MOLECULAR TARGETS OF ANTI-OPPORTUNISTIC INFECTION AGENTS

抗机会性感染剂的分子靶标

基本信息

批准号：
6099612
负责人：
CHRISTINE C DYKSTRA
金额：
$ 8.84万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2000-01-31
项目状态：
已结题

项目摘要

Based on the discoveries during the initial funding period, a former core has been converted to a Project for the renewal application. It has become increasingly important to the program to determine how the dicationic molecules exert their effects on the targeted pathogens. It has also become clear that while there are many similarities in the mode of action of compounds from both Tidwell and Boykin, their differences (ie. in toxicity and selectivity for different parasites) in their action is what makes them so amenable to development as therapeutic agents. The inclusion of a new target organism (Cryptococcus neoformans) that can be grown easily in vitro and can be genetically manipulated permits a level of analysis that still can only be contemplated for Pneumocystis carinii and Cryptosporidium parvum. Many of the ongoing studies will continue as new compounds are synthesized by Tidwell and Boykin; so that the database containing the activity of the compounds on target enzymes and their effects on DNA will be continuously updated. The compounds under development are all DNA minor groove binding agents. But, it is clear that topoisomerases are affected as well. For this reason, we will focus our studies more on the mechanism of inhibition of the enzymes that are selectively inhibited by the dicationic compounds, topoisomerases. The major objective of this project during the previous funding period was to identify the DNA-dependent enzyme(s) that is the most likely target(s) of the dicationic compounds. This goal has been achieved for most of the compound series with the identification of topoisomerases as the most likely target.. The question that needs to be addressed now is how this enzyme is affected. This will be achieved through a combination of in vitro and in vivo analysis of topoisomerase action in the presence of the compound. As new types of molecules are produced by Tidwell and Boykin, however, they will continue to be tested for more generalized effects. An important objective will be to determine structure-activity relationships between the existing compounds and C. neoformans topoisomerases in vitro. This will be accomplished in collaboration with Perfect. Once this is accomplished, drug resistant mutants isolated by Perfect will be used to identify regions on the genes important to drug action. Concurrently, the effect of the dicationic drugs on the topoisomerase dependent DNA cleavage activity ex vivo and in vitro will be determined. This analysis will performed for P. carinii, C. parvum, and mammalian cells. In summary, we have found that there is selectivity for inhibition of the parasite topoisomerases over those from mammalian cells, and consequently the major objective of the proposed studies is to determine the molecular basis of this selectivity.

根据最初资助期间的发现，前核心已转换为项目以供续订申请。它已经成为对于该计划来说越来越重要的是确定如何进行指示分子对目标病原体发挥作用。它还具有清楚的是，虽然作用方式有很多相似之处 Tidwell 和 Boykin 的化合物，它们的差异（即在对不同寄生虫的毒性和选择性）在其作用中是什么使它们非常适合作为治疗剂进行开发。包容性一种可以生长的新目标生物体（新型隐球菌）很容易在体外进行，并且可以进行基因操纵，从而允许一定程度的仍然只能考虑卡氏肺孢子虫的分析和小隐孢子虫。随着新化合物的合成，许多正在进行的研究将继续进行作者：蒂德韦尔和博伊金；以便包含该活动的数据库目标酶上的化合物及其对 DNA 的影响将持续不断已更新。正在开发的化合物均为DNA小沟结合代理。但是，很明显拓扑异构酶也会受到影响。为了因此，我们将更多的研究重点放在抑制机制上被二价化合物选择性抑制的酶，拓扑异构酶。上一时期该项目的主要目标资助期的目的是确定 DNA 依赖性酶，即双剂化合物最可能的目标。这个目标已经大多数化合物系列均已实现，并鉴定出拓扑异构酶是最有可能的目标。需要解决的问题现在要解决的是这种酶是如何受到影响的。这将实现通过拓扑异构酶的体外和体内分析相结合在化合物存在下发生作用。随着新型分子的出现由 Tidwell 和 Boykin 生产，但是，它们将继续进行测试以获得更普遍的效果。一个重要的目标是确定结构-活性现有化合物与新型隐球菌之间的关系体外拓扑异构酶。这将与以下机构合作完成完美的。一旦完成，就会分离出耐药突变体 Perfect 将用于识别对药物重要的基因区域行动。同时，药物对患者的影响离体和体外拓扑异构酶依赖性 DNA 切割活性将决定。该分析将对 P. carinii、C. parvum 和哺乳动物细胞。综上所述，我们发现存在选择性与来自哺乳动物细胞的拓扑异构酶相比，对寄生虫拓扑异构酶的抑制，因此，拟议研究的主要目标是确定这种选择性的分子基础。