Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

The Streptococcus pyogenes cell envelope protease (SpyCEP) is a vital virulence factor in streptococcal pathogenesis. Despite its key role in disease progression and strong association with invasive disease, little is known about the enzymatic function beyond the ELR+ CXC chemokine substrate range. We utilised multiple SpyCEP constructs to interrogate the protein domains and catalytic residues necessary for enzyme function. We leveraged high-throughput mass spectrometry to describe the Michaelis-Menton parameters of active SpyCEP, revealing a Michaelis-Menton constant (KM) of 53.49 nM and a turnover of 1.34 molecules per second, for the natural chemokine substrate CXCL8. Unexpectedly, we found that an N-terminally-truncated SpyCEP C-terminal construct consisting of only the H279 and S617 catalytic dyad had specific CXCL8 cleaving activity, albeit with a reduced substrate turnover of 2.45 molecules per hour, representing a ~2000-fold reduction in activity. In contrast, the KM of the C-terminal SpyCEP construct and full-length enzyme did not differ. We conclude that the SpyCEP C-terminus plays a key role in substrate binding and recognition with key implications for both current and future streptococcal vaccine designs.

Exploring the Binding Mechanism of PF-07321332 SARS-CoV-2 Protease Inhibitor through Molecular Dynamics and Binding Free Energy Simulations

The novel coronavirus disease, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), rapidly spreading around the world, poses a major threat to the global public health. Herein, we demonstrated the binding mechanism of PF-07321332, α-ketoamide, lopinavir, and ritonavir to the coronavirus 3-chymotrypsin-like-protease (3CLpro) by means of docking and molecular dynamic (MD) simulations. The analysis of MD trajectories of 3CLpro with PF-07321332, α-ketoamide, lopinavir, and ritonavir revealed that 3CLpro–PF-07321332 and 3CLpro–α-ketoamide complexes remained stable compared with 3CLpro–ritonavir and 3CLpro–lopinavir. Investigating the dynamic behavior of ligand–protein interaction, ligands PF-07321332 and α-ketoamide showed stronger bonding via making interactions with catalytic dyad residues His41–Cys145 of 3CLpro. Lopinavir and ritonavir were unable to disrupt the catalytic dyad, as illustrated by increased bond length during the MD simulation. To decipher the ligand binding mode and affinity, ligand interactions with SARS-CoV-2 proteases and binding energy were calculated. The binding energy of the bespoke antiviral PF-07321332 clinical candidate was two times higher than that of α-ketoamide and three times than that of lopinavir and ritonavir. Our study elucidated in detail the binding mechanism of the potent PF-07321332 to 3CLpro along with the low potency of lopinavir and ritonavir due to weak binding affinity demonstrated by the binding energy data. This study will be helpful for the development and optimization of more specific compounds to combat coronavirus disease.

Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease

Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145–His41 catalytic dyad. We here characterized the catalytic roles of the cysteine–histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine–histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

The temperature-dependent conformational ensemble of SARS-CoV-2 main protease (Mpro)

The COVID-19 pandemic, instigated by the SARS-CoV-2 coronavirus, continues to plague the globe. The SARS-CoV-2 main protease, or Mpro, is a promising target for development of novel antiviral therapeutics. Previous X-ray crystal structures of Mpro were obtained at cryogenic temperature or room temperature only. Here we report a series of high-resolution crystal structures of unliganded Mpro across multiple temperatures from cryogenic to physiological, and another at high humidity. We interrogate these datasets with parsimonious multiconformer models, multi-copy ensemble models, and isomorphous difference density maps. Our analysis reveals a temperature-dependent conformational landscape for Mpro, including a mobile water interleaved between the catalytic dyad, mercurial conformational heterogeneity in a key substrate-binding loop, and a far-reaching intramolecular network bridging the active site and dimer interface. Our results may inspire new strategies for antiviral drug development to counter-punch COVID-19.

Rutin Is a Low Micromolar Inhibitor of SARS-CoV-2 Main Protease 3CLpro: Implications for Drug Design of Quercetin Analogs

The pandemic, due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has stimulated the search for antivirals to tackle COVID-19 infection. Molecules with known pharmacokinetics and already approved for human use have been demonstrated or predicted to be suitable to be used either directly or as a base for a scaffold-based drug design. Among these substances, quercetin is known to be a potent in vitro inhibitor of 3CLpro, the SARS-CoV-2 main protease. However, its low in vivo bioavailability calls for modifications to its molecular structure. In this work, this issue is addressed by using rutin, a natural flavonoid that is the most common glycosylated conjugate of quercetin, as a model. Combining experimental (spectroscopy and calorimetry) and simulation techniques (docking and molecular dynamics simulations), we demonstrate that the sugar adduct does not hamper rutin binding to 3CLpro, and the conjugated compound preserves a high potency (inhibition constant in the low micromolar range, Ki = 11 μM). Although showing a disruption of the pseudo-symmetry in the chemical structure, a larger steric volume and molecular weight, and a higher solubility compared to quercetin, rutin is able to associate in the active site of 3CLpro, interacting with the catalytic dyad (His41/Cys145). The overall results have implications in the drug-design of quercetin analogs, and possibly other antivirals, to target the catalytic site of the SARS-CoV-2 3CLpro.

Identification of Potential Mpro Inhibitors for the Treatment of COVID-19 by Targeted Covalent Inhibition

A novel coronavirus is the causative agent identified for the current COVID-19 outbreak. Globally, more than 43 million people have been infected by this virus. The total number of deaths has surpassed 1.6 million across 210 countries due to the current pandemic. Till date, there is no specific therapeutic agent available for its treatment. Mpro, a non-structural protein cleaves viral polyproteins into other non-structural proteins. Inhibition of Mpro could prevent the virus replication projecting it as a potential candidate for anti-COVID-19 drug development. The authors report herein 10 top-ranked curcumin derivatives as non-peptide covalent-binding Mpro inhibitors using systematic virtual screening approach. Detailed ligand-receptor interaction analysis conferred that the α,β-unsaturated carbonyl moiety of curcumin functions as a warhead to yield a Michael adduct with Cys145 of the catalytic dyad of Mpro. Collectively, these results have offered new high affinity molecules for the development of potential drugs for the treatment of COVID-19.

Asymmetry in catalysis: ‘unidirectional’ amino acid racemases

d-Amino acids play widespread structural, functional and regulatory roles in organisms. These d-amino acids often arise through the stereoinversion of the more plentiful l-amino acids catalysed by amino acid racemases and epimerases. Such enzymes are of interest since many are recognized targets for the development of drugs or may be employed industrially in biotransformation reactions. Despite their enzyme–substrate complexes being diastereomers, some racemases and epimerases exhibit a kinetic pseudo-symmetry, binding their enantiomeric or epimeric substrate pairs with roughly equal affinities and catalyzing their stereoinversion with similar turnover numbers. In other cases, this kinetic pseudo-symmetry is absent or may be ‘broken’ by substitution of a catalytic Cys by Ser at the active site of cofactor-independent racemases and epimerases, or by altering the Brønsted base of the catalytic dyad that facilitates deprotonation of the Cys residue. Moreover, a natural Thr-containing l-Asp/Glu racemase was discovered that catalyses ‘unidirectional’ substrate turnover, unlike the typical bidirectional racemases and epimerases. These observations suggest that bidirectional Cys–Cys racemases may be re-engineered into ‘unidirectional’ racemases through substitution of the thiol by a hydroxyl group. Catalysis by such ‘unidirectional’ racemase precursors could then be optimized further by site-directed mutagenesis and directed evolution to furnish useful enzymes for biotechnological applications.