EADB: an estrogenic activity database for assessing potential endocrine activity.

Endocrine-active chemicals can potentially have adverse effects on both humans and wildlife. They can interfere with the body's endocrine system through direct or indirect interactions with many protein targets. Estrogen receptors (ERs) are one of the major targets, and many endocrine disruptors are estrogenic and affect the normal estrogen signaling pathways. However, ERs can also serve as therapeutic targets for various medical conditions, such as menopausal symptoms, osteoporosis, and ER-positive breast cancer. Because of the decades-long interest in the safety and therapeutic utility of estrogenic chemicals, a large number of chemicals have been assayed for estrogenic activity, but these data exist in various sources and different formats that restrict the ability of regulatory and industry scientists to utilize them fully for assessing risk-benefit. To address this issue, we have developed an Estrogenic Activity Database (EADB; http://www.fda.gov/ScienceResearch/BioinformaticsTools/EstrogenicActivityDatabaseEADB/default.htm) and made it freely available to the public. EADB contains 18,114 estrogenic activity data points collected for 8212 chemicals tested in 1284 binding, reporter gene, cell proliferation, and in vivo assays in 11 different species. The chemicals cover a broad chemical structure space and the data span a wide range of activities. A set of tools allow users to access EADB and evaluate potential endocrine activity of chemicals. As a case study, a classification model was developed using EADB for predicting ER binding of chemicals.

U.s. Food and Drug Administration approval: carfilzomib for the treatment of multiple myeloma.

The U.S. Food and Drug Administration (FDA) review leading to accelerated approval of carfilzomib is described. A single-arm trial enrolled 266 patients with multiple myeloma refractory to the most recent therapy who had received prior treatment with bortezomib and an immunomodulatory agent (IMID). Patients received carfilzomib by intravenous infusion over 2 to 10 minutes at a dose of 20 mg/m2 on days 1, 2, 8, 9, 15, and 16 of the 28 days of cycle 1, and at a dose of 27 mg/m2 on the same schedule in cycle 2 and subsequent cycles. The primary efficacy endpoint was overall response rate (ORR) as determined by an independent review committee using International Myeloma Working Group Uniform Response Criteria. The safety of carfilzomib was evaluated in 526 patients with multiple myeloma treated with various dosing regimens. The ORR was 23%. The median duration of response was 7.8 months. The most common adverse reactions associated with carfilzomib infusion were fatigue, anemia, nausea, thrombocytopenia, dyspnea, diarrhea, and fever. The most common serious adverse events were pneumonia, acute renal failure, fever, and congestive heart failure. Infusion reactions to carfilzomib could be reduced by pretreatment with dexamethasone and intravenous fluids. On July 20, 2012, the FDA granted accelerated approval of carfilzomib for the treatment of patients with multiple myeloma who have received at least two prior therapies including bortezomib and an IMID and who have shown disease progression while on therapy or within 60 days of completion of the last therapy.

Activated glucocorticoid and eicosanoid pathways in endometriosis.

OBJECTIVE: To define altered gene expression networks in endometriosis. DESIGN: Experiments using endometriotic tissues and primary cells. SETTING: Division of Reproductive Biology Research, Northwestern University. PATIENT(S): Premenopausal women. INTERVENTION(S): Matched samples of eutopic endometrium and ovarian endometriosis (n = 8 patients) were analyzed by microarray and verified in a separate set of tissues (n = 6 patients). Experiments to define signaling pathways were performed in primary endometriotic stromal cells (n = 12 patients). MAIN OUTCOMES MEASURE(S): Using a genome-wide in vivo approach, we identified 1,366 differentially expressed genes and a new gene network favoring increased glucocorticoid levels and action in endometriosis. RESULT(S): Transcript and protein levels of 11ß-hydroxysteroid dehydrogenase (HSD11B1), which produces cortisol, the biologically active glucocorticoid, were strikingly higher, whereas messenger RNA (mRNA) levels of the cortisol-degrading HSD11B2 enzyme were significantly lower in endometriotic tissue. Glucocorticoid receptor mRNA and protein levels were significantly higher in endometriosis. The inflammatory cytokine tumor necrosis factor robustly induced mRNA and protein levels of HSD11B1 and glucocorticoid receptor but suppressed HSD11B2 mRNA in primary endometriotic stromal cells, suggesting that tumor necrosis factor stimulates cortisol production and action. We also uncovered a subset of genes critical for prostaglandin synthesis and degradation, which favor high eicosanoid levels and activity in endometriosis. CONCLUSION(S): The proinflammatory milieu of the endometriotic lesion stimulates cortisol synthesis and action in endometriotic lesions.

Improving the developability profile of pyrrolidine progesterone receptor partial agonists.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

2-Amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[2',3':4,5]thieno[2,3-b]pyridine derivatives as selective progesterone receptor agonists.

High throughput screening of the corporate compound collection led to the identification of a novel series of 2-amino-9-aryl-3-cyano-4-methyl-7-oxo-6,7,8,9-tetrahydropyrido[2',3':4,5]thieno[2,3-b]pyridine derivatives as selective PR agonists. Initial SAR exploration leading to potent and selective agonists 9 and 11, X-ray crystal structure of 9 bound to PR-LBD and preliminary developability data are described.

Estrogen-induced stromal cell-derived factor-1 (SDF-1/Cxcl12) expression is repressed by progesterone and by Selective Estrogen Receptor Modulators via estrogen receptor alpha in rat uterine cells and tissues.

Endometriosis, defined as the presence of endometrial glands and stroma at extra-uterine sites, is a gynecological condition that affects women of reproductive age. Consistent with its uterine origins, endometriotic lesions and resulting symptoms are hormonally responsive. To investigate Progesterone Receptor (PR)-based therapies, we measured physiological endpoints and gene expression in rat models of uterine cell estrogenic activity. Estrogen-induced ELT-3 rat leiomyoma cell proliferation was significantly inhibited by progesterone (P4), while the antiprogestin RU486 or the Selective PR Modulator (SPRM) asoprisnil, did not block proliferation. Stromal cell-derived factor-1 (SDF-1/Cxcl12) gene expression was induced by estrogen, and was repressed by the Selective Estrogen Receptor Modulators (SERMs), the antiestrogen ICI 182,780, and P4, but not by RU486 or the ERbeta-selective ligand ERB-041. In ELT-3 cells, asoprisnil demonstrated partial PR agonism on SDF-1 gene repression. Magnetic Resonance Imaging was used to monitor development of ectopic cysts in a rat surgical model of endometriosis. SERMs and P4 significantly decreased cyst volumes comparably by approximately 60%. However, ERB-041 and asoprisnil had no effect on cyst volume, and RU486 increased cyst volume by 20%. SDF-1 expression was modestly, but significantly, increased in the cyst compared to eutopic uterus, and P4 and raloxifene could repress the expression. We showed that SDF-1 was similarly regulated in human cells. These data suggest that transcriptional regulation of SDF-1 is a surrogate marker of estrogenic activities via ERalpha in rat uterine cells, and that SDF-1 repression by PR agonists can predict the ability to oppose the actions of estrogen in vivo.

Discovery of orally active, pyrrolidinone-based progesterone receptor partial agonists.

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity against hERG channel inhibition.

Rational design of orally-active, pyrrolidine-based progesterone receptor partial agonists.

Using the X-ray crystal structure of an amide-based progesterone receptor (PR) partial agonist bound to the PR ligand binding domain, a novel PR partial agonist class containing a pyrrolidine ring was designed. Members of this class of N-alkylpyrrolidines demonstrate potent and highly selective partial agonism of the progesterone receptor, and one of these analogs was shown to be efficacious upon oral dosing in the OVX rat model of estrogen opposition.

Synthesis and SAR of amino acid-derived heterocyclic progesterone receptor full and partial agonists.

Two classes of amino acid-derived heterocyclic progesterone receptor ligands were developed to address the metabolic issues posed by the dimethyl amide functionality of the lead compound (1). The tetrazole-derived ligands behaved as potent partial agonists, while the 1,2,4-triazole ligands behaved as potent full agonists.

Development of a small-molecule serum- and glucocorticoid-regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic.

Androgens, through their actions on the androgen receptor (AR), are required for the development of the prostate and contribute to the pathologic growth dysregulation observed in prostate cancers. Consequently, androgen ablation has become an essential component of the pharmacotherapy of prostate cancer. In this study, we explored the utility of targeting processes downstream of AR as an alternate approach for therapy. Specifically, we show that the serum and glucocorticoid-regulated kinase 1 (SGK1) gene is an androgen-regulated target gene in cellular models of prostate cancer. Furthermore, functional serum- and glucocorticoid-regulated kinase 1 (SGK1) protein, as determined by the phosphorylation of its target Nedd4-2, was also increased with androgen treatment. Importantly, we determined that RNA interference-mediated knockdown of SGK1 expression attenuates the androgen-mediated growth of the prostate cancer cell line LNCaP. Given these findings, we explored the utility of SGK1 as a therapeutic target in prostate cancer by developing and evaluating a small-molecule inhibitor of this enzyme. From these studies emerged GSK650394, a competitive inhibitor that quantitatively blocks the effect of androgens on LNCaP cell growth. Thus, in addition to androgen ablation, inhibition of pathways downstream of AR is likely to have therapeutic utility in prostate cancer.

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator.

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

Noninvasive assessment of ectopic uterine tissue development in rats using magnetic resonance imaging.

OBJECTIVE: To non-invasively characterize ectopic uterine tissue (EUT) development in a modified autologous rat surgical model of endometriosis using magnetic resonance imaging (MRI). DESIGN: Investigational MRI study. SETTING: A pharmaceutical company. ANIMAL(S): Female Sprague Dawley rats. INTERVENTION(S): Uterine tissue was autotransplanted on the right peritoneal wall of rats. Rats were serially imaged after surgery and after endogenous hormone suppression, hormone supplementation, or ovariectomy. In addition, an MRI contrast agent was administered to examine EUT perfusion characteristics. MAIN OUTCOME MEASURE(S): Changes in transplanted EUT volume and perfusion were monitored using MRI. RESULT(S): The EUT growth could be readily monitored non-invasively by MRI. Although EUT growth was rapid during the initial 4 days after surgery, volume stabilized by the third week and maintained for at least 9 weeks after transplantation. The EUT volumes varied with the estrous cycle and were hormonally sensitive to ovariectomy, to Antide (GnRH antagonist), and to Antide followed by 17beta-E(2) supplementation. The use of an MRI contrast agent facilitated visualization of EUT wall perfusion. CONCLUSION(S): MRI allows for noninvasive, dynamic evaluation of transplanted EUT growth in the rat. This reproducible model will allow for performing quantifiable pharmacologic studies in pre-clinical drug discovery for therapies targeting endometriosis.

Quantitative analysis of gene regulation by seven clinically relevant progestins suggests a highly similar mechanism of action through progesterone receptors in T47D breast cancer cells.

Progesterone (P4) is an essential reproductive steroid hormone required for many aspects of female reproductive physiology. Progestins are compounds that demonstrate progesterone-like activity and are used in oral contraception, hormone therapy, and treatment of some reproductive disorders, but differ widely in their chemical structures, potency, and pharmacokinetics. While numerous studies have assessed progestins on specific endpoints, little is known about the activation of global gene expression by progestins. We used Affymetrix GeneChip U133A expression arrays to examine the action of P4 and six clinically relevant synthetic progestins (3-ketodesogestrel, drospirenone, levonorgestrel, medroxyprogesterone acetate, norethindrone acetate, and trimegestone) on the progesterone receptor (PR)-positive T47Dco and the PR-negative T47D-Y breast cancer cell lines. Excluding drospirenone, one or more of the progestins-regulated 329 genes, with 30 genes regulated by at least 2.0-fold by all progestins in the T47Dco cells. The synthetic progestins show a high degree of similarity in their transcriptional responses, and each progestin regulates between 77 and 91% of the genes regulated by P4. Independent quantitative RT-PCR analysis confirmed a similar regulation for S100P, PPL, IL20RA, NET1, ATP1A1, HIG2, and CXCL12 (SDF-1) by all seven progestins. Attempts to find differentially regulated genes by any progestin compared to all other treatments failed, suggesting any differences are quantitative, not qualitative. This analysis demonstrates a high degree of similarity among these progestins on PR-regulated gene expression in T47D cells, suggesting a similar and fairly specific mode of action.

KIF2Abeta: A kinesin family member enriched in mouse male germ cells, interacts with translin associated factor-X (TRAX).

Translin associated factor X (TRAX) is a binding partner of TB-RBP/Translin. A cDNA encoding the 260 C-terminal amino acids of KIF2Abeta was isolated from mouse testis cDNAs in a yeast two-hybrid library screen for specific TRAX-interacting proteins. KIF2Abeta was expressed predominantly in the mouse testis and enriched in germ cells. The interaction of full-length KIF2Abeta or its C-terminus with TRAX was verified using in vitro synthesized fusion proteins. Deletion mapping of the TRAX-binding region of KIF2Abeta indicated that amino acids 514-659 were necessary and sufficient for the interaction in vivo. Confocal microscopy studies using GFP-fusion proteins demonstrated that KIF2Abeta colocalizes with TRAX in a perinuclear location. KIF2Abeta does not interact with TB-RBP, suggesting that either TRAX can function as an adaptor molecule for motor proteins and TB-RBP, or that this interaction reveals an undescribed role for TRAX in germ cells. The interaction with KIF2Abeta suggests a role for TRAX in microtubule-based functions during spermatogenesis.

Regulation of gene expression by PRA-910, a novel progesterone receptor modulator, in T47D cells.

Progestins play an important role in women's health and are used in oral contraception, hormone therapy, and treatment of reproductive disorders. The effects of progestins upon gene expression in breast epithelium are poorly understood. In an attempt to characterize the molecular mechanism of progestin action, we used a gene expression profiling approach to examine the action of a novel progestin in the T47D cell model, a human breast cancer cell line. PRA-910 is a novel, nonsteroidal progesterone receptor modulator (PRM) with species-specific activities identified in a screen for selective PRMs. To understand the mechanism of action for PRA-910 in T47D cells, we compared its gene regulation to progesterone (P4) and RU486 through Affymetrix U95A GeneChip analysis and TaqMan RT-PCR. PRA-910, P4, and RU486 regulated 50, 108, and 16 genes by threefold or greater versus vehicle, respectively, with 18 genes having similar regulation for P4 and PRA-910. These data confirm and extend previous findings for T47D cells. We also obtained time course, concentration-response, cyclohexamide sensitivity, and PR-specificity data for two progestin-regulated genes, ATP1A1 and CLDN8. Our data demonstrate that PRA-910 has a unique gene regulation profile distinct from both P4 and RU486. Further investigation of the underlying mechanism for these differences is ongoing.

Identification and characterization of cDNAs encoding four novel proteins that interact with translin associated factor-X.

Translin-associated factor X (TRAX) is the predominantly cytoplasmic binding partner of TB-RBP/translin in mouse testis. Four mouse testis cDNAs encoding specific TRAX-interacting proteins were isolated from a yeast two-hybrid library screen. One novel cDNA designated Tsnaxip1 (TRAX-interacting protein-1) encodes 709 amino acids. We isolated a cDNA encoding the 427 carboxy-terminal amino acids of MEA-2, a Golgi-associated, maleenhanced autoantigen; a cDNA encoding 429 amino acids with 73% homology to centrosomal Akap9; and a cDNA encoding 346 amino acids with 75% homology to SUN1, a predicted human protein that contains a SUN domain (which is present in some perinuclear proteins). Interactions were verified using in vitro synthesized fusion proteins. All four genes were expressed in the testis and enriched in germ cells. Confocal microscopy studies using green fluorescent protein fusion proteins determined that these TRAX-interacting proteins colocalize with TRAX. The data suggest that TRAX may have a function associated with perinuclear organelles during spermatogenesis.