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1.
Chemical science ; 12(4):1513-1527, 2020.
Article in English | EuropePMC | ID: covidwho-1766761

ABSTRACT

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41–Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored Nδ (HD) and Nϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts. The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics.

2.
J Med Chem ; 64(8): 4991-5000, 2021 04 22.
Article in English | MEDLINE | ID: covidwho-1574766

ABSTRACT

The main protease (3CL Mpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 Mpro. To aid rational drug design, we determined a neutron structure of Mpro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free Mpro, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the Mpro active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.


Subject(s)
Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/chemistry , Oligopeptides/chemistry , Binding Sites , Coronavirus 3C Proteases/metabolism , Crystallography/methods , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Models, Molecular , Neutrons , Oligopeptides/metabolism , Protein Conformation , Protons
3.
IUCrJ ; 7(Pt 6)2020 Sep 21.
Article in English | MEDLINE | ID: covidwho-1546124

ABSTRACT

The emergence of the novel coronavirus SARS-CoV-2 has resulted in a worldwide pandemic not seen in generations. Creating treatments and vaccines to battle COVID-19, the disease caused by the virus, is of paramount importance in order to stop its spread and save lives. The viral main protease, 3CL Mpro, is indispensable for the replication of SARS-CoV-2 and is therefore an important target for the design of specific protease inhibitors. Detailed knowledge of the structure and function of 3CL Mpro is crucial to guide structure-aided and computational drug-design efforts. Here, the oxidation and reactivity of the cysteine residues of the protease are reported using room-temperature X-ray crystallography, revealing that the catalytic Cys145 can be trapped in the peroxysulfenic acid oxidation state at physiological pH, while the other surface cysteines remain reduced. Only Cys145 and Cys156 react with the alkylating agent N-ethylmaleimide. It is suggested that the zwitterionic Cys145-His45 catalytic dyad is the reactive species that initiates catalysis, rather than Cys145-to-His41 proton transfer via the general acid-base mechanism upon substrate binding. The structures also provide insight into the design of improved 3CL Mpro inhibitors.

4.
IUCrJ ; 8(Pt 6): 973-979, 2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1522472

ABSTRACT

SARS-CoV-2 emerged at the end of 2019 to cause an unprecedented pandemic of the deadly respiratory disease COVID-19 that continues to date. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and is therefore an important drug target. Understanding the catalytic mechanism of Mpro, a cysteine protease with a catalytic site comprising the noncanonical Cys145-His41 dyad, can help in guiding drug design. Here, a 2.0 Šresolution room-temperature X-ray crystal structure is reported of a Michaelis-like complex of Mpro harboring a single inactivating mutation C145A bound to the octapeptide Ac-SAVLQSGF-CONH2 corresponding to the nsp4/nsp5 autocleavage site. The peptide substrate is unambiguously defined in subsites S5 to S3' by strong electron density. Superposition of the Michaelis-like complex with the neutron structure of substrate-free Mpro demonstrates that the catalytic site is inherently pre-organized for catalysis prior to substrate binding. Induced fit to the substrate is driven by P1 Gln binding in the predetermined subsite S1 and rearrangement of subsite S2 to accommodate P2 Leu. The Michaelis-like complex structure is ideal for in silico modeling of the SARS-CoV-2 Mpro catalytic mechanism.

5.
J Chem Inf Model ; 62(1): 116-128, 2022 01 10.
Article in English | MEDLINE | ID: covidwho-1521685

ABSTRACT

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 noncovalent 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 µM (95% CI 2.2, 4.0). Furthermore, 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-time scale 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.


Subject(s)
COVID-19 , Protease Inhibitors , Antiviral Agents , Coronavirus 3C Proteases , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Orotic Acid/analogs & derivatives , Piperazines , SARS-CoV-2
6.
J Med Chem ; 64(23): 17366-17383, 2021 12 09.
Article in English | MEDLINE | ID: covidwho-1493002

ABSTRACT

Creating small-molecule antivirals specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins is crucial to battle coronavirus disease 2019 (COVID-19). SARS-CoV-2 main protease (Mpro) is an established drug target for the design of protease inhibitors. We performed a structure-activity relationship (SAR) study of noncovalent compounds that bind in the enzyme's substrate-binding subsites S1 and S2, revealing structural, electronic, and electrostatic determinants of these sites. The study was guided by the X-ray/neutron structure of Mpro complexed with Mcule-5948770040 (compound 1), in which protonation states were directly visualized. Virtual reality-assisted structure analysis and small-molecule building were employed to generate analogues of 1. In vitro enzyme inhibition assays and room-temperature X-ray structures demonstrated the effect of chemical modifications on Mpro inhibition, showing that (1) maintaining correct geometry of an inhibitor's P1 group is essential to preserve the hydrogen bond with the protonated His163; (2) a positively charged linker is preferred; and (3) subsite S2 prefers nonbulky modestly electronegative groups.


Subject(s)
Coronavirus 3C Proteases , Protease Inhibitors , Orotic Acid/analogs & derivatives , Piperazines , Protein Conformation , Static Electricity
7.
Chem Sci ; 12(4): 1513-1527, 2021 Jan 28.
Article in English | MEDLINE | ID: covidwho-1083334

ABSTRACT

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an attractive target for antiviral therapeutics. Recently, many high-resolution apo and inhibitor-bound structures of Mpro, a cysteine protease, have been determined, facilitating structure-based drug design. Mpro plays a central role in the viral life cycle by catalyzing the cleavage of SARS-CoV-2 polyproteins. In addition to the catalytic dyad His41-Cys145, Mpro contains multiple histidines including His163, His164, and His172. The protonation states of these histidines and the catalytic nucleophile Cys145 have been debated in previous studies of SARS-CoV Mpro, but have yet to be investigated for SARS-CoV-2. In this work we have used molecular dynamics simulations to determine the structural stability of SARS-CoV-2 Mpro as a function of the protonation assignments for these residues. We simulated both the apo and inhibitor-bound enzyme and found that the conformational stability of the binding site, bound inhibitors, and the hydrogen bond networks of Mpro are highly sensitive to these assignments. Additionally, the two inhibitors studied, the peptidomimetic N3 and an α-ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N δ (HD) and N ϵ (HE) protonation of His41 and His164, respectively, the α-ketoamide was not stably bound in this state. Our results illustrate the importance of using appropriate histidine protonation states to accurately model the structure and dynamics of SARS-CoV-2 Mpro in both the apo and inhibitor-bound states, a necessary prerequisite for drug-design efforts.

8.
Structure ; 28(12): 1313-1320.e3, 2020 12 01.
Article in English | MEDLINE | ID: covidwho-997553

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 requires rapid development of specific therapeutics and vaccines. The main protease of SARS-CoV-2, 3CL Mpro, is an established drug target for the design of inhibitors to stop the virus replication. Repurposing existing clinical drugs can offer a faster route to treatments. Here, we report on the binding mode and inhibition properties of several inhibitors using room temperature X-ray crystallography and in vitro enzyme kinetics. The enzyme active-site cavity reveals a high degree of malleability, allowing aldehyde leupeptin and hepatitis C clinical protease inhibitors (telaprevir, narlaprevir, and boceprevir) to bind and inhibit SARS-CoV-2 3CL Mpro. Narlaprevir, boceprevir, and telaprevir are low-micromolar inhibitors, whereas the binding affinity of leupeptin is substantially weaker. Repurposing hepatitis C clinical drugs as COVID-19 treatments may be a useful option to pursue. The observed malleability of the enzyme active-site cavity should be considered for the successful design of specific protease inhibitors.


Subject(s)
Antiviral Agents , Betacoronavirus , COVID-19 , Coronavirus Infections , Antiviral Agents/pharmacology , Betacoronavirus/metabolism , Catalytic Domain , Coronavirus Infections/drug therapy , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Humans , Pandemics , Protease Inhibitors/pharmacology , SARS-CoV-2 , Temperature , Viral Nonstructural Proteins
9.
Nat Commun ; 11(1): 3202, 2020 06 24.
Article in English | MEDLINE | ID: covidwho-981316

ABSTRACT

The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.


Subject(s)
Betacoronavirus/enzymology , Cysteine Endopeptidases/chemistry , Viral Nonstructural Proteins/chemistry , Catalytic Domain , Coronavirus 3C Proteases , Crystallography, X-Ray , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/metabolism , Ligands , Models, Molecular , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Domains , Protein Structure, Secondary , SARS-CoV-2 , Temperature , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/metabolism
10.
J Biol Chem ; 295(50): 17365-17373, 2020 12 11.
Article in English | MEDLINE | ID: covidwho-872797

ABSTRACT

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 in which 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.


Subject(s)
Coronavirus 3C Proteases/chemistry , Models, Molecular , Neutrons , SARS-CoV-2/genetics , Catalytic Domain , Crystallography, X-Ray
11.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 10): 483-487, 2020 Oct 01.
Article in English | MEDLINE | ID: covidwho-817571

ABSTRACT

The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro) into a series of smaller functional proteins. At the heart of 3CL Mpro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-binding cavity, and to visualize the hydrogen-bonding networks throughout the enzyme, room-temperature neutron and X-ray data were collected from a large H/D-exchanged crystal of ligand-free (apo) 3CL Mpro.


Subject(s)
Betacoronavirus/enzymology , Coronavirus Infections/virology , Cysteine Endopeptidases/chemistry , Pneumonia, Viral/virology , Viral Nonstructural Proteins/chemistry , Betacoronavirus/chemistry , Betacoronavirus/genetics , COVID-19 , Catalytic Domain , Coronavirus 3C Proteases , Crystallography, X-Ray , Cysteine Endopeptidases/genetics , Humans , Models, Molecular , Neutron Diffraction , Pandemics , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , SARS-CoV-2 , Temperature , Viral Nonstructural Proteins/genetics
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