Design and synthesis of benzofused heterocyclic RXR modulators.

Benzofused heterocyclic analogs of the RXR selective modulator 1 (LG101506) were synthesized, and tested for their ability to bind RXRalpha and activate RXR homo and heterodimers. Potency and efficacy were observed to be dependent upon the choice of heterocycle as well as the sidechain employed.

Design and synthesis of fluorinated RXR modulators.

Fluorinated trienoic acid analogues of the RXR selective modulator 1 (LG101506) were synthesized, and tested for their ability to bind RXRalpha and activate RXR homo and heterodimers. Potency and efficacy were observed to be dependent upon the position of fluorination, and improvement in pharmacological profile was demonstrated in some cases.

Novel (2E,4E,6Z)-7-(2-alkoxy-3,5-dialkylbenzene)-3-methylocta-2,4,6-trienoic acid retinoid X receptor modulators are active in models of type 2 diabetes.

Previous data have shown that RXR-selective agonists (e.g., 3 and 4) are insulin sensitizers in rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Unfortunately, they also produce dramatic increases in triglycerides and profound suppression of the thyroid hormone axis. Here we describe the design and synthesis of new RXR modulators that retain the insulin-sensitizing activity of RXR agonists but produce substantially reduced side effects. These molecules bind selectively and with high affinity to RXR and, unlike RXR agonists, do not activate RXR homodimers. To further evaluate the antidiabetic activity of these RXR modulators, we have designed a concise and systematic structure-activity relationship around the 2E,4E,6Z-7-aryl-3-methylocta-2,4,6-trienoic acid scaffold. Selected compounds have been evaluated using insulin-resistant rodents (db/db mice) to characterize effects on glucose homeostasis. Our studies demonstrate the effectiveness of RXR modulators in lowering plasma glucose in the db/db mouse model.

The rexinoid LG100754 is a novel RXR:PPARgamma agonist and decreases glucose levels in vivo.

The RXR serves as a heterodimer partner for the PPARgamma and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPARgamma agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPARalpha agonist, also functions as a RXR:PPARgamma agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptoralpha, RXR:liver X receptorbeta, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPARgamma-dependent pathways including adipocyte differentiation and inhibition of TNFalpha-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPARgamma by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.

Selective activation of an apoptotic retinoid precursor in macrophage cell lines.

Advances in the understanding of the retinoid signaling mechanism has allowed the discovery of highly selective retinoids that activate only one specific receptor class, subtype, or signaling pathway. These novel compounds lack certain of the common retinoid toxicities and therefore suggest promising new approaches for therapeutic applications. We describe here a new compound, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid methyl ester (MX84), that is selectively activated in macrophages, leading to killing of only macrophage monocyte type cells in vitro. We provide evidence that MX84 is an inactive precursor that is converted into an active apoptosis-inducing retinoid in macrophages. The macrophage activity is also secreted, and our data suggest that the secreted activity is a phospholipase D type activity. Our observation may lead to the development of molecules that are highly macrophage-selective apoptosis inducers in vivo and that could represent important novel therapeutics against diseases caused by excessive macrophage activity.

Novel retinoid-related molecules as apoptosis inducers and effective inhibitors of human lung cancer cells in vivo.

Lung cancer causes more than 140,000 deaths annually in the United States alone, and the prognosis for non-small cell lung cancer (NSCLC) is particularly poor. Therapies using small molecules that preferentially kill lung tumor cells by inducing cellular suicide (apoptosis) would therefore be highly desirable. Retinoids have shown promise as cancer preventive and cancer therapeutic agents. Retinoid signals are mediated by two classes of nuclear receptors: the retinoic acid receptors (RAR alpha, beta, and gamma) and the retinoid X receptors (RXR alpha, beta and gamma). These receptors usually bind as heterodimers to specific DNA sequences and/or interact with other transcriptional regulators, such as AP-1 (ref. 10) to regulate gene transcription. Synthetic retinoids can be made that activate only specific portions of the complex retinoid response network and activate selective biological programs. To identify retinoids with novel biological activities, we used a high-throughput "biological activity fingerprint" screen on a large library of retinoids and retinoid-related molecules (RRMs). We identified new structures that are highly effective against lung cancer cells in vitro, inducing apoptosis. We show here for one of these compounds that it is very effective against a human NSCLC in vivo in an animal model. These new molecules show a distinct pattern of receptor signaling.

Regulation of tyrosinase mRNA in mouse melanoma cells by alpha-melanocyte-stimulating hormone.

Cloudman S-91 mouse melanoma cells respond to alpha-melanocyte-stimulating hormone) by demonstrating a marked increase in tyrosinase activity (O-diphenol-O2 oxidoreductase, EC 1.14.18.1). This increase is the result of increased levels of tyrosinase mRNA with a subsequent increase in tyrosinase abundance. Our studies were carried out to determine the effect of melanocyte-stimulating hormone on tyrosinase gene transcription and to measure the kinetics of the hormone-induced increase in tyrosinase mRNA. When melanoma cells were exposed continuously to melanocyte-stimulating hormone for 6 d, a large but transient increase in both tyrosinase mRNA abundance and enzyme activity were observed. The maximum increase in tyrosinase mRNA occurred 60 h after melanocyte-stimulating hormone stimulation and was followed by a decline in message levels even though cells were continuously exposed to hormone. Results of nuclear run-off transcription assays showed that melanocyte-stimulating hormone caused a slow increase in the rate of transcription of the tyrosinase gene with a maximal 6-fold stimulation occurring at 48 h. In cells treated with the ribonucleic acid synthesis inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, tyrosinase mRNA levels decayed with a half-life of 4-5 h. This decay rate was unaffected by treatment of cells with melanocyte-stimulating hormone, indicating that the hormone does not act to stabilize tyrosinase ribonucleic acid. Inhibition of protein synthesis by treatment with cycloheximide had no effect on the melanocyte-stimulating hormone-induced increase in tyrosinase messenger ribonucleic acid levels suggesting that ongoing protein synthesis is not required for, at least, the initial stimulation of tyrosinase gene transcription by melanocyte-stimulating hormone.