Once activated at the surface of cells, G protein-coupled receptors (GPCRs) redistribute to endosomes, where they can continue to signal. Whether GPCRs in endosomes generate signals that contribute to human disease is unknown. We evaluated endosomal signaling of protease-activated receptor-2 (PAR), which has been proposed to mediate pain in patients with irritable bowel syndrome (IBS). Trypsin, elastase, and cathepsin S, which are activated in the colonic mucosa of patients with IBS and in experimental animals with colitis, caused persistent PAR-dependent hyperexcitability of nociceptors, sensitization of colonic afferent neurons to mechanical stimuli, and somatic mechanical allodynia. Inhibitors of clathrin- and dynamin-dependent endocytosis and of mitogen-activated protein kinase kinase-1 prevented trypsin-induced hyperexcitability, sensitization, and allodynia. However, they did not affect elastase- or cathepsin S-induced hyperexcitability, sensitization, or allodynia. Trypsin stimulated endocytosis of PAR, which signaled from endosomes to activate extracellular signal-regulated kinase. Elastase and cathepsin S did not stimulate endocytosis of PAR, which signaled from the plasma membrane to activate adenylyl cyclase. Biopsies of colonic mucosa from IBS patients released proteases that induced persistent PAR-dependent hyperexcitability of nociceptors, and PAR association with β-arrestins, which mediate endocytosis. Conjugation to cholestanol promoted delivery and retention of antagonists in endosomes containing PAR A cholestanol-conjugated PAR antagonist prevented persistent trypsin- and IBS protease-induced hyperexcitability of nociceptors. The results reveal that PAR signaling from endosomes underlies the persistent hyperexcitability of nociceptors that mediates chronic pain of IBS. Endosomally targeted PAR antagonists are potential therapies for IBS pain. GPCRs in endosomes transmit signals that contribute to human diseases.

A series of 4-substituted 2-amino-3-benzoylthiophenes was screened using a functional assay of A(1)AR-mediated phosphorylation of ERK1/2 in intact CHO cells to identify both potential agonistic effects as well the ability to allosterically modulate the activity of the orthosteric agonist, R-PIA. More detailed concentration-response experiments were subsequently performed on two compounds (9a and 9o) utilizing both the ERK1/2 assay as well as a second assay of [(35)S]GTPgammaS binding to activated G proteins.

Benzo[b]furan and indole analogues of some recently identified benzo[b]thiophene inhibitors of tubulin polymerization have been prepared, and their biological activity has been assessed. Several very potent analogues were identified.

No abstract

A structure–activity relationship (SAR) guided design of novel tubulin polymerization inhibitors has resulted in a series of benzo[b]furans with exceptional potency toward cancer cells and activated endothelial cells. The potency of early lead compounds has been substantially improved through the synergistic effect of introducing a conformational bias and additional hydrogen bond donor to the pharmacophore. Screening of a focused library of potent tubulin polymerization inhibitors for selectivity against cancer cells and activated endothelial cells over quiescent endothelial cells has afforded 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo-[b]furan (BNC105, 8) as a potent and selective antiproliferative. Because of poor solubility, 8 is administered as its disodium phosphate ester prodrug 9 (BNC105P), which is rapidly cleaved in vivo to return the active 8. 9 exhibits both superior vascular disrupting and tumor growth inhibitory properties compared with the benchmark agent combretastatin A-4 disodium phosphate 5 (CA4P).

Vascular disruption agents (VDA) cause occlusion of tumor vasculature, resulting in hypoxia-driven tumor cell necrosis. Tumor vascular disruption is a therapeutic strategy of great potential; however, VDAs currently under development display a narrow therapeutic margin, with cardiovascular toxicity posing a dose-limiting obstacle. Discovery of new VDAs, which display a wider therapeutic margin, may allow attainment of improved clinical outcomes. To identify such compounds, we used an in vitro selectivity screening approach that exploits the fact that tumor endothelial cells are in a constant state of activation and angiogenesis and do not undergo senescence. Our effort yielded the compound BNC105. This compound acts as a tubulin polymerization inhibitor and displays 80-fold higher potency against endothelial cells that are actively proliferating or are engaged in the formation of in vitro capillaries compared with nonproliferating endothelial cells or endothelium found in stable capillaries. This selectivity was not observed with CA4, a VDA currently under evaluation in phase III clinical trials. BNC105 is more potent and offers a wider therapeutic window. CA4 produces 90% vascular disruption at its no observed adverse event level (NOAEL), whereas BNC105 causes 95% vascular disruption at 1/8th of its NOAEL. Tissue distribution analysis of BNC105 in tumor-bearing mice showed that while the drug is cleared from all tissues 24 hours after administration, it is still present at high concentrations within the solid tumor mass. Furthermore, BNC105 treatment causes tumor regressions with complete tumor clearance in 20% of treated animals. Mol Cancer Ther; 9(6); 1562-73. ©2010 AACR.

[structure: see text]. Flexible, convergent access to 2,3-disubstituted benzo[b]thiophenes has been developed. The most concise approach involves sequential coupling of o-bromoiodobenzenes with benzylmercaptan and zinc acetylides to give benzyl o-ethynylphenyl sulfides which react with iodine to give 3-iodobenzo[b]thiophenes in a 5-endo-dig iodocyclization. These iodides can be further elaborated using palladium-mediated coupling and/or metalation techniques. This method has been applied to the synthesis of some novel tubulin binding agents.

[reaction: see text] Readily accessible propynols with a 2-thioxyphenyl substituent selectively undergo 5-exo-iodocyclization followed by tandem rearrangement and elimination or substitution processes to give selective access to either 2-acyl- or 2-(1-iodoalkeny)-benzo[b]thiophene systems.