; The goal of Project 3 is to provide high-resolution structural information that will: (1) further our understanding ofthe mechanism by which allosteric modulators regulate GPCR function, (2) form the basis for docking experiments to identify new subtype selective allosteric modulators, and (3) provide structural information for optimizing docking hits. The X-ray structures of GPCRs such as the P2AR (in both inactive and active states), the muscarinic M2, and M3, and the p- and 5-opioid receptors have enabled this project. These targets were initially selected for structural studies in the Kobilka lab based on their interesting pharmacological properties: the P2AR, an established model system for GPCR signaling;the muscarinic receptors, model systems for allosteric regulation;and the opioid receptors, which have been shown to function as homo- and hetero-oligomers, and for which there is a wealth of both peptide and small molecule ligands. The existing structures will form the basis for initial docking studies proposed in Project 1. We will work with Project 1, Project 2, and the Medicinal Chemistry core to identify and optimize novel ligands through iterative rounds of crystallography and chemical optimization. Finally, the Kobilka lab will work with the Sunahara (Project 2) and Shoichet (Project 1) labs to correlate chemical structure and functional properties with structural changes and protein dynamics determined by biochemical and biophysical approaches.
Specific Aims :
Aim 1. Determine active-state structures ofthe M2 and M3 muscarinic receptors.
Aim 2. Obtain structures of receptors bound to positive and negative allosteric modulators.
Aim 3. Determine structures of intermediate conformational states ofthe B2AR.
Aim 4. Determine the structure of ligands bound to the B-opioid receptor oligomerization interface.
Aim 5. Characterize the effect of allosteric modulators on receptor structure and dynamics.

Public Health Relevance

This Project will obtain high resolution structural information that will enable the development of more selective and effective drugs for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZRG1-BST-J (40))
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Stanford University
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Korczynska, Magdalena; Clark, Mary J; Valant, Celine et al. (2018) Structure-based discovery of selective positive allosteric modulators of antagonists for the M2 muscarinic acetylcholine receptor. Proc Natl Acad Sci U S A 115:E2419-E2428
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Thal, David M; Sun, Bingfa; Feng, Dan et al. (2016) Crystal structures of the M1 and M4 muscarinic acetylcholine receptors. Nature 531:335-40
Mahoney, Jacob P; Sunahara, Roger K (2016) Mechanistic insights into GPCR-G protein interactions. Curr Opin Struct Biol 41:247-254
Kruse, Andrew C; Hu, Jianxin; Kobilka, Brian K et al. (2014) Muscarinic acetylcholine receptor X-ray structures: potential implications for drug development. Curr Opin Pharmacol 16:24-30

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