Jeffrey P. Mitchell

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has highlighted the vulnerability of a globally connected population to zoonotic viruses. The FDA-approved coronavirus antiviral Paxlovid targets the essential SARS-CoV-2 main protease, Mpro. Whilst effective in the acute phase of a COVID infection, Paxlovid cannot be used by all patients, can lead to viral recurrence, and does not protect against post-acute sequelae of COVID-19 (PASC), commonly known as long COVID, an emerging significant health burden that remains poorly understood and untreated. Alternative antivirals that are addressing broader patient needs are urgently required. We here report our drug discovery efforts to target PLpro, a further essential coronaviral protease, for which we report a novel chemical scaffold that targets SARS-CoV-2 PLpro with low nanomolar activity, and which exhibits activity against PLpro of other pathogenic coronaviruses. Our lead compound shows excellent in vivo efficacy in a mouse model of severe acute disease. Importantly, our mouse model recapitulates long-term pathologies matching closely those seen in PASC patients. Our lead compound offers protection against a range of PASC symptoms in this model, prevents lung pathology and reduces brain dysfunction. This provides proof-of-principle that PLpro inhibition may have clinical relevance for PASC prevention and treatment going forward.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has highlighted the vulnerability of a globally connected population to zoonotic viruses. The only FDA-approved coronavirus antiviral, Paxlovid, targets the essential SARS-CoV-2 main protease, Mpro. Whilst effective in the acute phase of an infection, Paxlovid cannot be used by all patients, can lead to viral recurrence, and does not protect against post-acute sequelae of COVID-19 (PASC), commonly known as Long COVID, an emerging significant health burden that remains poorly understood and untreated. Alternative antivirals that are addressing broader patient needs are urgently required. We here report our drug discovery efforts to target PLpro, a further essential coronaviral protease, for which we report a novel chemical scaffold that targets SARS-CoV-2 PLpro with low nanomolar activity, and which exhibits activity against PLpro of other pathogenic coronaviruses. Our lead compound shows excellent in vivo efficacy in a mouse model of severe acute disease. Importantly, our mouse model recapitulates long-term pathologies matching closely those seen in PASC patients, including lung, heart, gut and brain dysfunction. Our lead compound offers protection against PASC in this model, prevents lung pathology and reduces brain dysfunction, providing a potential treatment option for PASC sufferers going forward.

Papain-like protease (PLpro) is an attractive drug target for SARS-CoV-2 because it is essential for viral replication, cleaving viral poly-proteins pp1a and pp1ab, and has de-ubiquitylation and de-ISGylation activities, affecting innate immune responses. We employ Deep Mutational Scanning to evaluate the mutational effects on PLpro enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify mutations in neighboring residues that alter activity. We characterize residues responsible for substrate binding and demonstrate that although residues in the blocking loop are remarkably tolerant to mutation, blocking loop flexibility is important for function. We additionally find a connected network of mutations affecting activity that extends far from the active site. We leverage our library to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

SARS-CoV-2, the causative agent of COVID-19, is responsible for the recent global pandemic and remains a major source of mortality. Papain-like protease (PLpro) is a target for SARS-CoV-2 inhibitor development, as it is not only essential for viral replication through cleavage of the viral poly-proteins pp1a and pp1ab, but also has de-ubiquitylation and de-ISGylation activities, which can affect innate immune responses. To understand the features of PLpro that dictate activity and anticipate how emerging PLpro variants will affect function, we employed Deep Mutational Scanning to evaluate the mutational effects on enzymatic activity and protein stability in mammalian cells. We confirm features of the active site and identify all mutations in neighboring residues that support or ablate activity. We characterize residues responsible for substrate binding and demonstrate that although the blocking loop is remarkably tolerant to nearly all mutations, its flexibility is important for enzymatic function. We additionally find a connected network of mutations affecting function but not structure that extends far from the active site. Using our DMS libraries we were able to identify drug-escape variants to a common PLpro inhibitor scaffold and predict that plasticity in both the S4 pocket and blocking loop sequence should be considered during the drug design process.

The fungal genus Fusarium contains plant pathogens, mutualists, and commensalists. Because nonpathogenic Fusarium strains can contribute to disease suppression in soil by competition or induced plant resistance, identifying factors that influence the abundance of nonpathogenic strains could lead to strategies that enhance plant-beneficial interactions and deter pathogen invasion. We developed a Fusarium-specific high-throughput translation elongation factor 1α (EF-1α) amplicon-sequencing method with subspecies resolution. Newly designed primers enabled reads to be joined for improved quality metrics after Illumina MiSeq sequencing. By including the ‘oligotyping’ program in our bioinformatic workflow, we reconstructed Fusarium EF-1α haplotypes with 100% accuracy. We conducted simulated crop rotation experiments in soil infested with the strawberry pathogen Fusarium oxysporum f. sp. fragariae ( Fof) and the following treatments: strawberry, blackberry, raspberry, lettuce, broccoli, and fallow. We profiled root Fusarium communities with EF-1α amplicons and observed that nonpathogenic strains of F. oxysporum did not colonize crops uniformly; some strains exhibited asymptomatic host preferences. In susceptible strawberry roots, >90% of sequences corresponded to Fof. Bulk soil communities of Fusarium were remarkably stable and had minimal treatment-dependent effects after 1 year. Similarly, in a long-term agricultural research experiment, 18 years of conservation tillage and cover cropping did not lead to bulk soil Fusarium communities that were significantly different than controls. Although the roots of rotation crops can augment the abundance of nonpathogenic F. oxysporum strains, their abundance in bulk soil is unlikely to be altered after a single year. Climate and soil physicochemical properties may have a greater effect on Fusarium community structure.[ ] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

California has an aging vegetable-crop producing workforce and well-trained workers are urgently needed. The State’s public universities with agricultural programs thus have a critical role in preparing the future workforce to sustain the State’s economy. The aim of this project was to develop educational materials to train the next generation of workers with the technical, agronomic, economic and ecological skills required to keep California’s vegetable crop sector innovative and competitive. Thirty high-quality instructional videos on vegetable crop production, marketing, and postharvest processing and handling systems were created. Students were surveyed on the utility of selected videos, by assessing various parameters of knowledge before and after watching the videos. The videos made positive and significant changes in the knowledge of the topics among students. Therefore, incorporation of videos into agricultural curricula should contribute to effective communication and understanding of concepts by post-secondary students.

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications ^1,2 . Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses ³ . We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.