MULTITARGET DRUGS: VERIFICATION-OF-PRINCIPLE STUDIES

多靶点药物：原理验证研究

• 批准号: 6342202
• 负责人: ALEXANDER J VARSHAVSKY
• 金额: $ 15.6万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-06 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342202
• 关键词: Saccharomyces cerevisiae; chemical binding; chemical structure; chemical structure function; cytotoxicity; drug design /synthesis /production; gene expression; methotrexate; novobiocin; trimetrexate

The insufficient selectivity of drugs is a bane of present-day therapies. This problem is significant for antibacterial drugs, difficult for antivirals, and utterly unsolved for anti-cancer drugs, which remain ineffective against major cancers, and in addition cause severe side effects. T he problem may be solved if therapeutic agents could acquire a multi-target, combinatorial selectivity, killing (or otherwise modifying) a cell if, and only if, it contains a predetermined set of molecular targets and lacks another predetermined set of targets. Recently, a solution to the problem of drug selectivity was proposed that is applicable to small (less than or equal to 1 kD) compounds (Varshavsky, A. PNAS 95, 2094-2099 (1998)). Two ideas, interference/co-dominance (IC) and anti-effectors, should allow a therapeutic regimen to possess combinatorial selectivity, in which the number of positively and negatively sensed macromolecular targets can be two, three, or more. The in vivo effects of such drugs would be analogous to the outcomes of the Boolean operations "and", "or" "not", and combinations thereof. The proposed studies, to be supported by the present R21 application, will verify the principles of multi-target designs using the yeast Saccharomyces cerevisiae as a model organism. Specific Aims 1) The ubiquitin fusion technique will be used to express and analyze, in the yeast S. cerevisiae, a set of multi-moiety linear peptides that act as IC- type compounds in this in vivo setting. 2) Bipartite, IC-type compounds that comprise the anhydrotetracycline (at) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety linked to at will be synthesized, and thereafter characterized in vitro (mutual exclusivity of binding to the purified TetR or DHFR) and in vivo (differential toxicity to S. cerevisiae that either lack or express the at- binding E. coli TetR repressor). 3) Bipartite, IC-type compounds containing the novobiocin (nov) moiety and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential cytotoxicity with S. cerevisiae that either lack or express the nov-binding B-subunit of E. coli DNA gyrates (GyrB). 4) Tripartite, IC-type compounds containing the at moiety, the beta- ecdysone (ec) moiety, and either the methotrexate (mtx) or the trimetrexate (tm) moiety will be synthesized and tested for differential toxicity with S. cerevisiae that lack or express the corresponding macromolecular targets and their pairwise combinations.

药物选择性不足是当今治疗的一大祸根。这个问题对于抗菌药物来说很重要，对于抗病毒药物来说是困难的，对于抗癌药物来说则完全没有解决，抗癌药物对主要癌症仍然无效，而且还会导致严重的副作用。如果治疗剂能够获得多靶点、组合选择性，当且仅当它包含一组预定的分子靶点并且缺乏另一组预定的靶点时杀死（或以其他方式修饰）细胞，那么问题就可以得到解决。最近，提出了一种适用于小（小于或等于1 kD）化合物的药物选择性问题的解决方案（Varshavsky, A. PNAS 95, 2094-2099 (1998)）。干扰/共显性（IC）和抗效应物这两种想法应该允许治疗方案具有组合选择性，其中阳性和阴性感应大分子靶标的数量可以是两个、三个或更多。此类药物的体内作用类似于布尔运算“和”、“或”、“非”及其组合的结果。拟议的研究将得到当前 R21 申请的支持，将验证使用酿酒酵母作为模式生物的多目标设计的原理。具体目标 1) 泛素融合技术将用于在酿酒酵母中表达和分析一组多部分线性肽，这些肽在体内环境中充当 IC 型化合物。 2) 合成包含脱水四环素 (at) 部分和连接到 at 的甲氨蝶呤 (mtx) 或三甲曲沙 (tm) 部分的二部分 IC 型化合物，然后在体外进行表征（与纯化的结合的相互排他性） TetR 或 DHFR）和体内（对缺乏或表达 at 结合的大肠杆菌 TetR 的酿酒酵母的不同毒性）抑制器）。 3) 将合成含有新生霉素 (nov) 部分和甲氨蝶呤 (mtx) 或三甲曲沙 (tm) 部分的二分 IC 型化合物，并测试与缺乏或表达 nov 结合的酿酒酵母的不同细胞毒性大肠杆菌 DNA 旋转的 B 亚基 (GyrB)。 4) 将合成含有 at 部分、β-蜕皮激素 (ec) 部分以及甲氨蝶呤 (mtx) 或三甲曲沙 (tm) 部分的三方 IC 型化合物，并测试与缺乏的酿酒酵母的不同毒性。或表达相应的大分子靶标及其成对组合。