Oxidized analogs of Di(1H-indol-3-yl)methyl-4-substituted benzenes are NR4A1-dependent UPR inducers with potent and safe anti-cancer activity.

Di(1H-indol-3-yl)(4-trifluoromethylphenyl)methane (DIM-Ph-4-CF3) is an analog of orphan nuclear receptor 4A1 (NR4A1) ligand cytosporone B. We have synthesized several oxidation products of DIM-Ph-4-CF3, focusing on analogs with electron-withdrawing or donating groups at their phenyl ring 4-positions, and examined their anti-cancer activity and mechanism-of-action. Mesylates (DIM-Ph-4-X+ OMs-s) having CF3, CO2Me and Cl groups were more effective inhibitors of cancer cell viability than their precursors. 19F NMR spectroscopy and differential scanning calorimetry strongly indicated interactions of DIM-Ph-4-CF3+ OMs- with the NR4A1 ligand binding domain, and compound-induced apoptosis of prostate cancer cells was dependent on NR4A1. DIM-Ph-4-CF3+ OMs- showed robust inhibition of LNCaP prostate cancer xenografts with no apparent toxicity. In vitro and in vivo, DIM-Ph-4-CF3+ OMs- activated proapoptotic unfolded protein response (UPR) signaling in prostate cancer cells. Independently of DIM-Ph-4-CF3+ OMs-, the bulk of NR4A1 localized to the cytoplasm in various cancer cell lines, suggesting a cytoplasmic mechanism-of-action of DIM-Ph-4-CF3+ OMs- in UPR induction and cell death. In summary, the data suggest that oxidized analogs of DIM-Ph-4-CF3 possess potent and safe anti-cancer activity which is mediated through UPR signaling downstream of NR4A1 binding.

Down regulation of miR-202 modulates Mxd1 and Sin3A repressor complexes to induce apoptosis of pancreatic cancer cells.

Aberrant regulation of microRNA expression in pancreatic cancers has been shown to play an important role in its inherent poor prognosis and malignant potential. MicroRNAs have also been shown to inhibit translation of genes by targeting the 3'-untranslated region (3-UTR) of mRNAs resulting in the inhibition of translation and often destruction of the mRNA. In the present study we investigated the role of the microRNA miR-202 in the apoptotic pathways of pancreatic cancer cells. The adamantyl-related molecule, 3-Cl-AHPC down-regulated expression of miR-202 and miR-578 resulting in the increased expression of mRNA and protein expression of their target genes, Max dimerization protein 1 (Mxd1/Mad1) and the Sin3A associated protein 18 (SAP18). Overexpression of pre-miR-202 led to diminished levels of Mxd1 and blocked the 3-Cl-AHPC-mediated increase in Mxd1 mRNA expression. The addition of the microRNA inhibitor 2'-O-methylated miR-202 enhanced the 3-Cl-AHPC-mediated increase of Mxd1 mRNA levels as well as 3-CI-AHPC-mediated apoptosis. We found increased Mxd1 bound to the Sin3A repressor protein complex through its increased binding with HDAC-2 and subsequently enhanced transcriptional repression in cells as evidenced by increased HDAC activity. Mxd1 also repressed human telomerase reverse transcriptase (hTERT) mRNA expression through its increased binding to the hTERT promoter site and resulted in decreased telomerase activity in cells. Our results demonstrate that down regulation of miR-202 increased the expression of its target Mxd1, followed by Mxd1 recruitment to the Sin3A repressor complex and through its dimerization with Max, and increased repression of Myc-Max target proteins.

NF-κB p65 recruited SHP regulates PDCD5-mediated apoptosis in cancer cells.

Transcription factor NF-κB promotes cell proliferation in response to cell injury. Increasing evidence, however, suggests that NF-κB can also play an apoptotic role depending on the stimulus and cell type. We have previously demonstrated that novel retinoid 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated apoptosis in breast carcinoma cells requires activation of canonical and non-canonical NF-κB pathways. The mechanism NF-κB uses to induce apoptosis remains largely unknown. NF-κB subunit p65 (RelA) was identified as one potent transcriptional activator in 3-Cl-AHPC-mediated apoptosis in cells. Here we used ChIP-on-chip to identify NF-κB p65 genes activated in 3-Cl-AHPC mediated apoptosis. This paper focuses on one hit: pro-apoptotic protein programmed cell death 5 (PDCD5). 3-Cl-AHPC mediated apoptosis in MDA-MB-468 had three related effects on PDCD5: NF-κB p65 binding to the PDCD5 gene, enhanced PDCD5 promoter activity, and increased PDCD5 protein expression. Furthermore, 3-Cl-AHPC increased orphan nuclear receptor small heterodimer partner (SHP) mRNA expression, increased SHP protein bound to NF-κB p65, and found the SHP/NF-κB p65 complex attached to the PDCD5 gene. PDCD5 triggered apoptosis through increased Bax protein and release of cytochrome C from mitochondria to cytosol. Lastly, knockdown of PDCD5 protein expression blocked 3-Cl-AHPC mediated apoptosis, while over-expression of PDCD5 enhanced apoptosis, suggesting PDCD5 is necessary and sufficient for NF-κB p65 mediated apoptosis. Our results demonstrate a novel pathway for NF-κB p65 in regulating apoptosis through SHP and PDCD5.

Upregulation of miR-150* and miR-630 induces apoptosis in pancreatic cancer cells by targeting IGF-1R.

MicroRNAs have been implicated in many critical cellular processes including apoptosis. We have previously found that apoptosis in pancreatic cancer cells was induced by adamantyl retinoid-related (ARR) molecule 3-Cl-AHPC. Here we report that 3-Cl-AHPC-dependent apoptosis involves regulating a number of microRNAs including miR-150* and miR-630. 3-Cl-AHPC stimulated miR-150* expression and caused decreased expression of c-Myb and IGF-1R in the pancreatic cancer cells. 3-Cl-AHPC-mediated reduction of c-Myb resulted in diminished binding of c-Myb with IGF-1R and Bcl-2 promoters, thereby causing repression of their transcription and protein expression. Over-expression of miR-150* also resulted in diminished levels of c-Myb and Bcl-2 proteins. Furthermore, the addition of the miRNA inhibitor 2'-O-methylated miR-150 blocked 3-Cl-AHPC-mediated increase in miR-150* levels and abrogated loss of c-Myb protein. Knockdown of c-Myb in PANC-1 cells resulted in enhanced apoptosis both in the presence or absence of 3-Cl-AHPC confirming the anti-apoptotic property of c-Myb. Overexpression of miR-630 also induced apoptosis in the pancreatic cancer cells and inhibited target protein IGF-1R mRNA and protein expression. Together these results implicate key roles for miR-150* and miR-630 and their targeting of IGF-1R to promote apoptosis in pancreatic cancer cells.

Relative impact of 3- and 5-hydroxyl groups of cytosporone B on cancer cell viability.

A novel and the shortest route, thus far, for preparing cytosporone B (Csn-B) is reported. Csn-B and two analogs were used to probe the importance of hydroxyl groups at the 3- and 5-positions of the Csn-B benzene ring in inhibiting the viability of human H460 lung cancer and LNCaP prostate cancer cells, inducing H460 cell apoptosis, and interacting with the NR4A1 (TR3) ligand-binding domain (LBD). These studies indicate that Csn-B and 5-Me-Csn-B, having a phenolic hydroxyl at the 3-position of their aromatic rings, had similar activities in inhibiting cancer cell viability and in inducing apoptosis, whereas 3,5-(Me)2-Csn-B was unable to do so. These results are in agreement with ligand-binding experiments showing that the interaction with the NR4A1 LBD required the presence of the 3-hydroxyl group.

Reversal by RARα agonist Am580 of c-Myc-induced imbalance in RARα/RARγ expression during MMTV-Myc tumorigenesis.

INTRODUCTION: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently, the nuclear retinoic acid receptor (RAR) isotypes α, ß and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance, RARγ appears to be involved in stem cell compartment expansion, while RARα and RARß are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer, disrupts the balance between RARγ and RARα/ß in favor of RARγ. METHODS: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium, mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis. RESULTS: Modulation of the RARα/ß to RARγ expression in mammary glands of normal mice, oncomice, and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation, survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (~90%, P<0.001), lung metastasis (P<0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice, RARα responsive genes such as Cyp26A1, E-cadherin, cellular retinol-binding protein 1 (CRBP1) and p27, were up-regulated. In contrast, the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus, activation of the RARα pathway is linked to tumor growth inhibition, differentiation and cell death. CONCLUSIONS: The functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/ß balance suggests that prevalence of RARγ over-RARα/ß expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials.

HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic ß cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.

Adamantyl Retinoid-Related Molecules Induce Apoptosis in Pancreatic Cancer Cells by Inhibiting IGF-1R and Wnt/ß-Catenin Pathways.

Pancreatic carcinoma has a dismal prognosis as it often presents as locally advanced or metastatic. We have found that exposure to adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 resulted in growth inhibition and apoptosis induction in PANC-1, Capan-2, and MiaPaCa-2 pancreatic cancer cell lines. In addition, AHP3 and 3-Cl-AHPC inhibited growth and induced apoptosis in spheres derived from the CD44(+)/CD24(+) (CD133(+)/EpCAM(+)) stem-like cell population isolated from the pancreatic cancer cell lines. 3-Cl-AHPC-induced apoptosis was preceded by decreasing expression of IGF-1R, cyclin D1, ß-catenin, and activated Notch-1 in the pancreatic cancer cell lines. Decreased IGF-1R expression inhibited PANC-1 proliferation, enhanced 3-Cl-AHPC-mediated apoptosis, and significantly decreased sphere formation. 3-Cl-AHPC inhibited the Wnt/ß-catenin pathway as indicated by decreased ß-catenin nuclear localization and inhibited Wnt/ß-catenin activation of transcription factor TCF/LEF. Knockdown of ß-catenin using sh-RNA also induced apoptosis and inhibited growth in pancreatic cancer cells. Thus, 3-Cl-AHPC and AHP3 induce apoptosis in pancreatic cancer cells and cancer stem-like cells and may serve as an important potential therapeutic agent in the treatment of pancreatic cancer.

The retinoid X receptors and their ligands.

This chapter presents an overview of the current status of studies on the structural and molecular biology of the retinoid X receptor subtypes α, ß, and γ (RXRs, NR2B1-3), their nuclear and cytoplasmic functions, post-transcriptional processing, and recently reported ligands. Points of interest are the different changes in the ligand-binding pocket induced by variously shaped agonists, the communication of the ligand-bound pocket with the coactivator binding surface and the heterodimerization interface, and recently identified ligands that are natural products, those that function as environmental toxins or drugs that had been originally designed to interact with other targets, as well as those that were deliberately designed as RXR-selective transcriptional agonists, synergists, or antagonists. Of these synthetic ligands, the general trend in design appears to be away from fully aromatic rigid structures to those containing partial elements of the flexible tetraene side chain of 9-cis-retinoic acid. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Analogues of orphan nuclear receptor small heterodimer partner ligand and apoptosis inducer (E)-4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid. 2. Impact of 3-chloro group replacement on inhibition of proliferation and induction of apoptosis of leukemia and cancer cell lines.

The parent phenol of adapalene and its (E)-cinnamic acid analogue were found to induce cancer cell apoptosis but cause adverse systemic effects when administered to mice. In contrast, their respective 5-Cl- and 3-Cl-substituted analogues had their adverse effects mitigated without a comparable loss of cancer cell inhibitory activity. As a result, pharmacologic space in this region of the cinnamic phenyl ring scaffold was explored. Various substituents were introduced, and their effects on cancer cell proliferation and viability were evaluated. Cinnamic acids having 3-Br, CN, NO(2), NH(2), OMe, and N(3) groups had activity comparable to that of 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid. A comparative molecular field analysis study indicated that introduction of an H-bond acceptor at position 3 of the central phenyl ring would favor inhibition of leukemia cell viability, and docking suggested its hydrogen bonding with a polar group in a small heterodimer partner homology model. The 3-CN, NO(2), NH(2), and OH analogues also inhibited MMTV-Wnt1 murine mammary stem cell viability.

Heteroatom-substituted analogues of orphan nuclear receptor small heterodimer partner ligand and apoptosis inducer (E)-4-[3-(1-Adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid.

(E)-4-[3'-(1-Adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces the cell cycle arrest and apoptosis of cancer cells. Because its pharmacologic properties-solubility, bioavailability, and toxicity-required improvement for translation, structural modifications were made by introducing nitrogen atoms into the cinnamyl ring and replacing its E-double bond with XCH(2) (X = O, N, and S) with the objective of enhancing these properties without impacting apoptosis-inducing activity. Analogues having nitrogen atoms in heterocyclic rings corresponding to the cinnamyl phenyl ring displayed equal or higher biological activities. The pyrimidine and pyridine analogues were more soluble in both phosphate-buffered saline and water. While the 2,5-disubstituted pyridine analogue was the most potent inducer of KG-1 acute myeloid leukemia cell apoptosis, on the basis of apoptotic activity in KG-1 cells and solubility, the 2,5-disubstituted pyrimidine proved to be the more promising candidate for treatment of acute myeloid leukemia.

A strong case for personalized, targeted cancer prevention.

The study reported by Lee and colleagues in this issue of the journal (beginning on page 185) incorporated global genetic variation within a new assessment of the outcome of a previously reported phase-III trial of low-dose 13-cis-retinoic acid (13-cRA) for preventing second primary tumors (SPT) or the recurrence of head-and-neck cancer. This analysis identified genotypes of common single-nucleotide polymorphisms (SNP) and cumulative effect and potential gene-gene interactions that were highly associated with increased placebo-arm risk (prognostic) and/or with reduced treatment-arm risk and longer event-free survival (predictive). For example, the wild-type rs3118570 SNP of the retinoid X receptor alpha gene (carried by 71% of the 13-cRA trial population) marked a 3.33-fold increased SPT/recurrence risk in the placebo arm and a 38% reduced risk in the treatment arm. Adding two other informative genotypes strengthened the treatment-arm risk reduction to 76%, although the genotype trio reflected only 13% of the trial population. This report extends the concept of personalized therapy to cancer prevention.

Adamantyl-substituted retinoid-related molecules induce apoptosis in human acute myelogenous leukemia cells.

The adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 induce apoptosis in vitro and in vivo in a newly established human acute myelogenous leukemia (AML) cell line, FFMA-AML, and in the established TF(v-SRC) AML cell line. FFMA-AML and TF(v-SRC) cells displayed resistance to apoptosis mediated by the standard retinoids (including trans-retinoic acid, 9-cis-retinoic acid, and the synthetic retinoid TTNPB) but showed sensitivity to apoptosis mediated by 3-Cl-AHPC- and AHP3 in vitro and in vivo as documented by poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. 3-Cl-AHPC or AHP3 exposure in vitro resulted in decreased expression of the antiapoptotic proteins (cellular inhibitor of apoptosis 1, X-linked inhibitor of apoptosis protein) and phospho-Bad and activated the NF-κB canonical pathway. A significant prolongation of survival was observed both in nonobese diabetic severe combined immunodeficient mice carrying FFMA-AML cells and treated with either 3-Cl-AHPC or AHP3 and in severe combined immunodeficient mice carrying TF(v-SRC) cells and treated with AHP3. We have previously shown that ARRs bind to the orphan nuclear receptor small heterodimer partner (SHP) and that the expression of SHP is required for ARR-mediated apoptosis. Induced loss of SHP in these AML cells blocked 3-Cl-AHPC- and AHP3-mediated induction of apoptosis. These results support the further development of 3-Cl-AHPC and AHP3 as potential therapeutic agents in the treatment of AML patients.

Novel cis-trans interactions are involved in post-transcriptional regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 mRNA.

BACKGROUND: A variety of pathways target CDKI p21WAF1/CIP1 expression at transcriptional, post-transcriptional as well as translational levels. We previously found that cell growth suppressing retinoid CD437 enhanced expression of p21WAF1/CIP1 and DNA damage inducible GADD45 proteins in part by elevating their mRNA stability. RESULTS: Here, we investigated molecular mechanisms of CD437-dependent post-transcriptional regulation of p21WAF1/CIP1 expression. By utilizing MDA-MB-468 HBC cells expressing chimeric rabbit beta-globin-p21WAF1/CIP1 transcripts we mapped multiple CD437-responsive sequences located within positions 1195 to 1795 of the 3'-untranslated region of p21WAF1/CIP1 mRNA. Several cytoplasmic proteins present in MDA-MB-468, MCF-7 HBC as well as HL-60R leukemia cells bound specifically, in vitro, with these CD437-responsive sequences. CD437 treatment of cells resulted in elevated binding of ~85 kD and ~55 kD cytoplasmic proteins with putative CD437-responsive sequences. A 12 nt RNA sequence (5'-UGUGGUGGCACA-3') present within CD437-responsive region of p21WAF1/CIP1 mRNA displayed specific and elevated binding with the above noted proteins. Treatment of cells with ActD or CHX prior to CD437 exposure did not abrogate RNA-protein interactions. However, treatment of cytoplasmic protein extracts with proteinase K or alkaline phosphatase resulted in loss of RNA-protein interactions. CONCLUSIONS: CD437 regulates cell growth in part by regulating stability of p21WAF1/CIP1 mRNA that involves specific RNA-protein interactions that are phosphorylation-dependent, while not requiring nascent transcription or protein synthesis.

The peptidomimetic, 1-adamantyl-substituted, and flex-het classes of retinoid-derived molecules: structure-activity relationships and retinoid receptor-independent anticancer activities.

Increasing evidence demonstrates that three classes of molecules originally derived from all-trans-retinoic acid and its synthetic analogues, which function by interacting with the retinoid nuclear receptors, exert their anticancer activities through alternative signaling pathways. Thus, the methylene-linked analogues (4-HBR, 4-HPRCG, and 4-HBRCG) of N-(4-hydroxyphenyl) retinamide (4-HPR) and its O-glucuronide metabolite (4-HPROG), the cinnamic acid analogues (3-Cl-AHPC and AHPC/ST1926) of 6-[3'-(1-adamantyl)-4'-hydroxyphenyl)]-2-naphthalenecarboxylic acid, and N-(2,3-dihydro-2,2,4,4-tetramethyl-6-benzothiopyranyl),N'-(4-nitrophenyl)thiourea (SHetA2) induce cancer cell-cycle arrest and apoptosis mediated most likely through mitochondrial and/or endoplasmic reticulum stress responses. Structure-activity relationships and potential for clinical translation as anticancer therapeutics are presented.

Inhibition of mammary carcinoma cell growth by RXR is mediated by the receptor's oligomeric switch.

Ligands that activate the nuclear receptor retinoid X receptor (RXR) display potent anticarcinogenic activities, but the mechanisms by which these compounds inhibit carcinoma cell growth are poorly understood. While RXR can regulate gene expression due to its intrinsic ligand-activated transcription function, this receptor can also regulate transcription by functioning as a ligand-controlled DNA architectural factor. It was thus reported that apo-RXR self-associates into tetramers and that each dimer within these tetramers can separately bind to an RXR response element. Hence, DNA binding by RXR tetramers may bring distant genomic regions into close physical proximity. As ligand binding induces the dissociation of RXR tetramers into dimers, it can alter gene expression by modulating the DNA architecture. Here, we show that inhibition of mammary carcinoma cell growth by RXR ligands stems from the ability of these compounds to regulate the oligomeric state of RXR and is independent of the direct intrinsic transcriptional activity of the receptor. The data suggest that compounds that trigger dissociation of RXR tetramers may comprise a novel class of anticarcinogenic agents.

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand-binding domain.

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand-binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRalpha LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator-activated receptor gamma (PPARgamma) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line-broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo- or hetero-dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor-interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRalpha LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C-terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line-broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRalpha LBD-antagonist-peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRalpha LBD conformations in favor of a compact state, and that a PPARgamma LBD-agonist complex can prevent the antagonist from enhancing the RXRalpha LBD-coactivator binding interaction.

SHP and Sin3A expression are essential for adamantyl-substituted retinoid-related molecule-mediated nuclear factor-kappaB activation, c-Fos/c-Jun expression, and cellular apoptosis.

We previously found that the adamantyl-substituted retinoid-related molecules bind to the small heterodimer partner (SHP) as well as the Sin3A complex. In this report, we delineated the role of SHP and the Sin3A complex in 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC)-mediated inhibition of cell growth and apoptosis. We examined the effect of loss of SHP and Sin3A expression in a number of cell types on 3-Cl-AHPC-mediated growth inhibition and apoptosis induction, 3-Cl-AHPC-mediated nuclear factor-kappaB (NF-kappaB) activation, and 3-Cl-AHPC-mediated increase in c-Fos and c-Jun expression. We found that loss of SHP or Sin3A expression, while blocking 3-Cl-AHPC-mediated apoptosis, had little effect on 3-Cl-AHPC inhibition of cellular proliferation. We have previously shown that 3-Cl-AHPC-mediated NF-kappaB activation is necessary for apoptosis induction. We have now shown that 3-Cl-AHPC-enhanced c-Fos and c-Jun expression is also essential for maximal 3-Cl-AHPC-mediated apoptosis. 3-Cl-AHPC induction of c-Fos and c-Jun expression as well as NF-kappaB activation was dependent on SHP protein levels. In turn, SHP levels are regulated by Sin3A because ablation of Sin3A resulted in a decrease in SHP expression. Thus, SHP and Sin3A play an important role in adamantyl-substituted retinoid-related induction of cellular apoptosis.

Studies of cannabinoid-1 receptor antagonists for the treatment of obesity: hologram QSAR model for biarylpyrazolyl oxadiazole ligands.

Hologram QSAR studies were conducted on a series of 60 training set of cannabinoid-1 receptor (CB(1)) antagonists. Significant cross-validated correlation coefficients (q(2)=0.763) and noncross-validated correlation coefficients (r(2)=0.897) were obtained. The model was then employed to predict the biological activities of 15 test set compounds, and a good agreement between the experimental and predicted values was verified exhibiting a powerful predictable capability of this model (q(pred)(2)=0.868). Contribution map shows that 1,2,4-trizole and cyclopropane moieties make big contributions for the activities. Both the HQSAR model and analysis from the contribution map should be useful for the further design of novel structurally related CB(1) antagonists.

Derivation of a retinoid X receptor scaffold from peroxisome proliferator-activated receptor gamma ligand 1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene.

PPARgamma agonist DIM-Ph-4-CF(3), a template for RXRalpha agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl] acrylic acid: DIM-Ph-CF(3) is reported to inhibit cancer growth independent of PPARgamma and to interact with NR4A1. As both receptors dimerize with RXR, and natural PPARgamma ligands activate RXR, DIM-Ph-4-CF(3) was investigated as an RXR ligand. It displaces 9-cis-retinoic acid from RXRalpha but does not activate RXRalpha. Structure-based direct design led to an RXRalpha agonist.1-Di(1H-indol-3-yl)methyl-4-trifluoromethylbenzene (DIM-Ph-4-CF(3)) is reported to inhibit cancer cell growth and to act as a transcriptional agonist of peroxisome proliferator-activated receptor gamma (PPARgamma) and nuclear receptor 4A subfamily member 1 (NR4A1). In addition, DIM-Ph-4-CF(3) exerts anticancer effects independent of these receptors because PPARgamma antagonists do not block its inhibition of cell growth, and the small pocket in the NR4A1 crystal structure suggests no ligand can bind. Because PPARgamma and NR4A1 heterodimerize with retinoid X receptor (RXR), and several PPARgamma ligands transcriptionally activate RXR, DIM-Ph-4-CF(3) was investigated as an RXR ligand. DIM-Ph-4-CF(3) displaces 9-cis-retinoic acid from RXRalpha but does not transactivate RXRalpha. Structure-based design using DIM-Ph-4-CF(3) as a template led to the RXRalpha transcriptional agonist (E)-3-[5-di(1-methyl-1H-indol-3-yl)methyl-2-thienyl]acrylic acid. Its docked pose in the RXRalpha ligand binding domain suggests that binding is stabilized by interactions of its carboxylate group with arginine 316, its indoles with cysteines 269 and 432, and its 1-methyl groups with hydrophobic residues lining the binding pocket. As is expected of a selective activator of RXRalpha, but not of RARs and PPARgamma, this RXRalpha agonist, unlike DIM-Ph-4-CF(3), does not appreciably decrease cancer cell growth or induce apoptosis at pharmacologically relevant concentrations.