Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic ␦-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human ␦-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 Ϯ 91, 399 Ϯ 2, and 433 Ϯ 73% after chronic treatment with the ␦-opioid-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 M, 4-h) treatment augmented 32 P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 Ϯ 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic ␦-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.Chronic opioid receptor activation frequently leads to the sensitization of adenylyl cyclase to stimulators after the inhibitory agonist has been removed (AC superactivation). AC superactivation after chronic opioid agonist exposure is thought to contribute to the development of opioid tolerance, dependence, and withdrawal (Williams et al., 2001). A better understanding of the molecular mechanisms of chronic ␦-opioid agonist treatment-mediated AC superactivation should aid in the development of longer acting analgesics with fewer side effects.We have reported previously that in Chinese hamster ovary (CHO) cells stably expressing the human ␦-opioid receptor (hDOR/CHO), chronic ␦-opioid agonist treatment gives rise to AC superactivation (Malatynska et al., 1996). In addition, we also demonstrated that chronic ␦-opioid agonist treatment of the hDOR/CHO cells augments 32 P incorporation into proteins immunoreactive with an AC V/VI-specific antibody (Varga et al., 1999). The identity of protein kinase(s) involved in chronic ␦-opioid agonist-mediated phosphorylation in hDOR/CHO cells, and the role of the phosphorylation in AC superactivation, however, have not been investigated previously. In the present work, we studied the effect of protein kinase inhibitors on chronic ␦-opioid agonistmediated AC superactivation.Recent data (Tan et al., 2001) indicate the important role of the protein kinase p74Raf-1 in phosphorylation of adenylyl cyclase VI in transfected human embryonic kidney 293 cells. Moreover, Raf-1-mediated phosphorylation led to the sensitization of AC VI to s...

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An enantioselective synthesis of the CGRP antagonist BMS-846372, amenable to large scale preparation, is presented. This new synthesis showcases a chemo- and enantioselective reduction of a cyclohepta[b]pyridine-5,9-dione as well as a Pd-catalyzed alpha-arylation reaction to form the key carbon-carbon bond and set the absolute and relative stereochemistry.

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A new and efficient synthesis of chloromethyl chlorosulfate (CMCS) from chloroiodomethane and chlorosulfonic acid is described. This process leverages a chlorosulfonic acid-mediated iodide oxidation to drive the equilibrating displacement process to full conversion. The resulting iodine byproduct is further oxidized and removed as iodate, to prevent iodide-induced decomposition of CMCS. This new process provides an efficient and scalable protocol for the preparation of CMCS in 92% solution yield and high purity (>99 GC area %).