Chemical Genetic Approach to Melanocyte Chemosensitivity

描述(申请人提供)：黑色素瘤通常对多种化疗药物耐药。虽然人们普遍了解一些肿瘤化疗耐药的基础，但导致黑色素瘤化疗耐药的机制尚不清楚。此外，黑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，黑素细胞特有的粉眼稀释(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。来自缺失该基因的小鼠的黑素细胞对这些药物具有抵抗力，p基因在酵母中的表达导致对砷化合物和其他类金属的敏感性增加。小分子可用于剖析控制黑素细胞对细胞毒剂敏感性的操作途径，这将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。这项研究的具体目标是：1.在一种简单的细胞毒性试验中筛选包含10,000种化合物的不同的基于三嗪的标记库，以鉴定那些本身不具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抵抗力的化合物。2.组合化学，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.用固定化化合物亲和层析分离活性物质的细胞靶标。这个独特的文库中的所有分子都在3个R位之一包含几个长连接子之一，从而允许构建亲和力矩阵来快速分离先导化合物的目标。将对亲和力分离的靶标进行微测序，并用于从蛋白质数据库中识别靶标。拟议的研究结果将使人们更深入地了解黑素细胞化疗敏感性的细胞途径，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脱色的治疗提供基础。

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.