Biomedical subjects
Publications and source records attributed to S P Rohrer.
Synthesis of a series of fused pyrazole tetrahydrofluorenone analogs which are potent, ERbeta subtype selective ligands is described. Analogs possessing subnanomolar ERbeta binding, greater than 100-fold ERbeta-selectivity, and oral bioavailability are reported.
Synthesis and derivatization of a series of substituted tetrahydrofluorenone analogs giving potent, ERbeta subtype selective ligands are described. Several analogs possessing ERbeta binding affinities comparable to 17beta-estradiol but with greater than 75-fold selectivity over ERalpha are reported.
The effects of estrogen receptor (ER) ligands on the stability and transcriptional activity of ERbeta in the breast cancer cell lines MCF-7 and HeLa were examined. We found that ERbeta was degraded in the presence of 17beta-estradiol. Tamoxifen and Faslodex (ICI 182,780) prevented ERbeta receptor destabilization. In contrast to ERalpha, ERbeta degradation was not abolished by inhibitors of the proteasome-mediated protein degradation pathway. Furthermore, single point mutations in helix 12 of the receptor dramatically affected the stability and subsequent transcriptional activation of ERbeta.
Somatostatin (SRIH) functions as an endocrine mediator in processes such as growth, immune resistance and reproduction. Five SRIH receptors (sstr1-5) have been identified in mammals, where they are expressed in both the brain and peripheral tIssues. To study the specific function of each receptor subtype, specific agonists (ag1-5) have been synthesized. The high degree of homology between mammalian and avian SRIH receptors suggests that these agonists might also be used in chickens. In this paper we describe two in vitro protocols (static incubation and perifusion system) to identify the SRIH receptors controlling the secretion of GH and TSH from the chicken pituitary. We found that basal GH or TSH secretion were never affected when SRIH or an agonist (1 microM) were added. SRIH diminished the GH as well as the TSH response to TSH-releasing hormone (TRH; 100 nM) in both systems. Our results have indicated that the SRIH actions at the level of the pituitary are regulated through specific receptor subtypes. In both the static and flow incubations, ag2 lowered the GH response to TRH, whereas stimulated TSH release was diminished by both ag2 and ag5. Ag3 and ag4 tended to increase rather than decrease the responsiveness of both pituitary cell types to TRH in perifusion studies. Our data have indicated that SRIH inhibits chicken pituitary function through sstr2 and sstr5. Only sstr2 seems to be involved in the control of chicken GH release, whereas both sstr2 and sstr5 inhibit induced GH secretion in mammals. The possible stimulatory action of ag3 and ag4 may point towards a species-specific function of sstr3 and sstr4.
Extensive development of the structure-activity relationships of a screening lead determined three important pharmacophores for gonadotropin-releasing hormone (GnRH) receptor antagonist activity. Incorporation of the 3,4,5-trimethylphenyl group at the 3-position, 2-(2(S)-azetidinyl)ethoxy group at the 4-position, and N-4-pyrimidinylcarboxamide at the 6-position of the quinolone core resulted in the identification of 4-(2-(azetidin-2(S)-yl)ethoxy)-7-chloro-2-oxo-3-(3,4,5-trimethylphenyl)-1,2-dihydroquinoline-6-carboxylic acid pyrimidin-4-ylamide (1) as a potent antagonist of the GnRH receptor. A 10(4)-fold increase in in vitro binding affinity is observed for the GnRH receptor as compared to the initial screening lead. Compound 1 exhibits nanomolar binding activity and functional antagonism at the human receptor and is 7-fold less active at the rhesus receptor. Intravenous administration of compound 1 to rhesus monkeys results in a significant decrease of the serum levels of downstream hormones, luteinizing hormone (79% decrease in area under the curve) and testosterone (92% decrease in area under the curve), at a dose of 3 mg/kg. Quinolone 1 is a potent nonpeptidyl antagonist for the human GnRH receptor that is efficacious for the suppression of luteinizing hormone and testosterone in primates.
N-Substituted nipecotic and iso-nipecotic amides of beta-methylTrpLys tert-butyl ester were found to be novel, selective and potent agonists of the somatostatin subtype-2 receptor in vitro. For example iso-nipecotic amide 8a showed high hsst2 binding affinity (Ki = 0.5 nM) and good selectivity (h5/h2 = 832).
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Quinazolinone derivatives were synthesized and evaluated as non-peptidic growth hormone secretagogues. Modeling guided design of quinazolinone compound 21 led to a potency enhancement of greater than 200-fold compared to human growth hormone secretagogue affinity of a screening lead 4.
Somatostatin (SRIF) controls many physiological and pathological processes in the central nervous system but the respective roles of the five receptor isotypes (sst1-5) that mediate its effects are yet to be defined. In the present study, we attempted to identify functions of the sst2 receptor using mice with no functional copy of this gene (sst2 KO mice). In contrast with control 129Sv/C57Bl6 mice, sst2 mRNA was no longer detectable in the brain of sst2 KO mice; 125I-labeled Tyr0DTrp8-SRIF14 binding was also greatly reduced in almost all brain structures except for the hippocampal CA1 area, demonstrating that sst2 accounts for most SRIF binding in mouse brain. Invalidation of this subtype generated an increased anxiety-related behaviour in a number of behavioural paradigms, while locomotor and exploratory activity was decreased in stress-inducing situations. No major motor defects could be detected. sst2 KO mice also displayed increased release of pituitary ACTH, a main regulator of the stress response. Thus, somatostatin, via sst2 receptor isotype pathways, appears involved in the modulation of locomotor, exploratory and emotional reactivity in mice.
Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P < 0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.
Backbone cyclization of urea-based somatostatin agonists resulted in novel, orally bioavailable agonists. Binding assays confirmed that the resulting conformationally constrained cyclic ureas retained the potency of their acyclic counterparts. SAR studies subsequently led to highly potent analogs, selective for receptor subtype 2, and having good oral bioavailability.
Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.
A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 microgram/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054, 522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.
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A series of ophiobolins were isolated from a fungal extract based on their nematocidal activity. These compounds are non-competitive inhibitors of ivermectin binding to membranes prepared from the free-living nematode, Caenorhabditis elegans, with an inhibition constant of 15 microM. The ophiobolins which were most potent in the biological assays, ophiobolin C and ophiobolin M, were also the most potent compounds when evaluated in a C. elegans motility assay. These data suggest that the nematocidal activity of the ophiobolins is mediated via an interaction with the ivermectin binding site. The isolation, structure and biological activity of ophiobolins have been described.
Few studies have examined activity against trematodes for the avermectin/milbemycin class of anthelmintics. To gain insight into this, 12 different members of the avermectin/milbemycin mode of action class were tested against juvenile Fasciola hepatica in a mouse model. The compounds chosen were Avermectin A1, Avermectin A2, Avermectin B1, Avermectin B2, Ivermectin, Ivermectin monosaccharide, Ivermectin aglycone, 13-deoxy ivermectin aglycone, Moxidectin, 13-O-methoxyethoxymethyl ivermectin aglycone, 4"-deoxy-4"-epi-methylamino avermectin B1, and 4"-deoxy-4"-epi-acetylamino avermectin B1 5-oxime. Each of these compounds was administered orally to 4 mice at 2.0 mg kg-1. These mice had been administered 3 metacercariae of F. hepatica 14 days prior to treatment and all mice were necropsied 4 days after treatment. At necropsy, none of the individual avermectin or milbemycin-treated groups showed any significant activity (P > 0.05) against juvenile F. hepatica relative to a vehicle-treated control. In a receptor binding study, adult F. hepatica that had been obtained from sheep were homogenized, their membranes incubated in the presence of 3H-ivermectin, and then measured for high affinity binding sites. The same was done with the free-living nematode, Caenorhabditis elegans. While the C. elegans membranes displayed high affinity 3H-ivermectin binding sites over the range of ivermectin concentrations tested (5-100 nM), no significant 3H-ivermectin binding sites were detected in the F. hepatica membranes. Based on these data, it seems unlikely that any avermectin or milbemycin will show activity against F. hepatica, and certainly makes one pessimistic about possible activity of this mode of action class against trematodes in general.
High affinity avermectin binding sites have been identified and partially characterized in membranes from two insect species, Drosophila melanogaster and the locus Schistocerca americana. There is a 10-fold increase in the density of ivermectin binding sites associated with membranes isolated from Drosophila heads (a neuronally enriched tissue source) compared to the bodies (Bmax values were 3.5 and 0.22 pmol/mg, respectively) with only a small difference in the apparent dissociation constant (Kd values of 0.20 and 0.34 nM for heads and bodies, respectively). Membranes prepared from metathoracic ganglia of the locust, Schistocerca americana, were highly enriched in high affinity avermectin binding sites (Kd = 0.2 nM and Bmax = 42 pmol/mg). Using an [125I]arylazido-avermectin analog as a photoaffinity probe, a 45 kDa protein was identified in both the Drosophila head and body tissue preparations. A 45 kDa protein was also specifically labeled with [125I]azido-avermectin in the locust neuronal membranes.
Avermectin-binding proteins from the free-living nematode worm Caenorhabditis elegans and from the fruitfly Drosophila melanogaster were purified to homogeneity via a three-step procedure. The binding proteins were covalently labelled using a radioactive photoaffinity probe and then partially purified on a Sephacryl S-300 gel-filtration column. The radiolabelled binding proteins were then purified by immunoaffinity chromatography using a monoclonal antibody to avermectin covalently attached to Protein A-Sepharose beads. Three affinity-labelled Drosophila proteins with molecular masses between 45 and 50 kDa were isolated in this way and then separated from each other by electroelution. This three-step protocol provides a rapid technique for receptor purification which may be of use in the purification of other binding proteins.