Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor Mpro. We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains Mpro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall Mpro inhibition and the fraction of uncleaved precursor form of Mpro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature Mpro. Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature Mpro, indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of Mpro for inhibition or premature activation of Mpro.

• Klíčová slova
• Förster resonance energy transfer (FRET), SARS-CoV-2 main protease, activation, autoprocessing, cell-based assay, fluorescence cross-correlation spectroscopy (FCCS), fluorescence life-time imaging, inhibitor, maturation, nsp5, precursor, protease, virus,
• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• COVID-19 virologie   MeSH
• farmakoterapie COVID-19 * MeSH
• HEK293 buňky MeSH
• inhibitory proteas * farmakologie   chemie   MeSH
• koronavirové proteasy 3C * metabolismus   genetika   antagonisté a inhibitory   chemie   MeSH
• lidé MeSH
• mutace MeSH
• objevování léků * MeSH
• SARS-CoV-2 * enzymologie   účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
• antivirové látky * MeSH
• inhibitory proteas * MeSH
• koronavirové proteasy 3C * MeSH

The SARS-CoV-2 virus is highly contagious to humans and has caused a pandemic of global proportions. Despite worldwide research efforts, efficient targeted therapies against the virus are still lacking. With the ready availability of the macromolecular structures of coronavirus and its known variants, the search for anti-SARS-CoV-2 therapeutics through in silico analysis has become a highly promising field of research. In this study, we investigate the inhibiting potentialities of triazole-based compounds against the SARS-CoV-2 main protease (Mpro). The SARS-CoV-2 main protease (Mpro) is known to play a prominent role in the processing of polyproteins that are translated from the viral RNA. Compounds were pre-screened from 171 candidates (collected from the DrugBank database). The results showed that four candidates (Bemcentinib, Bisoctrizole, PYIITM, and NIPFC) had high binding affinity values and had the potential to interrupt the main protease (Mpro) activities of the SARS-CoV-2 virus. The pharmacokinetic parameters of these candidates were assessed and through molecular dynamic (MD) simulation their stability, interaction, and conformation were analyzed. In summary, this study identified the most suitable compounds for targeting Mpro, and we recommend using these compounds as potential drug molecules against SARS-CoV-2 after follow up studies.

• Klíčová slova
• MD simulation, SARS-CoV-2, docking, drug, main protease, triazole,
• MeSH
• antivirové látky chemie   metabolismus   terapeutické užití   MeSH
• benzocyklohepteny chemie   metabolismus   MeSH
• chemické databáze MeSH
• COVID-19 virologie   MeSH
• farmakoterapie COVID-19 MeSH
• inhibitory proteas chemie   metabolismus   terapeutické užití   MeSH
• koronavirové proteasy 3C antagonisté a inhibitory   metabolismus   MeSH
• kvantitativní vztahy mezi strukturou a aktivitou MeSH
• lidé MeSH
• ligandy MeSH
• poločas MeSH
• SARS-CoV-2 enzymologie   izolace a purifikace   MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• triazoly chemie   metabolismus   terapeutické užití   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3C-like proteinase, SARS-CoV-2 MeSH Prohlížeč
• antivirové látky MeSH
• bemcentinib MeSH Prohlížeč
• benzocyklohepteny MeSH
• inhibitory proteas MeSH
• koronavirové proteasy 3C MeSH
• ligandy MeSH
• triazoly MeSH