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1.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-328785

ABSTRACT

The COVID-19 pandemic continues to disrupt everyday life, with constantly emerging SARS-CoV-2 variants threatening to render current vaccines ineffective. Small-molecule antivirals can provide an important therapeutic treatment option that is subject to challenges caused by the virus variants. The viral main protease (M pro ) is critical for the virus replication and thus is considered an attractive drug target for specific protease inhibitors. We performed the design and characterization of three reversible covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2, whose structures were derived from those of hepatitis C protease inhibitors boceprevir and narlaprevir. A joint X-ray/neutron structure of the M pro /BBH-1 complex demonstrated that a Cys145 thiolate reaction with the inhibitor’s keto-warhead creates a negatively charged oxyanion, similar to that proposed for the M pro -catalyzed peptide bond hydrolysis. Protonation states of the ionizable residues in the M pro active site adapt to the inhibitor, which appears to be an intrinsic property of M pro . Structural comparisons of the hybrid inhibitors with PF-07321332 revealed unconventional interactions of PF-07321332 with M pro which may explain its more favorable enthalpy of binding and consequently higher potency. BBH-1, BBH-2 and NBH-2 demonstrated comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

2.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327493

ABSTRACT

The NSP3 macrodomain of SARS CoV 2 (Mac1) removes ADP-ribosylation post-translational modifications, playing a key role in the immune evasion capabilities of the virus responsible for the COVID-19 pandemic. Here, we determined neutron and X-ray crystal structures of the SARS-CoV-2 NSP3 macrodomain using multiple crystal forms, temperatures, and pHs, across the apo and ADP-ribose-bound states. We characterize extensive solvation in the Mac1 active site, and visualize how water networks reorganize upon binding of ADP-ribose and non-native ligands, inspiring strategies for displacing waters to increase potency of Mac1 inhibitors. Determining the precise orientations of active site water molecules and the protonation states of key catalytic site residues by neutron crystallography suggests a catalytic mechanism for coronavirus macrodomains distinct from the substrate-assisted mechanism proposed for human MacroD2. These data provoke a re-evaluation of macrodomain catalytic mechanisms and will guide the optimization of Mac1 inhibitors.

3.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-314309

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 M pro 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 M pro , revealing the resting structure of the active site and the conformation of the catalytic site cavity. 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.

4.
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
5.
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.

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.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-291462

ABSTRACT

Direct-acting antivirals for the treatment of COVID-19, which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), are needed to complement vaccination efforts. The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral proliferation. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophilic “warheads” onto analogs of the noncovalent PLpro inhibitor GRL0617. We show that the most promising PLpro inhibitor is potent and selective, with activity in cell-based antiviral assays rivaling that of the RNA-dependent RNA polymerase inhibitor remdesivir. An X-ray crystal structure of the most promising lead compound bound covalently to PLpro establishes the molecular basis for protease inhibition and selectivity against structurally similar human deubiquitinases. These findings present an opportunity for further development of potent and selective covalent PLpro inhibitors.

8.
J Phys Chem Lett ; 12(23): 5608-5615, 2021 Jun 17.
Article in English | MEDLINE | ID: covidwho-1263456

ABSTRACT

Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.


Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , Cytokines/chemistry , Cytokines/metabolism , Interferons/metabolism , SARS-CoV-2/metabolism , Ubiquitins/chemistry , Ubiquitins/metabolism , Coronavirus Papain-Like Proteases/genetics , Cytokines/genetics , Humans , Neutron Diffraction , Protein Conformation , SARS-CoV-2/enzymology , SARS-CoV-2/genetics , Scattering, Small Angle , Ubiquitins/genetics , X-Ray Diffraction
9.
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
10.
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
11.
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
12.
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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