Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids.

N6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and N6-trifluoroacetyl-l-lysine (TFAK) are non-canonical amino acids, which can be installed in proteins by genetic encoding. In addition, we describe a new aminoacyl-tRNA synthetase specific for N6-(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK), which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2 main protease (Mpro) as a model system with three different ligands, we show that the 1H and 19F nuclei of the solvent-exposed trimethylsilyl and CF3 groups produce intense signals in the nuclear magnetic resonance (NMR) spectrum. Their response to active-site ligands differed significantly when positioned near rather than far from the active site. Conversely, the NMR probes failed to confirm the previously reported binding site of the ligand pelitinib, which was found to enhance the activity of Mpro by promoting the formation of the enzymatically active dimer. In summary, the amino acids TMSK, TMSNK, and TFAK open an attractive path for site-specific NMR analysis of ligand binding to large proteins of limited stability and at low concentrations.

Predicting Antiviral Resistance Mutations in SARS-CoV-2 Main Protease with Computational and Experimental Screening.

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 Mpro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of Mpro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of Mpro. The E166M mutation reduced the half-maximal inhibitory concentration (IC50) of NTV 24-fold and 118-fold for the non-covalent peptide inhibitor. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages.

Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP);(ii) reaction of the cyano groups withα-cysteine, penicillamine or β-cysteine to complete the lanthanoid chelating moiety;and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with Gd3+ ions.

SARS-CoV-2 Papain-Like Protease: Structure, Function and Inhibition.

Emerging variants of SARS-CoV-2 and potential novel epidemic coronaviruses underline the importance of investigating various viral proteins as potential drug targets. The papain-like protease of coronaviruses has been less explored than other viral proteins; however, its substantive role in viral replication and impact on the host immune response make it a suitable target to study. This review article focuses on the structure and function of the papain-like protease (PLpro ) of SARS-CoV-2, including variants of concern, and compares it to those of other coronaviruses, such as SARS-CoV-1 and MERS-CoV. The protease's recognition motif is mirrored in ubiquitin and ISG15, which are involved in the antiviral immune response. Inhibitors, including GRL0617 derivatives, and their prospects as potential future antiviral agents are also discussed.

Antiviral cyclic peptides targeting the main protease of SARS-CoV-2.

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir.

The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics reveal that these Mpro variants are catalytically competent to a similar degree as the wildtype. We show that nirmatrelvir has similar potency against the variants as the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.

Challenges of short substrate analogues as SARS-CoV-2 main protease inhibitors.

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.

The SARS-CoV-2 main protease as drug target.

The unprecedented pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is threatening global health. The virus emerged in late 2019 and can cause a severe disease associated with significant mortality. Several vaccine development and drug discovery campaigns are underway. The SARS-CoV-2 main protease is considered a promising drug target, as it is dissimilar to human proteases. Sequence and structure of the main protease are closely related to those from other betacoronaviruses, facilitating drug discovery attempts based on previous lead compounds. Covalently binding peptidomimetics and small molecules are investigated. Various compounds show antiviral activity in infected human cells.