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

描述(申请人提供)：每年约有200万人死于感染疟疾寄生虫恶性疟原虫，这是人类最致命的寄生虫病。这种寄生虫的抗药性菌株已经出现，并威胁到用于抗击这种疾病的传统抗疟疾化合物的效用。在多种真菌中，已经证明Hsp90抑制剂可以逆转耐药性，这可能适用于其他真核病原体，如恶性疟原虫。此外，Hsp90抑制剂在培养中对疟原虫菌株显示出强大的抗疟疾活性。由于Hsp90的全局抑制可能对疾病易感性的个体有害，在此我们提出了一种策略，以发现专门抑制该蛋白的疟疾同源物的化合物。我们计划对具有预期选择性的化合物进行高通量筛选，方法是使用经过基因工程改造的酵母菌株，这些菌株依靠恶性疟原虫(Pf)或人类(HS)Hsp90同系物生存，取代了天然的真菌蛋白。将对显著抑制PfHsp90酵母生长的化合物进行反筛选，以找到那些不显著抑制HsHsp90菌株生长的化合物。针对已知的Hsp90抑制剂以及一组测试化合物的初步结果表明，这是一种非常强大、可重复和成本效益高的分析策略。此外，测试集的命中率显示了所需的物种特异性，并提供了极好的概念证明。二次测试将包括测试HIT化合物对恶性疟原虫感染的红细胞的作用，以确定这些化合物的抗寄生虫活性，以及标准的生存能力测试，以确定对人类细胞系的细胞毒性。这些化合物对Hsp90的影响将通过基于酵母的检测来探索，以评估已知需要Hsp90折叠的蛋白质的功能。将选择最多10个匹配项进行进一步的生物学研究，并将组装相关类似物的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对探针的迫切需要，以剖析恶性疟原虫复杂生活史中寄生虫编码的Hsp90与宿主红细胞的贡献。在今后的工作中，它们很可能成为开发具有以前未曾开发过的行动模式的抗疟疾药物的宝贵线索。 公共卫生相关性：该项目的目标是确定恶性疟原虫特定蛋白Hsp90的抑制物。随着恶性疟原虫菌株对常规抗疟疾药物产生抗药性，并在全球范围内传播，创造通过新机制发挥作用的药物至关重要。以往的工作表明，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，因此针对Hsp90的新药可能提供一种很好的方法来对抗这种致命的疾病。

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.