assays. These results suggest that the conjugation of 4-hydroxytamoxifen to a polymer results in a macromolecular conjugate that can display ligand in a manner that can be recognized by estrogen receptor and still act as a potent antiestrogen in cells.