The Nonpeptide Agonist MK-5046 Functions As an Allosteric Agonist for the Bombesin Receptor Subtype-3.

Allosteric ligands of various G-protein-coupled receptors are being increasingly described and are providing important advances in the development of ligands with novel selectivity and efficacy. These unusual properties allow expanded opportunities for pharmacologic studies and treatment. Unfortunately, no allosteric ligands are yet described for the bombesin receptor family (BnRs), which are proposed to be involved in numerous physiologic/pathophysiological processes in both the central nervous system and peripheral tissues. In this study, we investigate the possibility that the bombesin receptor subtype-3 (BRS-3) specific nonpeptide receptor agonist MK-5046 [(2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-[[1-(trifluoromethyl)cyclopropyl]methyl]-1H-imidazol-2-yl)propan-2-ol] functions as a BRS-3 allosteric receptor ligand. We find that in BRS-3 cells, MK-5046 only partially inhibits iodine-125 radionuclide (125I)-Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate] binding and that both peptide-1 (a universal BnR-agonist) and MK-5046 activate phospholipase C; however, the specific BRS-3 peptide antagonist Bantag-1 inhibits the action of peptide-1 competitively, whereas for MK-5046 the inhibition is noncompetitive and yields a curvilinear Schild plot. Furthermore, MK-5046 shows other allosteric behaviors, including slowing dissociation of the BRS-3 receptor ligand 125I-Bantag-1, dose-inhibition curves being markedly affected by increasing ligand concentration, and MK-5046 leftward shifting the peptide-1 agonist dose-response curve. Lastly, receptor chimeric studies and site-directed mutagenesis provide evidence that MK-5046 and Bantag-1 have different binding sites determining their receptor high affinity/selectivity. These results provide evidence that MK-5046 is functioning as an allosteric agonist at the BRS-3 receptor, which is the first allosteric ligand described for this family of receptors. SIGNIFICANCE STATEMENT: G-protein-coupled receptor allosteric ligands providing higher selectivity, selective efficacy, and safety that cannot be obtained using usual orthosteric receptor-based strategies are being increasingly described, resulting in enhanced usefulness in exploring receptor function and in treatment. No allosteric ligands exist for any of the mammalian bombesin receptor (BnR) family. Here we provide evidence for the first such example of a BnR allosteric ligand by showing that MK-5046, a nonpeptide agonist for bombesin receptor subtype-3, is functioning as an allosteric agonist.

Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia.

Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.

Adipocyte exosomes induce transforming growth factor beta pathway dysregulation in hepatocytes: a novel paradigm for obesity-related liver disease.

BACKGROUND: The pathogenesis of nonalcoholic fatty liver disease (NAFLD) has been attributed to increased systemic inflammation and insulin resistance mediated by visceral adipose tissue (VAT), although the exact mechanisms are undefined. Exosomes are membrane-derived vesicles containing messenger RNA, microRNA, and proteins, which have been implicated in cancer, neurodegenerative, and autoimmune diseases, which we postulated may be involved in obesity-related diseases. We isolated exosomes from VAT, characterized their content, and identified their potential targets. Targets included the transforming growth factor beta (TGF-ß) pathway, which has been linked to NAFLD. We hypothesized that adipocyte exosomes would integrate into HepG2 and hepatic stellate cell lines and cause dysregulation of the TGF-ß pathway. METHODS: Exosomes from VAT from obese and lean patients were isolated and fluorescently labeled, then applied to cultured hepatic cell lines. After incubation, culture slides were imaged to detect exosome uptake. In separate experiments, exosomes were applied to cultured cells and incubated 48-h. Gene expression of TGF-ß pathway mediators was analyzed by polymerase chain reaction, and compared with cells, which were not exposed to exosomes. RESULTS: Fluorescent-labeled exosomes integrated into both cell types and deposited in a perinuclear distribution. Exosome exposure caused increased tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and integrin ανß-5 expression and decreased matrix metalloproteinase-7 and plasminogen activator inhibitor-1 expression in to HepG2 cells and increased expression of TIMP-1, TIMP-4, Smad-3, integrins ανß-5 and ανß-8, and matrix metalloproteinase-9 in hepatic stellate cells. CONCLUSIONS: Exosomes from VAT integrate into liver cells and induce dysregulation of TGF-ß pathway members in vitro and offers an intriguing possibility for the pathogenesis of NAFLD.

Integrin ß-8, but not ß-5 or -6, protein expression is increased in livers of children with biliary atresia.

OBJECTIVES: Our previous work demonstrated altered messenger RNA expression of integrin ß-5 and -8, using an in silico analysis of publically available data from patients with biliary atresia (BA); however, we were unable to demonstrate statistically significant differences in protein expression because of sample size. In the present study, we repeated the analysis of liver fibrosis and protein expression of the integrins in a larger cohort of patients with BA and compared them with patients undergoing liver biopsy for other diagnoses, with the hypothesis that ≥ 1 of the integrins would be differentially expressed. METHODS: Liver specimens were obtained at 2 collaborating institutions. Samples from infants with BA (n = 23) were compared with samples from those who underwent liver biopsy for neonatal hepatitis (n = 9). All of the specimens were analyzed by 2 pathologists (C.R. and R.A.), who were blinded to the diagnoses. Standard Ishak scoring was performed to evaluate fibrosis and inflammation, and immunohistochemical (IHC) positivity was graded from 0 to 4. Comparisons between the IHC positivity and Ishak scoring for the BA and control groups were performed using the Student t test with P < 0.01 considered significant because of the multiple comparisons. Interobserver variability was assessed by intraclass correlation (ICC). RESULTS: Pooled analysis from specimens from patients with BA showed significantly more fibrosis than controls based on Ishak scores (3.21 ± 1.82 vs 1.17 ± 1.00, P < 0.005). IHC evaluation showed increased integrin ανß8 protein expression when compared with controls (2.67 ± 0.81 vs 1.72 ± 0.62, P < 0.005); however, there were no significant differences in integrin ανß5 (1.93 ± 0.84 vs 1.50 ± 0.90, P = 0.23) or integrin ανß6 (0.85 ± 1.20 vs 0.94 ± 0.85, P = 0.82) expression. These data were confirmed on individual analysis. Interobserver agreement was fair for integrin ανß5 (ICC 0.52), good for integrin ανß6 (ICC 0.72), and excellent for integrin ανß8 (ICC 0.79) and fibrosis (ICC 0.89). CONCLUSIONS: Our data show that integrin ανß8, but not integrin ανß5 or integrin ανß6, protein expression is increased in liver specimens of patients with BA. These data support the mounting evidence that transforming growth factor-ß (TGF-ß) activation is responsible for the fibrosis found in BA. Anti-integrin ανß8 or more global integrin blocking strategies may be therapeutic options in BA, but further work is clearly needed.

Mechanisms of transforming growth factor ß induced cell cycle arrest in palate development.

Immaculate and complete palatal seam disintegration, which takes place at the last phase of palate development, is essential for normal palate development. And in absence of palatal midline epithelial seam (MES) disintegration, cleft palate may arise. It has been established that transforming growth factor (TGF) ß induces both epithelial mesenchymal transition (EMT) and/or apoptosis during MES disintegration. It is likely that MES might cease cell cycle to facilitate cellular changes prior to undergoing transformation or apoptosis, which has never been studied before. This study was designed to explore whether TGFß, which is crucial for palatal MES disintegration, is capable of inducing cell cycle arrest. We studied the effects of TGFß1 and TGFß3, potent negative regulators of the cell cycle, on p15ink4b activity in MES cells. We surprisingly found that TGFß1, but not TGFß3, plays a major role in activation of the p15ink4b gene. In contrast, following successful cell cycle arrest by TGFß1, it is TGFß3 but not TGFß1 that causes later cellular morphogenesis, such as EMT and apoptosis. Since TGFß signaling activates Smads, we analyzed the roles of three Smad binding elements (SBEs) on the p15ink4b mouse promoter by site specific mutagenesis and found that these binding sites are functional. The ChIP assay demonstrated that TGFß1, not TGFß3, promotes Smad4 binding to two 5' terminal SBEs but not the 3' terminal site. Thus, TGFß1 and TGFß3 play separate yet complimentary roles in achieving cell cycle arrest and EMT/apoptosis and cell cycle arrest is a prerequisite for later cellular changes.

Bombesin Receptor Family Activation and CNS/Neural Tumors: Review of Evidence Supporting Possible Role for Novel Targeted Therapy.

G-protein-coupled receptors (GPCRs) are increasingly being considered as possible therapeutic targets in cancers. Activation of GPCR on tumors can have prominent growth effects, and GPCRs are frequently over-/ectopically expressed on tumors and thus can be used for targeted therapy. CNS/neural tumors are receiving increasing attention using this approach. Gliomas are the most frequent primary malignant brain/CNS tumor with glioblastoma having a 10-year survival <1%; neuroblastomas are the most common extracranial solid tumor in children with long-term survival<40%, and medulloblastomas are less common, but one subgroup has a 5-year survival <60%. Thus, there is an increased need for more effective treatments of these tumors. The Bombesin-receptor family (BnRs) is one of the GPCRs that are most frequently over/ectopically expressed by common tumors and is receiving particular attention as a possible therapeutic target in several tumors, particularly in prostate, breast, and lung cancer. We review in this paper evidence suggesting why a similar approach in some CNS/neural tumors (gliomas, neuroblastomas, medulloblastomas) should also be considered.

PAC1 regulates receptor tyrosine kinase transactivation in a reactive oxygen species-dependent manner.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a growth factor for lung cancer cells. PACAP-27 or PACAP-38 binds with high affinity to non-small cell lung cancer (NSCLC) cells, causing elevated cytosolic Ca2+, increased proliferation and increased phosphorylation of extracellular regulated kinase (ERK) and the epidermal growth factor receptor (EGFR). The role of reactive oxygen species (ROS) was investigated in these processes. Addition of PACAP-38 to NCI-H838 or A549 cells increased the tyrosine phosphorylation of the EGFR, HER2 and ERK significantly by 4-, 3-, and 2-fold, respectively. The transactivation of the EGFR and HER2 was inhibited by gefitinib or lapatinib (tyrosine kinase inhibitors), PACAP (6-38) (PAC1 antagonist), N-acetylcysteine (NAC is an anti-oxidant) or dipheyleneiodonium (DPI is an inhibitor of Nox and Duox enzymes). PACAP-38 addition to NSCLC cells increased ROS which was inhibited by PACAP (6-38), NAC or DPI. Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2 mRNA was present in many NSCLC cell lines. PACAP-38 stimulated the growth of NSCLC cells whereas PACAP (6-38), gefitinib or DPI inhibited proliferation. The results show that ROS are essential for PAC1 to regulate EGFR and HER2 transactivation as well as proliferation of NSCLC cells.

Molecular basis for high affinity and selectivity of peptide antagonist, Bantag-1, for the orphan BB3 receptor.

Bombesin-receptor-subtype-3 (BB3 receptor) is a G-protein-coupled-orphan-receptor classified in the mammalian Bombesin-family because of high homology to gastrin-releasing peptide (BB2 receptor)/neuromedin-B receptors (BB1 receptor). There is increased interest in BB3 receptor because studies primarily from knockout-mice suggest it plays roles in energy/glucose metabolism, insulin-secretion, as well as motility and tumor-growth. Investigations into its roles in physiological/pathophysiological processes are limited because of lack of selective ligands. Recently, a selective, peptide-antagonist, Bantag-1, was described. However, because BB3 receptor has low-affinity for all natural, Bn-related peptides, little is known of the molecular basis of its high-affinity/selectivity. This was systematically investigated in this study for Bantag-1 using a chimeric-approach making both Bantag-1 loss-/gain-of-affinity-chimeras, by exchanging extracellular (EC) domains of BB3/BB2 receptor, and using site-directed-mutagenesis. Receptors were transiently expressed and affinities determined by binding studies. Bantag-1 had >5000-fold selectivity for BB3 receptor over BB2/BB1 receptors and substitution of the first EC-domain (EC1) in loss-/gain-of affinity-chimeras greatly affected affinity. Mutagenesis of each amino acid difference in EC1 between BB3 receptor/BB2 receptor showed replacement of His(107) in BB3 receptor by Lys(107) (H107K-BB3 receptor-mutant) from BB2 receptor, decreased affinity 60-fold, and three replacements [H107K, E11D, G112R] decreased affinity 500-fold. Mutagenesis in EC1's surrounding transmembrane-regions (TMs) demonstrated TM2 differences were not important, but R127Q in TM3 alone decreased affinity 400-fold. Additional mutants in EC1/TM3 explored the molecular basis for these changes demonstrated in EC1, particularly important is the presence of aromatic-interactions by His(107), rather than hydrogen-bonding or charge-charge interactions, for determining Bantag-1 high affinity/selectivity. In regard to Arg(127) in TM3, both hydrogen-bonding and charge-charge interactions contribute to the high-affinity/selectivity for Bantag-1.

Dysregulation of upstream and downstream transforming growth factor-ß transcripts in livers of children with biliary atresia and fibrogenic gene signatures.

INTRODUCTION: Our previous work demonstrated that the transforming-growth factor (TGF) ß pathway plays a central role in the liver fibrosis associated with experimental biliary atresia (BA). To confirm these findings in humans, we performed an in silico analysis of publicly available microarray data from liver specimens from children with BA, with the hypothesis that the TGF-ß pathway would be dysregulated. METHODS: We analyzed publicly available liver gene expression microarray data from 47 infants with BA. We re-analyzed the microarray image files and clinical data to compare gene expression differences between the fibrogenic and inflammatory cohorts identified in the initial study. Targets from the microarray analysis were confirmed using the animal model of BA. RESULTS: Analysis of variance (ANOVA) detected 6903 transcripts (2822 distinct genes) differentially regulated between groups (p < 0.01; fold change >1.2). We used a targeted approach to identified a subgroup of 24 TGF-ß-related transcripts. Expressions for procollagen transcripts were increased in the fibrogenic group (1.2-fold to 1.4-fold); expression of matrix metalloproteinase (MMP)-7 was similarly increased 2-fold, while MMP-9 and plasminogen activator inhibitor-1 were decreased 2-fold and 3-fold respectively. Integrins ß5 (1.18-fold) and ß8 (1.84-fold) also demonstrated increased expression in the fibrogenic group. Increased expression of ß5 (3-fold) and ß8 (5-fold) as well as Smad-3 (4-fold) and Smad interacting protein (SIP)-1 (3.5-fold) mRNA was confirmed in experimental BA. Phosphorylated Smad-3 protein in the experimental group was also nearly twice that of the control group, further implicating the TGF-ß pathway. CONCLUSION: Gene transcripts for known upstream and downstream TGF-ß mediators are differentially expressed in liver specimens from children with BA and a fibrogenic gene signature. The same integrins that were dysregulated in the human specimens were also found to be up-regulated in our animal BA model, as were other intermediaries in the TGF-ß pathway. Further investigation into whether these mediators may be attractive targets for future therapy in children with BA is warranted.