G-15

G-15 is a selective antagonist of GPR30 with Ki value of 20 nM [1].

G protein-coupled receptor 30 (GPR30) is an integral membrane protein that localizes to the endoplasmic reticulum and with high affinity for estradiol and aldosterone. GPR30 participates in multiple intracellular signaling pathways [1].

G-15 is a selective GPR30 antagonist with Ki value of 20 nM, While displayed little binding to ERα or ERβ up to 10 μM. In SKBr3 breast cancer cells that expressed only GPR30, G-1 or estrogen significantly increased intracellular calcium concentrations, while G15 inhibited the response to G-1 or estrogen with IC50 value of 185 and 190 nM respectively in a dose-dependent way. In GPR30-transfected COS7 cells, G-15 inhibited both estrogen and G-1 activation of PI3K and accumulation of PIP3 [1]. In endometriotic cells, G-1 increased cell proliferation and Akt phosphorylation, while G-15 reversed this stimulation and inhibited cell proliferation and induced Akt dephosphorylation [2].

In intact rats and ovariectomized (OVX) rats treated with estradiol, G-15 impaired acquisition of delayed matching-to-position (DMP) T-maze task with a persistent turn. The result suggested that GPR30 played an crucial role in mediating the effects of estradiol on spatial learning [3].

References:
[1].  Dennis MK, Burai R, Ramesh C, et al. In vivo effects of a GPR30 antagonist. Nat Chem Biol, 2009, 5(6): 421-427.
[2].  G-protein-coupled estrogen receptor and the GPER-antagonist G-15 inhibits proliferation in endometriotic cells. Fertil Steril, 2013, 100(3): 770-776.
[3].  Hammond R, Nelson D, Kline E, et al. Chronic treatment with a GPR30 antagonist impairs acquisition of a spatial learning task in young female rats. Horm Behav, 2012, 62(4): 367-374.

Assessment of the efficacy of Sephadex G-15 filtration of bovine spermatozoa for cryopreservation.[Pubmed:15589282]

Theriogenology. 2005 Jan 1;63(1):160-78.

Semen from five dairy AI bulls was split-filtered through a Sephadex G-15 filter and frozen in a Tris-citric acid buffer egg yolk-based extender. The effect of filtration was studied morphologically for individual sperm abnormalities. Computer-assisted sperm analysis (CASA) was used for motility and sperm motion assessment. Flow cytometry was used to disclose sperm viability (SYBR-14/PI), mitochondrial membrane potential (Mitotracker Deep Red/SYBR 14), acrosome integrity (SYBR 14/PE-PNA/PI), plasma membrane stability (Merocyanine 540/YO-PRO 1/Hoechst 333342), and chromatin stability (acridine orange staining). Filtration significantly reduced the concentration of recovered spermatozoa (P < 0.01), but improved semen quality, reducing the number of spermatozoa with various forms of morphological defects. Filtration also affected percentages of sperm motility after equilibration and after freezing/thawing. Sperm motion characteristics were, however, not significantly affected by filtration at any stage of the cryopreservation protocol, including post-extension, equilibration, or freezing/thawing. Filtration enhanced sperm viability after thawing (P < 0.05), but had no significant effect (P > 0.05) on recovery of spermatozoa with high mitochondrial potential, intact acrosomes, or preserved sperm chromatin structure. Sperm plasma membrane stability was also not affected by the filtration method used (P > 0.05). It can be concluded that filtration effectively separates weaken or abnormal spermatozoa in pre-freezing semen samples and therefore the procedure could be recommended to improve post-thaw sperm viability of selected, fertile sires.

Histone deacetylase inhibitors down-regulate G-protein-coupled estrogen receptor and the GPER-antagonist G-15 inhibits proliferation in endometriotic cells.[Pubmed:23755949]

Fertil Steril. 2013 Sep;100(3):770-6.

OBJECTIVE: To investigate whether histone deacetylase inhibitors reduce the expression of the G-protein-coupled estrogen receptor (GPER) and whether the functional inhibition of GPER by the antagonist G-15 decreases the proliferation of endometriotic cells. DESIGN: In vitro study. SETTING: University hospital. PATIENT(S): Immortalized epithelial endometriotic cells. INTERVENTION(S): Treatment with the histone deacetylase inhibitor romidepsin or suberoylanilide hydroxamic acid (SAHA), or with the GPER antagonist G-15. MAIN OUTCOME MEASURE(S): Western blot analysis and quantitative real-time polymerase chain reaction (PCR) were used to monitor the expression of GPER in response to drug treatment. Effects of GPER stimulation and inhibition on cell proliferation were investigated by the 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (Sigma) (MTT) assay. RESULT(S): Our results demonstrate that romidepsin and SAHA reduce GPER expression in a concentration-dependent manner. This reduction correlated with the accumulation of acetylated histones. No decreased expression of the estrogen receptor (ER)-alpha and ERbeta was found under comparable experimental conditions. Pretreatment of endometriotic cells with the GPER agonist G-1 stimulated cell proliferation accompanied by rapid Akt phosphorylation. G-15 reversed this stimulation and inhibited cell proliferation, which was accompanied by Akt dephosphorylation. CONCLUSION(S): G-protein-coupled estrogen receptor is proposed as a potential therapeutic target in endometriosis. The down-regulation of GPER and/or the impairment of its function may reduce the estrogen responsiveness in endometriosis, and therefore might be considered a possible treatment option of endometriosis.

Establishment of an efficient fermentation system of gamma-aminobutyric acid by a lactic acid bacterium, Enterococcus avium G-15, isolated from carrot leaves.[Pubmed:20930374]

Biol Pharm Bull. 2010;33(10):1673-9.

In the present study, we successfully isolated a carrot leaf-derived lactic acid bacterium that produces gamma-aminobutyric acid (GABA) from monosodium L-glutamate (L-MSG) at a hyper conversion rate. The GABA-producing bacterium, identified as Enterococcus (E.) avium G-15, produced 115.7+/-6.4 g/l GABA at a conversion rate of 86.0+/-5.0% from the added L-MSG under the optimum culture condition by a continuous L-MSG feeding method using a jar-fermentor, suggesting that the bacterium displays a great potential ability for the commercial-level fermentation production of GABA. Using the reverse transcription polymerase chain reaction (RT-PCR) method, we analyzed the expression of genes for the GABA transporter and glutamate decarboxylase, designated gadT and gadG, respectively, which were cloned from the E. avium G-15 chromosome. Both genes were expressed even without the added L-MSG, but their expression was enhanced by the addition of L-MSG.

Potassium and sodium chloride ion pairs are presumed to constitute a complex during elution from a Sephadex G-15 column with sodium phosphate buffer.[Pubmed:12613796]

J Chromatogr A. 2003 Feb 14;987(1-2):49-55.

When a mixed solution of 0.72 M potassium and sodium chloride was eluted from a Sephadex G-15 column with 0.025 M sodium phosphate buffer (pH 7.0), the elution profiles of ions showed that the potassium and chloride ion pair from the sample and the sodium and chloride ion pair produced by ion-exchange reaction, were eluted in the same fractions as if they constituted a complex. When a mixed solution of different concentrations of potassium and sodium chloride was eluted with the same buffer, the excess amount of one ion pair over the other was eluted freely from the presumed complex.

Estrogen protects renal endothelial barrier function from ischemia-reperfusion in vitro and in vivo.[Pubmed:22622457]

Am J Physiol Renal Physiol. 2012 Aug 1;303(3):F377-85.

Emerging evidence suggests that renal endothelial function may be altered in ischemia-reperfusion injury. Acute kidney injury is sexually dimorphic, and estrogen protects renal tubular function after experimental ischemic injury. This study tested the hypothesis that during ischemia-reperfusion, estrogen alters glomerular endothelial function to prevent hyperpermeability. Glomerular endothelial cells were exposed to 8-h oxygen-glucose deprivation (OGD) followed by 4- and 8-h reoxygenation-glucose repletion. After 4-h reoxygenation-glucose repletion, transendothelial permeability to Ficoll-70 was reduced, and transendothelial resistance increased, by 17beta-estradiol vs. vehicle treatment during OGD (OGD-vehicle: 91.0 +/- 11.8%, OGD-estrogen: 102.6 +/- 10.8%, P < 0.05). This effect was reversed by coadministration of G protein-coupled receptor 30 (GPR30) antagonist G15 with 17beta-estradiol (OGD-estrogen-G15: 89.5 +/- 6.9, P < 0.05 compared with 17beta-estradiol). To provide preliminary confirmation of this result in vivo, Ficoll-70 was administered to mice 24 h after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Blood urea nitrogen (BUN) and serum creatinine (SCr) in these mice were elevated within 12 h following CA/CPR and reduced at 24 h by pretreatment with 17beta-estradiol (BUN/SCr 17beta-estradiol: 34 +/- 19/0.2 +/- 0.1 vehicle: 92 +/- 49/0.5 +/- 0.3, n = 8-12, P < 0.05). Glomerular sieving of Ficoll 70 was increased by CA/CPR within 2 h of injury and 17beta-estradiol treatment (theta; 17beta-estradiol: 0.74 +/- 0.26 vs. vehicle: 1.05 +/- 0.53, n = 14-15, P < 0.05). These results suggest that estrogen reduces postischemic glomerular endothelial hyperpermeability at least in part through GPR30 and that estrogen may regulate post CA/CPR glomerular permeability in a similar fashion in vivo.

In vivo effects of a GPR30 antagonist.[Pubmed:19430488]

Nat Chem Biol. 2009 Jun;5(6):421-7.

Estrogen is central to many physiological processes throughout the human body. We have previously shown that the G protein-coupled receptor GPR30 (also known as GPER), in addition to classical nuclear estrogen receptors (ER and ER), activates cellular signaling pathways in response to estrogen. In order to distinguish between the actions of classical estrogen receptors and GPR30, we have previously characterized G-1 (1), a selective agonist of GPR30. To complement the pharmacological properties of G-1, we sought to identify an antagonist of GPR30 that displays similar selectivity against the classical estrogen receptors. Here we describe the identification and characterization of G15 (2), a G-1 analog that binds to GPR30 with high affinity and acts as an antagonist of estrogen signaling through GPR30. In vivo administration of G15 revealed that GPR30 contributes to both uterine and neurological responses initiated by estrogen. The identification of this antagonist will accelerate the evaluation of the roles of GPR30 in human physiology.