The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure.

Secretion of proteins and neurotransmitters from large dense core vesicles (LDCVs) is a highly regulated process. Adrenal LDCV formation involves the granin proteins chromogranin A (CgA) and chromogranin B (CgB); CgA- and CgB-derived peptides regulate catecholamine levels and blood pressure. We investigated function of the granin VGF (nonacronymic) in LDCV formation and the regulation of catecholamine levels and blood pressure. Expression of exogenous VGF in nonendocrine NIH 3T3 fibroblasts resulted in the formation of LDCV-like structures and depolarization-induced VGF secretion. Analysis of germline VGF-knockout mouse adrenal medulla revealed decreased LDCV size in noradrenergic chromaffin cells, increased adrenal norepinephrine and epinephrine content and circulating plasma epinephrine, and decreased adrenal CgB. These neurochemical changes in VGF-knockout mice were associated with hypertension. Germline knock-in of human VGF1-615 into the mouse Vgf locus rescued the hypertensive knockout phenotype, while knock-in of a truncated human VGF1-524 that lacks several C-terminal peptides, including TLQP-21, resulted in a small but significant increase in systolic blood pressure compared to hVGF1-615 mice. Finally, acute and chronic administration of the VGF-derived peptide TLQP-21 to rodents decreased blood pressure. Our studies establish a role for VGF in adrenal LDCV formation and the regulation of catecholamine levels and blood pressure.

Identification of three residues essential for 5-hydroxytryptamine 2A-metabotropic glutamate 2 (5-HT2A·mGlu2) receptor heteromerization and its psychoactive behavioral function.

Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT(2A)) receptor in the mouse frontal cortex. Substitution of these residues (Ala-677(4.40), Ala-681(4.44), and Ala-685(4.48)) leads to absence of 5-HT(2A)·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT(2A) agonists. Furthermore, the ligand binding interaction between the components of the 5-HT(2A)·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer.

Compensatory redistribution of neuroligins and N-cadherin following deletion of synaptic ß1-integrin.

ß1-containing integrins are required for persistent synaptic potentiation in hippocampus and regulate hippocampal-dependent learning. Based largely on indirect evidence, there is a prevailing assumption that ß1-integrins are localized at synapses, where they contribute to synapse adhesion and signaling, but this has not been examined directly. Here we investigate the fine localization of ß1-integrin in adult mouse hippocampus using high-resolution immunogold labeling, with a particular emphasis on synaptic labeling patterns. We find that ß1-integrins localize to synapses in CA1 and are concentrated postsynaptically. At the postsynaptic membrane, ß1-integrins are found more commonly clustered near active zone centers rather than at the peripheral edges. In mice harboring a conditional deletion of ß1-integrins, labeling for N-cadherin and neuroligins increases. Western blots show increased levels of N-cadherin in total lysates and neuroligins increase selectively in synaptosomes. These data suggest there is a dynamic, compensatory adjustment of synaptic adhesion. Such adjustment is specific only for certain cell adhesion molecules (CAMs), because labeling for SynCAM is unchanged. Together, our findings demonstrate unequivocally that ß1-integrin is an integral synaptic adhesion protein, and suggest that adhesive function at the synapse reflects a cooperative and dynamic network of multiple CAM families.

The antiangiogenic activity in xenograft models of brivanib, a dual inhibitor of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 kinases.

Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic factors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth factors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination and may work synergistically to promote angiogenesis. Brivanib alaninate is the orally active prodrug of brivanib, a selective dual inhibitor of FGF and VEGF signaling. Here, we show that brivanib demonstrates antitumor activity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate shows dose-dependent efficacy equivalent to brivanib in L2987 human tumor xenografts. Brivanib alaninate (107 mg/kg) reduced tumor cell proliferation as determined by a 76% reduction in Ki-67 staining and reduced tumor vascular density as determined by a 76% reduction in anti-CD34 endothelial cell staining. Furthermore, Matrigel plug assays in athymic mice showed that brivanib alaninate inhibited angiogenesis driven by VEGF or basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, showed a marked decrease in gadopentetate dimeglumine contrast agent uptake at 107 mg/kg dose, with a reduction in area under the plasma concentration-time curve from time 0 to 60 minutes at 24 and 48 hours of 54% and 64%, respectively. These results show that brivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities.

Discovery and preclinical studies of 5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737), an in vivo active potent VEGFR-2 inhibitor.

We report herein a series of substituted N-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amines as inhibitors of vascular endothelial growth factor receptor-2 tyrosine kinase. Through structure-activity relationship studies, biochemical potency, pharmacokinetics, and kinase selectivity were optimized to afford BMS-645737 (13), a compound with good preclinical in vivo activity against human tumor xenograft models.

Synthesis, SAR, and Evaluation of 4-[2,4-Difluoro-5-(cyclopropylcarbamoyl)phenylamino]pyrrolo[2,1-f][1,2,4]triazine-based VEGFR-2 kinase inhibitors.

Introduction of the 2,4-difluoro-5-(cyclopropylcarbamoyl)phenylamino group at the C-4 position of the pyrrolo[2,1-f][1,2,4] triazine scaffold led to the discovery of a novel sub-series of inhibitors of VEGFR-2 kinase activity. Subsequent SAR studies on the 1,3,5-oxadiazole ring appended to the C-6 position of this new sub-family of pyrrolotriazines resulted in the identification of low nanomolar inhibitors of VEGFR-2. Antitumor efficacy was observed with compound 37 against L2987 human lung carcinoma xenografts in athymic mice.

Discovery and preclinical studies of (R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5- methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan- 2-ol (BMS-540215), an in vivo active potent VEGFR-2 inhibitor.

A series of substituted 4-(4-fluoro-1H-indol-5-yloxy)pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of vascular endothelial growth factor receptor-2 kinase is reported. Structure-activity relationship studies revealed that a methyl group at the 5-position and a substituted alkoxy group at the 6-position of the pyrrolo[2,1-f][1,2,4]triazine core gave potent compounds. Biochemical potency, kinase selectivity, and pharmacokinetics of the series were optimized and in vitro safety liabilities were minimized to afford BMS-540215 (12), which demonstrated robust preclinical in vivo activity in human tumor xenograft models. The l-alanine prodrug of 12, BMS-582664 (21), is currently under evaluation in clinical trials for the treatment of solid tumors.

Synthesis and SAR of 4-(3-hydroxyphenylamino)pyrrolo[2,1-f][1,2,4]triazine based VEGFR-2 kinase inhibitors.

A versatile synthesis of the suitably functionalized pyrrolo[2,1-f][1,2,4]triazine nucleus is described. SAR at the C-5 and C-6 positions of the 4-(3-hydroxy-4-methylphenylamino)pyrrolo[2,1-f][1,2,4]triazine template led to compounds with good in vitro potency against VEGFR-2 kinase. Glucuronidation of the phenol group is mitigated by incorporation of a basic amino group on the C-6 side chain of the pyrrolotriazine nucleus.

Discovery of the pyrrolo[2,1-f][1,2,4]triazine nucleus as a new kinase inhibitor template.

The pyrrolo[2,1-f][1,2,4]triazine nucleus was identified as a novel kinase inhibitor template which effectively mimics the well-known quinazoline kinase inhibitor scaffold. Attachment of a 4-((3-chloro-4-fluorophenyl)amino) substituent to the template provided potent biochemical inhibitors of the tyrosine kinase activity of EGFR, as well as inhibition of cellular proliferation of the human colon tumor cell line DiFi. Attachment of a 4-((3-hydroxy-4-methylphenyl)amino) substituent provided potent inhibitors of VEGFR-2 which also showed effects on the VEGF-dependent proliferation of human umbilical vein endothelial cells. Biological activity was maintained with substitution at positions 5 or 6, but not 7, suggesting that the former positions are promising sites for introducing side chains which modulate physicochemical properties. Preliminary inhibition studies with varying ATP concentrations suggest that, like the quinazoline-based kinase inhibitors, the pyrrolotriazine-based inhibitors bind in the ATP pocket.