Targeting the host kinase DYRK1A to optimize reversal of HIV-1 latency in CD4 T cells

 描述(由申请人提供)：有一个重大的未得到满足的治疗需求，可以在抗逆转录病毒治疗期间通过消除病毒驻留的存储细胞来治愈艾滋病毒感染[抗逆转录病毒疗法]。这一需求之所以持续存在，是因为对艾滋病毒如何维持对储藏细胞的持续潜伏感染的认识存在空白。这些差距是一个重要的问题，因为在填补这些差距之前，治愈艾滋病毒仍然是不可能的。长期目标是更全面地了解调节HIV潜伏期与主动复制的宿主机制。这项应用的直接目标是证明双特异性酪氨酸-磷酸化调节激酶1A[DYRK1A]的药理抑制剂允许从CD4T细胞最佳地重新激活潜伏的HIV。为了实现这一目标，将利用潜伏感染HIV的细胞系和原代CD4T细胞的组合来研究DYRK1A在维持HIV潜伏期方面的作用。中心假设是DYRK1A通过负调控驱动HIV LTR启动子的宿主转录因子来促进HIV潜伏期。这一假设是基于在申请人的实验室中进行的初步研究，该研究表明DYRK1A抑制剂三尖杉酯碱可以提高现有潜伏期反转剂[LRA]的疗效，并增强LRA诱导的NFAT活性。此外，DYRK1A基因的敲除与骆驼蓬碱的作用相似。第二种假设是，DYRK1A抑制抗原诱导的CD8 T细胞的激活，从而导致患者CD8 T细胞无法杀死HIV储存细胞，其中HIV复制已由LRA触发。我们证明了骆驼蓬碱能增强抗原诱导的CD8 T细胞活化。因此，我们的药理学方法是完全新颖的，因为它同时促进了潜伏的HIV的重新激活，并提高了杀死HIV储存库所需的CD8效应细胞的活性。这项拟议研究的基本原理是，了解调节艾滋病毒潜伏和复制的宿主机制将导致新药的开发，以最大限度地重新激活潜伏的艾滋病毒，并最大限度地使携带艾滋病毒基因组的储存细胞死亡。在初步研究的指导下，这一假设将通过追求三个具体目标进行客观检验：1)确定DYRK1A抑制剂作为抗潜伏期药物的有效性；2)确定从潜伏感染的CD4T细胞中最佳地重新激活艾滋病毒所需的宿主转录因子；以及3)确定DYRK1A抑制剂促进重新激活的艾滋病毒储存细胞死亡的有效性。这种方法是创新的，因为它使用了特定宿主调节蛋白DYRK1A的抑制剂，以更好地了解控制HIV基因表达和储藏细胞活性以及CD8 T细胞激活的机制。这项拟议的研究具有重要意义，因为它有望推动一类新的抗潜伏期药物(DYRK1A抑制剂)的发展，并增加对艾滋病毒潜伏期的机制理解，作为基于预期结果的新研究的基础。最终，从这项工作中获得的知识有可能支持艾滋病毒感染的联合治疗。

 DESCRIPTION (provided by applicant): There is a significant unmet need to develop therapeutics that could cure HIV infection by eliminating reservoir cells where the virus resides during antiretroviral therapy [ART]. This need persists because of gaps in knowledge of how HIV maintains persistent latent infection of reservoir cells. These gaps represent an important problem because until they are filled a cure for HIV remains impossible. The long term goal is to more completely understand host mechanisms that regulate HIV latency versus active replication. The immediate objective of this application is to prove that pharmacologic inhibitors of dual specificity tyrosine- phosphorylation-regulated kinase 1A [DYRK1A] allow optimal reactivation of latent HIV from CD4 T cells. To achieve this objective, a combination of latently HIV-infected cell lines and primary CD4 T cells will be utilized to investigate the role of DYRK1A in maintaining HIV latency. The central hypothesis is that DYRK1A facilitates HIV latency by negatively regulating host transcription factors that drive the HIV LTR promoter. This hypothesis is based upon preliminary studies performed in the applicant's laboratory demonstrating that the DYRK1A inhibitor harmine boosts the efficacy of existing latency reversing agents [LRAs] and enhances LRA-induced NFAT activity. Furthermore, knockdown of DYRK1A mimics the effects of harmine. A secondary hypothesis is that DYRK1A inhibits antigen-induced activation of CD8 T cells, thus contributing to the failure of patient CD8 T cells to kill HIV reservoir cells wherei HIV replication has been triggered by LRAs. We demonstrate that harmine enhances antigen-induced CD8 T cell activation. Thus, our pharmacologic approach is entirely novel because it simultaneously improves reactivation of latent HIV and improves activity of the CD8 effector cells needed to kill off the HIV reservoir. The rationale for the proposed study is that understanding host mechanisms that regulate HIV latency and replication will lead to development of new drugs to maximize reactivation of latent HIV and to maximize death of reservoir cells that carry HIV genomes. Guided by preliminary studies, the hypothesis will be objectively tested by pursuing three specific aims: 1) Establish the efficacy of DYRK1A inhibitors as anti-latency drugs; 2) Identify host transcription factors that are required for optimal HIV reactivation from latently-infected CD4 T cells; and 3) Establish the efficacy of DYRK1A inhibitors for enhancing death of reactivated HIV reservoir cells. The approach is innovative because it uses inhibitors of a specific host regulatory protein, DYRK1A, to better understand mechanisms that control HIV gene expression and viability of reservoir cells as well as CD8 T cell activation. The proposed research is significant because it is expected to advance a new class of anti-latency drugs (DYRK1A inhibitors) and to increase mechanistic understanding of HIV latency as a basis for new research based on expected outcomes. Ultimately, the knowledge gained from this work has the potential to support a combination therapeutic cure for HIV infection.