Identification of serine hydrolases involved in Plasmodium host cell invasion

描述(申请人提供)：恶性疟原虫是导致疟疾的寄生虫，疟疾是一种继续肆虐世界所有热带地区的疾病。每年报告的疟疾病例有25万例，其中近100万例导致死亡。由于寄生虫的抗药性不断增强，控制这种疾病的努力变得复杂，导致迫切需要新的药物靶点。识别新靶点的一种方法是通过用小分子抑制寄生虫中的关键酶来发现和表征它们。丝氨酸水解酶是一大类酶，具有高度活性和共价修饰的活性部位，因此对小分子的抑制很有吸引力。此外，丝氨酸水解酶在包括人类在内的大多数生物体中都有很好的表达，它们占所有蛋白质的1%。在恶性疟原虫中，已鉴定出两种必需的丝氨酸水解酶，即枯草杆菌素样丝氨酸蛋白酶1(PfSUB1)和丝氨酸蛋白酶2(PfSUB2)。为了在恶性疟原虫中发现更多必需的丝氨酸水解酶，我们在恶性疟原虫中筛选了哺乳动物中高效丝氨酸水解酶抑制剂的文库。这个三氮唑尿素化合物文库揭示了几种减缓或阻止恶性疟原虫生长的化合物。最有效的化合物AA691似乎可以诱导宿主细胞的侵袭缺陷。此外，在竞争分析中已证实AA691与丝氨酸水解酶抑制剂氟磷酸盐(FP)至少有一个相同的靶点，表明这一缺陷是由于丝氨酸水解酶的抑制。在这个项目中，将使用基于AA691结构的探针来分离AA691抑制的丝氨酸水解酶，该探针可以共价修饰其靶标。然后，将使用串联的基于蛋白质分解活性的蛋白质轮廓蛋白(TOP-ABPP)和质谱学来鉴定目标。这些靶标在宿主细胞入侵中的作用(S)将通过在恶性疟原虫中创建直接和可调节的条件敲除来在培养中表征。这种侵袭缺陷将在体内通过伯氏疟原虫小鼠感染得到进一步验证。最后，将在肝细胞培养和体内评估AA691对肝脏分期的影响。间日疟原虫感染的流行率不断上升，这种感染在肝脏中处于休眠状态，导致在血液阶段治疗后复发，这推动了对肝期寄生虫的治疗。迫切需要新的疟疾疗法，这项研究的结果可能直接导致这些药物的开发。

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.