Identifying species-specific anti-malarial Hsp90 inhibitors using genetically eng

描述（由申请人提供）：每年约有200万人死于疟疾寄生虫恶性疟原虫感染，这是最致命的人类寄生虫病。这种寄生虫的抗药性菌株已经出现，并威胁到用于对抗这种疾病的常规抗疟疾化合物的效用。在多种真菌物种中，已经显示Hsp90抑制剂可以逆转耐药性，并且这对于其他真核病原体如恶性疟原虫可能是真实的。此外，Hsp90抑制剂显示出对培养物中的疟原虫菌株的有效抗疟疾活性。由于Hsp90的全局抑制可能对疾病受损的个体有害，因此我们提出了一种策略来发现特异性抑制该蛋白质的疟疾同系物的化合物。我们计划通过使用酵母菌株进行高通量筛选，以获得具有所需选择性的化合物，这些酵母菌株已被基因工程改造为在恶性疟原虫（Pf）或人类（Hs）Hsp90同源物上生存，该同源物取代了天然真菌蛋白。将对显著抑制PfHsp90酵母生长的化合物进行反筛选，以发现不显著抑制HsHsp90菌株生长的化合物。对已知的Hsp90抑制剂以及化合物的测试集的初步结果表明，这是一个非常强大的，可重复的，和成本效益的测定策略。此外，来自测试集的命中显示了所需的物种特异性，并提供了极好的概念证明。二级测定将包括测试针对恶性疟原虫感染的红细胞的命中化合物以确定这些化合物的抗寄生虫活性，以及标准活力测定以确定人细胞系中的细胞毒性。这些化合物对Hsp90的影响将通过基于酵母的测定来探测，以评估已知需要Hsp90折叠的蛋白质的功能。将选择多达10个命中物用于进一步的生物学研究，并且将组装相关类似物的文库以定义结构活性关系。通过这种筛选策略发现的化合物将满足探针的关键需求，以剖析寄生虫编码的Hsp90在恶性疟原虫复杂生命周期中的作用。在未来的工作中，它们很可能成为开发具有以前未开发的作用模式的抗疟疾药物的有价值的线索。 公共卫生相关性：该项目的目标是确定特异于恶性疟原虫的Hsp90蛋白的抑制剂。由于恶性疟原虫株已对传统抗疟疾药物产生耐药性并在全球范围内传播，因此创造通过新机制发挥作用的药物至关重要。先前的研究表明，抑制真菌中的Hsp90可以逆转耐药性，并且Hsp90的抑制剂在杀死感染的红细胞中的恶性疟原虫方面非常有效，因此靶向Hsp90的新药可能提供对抗这种致命疾病的极好方法。

DESCRIPTION (provided by applicant): Approximately two million deaths a year result from infections with the malaria parasite Plasmodium falciparum, the most deadly human parasitic disease. Drug-resistant strains of this parasite have emerged and threaten the utility of conventional anti-malarial compounds used to fight this disease. In a variety of fungal species, it has been shown that Hsp90 inhibitors can reverse drug resistance, and this may be true for other eukaryotic pathogens such as P. falciparum. Furthermore, Hsp90 inhibitors show potent anti-malarial activity against Plasmodium strains in culture. As global inhibition of Hsp90 may be harmful in disease-compromised individuals, here we propose a strategy to discover compounds that specifically inhibit the malarial homolog of this protein. We plan to execute a high throughput screen for compounds that possess the desired selectivity by using yeast strains that have been genetically engineered to survive on either the P. falciparum (Pf) or human (Hs) Hsp90 homolog that replaces the native fungal protein. Compounds that significantly inhibit the growth of PfHsp90 yeast will be counter-screened to find those that do not significantly inhibit growth of the HsHsp90 strain. Preliminary results against known Hsp90 inhibitors as well as a test set of compounds show that this is a very robust, repeatable, and cost efficient assay strategy. Additionally, hits from the test set show the desired species specificity and provide excellent proof of concept. Secondary assays will include testing hit compounds against P. falciparum-infected erythrocytes to define the anti-parasite activity of these compounds, as well as standard viability assays to determine cytotoxicity in human cell lines. The effect of these compounds on Hsp90 will be probed through yeast-based assays to assess the function of proteins known to require Hsp90 to fold. Up to ten hits will be selected for further biological study, and a library of related analogs will be assembled to define structure activity relationships. Compounds discovered through this screening strategy will meet a critical need for probes to dissect the contribution of parasite-encoded Hsp90 from that of the host red blood cell in the complex life cycle of P. falciparum. In future work they could well serve as valuable leads for the development of anti-malarial drugs with a previously unexploited mode of action. PUBLIC HEALTH RELEVANCE: The goal of this project is to identify inhibitors of the protein Hsp90 specific to the malarial species Plasmodium falciparum. As strains of P. falciparum have developed resistance to conventional anti-malarial drugs and spread worldwide, creating drugs that operate through a novel mechanism is critically important. Previous work shows both that inhibition of Hsp90 in fungi can reverse drug resistance and that inhibitors of Hsp90 are very potent in killing P. falciparum in infected red blood cells, therefore new drugs that target Hsp90 may provide an excellent method to combat this deadly disease.