G-protein coupled receptors (GPCRs) are among the most important proteins in humans because they are the 'gateway'for many signally pathways, including those that contribute to diseases, and their functions are amenable to intervention by drugs. By recognizing and binding specific chemicals, GPCRs transduce signals from the outside of cell to the inside where they trigger a cascade of biological events. Consequently, GPCRs are the most fecund class of proteins for structure determination. They are the largest class of membrane proteins;~800 GPCRs are encoded in the human genome, about half of which are potential drug targets. However, only ~60 of them are currently used as receptors for small molecule drugs and ~25 of them for bio- therapeutics based on the natural ligand;nonetheless, drugs that bind to GPCRs account for about one-third of all therapeutic drugs. The annual worldwide market for drugs that interact with GPCRs is predicted to be $118 Billion by 2014. More than 80 additional GPCRs are potentially amenable to antibody therapeutics, a novel avenue of attack that is receiving increasing attention, and one that we will explore. The difficulty in preparing samples for X-ray crystallography and Solution NMR from large membrane proteins in liquid crystalline phospholipid bilayers has been the principal roadblock to structure-based drug discovery. In order to overcome this roadblock we have simultaneously developed new methods for solid-state NMR spectroscopy and applied to structure determination of membrane proteins. Our principal target is the chemokine receptor CXCR1, and we have made substantial progress towards determining its structure during the first five years of this award. Our studies of interleukin-8 (IL-8) interacting with CXCR1 provide a framework for discovery of drugs that may affect inflammation, cancer metastasis, and other diseases by binding to CXCR1. In order to accelerate the design and discovery of drugs that interact with CXCR1 and other GPCRs, we are developing a general method for determining the three-dimensional structures of GPCRs in their native phospholipid environment under physiological conditions. This research is multidisciplinary, involving molecular biology, biochemistry, structural biology, NMR spectroscopy, and computation. It is highly effective at training scientists who can work and interact across chemical and biological boundaries. We recognize that it requires the highest levels of technology available;as a result, it is organized as a Bioengineering Research Partnership among the University of California, San Diego (UCSD) and two biotechnology companies (Cambridge Isotope Laboratories (CIL), Andover, Massachusetts and Membrane Receptor Technologies (MRT), San Diego, California. CIL's key technology is the synthesis of isotopically labeled amino acids and precursors, and the manufacture of unique isotopically labeled bacterial growth media;their effort is led by Joel Bradley, Ph.D. MRT has the technology for the expression, purification, and refolding of biologically active GPCRs developed originally by Hans Kiefer, Ph.D., at M-Fold Biotech and he remains involved in the research.

Public Health Relevance

Most diseases that afflict humans can be treated or cured with drugs. The majority of therapeutic drugs are chemicals targeted to protein receptors that reside in cell membranes, the largest class of which is G-protein coupled receptors (GPCRs). Determining the structures of GPCRs is a very high priority goal of biomedical research because it will accelerate the discovery of new drugs to treat a wide range of diseases. The research project described in this proposal will advance the method of NMR spectroscopy so that it can be used to determine the structures of GPCRs in their native environment of phospholipid bilayers under physiological conditions. This is a technically demanding project that requires the methods of chemistry, physics, and biology. This project is focused on the chemokine receptor CXCR1 and its interactions with its natural ligand interleukin-8 (IL-8) as an example that affects several diseases, including inflammatory disease and cancer metastasis. The methods demonstrated with this example should be applicable to many other GPCRs (and diseases) as well as other classes of membrane proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB005161-07
Application #
8461160
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Liu, Christina
Project Start
2005-04-01
Project End
2015-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
7
Fiscal Year
2013
Total Cost
$720,904
Indirect Cost
$249,404
Name
University of California San Diego
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Park, Sang Ho; Wang, Vivian S; Radoicic, Jasmina et al. (2015) Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA). J Biomol NMR 61:185-96
Lin, Eugene C; Opella, Stanley J (2014) Covariance spectroscopy in high-resolution multi-dimensional solid-state NMR. J Magn Reson 239:57-60
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Tian, Ye; Lu, George J; Marassi, Francesca M et al. (2014) Structure of the membrane protein MerF, a bacterial mercury transporter, improved by the inclusion of chemical shift anisotropy constraints. J Biomol NMR 60:67-71
Lu, George J; Opella, Stanley J (2014) Mechanism of dilute-spin-exchange in solid-state NMR. J Chem Phys 140:124201
Wu, Chin H; De Angelis, Anna A; Opella, Stanley J (2014) Magic angle Lee-Goldburg frequency offset irradiation improves the efficiency and selectivity of SPECIFIC-CP in triple-resonance MAS solid-state NMR. J Magn Reson 246:1-3
Radoicic, Jasmina; Lu, George J; Opella, Stanley J (2014) NMR structures of membrane proteins in phospholipid bilayers. Q Rev Biophys 47:249-83
Das, Bibhuti B; Zhang, Hua; Opella, Stanley J (2014) Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers. J Magn Reson 242:224-32
Lu, George J; Opella, Stanley J (2014) Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR. J Biomol NMR 58:69-81

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