Synthesis, biological evaluation and molecular docking studies of bis-chalcone derivatives as xanthine oxidase inhibitors and anticancer agents.

In this study, a series of B-ring fluoro substituted bis-chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and evaluated for their ability to inhibit xanthine oxidase (XO) and growth inhibitory activity against MCF-7 and Caco-2 human cancer cell lines, in vitro. According to the results obtained, the bis-chalcone with fluoro group at the 2 (4b) or 2,5-position (4g) of B-ring were found to be potent inhibitors of the enzyme with IC50 values in the low micromolar range. The effects of these compounds were about 7 fold higher than allopurinol. The binding modes of the bis-chalcone derivatives in the active site of xanthine oxidase were explained using molecular docking calculations. Also, compound 4g and 4h showed in vitro growth inhibitory activity against a panel of two human cancer cell lines 1.9 and 6.8 µM of IC50 values, respectively.

Investigation of Structural and Antibacterial Properties of WS2-Doped ZnO Nanoparticles.

ZnO nanoparticles, well-known for their structural, optical, and antibacterial properties, are widely applied in diverse fields. The doping of different materials to ZnO, such as metals or metal oxides, is known to ameliorate its properties. Here, nanofilms composed of ZnO doped with WS2 at 5, 15, and 25% ratios are synthesized, and their properties are investigated. Supported by molecular docking analyses, the enhancement of the bactericidal properties after the addition of WS2 at different ratios is highlighted and supported by the inhibitory interaction of residues playing a crucial role in the bacterial survival through the targeting of proteins of interest.

Newly synthesized 6-substituted piperazine/phenyl-9-cyclopentyl containing purine nucleobase analogs act as potent anticancer agents and induce apoptosis via inhibiting Src in hepatocellular carcinoma cells.

Newly synthesized 6-substituted piperazine/phenyl-9-cyclopentyl-containing purine nucleobase analogs were tested for their in vitro anticancer activity against human cancer cells. Compounds 15, 17-24, 49, and 56 with IC50 values less than 10 µM were selected for further examination on an enlarged panel of liver cancer cell lines. Experiments revealed that compound 19 utilizes its high cytotoxic potential (IC50 < 5 µM) to induce apoptosis in vitro. Compound 19 displayed a KINOMEscan selectivity score S35 of 0.02 and S10 of 0.01 and demonstrated a significant selectivity against anaplastic lymphoma kinase (ALK) and Bruton's tyrosine kinase (BTK) over other kinases. Compounds 19, 21, 22, 23, and 56 complexed with ALK, BTK, and (discoidin domain-containing receptor 2) DDR2 were analyzed structurally for binding site interactions and binding affinities via molecular docking and molecular dynamics simulations. Compounds 19 and 56 displayed similar interactions with the activation loop of the kinases, while only compound 19 reached toward the multiple subsites of the active site. Cell cycle and signaling pathway analyses exhibited that compound 19 decreases phosho-Src, phospho-Rb, cyclin E, and cdk2 levels in liver cancer cells, eventually inducing apoptosis.

Modelling of C-terminal tail of human STING and its interaction with tank-binding kinase 1.

Stimulator of interferon genes (STING) plays a significant role in a cell's intracellular defense against pathogens or self-DNA by inducing inflammation or apoptosis through a pathway known as cGAS-cGAMP-STING. STING uses one of its domains, the C-terminal tail (CTT) to recruit the members of the pathway. However, the structure of this domain has not been solved experimentally. STING conformation is open and more flexible when inactive. When STING gets activated by cGAMP, its conformation changes to a closed state covered by 4 beta-sheets over the binding site. This conformational change leads to its binding to Tank-binding kinase 1 (TBK1). TBK1 then phosphorylates STING aiding its entry to the cell's nucleus. In this study, we focused on the loop modeling of the CTT domain in both the active and inactive STING conformations. After the modeling step, the active and inactive STING structures were docked to one of the cGAS-cGAMP-STING pathway members, TBK1, to observe the differences of binding modes. CTT loop stayed higher in the active structure, while all the best-scored models, active or inactive, ended up around the same position with respect to TBK1. However, when the STING poses are compared with the cryo-EM image of the complex structure, the models in the active structure chain B displayed closer results to the complex structure.

Correlated conformational dynamics of the human GluN1-GluN2A type N-methyl-D-aspartate (NMDA) receptor.

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels found in the nerve cell membranes. As a result of overexcitation of NMDARs, neuronal death occurs and may lead to diseases such as epilepsy, stroke, Alzheimer's disease, and Parkinson's disease. In this study, human GluN1- GluN2A type NMDAR structure is modeled based on the X-ray structure of the Xenopus laevis template and missing loops are added by ab-initio loop modeling. The final structure is chosen according to two different model assessment scores. To be able to observe the structural changes upon ligand binding, glycine and glutamate molecules are docked into the corresponding binding sites of the receptor. Subsequently, molecular dynamics simulations of 1.3 µs are performed for both apo and ligand-bound structures. Structural parameters, which have been considered to show functionally important changes in previous NMDAR studies, are monitored as conformational rulers to understand the dynamics of the conformational changes. Moreover, principal component analysis (PCA) is performed for the equilibrated part of the simulations. From these analyses, the differences in between apo and ligand-bound simulations can be summarized as the following: The girdle right at the beginning of the pore loop, which connects M2 and M3 helices of the ion channel, partially opens. Ligands act like an adhesive for the ligand-binding domain (LBD) by keeping the bi-lobed structure together and consequently this is reflected to the overall dynamics of the protein as an increased correlation of the LBD with especially the amino-terminal domain (ATD) of the protein.

Dynamic linear response theory for conformational relaxation of proteins.

Dynamic linear response theory is adapted to the problem of computing the time evolution of the atomic coordinates of a protein in response to the unbinding of a ligand molecule from a binding pocket within the protein. When the ligand dissociates from the molecule, the protein molecule finds itself out of equilibrium and its configuration begins to change, ultimately coming to a new stable configuration corresponding to equilibrium in a force field that lacks the ligand-protein interaction terms. Dynamic linear response theory (LRT) relates the nonequilibrium motion of the protein atoms that ensues after the ligand molecule dissociates to equilibrium dynamics in the force field, or equivalently, on the potential energy surface (PES) relevant to the unliganded protein. In general, the connection implied by linear response theory holds only when the ligand-protein force field is small. However, in the case where the PES of the unliganded protein system is a quadratic (harmonic oscillator) function of the coordinates, and the force of the ligand upon the protein molecule in the ligand-bound conformation is constant (the force on each atom in the protein is independent of the location of the atom), dynamic LRT is exact for any ligand-protein force field strength. An analogous statement can be made for the case where the atoms in the protein are subjected to frictional and random noise forces in accord with the Langevin equation (to account for interaction of the protein with solvent, for example). We numerically illustrate the application of dynamic LRT for a simple harmonic oscillator model of the ferric binding protein, and for an analogous model of T4 lysozyme. Using a physically appropriate value of the viscosity of water to guide the choice of friction parameters, we find relaxation time scales of residue-residue distances on the order of several hundred ps. Comparison is made to relevant experimental measurements.

Discovery of new azoles with potent activity against Candida spp. and Candida albicans biofilms through virtual screening.

Systemic candidiasis is a rampant bloodstream infection of Candida spp. and C. albicans is the major pathogen isolated from infected humans. Azoles, the most common class of antifungals which suffer from increasing resistance, and especially intrinsically resistant non-albicans Candida (NAC) species, act by inhibiting fungal lanosterol 14α-demethylase (CYP51). In this study we identified a number of azole compounds in 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol/ethanone oxime ester structure through virtual screening using consensus scoring approach, synthesized and tested them for their antifungal properties. We reached several hits with potent activity against azole-susceptible and azole-resistant Candida spp. as well as biofilms of C. albicans. 5i's minimum inhibitor concentration (MIC) was 0.125 µg/ml against C. albicans, 0.5 µg/ml against C. krusei and 1 µg/ml against azole-resistant C. tropicalis isolate. Considering the MIC values of fluconazole against these fungi (0.5, 32 and 512 µg/ml, respectively), 5i emerged as a highly potent derivative. The minimum biofilm inhibitor concentration (MBIC) of 5c, 5j, and 5p were 0.5 µg/ml (and 5i was 2 µg/ml) against C. albicans biofilms, lower than that of amphotericin B (4 µg/ml), a first-line antifungal with antibiofilm activity. In addition, the active compounds showed neglectable toxicity to human monocytic cell line. We further analyzed the docking poses of the active compounds in C. albicans CYP51 (CACYP51) homology model catalytic site and identified molecular interactions in agreement with those of known azoles with fungal CYP51s and mutagenesis studies of CACYP51. We observed the stability of CACYP51 in complex with 5i in molecular dynamics simulations.

Antifungal screening and in silico mechanistic studies of an in-house azole library.

Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C. albicans is the major pathogen identified in candidiasis; however, non-albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first-choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in-house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C. albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C. albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.

The neural γ2α1ß2α1ß2 gamma amino butyric acid ion channel receptor: structural analysis of the effects of the ivermectin molecule and disulfide bridges.

While ~30% of the human genome encodes membrane proteins, only a handful of structures of membrane proteins have been resolved to high resolution. Here, we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors, human type A γ2α1ß2α1ß2 gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy, ataxia, migraine, schizophrenia and other neurodegenerative diseases. The structure of human γ2α1ß2α1ß2 has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here, our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule, ivermectin, from the template structure, helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts, there are three potential disulfide bridges between the M1 and M3 helices of the γ2 and 2 α1 subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models-one with the disulfide bridge and one with protonated Cys residues. In all subunit types, RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally, loop A, loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory, the two model structures displayed different coupling in between the M2-M3 linker region, protruding from the membrane, and the ß1-ß2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop, which collapses directly over the bound ligand molecule, were also affected by differences in the packing of transmembrane helices. Finally, more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.

A wrench in the works of human acetylcholinesterase: soman induced conformational changes revealed by molecular dynamics simulations.

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.

Computational Analysis of a Zn-Bound Tris(imidazolyl) Calix[6]arene Aqua Complex: Toward Incorporating Second-Coordination Sphere Effects into Carbonic Anhydrase Biomimetics.

Molecular dynamics simulations and quantum-mechanical calculations were performed to characterize a supramolecular tris(imidazolyl) calix[6]arene Zn(2+) aqua complex, as a biomimetic model for the catalyzed hydration of carbon dioxide to bicarbonate, H2O + CO2 → H(+) + HCO3(-). On the basis of potential-of-mean-force (PMF) calculations, stable conformations had distorted 3-fold symmetry and supported either one or zero encapsulated water molecules. The conformation with an encapsulated water molecule is calculated to be lower in free energy than the conformation with an empty cavity (ΔG = 1.2 kcal/mol) and is the calculated free-energy minimum in solution. CO2 molecule partitioning into the cavity is shown to be very facile, proceeding with a barrier of 1.6 kcal/mol from a weak encounter complex which stabilizes the species by about 1.0 kcal/mol. The stabilization energy of CO2 is calculated to be larger than that of H2O (ΔΔG = 1.4 kcal/mol), suggesting that the complex will preferentially encapsulate CO2 in solution. In contrast, the PMF for a bicarbonate anion entering the cavity is calculated to be repulsive in all nonbonding regions of the cavity, due to the diameter of the calix[6]arene walls. Geometry optimization of the Zn-bound hydroxide complex with an encapsulated CO2 molecule showed that multiple noncovalent interactions direct the reactants into optimal position for nucleophilic addition to occur. The calixarene complex is a structural mimic of the hydrophilic/hydrophobic divide in the enzyme, providing a functional effect for CO2 addition in the catalytic cycle. The results show that Zn-binding calix[6]arene scaffolds can be potential synthetic biomimetics for CO2 hydration catalysis, both in terms of preferentially encapsulating CO2 from solution and by spatially fixing the reactive species inside the cavity.

Langevin dynamics of molecules with internal rigid fragments in the harmonic regime.

An approximation scheme is developed to compute Brownian motion according to the Langevin equation for a molecular system moving in a harmonic force field (corresponding to a quadratic potential energy surface) and characterized by one or more rigid internal fragments. This scheme, which relies on elements of the rotation translation block (RTB) method for computing vibrational normal modes of large molecules developed by Sanejouand and co-workers [Biopolymers 34, 759 (1994); Proteins: Struct., Funct., Genet. 41, 1 (2000)], provides a natural and efficient way to freeze out the small amplitude, high frequency motions within each rigid fragment. The number of dynamical degrees of freedom in the problem is thereby reduced, often dramatically. To illustrate the method, the relaxation kinetics of the small membrane-bound ion channel protein gramicidin-A, subjected to an externally imposed impulse, is computed. The results obtained from all-atom dynamics are compared to those obtained using the RTB-Langevin dynamics approximation (treating eight indole moieties as internal rigid fragments): good agreement between the two treatments is found.

A rigid-body Newtonian propagation scheme based on instantaneous decomposition into rotation and translation blocks.

The rotation and translation block (RTB) method of Durand et al. [Biopolymers 34, 759 (1994)] and Tama et al. [Proteins 41, 1 (2000)] provides an appealing way to calculate low-frequency normal modes of large biomolecules by restricting the space of motions to exclude internal motions of preselected rigid fragments within the molecule. These fragments are modeled essentially as rigid bodies and the need to calculate high-frequency relative motions of the atoms that form them is obviated in a natural way. Here we extend the RTB approach into a method for computing the classical (Newtonian) dynamics of a biomolecule, or any large molecule, with effective rigid-body constraints applied to a prechosen set of internal molecular fragments. This method, to be termed RTB dynamics, is easy to implement, conserves the total energy of the system, does not require the construction of the matrix of second spatial derivatives of the potential-energy function (Hessian matrix), and can be used to compute the classical dynamics of a system moving in an arbitrary anharmonic force field. An elementary numerical application to signal propagation in the small membrane-bound polypeptide gramicidin-A is presented for illustration purposes.