Room-temperature neutron and X-ray data collection of 3CL M<sup>pro</sup>from SARS-CoV-2

Kneller, D.W.; Phillips, G.N.; Kovalevsky, Andrey; Coates, Leighton

doi:10.1107/s2053230x20011814

Cited by 22 publications

(24 citation statements)

References 33 publications

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“…Hence, a signature of the inhibiting power of a compound is its capability to form a covalent bond with Cys145 41 , as very recently confirmed by Dai et al 42 , who have found two promising inhibitors 11a and 11b . The importance of the protonation state of Cys145 as well as the network of hydrogen bonds between the catalytic site of M pro and inhibiting compounds has also been recently discussed by Kneller et al 45 by combining X-ray and neutron scattering data. On these grounds, the experimental results obtained in the present study, together with the structure of the seven inhibitors within the M pro active site determined by the refined molecular docking, can be discussed.…”

Section: Discussionmentioning

confidence: 99%

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

Silvestrini

Belhaj

Comez

et al. 2021

Sci Rep

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The maturation of coronavirus SARS-CoV-2, which is the etiological agent at the origin of the COVID-19 pandemic, requires a main protease Mpro to cleave the virus-encoded polyproteins. Despite a wealth of experimental information already available, there is wide disagreement about the Mpro monomer-dimer equilibrium dissociation constant. Since the functional unit of Mpro is a homodimer, the detailed knowledge of the thermodynamics of this equilibrium is a key piece of information for possible therapeutic intervention, with small molecules interfering with dimerization being potential broad-spectrum antiviral drug leads. In the present study, we exploit Small Angle X-ray Scattering (SAXS) to investigate the structural features of SARS-CoV-2 Mpro in solution as a function of protein concentration and temperature. A detailed thermodynamic picture of the monomer-dimer equilibrium is derived, together with the temperature-dependent value of the dissociation constant. SAXS is also used to study how the Mpro dissociation process is affected by small inhibitors selected by virtual screening. We find that these inhibitors affect dimerization and enzymatic activity to a different extent and sometimes in an opposite way, likely due to the different molecular mechanisms underlying the two processes. The Mpro residues that emerge as key to optimize both dissociation and enzymatic activity inhibition are discussed.

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Section: Discussionmentioning

confidence: 99%

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

Silvestrini

Belhaj

Comez

et al. 2021

Sci Rep

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“…The N-terminal flanking sequence is autocleaved during the expression in Escherichia coli (BL21 DE3), whereas the C-terminal flanking sequence is removed by the treatment with human rhinovirus 3C protease (Millipore–Sigma). A detailed protocol for the enzyme expression, purification, and crystallization has been published elsewhere ( 44 ).…”

Section: Methodsmentioning

confidence: 99%

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

Kneller

Phillips

Weiss

et al. 2020

Journal of Biological Chemistry

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113

168

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The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL Mpro possesses an unusual catalytic dyad composed of Cys145 and His41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL Mpro, which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form where Cys145 is in the negatively charged thiolate state, and His41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen bonding networks in the active site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme’s electrostatic environment.

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“…A gene construct encoding 3CL M pro (NSP5) from SARS-CoV-2 was cloned into plasmid pD451-SR (Atum, Newark, CA), which was developed in (11), and expressed and purified consistent with the protocols detailed in (34). Protein purification supplies were purchased from Cytiva (Piscataway, New Jersey, USA).…”

Section: Methodsmentioning

confidence: 99%

High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

Clyde

Galanie

Kneller

et al. 2021

Preprint

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Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 uM [95% CI 2.2, 4.0]. Further, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple μs-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design.

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Room-temperature neutron and X-ray data collection of 3CL M^profrom SARS-CoV-2

Cited by 22 publications

References 33 publications

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

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Room-temperature neutron and X-ray data collection of 3CL Mprofrom SARS-CoV-2

Cited by 22 publications

References 33 publications

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

The dimer-monomer equilibrium of SARS-CoV-2 main protease is affected by small molecule inhibitors

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

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Product

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Room-temperature neutron and X-ray data collection of 3CL M^profrom SARS-CoV-2