Recruitment of leukocytes by chemokines is central to the inflammatory responses that cause tissue damage in a wide range of diseases. Eotaxin is the only chemokine that specifically attracts eosinophils. Its activity is implicated in allergic asthma, tumor metastasis, parasitic infestations, and other diseases involving eosinophil accumulation. Blocking the interaction between eotaxin and its receptor may be an effective therapeutic strategy towards prevention or attenuation of these diseases. In this study, the three-dimensional structure of eotaxin will be determined using nuclear magnetic resonance (NMR) spectroscopy and alanine scanning mutagenesis will be used to analyze the structure-activity relationships of eotaxin. In particular, the regions responsible for binding and activating the receptor will be identified along with a number of eotaxin variants that function as receptor agonists and antagonists. The proposed study will address the hypothesis that dimerization inhibits the interaction of eotaxin with its receptor. Indeed dimerization may be a general mechanism for regulating the biological activity of chemokines. Covalenfly cross- linked variants will be used to test this hypothesis. In addition, cotaxin mutants that do not interact with receptor but still dimerize will be tested for their ability to inhibit wild type eotaxin by a heterodimerization mechanism. Affinity maturation of the mutant/wild type interaction by phage display technology will be used to select mutants that strongly inhibit the wild type chemokine by heterodimerization. This approach could potentially be extended to yield variants that inhibit other chemokines or that specifically inhibit one chemokine but not another. Such compounds may be useful as molecular probes for elucidating the functions of chemokines both in vitro and in animal models of disease and will have potential as therapeutic anti-inflammatory agents.
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