Simulating the Skin Permeation Process of Ionizable Molecules.

It is commonly assumed that ionizable molecules, such as drugs, permeate through the skin barrier in their neutral form. By using molecular dynamics simulations of the charged and neutral states separately, we can study the dynamic protonation behavior during the permeation process. We have studied three weak acids and three weak bases and conclude that the acids are ionized to a larger extent than the bases, when passing through the headgroup region of the lipid barrier structure, at pH values close to their pKa. It can also be observed that even if these dynamic protonation simulations are informative, in the cases studied herein they are not necessary for the calculation of permeability coefficients. It is sufficient to base the calculations only on the neutral form, as is commonly done.

On the Path to Optimal Alchemistry.

Alchemical free energy calculations have become a standard and widely used tool, in particular for calculating and comparing binding affinities of drugs. Although methods to compute such free energies have improved significantly over the last decades, the choice of path between the end states of interest is usually still the same as two decades ago. We will show that there is a fundamentally arbitrary, implicit choice of parametrization of this path. To address this, the notion of the length of a path or a metric is required. A metric recently introduced in the context of the accelerated weight histogram method also proves to be very useful here. We demonstrate that this metric can not only improve the efficiency of sampling along a given path, but that it can also be used to improve the actual choice of path. For a set of relevant use cases, the combination of these improvements can increase the efficiency of alchemical free energy calculations by up to a factor 16.

Understanding Drug Skin Permeation Enhancers Using Molecular Dynamics Simulations.

Our skin constitutes an effective permeability barrier that protects the body from exogenous substances but concomitantly severely limits the number of pharmaceutical drugs that can be delivered transdermally. In topical formulation design, chemical permeation enhancers (PEs) are used to increase drug skin permeability. In vitro skin permeability experiments can measure net effects of PEs on transdermal drug transport, but they cannot explain the molecular mechanisms of interactions between drugs, permeation enhancers, and skin structure, which limits the possibility to rationally design better new drug formulations. Here we investigate the effect of the PEs water, lauric acid, geraniol, stearic acid, thymol, ethanol, oleic acid, and eucalyptol on the transdermal transport of metronidazole, caffeine, and naproxen. We use atomistic molecular dynamics (MD) simulations in combination with developed molecular models to calculate the free energy difference between 11 PE-containing formulations and the skin's barrier structure. We then utilize the results to calculate the final concentration of PEs in skin. We obtain an RMSE of 0.58 log units for calculated partition coefficients from water into the barrier structure. We then use the modified PE-containing barrier structure to calculate the PEs' permeability enhancement ratios (ERs) on transdermal metronidazole, caffeine, and naproxen transport and compare with the results obtained from in vitro experiments. We show that MD simulations are able to reproduce rankings based on ERs. However, strict quantitative correlation with experimental data needs further refinement, which is complicated by significant deviations between different measurements. Finally, we propose a model for how to use calculations of the potential of mean force of drugs across the skin's barrier structure in a topical formulation design.

Skin permeability prediction with MD simulation sampling spatial and alchemical reaction coordinates.

A molecular-level understanding of skin permeation may rationalize and streamline product development, and improve quality and control, of transdermal and topical drug delivery systems. It may also facilitate toxicity and safety assessment of cosmetics and skin care products. Here, we present new molecular dynamics simulation approaches that make it possible to efficiently sample the free energy and local diffusion coefficient across the skin's barrier structure to predict skin permeability and the effects of chemical penetration enhancers. In particular, we introduce a new approach to use two-dimensional reaction coordinates in the accelerated weight histogram method, where we combine sampling along spatial coordinates with an alchemical perturbation virtual coordinate. We present predicted properties for 20 permeants, and demonstrate how our approach improves correlation with ex vivo/in vitro skin permeation data. For the compounds included in this study, the obtained log KPexp-calc mean square difference was 0.9 cm2 h-2.

The Skin's Barrier: A Cryo-EM Based Overview of its Architecture and Stepwise Formation.

A major role of the skin is to serve as a barrier toward the environment. The skin's permeability barrier consists of a lipid structure positioned in the stratum corneum. Recent progress in high-resolution cryo-electron microscopy (cryo-EM) has allowed for elucidation of the architecture of the skin's barrier and its stepwise formation process representing the final stage of epidermal differentiation. In this review, we present an overview of the skin's barrier structure and its formation process, as evidenced by cryo-EM.

Molecular Reorganization during the Formation of the Human Skin Barrier Studied In Situ.

In vertebrates, skin upholds homeostasis by preventing body water loss. The skin's permeability barrier is located intercellularly in the stratum corneum and consists of stacked lipid lamellae composed of ceramides, cholesterol, and free fatty acids. We have combined cryo-electron microscopy with molecular dynamics modeling and electron microscopy simulation in our analysis of the lamellae's formation, a maturation process beginning in stratum granulosum and ending in stratum corneum. Previously, we have revealed the lipid lamellae's initial- and end-stage molecular organizations. In this study, we reveal two cryo-electron microscopy patterns representing intermediate stages in the lamellae's maturation process: a single-band pattern with 2.0‒2.5 nm periodicity and a two-band pattern with 5.5‒6.0 nm periodicity, which may be derived from lamellar lipid structures with 4.0‒5.0 nm and 5.5‒6.0 nm periodicity, respectively. On the basis of the analysis of the data now available on the four maturation stages identified, we can present a tentative molecular model for the complete skin barrier formation process.

Heterogeneous parallelization and acceleration of molecular dynamics simulations in GROMACS.

The introduction of accelerator devices such as graphics processing units (GPUs) has had profound impact on molecular dynamics simulations and has enabled order-of-magnitude performance advances using commodity hardware. To fully reap these benefits, it has been necessary to reformulate some of the most fundamental algorithms, including the Verlet list, pair searching, and cutoffs. Here, we present the heterogeneous parallelization and acceleration design of molecular dynamics implemented in the GROMACS codebase over the last decade. The setup involves a general cluster-based approach to pair lists and non-bonded pair interactions that utilizes both GPU and central processing unit (CPU) single instruction, multiple data acceleration efficiently, including the ability to load-balance tasks between CPUs and GPUs. The algorithm work efficiency is tuned for each type of hardware, and to use accelerators more efficiently, we introduce dual pair lists with rolling pruning updates. Combined with new direct GPU-GPU communication and GPU integration, this enables excellent performance from single GPU simulations through strong scaling across multiple GPUs and efficient multi-node parallelization.

Predicting drug permeability through skin using molecular dynamics simulation.

Understanding and predicting permeability of compounds through skin is of interest for transdermal delivery of drugs and for toxicity predictions of chemicals. We show, using a new atomistic molecular dynamics model of the skin's barrier structure, itself validated against near-native cryo-electron microscopy data from human skin, that skin permeability to the reference compounds benzene, DMSO (dimethyl sulfoxide), ethanol, codeine, naproxen, nicotine, testosterone and water can be predicted. The permeability results were validated against skin permeability data in the literature. We have investigated the relation between skin barrier molecular organization and permeability using atomistic molecular dynamics simulation. Furthermore, it is shown that the calculated mechanism of action differs between the five skin penetration enhancers Azone, DMSO, oleic acid, stearic acid and water. The permeability enhancing effect of a given penetration enhancer depends on the permeating compound and on the concentration of penetration enhancer inside the skin's barrier structure. The presented method may open the door for computer based screening of the permeation of drugs and toxic compounds through skin.

Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation.

In the present study we have analyzed the molecular structure and function of the human skin's permeability barrier using molecular dynamics simulation validated against cryo-electron microscopy data from near native skin. The skin's barrier capacity is located to an intercellular lipid structure embedding the cells of the superficial most layer of skin - the stratum corneum. According to the splayed bilayer model (Iwai et al., 2012) the lipid structure is organized as stacked bilayers of ceramides in a splayed chain conformation with cholesterol associated with the ceramide sphingoid moiety and free fatty acids associated with the ceramide fatty acid moiety. However, knowledge about the lipid structure's detailed molecular organization, and the roles of its different lipid constituents, remains circumstantial. Starting from a molecular dynamics model based on the splayed bilayer model, we have, by stepwise structural and compositional modifications, arrived at a thermodynamically stable molecular dynamics model expressing simulated electron microscopy patterns matching original cryo-electron microscopy patterns from skin extremely closely. Strikingly, the closer the individual molecular dynamics models' lipid composition was to that reported in human stratum corneum, the better was the match between the models' simulated electron microscopy patterns and the original cryo-electron microscopy patterns. Moreover, the closest-matching model's calculated water permeability and thermotropic behaviour were found compatible with that of human skin. The new model may facilitate more advanced physics-based skin permeability predictions of drugs and toxicants. The proposed procedure for molecular dynamics based analysis of cellular cryo-electron microscopy data might be applied to other biomolecular systems.

Structural Transitions in Ceramide Cubic Phases during Formation of the Human Skin Barrier.

The stratum corneum is the outermost layer of human skin and the primary barrier toward the environment. The barrier function is maintained by stacked layers of saturated long-chain ceramides, free fatty acids, and cholesterol. This structure is formed through a reorganization of glycosylceramide-based bilayers with cubic-like symmetry into ceramide-based bilayers with stacked lamellar symmetry. The process is accompanied by deglycosylation of glycosylceramides and dehydration of the skin barrier lipid structure. Using coarse-grained molecular dynamics simulation, we show the effects of deglycosylation and dehydration on bilayers of human skin glycosylceramides and ceramides, folded in three dimensions with cubic (gyroid) symmetry. Deglycosylation of glycosylceramides destabilizes the cubic lipid bilayer phase and triggers a cubic-to-lamellar phase transition. Furthermore, subsequent dehydration of the deglycosylated lamellar ceramide system closes the remaining pores between adjacent lipid layers and locally induces a ceramide chain transformation from a hairpin-like to a splayed conformation.

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor.

Molecular simulation workflows as parallel algorithms: the execution engine of Copernicus, a distributed high-performance computing platform.

Computational chemistry and other simulation fields are critically dependent on computing resources, but few problems scale efficiently to the hundreds of thousands of processors available in current supercomputers-particularly for molecular dynamics. This has turned into a bottleneck as new hardware generations primarily provide more processing units rather than making individual units much faster, which simulation applications are addressing by increasingly focusing on sampling with algorithms such as free-energy perturbation, Markov state modeling, metadynamics, or milestoning. All these rely on combining results from multiple simulations into a single observation. They are potentially powerful approaches that aim to predict experimental observables directly, but this comes at the expense of added complexity in selecting sampling strategies and keeping track of dozens to thousands of simulations and their dependencies. Here, we describe how the distributed execution framework Copernicus allows the expression of such algorithms in generic workflows: dataflow programs. Because dataflow algorithms explicitly state dependencies of each constituent part, algorithms only need to be described on conceptual level, after which the execution is maximally parallel. The fully automated execution facilitates the optimization of these algorithms with adaptive sampling, where undersampled regions are automatically detected and targeted without user intervention. We show how several such algorithms can be formulated for computational chemistry problems, and how they are executed efficiently with many loosely coupled simulations using either distributed or parallel resources with Copernicus.

NMR chemical shift prediction of glycans: application of the computer program CASPER in structural analysis.

Carbohydrate molecules have highly complex structures and the constituent monosaccharides and substituents are linked to each other in a large number of ways. NMR spectroscopy can be used to unravel these structures, but the process may be tedious and time-consuming. The computerized approach based on the CASPER program can facilitate rapid structural determination of glycans with little user intervention, which results in the most probable primary structure of the investigated carbohydrate material. Additionally, (1)H and (13)C NMR chemical shifts of a user-defined structure can be predicted, and this tool may thus be employed in many aspects where NMR spectroscopy plays an important part of a study.

Automatic GROMACS topology generation and comparisons of force fields for solvation free energy calculations.

Free energy calculation has long been an important goal for molecular dynamics simulation and force field development, but historically it has been challenged by limited performance, accuracy, and creation of topologies for arbitrary small molecules. This has made it difficult to systematically compare different sets of parameters to improve existing force fields, but in the past few years several authors have developed increasingly automated procedures to generate parameters for force fields such as Amber, CHARMM, and OPLS. Here, we present a new framework that enables fully automated generation of GROMACS topologies for any of these force fields and an automated setup for parallel adaptive optimization of high-throughput free energy calculation by adjusting lambda point placement on the fly. As a small example of this automated pipeline, we have calculated solvation free energies of 50 different small molecules using the GAFF, OPLS-AA, and CGenFF force fields and four different water models, and by including the often neglected polarization costs, we show that the common charge models are somewhat underpolarized.

Rapid structural elucidation of polysaccharides employing predicted functions of glycosyltransferases and NMR data: application to the O-antigen of Escherichia coli O59.

A computerized method that uses predicted functions of glycosyltransferases (GTs) in conjunction with unassigned NMR data has been developed for the structural elucidation of bacterial polysaccharides (PSs). In this approach, information about the action of GTs (consisting of possible sugar residues used as donors and/or acceptors, as well as the anomeric configuration and/or substitution position in the respective glycosidic linkages) is extracted from the Escherichia coli O-antigen database and is submitted, together with the unassigned NMR data, to the CASPER program. This time saving methodology, which alleviates the need for chemical analysis, was successfully implemented in the structural elucidation of the O-antigen PS of E. coli O59. The repeating unit of the O-specific chain was determined using the O-deacylated PS and has a branched structure, namely, →6)[α-d-GalpA3Ac/4Ac-(1 → 3)]-α-d-Manp-(1 → 3)-α-d-Manp-(1 → 3)-ß-d-Manp-(1 → 3)-α-d-GlcpNAc-(1→. The identification of the O-acetylation positions was efficiently performed by comparison of the (1)H,(13)C HSQC NMR spectra of the O-deacylated lipopolysaccharide and the lipid-free PS in conjunction with chemical shift predictions made by the CASPER program. The side-chain d-GalpA residue carries one equivalent of O-acetyl groups at the O-3 and O-4 positions distributed in the LPS in a 3:7 ratio, respectively. The presence of O-acetyl groups in the repeating unit of the E. coli O59 PS is consistent with the previously proposed acetyltransferase WclD in the O-antigen gene cluster.

An efficient and extensible format, library, and API for binary trajectory data from molecular simulations.

Molecular dynamics simulations is an important application in theoretical chemistry, and with the large high-performance computing resources available today the programs also generate huge amounts of output data. In particular in life sciences, with complex biomolecules such as proteins, simulation projects regularly deal with several terabytes of data. Apart from the need for more cost-efficient storage, it is increasingly important to be able to archive data, secure the integrity against disk or file transfer errors, to provide rapid access, and facilitate exchange of data through open interfaces. There is already a whole range of different formats used, but few if any of them (including our previous ones) fulfill all these goals. To address these shortcomings, we present "Trajectory Next Generation" (TNG)--a flexible but highly optimized and efficient file format designed with interoperability in mind. TNG both provides state-of-the-art multiframe compression as well as a container framework that will make it possible to extend it with new compression algorithms without modifications in programs using it. TNG will be the new file format in the next major release of the GROMACS package, but it has been implemented as a separate library and API with liberal licensing to enable wide adoption both in academic and commercial codes.

An in silico virtual screening study for the design of norovirus inhibitors: fragment-based molecular docking and binding free energy calculations.

Gastrointestinal infections caused by noroviruses may be prevented by the inhibition of their binding to histo-blood group carbohydrate antigens. A fragment-based virtual screening approach was used, employing docking followed by molecular dynamics simulations in order to enable binding free energy calculations using the linear interaction energy method. The resulting structures, composed of high-affinity fragments, can be a good starting point for lead optimizations and four molecules that pass both REOS and SYLVIA filters, which can remove known toxic features and assess the synthetic accessibility, respectively, are proposed as inhibitors.

Small molecules containing hetero-bicyclic ring systems compete with UDP-Glc for binding to WaaG glycosyltransferase.

The α-1,3-glucosyltransferase WaaG is involved in the synthesis of the core region of lipopolysaccharides in E. coli. A fragment-based screening for inhibitors of the WaaG glycosyltrasferase donor site has been performed using NMR spectroscopy. Docking simulations were performed for three of the compounds of the fragment library that had shown binding activity towards WaaG and yielded 3D models for the respective complexes. The three ligands share a hetero-bicyclic ring system as a common structural motif and they compete with UDP-Glc for binding. Interestingly, one of the compounds promoted binding of uridine to WaaG, as seen from STD NMR titrations, suggesting a different binding mode for this ligand. We propose these compounds as scaffolds for the design of selective high-affinity inhibitors of WaaG. Binding of natural substrates, enzymatic activity and donor substrate selectivity were also investigated by NMR spectroscopy. Molecular dynamics simulations of WaaG were carried out with and without bound UDP and revealed structural changes compared to the crystal structure and also variations in flexibility for some amino acid residues between the two WaaG systems studied.

Structural studies of the O-antigen polysaccharide from Escherichia coli TD2158 having O18 serogroup specificity and aspects of its interaction with the tailspike endoglycosidase of the infecting bacteriophage HK620.

We have analyzed the O-antigen polysaccharide of the previously uncharacterized Escherichia coli strain TD2158 which is a host of bacteriophage HK620. This bacteriophage recognizes and cleaves the polysaccharide with its tailspike protein (TSP). The polysaccharide preparation as well as oligosaccharides obtained from HK620TSP endoglycosidase digests were analyzed with NMR spectroscopy. Additionally, sugar analysis was performed on the O-antigen polysaccharide and MALDI-TOF MS was used in oligosaccharide analysis. The present study revealed a heterogeneous polysaccharide with a hexasaccharide repeating unit of the following structure: α-D-Glcp-(1→6|) →2)-α-L-Rhap-91→6)-α-D-Glcp-(1→4)-α-D-Ga|lp-(1→3)-α-D-GlcpNAc-(1→ ß-D-Glcp/ß-D-GlcpNAc-(1→3) A repeating unit with a D-GlcNAc substitution of D-Gal has been described earlier as characteristic for serogroup O18A1. Accordingly, we termed repeating units with D-Glc substitution at D-Gal as O18A2. NMR analyses of the polysaccharide confirmed that O18A1- and O18A2-type repeats were present in a 1:1 ratio. However, HK620TSP preferentially bound the D-GlcNAc-substituted O18A1-type repeating units in its high affinity binding pocket with a dissociation constant of 140 µM and disfavored the O18A2-type having a ß-D-Glcp-(1→3)-linked group. As a result, in hexasaccharide preparations, O18A1 and O18A2 repeats were present in a 9:1 ratio stressing the clear preference of O18A1-type repeats to be cleaved by HK620TSP.

Structural studies and biosynthetic aspects of the O-antigen polysaccharide from Escherichia coli O174.

The structure of the repeating unit of the O-antigenic polysaccharide (PS) from Escherichia coli O174 has been determined. Component analysis together with (1)H and (13)C NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by (1)H,(13)C-heteronuclear multiple-bond correlation and (1)H,(1)H-NOESY experiments. The PS is composed of tetrasaccharide repeating units with the following structure: [formula see text] Cross-peaks of low intensity were present in the NMR spectra consistent with a ß-D-GlcpNAc-(1→2)-ß-D-GlcpA(1→ structural element at the terminal part of the polysaccharide, which on average is composed of ∼15 repeating units. Consequently the biological repeating unit has a 3-substituted N-acetyl-D-galactosamine residue at its reducing end.