RP3: Targeting ATG gene-dependent immunity for novel anti-infective therapeutics

项目摘要-RP3：RP3的目标是定义和药理靶向细胞内免疫 利用自噬(ATG)基因(但不是降解性自噬)控制感染和 发炎。我们在旨在了解抗感染药物的实验中发现了这些过程。 人和小鼠必不可少的细胞因子干扰素γ在巨噬细胞介导的抗肿瘤机制中的作用 病毒、细菌和寄生虫。干扰素γ用于治疗慢性肉芽肿性疾病和骨化症，但 干扰素γ的其他用途也受到毒性的限制，这一事实通常被归因于干扰素γ诱导a 大量的促炎基因。我们推测新的治疗方法可能是由 确定干扰素γ有效作用的效应机制，然后选择性地刺激 这些机制可以产生ATG基因导向的广谱抗感染药物。这导致了一个发现： 使用同一套干扰素γ触发抗弓形虫和诺如病毒感染的atg基因依赖免疫 ATG基因(ATG5、ATG7、ATG16L1、ATG12)，而不是ATG14或降解性自噬。事实是ATG基因- 依赖机制(S)控制了两种在系统发育上截然不同的病原体，向我们暗示我们可能是 能够制造出对多种病原体有效的化合物。在第一次CETRI资助期间 期间，我们通过鉴定atg基因导向的广谱抗感染化合物证实了这一预测。 具有抗诺如病毒、沙门氏菌、结核分枝杆菌和弓形虫的活性。这一发现 ATG基因在炎症调节中的作用的发现补充了这一点。RP3的重点是 杯状病毒科(最重要的是新城疫)是主要病原体，导致了90%的非细菌性疫情 世界上最严重的胃肠炎。我们首次发现了小鼠NOV(MNOV)和培养的NOV，提供了一种 在实验室小鼠中建立临床前模型，并允许发现ATG基因依赖免疫。关于人类的研究 Nov(Hnov)的感染一直是有限的，直到最近发现hnov在分化为 取自活组织切片的人结肠干细胞。这一点，再加上MNOV在人类体内茁壮成长的发现 表达适当受体的细胞，以及ATG基因依赖的免疫对弓形虫和 人类细胞中的MNOV强烈主张这些机制在进化上是保守的，并提供了工具 通过以下目标确定与人类重大疾病相关的化合物：目标1：开发ATG基因- 定向广谱抗感染化合物；目的2：确定ATG基因的分子基础- 依赖干扰素γ诱导诺如病毒复制的控制，并确定靶点和先导化合物 触发这种形式的细胞内免疫；目标3：确定干扰素γ调节的分子基础 ATG基因的应答和鉴定上调特异性干扰素γ效应的先导化合物 机制；目标4.与其他CETR项目合作，以确定自噬和ATG基因控制的 广谱抗感染化合物。

PROJECT SUMMARY – RP3: The goal of RP3 is to define and pharmacologically target intracellular immune mechanisms utilizing autophagy (Atg) genes (but not degradative autophagy) to control infection and inflammation. We discovered these processes in experiments designed to understand the anti-infective mechanisms of IFNγ, a cytokine essential in both humans and mice for macrophage-mediated resistance to viruses, bacteria, and parasites. IFNγ is used to treat chronic granulomatous disease and osteopetrosis, but other uses of IFNγ have been limited by toxicity, a fact generally attributed to IFNγ inducing the expression of a very large number of pro-inflammatory genes. We theorized that new therapeutics might be generated by defining the effector mechanisms responsible for the potent effects of IFNγ, and then selectively stimulating these mechanisms to generate Atg gene-directed broad-spectrum anti-infectives. This led to the discovery that IFNγ triggers Atg gene-dependent immunity against T. gondii and norovirus (NoV) infection using the same set of Atg genes (Atg5, Atg7, Atg16L1, Atg12), but not Atg14 or degradative autophagy. The fact that Atg gene- dependent mechanism(s) controlled two phylogenetically distinct pathogens suggested to us that we might be able to make compounds that are effective against a broad range of pathogens. During the first CETR funding period we confirmed this prediction by identifying Atg gene-directed broad-spectrum anti-infective compounds with activity against norovirus, salmonella, Mycobacterium tuberculosis and Toxoplasma gondii. This discovery was complemented by discoveries of the role of Atg genes in the regulation of inflammation. RP3 focuses on the Caliciviridae (most importantly NoVs) which are priority pathogens, causing >90% of the epidemic non-bacterial gastroenteritis in the world. We discovered murine NoV (MNoV) and cultured NoVs for the first time, providing a preclinical model in laboratory mice and allowing discovery of Atg gene-dependent immunity. Studies of human NoV (HNoV) infection had been limited until it was recently found that HNoV grows in cells differentiated from biopsy-derived human colonic stem cells. This, together with the discovery that MNoV grows robustly in human cells expressing the proper receptor, and that Atg gene-dependent immunity operates against both T. gondii and MNoV in human cells strongly argues that these mechanisms are evolutionarily conserved and provide the tools to identify compounds relevant to a major human disease via the following aims: Aim 1: Develop Atg gene- directed broad spectrum anti-infective compounds; Aim 2: Define the molecular basis of Atg gene- dependent IFNγ-induced control of norovirus replication, and identify targets and lead compounds that trigger this form of intracellular immunity; Aim 3: Define the molecular basis for the regulation of IFNγ responses by Atg genes and identify lead compounds that up-regulate specific IFNγ effector mechanisms; Aim 4. Collaborate with other CETR projects to identify autophagy- and Atg gene-directed broad-spectrum anti-infective compounds.