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.

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.

Highlights d X-ray structures of SARS-CoV-2 3CL M pro -inhibitor complexes at room temperature d Telaprevir, narlaprevir, and boceprevir bind and efficiently inhibit the enzyme d 3CL M pro active-site cavity is malleable, accommodating large inhibitors d Hepatitis C clinical protease inhibitors can be repurposed to treat COVID-19

Emerging SARS-CoV-2 variants continue to threaten the effectiveness of COVID-19 vaccines, and small-molecule antivirals can provide an important therapeutic treatment option. The viral main protease (Mpro) is critical for virus replication and thus is considered an attractive drug target. We performed the design and characterization of three covalent hybrid inhibitors BBH-1, BBH-2 and NBH-2 created by splicing components of hepatitis C protease inhibitors boceprevir and narlaprevir, and known SARS-CoV-1 protease inhibitors. A joint X-ray/neutron structure of the Mpro/BBH-1 complex demonstrates that a Cys145 thiolate reaction with the inhibitor’s keto-warhead creates a negatively charged oxyanion. Protonation states of the ionizable residues in the Mpro active site adapt to the inhibitor, which appears to be an intrinsic property of Mpro. Structural comparisons of the hybrid inhibitors with PF-07321332 reveal unconventional F···O interactions of PF-07321332 with Mpro which may explain its more favorable enthalpy of binding. BBH-1, BBH-2 and NBH-2 exhibit comparable antiviral properties in vitro relative to PF-07321332, making them good candidates for further design of improved antivirals.

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.

The main protease (3CL M pro ) 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 M pro . To aid rational drug design, we determined a neutron structure of M pro 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 M pro , 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 M pro 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 M pro .

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 (M pro ) 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 M pro 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 M pro 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.

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 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.