Cancer Therapeutics that Anchor Proteins to Membranes

描述(申请人提供)：多药耐药(MDR)癌症仍然是根治癌症化疗的主要障碍。MDR癌细胞与典型的肿瘤细胞不同，它显著上调了几种因子的产生，包括药物转运蛋白P-糖蛋白、胆固醇结合蛋白小窝和细胞膜脂筏微域的成分。这些RAFT结构域富含胆固醇和鞘磷脂，在信号转导过程中发挥关键作用。MDR癌的不同质膜成分可能使针对这些癌症的选择性化疗成为可能。在上一个资助周期中，合成了新的小分子，包括与生物素等蛋白质配体共价连接的胆固醇胺。这些化合物结合了癌细胞质膜上的脂筏。用合成的生物素-胆固醇胺配体(配体#1)和蛋白链霉亲和素(SA)有效地将SA靶向脂筏，导致这种蛋白质-配体复合体的快速内吞。这个新的系统模拟霍乱毒素对细胞的渗透，霍乱毒素与脂筏中的神经节苷脂GM1结合。该项目基于一种假设，即1号配体将调节癌细胞系中与内体激活毒素柔红霉素和外毒素有关的SA的内吞作用。由于配体#1结合脂筏，选择性地将SA连接的毒素输送到富含脂筏的多药耐药癌细胞，将在体外和体内进行小鼠癌症模型的研究。还将评估配基#1在增强针对神经母细胞瘤细胞上特定受体的神经肽Y与萨托辛融合的内吞作用方面的有效性。这一新的方法旨在通过靶向小分子脂筏来增强表面受体的内吞作用，可以解决非内化肿瘤抗原免疫治疗的主要问题。配体调节的SA递送到抗原提呈细胞(APC)将刺激免疫反应的假设也将得到检验。新型免疫刺激剂将通过将SA融合到卵清蛋白抗原，用1号配体调节APC的内吞作用，以及分析T细胞的激活来研究。这种方法可以在分子水平上控制免疫刺激，并为疫苗开发提供新的工具。为了有条件地调节细胞的生长和死亡，还将研究1号配体对细胞内亲和素融合蛋白到质膜的募集作用。

DESCRIPTION (provided by applicant): Multidrug resistant (MDR) cancer remains the primary impediment to curative cancer chemotherapy. MDR cancer cells differ from typical tumor cells by dramatically upregulating production of several factors including the drug transporter P-glycoprotein, the cholesterol binding protein caveolin, and components of lipid raft microdomains of cellular plasma membranes. These raft domains are enriched in cholesterol and sphingolipids and play key roles in signal transduction processes. The distinct composition of plasma membranes of MDR cancers may enable selective chemotherapy targeting these cancers. During the last grant cycle, novel small molecules were synthesized that comprise cholesterylamine covalently linked to protein ligands such as biotin. These compounds bind lipid rafts in plasma membranes of cancer cells.Treatment of cancer cell lines with a synthetic biotin-cholesterylamine ligand (ligand #1) and the protein Streptavidin (SA) efficiently targets SA to lipid rafts, resulting in rapid clathrin-mediated endocytosis of this protein-ligand complex. This novel system mimics penetration of cells by Cholera toxin, which binds ganglioside GM1 in lipid rafts. This project is based on the hypothesis that ligand #1 will regulate endocytosis of SA linked to endosome-activated toxins daunorubicin and exotoxin in cancer cell lines. Since ligand #1 binds lipid rafts, selective delivery of SA-linked toxins to lipid raft-rich MDR cancer cells will be investigated in vitro and in vivo in murine cancer models. The effectiveness of ligand #1 at enhancing endocytosis of Satoxins fused to neuropeptide Y, which targets specific receptors on neuroblastoma cells, will also be evaluated. This novel approach directed at enhancing endocytosis of surface receptors by targeting to lipid rafts with small molecules could address the major problem in immunotherapy of non-internalized tumor antigens. The hypothesis that ligand-regulated delivery of SA to antigen presenting cells (APCs) will stimulate immune responses will also be tested. Novel immunostimulants will be investigated by fusing SA to the ovalbumin antigen, regulating endocytosis in APCs with ligand #1, and analyzing T-cell activation. This approach could control immunostimulation at the molecular level and yield novel tools for vaccine development. Recruitment of intracellular avidin fusion proteins to plasma membranes by ligand #1 will also be studied in an effort to conditionally regulate cellular growth and death