Abstract
Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3′-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand–protein contacts and its signaling complexes.
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Funding
SM is supported by funds from NIH Grants DA045884 and W81XWH-17-1-0256 (Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program) and start-up funds from Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University to SM. This research was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748. Studies were also supported by NIDA grants DA042888 and DA046714 (YXP) and DA048379, DA007242 and DA006241 (GWP/YXP).
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SG and SM jointly supervised this work. SGG, RU, AV, JS and AH performed research and provided tool compounds. SGG, RU, YXP, GWP and SM analyzed data. SGG, RU, GWP and SM wrote the paper with input from all authors. All authors read and approved the final manuscript.
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GWP, YXP and SM are co‐founders of Sparian BioSciences. All authors report no other biomedical financial interests or potential conflicts of interest.
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Grinnell, S.G., Uprety, R., Varadi, A. et al. Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain. Cell Mol Neurobiol 41, 977–993 (2021). https://doi.org/10.1007/s10571-020-00867-6
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DOI: https://doi.org/10.1007/s10571-020-00867-6