Function and pharmacology of schistosome multidrug resistance proteins

描述(申请人提供)：项目摘要：血吸虫属扁虫是血吸虫病的病原体。新的抗血吸虫药物的一个潜在的生理靶点是蠕虫排泄废物和异物的系统。P-糖蛋白(P-gp)是三磷酸腺苷结合盒超家族中的一员，是一种依赖于三磷酸腺苷的外排泵，参与细胞内毒素和外源物质的转运。在脊椎动物中，Pgp是多药耐药1基因的产物，该基因在表现出广泛耐药性的肿瘤细胞中被扩增和过度表达。Pgp也可能在蠕虫的耐药性中发挥作用。血吸虫PGP基因(SMDR2)早在几年前就被测序，但尚未得到功能鉴定。这项提案的长期目标是剖析SMDR2和其他血吸虫药物转运蛋白的功能作用和药理敏感性。我们将采用钙黄绿素荧光法来检测表达的SMDR2和其他转运蛋白的底物和抑制物的特异性。我们还将研究当前选择的抗血吸虫药物吡喹酮对SMDR2表达的影响，以及对吡喹酮耐药的分离株是否改变了这一或其他转运蛋白的表达。最后，我们将研究SMDR2基因或药物干扰对寄生虫的影响。该项目的具体目标是回答以下问题：1.在哺乳动物细胞中表达的SMDR2和其他血吸虫药物转运蛋白的生化特性和底物特异性是什么？2.SMDR2和其他血吸虫多药转运蛋白的组织分布和发育概况是什么？3.暴露于PZQ等药物是否会导致SMDR2表达或分布的变化？4.对PZQ易感性降低的蠕虫分离株是否表现出SMDR2或其他多药转运蛋白表达水平的差异？5.SMDR2的遗传和药理干扰对寄生虫存活、生理和药理敏感性/相关性：血吸虫病是由称为血吸虫的寄生虫扁平蠕虫引起的一种主要热带疾病。抗血吸虫病新药的潜在生理靶点可能是从血吸虫细胞中清除废物和毒素的转运蛋白。我们建议使用几种方法来确定这种血吸虫分子P-糖蛋白的性质，在脊椎动物中，P-糖蛋白也与一系列广泛的药物耐药有关。

DESCRIPTION (provided by applicant): Project Summary: Trematode flatworms of the genus Schistosoma are the causative agents of schistosomiasis. One potential physiological target for new anti-schistosoma drugs is the worm's system for excretion of wastes and xenobiotics. P-glycoprotein (Pgp), a member of the ATP-binding cassette superfamily of proteins, is an ATP-dependent efflux pump involved in transport of toxins and xenobiotics from cells. In vertebrates, Pgp is the product of the multi-drug-resistance 1 gene, which is amplified and over-expressed in tumor cells that show broad drug resistance. Pgp may also play a role in drug resistance in helminths. A schistosome Pgp cDNA (SMDR2) was sequenced several years ago, but has not been functionally characterized. The long-term goal of this proposal is to dissect the functional role and pharmacological sensitivities of SMDR2 and other schistosome drug transporters. We will adapt a calcein fluorescence assay to examine the substrate and inhibitor specificities of expressed SMDR2 and other transporters. We will also examine the effects of praziquantel, the current antischistosomal of choice, on expression of SMDR2, and whether praziquantel-resistant isolates have altered expression of this or other transporters. Finally, we will examine the effect on the parasite of genetic or pharmacological disruption of SMDR2. The specific aims of the project are to answer the following questions: 1. What are the biochemical properties and substrate specificities of SMDR2 and other schistosome drug transporters expressed in mammalian cells? 2. What is the tissue distribution and developmental profile of SMDR2 and other schistosome multi-drug transporters? 3. Does exposure to agents such as PZQ result in changes in SMDR2 expression or distribution? 4. Do isolates of worms with reduced susceptibility to PZQ show differences in expression levels of SMDR2 or other multidrug transporters? 5. What effects do genetic and pharmacological disruption of SMDR2 have on parasite survival, physiology and pharmacological sensitivities/relevance: Schistosomiasis is a major tropical disease caused by parasitic flatworms called schistosomes. Potential physiological targets for new drugs against schistosomiasis might be the transporters that remove wastes and toxins from schistosome cells. We propose to use several approaches to determine the properties of one such schistosome molecule, P-glycoprotein, which, in vertebrates, is also involved in resistance to a broad array of drugs.