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: 2414276
• 负责人: Susan A. Rotenberg
• 金额: $ 11.39万
• 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/2414276
• 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.

抗肿瘤药物的改进设计是通过了解以下知识来实现​​的 药物药效团及其在细胞内靶标上的结合表面。 Dequalinium，一种抗肿瘤药，可抑制癌症生长 在许多动物模型中，已知可抑制蛋白激酶 C (PKC)，这是一种 信号转导途径的关键组成部分。 365照射 纳米光在体外光激活地喹啉，导致其修饰 PKC 共价结合，从而对 PKC 产生不可逆的抑制 活动。 以这种方式进行的药物交联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。 该提案的第一个目标是确定结合表面 使用遗传和化学方法对 PKC 进行 deg=qualinium（具体 目标1）。 缺失突变体和定点诱变将鉴定 PKC 调控域中的结合位点。 为了识别 催化结构域中的结合位点，PKC-beta1 与 将进行光活化 3H-地喹啉，然后进行蛋白水解 以及氚化肽片段的微测序。 功能性的 这些序列的重要性将通过定点验证 诱变。 地喹啉与肽的分子相互作用 通过放射性标记和基因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进行建模。 这 这些研究的目的是获得三维模型 地喹啉和 pKC 之间的相互作用，以便设计和合成 新型地喹啉类似物（具体目标 2）。 这些研究的第二个目标是使用光活化地喹啉 或更有效的类似物来抑制细胞内 PKC（具体目标 3）， 并干预 PKC 介导的 B16 黑色素瘤细胞转移 比较新型地喹啉类似物的效果（具体目标 4）。 在 以确定 PKC 活性对 B16 细胞的机制意义 转移时，B16 细胞将耗尽 PKCalpha（通过反义 方法），或通过基因工程过度产生 PKCalpha 或 缺乏地喹啉结合位点的突变体，以及转移活性 这些细胞将在同基因 B57CL/6 小鼠中进行检测；抗转移的 地喹啉/紫外线在这些改良细胞系统中的作用将验证是否 该药物选择 PKC 作为靶标（具体目标 5）。 阐明 地喹啉的分子药理学与研究 光激活地喹啉与完整的 B16 黑色素瘤细胞可能会导致 改善皮肤病变的化疗。