Structure determination of an amorphous drug through large-scale NMR predictions.

Knowledge of the structure of amorphous solids can direct, for example, the optimization of pharmaceutical formulations, but atomic-level structure determination in amorphous molecular solids has so far not been possible. Solid-state nuclear magnetic resonance (NMR) is among the most popular methods to characterize amorphous materials, and molecular dynamics (MD) simulations can help describe the structure of disordered materials. However, directly relating MD to NMR experiments in molecular solids has been out of reach until now because of the large size of these simulations. Here, using a machine learning model of chemical shifts, we determine the atomic-level structure of the hydrated amorphous drug AZD5718 by combining dynamic nuclear polarization-enhanced solid-state NMR experiments with predicted chemical shifts for MD simulations of large systems. From these amorphous structures we then identify H-bonding motifs and relate them to local intermolecular complex formation energies.

De novo tertiary structure prediction using RNA123--benchmarking and application to Macugen.

The present benchmarking study utilizes the RNA123 program for de novo prediction of tertiary structures of a set of 50 RNA molecules for which X-ray/NMR structures are available, based on the nucleic acid sequence only. All molecules contain a hairpin loop motif and a helical structure of canonical and non-canonical base pairs, interrupted by bulges and internal loops to various degrees. RNA molecules with double helices made up purely by canonical base pairing, and molecules containing symmetric internal loops of non-canonical base pairing are, overall, very well predicted. Structures containing bulges and asymmetric internal loops, and more complex structures containing multiple bulges and internal loops in the same molecule, result in larger deviations from their X-ray/NMR predicted structures due to higher degree of flexibility of the nucleotide bases in these regions. In a majority of the molecules included herein, the RNA123 program was, however, able to predict the tertiary structure with a heavy atom RMSD of less than 5 Å to the X-ray/NMR structure, and the models were in most cases structurally closer to the X-ray/NMR structures than models predicted by MC-Fold and MC-Sym. A set of RNA molecules containing pseudoknot tertiary structure motifs were included, but neither of the programs was able to predict the folding of the single-stranded stem onto the helix without additional structural input. The RNA123 program was then applied to predict the tertiary structure of the RNA segment of Macugen®, the first RNA aptamer approved for clinical use, and for which no tertiary structure has yet been solved. Four possible tertiary structures were predicted for this 27-nucleic-acid-long RNA molecule, which will be used in constructing a full model of the PEGylated aptamer and its interaction with the vascular endothelial growth factor target.

Molecular Dynamics Studies of Liposomes as Carriers for Photosensitizing Drugs: Development, Validation, and Simulations with a Coarse-Grained Model.

Liposomes are proposed as drug delivery systems and can in principle be designed so as to cohere with specific tissue types or local environments. However, little detail is known about the exact mechanisms for drug delivery and the distributions of drug molecules inside the lipid carrier. In the current work, a coarse-grained (CG) liposome model is developed, consisting of over 2500 lipids, with varying degrees of drug loading. For the drug molecule, we chose hypericin, a natural compound proposed for use in photodynamic therapy, for which a CG model was derived and benchmarked against corresponding atomistic membrane bilayer model simulations. Liposomes with 21-84 hypericin molecules were generated and subjected to 10 microsecond simulations. Distribution of the hypericins, their orientations within the lipid bilayer, and the potential of mean force for transferring a hypericin molecule from the interior aqueous "droplet" through the liposome bilayer are reported herein.

Identifying the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) as a potential target for hypericin--a theoretical study.

The exact cellular target for the potent anti-cancer agent hypericin has not yet been determined; this thus encourages the application of computational chemistry tools to be employed in order to provide insights that can be employed in further drug development studies. In the present study computational docking and molecular dynamics simulations are applied to investigate possible interactions between hypericin and the Ca(2+) pump SERCA as proposed in the literature. Hypericin was found to bind strongly both in pockets within the transmembrane region and in the cytosolic region of the protein, although the two studied isoforms of SERCA differ slightly in their preferred binding sites. The calculated binding energies for hypericin in the four investigated sites were of the same magnitude as for thapsigargin (TG), the most potent SERCA inhibitor, or in the range between TG and di-tert-butylhydroquinone (BHQ), which is also known to possess inhibitory activity. The hydrophobic character of hypericin indicates that the molecule initially binds in the ER membrane from which it diffuses into the transmembrane region of the protein and to binding pockets therein. The transmembrane TG and BHQ binding pockets provide suitable locations for hypericin as they allow for favourable interactions with the lipid tails that surround these. High binding energies were noted for hypericin in these pockets and are expected to constitute highly possible binding sites due to their accessibility from the ER membrane. Hypericin most likely binds to both isoforms of SERCA and acts as an inhibitor or, under light irradiation, as a singlet oxygen generator that in turn degrades the protein or induces lipid peroxidation.

7-Nitro-4-(phenylthio)benzofurazan is a potent generator of superoxide and hydrogen peroxide.

Here, we report on 7-nitro-4-(phenylthio)benzofurazan (NBF-SPh), the most potent derivative among a set of patented anticancer 7-nitrobenzofurazans (NBFs), which have been suggested to function by perturbing protein-protein interactions. We demonstrate that NBF-SPh participates in toxic redox-cycling, rapidly generating reactive oxygen species (ROS) in the presence of molecular oxygen, and this is the first report to detail ROS production for any of the anticancer NBFs. Oxygraph studies showed that NBF-SPh consumes molecular oxygen at a substantial rate, rivaling even plumbagin, menadione, and juglone. Biochemical and enzymatic assays identified superoxide and hydrogen peroxide as products of its redox-cycling activity, and the rapid rate of ROS production appears to be sufficient to account for some of the toxicity of NBF-SPh (LC(50) = 12.1 µM), possibly explaining why tumor cells exhibit a sharp threshold for tolerating the compound. In cell cultures, lipid peroxidation was enhanced after treatment with NBF-SPh, as measured by 2-thiobarbituric acid-reactive substances, indicating a significant accumulation of ROS. Thioglycerol rescued cell death and increased survival by 15-fold to 20-fold, but pyruvate and uric acid were ineffective protectants. We also observed that the redox-cycling activity of NBF-SPh became exhausted after an average of approximately 19 cycles per NBF-SPh molecule. Electrochemical and computational analyses suggest that partial reduction of NBF-SPh enhances electrophilicity, which appears to encourage scavenging activity and contribute to electrophilic toxicity.

1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119): a cytotoxic prodrug with two stable conformations differing in biological and physical properties.

The anticancer prodrug 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119) selectively releases a short-lived cytotoxin following enzymatic reduction in hypoxic environments found in solid tumors. KS119, in addition to two enantiomers, has two stable atropisomers (conformers differing in structure owing to hindered bond rotation) that interconvert at 37 °C in aqueous solution by first-order kinetics with t(1/2) values of ∼50 and ∼64 h. The atropisomers differ in physical properties such as partition coefficients that allow their chromatographic separation on non-chiral columns. A striking difference in the rate of metabolism of the two atropisomers occurs in intact EMT6 murine mammary carcinoma cells under oxygen-deficient conditions. A structurally related molecule, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(3-hydroxy-4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119WOH), was also found to exist in similar stable atropisomers. The ratio of the atropisomers of KS119 and structurally related agents has the potential to impact the bioavailability, activation, and therapeutic activity. Thus, thermally stable atropisomers/conformers in small molecules can result in chemically and enantiomerically pure compounds having differences in biological activities.

Computational design of chlorin based photosensitizers with enhanced absorption properties.

The porphyrin and chlorin parent compounds constitute the base of many potent photosensitizers aimed to be utilized in photodynamic therapy (PDT). However, the photosensitizers available on the market today are not ideal for use in PDT; many of them suffering from drawbacks such as long-lasting photosensitization or absorption at wavelengths below the optimal tissue penetration. This has emphasized the need of new photosensitizers with improved photodynamic properties. In the present study we have used density functional theory based methods to design new chlorin compounds with conjugated substituents such as vinyl groups and carboxylic acids, aiming for strong absorption in the therapeutic window of PDT. The specific substituent positions were found to have a significant effect on the spectra. A chlorin with four propenoic acids was able to red-shift the absorption the most compared with non-substituted chlorin, generating the red-most absorption at 755 nm, and with significantly enhanced oscillator strengths. The results from the present study constitute a useful starting point for further design of tetrapyrrole derivatives as improved photosensitizers.

Predictive power of long-range corrected functionals on the spectroscopic properties of tetrapyrrole derivatives for photodynamic therapy.

Porphyrin and chlorin based compounds possess promising properties to be utilized as photosensitizers in photodynamic therapy (PDT). However, the photosensitizers available on the market today are not ideal for use in PDT, which has emphasized the need for new photosensitizers with improved photodynamic properties to be developed. Computational drug-design can be utilized in the search for improved pharmaceutical compounds, provided that the methods used are able to reproduce experimental data. In the present study we investigated, by the use of time-dependent density functional theory (TD-DFT), the performance of the long-range corrected functionals ωB97, ωB97X and ωB97XD on their ability to predict low-lying singlet excitations (>600 nm) of a set of well-known photosensitizing compounds. It was found that ωB97X reproduced the experimental red-most absorption band most satisfactorily. The use of either B3LYP, ωB97XD or M06 in geometry optimizations has a minor effect on the spectra in most cases. Calculated energy differences between the optimized singlet ground states and optimized first excited triplet states show consistent and overall higher triplet state energies for B3LYP, M06, and PBE0 compared with ωB97, ωB97X, and ωB97XD. The calculated triplet state energies are, however, sufficient to generate singlet oxygen in most cases.

The Influence of Cholesterol on the Properties and Permeability of Hypericin Derivatives in Lipid Membranes.

The promising photosensitizing properties of hypericin, a natural quinine substituted with hydroxyl and alkyl groups, have led to the proposal that it can be utilized in photodynamic therapy. Neither the detailed mechanism behind the powerful action of hypericin, arising as a result of light excitation, nor the intracellular localization and transportation of the molecule is yet fully understood. The behavior of hypericin derivatives in a pure dipalmitoylphosphatidylcholine (DPPC) lipid membrane has recently been studied theoretically by means of molecular dynamics simulations. Natural membranes however contain many important constituents-cholesterol being one of the most essential-that influence the function and structure of the membrane, and thereby also the behavior of drug molecules therein. In the present study, we investigated hypericin and its brominated derivatives in membranes containing 9 and 25 mol % cholesterol. The results show that the presence of cholesterol in the membrane affects the permeability of the hypericin molecules and does so differently for the various molecules in the two membranes. Hypericin containing one bromine was found to exhibit the lowest free energy profile for the transport process into the lipids, and also the highest permeability coefficients, indicating that this molecule displays the fastest and easiest diffusion in the membranes. All three molecules were found to accumulate most preferably close to the polar headgroup region in both membranes.

Can Range-Separated and Hybrid DFT Functionals Predict Low-Lying Excitations? A Tookad Case Study.

The spectral properties of Tookad (Pd-bacteriopheophorbide, Pd-BPheid), an effective photosensitizer that targets mainly prostate tumors, and metal-free BPheid have been studied using time-dependent density functional theory (TD-DFT). The well-established B3LYP functional, which is known to overestimate excitation energies, was included in the study along with recently introduced range-separated and meta hybrid DFT functionals CAM-B3LYP, M06, M06-2X, M06HF, ωB97XD, ωB97X, ωB97, LC-ωPBE, and PBE0 (PBE1PBE). The main focus is the performance of the new functionals in predicting low-lying excitations (>600 nm), to explore their potential roles in drug development for photodynamic therapy. The data suggests that ωB97XD overall performs best for the Qy transition band (the red-most absorption), followed by CAM-B3LYP. LC-ωPBE, ωB97, B3LYP, and PBE1PBE all generated the Qy band far from the experimental position. The error in absorption energy for the Qy band was found to be at most 0.05 eV for ωB97XD, compared to 0.15-0.19 eV for B3LYP. The use of different basis sets used in excited-state calculations was shown to be of less importance as was the use of either B3LYP or M06 in geometry optimizations.

Properties and Permeability of Hypericin and Brominated Hypericin in Lipid Membranes.

The promising photosensitizing properties of hypericin, a substituted phenanthroperylene quinone naturally found in Saint John's wort, has led to the proposal that it can be utilized in photodynamic therapy. Structurally modified derivatives are at the present time being investigated to generate a more effective hypericin photosensitizer. Neither the detailed mechanism behind the powerful action of hypericin, arising as a result of light excitation, nor the intracellular localization and transportation is still fully understood. In the present work, molecular dynamics simulations have been performed to study the properties and the permeability of hypericin and modifications thereof, substituted with one or four bromine atoms, in a dipalmitoylphosphatidylcholine lipid membrane. The molecules were found to accumulate in the most dense region of the lipids due to competing interactions with the hydrophobic lipid interior and the polar aqueous environment. This was confirmed by analyzing the radial distribution functions and by the density profiles of the system components. Calculated free energy profiles display large negative changes in free energy for the transport process of the molecules into the lipids, which also support this finding. Permeability coefficients show overall fastest diffusion in the membrane system for hypericin containing one bromine.