Cyclophilins and the IFN Response

描述(由申请人提供)：亲环素(Cyp)抑制剂(CypI)在处于I期和II期研究的丙型肝炎患者中具有很高的临床效力。最近的一项I期研究表明，每天服用赛普利可减少病毒血症，并迅速增加干扰素α，λ1和λ3以及2‘5’OAS-1的血浆浓度。所有标志物的血浆浓度变化与CypI的血浆浓度变化是一致的。这些新颖而吸引人的数据揭示了细胞色素P450和干扰素反应之间的第一个联系。我们能够在体外部分复制病人的数据。具体地说，CypI促进了细胞的分泌 干扰素α，λ和复制子细胞中的2‘5’OAS-1。这些数据表明，激活干扰素反应可能代表了CypI发挥临床抗病毒活性的一种机制。我们研究了CypI的细胞内靶点CypA与干扰素反应成分结合的可能性。值得注意的是，我们发现CypA通过其异构酶口袋与干扰素调节因子9(IRF9)结合。CypI阻止IRF9-CypA相互作用。CypA还与IRF3、5和7结合，表明CypA与所有IRF成员结合。我们的工作首次证明了CypA特定地与干扰素反应的一类主要元件-IRF结合。这也揭示了一个调节干扰素反应的新机会。在本申请中，我们建议通过进行一系列旨在剖析CypA在干扰素反应中的作用的实验来扩展这项工作。这一应用应该阐明CypA中和如何触发干扰素的产生，并确定CypI介导的干扰素产生是否代表了CypI抗病毒作用的新机制。这个应用程序还应该确定CypA对IRF施加什么动作或功能。有趣的是，我们最新的工作表明，CypA通过与红外线分子相互作用，极大地影响了它们的泛素化程度。我们提出了一组新的实验，旨在确定CypA介导的IRF泛素化效应和CypI介导的干扰素反应激活之间的联系。我们的发现是，CypI触发了从丙型肝炎患者分离的PBMC释放干扰素，开启了令人兴奋的新调查路线。这种体外试验可能使我们能够确定CypI应答者是否比CypI非应答者产生更多的干扰素，以及CypI反应是否具有基因型或病毒滴度特异性。如果发生这种情况，我们将利用这些信息为受试者开发经验性的预筛查，这些受试者可能对包括Cypi的方案有反应。我们最近发现，给小鼠注射CypI可以显著抑制病毒的感染性，这与丙型肝炎病毒无关，但对干扰素也高度敏感。此外，我们发现经CypI治疗的感染小鼠的血浆干扰素水平高于未治疗的感染小鼠，这表明Cypi诱导干扰素是一种更广泛使用的机制。这些了不起的初步发现开启了令人兴奋的调查的新线索。这项应用的最终目的是提高我们对Cyps在干扰素对病毒感染的反应中的作用的理解。

DESCRIPTION (provided by applicant): Cyclophilin (Cyp) inhibitors (CypI) are clinically highly potent in HCV patients in phase I and II studies. A recent phase I study showed that the daily administration of a CypI reduces viremia and rapidly increases plasma concentrations of IFNα, λ1 and λ3, and 2'5'OAS-1. Changes in plasma concentrations for all markers were coincident with changes in plasma concentration of the CypI. These novel and attractive data revealed the first link between Cyps and the IFN response. We were able to partly reproduce the patient data in vitro. Specifically, CypI enhance secretion of IFNα, λ and 2'5'OAS-1 from replicon cells. These data suggest that activation of the IFN response may represent a mechanism through which CypI exert their clinical antiviral activity. We investigated the possibility that the intracellular target for CypI, CypA, binds to components of the IFN response. Remarkably, we found that CypA binds to the IFN regulatory factor 9 (IRF9) via its isomerase pocket. CypI prevent IRF9-CypA interactions. CypA also binds to IRF3, 5 and 7, suggesting that CypA binds to all IRF members. Our work demonstrates for the first time that CypA binds specifically to a major class of elements of the IFN response - IRFs. It also reveals a novel opportunity to modulate the IFN response. In this application, we propose to extend this work by conducting a series of experiments aimed at dissecting the roles of CypA in the IFN response. This application should elucidate how CypA neutralization triggers IFN production and determine whether the CypI-mediated IFN production represents a new mechanism of antiviral action of CypI. This application should also determine what action or function CypA exerts on IRFs. Interestingly, our most recent work suggests that CypA, by interacting with IRFs, greatly impacts their degree of ubiquitination. We propose a new set of experiments aimed at identifying the link between the CypA-mediated IRF ubiquitination effect and the CypI-mediated activation of the IFN response. Our finding that CypI triggers IFN release from PBMCs isolated from HCV patients opens exciting new lines of enquiries. This ex vivo assay may allow us to determine whether CypI responders produce more IFN than CypI non-responders and whether the CypI response is genotype- or virus titer-specific. If this occurs, we then would use this information t develop an empiric pre-screen for subjects, who are likely to respond to a regimen comprising a CypI. We recently found that CypI administration to mice significantly inhibits the infectivity of virus, which is unrelated to HCV, but which is also highly sensitive to IFN. Moreover, we found that the IFN plasma levels were superior in infected mice treated with CypI compared to those in untreated infected mice, suggesting that the CypI IFN induction is a more widely used mechanism. These remarkable preliminary finding opened new lines of exciting enquiries. The ultimate goal of this application is to improve our understanding of the role of Cyps in the IFN response to viral infection.