Metastatic breast cancer kills patients. Our research is focused on developing a novel therapeutic platform to stop the progression of metastatic breast cancer. There is considerable evidence for the involvement of chemokines in metastatic tumor cell disease spreading. We have discovered novel small molecule CX3CR1 antagonists that target CX3CR1 in tumor cells and have shown that this chemokine receptor is involved in metastasis. This proposal aims to evaluate chemokine mediated dissemination as a novel therapeutic approach to halt the homing, arrest, and extravasation of circulating tumor cells to the bone marrow. We have demonstrated target proof of concept in Fractalkine knock-out animals and by treatment with a neutralizing antibody directed toward CX3CR1 expressed on cancer cells in animal models of metastasis by showing a significant decrease in disseminated tumor cells to the skeleton. Targeting an inflammatory chemokine such as Fractalkine as opposed to a homeostatic chemokine is predicted to provide a safe mechanism of action. Both CX3CR1 and Fractalkine knock-out animals are viable and appear normal compared to wild type animals which suggests that CX3CR1 antagonism will be a relatively benign intervention. This approach will provide non- cytotoxic drugs against a novel molecular target, the chemokine receptor CX3CR1, to stop the progression of metastatic breast cancer. The target, CX3CR1, is over expressed in malignant tissue and on highly metastatic tumor cells, including those responsible for inflammatory breast cancer (IBC). IBC is a very aggressive metastatic disease in need of new approaches. In addition to IBC, the successful development of agents which slow or halt metastasis are envisioned to have broad beneficial use. We will develop proprietary small molecule CX3CR1 antagonists which target highly metastatic tumor cells. A CX3CR1 neutralizing monoclonal antibody and drug conjugate approaches are likely as follow-on products. Specific Aims of this proposal are: Aim 1: To scale up the synthesis of JMS-17-2 for preliminary evaluation in our in vivo model, and optimize the lead compounds, JMS-16-7 and JMS-17-2, to improve selectivity and drug-like properties such as; water solubility, plasma and liver microsomal stability, and microsomal intrinsic clearance. Aim 2: To determine the pharmacokinetic properties of our best CX3CR1 antagonist meeting potency and ADME criteria for water solubility, plasma stability, intrinsic clearance, and plasma protein binding by pharmacokinetic determination in the mouse, and to test this candidate in our translational animal models of metastasis.