RP1: Targeting Beclin 1 complexes for broad-spectrum anti-infective therapeutics

项目摘要-RP1：细胞内病原体的自噬传递到溶酶体以进行破坏是 先天免疫的核心机制。我们的目标是制定成功的战略，以增加 在感染了细胞内病原体的患者的细胞/组织中自噬，以治疗优先病原体，如 基孔肯雅病毒(CHIKV)、西尼罗河病毒(WNV)、寨卡病毒(ZIKV)等传染病。我们 发现了(1)第一个被证明在自噬中起作用的哺乳动物基因，贝克林1；(2)一个保守的作用 抗菌宿主防御中的自噬基因；(3)细胞穿透性自噬诱导肽(TAT-Beclin 1)体内具有广谱抗感染活性；(4)调节Beclin 1的分子机制 自噬活性，包括通过Bcl2结合抑制。在我们目前的CETR计划中，我们(1)优化了 Tat-Beclin 1肽(目前正在与我们的合作伙伴Casma Treeutics进行IND使能研究)；(2) 鉴定出具有纳摩尔效力的化学实体，可破坏Bc l-2/Beclin 1结合并诱导自噬； (3)使用全基因组siRNA筛选来识别在病毒感染过程中触发自噬的蛋白质，导致 SNX5是一种新发现的Beclin-1结合蛋白，在抗病毒免疫中起重要作用。 此外，我们产生了敲入Beclin 1突变的小鼠，这种突变扰乱了Bcl2的结合并增加了 基础自噬；这些小鼠的寿命和健康时间延长，结核分枝杆菌减少。 复制和对致死性CHIKV感染的易感性。这提供了重要的遗传原理证明 自噬增加是安全的，并且证实了破坏Bcl2/Beclin结合是自噬诱导的目标 治疗学。为了实现我们的目标，我们将利用在我们目前的CETR期间所做的发现 程序研究，以(1)推进我们目前的半优化化合物(TAT-Beclin 1，Bcl2/Beclin 1结合 干扰物)，以及(2)发现新的化学实体，通过模仿 TAT-Beclin 1提高Beclin 1-III类磷脂酰肌醇3-激酶活性的作用机制 (PI3K)自噬复合体，或增强SNX5的自噬诱导活性。这将会实现的 由UTSW的一个多学科团队在高通量化学筛选、药物筛选方面拥有专业知识 化学、结构生物学和临床前药理学，以及与其他领域的化学家合作 经济、社会和文化权利国际项目(Rp2、Rp5)。我们将评估从我们的新工作中产生的新化学物质的有效性 和CETR计划中的其他项目(RP2-RP5)在我们建立的WNV新生动物模型中， CHIKV和ZIKV。我们预计，这些方法(旨在激活Beclin 1自噬功能)将 结果鉴定了先导化合物，为未来的IND研究开发新的自噬途径- 定向广谱抗感染药物。

Project Summary – RP1: The autophagic delivery of intracellular pathogens to the lysosome for destruction is a central mechanism of innate immunity. Our objective is to develop successful strategies to increase autophagy in cells/tissues of patients infected with intracellular pathogens to treat priority pathogens such as chikungunya virus (CHIKV), West Nile virus (WNV), Zika virus (ZIKV) and other infectious diseases. We discovered (1) the first mammalian gene shown to function in autophagy, beclin 1; (2) a conserved role for autophagy genes in antimicrobial host defense; (3) a cell penetrating autophagy-inducing peptide (Tat-Beclin 1) with broad-spectrum anti-infective activity in vivo; and (4) molecular mechanisms that regulate Beclin 1 autophagy activity, including inhibition by Bcl-2 binding. In our current CETR program, we (1) optimized the Tat-Beclin 1 peptide (now moving into IND-enabling studies with our partner Casma Therapeutics); (2) identified nanomolar potency chemical entities that disrupt Bcl-2/Beclin 1 binding and induce autophagy; and (3) used a genome-wide siRNA screen to identify proteins that trigger autophagy during viral infection, leading to the discovery of SNX5 as a newly-identified Beclin 1 binding protein that is important for antiviral immunity. Furthermore, we generated mice with a knock-in Beclin 1 mutation that disrupts Bcl-2 binding and increases basal autophagy; these mice have extended lifespan and healthspan, and show decreased M. tuberculosis replication and susceptibility to lethal CHIKV infection. This provides important genetic proof-of-principle that increased autophagy is safe and validates disruption of Bcl-2/Beclin binding as a target for autophagy-inducing therapeutics. To accomplish our objective, we will leverage discoveries made during our current CETR Program research to (1) advance our current semi-optimized compounds (Tat-Beclin 1, Bcl-2/Beclin 1 binding disruptors), and (2) discover new chemical entities that induce anti-infective autophagy by mimicking the mechanism of action of Tat-Beclin 1, increasing the activity of Beclin 1 Class III phosphatidylinositol 3-kinase (PI3K) autophagy complexes, or enhancing the autophagy-inducing activity of SNX5. This will be accomplished by a multidisciplinary team at UTSW with expertise in high-throughput chemical screening, medicinal chemistry, structural biology, and pre-clinical pharmacology, as well as in collaboration with chemists in other CETR Projects (RP2, RP5). We will evaluate the efficacy of new chemical entities that arise from our new work and other projects in this CETR Program (RP2-RP5) in our established neonatal animal models of WNV, CHIKV, and ZIKV. We anticipate that these approaches (aimed at activating Beclin 1 autophagy function) will result in identification of lead compounds for future IND-enabling studies to develop new autophagy pathway- directed broad-spectrum anti-infectives.