The long-term goal of this work is the discovery of novel approaches to promote or inhibit chemokine-mediated cell migration in vivo. Chemokines function through at least three distinct types of interactions: dimerization, extracellular matrix glycosaminoglycan (GAG) binding, and GPCR activation. Any of these complexes may serve as targets for drug development, and engineered versions of the chemokine may have unique properties that can be exploited therapeutically. Stromal cell-derived factor-1 (SDF1/ CXCL12) and its cognate receptors CXCR4 and CXCR7 are essential for life and play unique roles in human development, HIV infection, stem cell homing and cancer progression. Development of new agents targeting SDF1 for diagnostic or therapeutic use requires a view of its functional interactions at atomic resolution. In the previous funding period, we found that interactions with GAGs and the CXCR4 receptor promote SDF1 dimerization. Unexpectedly, our functional data revealed that SDF1 dimerization creates a potent inhibitor of chemotaxis. It is now clear that, while dimerization is essential for chemokine function in vivo, only chemokine monomers are capable of full GPCR activation and chemotaxis. We recently solved the monomeric structure of SDF1 in order to resolve conflicting reports in the literature, and are now positioned to determine structures of soluble monomeric SDF1 complexes with CXCR4 and CXCR7 (specific aim 1). SDF1 is under active investigation as a treatment for prevention of myocardial ischemia/reperfusion injury. Experiments in aim 2 will define the GAG binding site on the SDF1 dimer and use this knowledge to construct monomeric SDF1 variants with enhanced cardioprotective properties. Molecules that bind SDF1 and block its activity in vivo may find utility as novel therapeutic agents. In solving the structure of a dimeric SDF1-CXCR4 complex using sulfotyrosine-modified receptor fragments, we provided the first detailed view of receptor recognition by a chemokine, paving the way for structure-based drug discovery. A new component of aim 3 proposes to screen compound libraries for inhibitors that target the chemokine ligand, using a combination of high-throughput docking calculations, validation of site-specific ligand binding by 2D NMR, and functional assays to test for inhibition of SDF1 signaling. We will also define the basis for SDF1 inhibition by a viral chemokine binding protein in aim 3. Like other chemokines, SDF1-mediated chemotaxis is maximal at 10-30 nM, but ceases above 100 nM. We speculate that, at high concentrations where chemokines no longer induce cell migration, dimerization of wild-type SDF1 promoted by receptor binding acts to suppress CXCR4-mediated chemotaxis. Experiments in Aim 4 will test our hypothesis and compare the responses for CXCR4 and CXCR7 to different oligomeric states of the chemokine ligand. We will also investigate the ability of engineered SDF1 monomers and dimers to alter disease progression in animal models for human diseases including multiple sclerosis, ulcerative colitis, and metastatic cancer.
Chemokines orchestrate innate immune responses injury or infection, and also participate in embryonic development, stem cell homing, chronic inflammation, HIV infection and cancer metastasis. The chemokine SDF1 and other ligands for its receptor CXCR4 can block HIV-1 infection of T cells. Over 20 cancer types express CXCR4 and metastasize to tissues that secrete SDF1, including bone marrow, lung, liver and lymph nodes. Treatment with CXCR4-neutralizing antibodies reduces metastatic tumor formation in a mouse model for human breast cancer. In the previous funding period, we discovered that the dimeric form of SDF1 is a potent inhibitor of chemotaxis that can also block HIV-1 infection. This project will identify the molecular structures required for SDF1-CXCR4 cell migration and define the mechanistic basis for this novel mode of chemokine inhibition. New inhibitors and variants of SDF developed in this project will likely find application as diagnostic and therapeutic agents in a wide range of human diseases, including cardiovascular disease, cancer and multiple sclerosis.
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|Whitney, Dustin S; Peterson, Francis C; Kovrigin, Evgenii L et al. (2013) Allosteric activation of the Par-6 PDZ via a partial unfolding transition. J Am Chem Soc 135:9377-83|
|Ziarek, Joshua J; Veldkamp, Christopher T; Zhang, Fuming et al. (2013) Heparin oligosaccharides inhibit chemokine (CXC motif) ligand 12 (CXCL12) cardioprotection by binding orthogonal to the dimerization interface, promoting oligomerization, and competing with the chemokine (CXC motif) receptor 4 (CXCR4) N terminus. J Biol Chem 288:737-46|
|Ziarek, Joshua J; Getschman, Anthony E; Butler, Stephen J et al. (2013) Sulfopeptide probes of the CXCR4/CXCL12 interface reveal oligomer-specific contacts and chemokine allostery. ACS Chem Biol 8:1955-63|
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|Tyler, Robert C; Murray, Nathan J; Peterson, Francis C et al. (2011) Native-state interconversion of a metamorphic protein requires global unfolding. Biochemistry 50:7077-9|
|Ziarek, Joshua J; Peterson, Francis C; Lytle, Betsy L et al. (2011) Binding site identification and structure determination of protein-ligand complexes by NMR a semiautomated approach. Methods Enzymol 493:241-75|
|Levison, Julie H; Orrell, Catherine; Losina, Elena et al. (2011) Early outcomes and the virological effect of delayed treatment switching to second-line therapy in an antiretroviral roll-out programme in South Africa. Antivir Ther 16:853-61|
|Drury, Luke J; Ziarek, Joshua J; Gravel, Stephanie et al. (2011) Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways. Proc Natl Acad Sci U S A 108:17655-60|
|Veldkamp, Christopher T; Ziarek, Joshua J; Peterson, Francis C et al. (2010) Targeting SDF-1/CXCL12 with a ligand that prevents activation of CXCR4 through structure-based drug design. J Am Chem Soc 132:7242-3|
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