Publications by authors named "Danny Ortuno"

3 Publications

  • Page 1 of 1

Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel Na1.7.

J Pharmacol Exp Ther 2017 07 4;362(1):146-160. Epub 2017 May 4.

Department of Neuroscience (T.J.K., R.Y., S.A, C.P.I., M.J., D.J., J.H.L., S.G.L., J.Li., D.L., J.Lu., D.M., D.O., K.T., J.W., V.Y., D.X.D.Z., R.T.F., B.D.M.), Department of Medicinal Chemistry (M.M.W.), and Department of Pharmacokinetics and Drug Metabolism (X.B., V.B., J.R.), Amgen Inc., Cambridge, Massachusetts and Thousand Oaks, California

Potent and selective antagonists of the voltage-gated sodium channel Na1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human Na1.7 channels with an IC of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other Na family members, including Na1.4 expressed in muscle and Na1.5 expressed in the heart, as well as TTX-resistant Na channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple Na1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective Na1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
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http://dx.doi.org/10.1124/jpet.116.239590DOI Listing
July 2017

Identification of modulators of autophagic flux in an image-based high content siRNA screen.

Autophagy 2016 ;12(4):713-26

b Department of Neuroscience Research , Amgen Inc. , Thousand Oaks , CA , USA.

Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration.
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http://dx.doi.org/10.1080/15548627.2016.1147669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836002PMC
December 2016

Anti-thrombotic and vascular effects of AR246686, a novel 5-HT2A receptor antagonist.

Eur J Pharmacol 2008 May 3;586(1-3):234-43. Epub 2007 Dec 3.

Arena Pharmaceuticals, Inc. San Diego, CA 92121 USA.

We have evaluated the anti-platelet and vascular pharmacology of AR246686, a novel 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. AR246686 displayed high affinity binding to membranes of HEK cells stably expressing recombinant human and rat 5-HT2A receptors (Ki=0.2 nM and 0.4 nM, respectively). Functional antagonism (IC50=1.9 nM) with AR246686 was determined by inhibition of ligand-independent inositol phosphate accumulation in the 5-HT2A stable cell line. We observed 8.7-fold and 1360-fold higher affinity of AR246686 for the 5-HT2A receptor vs. 5-HT2C and 5-HT2B receptors, respectively. AR246686 inhibited 5-HT-induced amplification of ADP-stimulated human platelet aggregation (IC50=21 nM). Similar potency was observed for inhibition of 5-HT stimulated DNA synthesis in rat aortic smooth muscle cells (IC(50)=10 nM) and 5-HT-mediated contraction in rat aortic rings. Effects of AR246686 on arterial thrombosis and bleeding time were studied in a rat model of femoral artery occlusion. Oral dosing of AR246686 to rats resulted in prolongation of time to occlusion at 1 mg/kg, whereas increased bleeding time was observed at a dose of 20 mg/kg. In contrast, both bleeding time and time to occlusion were increased at the same dose (10 mg/kg) of clopidogrel. These results demonstrate that AR246686 is a high affinity 5-HT2A receptor antagonist with potent activity on platelets and vascular smooth muscle. Further, oral administration results in anti-thrombotic effects at doses that are free of significant effects on traumatic bleeding time.
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http://dx.doi.org/10.1016/j.ejphar.2007.11.056DOI Listing
May 2008