Identification of serine hydrolases involved in Plasmodium host cell invasion

鉴定参与疟原虫宿主细胞侵袭的丝氨酸水解酶

• 批准号: 8594762
• 负责人: Brooke Anderson-White
• 金额: $ 4.92万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8594762
• 关键词: Active Sites; Affect; Affinity Chromatography; Automobile Driving; Binding; Biological Assay; Blood; Carbamates; Case Study; Cell physiology; Cells; Cessation of life; Data; Defect; Development; Disease; Drug Targeting; Drug resistance; Enzymes; Erythrocytes; Family; Generic Drugs; Growth; Hepatocyte; Human; Hydrolase; Hydrolysis; In Vitro; Infection; Knock-out; Label; Lead; Libraries; Ligands; Liver; Malaria; Mammals; Mass Spectrum Analysis; Metabolism; Methods; Modification; Mus; Mutate; Organism; Parasites; Peptide Hydrolases; Plasmodium; Plasmodium berghei; Plasmodium falciparum; Play; Prevalence; Proteins; Proteolysis; Proteome; Recombinant Proteins; Relapse; Reporting; Research; Role; Serine; Serine Hydrolase; Serine Protease; Site; Solid; Staging; Structure; Structure-Activity Relationship; Subtilisins; System; Therapeutic; Triazoles; Urea; activity-based protein profiling; base; disorder control; drug development; experience; in vivo; inhibitor/antagonist; interest; novel; novel therapeutics; obligate intracellular parasite; profilin; public health relevance; screening; small molecule; success; therapeutic target

DESCRIPTION (provided by applicant): Plasmodium falciparum is the parasite responsible for malaria, a disease that continues to devastate all tropical regions of the world. A quarter of a million cases of malaria are reported annually with close to a million of these cases resulting in death. Efforts to control this disease are complicated by growing drug resistance in the parasite, leading to an imminent need for new drug targets. One avenue to the identification of novel targets is to discover and characterize essential enzymes in the parasite by inhibiting them with small molecules. The serine hydrolases are a large family of enzymes with highly reactive and covalently modifiable active sites making them attractive for inhibition with small molecules. Furthermore serine hydrolases are well represented in most organisms including humans where they comprise 1% of all proteins. In P. falciparum two essential serine hydrolases have already been identified, subtilisin-like serine protease 1 (PfSUB1) and 2 (PfSUB2). In order to find additional essential serine hydrolases in the parasite, a library of highly effective serine hydrolase inhibitors in mammals was screened in P. falciparum. This library of triazole urea compounds revealed several compounds that slow or block the growth of P. falciparum. The most efficacious compound, AA691, appears to induce a host cell invasion defect. Furthermore it has been verified that AA691 binds to at least one of the same targets as fluorophosphonate (FP), a generic serine hydrolase inhibitor, in competition assays suggesting this defect is due to inhibition of serine hydrolases. In this project the serine hydrolases inhibited by AA691 will be isolated using a probe based on the structure of AA691 that covalently modifies its targets. The targets will then be identified using tandem orthogonal proteolysis-activity-based protein profilin (TOP-ABPP) and mass spectrometry. The role(s) of these targets in host cell invasion will be characterized in culture through the creation of direct and regulatable conditional knockouts in P. falciparum. The invasion defect will be further verified in vivo with P. berghei mouse infections. Finally the effects of AA691 on the liver stage will be assessed in hepatocyte culture and in vivo. The rising prevalence of P. vivax infection, which remains dormant in the liver leading to relapse after blood stage treatment, is driving a push for therapeutics against liver stage parasites. There is an urgent need for novel malaria therapeutics and the results of this research could lead directly to the development of these drugs.

描述（由申请人提供）：恶性疟原虫是负责疟疾的寄生虫，这种疾病继续破坏世界上所有热带地区。每年有近四分之一的疟疾病例，其中近100万病例导致死亡。寄生虫中耐药性的增长使控制这种疾病的努力变得复杂，从而导致对新药靶标的需求。鉴定新靶标的途径是通过用小分子抑制它们来发现和表征寄生虫中的必需酶。丝氨酸水解酶是一个大型酶家族，具有高反应性和共价可修改的活性位点，使其对用小分子抑制具有吸引力。此外，丝氨酸水解酶在包括所有蛋白质的1％的人类在内的大多数生物中都很好地表示。在恶性疟原虫中，已经鉴定出两个必需的丝氨酸水解酶，枯草蛋白样的丝氨酸蛋白酶1（PFSUB1）和2（PFSUB2）。为了在寄生虫中找到其他必需的丝氨酸水解酶，在恶性疟原虫中筛选了哺乳动物中高效的丝氨酸水解酶抑制剂的库。这种三唑尿素化合物的库揭示了几种化合物，这些化合物会减慢或阻断恶性疟原虫的生长。最有效的化合物AA691似乎会诱导宿主细胞浸润缺陷。此外，已证实AA691与荧光膦酸酯（FP）（一种通用丝氨酸水解酶抑制剂）在竞争测定中结合至少一个相同的靶标（FP），这表明该缺陷归因于丝氨酸水解酶的抑制。在该项目中，AA691抑制的丝氨酸水解酶将使用基于AA691的结构进行探针分离，该探针将共价修改其靶标。然后，将使用串联正交蛋白水解活性基于蛋白蛋白（TOP-ABPP）和质谱法鉴定靶标。这些靶标在宿主细胞侵袭中的作用将通过在恶性疟原虫中创建直接和可调节的条件敲除在培养中表征。侵入缺陷将在体内进一步证实，并在体内用伯格氏菌小鼠感染。最后，将在肝细胞培养和体内评估AA691对肝脏阶段的影响。肝脏感染的发生率上升，在肝脏中处于休眠状态，导致血液阶段治疗后复发，这推动了针对肝脏寄生虫的治疗方法。迫切需要新型的疟疾疗法，这项研究的结果可能直接导致这些药物的发展。