MECHANISM OF DEQUALINIUM ACTION IN METASTATIC MELANOMA

地喹啉治疗转移性黑色素瘤的作用机制

• 批准号: 2101365
• 负责人: Susan A. Rotenberg
• 金额: $ 10.47万
• 依托单位: QUEENS COLLEGE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-18 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2101365
• 关键词: antineoplastics; antisense nucleic acid; chemical binding; chemical synthesis; computer assisted sequence analysis; crosslink; drug screening /evaluation; enzyme activity; enzyme inhibitors; gene deletion mutation; laboratory mouse; melanoma; metastasis; neoplasm /cancer pharmacology; photoactivation; physical model; protein isoforms; protein kinase C; protein sequence; proteolysis; site directed mutagenesis; stoichiometry

The improved design of antineoplastic drugs is enabled by a knowledge of the drug pharmacophore and its binding surface on an intracellular target. Dequalinium, an antineoplastic agent that inhibits carcinoma growth in a number of animal models, is known to inhibit protein kinase C (PKC), a critical component in signal transduction pathways. Irradiation with 365 nm light photoactivates dequalinium in vitro causing it to modify PKC covalently and consequently to cause irreversible inhibition of PKC activity. Crosslinking of the drug in this manner provides a valuable tool for identifying drug recognition sites on the protein. The first objective of this proposal is to determine the binding surface(s) of deg=qualinium on PKC by use of genetic and chemical approaches (Specific Aim 1). Deletion mutants and site-directed mutagenesis will identify binding sites in the regulatory domain of PKC. In order to identify binding site(s) in the catalytic domain, crosslinking of PKC-beta1 with photoactivated 3H-dequalinium will be carried out, followed by proteolysis and microsequencing of tritiated peptide fragments. The functional significance of these sequences will be verified by site-directed mutagenesis. The molecular interaction of dequalinium with peptide sequences identified by the radiolabeling and genetic studies will be modeled by use of computer-assisted energy minimization analysis. The intention of these studies is to obtain a three-dimensional model of the interaction between dequalinium and pKC, in order to design and synthesize novel dequalinium analogues (Specific Aim 2). The second objective of these studies is to use photoactivated dequalinium or a more potent analogue to inhibit intracellular PKC (Specific Aim 3), and to intervene in PKC-mediated metastasis of B16 melanoma cells by comparing the effects of novel dequalinium analogues (Specific Aim 4). In order to determine the mechanistic significance of PKC activity to B16 cell metastasis, B16 cells will be either depleted of PKCalpha (by anti-sense methodology), or genetically engineered to overproduce PKCalpha or a mutant lacking the dequalinium binding site, and the metastatic activity of these cells will be assayed in syngeneic B57CL/6 mice; the anti-metastatic effect of dequalinium/UV in these modified cell systems will verify whether the drug is selecting PKC as a target (Specific Aim 5). Elucidation of the molecular pharmacology of dequalinium coupled with studies of photoactivated dequalinium with intact B16 melanoma cells could lead to improved chemotherapy for cutaneous lesions.

通过了解以下知识，能够改进新药的设计： 药物药效团及其在细胞内靶点上的结合表面。 地奎铵是一种抑制肿瘤生长的抗肿瘤药物 许多动物模型，已知抑制蛋白激酶C（PKC）， 信号转导途径中的关键成分。 辐射365 纳米光在体外光活化地奎铵，使其修饰PKC 从而导致PKC的不可逆抑制 活动 以这种方式交联药物提供了一种有价值的工具， 用于识别蛋白质上的药物识别位点。 本提案的第一个目标是确定结合面 的deg=qualinium对蛋白激酶C通过使用遗传和化学方法（具体 目标1）。 缺失突变体和定点突变将鉴定 PKC调控域中的结合位点。 以便识别 催化结构域中的结合位点，PKC-β 1与 将进行光活化的3 H-地喹铵，然后进行蛋白水解 和氚化肽片段的微测序。 功能 这些序列的意义将通过定点 诱变 地奎铵与肽的分子相互作用 通过放射性标记和遗传研究鉴定的序列将被 通过使用计算机辅助能量最小化分析建模。 的 这些研究的目的是获得一个三维模型， 地喹铵和pKC之间的相互作用，以便设计和合成 新的地喹类似物（Specific Aim 2）。 这些研究的第二个目的是使用光活化的地喹铵 或抑制细胞内PKC的更有效的类似物（特异性目的3）， 并干预PKC介导的B16黑色素瘤细胞转移， 比较新型地喹类似物的效果（具体目标4）。 在 探讨PKC活性对B16细胞的作用机制 转移，B16细胞将耗尽PKCalpha（通过反义寡核苷酸）， 方法学），或遗传工程化以过量生产PKCalpha或 缺乏地喹铵结合位点的突变体，以及 这些细胞将在同系B57 CL/6小鼠中进行测定;抗转移性 地喹铵/UV在这些修饰的细胞系统中的作用将验证是否 该药物选择PKC作为靶点（特异性目标5）。 阐明 地喹宁的分子药理学研究 光活化的地喹铵与完整的B16黑色素瘤细胞可能导致 改进了皮肤病变的化疗。