Chemical Genetic Approach to Melanocyte Chemosensitivity

描述（由申请人提供）：黑色素瘤通常对多种化疗药物具有耐药性。虽然许多肿瘤的化学耐药基础已被普遍了解，但导致黑色素瘤化学耐药的机制尚不清楚。此外，黑色素细胞对细胞毒性损伤的敏感性与白癜风的发病机制有关。最近，黑素细胞特异性粉眼稀释(p)基因产物被认为与控制黑素细胞对包括砷和顺铂在内的细胞毒性化合物的敏感性有关。缺失该基因的小鼠黑色素细胞对这些药物具有抗性，酵母中p基因的表达导致对砷化合物和其他类金属的敏感性增加。利用小分子来解剖控制黑素细胞对细胞毒性药物敏感性的手术途径，对基础研究人员有价值，并提供潜在的治疗线索。提出了一种基于系统的“化学遗传学”方法，以进一步了解控制这些过程的途径。本课题的具体研究目的是：1。在简单的细胞毒性试验中筛选多种基于三嗪的标记文库，其中包含10,000种化合物，这些化合物将识别那些本身没有细胞毒性但能够克服缺乏p基因表达的黑素细胞对砷和顺铂的抗性的化合物。2. 组合化学，以最大限度地发挥先导化合物的效力，并确定活性的结构要求。3. 固定化化合物亲和层析分离活性化合物的细胞靶点。这个独特的文库中的分子都在3r位置中的一个位置包含几个长连接体中的一个，允许构建亲和矩阵以快速分离先导化合物的目标。亲和分离的目标将进行微测序，并用于从蛋白质数据库中识别目标。这些研究的发现将导致对黑素细胞化学敏感性的细胞途径的深入了解，并为细胞生物学家提供新的研究工具。这些数据应该为未来治疗转移性黑色素瘤和色素脱失提供基础。

DESCRIPTION (provided by applicant): Melanomas are typically resistant to a variety of chemotherapeutic agents. While the basis for chemoresistance of a number of neoplasms is generally understood, the mechanisms leading to melanoma chemoresistance are unclear. Moreover, sensitivity of melanocytes to cytotoxic insult has been implicated in the pathogenesis of vitiligo. Recently, the melanocyte-specific pink-eyed dilution (p) gene product has been implicated in controlling melanocyte sensitivity to cytotoxic compounds including arsenicals and cisplatin. Melanocytes from mice with a deletion of this gene are resistant to these agents and expression of the p gene in yeast leads to increased sensitivity to arsenical compounds and other metalloids. The availability of small molecules to dissect the operative pathways controlling the sensitivity of melanocytic cells to cytotoxic agents would be of value to basic researchers as well as offering potential therapeutic leads. A systems-based "chemical genetics" approach is proposed to further understanding of the pathways controlling these processes. Specific aims of the proposed research are: 1. Screening of a diverse triazine-based tagged library of 10,000 compounds in a simple cytotoxicity assay that will identify those compounds not inherently cytotoxic but capable of overcoming the resistance of melanocytes lacking p gene expression to arsenic and cisplatin. 2. Combinatorial chemistry to maximize the potency of lead compounds and to define structural requirements for activity. 3. Isolation of cellular targets for active compounds by affinity chromatography using immobilized compound. The molecules in this unique library all contain one of several long linkers at one of 3 R positions, allowing the construction of affinity matrices to rapidly isolate the targets of the lead compounds. Microsequencing of affinity-isolated targets will be performed and used to identify targets from protein databases. Findings from the proposed studies will result in a deeper understanding of the cellular pathways underlying melanocyte chemosensitivity, and in new research tools for cell biologists. The data should provide the basis for future therapeutics to treat metastatic melanoma and for purposes of depigmentation.