Production of large quantities of hiighly purified receptors is an underlying requirement for all the projects in ttie Program Project -""""""""Structure-Function of Opioid Receptors for Drug Discovery. The production of these samples will be carried out using well established protocols that have now been optimized for use with GPCRs as part of GPCR Network's GPCR Structure Determination Pipeline. These production protocols now include the use of newly developed GPCR fusion partner toolchest for stabilization and crystallization that has increased the quantity and quality of structures that we are able to determine. Most notably, with the use of a new fusion partner, apo-cytochrome b562 (RIL) mutant, BRIL, we were able to determine the structure of the A2A adenosine receptor- ZM241385 to 1.8 A resolution, the structure of NOP, and more recently 2 agonist bound serotonin receptors in an activated state. Structural studies will demand the use of large volumes of highly purified protein for crystallization studies while others doing functional studies will need much less. Studies that led to the structure solution of the K-opioid receptor and the nociception/oprhanin FQ peptide receptor required a large number of constructs and an average of 25 mgs of highly purified protein or about 100 liters of biomass. For receptor-ligand complex structural studies we estimate that 5-10 mgs will be required per structure.
Large-scale structural studies of ligand bound complexes of all four opioid receptors will require the preparation of large volumes of highly purified receptors. Successful delivery of these samples will enable studies that could lead to the design of new generation therapeutics targeting opioid receptors that are highly selective and with reduced or minimal side-effects.
|Manglik, Aashish; Lin, Henry; Aryal, Dipendra K et al. (2016) Structure-based discovery of opioid analgesics with reduced side effects. Nature 537:185-190|
|Bruchas, Michael R; Roth, Bryan L (2016) New Technologies for Elucidating Opioid Receptor Function. Trends Pharmacol Sci 37:279-89|
|White, Thomas A; Barty, Anton; Liu, Wei et al. (2016) Serial femtosecond crystallography datasets from G protein-coupled receptors. Sci Data 3:160057|
|Roth, Bryan L (2016) DREADDs for Neuroscientists. Neuron 89:683-94|
|Kuhar, Jamie Rose; Bedini, Andrea; Melief, Erica J et al. (2015) Mu opioid receptor stimulation activates c-Jun N-terminal kinase 2 by distinct arrestin-dependent and independent mechanisms. Cell Signal 27:1799-806|
|Vardy, Eyal; Robinson, J Elliott; Li, Chia et al. (2015) A New DREADD Facilitates the Multiplexed Chemogenetic Interrogation of Behavior. Neuron 86:936-46|
|O'Connor, Casey; White, Kate L; Doncescu, Nathalie et al. (2015) NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor. Proc Natl Acad Sci U S A 112:11852-7|
|Vardy, Eyal; Sassano, Maria F; Rennekamp, Andrew J et al. (2015) Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides. Chem Biol 22:764-75|
|Isberg, Vignir; de Graaf, Chris; Bortolato, Andrea et al. (2015) Generic GPCR residue numbers - aligning topology maps while minding the gaps. Trends Pharmacol Sci 36:22-31|
|Robinson, J Elliott; Vardy, Eyal; DiBerto, Jeffrey F et al. (2015) Receptor Reserve Moderates Mesolimbic Responses to Opioids in a Humanized Mouse Model of the OPRM1 A118G Polymorphism. Neuropsychopharmacology 40:2614-22|
Showing the most recent 10 out of 15 publications