High-affinity peptidomimetic targeting ligands to activated alpha4beta1 (generated in Program 1) have an LDI motif, which is similar to the LDV alpha4beta1 -binding motif of the CS-1 peptide of fibronectin. Objectives of Program 2 are to establish the integrin specificity of the targeting ligands, to identify how the targeting ligands bind to activated alpha4beta1, and to test their potential to specifically target alpha4beta1 expressing tumor cells. For this purpose, 1) we will determine the integrin specificity of targeting ligands using cells expressing different recombinant integrins, and identify the amino acid residues critical for the binding of targeting ligands using cell lines expressing alpha4beta1 with mutations within the predicted ligand-binding site. These experiments will identify how the targeting ligands bind to activated alpha4beta1 and facilitate optimization of the agents for clinical studies. 2) We will test the ability of the targeting ligands to block alpha4beta1 positive tumor growth, and to deliver toxin to alpha4-positive tumor cells and kill them in vitro. We expect that the targeting ligands will be effective in blocking alpha4beta1 functions or delivering toxins to malignant cells. 3) We will identify how the LDI motif interacts with alpha4. Since mutating Trp-188 of alpha4 to Ala abolishes the binding of our targeting agent, we hypothesize that the indole side-chain of Trp-188 directly interacts with some components on the targeting agent. To address this hypothesis, we will screen chemical libraries based on the LDI motif, for modified targeting ligands that bind to the mutant alpha4. We expect that the targeting ligands isolated from such screen will have additional pharmacophore that compensates for the loss of Trp side-chain of alpha4. Structural analysis of original and modified targeting ligands, and molecular modeling of alpha4beta1 will provide insight into how the LDI motif binds to alpha4. The proposed experiments will establish the binding specificities of targeting ligands, test their therapeutic potentials in vitro, and provide insight into how targeting ligands interact with alpha4beta1.
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