Understanding the biology of schistosomes in response to praziquantel

描述(申请人提供)：曼氏血吸虫是人类血吸虫病最常见的病原体之一，估计在54个国家感染了8300多万人。吡喹酮(PZQ)是目前所有抗血吸虫药物中最便宜、最容易使用和最容易获得的药物。与PZQ治疗相关的一个问题是，在血吸虫感染宿主后的2-4周内，它不会杀死血吸虫。第二个潜在问题是自然蠕虫种群中存在抗药性特征。到目前为止，PZQ结合的分子和作用机制都还不清楚。在这里，我们建议采用两种互补的方法来解决这些问题。我们将使用一种工程的PZQ探针来识别PZQ的分子靶标，该探针包含一个二氮杂基以共价方式将药物与其靶标进行交联，以及一个炔基，使用点击化学方法可以将报告标签连接到该基团上。然后将通过化学发光检测来识别探测器结合的目标。总细胞蛋白以及不同的细胞蛋白组分将是该分析的结合靶标之一。此外，我们还将针对电压门控钙通道Cav21和Cav2亚基以及烯醇酶和3-磷酸甘油醛脱氢酶作为潜在的PZQ结合蛋白。我们还建议利用不同的生活周期阶段曼氏血吸虫对PZQ的不同敏感性这一事实，利用微阵列来比较PZQ敏感的毛虫、尾蚴和成熟血吸虫与相对不敏感的母孢子囊和幼虫的转录本。以前的经验表明，这应该会为进一步研究提供一小部分候选目标基因，也可能有助于识别由PZQ目标驱动的生化途径的成员。这些实验将在PZQ存在和不存在的情况下进行。我们将通过RNA抑制成熟血吸虫的基因表达来确认正确的靶点或生化途径，这应该会消除PZQ敏感的表型。首先被靶向的潜在通路组件将是钙调蛋白、肌球蛋白轻链激酶和死亡相关蛋白激酶，它们可能在PZQ相关的被膜破坏中发挥作用。PZQ类似物的小分子文库将用于确定PZQ分子靶标的药效团。最后，我们将研究曼氏沙门氏菌肯尼亚野外分离株对PZQ可变敏感性的分子基础。来自自然感染的对PZQ具有不同敏感性的曼氏葡萄球菌将在小鼠身上保留。这些人群的转录本将使用微阵列进行比较，以努力确定变量敏感性是基于对PZQ靶基因的突变，还是基于(I)靶基因、(Ii)由PZQ靶驱动的生化途径下游组件或(Iii)PZQ解毒/清除机制的表达差异。更清楚地了解PZQ的结合靶点和药物的作用机制将有助于我们设计出更好的检测方法来监测耐药的出现。 公共卫生相关性：估计有2.07亿人感染血吸虫病，其中大部分在非洲。只有一种药物，吡喹酮，可以对抗所有形式的血吸虫病，但我们还不知道这种药物是如何起作用的。该项目旨在更好地了解吡喹酮的作用机制，从而促进下一代治疗学的发展。

DESCRIPTION (provided by applicant): Schistosoma mansoni is one of the most common etiological agents of human schistosomiasis and is estimated to infect more than 83 million humans in 54 countries. Praziquantel (PZQ) is the least expensive, easiest to use and most readily available of all current anti-schistosomal drugs. One problem associated with PZQ treatment is that it does not kill schistosomes for a period of 2-4 weeks after they infect the host. A second potential problem is the presence of drug resistance traits in natural populations of worms. As yet, neither the molecule to which PZQ binds nor its mechanism of action have been identified. Here, we propose to employ two complementary approaches to resolve these issues. We will identify the molecular target of PZQ using an engineered PZQ probe containing a diazirine group to covalently cross link the drug to its target and an alkyne group to which a reporter tag can be attached using click chemistry. Probe bound target will then be identified by chemiluminescent detection. Total cell protein as well as different cellular protein fractions will be one source of binding targets for this assay. In addition, we will specifically target the voltage gated Ca2+ channel Cav21 and 2 subunits as well as enolase and glyceraldehyde-3-phosphate dehydrogenase as potential PZQ binding proteins. We also propose to exploit the fact that different life cycle stages of S. mansoni have differing susceptibilities to PZQ by employing microarrays to compare the transcriptomes of PZQ sensitive miracidia, cercariae and mature schistosomes with those of mother sporocysts and juvenile schistosomes which are relatively insensitive. Previous experience suggests that this should provide a small pool of candidate target genes for further study and may also help identify members of the biochemical pathway driven by the PZQ target. These experiments will be performed in the presence and absence of PZQ. We will confirm the correct target or biochemical pathway has been identified by RNA inhibition of gene expression in mature schistosomes which should abolish the PZQ sensitive phenotype. Among the first potential pathway components to be targeted will be calmodulin, myosin light chain kinase and death associated protein kinase which may play a role in PZQ associated tegumental disruption. Small molecule libraries of PZQ analogs will be used to define the pharmacophore of the molecular target of PZQ. Finally, we will investigate the molecular basis of variable sensitivity to PZQ in Kenyan field isolates of S. mansoni. S. mansoni derived from natural infections and with varied sensitivities to PZQ will be maintained in mice. The transcriptomes of these populations will be compared using microarrays in an effort to determine if variable sensitivity is based on mutations to the PZQ target gene or differences in the expression of either (i) the target, (ii) a downstream component of the biochemical pathway driven by the PZQ target or (iii) a PZQ detoxification/clearance mechanism. More explicit knowledge of the binding target of PZQ and the mechanism of action of the drug will help us to devise improved assays for monitoring the emergence of resistance. PUBLIC HEALTH RELEVANCE: Schistosomiasis infects an estimated 207 million people, mostly in Africa. There is only one available drug, praziquantel, that combats all forms of schistosomiasis but we do not yet know how this drug works. This project seeks to better understand the mechanism of action of praziquantel, thus facilitating the development of the next generation of therapeutics.