Imaging SARS-CoV-2 proteases for spatio-temporal insight into Covid-19

项目摘要/摘要： 这项工作将为SARS-CoV-2主蛋白酶(MPRO；也称为 3CLPRO)，并在动物模型中进行验证。这种造影剂将作为一种 这是一个研究工具，因为它将绘制和测量具有空间和时间分辨率的MPRO。 研究SARS-CoV-2的传统方法是基于聚合酶链式反应和血清学。这些是 用于全人群研究的强大且负担得起的工具，但研究价值有限 研究是因为它们是使用单一时间点采样的体外工具。他们无法监控 病毒载量在体内的生物分布和时间进程或检测病毒库的位置。 相比之下，活体成像提供了纵向跟踪生物现象的能力， 数量上，并且相对非侵入性的。因此，该提案的目标1将构建造影剂 用于基于MPRO的化学发光共振能量转移(CRET)。化学发光是一种非常 有用的活体成像工具，因为它测量对比度中的光子自发辐射 探员们。因此，正常组织的背景发射为零，从而导致高灵敏度成像 与高本底的体内荧光形成对比。在牢房里的时候 配置时，探针是静默的；通过可MPRO裂解的序列激活会导致高信号。 我们将用化学和重组MPRO来验证这些基于CRT的分子。目标2将 用SARS-CoV-2的Sindbis病毒模型验证该探针。我们正在使用这个辛德比斯模型 因为它适用于BSL-2实验室，允许工作立即进行，而不像野生型SARS- 需要BSL-3的CoV-2。我们将通过Sindbis病毒在组织培养和动物模型中表达MPRO 并想象病毒在成年和新生小鼠中的进展。在验证了该造影剂和 成像方法，社区将有一个强大的工具来回答许多重要的问题 与SARS-CoV-2感染有关：感染和生物分布的时间进程是什么？ 生物分布是否因感染途径而改变？是否存在潜伏的病源？；如何 蛋白水解酶水平会因治疗而改变吗？这项工作具有创新性，因为它将是第一个 活体MPRO成像的例子。其重要意义在于对新冠肺炎的深刻影响 对我们的社会产生了影响。重要的是，这项工作是可行的，因为它将利用约克斯特博士的 在化学和造影剂开发以及Siqueira-Neto博士的 在包括寨卡病毒在内的传染病方面具有专业知识。

Project summary/abstract: This work will build a contrast agent for the SARS-CoV-2 main protease (Mpro; also known as 3CLpro) and validate it in an animal model. This contrast agent will have significant value as a research tool because it will map and measure Mpro with spatial and temporal resolution. Conventional methods to studying SARS-CoV-2 are based on PCR and serology. These are powerful and affordable tools for population-wide studies but have limited value in research studies because they are in vitro tools that use single time-point sampling. They cannot monitor the biodistribution and time course of the viral load in vivo or detect the location of viral reservoirs. In contrast, in vivo imaging offers the ability to track biological phenomena longitudinally, quantitatively, and relatively non-invasively. Thus, Aim 1 of this proposal will build a contrast agent for Mpro based chemiluminescent resonant energy transfer (CRET). Chemiluminescence is a very useful in vivo imaging tool because it measures spontaneous emission of photons from contrast agents. Thus, the background emission of normal tissue is zero leading to high sensitivity imaging in contrast to in vivo fluorescence that suffers from high background. When in the CRET configuration, the probe is silent; activation via a Mpro-cleavable sequence leads to high signal. We will validate these CRET-based molecules with chemistry and recombinant Mpro. Aim 2 will validate this probe with a Sindbis virus models of SARS-CoV-2. We are using this Sindbis model because it is suitable for BSL-2 labs allowing work to proceed immediately unlike wild type SARS- CoV-2 requiring BSL-3. We will express Mpro via Sindbis virus in tissue culture and animal models and image viral progression in adult and neonate mice. After validating this contrast agent and imaging approach, the community will have a powerful tool to answer many important questions related to SARS-Cov-2 infection: What is the time course of infection and biodistribution?; How does biodistribution change by route of infection? Are there latent disease reservoirs?; How do protease levels change in response to therapy? This work is innovative because it will be the first example of in vivo Mpro imaging. The significance is motivated by the profound impact Covid-19 has had on our society. Importantly, the work is feasible based it will harness Dr. Jokerst’s extensive experience in chemistry and contrast agent development as well as Dr. Siqueira-Neto’s expertise in infectious disease including Zika virus.