The Role of Lysozyme in the Formation of Bioinspired Silicon Dioxide.

Several organisms are able to polycondensate tetraoxosilicic(IV) acid to form silicon(IV) dioxide using polycationic molecules. According to an earlier mechanistic proposal, these molecules undergo a phase separation and recent experimental evidence appears to confirm this model. At the same time, polycationic proteins like lysozyme can also promote polycondensation of silicon(IV) dioxide, and they do so under conditions that are not compatible with liquid-liquid phase separation. In this manuscript we investigate this conundrum by molecular simulations.

Identification of Potential Inhibitors of the SARS-CoV-2 NSP13 Helicase via Structure-Based Ligand Design, Molecular Docking and Nonequilibrium Alchemical Simulations.

We have assembled a computational pipeline based on virtual screening, docking techniques, and nonequilibrium molecular dynamics simulations, with the goal of identifying possible inhibitors of the SARS-CoV-2 NSP13 helicase, catalyzing by ATP hydrolysis the unwinding of double or single-stranded RNA in the viral replication process inside the host cell. The druggable sites for broad-spectrum inhibitors are represented by the RNA binding sites at the 5' entrance and 3' exit of the central channel, a structural motif that is highly conserved across coronaviruses. Potential binders were first generated using structure-based ligand techniques. Their potency was estimated by using four popular docking scoring functions. Common docking hits for NSP13 were finally tested using advanced nonequilibrium alchemical techniques for binding free energy calculations on a high-performing parallel cluster. Four potential NSP13 inhibitors with potency from submicrimolar to nanomolar were finally identified.

Upgrading of the general AMBER force field 2 for fluorinated alcohol biosolvents: A validation for water solutions and melittin solvation.

The standard GAFF2 force field parameterization has been refined for the fluorinated alcohols 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 1,1,1,3,3,3-hexafluoropropan-2-one (HFA), which are commonly used to study proteins and peptides in biomimetic media. The structural and dynamic properties of both proteins and peptides are significantly influenced by the biomimetic environment created by the presence of these cosolvents in aqueous solutions. Quantum mechanical calculations on stable conformers were used to parameterize the atomic charges. Different systems, such as pure liquids, aqueous solutions, and systems formed by melittin protein and cosolvent/water solutions, have been used to validate the new models. The calculated macroscopic and structural properties are in agreement with experimental findings, supporting the validity of the newly proposed models.

NE-RDFE: A protocol and toolkit for computing relative dissociation free energies with GROMACS between dissimilar molecules using bidirectional nonequilibrium dual topology schemes.

We describe a step-by-step protocol and toolkit for the computation of the relative dissociation free energy (RDFE) with the GROMACS molecular dynamics package, based on a novel bidirectional nonequilibrium alchemical approach. The proposed methodology does not require any intervention on the code and allows computing with good accuracy the RDFE between small molecules with arbitrary differences in volume, charge, and chemical topology. The procedure is illustrated for the challenging SAMPL9 batch of host-guest pairs. The article is supplemented by a detailed online tutorial, available at https://procacci.github.io/vdssb_gromacs/NE-RDFE and by a public Zenodo repository available at https://zenodo.org/record/6982932.

2-Butanol Aqueous Solutions: A Combined Molecular Dynamics and Small/Wide-Angle X-ray Scattering Study.

Structural properties of 2-butanol aqueous solutions at different concentrations have been studied using small- and wide-angle X-ray scattering and molecular dynamics simulations. The experimental structure factors have been accurately reproduced by the simulations, allowing one to explain their variation with concentration and to achieve a detailed description of the structural and dynamic properties of the studied systems. The analysis of experimental and computational data has shown that 2-butanol, the simplest aliphatic chiral alcohol, tends to form aggregates at a concentration above 1 M, affecting also both the structural and dynamic properties of the solvent.

Probing Liquid-Ordered and Disordered Phases in Lipid Model Membranes: A Combined Theoretical and Spectroscopic Study of a Fluorescent Molecular Rotor.

An integrated theoretical/experimental strategy has been applied to the study of environmental effects on the spectroscopic parameters of 4-(diphenylamino)phtalonitrile (DPAP), a fluorescent molecular rotor. The computational part starts from the development of an effective force field for the first excited electronic state of DPAP and proceeds through molecular dynamics simulations in solvents of different polarities toward the evaluation of Stokes shifts by quantum mechanics/molecular mechanics (QM/MM) approaches. The trends of the computed results closely parallel the available experimental results thus giving confidence to the interpretation of new experimental studies of the photophysics of DPAP in lipid bilayers. In this context, results show unambiguously that both flexible dihedral angles and global rotations are significantly retarded in a cholesterol/DPPC lipid matrix with respect to the DOPC matrix, thus confirming the sensitivity of DPAP to probe different environments and, therefore, its applicability as a probe for detecting different structures and levels of plasma membrane organization.

Characterization of the non-covalent interaction between the PF-07321332 inhibitor and the SARS-CoV-2 main protease.

We have studied the non-covalent interaction between PF-07321332 and SARS-CoV-2 main protease at the atomic level using a computational approach based on extensive molecular dynamics simulations with explicit solvent. PF-07321332, whose chemical structure has been recently disclosed, is a promising oral antiviral clinical candidate with well-established anti-SARS-CoV-2 activity in vitro. The drug, currently in phase III clinical trials in combination with ritonavir, relies on the electrophilic attack of a nitrile warhead to the catalytic cysteine of the protease. Nonbonded interaction between the inhibitor and the residues of the binding pocket, as well as with water molecules on the protein surface, have been characterized using two different force fields and the two possible protonation states of the main protease catalytic dyad HIS41-CYS145. When the catalytic dyad is in the neutral state, the non-covalent binding is likely to be stronger. Molecular dynamics simulations seems to lend support for an inhibitory mechanism in two steps: a first non-covalent addition with the dyad in neutral form and then the formation of the thiolate-imidazolium ion pair and the ligand relocation for finalising the electrophilic attack.

Virtual Double-System Single-Box for Absolute Dissociation Free Energy Calculations in GROMACS.

We describe a step-by-step protocol for the computation of absolute dissociation free energy with GROMACS code and PLUMED library, which exploits a combination of advanced sampling techniques and nonequilibrium alchemical methodologies. The computational protocol has been automated through an open source Python middleware (HPC_Drug) which allows one to set up the GROMACS/PLUMED input files for execution on high performing computing facilities. The proposed protocol, by exploiting its inherent parallelism and the power of the GROMACS code on graphical processing units, has the potential to afford accurate and precise estimates of the dissociation constants in drug-receptor systems described at the atomistic level. The procedure has been applied to the calculation of the absolute dissociation free energy of PF-07321332, an oral antiviral proposed by Pfizer, with the main protease (3CLpro) of SARS-CoV-2.

On the NS-DSSB unidirectional estimates in the SAMPL6 SAMPLing challenge.

In the context of the recent SAMPL6 SAMPLing challenge (Rizzi et al. 2020 in J Comput Aided Mol Des 34:601-633) aimed at assessing convergence properties and reproducibility of molecular dynamics binding free energy methodologies, we propose a simple explanation of the severe errors observed in the nonequilibrium switch double-system-single-box (NS-DSSB) approach when using unidirectional estimates. At the same time, we suggest a straightforward and minimal modification of the NS-DSSB protocol for obtaining reliable unidirectional estimates for the process where the ligand is decoupled in the bound state and recoupled in the bulk.

Adsorption Geometry of Alizarin on Silver Nanoparticles: A Computational and Spectroscopic Study.

The knowledge of the adsorption geometry of an analyte on a metal substrate employed in surface enhanced Raman scattering (SERS) spectroscopy is important information for the correct interpretation of experimental data. The adsorption geometry of alizarin on silver nanoparticles was studied through ab initio calculations in the framework of density functional theory (DFT) by modeling alizarin taking into account all the different charged species present in solution as a function of pH. The calculations allowed a faithful reproduction of the measured SERS spectra and to elucidate the adsorption geometry of this dye on the silver substrate.

Mycotoxins aptasensing: From molecular docking to electrochemical detection of deoxynivalenol.

This work proposes a voltammetric aptasensor to detect deoxynivalenol (DON) mycotoxin. The development steps of the aptasensor were partnered for the first time to a computational study to gain insights onto the molecular mechanisms involved into the interaction between a thiol-tethered DNA aptamer (80mer-SH) and DON. The exploited docking study allowed to find the binding region of the oligonucleotide sequence and to determine DON preferred orientation. A biotinylated oligonucleotide sequence (20mer-BIO) complementary to the aptamer was chosen to carry out a competitive format. Graphite screen-printed electrodes (GSPEs) were electrochemically modified with polyaniline and gold nanoparticles (AuNPs@PANI) by means of cyclic voltammetry (CV) and worked as a scaffold for the immobilization of the DNA aptamer. Solutions containing increasing concentrations of DON and a fixed amount of 20mer-BIO were dropped onto the aptasensor surface: the resulting hybrids were labeled with an alkaline phosphatase (ALP) conjugate to hydrolyze 1-naphthyl phosphate (1-NPP) substrate into 1-naphthol product, detected by differential pulse voltammetry (DPV). According to its competitive format, the aptasensor response was signal-off in the range 5.0-30.0 ng·mL-1 DON. A detection limit of 3.2 ng·mL-1 was achieved within a 1-hour detection time. Preliminary experiments on maize flour samples spiked with DON yielded good recovery values.

Virtual Double-System Single-Box: A Nonequilibrium Alchemical Technique for Absolute Binding Free Energy Calculations: Application to Ligands of the SARS-CoV-2 Main Protease.

In the context of drug-receptor binding affinity calculations using molecular dynamics techniques, we implemented a combination of Hamiltonian replica exchange (HREM) and a novel nonequilibrium alchemical methodology, called virtual double-system single-box, with increased accuracy, precision, and efficiency with respect to the standard nonequilibrium approaches. The method has been applied for the determination of absolute binding free energies of 16 newly designed noncovalent ligands of the main protease (3CLpro) of SARS-CoV-2. The core structures of 3CLpro ligands were previously identified using a multimodal structure-based ligand design in combination with docking techniques. The calculated binding free energies for four additional ligands with known activity (either for SARS-CoV or SARS-CoV-2 main protease) are also reported. The nature of binding in the 3CLpro active site and the involved residues besides the CYS-HYS catalytic dyad have been thoroughly characterized by enhanced sampling simulations of the bound state. We have identified several noncongeneric compounds with predicted low micromolar activity for 3CLpro inhibition, which may constitute possible lead compounds for the development of antiviral agents in Covid-19 treatment.

Interaction of hydroxychloroquine with SARS-CoV2 functional proteins using all-atoms non-equilibrium alchemical simulations.

Using a combination of enhanced sampling molecular dynamics techniques and non-equilibrium alchemical transformations with full atomistic details, we have shown that hydroxychloroquine (HCQ) may act as a mild inhibitor of important functional proteins for SARS-CoV2 replication, with potency increasing in the series PLpro, 3CLpro, RdRp. By analyzing the bound state configurations, we were able to improve the potency for the 3CLpro target, designing a novel HCQ-inspired compound, named PMP329, with predicted nanomolar activity. If confirmed in vitro, our results provide a molecular rationale for the use of HCQ or of strictly related derivatives in the treatment of Covid-19.

Upgraded AMBER Force Field for Zinc-Binding Residues and Ligands for Predicting Structural Properties and Binding Affinities in Zinc-Proteins.

We developed a novel force field in the context of AMBER parameterization for glutamate and aspartate zinc(II)-binding residues. The interaction between the zinc ion and the coordinating atoms is represented by a spherical nonbonded parameterization. The polarization effect due to the zinc ion has been taken into account by redefining the atomic charges on the residues through accurate quantum mechanical calculations. The new zinc-binding ASP and GLU residues, along with the CYS and HIS zinc-binding residues, parameterized in a recent work [Macchiagodena M.;J. Chem. Inf. Model.2019, 59, 3803-3816], allow users to reliably simulate 96% of the Zn-proteins available in the Protein Data Bank. The upgraded force field for zinc(II)-bound residues has been tested performing molecular dynamics simulations with an explicit solvent and comparing the structural information with experimental data for five different proteins binding zinc(II) with GLU, ASP, HIS, and CYS. We further validated our approach by evaluating the binding free energy of (R)-2-benzyl-3-nitropropanoic acid to carboxypeptidase A using a recently developed nonequilibrium alchemical method. We demonstrated that in this setting it is crucial to take into account polarization effects also on the metal-bound inhibitor.

Identification of potential binders of the main protease 3CLpro of the COVID-19 via structure-based ligand design and molecular modeling.

We have applied a computational strategy, using a combination of virtual screening, docking and molecular dynamics techniques, aimed at identifying possible lead compounds for the non-covalent inhibition of the main protease 3CLpro of the SARS-CoV2 Coronavirus. Based on the X-ray structure (PDB code: 6LU7), ligands were generated using a multimodal structure-based design and then docked to the monomer in the active state. Docking calculations show that ligand-binding is strikingly similar in SARS-CoV and SARS-CoV2 main proteases. The most potent docked ligands are found to share a common binding pattern with aromatic moieties connected by rotatable bonds in a pseudo-linear arrangement.

Evidence of a Low-High Density Turning Point in Liquid Water at Ordinary Temperature under Pressure: A Molecular Dynamics Study.

Water has a fundamental role in important processes spanning a wide range of pressure and temperature conditions. Knowledge of structural, dynamic and thermodynamic properties of water at nonstandard conditions is a primary concern since interest in astronomical, geological, and technological processes is continuously growing. Molecular dynamics simulations allow us to study thermodynamic conditions that require sophisticated techniques and instruments, while at the same time offering the interpretation of properties at the atomic level. It is established that the behavior of water is strongly affected by the temperature and pressure conditions, determining the existence of low and high density regimes. For the first time, a thermodynamic property, isothermal compressibility, has been adopted to detect the low-high density turning point at ambient temperature in liquid water due to pressure. Molecular dynamics simulations have been performed with five three-site models, allowing us to characterize the complexity of water nature at these conditions at the atomic level.

Upgrading and Validation of the AMBER Force Field for Histidine and Cysteine Zinc(II)-Binding Residues in Sites with Four Protein Ligands.

We developed and validated a novel force field in the context of the AMBER parameterization for the simulation of zinc(II)-binding proteins. The proposed force field assumes nonbonded spherical interactions between the central zinc(II) and the coordinating residues. A crucial innovative aspect of our approach is to account for the polarization effects of the cation by redefining the atomic charges of the coordinating residues and an adjustment of Lennard-Jones parameters of Zn-interacting atoms to reproduce mean distance distributions. The optimal transferable parametrization was obtained by performing accurate quantum mechanical calculations on a training set of high-quality protein structures, encompassing the most common folds of zinc(II) sites. The addressed sites contain a zinc(II) ion tetra-coordinated by histidine and cysteine residues and represent about 70% of all physiologically relevant zinc(II) sites in the Protein Data Bank. Molecular dynamics simulations with explicit solvent, carried out on several zinc(II)-binding proteins not included in the training set, show that our model for zinc(II) sites preserves the tetra-coordination of the metal site with remarkable stability, yielding zinc(II)-X mean distances similar to experimental data. Finally, the model was tested by evaluating the zinc(II)-binding affinities, using the alchemical free energy perturbation approach. The calculated dissociation constants correlate satisfactorily with the experimental counterpart demonstrating the validity and transferability of the proposed parameterization for zinc(II)-binding proteins.

Computational study of the DPAP molecular rotor in various environments: from force field development to molecular dynamics simulations and spectroscopic calculations.

Fluorescent molecular rotors (FMRs) belong to an important class of environment-sensitive dyes capable of acting as nanoprobes in the measurement of viscosity and polarity of their micro-environment. FMRs have found widespread applications in various research fields, ranging from analytical to biochemical sciences, for example in intracellular imaging studies or in volatile organic compound detection. Here, a computational investigation of a recently proposed FMR, namely 4-(diphenylamino)phthalonitrile (DPAP), in various chemical environments is presented. A purposely developed molecular mechanics force field is proposed and then applied to simulate the rotor in a high- and low-polar solvent (i.e., acetonitrile, tetrahydrofuran, o-xylene and cyclohexane), a polymer matrix and a lipid membrane. Subtle effects of the molecular interactions with the embedding medium, the structural fluctuations of the rotor and its rotational dynamics are analyzed in some detail. The results correlate with a previous work, thus supporting the reliability of the model, and provide further insights into the environment-specific properties of the dye. In particular, it is shown how molecular diffusion and rotational correlation times of the FMR are affected by the surrounding medium and how the molecular orientation of the dye becomes anisotropic once immersed in the lipid bilayer. Moreover, a qualitative correlation between the FMR rotational dynamics and the fluorescence lifetime is detected, a result in line with the observed viscosity dependence of its emission. Finally, optical absorption spectra are computed and successfully compared with their experimental counterparts.

Accurate prediction of bulk properties in hydrogen bonded liquids: amides as case studies.

In this contribution we show that it is possible to build accurate force fields for small organic molecules allowing the reliable reproduction of a large panel of bulk properties, which are seldom addressed in the same context. Starting from the results obtained in recent studies, we developed a protocol for charge estimation and virtual site generation for the amide class of molecules. The parametrization of electrostatic properties is based on population analysis and orbital localization of quantum mechanical computations rooted in density functional theory and the polarizable continuum model, without any additional external information. The new protocol, coupled to other recent studies in our group targeted at an accurate fitting of internal degrees of freedom, makes available a method for building force fields from scratch (excluding for the moment intermolecular van der Waals interactions) with focus on reproducing the structure and dynamics of hydrogen bonded liquids, yielding results that are in line or better than those delivered by current general force fields. The approach is tested on the demanding series formed by formamide and its two N-methyl derivatives, N-methylformamide and N,N-dimethylformamide. We show that the atomistic structure of the liquids arising from classical molecular dynamics (MD) simulations employing the new force field is in full agreement with X-ray and neutron diffraction experiments and the corresponding spatial distribution functions are in remarkable agreement with the results of ab initio MD simulations. It is noteworthy that the latter result has never been obtained before without using ad hoc (and system dependent) scale factors and that, in addition, our parameter-free procedure is able to reproduce static dielectric constants over a wide range of values without sacrificing the force field accuracy with respect to other observables. Finally, we are able to explain the trend of static dielectric constants followed by the three amides in terms of properties obtained from the simulations, namely hydrogen bond patterns and reorientational lifetimes.

Triphilic Ionic-Liquid Mixtures: Fluorinated and Non-fluorinated Aprotic Ionic-Liquid Mixtures.

We present here the possibility of forming triphilic mixtures from alkyl- and fluoroalkylimidazolium ionic liquids, thus, macroscopically homogeneous mixtures for which instead of the often observed two domains-polar and nonpolar-three stable microphases are present: polar, lipophilic, and fluorous ones. The fluorinated side chains of the cations indeed self-associate and form domains that are segregated from those of the polar and alkyl domains. To enable miscibility, despite the generally preferred macroscopic separation between fluorous and alkyl moieties, the importance of strong hydrogen bonding is shown. As the long-range structure in the alkyl and fluoroalkyl domains is dependent on the composition of the liquid, we propose that the heterogeneous, triphilic structure can be easily tuned by the molar ratio of the components. We believe that further development may allow the design of switchable, smart liquids that change their properties in a predictable way according to their composition or even their environment.