The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance and characterized Förster resonance energy transfer (FRET) substrates with a preference for 2-Abz/Tyr(3-NO2) FRET pairs. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at position P5 improved the catalytic efficiency. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

The β-lactam ring represents a valuable moiety that can induce covalent binding of an inhibitor to its target. In this study, we explored di- and tripeptides with β-lactam electrophilic warheads as inhibitors of dengue and West Nile virus NS2B-NS3 protease. Tripeptides with a (3S)-β-lactam moiety displayed the highest activity, with IC50 and EC50 values in the lower micromolar range in biochemical and cellular assays. The activity against dengue protease was in general higher than against West Nile virus protease. The compounds were inactive against the off-targets thrombin and trypsin. Liquid chromatography–mass spectrometry experiments revealed that tripeptide-β-lactam inhibitors bind to the protease in two distinct binding modes. Only one binding mode leads to a covalent, but reversible, interaction of the β-lactam ring with the catalytic serine, followed by release of the inhibitor with opened β-lactam ring. The other binding mode leads to the cleavage of the peptide backbone. This observation provides the first experimental evidence that benzyloxyphenylglycine in flaviviral protease inhibitors is positioned in the prime site of the enzyme.

Two new trans-(3R,4R)-amino-β-lactam derivatives and their diastereoisomeric mixtures were synthesized as ezetimibe bioisosteres and tested in in vitro and in vivo experiments as novel β-lactam cholesterol absorption inhibitors. Both compounds exhibited low cytotoxicity in MDCKII, hNPC1L1/MDCKII, and HepG2 cell lines and potent inhibitory effect in hNPC1L1/MDCKII cells. In addition, these compounds markedly reduced cholesterol absorption in mice, resulting in reduced cholesterol concentrations in plasma, liver, and intestine. We determined the crystal structure of one amino-β-lactam derivative to establish unambiguously both the absolute and relative configuration at the new stereogenic centre C17, which was assigned to be S. The pKa values for both compounds are 9.35, implying that the amino-β-lactam derivatives and their diastereoisomeric mixtures are in form of ammonium salt in blood and the intestine. The IC50 value for the diastereoisomeric mixture is 60 μM. In vivo, it efficiently inhibited cholesterol absorption comparable to ezetimibe.

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