The Discovery of a Potent, Selective, and Peripherally Restricted Pan-Trk Inhibitor (PF-06273340) for the Treatment of Pain

The neurotrophin family of growth factors, comprised of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), is implicated in the physiology of chronic pain. Given the clinical efficacy of anti-NGF monoclonal antibody (mAb) therapies, there is significant interest in the development of small molecule modulators of neurotrophin activity. Neurotrophins signal through the tropomyosin related kinase (Trk) family of tyrosine kinase receptors, hence Trk kinase inhibition represents a potentially “druggable” point of intervention. To deliver the safety profile required for chronic, nonlife threatening pain indications, highly kinase-selective Trk inhibitors with minimal brain availability are sought. Herein we describe how the use of SBDD, 2D QSAR models, and matched molecular pair data in compound design enabled the delivery of the highly potent, kinase-selective, and peripherally restricted clinical candidate PF-06273340

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

Non-neuronal, peripheral serotonin deficiency causes diabetes mellitus and identifies an intracellular role for serotonin in the regulation of insulin secretion

Functional coupling of endogenous serotonin (5-HT1B) and calcitonin (C1a) receptors in CHO cells to a cyclic AMP-responsive luciferase reporter gene

A cyclic AMP-responsive reporter cell line has been established through the stable expression of a luciferase reporter plasmid in Chinese hamster ovary (CHO) cells. Reporter cells showed a dose-dependent expression of luciferase in response to incubation with forskolin. These CHO cells were screened for endogenous G protein-coupled receptors capable of stimulating or inhibiting adenylyl cyclase, by monitoring changes in luciferase expression. Serotonin (5-HT) receptor agonist ligands caused an inhibition of forskolin-stimulated luciferase expression in the rank order 5-carboxamidotryptamine > 5-HT > sumatriptan > 8-hydroxy-2-(di-n-propylamino)tetralin, The response to 5-HT was reversed by the 5-HT1 receptor antagonists cyanopindolol and pindolol, but not the 5-HT2 receptor antagonist ketanserin. Calcitonin was more potent than calcitonin gene-related peptide (CGRP) at stimulating luciferase expression in this cell line, and these responses were insensitive to the CGRP receptor antagonist, CGRP (8-37). These results were consistent with the presence of 5-HT1B-like and calcitonin (C1a-like) receptors in CHO cells, with the responses to 5-HT and CGRP being pertussis and cholera toxin-sensilive, respectively, This reporter gene assay gave the expected pharmacological profile for these receptors when compared with cyclic AMP accumulation assays, confirming its value as a functional assay for G protein-coupled receptors linked to adenylyl cyclase

Partial agonism at serotonin 5-HT1B and dopamine D-2L receptors using a luciferase reporter gene assay

We have used a luciferase reporter gene assay to study the functional responses of two G-protein-coupled receptors in Chinese hamster ovary (CHO) cells. The rank order of potency of drugs for the endogenous 5-HT1B receptor was 5-Hydroxytryptamine (5-HT)> zolmitriptan > dihydroergocristine >(-)Lisuride (with no response to bromocriptine), However, only 5-HT and (-)lisuride produced a full functional response, with zolmitriptan and dihydroergocristine achieving 69 +/- 2% and 50 +/- 1% of the maximal response. In the same cells stably transfected with the rat dopamine D-2L receptor, dopamine and bromocriptine produced a full agonist functional response, whilst (-)lisuride produced a biphasic response curve, indicating activity at both the endogenous 5-HT1B and exogenous dopamine D-2L receptors. Using the receptor specific antagonists, pindolol and(+)butaclamol, (-)lisuride was shown to produce 52% of the maximal response at the dopamine D-2 receptor relative to dopamine. In comparison to a cAMP accumulation assay, the rank orders of potency and intrinsic activity were the same for all compounds used. These results demonstrate that this reporter gene assay is capable of discriminating both potency and efficacy of drugs and can be used to characterise partial agonists at endogenously and heterologously expressed receptors in CHO cells. (C) 1999 Elsevier Science B.V. All rights reserved