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Simulations are revealing detailed mechanisms of biomolecular systems and functionally relevant dynamics, and contributing to enzyme design. Biomolecular simulations can be used as computational ‘assays’ of biological activity, e.g. to predict drug resistance or the effects of mutation. Molecular simulation methods of various types are now capable of modelling processes ranging from biochemical reactions to membrane dynamics, and offer increasing predictive power. Recently, this has included identifying key features of SARS-CoV-2 proteins. Molecular dynamics (MD) simulations on long timescales can model substrate binding, and reveal dynamical changes associated with thermoadaptation and directed evolution of enzyme catalytic activity. MD simulations can calculate thermodynamic properties such as activation heat capacities. Increasingly, simulations are contributing to the design and engineering of natural enzymes and de novo biocatalysts. Interactive MD simulation in virtual reality allows direct manipulation of biological macromolecules, going beyond mere visualization to allow e.g. fully flexible docking of drugs into protein targets such as the SARS-CoV-2 main protease. Groups of researchers can work together in the same virtual environment. Mechanisms of signal transduction in receptors can be studied by a combination of equilibrium and nonequilibrium MD simulations, e.g. identifying a general mechanism of signal propagation in nicotinic acetylcholine receptors. Different types of application (e.g. ranging from chemical reactions to signal transduction) require different levels of treatment, which can be combined in multiscale models to tackle a range of time- and length-scales, e.g. to study drug metabolism by cytochrome P450 enzymes combining coarse-grained and atomistic MD and QM/MM methods. By coupling together different levels of description, multiscale methods can address e.g. how chemical changes in individual molecules cause changes at larger scales. QM/MM methods are an archetype of multiscale methods in biochemistry and can be used for modelling transition states and reaction intermediates, to identify catalytic interactions, and to analyse determinants of reactivity. QM/MM modelling can identify mechanisms of covalent inhibition and predict the activity of bacterial enzymes against antibiotics. References ‘Evolution of dynamical networks enhances catalysis in a designer enzyme’ H.A. Bunzel et al. Nature Chemistry, in press (2021). https://www.biorxiv.org/content/10.1101/2020.08.21.260885v1 ‘Designing better enzymes: Insights from directed evolution’ HA Bunzel, JLR Anderson, AJ Mulholland Current Opinion in Structural Biology 67, 212-218 (2021) ‘Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics’ I. Galdadas et al. eLife 10:e66567 DOI: 10.7554/eLife.66567 (2021) ‘Mechanism of covalent binding of ibrutinib to Bruton’s tyrosine kinase revealed by QM/MM calculations’ A Voice et al. Chemical Science https://doi.org/10.1039/D0SC06122K (2021) ‘Interactive Molecular Dynamics in Virtual Reality Is an Effective Tool for Flexible Substrate and Inhibitor Docking to the SARS-CoV-2 Main Protease’ HM Deeks et al. Journal of Chemical Information and Modeling 60, 5803-5814 (2020) https://doi.org/10.1021/acs.jcim.0c01030 ‘Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein’ D.K. Shoemark et al. 133, 7174-7186 (2021) ‘Biomolecular Simulations in the Time of COVID-19, and After’ R.E. Amaro & A.J. Mulholland Computing in Science & Engineering 22, 30-36 (2020) DOI: 10.1109/MCSE.2020.3024155

Synthetic reactions often involve solvents incompatible with biocatalysts. Here, we encapsulate de novo heme-containing enzymes in calcium-alginate beads to facilitate heterogeneous biocatalysis in organic solvents. After encapsulation, enzymes remained structured and retained activity even when the beads are suspended in organic solvents. Carbene transferase activity, brought about by the heme cofactor, was enhanced when reactions were performed in organic solvent with alginate-encapsulated enzymes. Activity-solvent dependencies revealed that the activity boost is due to beneficial partitioning of the substrate between the beads and organic phase. Encapsulation furthermore facilitates enzyme recycling after the reaction. Alginate encapsulation opens up novel opportunities for biocatalysis in organic solvent systems, combining desired solvent properties of organic chemistry with enzymatic selectivity and proficiency.

Following our recent identification of a fatty acid binding site in the SARS-CoV-2 spike protein (Toelzer et al., Science eabd3255 (2020)), we investigate the binding of linoleate and other potential ligands at this site using molecular dynamics simulations. The results support the hypothesis that linoleate stabilises the locked form of the spike, in which its interaction interface for the ACE2 receptor is occluded. The simulations indicate weaker binding of linoleate to the partially open conformation. Simulations of dexamethasone bound at this site indicate that it binds similarly to linoleate, and thus may also stabilize a locked spike conformation. In contrast, simulations suggest that cholesterol bound at this site may destabilize the locked conformation, and in the open conformation, may preferentially bind at an alternative site in the hinge region between the receptor binding domain and the domain below, which could have functional relevance. We also use molecular docking to identify potential ligands that may bind at the fatty acid binding site, using the Bristol University Docking Engine (BUDE). BUDE docking successfully reproduces the linoleate complex and also supports binding of dexamethasone at the spike fatty acid site. Virtual screening of a library of approved drugs identifies vitamins D, K and A, as well as retinoid ligands with experimentally demonstrated activity against SARS-CoV-2 replication in vitro, as also potentially able to bind at this site. Our data suggest that the fatty acid binding site of the SARS-CoV-2 spike protein may bind a diverse array of candidate ligands. Targeting this site with small molecules, including dietary components such as vitamins, which may stabilise its locked conformation and represents a potential avenue for novel therapeutics or prophylaxis for COVID-19.

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., QM/MM simulations identify the mechanism of reaction of N3, a covalent peptidyl inhibitor of SARS-CoV-2 main protease. Modelling of two novel proposed compounds, B1 and B2, suggests that reversibility of covalent inhibition could be tailored.

Changeux et al. recently suggested that the SARS-CoV-2 spike (S) protein may interact with nicotinic acetylcholine receptors (nAChRs). Such interactions may be involved in pathology and infectivity. Here, we use molecular simulations of validated atomically detailed structures of nAChRs, and of the S protein, to investigate this ‘nicotinic hypothesis’. We examine the binding of the Y674-R685 loop of the S protein to three nAChRs, namely the human α4β2 and α7 subtypes and the muscle-like αβγδ receptor from Tetronarce californica. Our results indicate that Y674-R685 has affinity for nAChRs and the region responsible for binding contains the PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. In particular, R682 has a key role in the stabilisation of the complexes as it forms interactions with loops A, B and C in the receptor’s binding pocket. The conformational behaviour of the bound Y674-R685 region is highly dependent on the receptor subtype, adopting extended conformations in the α4β2 and α7 complexes and more compact ones when bound to the muscle-like receptor. In the α4β2 and αβγδ 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 where 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, but has highest affinity for the muscle-type receptor. Analyses of the simulations of the full-length S protein show that the Y674-R685 region is accessible for binding, and suggest a potential binding orientation of the S protein with nAChRs.