Biomolecular Simulations in the Time of COVID19, and After.

COVID19 has changed life for people worldwide. Despite lockdowns globally, computational research has pressed on, working remotely and collaborating virtually on research questions in COVID19 and the virus it is caused by, SARS-CoV-2. Molecular simulations can help to characterize the function of viral and host proteins and have the potential to contribute to the search for vaccines and treatments. Changes in the modus operandi of research groups include broader adoption of the use of preprint servers, earlier and more open sharing of methods, models, and data, the use of social media to rapidly disseminate information, online seminars, and cloud-based virtual collaboration. Research funders and computing providers worldwide recognized the need to provide rapid and significant access to computational architectures. In this review, we discuss how the interplay of all of these factors is influencing the impact - both potential and realized - of biomolecular simulations in the fight against SARS-CoV-2.

SARS-CoV-2 spike variants differ in their allosteric response to linoleic acid.

The SARS-CoV-2 spike protein contains a functionally important fatty acid (FA) binding site, also found in some other coronaviruses (e.g. SARS-CoV and MERS-CoV), which binds linoleic acid. When occupied by linoleic acid, it reduces infectivity, by 'locking' the spike in a less infectious conformation. Here, we use dynamical-nonequilibrium molecular dynamics (D-NEMD) simulations to compare the response of spike variants to linoleic acid removal. D-NEMD simulations show that the FA site is coupled to other, some distant, functional regions of the protein, e.g. the receptor-binding motif, N-terminal domain, furin cleavage site, and regions surrounding the fusion peptide. D-NEMD simulations also identify the allosteric networks connecting the FA site to the functional regions. Comparison of the response of the wild-type spike with four variants (Alpha, Delta, Delta plus, and Omicron BA.1) shows that the variants differ significantly in their response to linoleic acid removal. The allosteric connections to the FA site on Alpha are generally similar to those on the wild-type protein, with the exception of the receptor-binding motif and the S71-R78 region, which show a weaker link to the FA site. In contrast, Omicron is the most affected variant exhibiting significant differences in the receptor-binding motif, N-terminal domain, V622-L629, and the furin cleavage site. These differences in allosteric modulation may be of functional relevance, potentially affecting transmissibility and virulence. Experimental comparison of the effects of linoleic acid on SARS-CoV-2 variants, including emerging variants, is warranted.

#COVIDisAirborne: AI-enabled multiscale computational microscopy of delta SARS-CoV-2 in a respiratory aerosol.

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus obscure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

#COVIDisAirborne: AI-enabled multiscale computational microscopy of delta SARS-CoV-2 in a respiratory aerosol

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus obscure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.

The emerging potential of interactive virtual reality in drug discovery.

INTRODUCTION: The potential of virtual reality (VR) to contribute to drug design and development has been recognized for many years. A recent advance is to use VR not only to visualize and interact with molecules, but also to interact with molecular dynamics simulations 'on the fly' (interactive molecular dynamics in VR, IMD-VR), which is useful for flexible docking and examining binding processes and conformational changes. AREAS COVERED: The authors use the term 'interactive VR' to refer to software where interactivity is an inherent part of the user VR experience e.g. in making structural modifications or interacting with a physically rigorous molecular dynamics (MD) simulation, as opposed to using VR controllers to rotate and translate the molecule for enhanced visualization. Here, they describe these methods and their application to problems relevant to drug discovery, highlighting the possibilities that they offer in this arena. EXPERT OPINION: The ease of viewing and manipulating molecular structures and dynamics, using accessible VR hardware, and the ability to modify structures on the fly (e.g. adding or deleting atoms) - and for groups of researchers to work together in the same virtual environment - makes modern interactive VR a valuable tool to add to the armory of drug design and development methods.

Impact of Warhead Modulations on the Covalent Inhibition of SARS-CoV-2 Mpro Explored by QM/MM Simulations.

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2, SARS-CoV-2, shows the need for effective antiviral treatments. Here, we present a simulation study of the inhibition of the SARS-CoV-2 main protease (Mpro), a cysteine hydrolase essential for the life cycle of the virus. The free energy landscape for the mechanism of the inhibition process is explored by QM/MM umbrella sampling and free energy perturbation simulations at the M06-2X/MM level of theory for two proposed peptidyl covalent inhibitors that share the same recognition motif but feature distinct cysteine-targeting warheads. Regardless of the intrinsic reactivity of the modeled inhibitors, namely a Michael acceptor and a hydroxymethyl ketone activated carbonyl, our results confirm that the inhibitory process takes place by means of a two-step mechanism, in which the formation of an ion pair C145/H41 dyad precedes the protein-inhibitor covalent bond formation. The nature of this second step is strongly dependent on the functional groups in the warhead: while the nucleophilic attack of the C145 sulfur atom on the Cα of the double bond of the Michael acceptor takes place concertedly with the proton transfer from H41 to Cß, in the compound with an activated carbonyl, the sulfur attacks the carbonyl carbon concomitant with a proton transfer from H41 to the carbonyl oxygen via the hydroxyl group. An analysis of the free energy profiles, structures along the reaction path, and interactions between the inhibitors and the different pockets of the active site on the protein shows a measurable effect of the warhead on the kinetics and thermodynamics of the process. These results and QM/MM methods can be used as a guide to select warheads to design efficient irreversible and reversible inhibitors of SARS-CoV-2 Mpro.

Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2.

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

Discovery of SARS-CoV-2 Mpro peptide inhibitors from modelling substrate and ligand binding.

The main protease (Mpro) of SARS-CoV-2 is central to viral maturation and is a promising drug target, but little is known about structural aspects of how it binds to its 11 natural cleavage sites. We used biophysical and crystallographic data and an array of biomolecular simulation techniques, including automated docking, molecular dynamics (MD) and interactive MD in virtual reality, QM/MM, and linear-scaling DFT, to investigate the molecular features underlying recognition of the natural Mpro substrates. We extensively analysed the subsite interactions of modelled 11-residue cleavage site peptides, crystallographic ligands, and docked COVID Moonshot-designed covalent inhibitors. Our modelling studies reveal remarkable consistency in the hydrogen bonding patterns of the natural Mpro substrates, particularly on the N-terminal side of the scissile bond. They highlight the critical role of interactions beyond the immediate active site in recognition and catalysis, in particular plasticity at the S2 site. Building on our initial Mpro-substrate models, we used predictive saturation variation scanning (PreSaVS) to design peptides with improved affinity. Non-denaturing mass spectrometry and other biophysical analyses confirm these new and effective 'peptibitors' inhibit Mpro competitively. Our combined results provide new insights and highlight opportunities for the development of Mpro inhibitors as anti-COVID-19 drugs.

The fatty acid site is coupled to functional motifs in the SARS-CoV-2 spike protein and modulates spike allosteric behaviour.

The SARS-CoV-2 spike protein is the first contact point between the SARS-CoV-2 virus and host cells and mediates membrane fusion. Recently, a fatty acid binding site was identified in the spike (Toelzer et al. Science 2020). The presence of linoleic acid at this site modulates binding of the spike to the human ACE2 receptor, stabilizing a locked conformation of the protein. Here, dynamical-nonequilibrium molecular dynamics simulations reveal that this fatty acid site is coupled to functionally relevant regions of the spike, some of them far from the fatty acid binding pocket. Removal of a ligand from the fatty acid binding site significantly affects the dynamics of distant, functionally important regions of the spike, including the receptor-binding motif, furin cleavage site and fusion-peptide-adjacent regions. Simulations of the D614G mutant show differences in behaviour between these clinical variants of the spike: the D614G mutant shows a significantly different conformational response for some structural motifs relevant for binding and fusion. The simulations identify structural networks through which changes at the fatty acid binding site are transmitted within the protein. These communication networks significantly involve positions that are prone to mutation, indicating that observed genetic variation in the spike may alter its response to linoleate binding and associated allosteric communication.

Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein*.

We investigate binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS-CoV-2 spike protein, using docking and molecular dynamics simulations. Simulations suggest that linoleate and dexamethasone stabilize the locked spike conformation, thus reducing the opportunity for ACE2 interaction. In contrast, cholesterol may expose the receptor-binding domain by destabilizing the closed structure, preferentially binding to a different site in the hinge region of the open structure. We docked a library of FDA-approved drugs to the fatty acid site using an approach that reproduces the structure of the linoleate complex. Docking identifies steroids (including dexamethasone and vitamin D); retinoids (some known to be active in vitro, and vitamin A); and vitamin K as potential ligands that may stabilize the closed conformation. The SARS-CoV-2 spike fatty acid site may bind a diverse array of ligands, including dietary components, and therefore provides a promising target for therapeutics or prophylaxis.

Frontispiece: Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein

Drug Docking The binding of linoleate and other potential ligands to the recently discovered fatty acid binding site in the SARS-CoV-2 spike protein is investigated by James Spencer, Adrian J. Mulholland et?al. in their Research Article on page?7098.

Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity.

The SARS-CoV-2 main protease (Mpro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 Mpro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 Mpro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of Mpro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors.

Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.

Interactive Molecular Dynamics in Virtual Reality Is an Effective Tool for Flexible Substrate and Inhibitor Docking to the SARS-CoV-2 Main Protease.

The main protease (Mpro) of the SARS-CoV-2 virus is one focus of drug development efforts for COVID-19. Here, we show that interactive molecular dynamics in virtual reality (iMD-VR) is a useful and effective tool for creating Mpro complexes. We make these tools and models freely available. iMD-VR provides an immersive environment in which users can interact with MD simulations and so build protein complexes in a physically rigorous and flexible way. Recently, we have demonstrated that iMD-VR is an effective method for interactive, flexible docking of small molecule drugs into their protein targets (Deeks et al. PLoS One 2020, 15, e0228461). Here, we apply this approach to both an Mpro inhibitor and an oligopeptide substrate, using experimentally determined crystal structures. For the oligopeptide, we test against a crystallographic structure of the original SARS Mpro. Docking with iMD-VR gives models in agreement with experimentally observed (crystal) structures. The docked structures are also tested in MD simulations and found to be stable. Different protocols for iMD-VR docking are explored, e.g., with and without restraints on protein backbone, and we provide recommendations for its use. We find that it is important for the user to focus on forming binding interactions, such as hydrogen bonds, and not to rely on using simple metrics (such as RMSD), in order to create realistic, stable complexes. We also test the use of apo (uncomplexed) crystal structures for docking and find that they can give good results. This is because of the flexibility and dynamic response allowed by the physically rigorous, atomically detailed simulation approach of iMD-VR. We make our models (and interactive simulations) freely available. The software framework that we use, Narupa, is open source, and uses commodity VR hardware, so these tools are readily accessible to the wider research community working on Mpro (and other COVID-19 targets). These should be widely useful in drug development, in education applications, e.g., on viral enzyme structure and function, and in scientific communication more generally.

Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.