An award is made to the University of Wisconsin-Milwaukee (UWM) to investigate the molecular mechanism of an essential coronavirus protease reaction in real time. This project addresses fundamental questions how the SARS coronavirus-2 (CoV-2) proliferates. CoV-2 is the source of the worldwide COVID-19 pandemic, which severely impacts public health and national prosperity. The CoV-2 main protease, also called 3CLpro, catalyzes an essential reaction for assembly of CoV-2 infectious particles. If the 3CLpro is blocked, the virus cannot assemble, and its spread is effectively suppressed. The goal is to characterize the 3CLpro?s catalytic cycle at ambient temperatures with X-ray structures. This research takes advantage of opportunities at X-ray Free Electron Lasers (XFELs) such as the Linac Coherent Light Source (LCLS) at Stanford Linear Accelerator Center in Menlo Park, CA. XFELs are the strongest X-ray sources in the world that make it possible to capture molecular reaction intermediates at near atomic resolution within biologically relevant temperatures and time scales. The unique opportunities at XFELs will advance the understanding of 3CLpro catalysis and its function in the proliferation of the virus and contribute to the elimination of the pandemic. This project will involve UWM graduate students and postdoctoral researchers who will be trained in newest data collection and data analysis methods at XFELs.
In host cells the SARS-CoV-2?s RNA genome is translated by host ribosomes into a long polypeptide strand that must be cleaved into functional proteins. This is achieved by the CoV-2 3CLpro. If the 3CLpro is inhibited, the newly formed virus particles cannot assemble correctly and become non-infectious. This project structurally characterizes the catalytic cycle of the 3CLpro at XFELs. XFELs are extremely powerful, femtosecond-pulsed X-ray sources, which became available to a wider community a decade ago. At XFELs, crystal structures that are essentially free of radiation damage can be determined at ambient (near physiological) temperatures. Within 3CLpro microcrystals the catalytic cycle will be initiated by diffusion of substrate. Since the crystals are so small, diffusion is not rate-limiting. Microcrystals are mixed with substrate at various time delays before the mixture is injected into the X-ray beam, a method known as ?mix-and-inject? serial crystallography (MISC). MISC will be used to follow the 3CLpro enzymatic reaction in real time with X-ray structures of reaction intermediates. The binding of small compounds that inhibit the 3CLpro will be probed at near atomic resolution at relevant temperatures. Results will (i) aid the design and discovery of new inhibitory compounds that affect the function of this essential protease and prevent the formation of infectious viral particles, and (ii) contribute to the development of MISC as an applicable method at XFELs, to be used for the structural characterization of reactions in biologically significant molecules.
This RAPID award is made by the Division of Biological Infrastructure (DBI) using funds from the Coronavirus Aid, Relief, and Economic Security (CARES) Act.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
