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

Project summary/abstract:This work will build a contrast agent for the SARS-CoV-2 main protease (Mpro; also known as3CLpro) and validate it in an animal model. This contrast agent will have significant value as aresearch 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 arepowerful and affordable tools for population-wide studies but have limited value in researchstudies because they are in vitro tools that use single time-point sampling. They cannot monitorthe 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 agentfor Mpro based chemiluminescent resonant energy transfer (CRET). Chemiluminescence is a veryuseful in vivo imaging tool because it measures spontaneous emission of photons from contrastagents. Thus, the background emission of normal tissue is zero leading to high sensitivity imagingin contrast to in vivo fluorescence that suffers from high background. When in the CRETconfiguration, 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 willvalidate this probe with a Sindbis virus models of SARS-CoV-2. We are using this Sindbis modelbecause 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 modelsand image viral progression in adult and neonate mice. After validating this contrast agent andimaging approach, the community will have a powerful tool to answer many important questionsrelated to SARS-Cov-2 infection: What is the time course of infection and biodistribution?; Howdoes biodistribution change by route of infection? Are there latent disease reservoirs?; How doprotease levels change in response to therapy? This work is innovative because it will be the firstexample of in vivo Mpro imaging. The significance is motivated by the profound impact Covid-19has had on our society. Importantly, the work is feasible based it will harness Dr. Jokerst'sextensive experience in chemistry and contrast agent development as well as Dr. Siqueira-Neto'sexpertise in infectious disease including Zika virus.