Characterization of Targeting Ligands-Integrin Interaction and in vitro targeting

靶向活化α 4 β 1配体的高亲和力拟肽（程序1中生成）具有LDI基序，其与纤连蛋白CS-1肽的LDV α 4 β 1结合基序相似。程序2的目的是建立靶向配体的整合素特异性，鉴定靶向配体如何结合活化的α 4 β 1，并测试它们特异性靶向表达α 4 β 1的肿瘤细胞的潜力。为此目的，1）我们将使用表达不同重组整合素的细胞来确定靶向配体的整合素特异性，并使用表达α 4 β 1的细胞系来鉴定对于靶向配体的结合至关重要的氨基酸残基，所述α 4 β 1具有在预测的整合素特异性范围内的突变。 配体结合位点。这些实验将确定靶向配体如何与活化的α 4 β 1结合，并促进用于临床研究的试剂的优化。2)我们将测试靶向配体阻断α 4 β 1阳性肿瘤生长的能力，以及将毒素递送至α 4阳性肿瘤细胞并在体外杀死它们的能力。我们期望靶向配体将有效地阻断α 4 β 1功能或将毒素递送至恶性细胞。3)我们将确定LDI基序如何与α 4相互作用。由于将α 4的Trp-188突变为Ala消除了我们的靶向剂的结合，我们假设α 4的吲哚侧链 Trp-188直接与靶向剂上的一些组分相互作用。为了解决这一假设，我们将筛选基于LDI基序的化学文库，用于结合突变体α 4的修饰的靶向配体。我们预期从这种筛选中分离的靶向配体将具有额外的药效团，其补偿α 4的Trp侧链的损失。原始和修饰的靶向配体的结构分析以及α 4 β 1的分子建模将提供对LDI基序如何与α 4结合的深入了解。拟议的实验将建立靶向配体的结合特异性，测试其体外治疗潜力，并提供洞察靶向配体如何与α 4 β 1相互作用。

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.