Understanding the biology of schistosomes in response to praziquantel

描述（由申请人提供）：曼氏血吸虫是人类血吸虫病最常见的病原之一，估计在54个国家感染超过8300万人。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。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，它不能杀死血吸虫。第二个潜在的问题是蠕虫自然种群中存在的耐药性特征。到目前为止，PZQ结合的分子及其作用机制都没有被确定。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化的PZQ探针来识别PZQ的分子靶标，该探针含有一个重氮嘧啶基团，可以将药物与其靶标共价交联，并使用点击化学将报告标签附着在炔基上。然后通过化学发光检测来识别探针结合的目标。总细胞蛋白以及不同的细胞蛋白组分将是该试验的结合靶标的一个来源。此外，我们将专门针对电压门控Ca2+通道Cav21和2亚基以及烯醇化酶和甘油醛-3-磷酸脱氢酶作为潜在的PZQ结合蛋白。我们还建议利用曼氏血吸虫不同生命周期阶段对PZQ的不同敏感性，利用微阵列技术比较PZQ敏感的毛蚴、尾蚴和成熟血吸虫与相对不敏感的母孢子囊和幼血吸虫的转录组。以往的经验表明，这应该为进一步研究提供一小部分候选靶基因，也可能有助于确定由PZQ靶点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抑制成熟血吸虫基因表达来确定正确的靶点或生化途径，从而消除PZQ敏感表型。第一个潜在的途径成分将是钙调蛋白、肌球蛋白轻链激酶和死亡相关蛋白激酶，它们可能在PZQ相关的被囊破坏中起作用。利用PZQ类似物的小分子文库来确定PZQ分子靶点的药效团。最后，我们将研究肯尼亚曼氏沙门氏菌对PZQ可变敏感性的分子基础。来自自然感染和对PZQ具有不同敏感性的曼氏梭菌将在小鼠中保持。这些群体的转录组将使用微阵列进行比较，以确定可变敏感性是基于PZQ靶基因的突变，还是基于(i)靶基因，（ii）由PZQ靶基因驱动的生化途径的下游组分，或（iii） PZQ解毒/清除机制的表达差异。更明确地了解PZQ的结合靶点和药物的作用机制将有助于我们设计出改进的检测方法来监测耐药性的出现。

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.