Modelling of the mechanism of covalent adduct formation by the inhibitor O-arylcarbamate URB524 in FAAH shows that only one of the two possible inhibitor binding orientations is consistent with the experimentally observed irreversible carbamoylation of the nucleophile serine: this is a potentially crucial insight for designing new covalent inhibitors of this promising drug target.

Protein functions are dynamically regulated by allostery, which enables conformational communication even between faraway residues, and expresses itself in many forms, akin to different languages: allosteric control pathways predominating in an unperturbed protein are often unintuitively reshaped whenever biochemical perturbations arise (e.g., mutations). To accurately model allostery, unbiased molecular dynamics (MD) simulations require integration with a reliable method able to, e.g., detect incipient allosteric changes or likely perturbation pathways; this is because allostery can operate at longer time scales than those accessible by plain MD. Such methods are typically applied singularly, but we here argue their joint application─as a multilingual approach─could work significantly better. We successfully prove this through unbiased MD simulations (∼100 μs) of the widely studied, allosterically active oncotarget K-Ras4B, solvated and embedded in a phospholipid membrane, from which we decrypt allostery using four showcase languages: Distance Fluctuation analysis and the Shortest Path Map capture allosteric hotspots at equilibrium; Anisotropic Thermal Diffusion and Dynamical Non-Equilibrium MD simulations assess perturbations upon, respectively, either superheating or hydrolyzing the GTP that oncogenically activates K-Ras4B. Chosen languages work synergistically, providing an articulate, mutually coherent, experimentally consistent picture of K-Ras4B allostery, whereby distinct traits emerge at equilibrium and upon GTP cleavage. At equilibrium, combined evidence confirms prominent allosteric communication from the membrane-embedded hypervariable region, through a hub comprising helix α5 and sheet β5, and up to the active site, encompassing allosteric switches I and II (marginally), and two proposed pockets. Upon GTP cleavage, allosteric perturbations mostly accumulate on the switches and documented interfaces.

Carbamate and urea derivatives are important classes of fatty acid amide hydrolase (FAAH) inhibitors that carbamoylate the active-site nucleophile Ser241. In the present work, the reactivation mechanism of carbamoylated FAAH is investigated by means of a quantum mechanics/molecular mechanics (QM/MM) approach. The potential energy surfaces for decarbamoylation of FAAH covalent adducts, derived from the O-aryl carbamate URB597 and from the N-piperazinylurea JNJ1661610, were calculated and compared to that for deacylation of FAAH acylated by the substrate oleamide. Calculations show that a carbamic group bound to Ser241 prevents efficient stabilization of transition states of hydrolysis, leading to large increments in the activation barrier. Moreover, the energy barrier for the piperazine carboxylate was significantly lower than that for the cyclohexyl carbamate derived from URB597. This is consistent with experimental data showing slowly reversible FAAH inhibition for the N-piperazinylurea inhibitor and irreversible inhibition for URB597.

Fatty acid amide hydrolase (FAAH), a promising target for the treatment of several central and peripheral nervous system disorders, such as anxiety, pain and hypertension, has an unusual catalytic site, and its mechanism has been uncertain; hybrid quantum mechanics/molecular mechanics (QM/MM) calculations reveal a new mechanism of nucleophile activation (involving a Lys-Ser-Ser catalytic triad), with potentially crucial insights for the design of potent and selective inhibitors.

Quantum mechanics/molecular mechanics and molecular dynamics simulations of fatty acid amide hydrolase show that reaction (amide hydrolysis) occurs via a distinct, high energy conformation. This unusual finding has important implications for fatty acid amide hydrolase, a key enzyme in the endocannabinoid system. These results demonstrate the importance of structural fluctuations and the need to include them in the modeling of enzyme reactions. They also show that approaches based simply on studying enzyme-substrate complexes can be misleading for understanding biochemical reactivity.

QM/MM modelling of FAAH inactivation by O-biphenyl-3-yl carbamates identifies the deprotonation of Ser241 as the key reaction step, explaining why FAAH is insensitive to the electron-donor effect of conjugated substituents; this may aid design of new inhibitors with improved selectivity and in vivo potency.

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.