Initial Stages of Spontaneous Binding of Folate-Based Vectors to Folate Receptor-α Observed by Unbiased Molecular Dynamics.

Active targeting is a prospective strategy for controlled drug delivery to malignant tumor tissues. One of the approaches relies on recognition of a bioactive ligand by a receptor expressed abundantly on the surface of cancer cell membranes. A promising ligand-receptor pair is folic acid (or its dianionic form, folate) combined with the folate receptor-α (FRα). A number of targeting drug delivery systems based on folate have been suggested, but the mechanism of binding of the ligand or its derivatives to the receptor is not fully known at the molecular level. The current study summarizes the results from unbiased all-atom molecular dynamics simulations at physiological conditions describing the binding of two forms of folate and four of its synthetically available derivatives to FRα. The models (ca. 185,000 atoms) contain one receptor molecule, embedded in the outer leaflet of a lipid bilayer, and one ligand, all immersed in saline. The bilayer represents a human cancer cell membrane and consists of 370 asymmetrically distributed lipid molecules from 35 types. The ability of the vector molecules to bind to the receptor, the position of binding, and the interactions between them are analyzed. Spontaneous binding on the nanosecond scale is observed for all molecules, but its time, position, and persistence depend strongly on the ligand. Only folate, 5-methyltetrahydrofolate, and raltitrexed bind selectively at the active site of the receptor. Two binding poses are observed, one of them (realized by raltitrexed) corresponding qualitatively to that reported for the crystallographic structure of the complex folate-FRα. Pemetrexed adsorbs nonspecifically on the protein surface, while methotrexate and pteroyl ornithine couple much less to the receptor. The molecular simulations reproduce qualitatively correctly the relative binding affinity measured experimentally for five of the ligands. Analysis of the interactions between the ligands and FRα shows that in order to accomplish specific binding to the active site, a combination of hydrogen bonding, π-stacking, and van der Waals and Coulomb attraction should be feasible simultaneously for the vector molecule. The reported results demonstrate that it is possible to observe receptor-ligand binding without applying bias by representing the local environment as close as possible and contain important molecular-level guidelines for the design of folate-based systems for targeted delivery of anticancer drugs.

Characteristics of a Folate Receptor-α Anchored into a Multilipid Bilayer Obtained from Atomistic Molecular Dynamics Simulations.

Thorough computational description of the properties of membrane-anchored protein receptors, which are important for example in the context of active targeting drug delivery, may be achieved by models representing as close as possible the immediate environment of these macromolecules. An all-atom bilayer, including 35 different lipid types asymmetrically distributed among the two monolayers, is suggested as a model neoplastic cell membrane. One molecule of folate receptor-α (FRα) is anchored into its outer leaflet, and the behavior of the system is explored by atomistic molecular dynamics simulations. The total number of atoms in the model is ∼185 000. Three 1-µs-long simulations are carried out, where physiological conditions (310 K and 1 bar) are maintained with three different pressure scaling schemes. To evaluate the structure and the phase state of the membrane, the density profiles of the system, the average area per lipid, and the deuterium order parameter of the lipid tails are calculated. The bilayer is in liquid ordered state, and the specific arrangement varies between the three trajectories. The changes in the structure of FRα are investigated and are found time- and ensemble-dependent. The volume of the ligand binding pocket fluctuates with time, but this variation remains independent of the more global structural alterations. The latter are mostly "waving" motions of the protein, which periodically approaches and retreats from the membrane. The semi-isotropic pressure scaling perturbs the receptor most significantly, while the isotropic algorithm induces rather slow changes. Maintaining constant nonzero surface tension leads to behavior closest to the experimentally observed one.

Molecular simulation of the structure of folate and antifolates at physiological conditions.

The study is focused on description of folate and several antifolates at physiological conditions. Knowledge of the molecular structure and dynamics is important for understanding their biological activity and therapeutic application. They are modelled in saline by atomistic molecular dynamics simulations and characterized in detail. In addition, quantum chemical calculations are used for determining the electronic structure of the six compounds. All molecules are highly flexible and have similar interactions with water. Specifics are found in some of their local backbone conformations, in the molecular shape, and in the electron density distribution. Most of the ligands have fairly folded geometry and prefer U- and Z-shapes. Two of them are quasi-linear. Key to the molecular shape are the bicyclic fragment, its bridge, and the charge of the terminal amino acid residue. Docking into the active site of folate receptor-α predicts a similar best binding pose for four of the ligands, which requires stretching of pterin and bending of glutamate/ornithine relative to the geometry in saline. The chemical modifications in the antifolates induce local electron density redistribution in comparison to folate, leading to increase of the positive charges of the neighboring fragments. The obtained results would help better tuning of the potential usage of the molecules in new bioactive materials, e.g., as vector-ligands for drug delivery.

Identification and computational characterization of isomers with cis and trans amide bonds in folate and its analogues.

Folate and its synthetic analogues, called antifolates, are known to have diverse bio-applications, for example as cell proliferation stimulators or anticancer drugs. Their molecular structure is important for performing the required biological activity. Since all folate-derived ligands contain a peptide-like amide bond, its configuration is one of the key components for the functional fitness of such compounds. During the modelling of folate and three of its derivatives - methotrexate, 5-methyl tetrahydrofolate, and pteroyl ornithine, we registered significant population of the cis isomers along the amide bond. The properties of the cis and trans forms of the ligands in saline are studied in detail by classical atomistic molecular dynamics and by quantum chemical methods. The calculations predict high probability for coexistence of the cis isomers for two of the ligands. The energetic instability of the cis form is explained with a σ-character admixture into the C[double bond, length as m-dash]O(π) bond, while its magnitude is attributed to the pattern of local electron density redistribution. The cis forms of all molecules have markedly slower structural dynamics than the trans ones, which might affect their behavior in vivo.

Unit cell structure of water-filled monoolein in inverted hexagonal mesophase in the presence of incorporated tricaprylin and entrapped lysozyme.

Molecular dynamics (MD) was employed by means of a specific simulation protocol to investigate the equilibrium structure at 25 °C of the hexagonal inverted (HII) mesophase composed from water, 1-monoolein (GMO), and tricaprylin, with or without entrapped lysozyme. Based on robust and fast MD simulations, the study provides a comprehensive analysis and visualization of the local structure of HII mesophase containing admixtures. The most important physical insight is the possibility to observe the strong self-recovery capacity of the GMO layer, which allows the HII mesophase tubes to reorganize and host lysozyme molecules with a size bigger than the diameter of the water channel. This is a direct message to the experimenters that the HII mesophase has the potential to host molecules larger than the diameter of the water channel. Collective character of the interlipid interactions is outlined, which is not affected by the presence of the cargo and may be the reason for the efficient GMO reorganization. Another important result is the possible explanation of the role of triacylglycerols on the low-temperature stabilization of the HII mesophase. The analysis shows that despite the low amount of tricaprylin, its molecules prevent the extreme inclination of the lipid tails and thus optimize the alignment capacity of the lipid tails layer. The study also reveals that the packing frustration does not depend on the temperature and the presence of admixtures. Hence, it might be numerically defined as a universal invariant parameter of a stable HII mesophase composed of a certain lipid.

Molecular Dynamics Simulation of the Aggregation Patterns in Aqueous Solutions of Bile Salts at Physiological Conditions.

Classical molecular dynamics simulations are employed to monitor the aggregation behavior of six bile salts (nonconjugated and glycine- and taurine-conjugated sodium cholate and sodium deoxycholate) with concentration of 10 mM in aqueous solution in the presence of 120 mM NaCl. There are 150 ns trajectories generated to characterize the systems. The largest stable aggregates are analyzed to determine their shape, size, and stabilizing forces. It is found that the aggregation is a hierarchical process and that its kinetics depends both on the number of hydroxyl groups in the steroid part of the molecules and on the type of conjugation. The micelles of all salts are similar in shape-deformed spheres or ellipsoids, which are stabilized by hydrophobic forces, acting between the steroid rings. The differences in the aggregation kinetics of the various conjugates are rationalized by the affinity for hydrogen bond formation for the glycine-modified salts or by the longer time needed to achieve optimum packing for the tauro derivatives. Evidence is provided for the hypothesis from the literature that the entirely hydrophobic core of all aggregates and the enhanced dynamics of the molecules therein should be among the prerequisites for their pronounced solubilization capacity for hydrophobic substances in vivo.

Unit cell structure of water-filled monoolein into inverted hexagonal (H(II)) mesophase modeled by molecular dynamics.

The study investigates the unit cell structure of inverted hexagonal (H(II)) mesophase composed of monoolein (1-monoolein, GMO) and water using atomistic molecular dynamics methods without imposing any restraints on lipid and water molecules. Statistically meaningful and very contrast images of the radial mass density distribution, scrutinizing also the separate components water, monoolein, the polar headgroups of the lipids, the double bond, and the termini of the hydrocarbon chain (the tail), are obtained. The lipid/water interface structure is analyzed based on the obtained water density distribution, on the estimated number of hydrogen bonds per monoolein headgroup, and on the headgroup-water radial distribution functions. The headgroup mass density distribution demonstrates hexagonal shape of the monoolein/water interface that is well-defined at higher water/monoolein ratios. Water interacts with the headgroups by forming a three-layer diffusive mass density distribution, and each layer's shape is close to hexagonal, which is an indication of long-range structural interactions. It is found that the monoolein headgroups form a constant number of hydrogen bonds leaving an excessive amount of water molecules outside the first lipid coordination sphere. Furthermore, the quantity of water at the monoolein/water interface increases steadily upon extension of the unit cell, so the interface should have a very dynamic structure. Investigation of the hydrocarbon residues reveals high compression and well-expressed structuring of the tails. The tails form a very compressed and constrained structure of defined layers across the unit cell with properties corresponding to a more densely packed nonpolar liquid (oil). Due to the hexagonal shape the 2D packing frustration is constant and does not depend on the water content. All reported structural features are based on averaging of the atomic coordinates over the time-length of the simulation trajectories. That kind of processing allows the observation of the water/GMO interface shape and its stability and mobility at a time scale close to the ones of the intermolecular interactions.

Absorption spectra of model single chains of conducting polyaniline.

The theoretical study addresses the type and nature of the transitions in the absorption spectra of octamers, dodecamers, and hexadecamers of the emeraldine salt-the conducting form of polyaniline. Each of the fully protonated oligomers is modeled in its lowest singlet (bipolaronic form) and highest possible multiplicity (polaronic form). Two configurations of the chloride counterions with respect to the oligomer chains are considered. All structures are optimized with BLYP/6-31G*/PCM, while the spectra are evaluated with CIS/6-31G*/PCM. The optical behavior of the bipolaronic and polaronic forms of the investigated systems is discussed and compared to relevant experimental data. The optical transitions at about 400 and 800 nm characteristic for the emeraldine salt are registered for all model structures. Weighed against experimental and earlier theoretical findings the results prove that CIS gives qualitatively correct electronic spectra of these conjugated species. While the two configurations have almost identical spectra in the highest multiplicity, the singlets' absorption conduct turns out to be sensitive to the counterions position. In all cases the most intensive absorption is the longest wavelength one in the near-IR region, but the number and oscillator strengths of the polaronic and bipolaronic bands are noticeably dissimilar. The bands of the low-spin oligomers are grouped, while those of the high-spin species cover the entire visible region. Each extension of the chain with one elementary unit contributes systematically a set of new bands to the spectrum. The possibility for a solvatochromic effect is estimated.

Molecular dynamics approach to water structure of H(II) mesophase of monoolein.

The goal of the present work is to study theoretically the structure of water inside the water cylinder of the inverse hexagonal mesophase (H(II)) of glyceryl monooleate (monoolein, GMO), using the method of molecular dynamics. To simplify the computational model, a fixed structure of the GMO tube is maintained. The non-standard cylindrical geometry of the system required the development and application of a novel method for obtaining the starting distribution of water molecules. A predictor-corrector schema is employed for generation of the initial density of water. Molecular dynamics calculations are performed at constant volume and temperature (NVT ensemble) with 1D periodic boundary conditions applied. During the simulations the lipid structure is kept fixed, while the dynamics of water is unrestrained. Distribution of hydrogen bonds and density as well as radial distribution of water molecules across the water cylinder show the presence of water structure deep in the cylinder (about 6 Å below the GMO heads). The obtained results may help understanding the role of water structure in the processes of insertion of external molecules inside the GMO∕water system. The present work has a semi-quantitative character and it should be considered as the initial stage of more comprehensive future theoretical studies.

Fully doped oligomers of emeraldine salt: polaronic versus bipolaronic configuration.

Calculations for model oligomers of the emeraldine salt with UBLYP/6-31G*/PCM are performed. The models differ in number of monomers, in the position of the counterions (Cl(-)), and in multiplicity. The molecular features affected most prominently by the protonation, namely, structure, energetics, and electron and spin density partitioning are analyzed. The results show unequivocally that the studied molecular characteristics are essentially size independent. The octamer profiles of all parameters are repeated in the dodecamer and the hexadecamer. The bipolaronic forms are energetically more favorable than the polaronic ones within the chosen protocol. The electronic structure in the intermediate multiplicities differs from the bipolaronic and polaronic periodicity. The geometrical changes and electron density redistribution upon increase of multiplicity illustrate the pathway of intramolecular bipolaron-polaron conversion. The orbital analysis rationalizes the observed behavior of the oligomers.