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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Program Projects (P01)
Project #
1P01DA035764-01A1
Application #
8667569
Study Section
Special Emphasis Panel (ZRG1-BCMB-S (40))
Project Start
Project End
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
1
Fiscal Year
2014
Total Cost
$375,629
Indirect Cost
$177,408
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Schattauer, Selena S; Land, Benjamin B; Reichard, Kathryn L et al. (2017) Peroxiredoxin 6 mediates G?i protein-coupled receptor inactivation by cJun kinase. Nat Commun 8:743
Wacker, Daniel; Stevens, Raymond C; Roth, Bryan L (2017) How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170:414-427
Huang, Xi-Ping; Che, Tao; Mangano, Thomas J et al. (2017) Fentanyl-related designer drugs W-18 and W-15 lack appreciable opioid activity in vitro and in vivo. JCI Insight 2:
Zheng, Zhong; Huang, Xi-Ping; Mangano, Thomas J et al. (2017) Structure-Based Discovery of New Antagonist and Biased Agonist Chemotypes for the Kappa Opioid Receptor. J Med Chem 60:3070-3081
Lansu, Katherine; Karpiak, Joel; Liu, Jing et al. (2017) In silico design of novel probes for the atypical opioid receptor MRGPRX2. Nat Chem Biol 13:529-536
Bruchas, Michael R; Roth, Bryan L (2016) New Technologies for Elucidating Opioid Receptor Function. Trends Pharmacol Sci 37:279-89
Manglik, Aashish; Lin, Henry; Aryal, Dipendra K et al. (2016) Structure-based discovery of opioid analgesics with reduced side effects. Nature 537:185-190
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
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

Showing the most recent 10 out of 20 publications