The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its...

A high-throughput mass spectrometry based Mpro assay identifies penicillin esters as new SARS-CoV-2 Mpro inhibitors.

The fluorescein-labeled E166C mutant of the PenPC beta-lactamase (E166Cf) represents a successful model in the construction of "switch-on" fluorescent biosensors from nonallosteric proteins (Chan P.-H. et al.; J. Am Chem. Soc., 2004, 126, 4074). This paper focuses on the study of the biosensing mechanism by which the E166Cf biosensor changes its fluorescence upon beta-lactam binding and hydrolysis. Mass spectrometric and stopped-flow fluorescence studies of E166Cf with cefuroxime, penicillin G, and 6-aminopenicillanic acid reveal that the formation of enzyme-substrate complex enhances the fluorescence of E166Cf, and the subsequent regeneration of the free enzyme restores the weak fluorescence of E166Cf. Molecular modeling studies of E166Cf with penicillin G show that the fluorescein label is likely to share a common space with the beta-lactam and thiazolidine rings of the antibiotic in the active site. This spatial clash appears to cause the fluorescein label to move from the active site to the external aqueous environment upon substrate binding and hence experience higher water exposure. Steady-state fluorescence measurements indicate that the fluorescence of E166Cf can be enhanced by 6-aminopenicillanic acid, which consists of the beta-lactam and thiazolidine rings only. Thermal denaturation experiments of the wild-type enzyme, E166C, and E166Cf reveal that the E166C mutation is likely to increase the flexibility of the Omega-loop. This "modified" structural property might compensate for the possible steric effect of the fluorescein label on substrate binding.

An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining βlactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e., Trp105, Val120, and Leu158, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e., the OXA-48 V120L and OXA-23 V128L variants, catalyze increased βlactone formation compared to the wild-type enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show some Class D SBLs variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or not of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenems derived acylenzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBLs variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism.

The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalysing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behaviour of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.

Enzymes often use nucleophilic serine, threonine, and cysteine residues to achieve the same type of reaction; the underlying reasons for this are not understood. While bacterial d,d ‐transpeptidases (penicillin‐binding proteins) employ a nucleophilic serine, l,d ‐transpeptidases use a nucleophilic cysteine. The covalent complexes formed by l,d ‐transpeptidases with some β‐lactam antibiotics undergo non‐hydrolytic fragmentation. This is not usually observed for penicillin‐binding proteins, or for the related serine β‐lactamases. Replacement of the nucleophilic serine of serine β‐lactamases with cysteine yields enzymes which fragment β‐lactams via a similar mechanism as the l,d ‐transpeptidases, implying the different reaction outcomes are principally due to the formation of thioester versus ester intermediates. The results highlight fundamental differences in the reactivity of nucleophilic serine and cysteine enzymes, and imply new possibilities for the inhibition of nucleophilic enzymes.

The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalyzing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behavior of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.

The main protease (Mpro) of SARS-CoV-2 is central to its viral lifecycle and is a promising drug target, but little is known concerning structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of classical molecular mechanics and quantum mechanical techniques, including automated docking, molecular dynamics (MD) simulations, linear-scaling DFT, QM/MM, and interactive MD in virtual reality, to investigate the molecular features underlying recognition of the natural Mpro substrates. Analyses of the subsite interactions of modelled 11-residue cleavage site peptides, ligands from high-throughput crystallography, and designed covalently binding inhibitors were performed. Modelling studies reveal remarkable conservation of hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular at the P2/S2 sites. The binding modes of the natural substrates, together with extensive interaction analyses of inhibitor and fragment binding to Mpro, reveal new opportunities for inhibition. Building on our initial Mpro-substrate models, computational mutagenesis scanning was employed to design peptides with improved affinity and which inhibit Mpro competitively. The combined results provide new insight useful for the development of Mpro inhibitors.