Pioneering government-sponsored drug repositioning collaborations: progress and learning.

A new model for translational research and drug repositioning has recently been established based on three-way partnerships between public funders, the pharmaceutical industry and academic investigators. Through two pioneering initiatives - one involving the Medical Research Council in the United Kingdom and one involving the National Center for Advancing Translational Sciences of the National Institutes of Health in the United States - new investigations of highly characterized investigational compounds have been funded and are leading to the exploration of known mechanisms in new disease areas. This model has been extended beyond these first two initiatives. Here, we discuss the progress to date and the unique requirements and challenges for this model.

Overcoming obstacles to repurposing for neurodegenerative disease.

Repurposing Food and Drug Administration (FDA)-approved drugs for a new indication may offer an accelerated pathway for new treatments to patients but is also fraught with significant commercial, regulatory, and reimbursement challenges. The Alzheimer's Drug Discovery Foundation (ADDF) and the Michael J. Fox Foundation for Parkinson's Research (MJFF) convened an advisory panel in October 2013 to understand stakeholder perspectives related to repurposing FDA-approved drugs for neurodegenerative diseases. Here, we present opportunities on how philanthropy, industry, and government can begin to address these challenges, promote policy changes, and develop targeted funding strategies to accelerate the potential of FDA-approved repurposed drugs.

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Identification of novel estrogen receptor alpha antagonists.

We have identified novel estrogen receptor alpha (ERalpha) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERalpha ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERalpha LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-beta-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-beta-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.

Full-length estrogen receptor alpha and its ligand-binding domain adopt different conformations upon binding ligand.

The binding of ligand to a nuclear receptor causes conformational changes that can result in coactivator or corepressor recruitment and subsequent regulation of transcription. Several peptides have previously been identified that bind to the liganded estrogen receptor (ER). One interacting peptide, pepalphaII, was used in the present studies to assess the ability of ligands to induce spatial changes within both the full-length human estrogen receptor alpha (ER-alpha) and a truncated receptor containing the ligand-binding domain (LBD). pepalphaII interacted weakly with the full-length estrogen receptor alpha in the presence of both agonists and antagonists. In contrast, the interaction of pepalphaII with the truncated receptor containing the ligand-binding domain was strongly induced by antagonists and only weakly induced by agonists. Thus, the same ligand can induce different spatial configurations of the full-length and ligand-binding domain of estrogen receptor alpha as measured by pepalphaII affinity. Crystal structures of nuclear hormone receptors solved to date have used ligand-binding domains and therefore may not accurately predict surface interaction domains present in the liganded full-length receptor. Furthermore, the ability of a ligand to induce a strong interaction of pepalphaII with the estrogen receptor alpha ligand-binding domain predicts that the ligand will have greater antagonist activity on the full-length receptor.

Evaluation of an estrogen receptor-beta agonist in animal models of human disease.

The discovery of a second estrogen receptor (ER), called ERbeta, in 1996 sparked intense interest within the scientific community to discover its role in mediating estrogen action. However, despite more than 6 yr of research into the function of this receptor, its physiological role in mediating estrogen action remains unclear and controversial. We have developed a series of highly selective agonists for ERbeta and have characterized their activity in several clinically relevant rodent models of human disease. The activity of one such compound, ERB-041, is reported here. We conclude from these studies that ERbeta does not mediate the bone-sparing activity of estrogen on the rat skeleton and that it does not affect ovulation or ovariectomy-induced weight gain. In addition, these compounds are nonuterotrophic and nonmammotrophic. However, ERB-041 has a dramatic beneficial effect in the HLA-B27 transgenic rat model of inflammatory bowel disease and the Lewis rat adjuvant-induced arthritis model. Daily oral doses as low as 1 mg/kg reverse the chronic diarrhea of HLA-B27 transgenic rats and dramatically improve histological disease scores in the colon. The same dosing regimen in the therapeutic adjuvant-induced arthritis model reduces joint scores from 12 (maximal inflammation) to 1 over a period of 10 d. Synovitis and Mankin (articular cartilage) histological scores are also significantly lowered (50-75%). These data suggest that one function of ERbeta may be to modulate the immune response, and that ERbeta-selective ligands may be therapeutically useful agents to treat chronic intestinal and joint inflammation.

Roles of NPM2 in chromatin and nucleolar organization in oocytes and embryos.

Upon fertilization, remodeling of condensed maternal and paternal gamete DNA occurs to form the diploid genome. In Xenopus laevis, nucleoplasmin 2 (NPM2) decondenses sperm DNA in vitro. To study chromatin remodeling in vivo, we isolated mammalian NPM2 orthologs. Mouse NPM2 accumulates in oocyte nuclei and persists in preimplantation embryos. Npm2 knockout females have fertility defects owing to failed preimplantation embryo development. Although sperm DNA decondensation proceeds without NPM2, abnormalities are evident in oocyte and early embryonic nuclei. These defects include an absence of coalesced nucleolar structures and loss of heterochromatin and deacetylated histone H3 that normally circumscribe nucleoli in oocytes and early embryos, respectively. Thus, Npm2 is a maternal effect gene critical for nuclear and nucleolar organization and embryonic development.

Estradiol differentially regulates lipocalin-type prostaglandin D synthase transcript levels in the rodent brain: Evidence from high-density oligonucleotide arrays and in situ hybridization.

Microarrays comprise an efficient approach to discovering large numbers of differentially expressed mRNA transcripts in the CNS resulting from changes in hormonal milieu. We used high-density oligonucleotide microarrays to examine the short- and long-term actions of estradiol (E(2)) on the transcriptomes from the medial basal hypothalamus and other brain regions of E(2)-treated (10 microg) adult female mice. Our results have revealed several unanticipated gene regulations. Most striking is lipocalin prostaglandin D(2) synthase (L-PGDS), which catalyzes the conversion of prostaglandin (PG) H(2) to PGD(2), a neuromodulator involved in a variety of functions, including sleep, pain, and odor responses. In situ hybridization revealed significant increases in L-PGDS expression in the arcuate and ventromedial nucleus of the medial basal hypothalamus compared with vehicle controls. The magnitude of these changes is approximately 2-fold and suggests a modulatory role for PGD(2) in E(2)-controlled neuroendocrine secretions and behaviors. Surprisingly, L-PGDS gene expression is reduced 2-fold after E(2) treatment in the ventrolateral preoptic area (VLPO), the suspected site of action for the sleep-promoting effects of PGD(2). Finally, whereas L-PGDS has been reported to be expressed primarily in oligodendrocytes of the adult rodent brain, we demonstrate, immunocytochemically, that L-PGDS is also expressed in a population of VLPO neurons. Thus, our data suggest the intriguing possibility that E(2) modulation of L-PGDS plays a role in the regulation of sleep-wake states through hitherto unknown mechanisms in VLPO neurons and through hormone-dependent neuronal-glial cooperation.

Characterization of a membrane-associated estrogen receptor in a rat hypothalamic cell line (D12).

The ability of estrogens to produce rapid changes in cellular function has been firmly established. The question remains whether these changes are mediated by a modified form of the nuclear estrogen receptor (ER) that is associated with the plasma membrane (mER) or by a completely novel membrane receptor. Therefore, we characterized the biochemical properties of the nuclear and membrane-associated ERs expressed endogenously in a rat hypothalamic endothelial cell line (D12). Radioligand binding experiments using D12 membrane fractions showed that these cells exhibit properties consistent with a binding site specific for estrogens (mER). Equilibrium binding assays using [125I]16-alpha-iodo-3,17- beta-estradiol revealed saturable binding to mER, an affinity value similar to nuclear ER, with differing receptor expression levels. Competition assays revealed that 9 of 12 ER ligands tested had comparable affinities for mER and ER. For example, 17-alpha-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for raloxifene, 17beta-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for ERalpha confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERalpha that colocalized with caveolae-enriched membranes. A rapid increase in intracellar Ca2+ levels in the presence of E2 suggests that mER can mediate physiologic changes through calcium mobilization. These data support the expression of mER in these brain-derived endothelial cells that is similar to, but biochemically distinguishable from, nuclear ERalpha.

Identification and characterization of a selective, nonpeptide follicle-stimulating hormone receptor antagonist.

The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-[4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene]-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [(125)I]hFSH with an IC(50) value of 5.4 +/- 2.3 micro M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [(125)I]hFSH binding to BV-tFSHR with an IC(50) value of 10 +/- 2.8 micro M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC(50) value of 3 +/- 0.6 micro M. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.

The ligand binding profiles of estrogen receptors alpha and beta are species dependent.

Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-alpha and ER-beta. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-alpha and ER-beta. Certain compounds show species dependent binding preferences for ER-alpha or ER-beta, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.