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When faced with the investigation of the preferential binding of a series of ligands against a known target, the solution is not always evident from single structure analysis. An ensemble of structures generated from computer simulations is valuable; however, visual analysis of the extensive structural data can be overwhelming. Rapid analysis of trajectory data, with tools available in the Galaxy platform, can be used to understand key features and compare differences that inform the preferential ligand structure that favors binding. We illustrate this informatics approach by investigating the in-silico binding of a peptide and glycopeptide epitope of the glycoprotein Mucin 1 (MUC1) binding with the antibody AR20.5. To study the binding, we performed molecular dynamics simulations using OpenMM and then used the Galaxy platform for data analysis. The same analysis tools are applied to each of the simulation trajectories and this process was streamlined by using Galaxy workflows. The conformations of the antigens were analyzed using root-mean-square deviation, end-to-end distance, Ramachandran plots, and hydrogen bonding analysis. Additionally, RMSF and clustering analysis were carried out. These analyses were used to rapidly assess key features of the system, interrogate the dynamic structure of the ligand, and determine the role of glycosylation on the conformational equilibrium. The glycopeptide conformations in solution change relative to the peptide; thus a partially pre-structuring is seen prior to binding. Although the bound conformation of peptide and glycopeptide is similar, the glycopeptide fluctuates less and resides in specific conformers for more extended periods. This structural analysis which gives a high-level view of the features in the system under observation, could be readily applied to other binding problems as part of a general strategy in drug design or mechanistic analysis.

This essay aims to show that baseball's time-honored emphases on physical and spiritual discipline follow from its metaphysical imaginary. In turn, it will reason that Christian life and thought are capable of illuminating baseball—and vice versa. The argument will proceed as follows: First, both Christianity and baseball frame their worlds in terms of emanation (exitus) and return (reditus): “players” leave home and aim to return home; second, though players belong to a team or community (ecclesia), the task of returning home is ultimately a solitary one; it has to be done by the individual player, even if the team, too, benefits from the individual's undertaking; and third, the spiritual or attitudinal development of the individual is thus crucial: players have to attend to how they approach the “game,” particularly in terms of their internal comportment. This last point will receive special attention: it will be reasoned that Søren Kierkegaard's spiritual writings, tendered for the existential “upbuilding” (Opbyggelse) of “the single individual” (den Enkelte), might likewise offer upbuilding insights for the individuals who play baseball—a sport that John Updike once called “an essentially lonely game.”

Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled ajoene analogue called dansyl-ajoene, ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to ajoene was performed using Schrödinger and pKa calculations by Epik software. Cytotoxicity of ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that ajoene and dansyl-ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by ajoene. Conclusions The garlic-derived phytochemical ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of ajoene in cancer cells.

Biomolecular Reaction and Interaction Dynamics Global Environment (BRIDGE) is an open-source web platform developed with the aim to provide an environment for the design of reliable methods to conduct reproducible biomolecular simulations. It is built on the well-known Galaxy bioinformatics platform. Through this, BRIDGE hosts computational chemistry tools on public web servers for internet use and provides machine- and operating-system-independent portability using the Docker container platform for local use. This construction improves the accessibility, shareability, and reproducibility of computational methods for molecular simulations. Here we present integrated free energy tools (or apps) to calculate absolute binding free energies (ABFEs) and relative binding free energies (RBFEs), as illustrated through use cases. We present free energy perturbation (FEP) methods contained in various software packages such as GROMACS and YANK that are independent of hardware configuration, software libraries, or operating systems when ported in the Galaxy-BRIDGE Docker container platform. By performing cyclin-dependent kinase 2 (CDK2) FEP calculations on geographically dispersed web servers (in Africa and Europe), we illustrate that large-scale computations can be performed using the exact same codes and methodology by collaborating groups through publicly shared protocols and workflows. The ease of public sharing and independent reproduction of simulations via BRIDGE makes possible an open review of methods and complete simulation protocols. This makes the discovery of inhibitors for drug targets accessible to nonexperts and the computer experiments that are used to arrive at leads verifiable by experts and reviewers. We illustrate this on β-galactoside α-2,3-sialyltransferase I (ST3Gal-I), a breast cancer drug target, where a combination of RBFE and ABFE methods are used to compute the binding free energies of three inhibitors.

Iridium(iii) half-sandwich complexes containing 7-chloroquinoline-1,2,3-triazole hybrid ligands were synthesised and their inhibitory activities evaluated against the Plasmodium falciparum malaria parasite. Supporting computational analysis revealed that metal coordination to the quinoline nitrogen occurs first, forming a kinetic product that, upon heating over time, forms a more stable cyclometallated thermodynamic product. Single crystal X-ray diffraction confirmed the proposed molecular structures of both isolated kinetic and thermodynamic products. Complexation with iridium significantly enhances the in vitro activity of selected ligands against the chloroquine-sensitive (NF54) Plasmodium falciparum strain, with selected complexes being over one hundred times more active than their respective ligands. No cross-resistance was observed in the chloroquine-resistant (K1) strain. No cytotoxicity was observed for selected complexes tested against the mammalian Chinese Hamster Ovarian (CHO) cell line. In addition, speed-of-action assays and β-haematin inhibition studies were performed. Through preliminary qualitative and quantitative cell-free experiments, it was found that the two most active neutral, cyclometallated complexes can act as transfer hydrogenation catalysts, by reducing β-nicotinamide adenine dinucleotide (NAD+) to NADH in the presence of a hydrogen source, sodium formate.

This paper is a tutorial developed for the data analysis platform Galaxy. The purpose of Galaxy is to make high-throughput computational data analysis, such as molecular dynamics, a structured, reproducible and transparent process. In this tutorial we focus on 3 questions: How are protein-ligand systems parameterized for molecular dynamics simulation? What kind of analysis can be carried out on molecular trajectories? How can high-throughput MD be used to study multiple ligands? After finishing you will have learned about force-fields and MD parameterization, how to conduct MD simulation and analysis for a protein-ligand system, and understand how different molecular interactions contribute to the binding affinity of ligands to the Hsp90 protein.

Protein-ligand binding prediction is central to the drug-discovery process. This often follows an analysis of genomics data for protein targets and then protei n structure discovery. However, the complexity of performing reproducible protein conformational analysis and ligand binding calculations, using vetted methods and protocols can be a challenge. Here we show how Biomolecular Reaction and Interaction Dynamics Global Environment (BRIDGE), an open-source web-based compute and analytics platform for computational chemistry developed based on the Galaxy bioinformatics platform, makes protocol sharing seamless following genomics and proteomics. BRIDGE makes available tools and workflows to carry out protein molecular dynamics simulations and accurate free energy computations of protein-ligand binding. We illustrate the dynamics and simulation protocols for predicting protein-ligand binding affinities in silico on the T4 lysozyme system. This protocol is suitable for both novice and experienced practitioners. We show that with BRIDGE, protocols can be shared with collaborators or made publicly available, thus making simulation results and computations independently verifiable and reproducible.

Scope Garlic (Allium sativum) has been used for centuries as both a prophylactic and therapeutic medicinal agent to control inflammation-associated pathologies. To investigate the underlying mechanisms, we established an in vitro inflammatory model using RAW264.7 murine macrophages exposed to low-doses of lipopolysaccharide (LPS) in the presence of the garlic compounds allicin and Z-ajoene, mimicking regular garlic consumption. Methods and results Both allicin and Z-ajoene dampened both transcript and protein expression of the pro-inflammatory cytokines IL1β, IL6 and IL12β, and upregulated the expression of the anti-inflammatory cytokine IL10. Protein arrays of selected secreted inflammatory mediators confirmed that Z-ajoene has a pronounced down-regulatory effect on LPS-induced inflammatory cytokines and chemokines. Many of these proteins are known targets of the transcription factor STAT3; and indeed, Z-ajoene or its analogue dansyl-ajoene (DP) was found to decrease the phosphorylation and nuclear translocation of STAT3, and to covalently modify the protein by S-thiolation at Cys108, Cys367 and Cys687. It was further found that Z-ajoene dose-dependently and non-competitively inhibited the activity of COX2, possibly attributed to S-thiolation at Cys9 and Cys299. Conclusion The characterisation of Z-ajoene's activity of targeting and covalently modifying STAT3 and COX2, both important regulators of inflammation, may contribute to the health benefits of regular dietary garlic consumption. This article is protected by copyright. All rights reserved.

Motivation The pathway from genomics through proteomics and onto a molecular description of biochemical processes makes the discovery of drugs and biomaterials possible. A research framework common to genomics and proteomics is needed to conduct biomolecular simulations that will connect biological data to the dynamic molecular mechanisms of enzymes and proteins. Novice biomolecular modelers are faced with the daunting task of complex setups and a myriad of possible choices preventing their use of molecular simulations and their ability to conduct reliable and reproducible computations that can be shared with collaborators and verified for procedural accuracy. Results We present the foundations of Biomolecular Reaction and Interaction Dynamics Global Environment (BRIDGE) developed on the Galaxy platform that makes possible fundamental molecular dynamics of proteins through workflows and pipelines via commonly used packages, such as NAMD, GROMACS and CHARMM. BRIDGE can be used to set up and simulate biological macromolecules, perform conformational analysis from trajectory data and conduct data analytics of large scale protein motions using statistical rigor. We illustrate the basic BRIDGE simulation and analytics capabilities on a previously reported CBH1 protein simulation. Availability and implementation Publicly available at https://github.com/scientificomputing/BRIDGE and https://usegalaxy.eu Supplementary information Supplementary data are available at Bioinformatics online.

Understanding carbohydrate ring pucker is critical to rational design in materials and pharmaceuticals. Recently we have generalized our adaptive reaction coordinate force biasing method to perform calculations on multidimensional reaction coordinates. We termed this the Free Energies from Adaptive Reaction Coordinate Forces (FEARCF) method. Using FEARCF in SCC-DFTB QM/MM non-Boltzmann simulations, we are able to calculate multidimensional ring pucker free energies of conformation. Here we apply this to the six-membered glucopyranose ring located in an eight-membered β 1-4 linked octaose oligosaccharide (cellooctaose). The cellooctaose was built following the conformation of the saccharides bound to cellobiohydrolase I (CBHI) of Trichoderma reesei as reported in the 7CEL crystal structure obtained from the PDB. We calculate the free energy of ring puckering of the glucopyranose ring at the -1 position in vacuum, in water, and bound to the protein. We find that the protein induces (4)E and (4)H(3) conformations that are much more stable than the usually preferred (4)C(1) conformer. Furthermore, for the (4)H(3) conformation in the catalytic binding domain, there is significant electronic rearrangement that drives the structure toward the transition state of the glycosylation reaction.

Previously an adaptive reaction coordinate force biasing method based on probability distributions has been used to calculate the free energy of conformation, configuration and chemical reactions. This method has recently been generalised to perform calculations on multidimensional reaction coordinates. This paper presents details of this method, termed Free Energies from Adaptive Reaction Coordinate Forces (FEARCF). The efficiency of sampling of this method is demonstrated by applying it to the problem of sampling the many characteristic pucker conformations of a β-D-glucose pyranose using a semi-empirical PM3 Hamiltonian. The sampling ratio of the global minimum conformer (4C1) to the highest energy conformer (a planar hexapyranose ring) is 1.7:1. Pucker free energy surfaces such as the one presented here can be a useful tool in the analysis of enzymatic reactions involving molecular ring puckers in the Michaelis complex.

Motivation: Complex carbohydrates play a central role in cellular communication and in disease development. O- and N-glycans, which are post-translationally attached to proteins and lipids, are sugar chains that are rooted, tree structures. Independent efforts to develop computational tools for analyzing complex carbohydrate structures have been designed to exploit specific databases requiring unique formatting and limited transferability. Attempts have been made at integrating these resources, yet it remains difficult to communicate and share data across several online resources. A disadvantage of the lack of coordination between development efforts is the inability of the user community to create reproducible analyses (workflows). The latter results in the more serious unreliability of glycomics metadata. Results: In this paper, we realize the significance of connecting multiple online glycan resources that can be used to design reproducible experiments for obtaining, generating and analyzing cell glycomes. To address this, a suite of tools and utilities, have been integrated into the analytic functionality of the Galaxy bioinformatics platform to provide a Glycome Analytics Platform (GAP). Using this platform, users can design in silico workflows to manipulate various formats of glycan sequences and analyze glycomes through access to web data and services. We illustrate the central functionality and features of the GAP by way of example; we analyze and compare the features of the N-glycan glycome of monocytic cells sourced from two separate data depositions. This paper highlights the use of reproducible research methods for glycomics analysis and the GAP presents an opportunity for integrating tools in glycobioinformatics. Availability and Implementation: This software is open-source and available online at https://bitbucket.org/scientificomputing/glycome-analytics-platform Contacts: chris.barnett@uct.ac.za or kevin.naidoo@uct.ac.za Supplementary information: Supplementary data are available at Bioinformatics online.

The conformations and shapes of macromolecular structures in biological and synthetic materials often define the macroscopic functions of the systems. Tessellate and Montage provide a standardized toolset for rapid reporting of large datasets allowing comparisons of cyclic molecule conformations (ring pucker) from structural databases and simulation trajectory data. This facilitates an understanding of the dynamic transition between common conformations and the flexible range in a ring that underlies molecular behaviour and recognition properties.

Purine nucleoside phosphorylase is a transferase that catalyzes the addition of phosphate and removal of a purine base from guanosine and similar nucleosides. Here the interplay between sugar puckering conformation, the enzyme, and the perceived course of the reaction is examined using QM/MM FEARCF dynamics simulations. The enzyme biases the guanosine sugar ring toward a flattened (4)E conformer as a step that is critical to the success of the phosphorylation reaction. The C4' endo conformer allows the nonbonded ring oxygen orbital to align and donate electrons into the antibonding glycosidic bond orbital, thus weakening the bond. This conformational preference is due to sustained and directed noncovalent interactions anchored by the phosphate nucleophile's hydrogen bonds to the sugar C2' and C3' hydroxyls. In so doing, PNP alters the solution sugar ring pucker preferences as part of its catalytic reaction barrier lowering function.

Cyclic conformational coordinates are essential for the distinction of molecular ring conformers as the use of Cremer-Pople coordinates have illustrated for five- and six-membered rings. Here, by tessellating medium rings into triangular planes and using the relative angles made between triangular planes we are able to assign macrocyclic pucker conformations into canonical pucker conformers such as chairs, boats, etc. We show that the definition is straightforward compared with other methods popularly used for small rings and that it is computationally simple to implement for complex macrocyclic rings. These cyclic conformational coordinates directly couple to the motion of individual nodes of a ring. Therefore, they are useful for correlating the physical properties of macrocycles with their ring pucker and measuring the dynamic ring conformational behavior. We illustrate the triangular tessellation, assignment, and pucker analysis on 7- and 8-membered rings. Sets of canonical states are given for cycloheptane and cyclooctane that have been previously experimentally analysed.

Glycosylation of cellobiose hydrolase I (CBHI), is a key step in the processing and degradation of cellulose. Here the pathways and barriers of the reaction are explored using the free energy from adaptive reaction coordinate forces (FEARCF) reaction dynamics method coupled with SCC-DFTB/MM. In many respects CBHI follows the expected general GH7 family mechanism that involves the Glu-X-Asp-X-X-Glu motif. However, critical electronic and conformational details, previously not known, were discovered through our computations. The central feature that ensures the success of the glycosylation reaction are the Glu212 nucleophile's hydrogen bond to the hydroxyl on C2, of the glucose in the -1 position of the cellulosic strand. This Glu212 function restricts the C2 hydroxyl in such a way as to favor the formation of the (4)E ring pucker of the -1 position glucose. A frontier molecular orbital analysis of the structures along the reaction surface proves the existence of an oxocarbenium ion, which has both transition state and intermediate character. The transition state structure is able to descend down the glycosylation pathway through the critical combination of Asp214 (HOMO), ring oxygen (LUMO), and Glu212 (HOMO), anomeric carbon (LUMO) interactions. Using the fully converged FEARCF SCC-DFTB/MM reaction surface, we find a barrier of 17.48 kcal/mol separating bound cellulose chain from the glycosylated CBHI. Taking recrossing into account gives k(cat) = 0.415 s(-1) for cellobiohydrolase glycosylation.

The puckered conformations of furanose and pyranose carbohydrate rings are central to analyzing the action of enzymes on carbohydrates. Enzyme reaction mechanisms are generally inaccessible to experiments and so have become the focus of QM(semiempirical)/MM simulations. We show that the complete free energy of puckering is required to evaluate the accuracy of semiempirical methods used to study reactions involving carbohydrates. Interestingly, we find that reducing the free energy space to lower dimensions results in near meaningless minimum energy pathways. We analyze the furanose and pyranose free energy pucker surfaces and volumes using AM1, PM3, PM3CARB-1, and SCC-DFTB. A comparison with DFT optimized structures and a HF free energy surface reveals that SCC-DFTB provides the best semiempirical description of five- and six-membered carbohydrate ring deformation.

We implemented the free energy from adaptive reaction coordinate forces (FEARCF) method and applied it to the conformational investigation of carbohydrate ring puckering. The method allows for significantly enhanced sampling of reaction coordinate space. The free energies associated with the ring pucker motion of β‐D‐ribose (a furanose) and β‐D‐glucose (a pyranose) were calculated. These can be used to interpret catalytic mechanisms of glycosylases.

Although the conformational preferences in glucose and galactose have been studied since the early 1970s, only recently have the glucose and galactose hydroxymethyl populations been resolved by combining (3)J(HH) and (2)J(HH) NMR coupling data using a modified Karplus equation. A preference for gauche conformations is observed in monosaccharides, but the reasons for this are not understood. We calculated the free energy of rotation profiles for glucose and galactose primary alcohols using a semiempirical description of the monosaccharides in QM/MM simulations. From this we observed excellent agreement between our simulated population distributions for glucose gg/gt/tg = 35:57:3 and galactose gg/gt/tg = 4:86:7 with those measured from NMR. A stereoelectronic analysis of the minimum energy conformations using natural bond orbitals provides a clear description of the stabilizing contribution to the gauche conformers stemming from the C-H bonding and the C-O antibonding orbital interactions, specifically sigma(C6-H) --> sigma*(C5-O5) and sigma(C5-H) --> sigma*(C6-O6). Analysis of the solution trajectories reveals that persistent intramolecular hydrogen bonds and intermolecular bridging hydrogen bonds formed by water molecules between the ring oxygen and the hydroxymethyl group further stabilizes the gt conformation making it the preferred rotamer in both hydrated glucose and galactose. The hydroxymethyl quantum mechanics/molecular mechanics molecular dynamics trajectories and derived rotational free energies for these monosaccharides in water solutions explain that the experimental observations are due to a combination of competing stereoelectronic (gauche), electronic (intramolecular hydrogen bonding), and electrostatic (solvent-saccharide hydrogen bonding) factors.