Sulfotyrosine-guided discovery of small molecule chemokine inhibitors

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ABSTRACT The goal of this administrative supplement request is to provide a microscale thermophoresis instrument for measuring protein-ligand and protein-protein interactions for chemokine drug discovery for R01 GM097381. Chemokines direct cell migration primarily by binding and activating a family of G protein-coupled receptors that are post- translationally modified by tyrosine sulfation. Sulfation is required for chemokine function, suggesting that receptor sulfotyrosines participate directly in specific binding of the chemokine ligand. We are identifying small molecule ligands of the three main pro-metastatic chemokines (CXCL12, CCL19 and CCL21) and optimizing them as competitive inhibitors of receptor binding that block cancer cell migration. The proposed instrument will allow determination of affinities between small molecules and chemokines as well as chemokine- chemokine and chemokine-receptor interactions. Microscale thermophoresis (MST) offers an attractive complement to the tools currently employed because of its ability to use one to five orders of magnitude less sample, collect data in minutes rather than hours, and the ability to use MST with membranes and cell lysates. This instrument will be installed in the PIs laboratory as part of the Program in Chemical Biology (PCB) at the Medical College of Wisconsin. The PCB is already equipped with the necessary infrastructure for successful installation and operation of this instrument. The new instrument will be maintained by PhD-level staff with experience in the operation and maintenance of biophysical instruments. Consistent with its record of major investments in biophysical research infrastructure and facilities, MCW has committed space to house the requested instrument and partial salary for its maintenance.