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 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.

Some of the improvements inSHELX2013makeSHELXLconvenient to use for refinement of macromolecular structures against neutron data without the support of X-ray data. The new NEUT instruction adjusts the behaviour of the SFAC instruction as well as the default bond lengths of the AFIX instructions. This work presents a protocol on how to useSHELXLfor refinement of protein structures against neutron data. It includes restraints extending the Engh & Huber [Acta Cryst.(1991), A47, 392–400] restraints to H atoms and discusses several of the features ofSHELXLthat make the program particularly useful for the investigation of H atoms with neutron diffraction.SHELXL2013is already adequate for the refinement of small molecules against neutron data, but there is still room for improvement, like the introduction of chain IDs for the refinement of macromolecular structures.

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 (M^pro) 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 M^pro inhibitor with an inhibition constant (K_i) 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 M^pro 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 M^pro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits M^pro and offers a springboard for further therapeutic design.

The main protease (3CL M^pro) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL M^pro 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 M^pro, 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.