Bombesin-like receptor 3 expression induced by bisphenol A is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing inflammation in liver cells.

BACKGROUND: Bisphenol A (BPA) is an exogenous endocrine disruptor mimicking hormones closely associated with health complications, such as cancer progression. BPA is also related to an increase in the prevalence of obesity-related diseases due to its obesogenic action. Bombesin-like receptor 3 (BRS3) is an important factor that should be considered in the adipogenic gene network, as depletion of this gene alters adiposity. METHODS: Therefore, the present study aimed to investigate the messenger ribonucleic acid (mRNA) expression of BRS3 in human liver THLE-2 cells post-BPA treatment by real-time polymerase chain reaction. The effects of BPA on the levels of pro-inflammatory proteins, interleukin 6 (IL6) and CC motif chemokine ligand 2 (CCL2), in conditioned media of BPA-treated THLE-2 cells and deoxyribonucleic acid (DNA) synthesis in replicating BPA-treated THLE-2 cells during the cell cycle were also examined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, respectively. RESULTS: The study found that the mRNA expression of BRS3 was increased in THLE-2 cells treated with BPA. The study also showed that the expression levels of IL6 and CCL2 reached an optimum level in the conditioned media of BPA-treated THLE-2 cells after 48 h of treatment. Subsequently, the DNA synthesis analysis showed that bromodeoxyuridine/propidium iodide (BrdU/PI) stained positive cells were decreased in BPA-treated THLE-2 cells at 72 h of treatment. CONCLUSION: The study demonstrates that BRS3 expression induced by BPA is likely associated with reduced cell proliferation by inhibiting DNA synthesis and inducing cellular inflammation in liver cells.

[Discovery of exogenous ligands for orphan receptor BRS-3 from Chinese herbs].

Bombesin receptor subtype-3(BRS-3) is an orphan receptor in the bombesin receptor family. Its signal transduction mechanism and biological function have attracted much attention. Seeking the ligand for BRS-3 is of great significance for exploring its function. Considering the fact that the activation of BRS-3 receptor can induce the change in intracellular Ca~(2+) concentration, the fluo-rometric imaging plate reader(FLIPR) was utilized for ligand screening at the cellular level. Among more than 400 monomeric compounds isolated from Chinese herbs, yuanhunine from Corydalis Rhizoma and sophoraisoflavanone A and licoriphenone from Glycyrrhizae Radix et Rhizoma antagonized BRS-3 to varying degrees. It was confirmed in HEK293 cells expressing BRS-3 that yuanhunine, sophoraisoflavanone A, and licoriphenone inhibited the calcium current response after the activation of BRS-3 by [D-Phe~6,ß-Ala~(11),Phe~(13),Nle~(14)]bombesin-(6-14) in a dose-dependent manner with the IC_(50) values being 8.58, 4.10, and 2.04 µmol·L~(-1), respectively. Further study indicated that yuanhunine and sophoraisoflavanone A exhibited good selectivity for BRS-3. In this study, it was found for the first time that monomers derived from Chinese herbs had antagonistic activity against orphan receptor BRS-3, which has provided a tool for further study of BRS-3 and also the potential lead compounds for new drug discovery. At the same time, it provides reference for the research and development of innovative drugs based on the active ingredients of Chinese herbs.

Accelerating the identification of subtype selective inhibitors via Three-Dimensional Biologically Relevant Spectrum (BRS-3D): The monoamine oxidase subtypes as a case study.

Subtype-selective drugs are of great therapeutic importance as they are expected to be more effective and with less side-effects. However, discovery of subtype selective inhibitors was hampered by the high similarity of the binding sites within subfamilies. In this study, we further evaluated the applicability of "Three-Dimensional Biologically Relevant Spectrum (BRS-3D)" for the identification of subtype-selective inhibitors. A case study was performed on monoamine oxidase, which has two subtypes related to distinct diseases. The inhibitory activity against MAO-A/B of 347 compounds experimentally tested in this research was reported. Compound M124 (5H-thiazolo[3,2-a]pyrimidin-5-one) with IC50 less than 100 nM (SI = 23) was selected as a probe to investigate the structure selectivity relationship. Similarity search led to the identification of compound M229 and M249 with IC50 values of 7.4 nM, 4 nM and acceptable selectivity index over MAO-A (M229 SI > 1351, M249 SI > 2500). The molecular basis for subtype selectivity was explored through docking study and attention based DNN model. Additionally, in silico ADME properties were characterized. Accordingly, it is found that BRS-3D is a robust method for subtype selectivity in the early stage of drug discovery and the compounds reported here can be promising leads for further experimental analysis.

Proteomics Analysis of Cellular BRS3 Receptor Activation Reveals Potential Mechanism for Signal Transduction and Cell Proliferation.

Bombesin-like receptor 3 (BRS3), an orphan G protein-coupled receptor (GPCR), plays important roles in our biological system while the exact mechanisms behind it are less known. To get insights of the biological effects upon BRS3 activation, we utilized quantitative proteomics approach to explore the dynamic protein profiling during the stimulation by its ligand. At different time points after stimulation with BRS3 surrogate agonist, the protein profiling in BRS3 overexpressed HEK 293 cells BRS3 (HEK 293-BRS3) was analyzed by nano-LC-MS/MS. In total, 1593 cellular proteins were confidently identified and quantified, including 146 proteins dysregulated at multiple time points and 319 proteins only altered at one time point. Data analysis indicated that BRS3 activation could regulate cell death, survival, and protein synthesis, particularly mRNA translation. Key signaling pathways were revealed for BRS3 signal transduction. In particular, 21 of our identified proteins are involved in the rapamycin (mTOR) signaling pathway. The promotion of mTOR was further confirmed through monitoring its indicative targets upon BRS3 activation. Upon the inhibition of mTOR by rapamycin, cell proliferation was dramatically reversed. Our proteomics data collectively demonstrate that BRS3 activation will lead to cascades of signal transduction and promote cell proliferation. The developed strategy might be utilized to discover the roles of other GPCRs and improve our understanding of their unknown functions.

Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Bombesin receptor subtype 3 (BRS-3) is a GPCR that is expressed in the CNS, peripheral tissues, and tumors. Our understanding of BRS-3's role in physiology and pathophysiology is limited because its natural ligand is unknown. In an attempt to identify this ligand, we screened toad skin ( Bufo bufo gargarizans Cantor) extracts and identified prostaglandins as putative ligands. In BRS-3-transfected human embryonic kidney (HEK) cells, we found that prostaglandins, with prostaglandin E2 (PGE2) being the most potent, fulfill the pharmacologic criteria of affinity, selectivity, and specificity to be considered as agonists to the BRS-3 receptor. However, PGE2 is unable to activate BRS-3 in different cellular environments. We speculated that EP receptors might be the cause of this cellular selectivity, and we found that EP3 is the receptor primarily responsible for the differential PGE2 effect. Consequently, we reconstituted the HEK environment in Chinese hamster ovary (CHO) cells and found that BRS-3 and EP3 interact to potentiate PGE2 signaling. This potentiating effect is receptor specific, and it occurs only when BRS-3 is paired to EP3. Our study represents an example of functional crosstalk between two distantly related GPCRs and may be of clinical importance for BRS-3-targeted therapies.-Zhang, Y., Liu, Y., Wu, L., Fan, C., Wang, Z., Zhang, X., Alachkar, A., Liang, X., Civelli, O. Receptor-specific crosstalk between prostanoid E receptor 3 and bombesin receptor subtype 3.

Novel C7-Substituted Coumarins as Selective Monoamine Oxidase Inhibitors: Discovery, Synthesis and Theoretical Simulation.

There is a continued need to develop new selective human monoamine oxidase (hMAO) inhibitors that could be beneficial for the treatment of neurological diseases. However, hMAOs are closely related with high sequence identity and structural similarity, which hinders the development of selective MAO inhibitors. "Three-Dimensional Biologically Relevant Spectrum (BRS-3D)" method developed by our group has demonstrated its effectiveness in subtype selectivity studies of receptor and enzyme ligands. Here, we report a series of novel C7-substituted coumarins, either synthesized or commercially purchased, which were identified as selective hMAO inhibitors. Most of the compounds demonstrated strong activities with IC50 values (half-inhibitory concentration) ranging from sub-micromolar to nanomolar. Compounds, FR1 and SP1, were identified as the most selective hMAO-A inhibitors, with IC50 values of 1.5 nM (selectivity index (SI) < -2.82) and 19 nM (SI < -2.42), respectively. FR4 and FR5 showed the most potent hMAO-B inhibitory activity, with IC50 of 18 nM and 15 nM (SI > 2.74 and SI > 2.82). Docking calculations and molecular dynamic simulations were performed to elucidate the selectivity preference and SAR profiles.

Synthesis and biological evaluation of novel imidazol-1-ylacetic acid derivatives as non-brain penetrant bombesin receptor subtype-3 (BRS-3) agonists.

Novel compounds based on 1a were synthesized with the focus of obtaining agonists acting upon peripheral BRS-3. To identify potent anti-obesity compounds without adverse effects on the central nervous system (CNS), a carboxylic acid moiety and a labile carboxylic ester with an antedrug functionality were introduced. Through the extensive synthetic exploration and the pharmacokinetic studies of intravenous administration in mice, the ester 2b was selected owing to its most suitable pharmacological profile. In the evaluation of food intake suppression in C57BL/6N mice, 2b showed significant in vivo efficacy and no clear adverse effects on blood pressure change in dogs administered the compound by intravenous infusion.

Three-Dimensional Biologically Relevant Spectrum (BRS-3D): Shape Similarity Profile Based on PDB Ligands as Molecular Descriptors.

The crystallized ligands in the Protein Data Bank (PDB) can be treated as the inverse shapes of the active sites of corresponding proteins. Therefore, the shape similarity between a molecule and PDB ligands indicated the possibility of the molecule to bind with the targets. In this paper, we proposed a shape similarity profile that can be used as a molecular descriptor for ligand-based virtual screening. First, through three-dimensional (3D) structural clustering, 300 diverse ligands were extracted from the druggable protein-ligand database, sc-PDB. Then, each of the molecules under scrutiny was flexibly superimposed onto the 300 ligands. Superimpositions were scored by shape overlap and property similarity, producing a 300 dimensional similarity array termed the "Three-Dimensional Biologically Relevant Spectrum (BRS-3D)". Finally, quantitative or discriminant models were developed with the 300 dimensional descriptor using machine learning methods (support vector machine). The effectiveness of this approach was evaluated using 42 benchmark data sets from the G protein-coupled receptor (GPCR) ligand library and the GPCR decoy database (GLL/GDD). We compared the performance of BRS-3D with other 2D and 3D state-of-the-art molecular descriptors. The results showed that models built with BRS-3D performed best for most GLL/GDD data sets. We also applied BRS-3D in histone deacetylase 1 inhibitors screening and GPCR subtype selectivity prediction. The advantages and disadvantages of this approach are discussed.

Synthesis and biological evaluation of novel chiral diazepine derivatives as bombesin receptor subtype-3 (BRS-3) agonists incorporating an antedrug approach.

Novel compounds based on the lead BRS-3 agonists from our HTS compounds 2a and 2b have been synthesized with the focus on obtaining peripheral BRS-3 agonists. To identify potent anti-obesity compounds without adverse effects on the central nerve system, a labile carboxylic ester with an antedrug functionality was introduced onto the terminal position. Through the extensive synthetic exploration and the pharmacokinetic studies of oral administration in mice, the phenol ester 17c was selected due to the most suitable pharmacological profile. In the evaluation of food intake suppression in B6 mice, 17c showed significant in vivo efficacy and no clear adverse effect on heart rate and blood pressure change in dog iv infusion. Our study paved the way for development of anti-diabetes and obesity drugs with a safer profile.

Discovery of novel chiral diazepines as bombesin receptor subtype-3 (BRS-3) agonists with low brain penetration.

The discovery and optimization of a novel series of BRS-3 agonists are described. We explored a potent BRS-3 agonist with low brain penetration to avoid an adverse effect derived from central nervous system exposure. Through the derivatization process, chiral diazepines 9f and 9g were identified as possessing low brain penetration as well as potent in vitro activity against human and mouse BRS-3s.

Structural insights into ligand recognition, selectivity, and activation of bombesin receptor subtype-3.

Bombesin receptor subtype-3 (BRS3) is an important orphan G protein-coupled receptor that regulates energy homeostasis and insulin secretion. As a member of the bombesin receptor (BnR) family, the lack of known endogenous ligands and high-resolution structure has hindered the understanding of BRS3 signaling and function. We present two cryogenic electron microscopy (cryo-EM) structures of BRS3 in complex with the heterotrimeric Gq protein in its active states: one bound to the pan-BnR agonist BA1 and the other bound to the synthetic BRS3-specific agonist MK-5046. These structures reveal the architecture of the orthosteric ligand pocket underpinning molecular recognition and provide insights into the structural basis for BRS3's selectivity and low affinity for bombesin peptides. Examination of conserved micro-switches suggests a shared activation mechanism among BnRs. Our findings shed light on BRS3's ligand selectivity and signaling mechanisms, paving the way for exploring its therapeutic potential for diabetes, obesity, and related metabolic disorders.

Cre Recombinase Driver Mice Reveal Lineage-Dependent and -Independent Expression of Brs3 in the Mouse Brain.

Bombesin receptor subtype-3 (BRS3) is an orphan receptor that regulates energy homeostasis. We compared Brs3 driver mice with constitutive or inducible Cre recombinase activity. The constitutive BRS3-Cre mice show a reporter signal (Cre-dependent tdTomato) in the adult brain because of lineage tracing in the dentate gyrus, striatal patches, and indusium griseum, in addition to sites previously identified in the inducible BRS3-Cre mice (including hypothalamic and amygdala subregions, and parabrachial nucleus). We detected Brs3 reporter expression in the dentate gyrus at day 23 but not at postnatal day 1 or 5 months of age. Hypothalamic sites expressed reporter at all three time points, and striatal patches expressed Brs3 reporter at 1 day but not 5 months. Parabrachial nucleus Brs3 neurons project to the preoptic area, hypothalamus, amygdala, and thalamus. Both Cre recombinase insertions reduced Brs3 mRNA levels and BRS3 function, causing obesity phenotypes of different severity. These results demonstrate that driver mice should be characterized phenotypically and illustrate the need for knock-in strategies with less effect on the endogenous gene.

Preoptic BRS3 neurons increase body temperature and heart rate via multiple pathways.

The preoptic area (POA) is a key brain region for regulation of body temperature (Tb), dictating thermogenic, cardiovascular, and behavioral responses that control Tb. Previously characterized POA neuronal populations all reduced Tb when activated. Using mice, we now identify POA neurons expressing bombesin-like receptor 3 (POABRS3) as a population whose activation increased Tb; inversely, acute inhibition of these neurons reduced Tb. POABRS3 neurons that project to either the paraventricular nucleus of the hypothalamus or the dorsomedial hypothalamus increased Tb, heart rate, and blood pressure via the sympathetic nervous system. Long-term inactivation of POABRS3 neurons caused increased Tb variability, overshooting both increases and decreases in Tb set point, with RNA expression profiles suggesting multiple types of POABRS3 neurons. Thus, POABRS3 neuronal populations regulate Tb and heart rate, contribute to cold defense, and fine-tune feedback control of Tb. These findings advance understanding of homeothermy, a defining feature of mammalian biology.

The effects of housing density on mouse thermal physiology depend on sex and ambient temperature.

OBJECTIVE: To improve understanding of mouse energy homeostasis and its applicability to humans, we quantitated the effects of housing density on mouse thermal physiology in both sexes. METHODS: Littermate wild type and Brs3-null mice were single- or group- (three per cage) housed and studied by indirect calorimetry with continuous measurement of core body temperature, energy expenditure, physical activity, and food intake. RESULTS: At 23 °C, below thermoneutrality, single-housed males had a lower body temperature and unchanged metabolic rate compared to group-housed controls. In contrast, single-housed females maintained a similar body temperature to group-housed controls by increasing their metabolic rate. With decreasing ambient temperature below 27 °C, only group-housed mice decreased their heat conductance, likely due to huddling, thus interfering with the energy expenditure vs ambient temperature relationship described by Scholander. In a hot environment (35 °C), the single-housed mice were less heat stressed. Upon fasting, single-housed mice had larger reductions in body temperature, with male mice having more torpor episodes of similar duration and female mice having a similar number of torpor episodes that lasted longer. Qualitatively, the effects of housing density on thermal physiology of Brs3-null mice generally mimicked the effects in controls. CONCLUSIONS: Single housing is more sensitive than group housing for detecting thermal physiology phenotypes. Single housing increases heat loss and amplifies the effects of fasting or a cold environment. Male and female mice utilize different thermoregulatory strategies to respond to single housing.

BRS3 in both MC4R- and SIM1-expressing neurons regulates energy homeostasis in mice.

OBJECTIVE: Bombesin-like receptor 3 (BRS3) is an orphan receptor and Brs3 knockout mice develop obesity with increased food intake and reduced resting metabolic rate and body temperature. The neuronal populations contributing to these effects were examined. METHODS: We studied energy metabolism in mice with Cre-mediated recombination causing 1) loss of BRS3 selectively in SIM1- or MC4R-expressing neurons or 2) selective re-expression of BRS3 from a null background in these neurons. RESULTS: The deletion of BRS3 in MC4R neurons increased body weight/adiposity, metabolic efficiency, and food intake, and reduced insulin sensitivity. BRS3 re-expression in these neurons caused partial or no reversal of these traits. However, these observations were confounded by an obesity phenotype caused by the Mc4r-Cre allele, independent of its recombinase activity. The deletion of BRS3 in SIM1 neurons increased body weight/adiposity and food intake, but not to the levels of the global null. The re-expression of BRS3 in SIM1 neurons reduced body weight/adiposity and food intake, but not to wild type levels. The deletion of BRS3 in either MC4R- or SIM1-expressing neurons affected body temperature, with re-expression in either population reversing the null phenotype. MK-5046, a BRS3 agonist, increases light phase body temperature in wild type, but not Brs3 null, mice and BRS3 re-expression in either population restored response to MK-5046. CONCLUSIONS: BRS3 in both MC4R- and SIM1-expressing neurons contributes to regulation of body weight/adiposity, insulin sensitivity, food intake, and body temperature.

Identification of novel monoamine oxidase selective inhibitors employing a hierarchical ligand-based virtual screening strategy.

Aim: Due to the pivotal role in the oxidative deamination of monoamine neurotransmitters, two distinct monoamine oxidase (MAO) subtypes, MAO-A and MAO-B, present a significant pharmacological interest. Here, we reported a hierarchical and time-efficient ligand-based virtual screening strategy to identify potent selective and reversible MAO inhibitors. Result: A total of 130 compounds were assessed in dose-response biochemical assay against MAOs. Among them, 70 compounds were active with inhibition higher than 70%, involving 25 compounds with IC50 values less than 1 µM. Conclusion: Our research demonstrated the validity of Biologically Relevant Spectrum (BRS-3D) in predicting subtype-selective ligands and afforded a novel highly efficient way to develop selective inhibitors in the early stage of drug discovery.

Activation of bombesin receptor Subtype-3 by [D-Tyr6,ß-Ala11,Phe13,Nle14]bombesin6-14 increased glucose uptake and lipogenesis in human and rat adipocytes.

BRS-3 has an important role in glucose homeostasis. Its expression was reduced in skeletal muscle from obese and/or diabetic patients, and BRS-3 KO-mice developed obesity. In this work, focused on rat/human adipose tissue, BRS-3 gene-expression was lower than normal-levels in hyperlipidemic, type-2-diabetic (T2D), and type-1-diabetic rats and also in obese (OB) and T2D patients. Moreover, BRS-3 protein levels were decreased in diabetic rat and in obese and diabetic human fat pieces; but neither mutation nor even polymorphism in the BRS-3-gene was found in OB or T2D patients. Interestingly, in rat and human adipocytes, without metabolic alterations, [D-Tyr6,ß-Ala11,Phe13,Nle14]bombesin6-14 -BRS-3-agonist-, as insulin, enhanced BRS-3 gene/protein expression, increased, PKB, p70s6K, MAPKs and p90RSK1 phosphorylation-levels, and induced a concentration-related stimulation of glucose transport, GLUT-4 membrane translocation and lipogenesis, exclusively mediated by BRS-3, and abolished by wortmannin, PD98059 or rapamacyn. These results confirm that BRS-3 and/or its agonist are a potential therapeutic tool for obesity/diabetes.

A possible new target in lung-cancer cells: The orphan receptor, bombesin receptor subtype-3.

Human bombesin receptors, GRPR and NMBR, are two of the most frequently overexpressed G-protein-coupled-receptors by lung-cancers. Recently, GRPR/NMBR are receiving considerable attention because they act as growth factor receptors often in an autocrine manner in different lung-cancers, affect tumor angiogenesis, their inhibition increases the cytotoxic potency of tyrosine-kinase inhibitors reducing lung-cancer cellular resistance/survival and their overexpression can be used for sensitive tumor localization as well as to target cytotoxic agents to the cancer. The orphan BRS-3-receptor, because of homology is classified as a bombesin receptor but has received little attention, despite the fact that it is also reported in a number of studies in lung-cancer cells and has growth effects in these cells. To address its potential importance, in this study, we examined the frequency/relative quantitative expression of human BRS-3 compared to GRPR/NMBR and the effects of its activation on cell-signaling/growth in 13 different human lung-cancer cell-lines. Our results showed that BRS-3 receptor is expressed in 92% of the cell-lines and that it is functional in these cells, because its activation stimulates phospholipase-C with breakdown of phosphoinositides and changes in cytosolic calcium, stimulates ERK/MAPK and stimulates cell growth by EGFR transactivation in some, but not all, the lung-cancer cell-lines. These results suggest that human BRS-3, similar to GRPR/NMBR, is frequently ectopically-expressed by lung-cancer cells in which, it is functional, affecting cell signaling/growth. These results suggest that similar to GRPR/NMBR, BRS-3 should receive increased attention as possible approach for the development of novel treatments and/or diagnosis in lung-cancer.

Characterization of NMB, GRP and their receptors (BRS3, NMBR and GRPR) in chickens.

The two structurally and functionally related peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) play critical roles in many physiological/pathological processes in mammals. However, the information regarding the expression and functionality of avian NMB, GRP and their receptors is limited. Here, we characterized cNMB, cGRP and their receptors (cNMBR, cGRPR and cBRS3) in chickens. Our results showed that: (1) cNMBR and cGRPR expressed in CHO cells could be potently activated by cNMB and cGRP, respectively, as monitored by cell-based luciferase reporter assays, indicating that cNMBR and cGRPR are cNMB- and cGRP-specific receptors; strikingly, BRS3 of chickens (/spotted gars), which is orthologous to mouse bombesin receptor subtype-3 (BRS3), could be potently activated by GRP and NMB, demonstrating that both peptides are the endogenous ligands for chicken (/spotted gar) BRS3; (2) quantitative real-time PCR (qPCR) revealed that cGRPR is widely expressed in chicken tissues with abundant expression in the ovary, pancreas, proventriculus, spinal cord and brain, whereas cNMB, cNMBR and cBRS3 are mainly expressed in the brain and testes; (3) interestingly, qPCR, Western blot and immunostaining revealed that cGRP is predominantly expressed in the anterior pituitary and mainly localized to LH-cells, suggesting that cGRP is likely a novel pituitary hormone in chickens. In summary, our data help to uncover the roles of GRP, NMB and their receptors in birds, and provide the first persuasive evidence from an evolutionary prospective that in vertebrates, GRP and NMB are the endogenous ligands for BRS3, an orphan receptor that has puzzled endocrinologists for more than two decades.

Gastrin-releasing peptide receptor mediates the excitation of preoptic GABAergic neurons by bombesin.

Bombesin, a pan agonist of the bombesin-like peptide receptor family, elicits potent hypothermia when applied centrally. The signaling mechanisms involved are not known. Here we report that GABAergic preoptic neurons express gastrin-releasing peptide (GRP) receptors and are directly excited by GRP or bombesin. This effect was abolished by a GRP receptor antagonist. A partially overlapping group of preoptic GABAergic neurons express bombesin-like receptor 3 (BRS3), however their activation results in a decrease in firing rate. The excitatory effects of bombesin or GRP were not affected by BRS3 antagonist. GRP activated a Ca2+-dependent inward nonselective cationic current and Ca2+ release from intracellular stores. Our data indicate that GRP receptors mediate the excitatory effects of bombesin in preoptic neurons.