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

对 SARS-CoV-2 蛋白酶进行成像以时空洞察 Covid-19

• 批准号: 10167571
• 负责人: Jesse Vincent Jokerst
• 金额: $ 43.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10167571
• 关键词: 2019-nCoV; Adult; Aftercare; Animal Model; Antibodies; Biodistribution; Biological Assay; Biological Phenomena; COVID-19; Cells; Chemistry; Chromatography; Communicable Diseases; Communities; Complex; Contrast Media; Coronavirus Infections; Coupled; DegP protease; Detection; Development; Disease; Disease Reservoirs; Drug Targeting; Energy Transfer; Excision; Exposure to; Fluorescence; Genes; Goals; Image; Imaging Device; In Vitro; Infection; Light; Literature; Location; Maps; Mass Chromatography; Mass Spectrum Analysis; Measures; Mediating; Methods; Modeling; Monitor; Mus; Nanotechnology; Nature; Neonatal; Noise; Normal tissue morphology; Organ; Organism; Paper; Patients; Peptide Hydrolases; Photons; Population; Production; Proteins; Publishing; Reaction; Reagent; Recombinants; Reporting; Research; Rodent Model; Route; Saliva; Sampling; Screening procedure; Serologic tests; Serum; Severe Acute Respiratory Syndrome; Signal Transduction; Sindbis Virus; Societies; Swab; Testing; Time; Tissues; Viral; Viral Load result; Viral Vector; Viral reservoir; Virus; Virus Replication; Work; Zika Virus; acoustic imaging; animal imaging; base; chemical synthesis; contrast imaging; cost; design; experience; human disease; human model; imaging agent; imaging approach; imaging modality; in vitro testing; in vivo; in vivo Model; in vivo imaging; innovation; insight; latent infection; nano; neonate; optical imaging; pathogen; research study; response; spatiotemporal; targeted imaging; temporal measurement; tissue culture; tool

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.

项目总结/摘要： 这项工作将建立SARS-CoV-2主要蛋白酶（Mpro;也称为 3CLpro）并在动物模型中验证。这种造影剂将具有显著的价值， 研究工具，因为它将映射和测量Mpro的空间和时间分辨率。 传统的SARS-CoV-2研究方法是基于PCR和血清学。这些是 用于全人群研究的强大和负担得起的工具，但在研究中的价值有限 研究，因为它们是使用单时间点采样的体外工具。他们无法监控 体内病毒载量的生物分布和时间进程或检测病毒储存库的位置。 相比之下，体内成像提供了纵向跟踪生物现象的能力， 定量的，相对非侵入性的。因此，本提案的目标1将构建造影剂 用于基于Mpro的荧光共振能量转移（CRET）。化学发光是一种非常 这是一种有用的体内成像工具，因为它可以测量来自造影剂的光子的自发发射 剂.因此，正常组织的背景发射为零，导致高灵敏度成像 这与遭受高背景的体内荧光相反。在Cret中， 在该构型中，探针是沉默的;经由Mpro可切割序列的激活导致高信号。 我们将用化学和重组Mpro来验证这些基于CRET的分子。目标2将 用SARS-CoV-2的Sindbis病毒模型验证该探针。我们使用辛德毕斯模型 因为它适合BSL-2实验室，允许工作立即进行，不像野生型SARS- CoV-2需要BSL-3。我们将通过辛德毕斯病毒在组织培养和动物模型中表达Mpro 并对成年和新生小鼠的病毒进展进行成像。在验证该造影剂后， 成像的方法，社会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过程是什么？如何 生物分布是否因感染途径而改变？是否存在潜在的病源？怎么 蛋白酶水平会因治疗而改变吗？这项工作是创新的，因为它将是第一个 体内Mpro成像的实例。这一意义是由新型冠状病毒肺炎（COVID-19）的深远影响所激发的 对我们社会的影响重要的是，这项工作是可行的，它将利用Jokerst博士的 在化学和造影剂开发方面的丰富经验以及Siqueira-Neto博士的 寨卡病毒是一种常见的传染病。

项目成果

Goldilocks Energy Minimum: Peptide-Based Reversible Aggregation and Biosensing.

金发姑娘能量最低：基于肽的可逆聚集和生物传感。

• DOI: 10.1021/acsami.3c09627
• 发表时间: 2023
• 期刊: ACS applied materials & interfaces
• 影响因子: 9.5
• 作者: Yim,Wonjun;Retout,Maurice;Chen,AmandaA;Ling,Chuxuan;Amer,Lubna;Jin,Zhicheng;Chang,Yu-Ci;Chavez,Saul;Barrios,Karen;Lam,Benjamin;Li,Zhi;Zhou,Jiajing;Shi,Lingyan;Pascal,TodA;Jokerst,JesseV
• 通讯作者: Jokerst,JesseV