A multidisciplinary approach yields novel inhibitors and reveals substrate specificity, structure, and mechanisms of CoV-2 PLpro.

Significance: The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Using this approach, we have designed an extremely active substrate of NE and subsequently converted it into an ultrasensitive activity-based probe for imaging active elastase during the process of neutrophil extracellular trap formation. Our study could have a substantial effect on the design of substrates, inhibitors, and probes for any endopeptidase.

COVID-19 vaccinations are about to begin in various countries or are already ongoing. This is an unprecedented operation that is also met with a loud response from anti-vaccine communities—currently using all available channels to manipulate public opinion. At the same time, the strategy to educate on vaccinations, explain their mechanism of action, and build trust in science is subdued in different world parts. Such actions should go much beyond campaigns promoting the COVID-19 vaccines solely on the information provided by the health institutions and national authorities. In this paper, actions provided by independent expert groups needed to counteract the anti-vaccine propaganda and provide scientific-based information to the general public are offered. These actions encompass organizing groups continuously communicating science on COVID-19 vaccines to the general public; tracking and tackling emerging and circulating fake news; and equipping celebrities and politicians with scientific information to ensure the quality of messages they communicate, as well as public letters, and statements of support for vaccination by healthcare workers, recognized scientists, VIPs, and scientific societies; and no tolerance to false and manipulated claims on vaccination spread via traditional and social media as well as by health professionals, scientists, and academics. These activities should be promptly implemented worldwide, regardless of the current status and availability of the COVID-19 vaccine in a particular region. If we are about to control the pandemic for the sake of public benefit, it is high time to collectively speak out as academic and medical societies with support from decision-makers. Otherwise, the battle will be lost to those who stand against scientific evidence while offering no feasible solution to the problem.

The main viral protease (3CL^pro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL^pro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CL^pro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CL^pro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CL^pro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.