Exploring the mechanism of action of spirooxindoles as a class of CDK2 inhibitors: a structure-based computational approach.

Cyclin-dependent kinase 2 (CDK2) regulates cell cycle checkpoints in the synthesis and mitosis phases and plays a pivotal role in cancerous cell proliferation. The activation of CDK2, influenced by various protein signaling pathways, initiates the phosphorylation process. Due to its crucial role in carcinogenesis, CDK2 is a druggable hotspot target to suppress cancer cell proliferation. In this context, several studies have identified spirooxindoles as an effective class of CDK2 inhibitors. In the present study, three spirooxindoles (SOI1, SOI2, and SOI3) were studied to understand their inhibitory mechanism against CDK2 through a structure-based approach. Molecular docking and molecular dynamics (MD) simulations were performed to explore their interactions with CDK2 at the molecular level. The calculated binding free energy for the spirooxindole-based CDK2 inhibitors aligned well with experimental results regarding CDK2 inhibition. Energy decomposition (ED) analysis identified key binding residues, including I10, G11, T14, R36, F82, K89, L134, P155, T158, Y159, and T160, in the CDK2 active site and T-loop phosphorylation. Molecular mechanics (MM) energy was identified as the primary contributor to stabilizing inhibitor binding in the CDK2 protein structure. Furthermore, the analysis of binding affinity revealed that the inhibitor SOI1 binds more strongly to CDK2 compared to the other inhibitors under investigation. It demonstrated a robust interaction with the crucial residue T160 in the T-loop phosphorylation site, responsible for kinase activation. These insights into the inhibitory mechanism are anticipated to contribute to the development of potential CDK2 inhibitors using the spirooxindole scaffold.

Complete Virion Simulated: All-Atom Model of an MS2 Bacteriophage with Native Genome.

For the first time, a complete all-atom molecular dynamics (MD) model of a virus, bacteriophage MS2, in its entirety, including a protein outer shell, native genomic RNA with necessary divalent ions, and surrounding explicit aqueous solution with ions at physiological concentration, was built. The model is based on an experimentally measured cryo-EM structure, which was substantially augmented by reconstructing missing or low-resolution parts of the measured density (where the atomistic structure cannot be fit unambiguously). The model was tested by a quarter of a microsecond MD run, and various biophysical characteristics are obtained and analyzed. The developed methodology of building the model can be used for reconstructing other large biomolecular structures when experimental data are fragmented and/or of varying resolution, while the model itself can be used for studying the biology of MS2, including the dynamics of its interaction with the host bacteria.

The nut-and-bolt motion of a bacteriophage sliding along a bacterial flagellum: a complete hydrodynamics model.

The 'nut-and-bolt' mechanism of a bacteriophage-bacteria flagellum translocation motion is modelled by numerically integrating the 3D Stokes equations using a Finite-Element Method (FEM). Following the works by Katsamba and Lauga (Phys Rev Fluids 4(1): 013101, 2019), two mechanical models of the flagellum-phage complex are considered. In the first model, the phage fiber wraps around the smooth flagellum surface separated by some distance. In the second model, the phage fiber is partly immersed in the flagellum volume via a helical groove imprinted in the flagellum and replicating the fiber shape. In both cases, the results of the Stokes solution for the translocation speed are compared with the Resistive Force Theory (RFT) solutions (obtained in Katsamba and Lauga Phys Rev Fluids 4(1): 013101, 2019) and the asymptotic theory in a limiting case. The previous RFT solutions of the same mechanical models of the flagellum-phage complex showed opposite trends for how the phage translocation speed depends on the phage tail length. The current work uses complete hydrodynamics solutions, which are free from the RFT assumptions to understand the divergence of the two mechanical models of the same biological system. A parametric investigation is performed by changing pertinent geometrical parameters of the flagellum-phage complex and computing the resulting phage translocation speed. The FEM solutions are compared with the RFT results using insights provided from the velocity field visualisation in the fluid domain.

Covalent conjugates based on nanodiamonds with doxorubicin and a cytostatic drug from the group of 1,3,5-triazines: Synthesis, biocompatibility and biological activity.

We report the synthesis of covalent conjugates of nanodiamonds with doxorubicin and a cytostatic drug from the class of 1,3,5-triazines. The obtained conjugates were identified using a number of physicochemical methods (IR-spectroscopy, NMR-spectroscopy, XRD, XPS, TEM). As a result of our study, it was found that ND-СONH-Dox and ND-COO-Diox showed good hemocompatibility, since they did not affect plasma coagulation hemostasis, platelet functional activity, and erythrocyte membrane. The ND-COO-Diox conjugates are also capable of binding to human serum albumin due to the presence of ND in their composition. In the study of the cytotoxic properties of ND-СONH-Dox and ND-COO-Diox in the T98G glioblastoma cell line, indicating that ND-СONH-Dox and ND-COO-Diox demonstrate greater cytotoxicity at lower concentrations of Dox and Diox in the composition of the conjugates compared to individual drugs; the cytotoxic effect of ND-COO-Diox was statistically significantly higher than that of ND-СONH-Dox at all concentrations studied. Greater cytotoxicity at lower concentrations of Dox and Diox in the composition of conjugates compared to individual cytostatics makes it promising to further study the specific antitumor activity and acute toxicity of these conjugates in models of glioblastoma in vivo. Our results demonstrated that ND-СONH-Dox and ND-COO-Diox enter HeLa cells predominantly via a nonspecific actin-dependent mechanism, while for ND-СONH-Dox a clathrin-dependent endocytosis pathway. All data obtained provide that the synthesized nanomaterials show a potential application as the agents for intertumoral administration.

Estimation of Nanoparticle's Surface Electrostatic Potential in Solution Using Acid-Base Molecular Probes I: In Silico Implementation for Surfactant Micelles.

Surface electrostatic potential Ψ is a key characteristic of colloid particles. Since the surface of the particles adsorbs various compounds and facilitates chemical reactions between them, Ψ largely affects the properties of adsorbed reactants and governs the flow of chemical reactions occurring between them. One of the most popular methods for estimating Ψ in hydrophilic colloids, such as micellar surfactant solutions and related systems, is the application of molecular probes, predominantly acid-base indicator dyes. The Ψ value is calculated from the difference of the probe's indices of the apparent acidity constant between the examined colloid solution and, usually, some other colloid solution with noncharged particles. Here, we show how to implement this method in silico using alchemical free energy calculations within the framework of molecular dynamics simulations. The proposed implementation is tested on surfactant micelles and is shown to predict experimental Ψ values with quantitative accuracy depending on the kind of surfactant. The sources of errors in the method are discussed, and recommendations for its application are given.

Estimation of Nanoparticle's Surface Electrostatic Potential in Solution Using Acid-Base Molecular Probes II: Insight from Atomistic Simulations of Micelles.

Exploiting acid-base indicators as molecular probes is one of the most popular methods for determining the surface electrostatic potential Ψ in hydrophilic colloids like micellar surfactant solutions and related systems. Specifically, the indicator's apparent acidity constant index is measured in the colloid solution of interest and, as a rule, in a nonionic surfactant solution; the difference between the two is proportional to Ψ. Despite the widespread use of this approach, a major problem remains unresolved, namely, the dissimilarity of Ψ values obtained with different indicators for the same system. The common point of view recognizes the effect of several factors (the choice of the nonionic surfactant, the probe's localization, and the degree of hydration of micellar pseudophase) but does not allow to quantitatively assess their impact and decide which indicator reports the most correct Ψ value. Here, based on the ability to predict the reported Ψ values in silico, we examined the role of these factors using molecular dynamics simulations for five probes and two surfactants. The probe's hydration in the Stern layer was found responsible for approximately half of the dissimilarity range. The probe's localization is found important but hard to quantify because of the irregular structure of the Stern layer. The most accurate indicators among the examined set were identified. Supplementing experiments on measuring Ψ with molecular dynamics simulation is proposed as a way of improving the efficacy of the indicator method: the simulations can guide the choice of the most suitable probe and nonionic surfactant for the given nanoparticles.

Estimation of Nanoparticle's Surface Electrostatic Potential in Solution Using Acid-Base Molecular Probes. III. Experimental Hydrophobicity/Hydrophilicity and Charge Distribution of MS2 Virus Surface.

MS2 bacteriophage is often used as a model for evaluating pathogenic viruses' behavior in aqueous solution. However, the questions of the virus surface's hydrophilic/hydrophobic balance, the charge distribution, and the binding mechanism are open. Using the dynamic light scattering method and laser Doppler electrophoresis, the hydrodynamic diameter and the ζ-potential of the virus particles were measured at their concentration of 5 × 1011 particles per mL and ionic strength 0.03 M. The values were found to be 30 nm and -29 or -34 mV (by Smoluchowski or Ohshima approximations), respectively. The MS2 bacteriophage surface was also investigated using a series of acid-base indicator dyes of various charge type, size, and structure. Their spectral and acid-base properties (pKa) are very sensitive to the microenvironment in aqueous solution, including containing nanoparticles. The electrostatic potential of the surface Ψ was estimated using the common formula: Ψ = 59 × (pKai - pKa) in mV at 25 °C. The Ψ values were -50 and +10 mV, respectively, which indicate the "mosaic" way of the charge distribution on the surface. These data are in good agreement with the obtained ζ-potential values and provide even more information about the virus surface. It was found that the surface of the MS2 virus is hydrophilic in solution in contrast to the commonly accepted hypothesis of the hydrophobicity of virus particles. No hydrophobic interactions between various molecular probes and the capsid were observed.

Reconstruction and validation of entire virus model with complete genome from mixed resolution cryo-EM density.

It is very difficult to reconstruct computationally a large biomolecular complex in its biological entirety from experimental data. The resulting atomistic model should not contain gaps structurally and it should yield stable dynamics. We, for the first time, reconstruct from the published incomplete cryo-EM density a complete MS2 virus at atomistic resolution, that is, the capsid with the genome, and validate the result by all-atom molecular dynamics with explicit water. The available experimental data includes a high resolution protein capsid and an inhomogeneously resolved genome map. For the genomic RNA, apart from 16 hairpins with atomistic resolution, the strands near the capsid's inner surface were resolved up to the nucleic backbone level, and the innermost density was completely unresolved. As a result, only 242 nucleotides (out of 3569) were positioned, while only a fragmented backbone was outlined for the rest of the genome, making a detailed model reconstruction necessary. For model reconstruction, in addition to the available atomistic structure information, we extensively used the predicted secondary structure of the genome (base pairing). The technique was based on semi-automatic building of relatively large strands of RNA with subsequent manual positioning over the traced backbone. The entire virus structure (capsid + genome) was validated by a molecular dynamics run in physiological solution with ions at standard conditions confirming the stability of the model.

Constant pH molecular dynamics of porcine circovirus 2 capsid protein reveals a mechanism for capsid assembly.

Spatiotemporal regulation of viral capsid assembly ensures the selection of the viral genome for encapsidation. The porcine circovirus 2 is the smallest autonomously replicating pathogenic virus, yet how PCV2 capsid assembly is regulated to occur within the nucleus remains unknown. We report that pure PCV2 capsid proteins, in the absence of nucleic acids, require acidic conditions to assemble into empty capsids in vitro. By employing constant pH replica exchange molecular dynamics, we unveil the atomistic mechanism of pH-dependency for capsid assembly. The results show that an appropriate protonation configuration for a cluster of acidic amino acids is necessary to appropriately position the GH-loop for driving the capsid assembly. We demonstrate that assembly is prohibited at neutral pH because deprotonation of these residues results in their electrostatic repulsion, shifting the GH-loop to a position incompatible with capsid assembly. We propose that encapsulation of nucleic acids overcomes this repulsion to suitably position the GH-loop. Our findings provide the first atomic resolution mechanism of capsid assembly regulation. These findings are useful for the development of therapeutics that inhibit PCV2 self-assembly.

Graphene Oxide of Extra High Oxidation: A Wafer for Loading Guest Molecules.

We present a new modification of graphene oxide with very high content (85 wt %) of oxygen-containing functional groups (hydroxy, epoxy, lactol, carboxyl, and carbonyl groups) that forms stable aqueous dispersion in up to 9 g·L-1 concentration solutions. A novel faster method of the synthesis is described that produces up to 1 kg of the material and allows controlling the particle size in solution. The synthesized compound was characterized by various physicochemical methods and molecular dynamics modeling, revealing a unique structure in the form of a multilayered wafer of several sheets thick, where each sheet is highly corrugated. The ragged structure of the sheets forms pockets with hindered mobility of water that leads to the possibility of trapping guest molecules.

Protein-Ligand Dissociation Rate Constant from All-Atom Simulation.

Dissociation of a ligand isoniazid from a protein catalase was investigated using all-atom molecular dynamics (MD) simulations. Random acceleration MD (τ-RAMD) was used, in which a random artificial force applied to the ligand facilitates its dissociation. We have suggested a novel approach to extrapolate such obtained dissociation times to the zero-force limit assuming never before attempted universal exponential dependence of the bond strength on the applied force, allowing direct comparison with experimentally measured values. We have found that our calculated dissociation time was equal to 36.1 s with statistically significant values distributed in the interval of 0.2-72.0 s, which quantitatively matches the experimental value of 50 ± 8 s despite the extrapolation over 9 orders of magnitude in time.

In Vitro and In Silico Investigation of Water-Soluble Fullerenol C60(OH)24: Bioactivity and Biocompatibility.

Light fullerenes, C60 and C70, have significant potential in biomedical applications due to their ability to absorb reactive oxygen species, inhibit the development of tumors, inactivate viruses and bacteria, and as the basis for developing systems for targeted drug delivery. However, the hydrophobicity of individual fullerenes complicates their practical use; therefore, creating water-soluble derivatives of fullerenes is increasingly important. Currently, the most studied soluble adducts of fullerenes are polyhydroxy fullerenes or fullerenols. Unfortunately, investigations of fullerenol biocompatibility are fragmental. They often lack reproducibility both in the synthesis of the compounds and their biological action. We here investigate the biocompatibility of a well-defined fullerenol C60(OH)24 obtained using methods that minimize the content of impurities and quantitatively characterize the product's composition. We carry out comprehensive biochemical and biophysical investigations of C60(OH)24 that include photodynamic properties, cyto- and genotoxicity, hemocompatibility (spontaneous and photo-induced hemolysis, platelet aggregation), and the thermodynamic characteristics of C60(OH)24 binding to human serum albumin and DNA. The performed studies show good biocompatibility of fullerenol C60(OH)24, which makes it a promising object for potential use in biomedicine.

Structural insight into TNF-α inhibitors through combining pharmacophore-based virtual screening and molecular dynamic simulation.

Tumor Necrosis Factor-alpha (TNF-α), a multifunctional cytokine responsible for providing resistance against infections, inflammation, and cancers. TNF-α has emerged as a promising drug target against several autoimmune and inflammatory disorders. Several synthetic antibodies (Infliximab, Etanercept, and Adalimumab) are available, but their potential to cause severe side effects has prompted them to develop alternative small molecules-based therapies for inhibition of TNF-α. In the present study, combined in silico approaches based on pharmacophore modeling, virtual screening, molecular docking, and molecular dynamics studies were employed to understand significant direct interactions between TNF-α protein and small molecule inhibitors. Initially, four different small molecule libraries (∼17.5 million molecules) were virtually screened against the selected pharmacophore model. The identified hits were further subjected to molecular docking studies. The three potent lead compounds (ZINC05848961, ZINC09402309, ZINC04502991) were further subjected to 100 ns molecular dynamic studies to examine their stability. Our docking and molecular dynamic analysis revealed that the selected lead compounds target the TNF receptor (TNFR) and efficiently block the production of TNF. Moreover, in silico ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) analysis revealed that all the predicted compounds have good pharmacokinetic properties with high gastrointestinal absorption and a decent bioavailability score. Furthermore, toxicity profiles further evidenced that these compounds have no risk of being mutagenic, tumorigenic, reproductive and irritant except ZINC11915498. In conclusion, the present study could serve as the starting point to develop new therapeutic regimens to treat various TNF- related diseases. Communicated by Ramaswamy H. Sarma.

Disordered protein-graphene oxide co-assembly and supramolecular biofabrication of functional fluidic devices.

Supramolecular chemistry offers an exciting opportunity to assemble materials with molecular precision. However, there remains an unmet need to turn molecular self-assembly into functional materials and devices. Harnessing the inherent properties of both disordered proteins and graphene oxide (GO), we report a disordered protein-GO co-assembling system that through a diffusion-reaction process and disorder-to-order transitions generates hierarchically organized materials that exhibit high stability and access to non-equilibrium on demand. We use experimental approaches and molecular dynamics simulations to describe the underlying molecular mechanism of formation and establish key rules for its design and regulation. Through rapid prototyping techniques, we demonstrate the system's capacity to be controlled with spatio-temporal precision into well-defined capillary-like fluidic microstructures with a high level of biocompatibility and, importantly, the capacity to withstand flow. Our study presents an innovative approach to transform rational supramolecular design into functional engineering with potential widespread use in microfluidic systems and organ-on-a-chip platforms.

MS2 bacteriophage capsid studied using all-atom molecular dynamics.

The all-atom model of an MS2 bacteriophage particle without its genome (the capsid) was built using high-resolution cryo-electron microscopy (EM) measurements for initial conformation. The structural characteristics of the capsid and the dynamics of the surrounding solution were examined using molecular dynamics simulation. The model demonstrates the overall preservation of the cryo-EM structure of the capsid at physiological conditions (room temperature and ions composition). The formation of a dense anion layer near the inner surface and a diffuse cation layer near the outer surface of the capsid was detected. The flow of water molecules and ions across the capsid through its pores were quantified, which was considerable for water and substantial for ions.

All-Atom Molecular Dynamics Simulations of Whole Viruses.

Classical molecular dynamics modeling of whole viruses or their capsids in explicit water is discussed, and known examples from the literature are analyzed. Only works on all-atom modeling in explicit water are included. Physical chemistry of the whole system is the focus, which includes the structure and dynamics of the biomolecules as well as water and ion behavior in and around the virus particle. It was demonstrated that in most investigations molecular phenomena that currently can not be studied experimentally are successfully reproduced and explained by the simulations. These include, for example, transport and distribution of ions inside viruses that ultimately connected to their stability, the hydrodynamic pressure in the capsid related to viruses' elastic properties, the role of metal ions in virus swelling, and others. Current and future tendencies in the development of all-atom virus simulations are outlined.

All-Atom Molecular Dynamics Simulations of Entire Virus Capsid Reveal the Role of Ion Distribution in Capsid's Stability.

Present experimental methods do not have sufficient resolution to investigate all processes in virus particles at atomistic details. We report the results of molecular dynamics simulations and analyze the connection between the number of ions inside an empty capsid of PCV2 virus and its stability. We compare the crystallographic structures of the capsids with unresolved N-termini and without them in realistic conditions (room temperature and aqueous solution) and show that the structure is preserved. We find that the chloride ions play a key role in the stability of the capsid. A low number of chloride ions results in loss of the native icosahedral symmetry, while an optimal number of chloride ions create a neutralizing layer next to the positively charged inner surface of the capsid. Understanding the dependence of the capsid stability on the distribution of the ions will help clarify the details of the viral life cycle that is ultimately connected to the role of packaged viral genome inside the capsid.

A hybrid molecular dynamics/fluctuating hydrodynamics method for modelling liquids at multiple scales in space and time.

A new 3D implementation of a hybrid model based on the analogy with two-phase hydrodynamics has been developed for the simulation of liquids at microscale. The idea of the method is to smoothly combine the atomistic description in the molecular dynamics zone with the Landau-Lifshitz fluctuating hydrodynamics representation in the rest of the system in the framework of macroscopic conservation laws through the use of a single "zoom-in" user-defined function s that has the meaning of a partial concentration in the two-phase analogy model. In comparison with our previous works, the implementation has been extended to full 3D simulations for a range of atomistic models in GROMACS from argon to water in equilibrium conditions with a constant or a spatially variable function s. Preliminary results of simulating the diffusion of a small peptide in water are also reported.

Visualising and controlling the flow in biomolecular systems at and between multiple scales: from atoms to hydrodynamics at different locations in time and space.

A novel framework for modelling biomolecular systems at multiple scales in space and time simultaneously is described. The atomistic molecular dynamics representation is smoothly connected with a statistical continuum hydrodynamics description. The system behaves correctly at the limits of pure molecular dynamics (hydrodynamics) and at the intermediate regimes when the atoms move partly as atomistic particles, and at the same time follow the hydrodynamic flows. The corresponding contributions are controlled by a parameter, which is defined as an arbitrary function of space and time, thus, allowing an effective separation of the atomistic 'core' and continuum 'environment'. To fill the scale gap between the atomistic and the continuum representations our special purpose computer for molecular dynamics, MDGRAPE-4, as well as GPU-based computing were used for developing the framework. These hardware developments also include interactive molecular dynamics simulations that allow intervention of the modelling through force-feedback devices.