The mechanisms of catalysis and ligand binding for the SARS-CoV-2 NSP3 macrodomain from neutron and X-ray diffraction at room temperature (preprint)

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.

Covalent narlaprevir- and boceprevir-derived hybrid inhibitors of SARS-CoV-2 main protease: room-temperature X-ray and neutron crystallography, binding thermodynamics, and antiviral activity (preprint)

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 (Mpro) 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 Mpro/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 Mpro-catalyzed peptide bond hydrolysis. 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 revealed unconventional interactions of PF-07321332 with Mpro 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.

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

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 s-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.

Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease (preprint)

The main protease (3CL Mpro) from 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. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation 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.

Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography (preprint)

The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication, possessing an unusual catalytic dyad composed of His41 and Cys145. A long-standing question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are. Here, we present the room-temperature neutron structure of 3CL Mpro from SARS-CoV-2, which allows direct determination of hydrogen atom positions and, hence, protonation states. The catalytic site natively adopts a zwitterionic reactive state where His41 is doubly protonated and positively charged, and Cys145 is in the negatively charged thiolate state. The neutron structure also identified the protonation states of other amino acid residues, mapping electrical charges and intricate hydrogen bonding networks in the SARS-CoV-2 3CL Mpro active site cavity and dimer interface. This structure highlights the ability of neutron protein crystallography for experimentally determining protonation states at near-physiological temperature -- the critical information for structure-assisted and computational drug design.

Susceptibility of domestic swine to experimental infection with SARS-CoV-2 (preprint)

SARS-CoV-2, the agent responsible for COVID-19 has been shown to infect a number of species. The role of domestic livestock and the risk associated for humans in close contact remains unknown for many production animals. Determination of the susceptibility of pigs to SARS-CoV-2 is critical towards a One Health approach to manage the potential risk of zoonotic transmission. Here, we show pigs are susceptible to SARS-CoV-2 following oronasal inoculation. Viral RNA was detected in group oral fluids and nasal wash from at least two animals while live virus was isolated from a pig. Further, antibodies could be detected in two animals at 11 and 13 days post infection, while oral fluid samples at 6 days post inoculation indicated the presence of secreted antibodies. These data highlight the need for additional livestock assessment to determine the potential role domestic animals may contribute towards the SARS-CoV-2 pandemic.

Inhibitor binding influences the protonation states of histidines in SARS-CoV-2 main protease (preprint)

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 nu-cleophile 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 -ketoamide, display distinct His41/His164 protonation-state-dependent stabilities. While the apo and the N3-bound systems favored N{delta} (HD) and N{epsilon} (HE) protonation of His41 and His164, respectively, the -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.

Supercomputer-Based Ensemble Docking Drug Discovery Pipeline with Application to Covid-19 (preprint)

We present a supercomputer-driven pipeline for in-silico drug discovery using enhanced sampling molecular dynamics (MD) and ensemble docking. We also describe preliminary results obtained for 23 systems involving eight protein targets of the proteome of SARS CoV-2. THe MD performed is temperature replica-exchange enhanced sampling, making use of the massively parallel supercomputing on the SUMMIT supercomputer at Oak Ridge National Laboratory, with which more than 1ms of enhanced sampling MD can be generated per day. We have ensemble docked repurposing databases to ten configurations of each of the 23 SARS CoV-2 systems using AutoDock Vina. We also demonstrate that using Autodock-GPU on SUMMIT, it is possible to perform exhaustive docking of one billion compounds in under 24 hours. Finally, we discuss preliminary results and planned improvements to the pipeline, including the use of quantum mechanical (QM), machine learning, and AI methods to cluster MD trajectories and rescore docking poses.

Structural Plasticity of the SARS-CoV-2 3CL Mpro Active Site Cavity Revealed by Room Temperature X-ray Crystallography (preprint)

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.