Potential inhibitors of chemokine function: analysis of noncovalent complexes of CC chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry.
Chemokines play a critical role in inducing chemotaxis, extravasation, and activation of leukocytes both in routine immunosurveillance and autoimmune diseases. Traditionally, to disrupt chemokine function, strategies have focused on blockage of its interaction with the receptor. Recently, it has been demonstrated that binding to glycosaminoglycans (GAGs) is also required for the in vivo activity of many chemokines. Thus, interference with the GAG-binding of chemokines may offer an alternative, valid, anti-inflammatory strategy. However, the potential of using small polyanions to inhibit the interactions between chemokines and cell surface GAGs has not been fully explored. In this study, a mass spectrometry based filtration trapping assay was utilized to study the interactions between two CCR 2 ligands (MCP-1/CCL2 and MCP-3/CCL7) and a series of low molecular weight, polyanionic molecules. Findings were confirmed by using a hydrophobic trapping assay. The results indicated that Arixtra (fondaparinux sodium), sucrose octasulfate, and suramin were specific binders of the chemokines, while cyclodextrin sulfate, although the most highly sulfated molecule among the ones investigated, showed no binding. The binding stoichiometry of the small molecule ligand was determined from the measured molecular weight of the noncovalent complex. Furthermore, the dissociation constant between MCP-3 and Arixtra was determined by using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, which compared favorably with the result of the isothermal titration calorimetry (ITC) assay. The relative binding affinity of these ligands to MCP-3 was also determined using a competitive filtration trapping assay.
Yu
Yonghao
Y
Genome Center, Department of Chemistry and Molecular Cell Biology, University of California at Davis, One Shields Road, 95616, Davis, CA, USA.|the Department of Chemistry, University of California at Berkeley, Berkeley, CA.
Sweeney
Matthew D
MD
Genome Center, Department of Chemistry and Molecular Cell Biology, University of California at Davis, One Shields Road, 95616, Davis, CA, USA.|the Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA.
Saad
Ola M
OM
Genome Center, Department of Chemistry and Molecular Cell Biology, University of California at Davis, One Shields Road, 95616, Davis, CA, USA.
Leary
Julie A
JA
Genome Center, Department of Chemistry and Molecular Cell Biology, University of California at Davis, One Shields Road, 95616, Davis, CA, USA. jaleary@ucdavis.edu.
D002151:Calorimetry; D002240:Carbohydrate Sequence; D018932:Chemokine CCL2 / Q000037:antagonists & inhibitors / Q000737:chemistry; D054410:Chemokine CCL7; D019742:Chemokines, CC / Q000037:antagonists & inhibitors* / Q000737:chemistry; D003505:Cyclodextrins / Q000737:chemistry / Q000494:pharmacology; D000077425:Fondaparinux; D005583:Fourier Analysis; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015394:Molecular Structure; D018945:Monocyte Chemoattractant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D000071228:Polyelectrolytes; D011108:Polymers / Q000737:chemistry / Q000494:pharmacology*; D011134:Polysaccharides / Q000737:chemistry / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013395:Sucrose / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology
D018932:Chemokine CCL2; D054410:Chemokine CCL7; D019742:Chemokines, CC; D003505:Cyclodextrins; D000077425:Fondaparinux; D046911:Macromolecular Substances; D018945:Monocyte Chemoattractant Proteins; D000071228:Polyelectrolytes; D011108:Polymers; D011134:Polysaccharides; D013395:Sucrose; C111835:CCL2 protein, human; C515450:CCL7 protein, human; C053081:sucrose octasulfate