G protein coupled receptors (GPCRs) represent the largest protein superfamily in humans, with nearly 1000 members. They are seven transmembrane receptors that coordinate intercellular communication via the transduction of a wide range of stimuli involved in sensation, neurotransmission, development, emotion, cognition, and function in the CNS, endocrine and immune systems. Chemokine receptors are an important class of GPCRs that are best known for their pivotal role in immune surveillance, where they control the migration and activation of leukocytes in an effort to detect and resolve physiological abnormalities such as cancer and infection. However, inappropriate expression or regulation of these receptors is associated with an extraordinary number of pathologies including inflammatory diseases, cancer and AIDS;thus there is significant interest in developing small molecule receptor antagonists that block the function of specific chemokine receptors. Until recently, GPCRs had eluded structure determination due to challenges in receptor expression and crystallization. However, new technologies have emerged which has made the viability of determining GPCR structures indisputable. To this end, our primary goal is to obtain structural information on chemokine receptors and receptor complexes that can aid drug discovery efforts aimed at improving affinity, efficacy, and selectivity. Accordingly, in collaboration with the PSI network, we will apply novel technologies for the expression, purification and crystallization of GPCRs from the chemokine receptor family, with the goal of determining at least two different receptor structures and multiple co-complexes by the five-year endpoint. To maximize the capabilities of the PSI centers in generating purified protein, and to acquire insights into the dynamic aspects of receptor function, the crystallographic work will be complemented with biophysical studies. Radiolytic footprinting will be developed and applied to map the binding interfaces between chemokines and receptors and to determine information on activation mechanisms. The interaction of pathogenic proteins with chemokine receptors will also be investigated, specifically, the CCR5 receptor with the HIV glycoprotein gp120, and the DARC receptor with the malarial docking protein, DBP. Site Directed Spin Labeling with Electron Paramagnetic Resonance (SDSL-EPR) will be used to characterize the conformational changes associated with ligand binding. All of these studies will be augmented with computational modeling methods in order to rationally guide the experimental construct design and to interpret the biophysical data in a 3D context.

Public Health Relevance

Chemokine receptors are involved in many human diseases including asthma, multiple sclerosis, rheumatoid arthritis, heart disease, cancer and HIV, Determining structures of chemokine receptors in complex with small molecule drugs and ligands, and information on ligand-induced conformational changes, will significantly aid the development of drugs targeting these receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01GM094612-03
Application #
8298576
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (BC))
Program Officer
Chin, Jean
Project Start
2010-09-30
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2012
Total Cost
$898,335
Indirect Cost
$411,763
Name
University of California San Diego
Department
None
Type
Schools of Pharmacy
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Gustavsson, Martin; Zheng, Yi; Handel, Tracy M (2016) Production of Chemokine/Chemokine Receptor Complexes for Structural Biophysical Studies. Methods Enzymol 570:233-60
Ngo, Tony; Kufareva, Irina; Coleman, James L J et al. (2016) Identifying ligands at orphan GPCRs: current status using structure-based approaches. Br J Pharmacol :
Kufareva, Irina; Gustavsson, Martin; Holden, Lauren G et al. (2016) Disulfide Trapping for Modeling and Structure Determination of Receptor: Chemokine Complexes. Methods Enzymol 570:389-420
Zheng, Yi; Qin, Ling; Zacarías, Natalia V Ortiz et al. (2016) Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature 540:458-461
Leach, Katie; Gregory, Karen J; Kufareva, Irina et al. (2016) Towards a structural understanding of allosteric drugs at the human calcium-sensing receptor. Cell Res 26:574-92
Hanes, Melinda S; Salanga, Catherina L; Chowdry, Arnab B et al. (2015) Dual targeting of the chemokine receptors CXCR4 and ACKR3 with novel engineered chemokines. J Biol Chem 290:22385-97
Geidl, Stanislav; Svobodová Vařeková, Radka; Bendová, Veronika et al. (2015) How Does the Methodology of 3D Structure Preparation Influence the Quality of pKa Prediction? J Chem Inf Model 55:1088-97
Husby, Jarmila; Bottegoni, Giovanni; Kufareva, Irina et al. (2015) Structure-based predictions of activity cliffs. J Chem Inf Model 55:1062-76
Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben et al. (2015) Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily. Membranes (Basel) 5:646-63
Kufareva, Irina; Handel, Tracy M; Abagyan, Ruben (2015) Experiment-Guided Molecular Modeling of Protein-Protein Complexes Involving GPCRs. Methods Mol Biol 1335:295-311

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