Funds are requested to carry out NMR studies to develop a deeper understanding of the dynamic behavior of G-Protein Coupled Receptors (GPCRs) and their signaling complexes to enable the construction of a better model of their mechanism of action. GPCRs are critical eukaryotic signal transduction gatekeepers which recognize a variety of extracellular stimuli, including photons, ions, small molecules, peptides and proteins. They transmit the resulting extracellular signal across the membrane by coupling to different intracellular proteins which then activate downstream effectors and trigger cascades of cellular and physiological responses. This has profound therapeutic implications, making these receptors the targets of ~30% of currently prescribed drugs. Developing a better understanding of how they work is, therefore, expected to help in developing new and better drugs targeting them. Although there has been a dramatic growth in the number of structures of GPCRs bound to antagonists, agonists including biased ligands, inverse agonists, and allosteric modulators, the molecular mechanisms of activation remain poorly understood. The proposed study rests on the premise that needed clarity can be achieved with a better understanding of the dynamic behavior of the system. We plan to characterize the dynamic behavior of all the members of the opioid family of receptors (Class A, ?-subfamily). There are four members in this family - the classical opioid receptors (OR) ?-, ?-, ?, (MOR, KOR, DOR) and the nociceptn receptor (NOP), all of which are coupled predominantly to heterotrimeric Gi/Go proteins. ORs are acted on by opioids which have had a long history in both chronic and acute pain control and addiction. This funding request is to support studies on KOR and the development of new knowledge and protocols that could be applied to the rest of the family. We will achieve a better understanding of the mechanism of action including the activation process through three specific aims: (1) Develop an understanding of the dynamic conformational landscape of KOR in the active and inactive state, (2) Develop an understanding of the dynamic behavior of the KOR signaling complex, and (3) Develop an understanding of the KOR-dynorphin interaction in the low and high affinity states. In this proposal, we will use our newly developed 19F NMR approach to characterize the dynamic behavior of KOR and standard NMR techniques for 2H/13C/15N NMR studies of the peptide dynorphin and the signaling partners to characterize changes to their dynamic behavior on binding to KOR. Studies will allow us to explore various questions, answers to which will help us build a better model describing the activation process. In addition, protocols and reagents generated in the study should help establish a new platform for developing new drugs targeting opioid receptors with reduced or minimal side-effects.
We will develop a better understanding of how receptors that interact with opioids work. These receptors have been the target of both chronic and acute pain control but their overuse has led to tolerance and addiction. Our study rests on the premise that dynamics plays an important role in receptor mechanisms and their characterization will generate new understanding that can be used to generate new and better therapeutics.
| Eddy, Matthew T; Gao, Zhan-Guo; Mannes, Philip et al. (2018) Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A2A Adenosine Receptor. J Am Chem Soc 140:8228-8235 |
| Eddy, Matthew T; Lee, Ming-Yue; Gao, Zhan-Guo et al. (2018) Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor. Cell 172:68-80.e12 |
| 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 |
| SuĊĦac, Lukas; O'Connor, Casey; Stevens, Raymond C et al. (2015) In-Membrane Chemical Modification (IMCM) for Site-Specific Chromophore Labeling of GPCRs. Angew Chem Int Ed Engl 54:15246-9 |
| Lamichhane, Rajan; Liu, Jeffrey J; Pljevaljcic, Goran et al. (2015) Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor ?2AR. Proc Natl Acad Sci U S A 112:14254-9 |
