Inhibition mechanism of SARS-CoV-2 main protease by ebselen and its derivatives

AbstractThe global emergence of SARS-CoV-2 has triggered numerous efforts to develop therapeutic options for COVID-19 pandemic. The main protease of SARS-CoV-2 (Mpro), which is a critical enzyme for transcription and replication of SARS-CoV-2, is a key target for therapeutic development against COVID-19. An organoselenium drug called ebselen has recently been demonstrated to have strong inhibition against Mpro and antiviral activity but its molecular mode of action is unknown preventing further development. We have examined the binding modes of ebselen and its derivative in Mpro via high resolution co-crystallography and investigated their chemical reactivity via mass spectrometry. Stronger Mpro inhibition than ebselen and potent ability to rescue infected cells were observed for a number of ebselen derivatives. A free selenium atom bound with cysteine 145 of Mpro catalytic dyad has been revealed by crystallographic studies of Mpro with ebselen and MR6-31-2 suggesting hydrolysis of the enzyme bound organoselenium covalent adduct, formation of a phenolic by-product is confirmed by mass spectrometry. The target engagement of these compounds with an unprecedented mechanism of SARS-CoV-2 Mpro inhibition suggests wider therapeutic applications of organo-selenium compounds in SARS-CoV-2 and other zoonotic beta-corona viruses.

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Mechanism of Inhibition of SARS-CoV-2 Mpro by N3 Peptidyl Michael Acceptor Explained by QM/MM Simulations and Design of New Derivatives with Tunable Chemical Reactivity

10.26434/chemrxiv.12941819.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kemel Arafet ◽

Natalia Serrano-Aparicio ◽

Alessio Lodola ◽

Adrian Mulholland ◽

Florenci V. González ◽

...

Keyword(s):

Enzyme Inhibitor ◽

Chemical Reactivity ◽

Reversible Inhibitor ◽

Irreversible Inhibitor ◽

Mechanism Of Inhibition ◽

Main Protease ◽

Michael Acceptor ◽

Molecular Dynamics Methods ◽

Potential Inhibitors ◽

Drug Leads

The SARS-CoV-2 main protease (Mpro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 Mpro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 Mpro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of Mpro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

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Seleno-Functionalization of Quercetin Improves the Non-Covalent Inhibition of Mpro and Its Antiviral Activity in Cells against SARS-CoV-2

International Journal of Molecular Sciences ◽

10.3390/ijms22137048 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7048

Author(s):

Francesca Mangiavacchi ◽

Pawel Botwina ◽

Elena Menichetti ◽

Luana Bagnoli ◽

Ornelio Rosati ◽

...

Keyword(s):

Selenium Atom ◽

Quercetin Derivatives ◽

Main Protease ◽

Infected Cells ◽

Flavonoid Quercetin ◽

Flavone Derivatives ◽

Toxic Concentrations ◽

First Time

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 μM and 8 μM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.

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Mechanism of Inhibition of SARS-CoV-2 Mpro by N3 Peptidyl Michael Acceptor Explained by QM/MM Simulations and Design of New Derivatives with Tunable Chemical Reactivity

10.26434/chemrxiv.12941819 ◽

2020 ◽

Author(s):

Kemel Arafet ◽

Natalia Serrano-Aparicio ◽

Alessio Lodola ◽

Adrian Mulholland ◽

Florenci V. González ◽

...

Keyword(s):

Enzyme Inhibitor ◽

Chemical Reactivity ◽

Reversible Inhibitor ◽

Irreversible Inhibitor ◽

Mechanism Of Inhibition ◽

Main Protease ◽

Michael Acceptor ◽

Molecular Dynamics Methods ◽

Potential Inhibitors ◽

Drug Leads

The SARS-CoV-2 main protease (Mpro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 Mpro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 Mpro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of Mpro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

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Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity

Chemical Science ◽

10.1039/d0sc06195f ◽

2021 ◽

Author(s):

Kemel Arafet ◽

Natalia Serrano-Aparicio ◽

Alessio Lodola ◽

Adrian J. Mulholland ◽

Florenci V. González ◽

...

Keyword(s):

Chemical Reactivity ◽

Covalent Inhibition ◽

Mechanism Of Inhibition ◽

Main Protease ◽

Mechanism Of Reaction ◽

Michael Acceptor

QM/MM simulations identify the mechanism of reaction of N3, a covalent peptidyl inhibitor of SARS-CoV-2 main protease. Modelling of two novel proposed compounds, B1 and B2, suggests that reversibility of covalent inhibition could be tailored.

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On the Adsorption of Aspartate Derivatives to Calcite Surfaces in Aqueous Environment

10.26434/chemrxiv.11558430.v1 ◽

2020 ◽

Author(s):

Robert Stepic ◽

Lara Jurković ◽

Ksenia Klementyeva ◽

Marko Ukrainczyk ◽

Matija Gredičak ◽

...

Keyword(s):

Active Sites ◽

Realistic Model ◽

Binding Energies ◽

Inhibition Mechanism ◽

Aqueous Environment ◽

Binding Modes ◽

Carboxylate Groups ◽

Dissolved Ions ◽

Living Organisms ◽

Dynamics Simulations

In many living organisms, biomolecules interact favorably with various surfaces of calcium carbonate. In this work, we have considered the interactions of aspartate (Asp) derivatives, as models of complex biomolecules, with calcite. Using kinetic growth experiments, we have investigated the inhibition of calcite growth by Asp, Asp2 and Asp3.This entailed the determination of a step-pinning growth regime as well as the evaluation of the adsorption constants and binding free energies for the three species to calcite crystals. These latter values are compared to free energy profiles obtained from fully atomistic molecular dynamics simulations. When using a flat (104) calcite surface in the models, the measured trend of binding energies is poorly reproduced. However, a more realistic model comprised of a surface with an island containing edges and corners, yields binding energies that compare very well with experiments. Surprisingly, we find that most binding modes involve the positively charged, ammonium group. Moreover, while attachment of the negatively charged carboxylate groups is also frequently observed, it is always balanced by the aqueous solvation of an equal or greater number of carboxylates. These effects are observed on all calcite features including edges and corners, the latter being associated with dominant affinities to Asp derivatives. As these features are also precisely the active sites for crystal growth, the experimental and theoretical results point strongly to a growth inhibition mechanism whereby these sites become blocked, preventing further attachment of dissolved ions and halting further growth.

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Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽

10.3390/molecules26123732 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3732

Author(s):

Agnieszka Dabrowska ◽

Aleksandra Milewska ◽

Joanna Ner-Kluza ◽

Piotr Suder ◽

Krzysztof Pyrc

Keyword(s):

Mass Spectrometry ◽

Zika Virus ◽

Viral Infections ◽

Protein Detection ◽

Extensive Discussion ◽

Protein Targets ◽

Highly Sensitive ◽

Infected Cells ◽

Ns3 Protease ◽

To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

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Atlas der SARS-CoV-2-RNA-Protein-Interaktionen in infizierten Zellen

BIOspektrum ◽

10.1007/s12268-021-1587-3 ◽

2021 ◽

Vol 27 (4) ◽

pp. 376-379

Author(s):

Nora Schmidt ◽

Mathias Munschauer

Keyword(s):

Mass Spectrometry ◽

Viral Rna ◽

Defense Strategies ◽

Pharmacological Inhibition ◽

Novel Therapeutics ◽

Contact Sites ◽

Human Proteins ◽

Infected Cells

AbstractUsing RNA antisense purification and mass spectrometry, we identified more than 100 human proteins that directly and specifically bind SARS-CoV-2 RNA in infected cells. To gain insights into the functions of selected RNA interactors, we applied genetic perturbation and pharmacological inhibition experiments, and mapped the contact sites on the viral RNA. This led to the identification of host dependency factors and defense strategies, which can guide the design of novel therapeutics against SARS-CoV-2.

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Allosteric inhibition of the SARS-CoV-2 main protease – insights from mass spectrometry-based assays

10.1101/2020.07.29.226761 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tarick J. El-Baba ◽

Corinne A. Lutomski ◽

Anastassia L. Kantsadi ◽

Tika R. Malla ◽

Tobias John ◽

...

Keyword(s):

Mass Spectrometry ◽

Native Mass Spectrometry ◽

Turnover Rates ◽

Kinetic Assay ◽

Antiviral Therapies ◽

Enzyme Substrate ◽

Main Protease ◽

Starting Point ◽

Catalytically Active ◽

Substrate Processing

AbstractFollowing translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 ± 0.03 μM, revealing MPro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively “flash-frozen” as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

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Crystal structure of SARS-CoV-2 main protease in complex with a Chinese herb inhibitor shikonin

10.1101/2020.06.16.155812 ◽

2020 ◽

Author(s):

Jian Li ◽

Xuelan Zhou ◽

Yan Zhang ◽

Fanglin Zhong ◽

Cheng Lin ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Drug Targets ◽

Chinese Herb ◽

Binding Modes ◽

High Potency ◽

Main Protease ◽

Covalent Inhibitors ◽

Catalytic Dyad ◽

Important Drug

AbstractMain protease (Mpro, also known as 3CLpro) has a major role in the replication of coronavirus life cycle and is one of the most important drug targets for anticoronavirus agents. Here we report the crystal structure of main protease of SARS-CoV-2 bound to a previously identified Chinese herb inhibitor shikonin at 2.45 angstrom resolution. Although the structure revealed here shares similar overall structure with other published structures, there are several key differences which highlight potential features that could be exploited. The catalytic dyad His41-Cys145 undergoes dramatic conformational changes, and the structure reveals an unusual arrangement of oxyanion loop stabilized by the substrate. Binding to shikonin and binding of covalent inhibitors show different binding modes, suggesting a diversity in inhibitor binding. As we learn more about different binding modes and their structure-function relationships, it is probable that we can design more effective and specific drugs with high potency that can serve as effect SARS-CoV-2 anti-viral agents.

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