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Enterovirus-D68 (EV68) continues to present as a global health issue causing respiratory illness and outbreaks associated with long-lasting neurological disease, with no antivirals or specific treatment options. The development of antiviral therapeutics, such as small-molecule inhibitors that target conserved proteins like the enteroviral 3C protease, remains to be achieved. While various 3C inhibitors have been investigated, their design does not consider the potential emergence of drug resistance mutations. For other antivirals where resistance has been a challenge, we have demonstrated that the likelihood of resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of the target. Here, we characterize a series of 3C inhibitors against EV68-3C protease through enzyme inhibition, protein crystallography, and structural analysis. We have determined and analyzed three high-resolution inhibitor-bound crystal structures of EV68-3C protease, which revealed possible sites of resistance mutations, a key structural water molecule conserved during ligand binding, and the conformational flexibility of the catalytic histidine H40. This structural analysis combined with enzymatic assays provides insights for the rational design of inhibitors that are robust against resistance toward developing antiviral treatments for EV68 infections. [ABSTRACT FROM AUTHOR]

The extremely rapid development of understanding and technology that led to the containment of the COVID-19 pandemic resulted from collaborative efforts in the fields of Betacoronavirus pandemicum (SARS-CoV-2) biology, pharmacology, vaccinology, and medicine. Perhaps surprisingly, much of the research was conducted using simple and efficient yeast models. In this manuscript, we describe how yeast, eukaryotic microorganisms, have been used to research this global challenge, focusing on the therapeutic potential of the studies discussed herein. Thus, we outline the role of yeast in studying viral protein interactions with the host cell proteome, including the binding of the SARS-CoV-2 virus spike protein to the human ACE2 receptor and its modulation. The production and exploration of viral antigens in yeast systems, which led to the development of two approved COVID-19 vaccines, are also detailed. Moreover, yeast platforms facilitating the discovery and production of single-domain antibodies (nanobodies) against SARS-CoV-2 are described. Methods guiding modern and efficient drug discovery are explained at length. In particular, we focus on studies designed to search for inhibitors of the main protease (Mpro), a unique target for anti-coronaviral therapies. We highlight the adaptability of the techniques used, providing opportunities for rapid modification and implementation alongside the evolution of the SARS-CoV-2 virus. Approaches introduced in yeast systems that may have universal potential application in studies of emerging viral diseases are also described. [ABSTRACT FROM AUTHOR]

Enterovirus-D68 (EV68) has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Unfortunately, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. For other viral proteases, we have demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. However, the structural characterization of EV68-3C protease bound to its substrates has been lacking. Here, we have determined the substrate specificity of EV68-3C protease through molecular modeling, molecular dynamics (MD) simulations, and co-crystal structures. Molecular models enabled us to successfully characterize the conserved hydrogen-bond networks between EV68-3C protease and the peptides corresponding to the viral cleavage sites. In addition, co-crystal structures we determined have revealed substrate-induced conformational changes of the protease which involved new interactions, primarily surrounding the S1 pocket. We calculated the substrate envelope, the three-dimensional consensus volume occupied by the substrates within the active site. With the elucidation of the EV68-3C protease substrate envelope, we evaluated how 3C protease inhibitors, AG7088 and SG-85, fit within the active site to predict potential resistance mutations. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 Main Protease (Mpro) is an enzyme that cleaves viral polyproteins translated from the viral genome, which is critical for viral replication. Mpro is a target for anti-SARS-CoV-2 drug development. Herein, we performed a large-scale virtual screening by comparing multiple structural descriptors of reference molecules with reported anti-coronavirus activity against a library with >17 million compounds. Further filtering, performed by applying two machine learning algorithms, identified eighteen computational hits as anti-SARS-CoV-2 compounds with high structural diversity and drug-like properties. The activities of twelve compounds on Mpro's enzymatic activity were evaluated by fluorescence resonance energy transfer (FRET) assays. Compound 13 (ZINC13878776) significantly inhibited SARS-CoV-2 Mpro activity and was employed as a reference for an experimentally hit expansion. The structural analogues 13a (ZINC4248385), 13b (ZNC13523222), and 13c (ZINC4248365) were tested as Mpro inhibitors, reducing the enzymatic activity of recombinant Mpro with potency as follows: 13c > 13 > 13b > 13a. Then, their anti-SARS-CoV-2 activities were evaluated in plaque reduction assays using Vero CCL81 cells. Subtoxic concentrations of compounds 13a, 13c, and 13b displayed in vitro antiviral activity with IC50 in the mid micromolar range. Compounds 13a–c could become lead compounds for the development of new Mpro inhibitors with improved activity against anti-SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The main protease (Mpro) is a promising drug target for inhibiting the coronavirus due to its conserved properties and lack of homologous genes in humans. However, previous studies on Mpro's kinetic parameters have been confusing, hindering the selection of accurate inhibitors. Therefore, obtaining a clear view of Mpro's kinetic parameters is necessary. In our study, we investigated the kinetic behaviors of Mpro from SARS-CoV-2 and SARS-CoV using both FRET-based cleavage assay and the LC-MS method, respectively. Our findings indicate that the FRET-based cleavage assay could be used for preliminary screening of Mpro inhibitors, while the LC-MS method should be applied to select the effective inhibitors with higher reliability. Furthermore, we constructed the active site mutants (H41A and C145A) and measured the kinetic parameters to gain a deeper understanding of the atomic-level enzyme efficiency reduction compared to the wild type. Overall, our study provides valuable insights for inhibitor screening and design by offering a comprehensive understanding of Mpro's kinetic behaviors. [ABSTRACT FROM AUTHOR]

With the spread of SARS-CoV-2 throughout the globe causing the COVID-19 pandemic, the threat of zoonotic transmissions of coronaviruses (CoV) has become even more evident. As human infections have been caused by alpha- and beta-CoVs, structural characterization and inhibitor design mostly focused on these two genera. However, viruses from the delta and gamma genera also infect mammals and pose a potential zoonotic transmission threat. Here, we determined the inhibitor-bound crystal structures of the main protease (Mpro) from the delta-CoV porcine HKU15 and gamma-CoV SW1 from the beluga whale. A comparison with the apo structure of SW1 Mpro, which is also presented here, enabled the identification of structural arrangements upon inhibitor binding at the active site. The cocrystal structures reveal binding modes and interactions of two covalent inhibitors, PF-00835231 (active form of lufotrelvir) bound to HKU15, and GC376 bound to SW1 Mpro. These structures may be leveraged to target diverse coronaviruses and toward the structure-based design of pan-CoV inhibitors. [ABSTRACT FROM AUTHOR]