A concept of dual-responsive prodrugs based on oligomerization-controlled reactivity of ester groups: an improvement of cancer cells versus neutrophils selectivity of camptothecin.

Many known chemotherapeutic anticancer agents exhibit neutropenia as a dose-limiting side effect. In this paper we suggest a prodrug concept solving this problem for camptothecin (HO-cpt). The prodrug is programmed according to Boolean "AND" logic. In the absence of H2O2 (trigger T1), e.g. in the majority of normal cells, it exists as an inactive oligomer. In cancer cells and in primed neutrophils (high H2O2), the oligomer is disrupted forming intermediate (inactive) lipophilic cationic species. These are accumulated in mitochondria (Mit) of cancer cells, where they are activated by hydrolysis at mitochondrial pH 8 (trigger T2) with formation of camptothecin. In contrast, the intermediates remain stable in neutrophils lacking Mit and therefore a source of T2. In this paper we demonstrated a proof-of-concept. Our prodrug exhibits antitumor activity both in vitro and in vivo, but is not toxic to normal cell and neutrophils in contrast to known single trigger prodrugs and the parent drug HO-cpt.

In Silico Study of Camptothecin-Based Pro-Drugs Binding to Human Carboxylesterase 2.

Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme-ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue.

Oxidation Enhances Binding of Extrahelical 5-Methyl-Cytosines by Thymine DNA Glycosylase.

The DNA repair protein thymine DNA glycosylase (TDG) removes mispaired or damaged bases, such as oxidized methyl-cytosine, from DNA by cleavage of the glycosidic bond between the sugar and the target base flipped into the enzyme's active site. The enzyme is active against formyl-cytosine and carboxyl-cytosine, whereas the lower oxidized hydroxymethyl-cytosine and methyl-cytosine itself are not processed by the enzyme. Molecular dynamics simulations with thermodynamic integration of TDG complexed to DNA carrying one of four different (oxidized) methyl-cytosine bases in extrahelcial conformation, methyl-cytosine (mC), hydroxymethyl-cytosine (hmC), formyl-cytosine (fC), or carboxyl-cytosine (caC), show a more favorable binding affinity of the higher oxidized forms, fC and caC, than the nonsubstrate bases hmC and mC. Despite rather comparable, reaction-competent conformations of the flipped bases in the active site of the enzyme, more and stronger interactions with active site residues account for the preferred binding of the higher oxidized bases. Binding of the negatively charged caC and the neutral fC are strengthened by interactions with positively charged His151. Our calculated proton affinities find this protonation state of His151 the preferred one in the presence of caC and conceivable in the presence of fC as well as increasing the binding affinity toward the two bases. Discrimination of the substrate bases is further achieved by the backbone of Tyr152 that forms a strong hydrogen bond to the carboxyl and formyl oxygen atoms of caC and fC, respectively, a contact that is completely lacking in mC and much weaker in hmC. Overall, our computational results indicate that the enzyme discriminates the different oxidation forms of methyl-cytosine already at the formation of the extrahelical complexes.

Interaction of Thymine DNA Glycosylase with Oxidised 5-Methyl-cytosines in Their Amino- and Imino-Forms.

Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson-Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation.

Tautomeric Equilibria of Nucleobases in the Hachimoji Expanded Genetic Alphabet.

Evolution has yielded biopolymers that are constructed from exactly four building blocks and are able to support Darwinian evolution. Synthetic biology aims to extend this alphabet, and we recently showed that 8-letter (hachimoji) DNA can support rule-based information encoding. One source of replicative error in non-natural DNA-like systems, however, is the occurrence of alternative tautomeric forms, which pair differently. Unfortunately, little is known about how structural modifications impact free-energy differences between tautomers of the non-natural nucleobases used in the hachimoji expanded genetic alphabet. Determining experimental tautomer ratios is technically difficult, and so, strategies for improving hachimoji DNA replication efficiency will benefit from accurate computational predictions of equilibrium tautomeric ratios. We now report that high-level quantum-chemical calculations in aqueous solution by the embedded cluster reference interaction site model, benchmarked against free-energy molecular simulations for solvation thermodynamics, provide useful quantitative information on the tautomer ratios of both Watson-Crick and hachimoji nucleobases. In agreement with previous computational studies, all four Watson-Crick nucleobases adopt essentially only one tautomer in water. This is not the case, however, for non-natural nucleobases and their analogues. For example, although the enols of isoguanine and a series of related purines are not populated in water, these heterocycles possess N1-H and N3-H keto tautomers that are similar in energy, thereby adversely impacting accurate nucleobase pairing. These robust computational strategies offer a firm basis for improving experimental measurements of tautomeric ratios, which are currently limited to studying molecules that exist only as two tautomers in solution.

2'-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis.

The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2-10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2' position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2'-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.

Hybrids of a 9-anthracenyl moiety and fluorescein as chemodosimeters for the detection of singlet oxygen in live cells.

Singlet oxygen (1O2) plays an important role in human innate immune response, plant physiology and anticancer photodynamic therapy (PDT). Therefore, its monitoring by convenient and sensitive methods (e.g. by detecting a fluorescence signal) by using non-toxic reagents would be advantageous. Known fluorogenic 1O2-chemodosimeters can potentially consume reducing agents in cells leading to the generation of toxic side products that limit their applications. In this paper we report on a series of 9-anthracenyl-fluorescein hybrids, which do not require any reducing agents for their reaction with 1O2. The selected compound 8d at a very low concentration of 100 nM is able to detect 1O2 in live human promyelocytic leukemia HL-60 cells with over 35-fold fluorescence signal enhancement within only 20 min assay time. This chemodosimeter is not toxic to HL-60 cells at concentrations ≤1 µM (higher concentrations were not tested) even at long incubation times ≤48 h.

Propagation of Holes and Electrons in Metal-Organic Frameworks.

Charge transport in two zinc metal-organic frameworks (MOFs) has been investigated using periodic semiempirical molecular orbital calculations with the AM1* Hamiltonian. Restricted Hartree-Fock calculations underestimate the band gap using Koopmans theorem (ca. 2 eV compared to the experimental value of 2.8 eV). However, it almost doubles when the constraint on the wave function to remain spin-restricted is removed and the energies of the UHF Natural Orbitals are used. Charge-transport simulations using propagation of the electron- or hole-density in imaginary time allow charge-transport paths and mechanisms to be determined. The calculated relative mobilities in the directions of the three crystal axes agree with experimental expectations, but the absolute values are not reliable using the current technique. Hole-mobility along the crystal c-axis (along the metal stacks) is found to be 13 times higher in the zinc MOF with anthracene linker (Zn-ANMOF-74) than in the other directions, whereas the factor is far smaller (1.7) for electron mobility. Directional preferences are far less distinct in the equivalent structure with phenyl linkers (Zn-MOF-74). The imaginary-time simulation technique does not give quantitative mobilities. The simulations reveal a change in mechanism between the different directions: Coherent polaron migration is observed along the stacks but tunneling hops between them.

Red light-triggered nucleic acid-templated reaction based on cyclic oligonucleotide substrates.

A red light-triggered reaction based on cyclic oligonucleotide substrates that is accelerated over 30-fold by specific nucleic acid templates and generates a bright fluorescent probe was developed. We confirmed that this reaction is compatible with fluorescence correlation spectroscopy (FCS) thereby allowing detection of nucleic acids down to 1 nM.

Identification of Boronic Acid Derivatives as an Active Form of N-Alkylaminoferrocene-Based Anticancer Prodrugs and Their Radiolabeling with 18F.

N-Alkylaminoferrocene (NAAF)-based prodrugs are activated in the presence of elevated amounts of reactive oxygen species (ROS), which corresponds to cancer specific conditions, with formation of NAAF and p-quinone methide. Both products act synergistically by increasing oxidative stress in cancer cells that causes their death. Though it has already been demonstrated that the best prodrugs of this type retain their antitumor activity in vivo, the effects were found to be substantially weaker than those observed in cell cultures. Moreover, the mechanistic studies of these compounds in vivo are missing. For clarification of these important questions, labeling of the prodrugs with radioactive moieties would be necessary. In this paper, we first observed that the representative NAAF-based prodrugs are hydrolyzed in dilute aqueous solutions to the corresponding arylboronic acids. We confirmed that these products are responsible for ROS amplification and anticancer properties of the parent prodrugs. Next, we developed the efficient synthetic protocol for radiolabeling the hydrolyzed NAAF-based prodrugs by [18F]fluoroglucosylation under the conditions of the copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition and used this protocol to prepare one representative hydrolyzed NAAF-based prodrug radiolabeled with 18F. Finally, we studied the stability of the 18F-labeled compound in human serum in vitro and in rat blood in vivo and obtained preliminary data on its biodistribution in vivo in mice carrying pancreatic (AR42J) and prostate (PC3) tumors by applying PET imaging studies. The compound described in this paper will help to understand in vivo effects (e.g., pharmacokinetics, accumulation in organs, the nature of side effects) of these prodrugs that will strongly contribute to their advancement to clinical trials.

DNA-Dye-Conjugates: Conformations and Spectra of Fluorescence Probes.

Extensive molecular-dynamics (MD) simulations have been used to investigate DNA-dye and DNA-photosensitizer conjugates, which act as reactants in templated reactions leading to the generation of fluorescent products in the presence of specific desoxyribonucleic acid sequences (targets). Such reactions are potentially suitable for detecting target nucleic acids in live cells by fluorescence microscopy or flow cytometry. The simulations show how the attached dyes/photosensitizers influence DNA structure and reveal the relative orientations of the chromophores with respect to each other. Our results will help to optimize the reactants for the templated reactions, especially length and structure of the spacers used to link reporter dyes or photosensitizers to the oligonucleotides responsible for target recognition. Furthermore, we demonstrate that the structural ensembles obtained from the simulations can be used to calculate steady-state UV-vis absorption and emission spectra. We also show how important quantities describing the quenching of the reporter dye via fluorescence resonance energy transfer (FRET) can be calculated from the simulation data, and we compare these for different relative chromophore geometries.

Carboxylate Ion Pairing with Alkali-Metal Ions for ß-Lactoglobulin and Its Role on Aggregation and Interfacial Adsorption.

We report a combined experimental and computational study of the whey protein ß-lactoglobulin (BLG) in different electrolyte solutions. Vibrational sum-frequency generation (SFG) and ellipsometry were used to investigate the molecular structure of BLG modified air-water interfaces as a function of LiCl, NaCl, and KCl concentrations. Molecular dynamics (MD) simulations and thermodynamic integration provided details of the ion pairing of protein surface residues with alkali-metal cations. Our results at pH 6.2 indicate that BLG at the air-water interface forms mono- and bilayers preferably at low and high ionic strength, respectively. Results from SFG spectroscopy and ellipsometry are consistent with intimate ion pairing of alkali-metal cations with aspartate and glutamate carboxylates, which is shown to be more effective for smaller cations (Li(+) and Na(+)). MD simulations show not only carboxylate-alkali-metal ion pairs but also ion multiplets with the alkali-metal ion in a bridging position between two or more carboxylates. Consequently, alkali-metal cations can bridge carboxylates not only within a monomer but also between monomers, thus providing an important dimerization mechanism between hydrophilic surface patches.

Molecular dynamics simulations of liquid phase interfaces: understanding the structure of the glycerol/water-dodecane system.

Modern spectroscopic techniques such as time-resolved second-harmonic-generation spectroscopy allow molecules to be examined selectively directly at phase interfaces. Two-phase systems formed by glycerol/water and alkane layers have previously been studied by time-resolved second-harmonic-generation spectroscopic measurements. In this molecular dynamics study, a triphenylmethane dye was inserted at the glycerol/water-alkane interface and was used as a probe for local properties such as viscosity. We now show how extensive simulations over a wide range of concentrations can be used to obtain a detailed view of the molecular structure at the glycerol/water-alkane interface. Glycerol is accumulated in a double layer adjacent to the alkane interface, which results in increased viscosity of the glycerol/water phase in the direct vicinity of the interface. We also show that conformational ensembles created by classical molecular-dynamics simulations can serve as input for QM/MM calculations, yielding further information such as transition dipoles, which can be compared with spectroscopic measurements.

A test of improved force field parameters for urea: molecular-dynamics simulations of urea crystals.

Molecular-dynamics (MD) simulations of urea crystals of different shapes (cubic, rectangular prismatic, and sheet) have been performed using our previously published force field for urea. This force field has been validated by calculating values for the cohesive energy, sublimation temperature, and melting point from the MD data. The cohesive energies computed from simulations of cubic and rectangular prismatic urea crystals in vacuo at 300 K agreed very well with the experimental sublimation enthalpies reported at 298 K. We also found very good agreement between the melting points as observed experimentally and from simulations. Annealing the crystals just below the melting point leads to reconstruction to form crystal faces that are consistent with experimental observations. The simulations reveal a melting mechanism that involves surface (corner/edge) melting well below the melting point, and rotational disordering of the urea molecules in the corner/edge regions of the crystal, which then facilitates the translational motion of these molecules.

Predicting the effects of basepair mutations in DNA-protein complexes by thermodynamic integration.

Thermodynamically rigorous free energy methods in principle allow the exact computation of binding free energies in biological systems. Here, we use thermodynamic integration together with molecular dynamics simulations of a DNA-protein complex to compute relative binding free energies of a series of mutants of a protein-binding DNA operator sequence. A guanine-cytosine basepair that interacts strongly with the DNA-binding protein is mutated into adenine-thymine, cytosine-guanine, and thymine-adenine. It is shown that basepair mutations can be performed using a conservative protocol that gives error estimates of ∼10% of the change in free energy of binding. Despite the high CPU-time requirements, this work opens the exciting opportunity of being able to perform basepair scans to investigate protein-DNA binding specificity in great detail computationally.

A simple QM/MM approach for capturing polarization effects in protein-ligand binding free energy calculations.

We present a molecular simulation protocol to compute free energies of binding, which combines a QM/MM correction term with rigorous classical free energy techniques, thereby accounting for electronic polarization effects. Relative free energies of binding are first computed using classical force fields, Monte Carlo sampling, and replica exchange thermodynamic integration. Snapshots of the configurations at the end points of the perturbation are then subjected to DFT-QM/MM single-point calculations using the B3LYP functional and a range of basis sets. The resulting quantum mechanical energies are then processed using the Zwanzig equation to give free energies incorporating electronic polarization. Our approach is conceptually simple and does not require tightly coupled QM and MM software. The method has been validated by calculating the relative free energies of hydration of methane and water and the relative free energy of binding of two inhibitors of cyclooxygenase-2. Closed thermodynamic cycles are obtained across different pathways, demonstrating the correctness of the technique, although significantly more sampling is required for the protein-ligand system. Our method offers a simple and effective way to incorporate quantum mechanical effects into computed free energies of binding.

Molecular dynamics characterization of the structures and induction mechanisms of a reverse phenotype of the tetracycline receptor.

Molecular-dynamics simulations have been used to investigate the mechanism of induction of a mutant (revTetR) of the tetracycline repressor protein (TetR) that shows the reverse phenotype (i.e., it is induced in the absence of tetracyclines and not in their presence). Low-frequency, normal-mode analyses demonstrate that the reverse phenotype is reproduced by the simulations on the basis of criteria established for wild-type TetR. The reverse phenotype is caused by the fact that the DNA-binding heads in revTetR are closer than the ideal distance needed for DNA-binding when no inducer is present. This distance increases on binding an inducer. Whereas this distance increase makes the interhead distance too large in wild-type TetR, it increases to the ideal value in revTetR. Thus, the mechanism of induction is the same for the two proteins, but the consequences are reversed because of the smaller interhead distance in revTetR when no inducer is present.

Structural changes and binding characteristics of the tetracycline-repressor binding site on induction.

The binding motif (pharmacophore) for induction and the changes in the structure of the binding site that accompany induction have been determined from molecular-dynamics simulations on the tetracycline-repressor signal-transduction protein. The changes and the induction mechanism are discussed and compared with conclusions drawn from earlier X-ray structures. The differences in inducer strength of tetracycline and 5a,6-anhydrotetracycline are discussed with respect to their interaction in the MD simulations.

Molecular dynamics simulations of the tetracycline-repressor protein: the mechanism of induction.

Molecular dynamics simulations on the tetracycline-repressor (TetR) protein, both in the absence of an inducer and complexed with the inducers tetracycline and 5a,6-anhydrotetracycline, show significant differences in the structures and dynamics of the induced and non-induced forms of the protein. Calpha-density-difference plots, low-frequency normal vibrations and inter-residue interaction energies all point to a common mechanism of induction. The inducer displaces Asp156 from the magnesium ion in the binding pocket, leading to a short cascade of rearrangements of salt bridges that results in the allosteric change. The increased flexibility of the induced form of the protein is suggested to contribute to the decrease in binding affinity to DNA on induction.

Simulating FRET from tryptophan: is the rotamer model correct?

We present a computational model study designed to simulate the results of time-resolved fluorescence spectra of tryptophan in proteins. In such measurements, the occurrence of more than one fluorescence lifetime is generally attributed to the existence of several tryptophan rotamers and/or structural conformations of the protein structure. The protein system we chose for this initial study is the tetracycline repressor (TetR), an interesting model system for the investigation of the mechanisms of transcriptional regulation. Fluorescence resonance energy transfer (FRET) from tryptophan to tetracycline is frequently observed in complexes of the TetR with the antibiotic tetracycline. We use a combined classical/quantum mechanical approach to model the structure and the spectroscopic properties of the TetR-tetracycline complex. A classical molecular dynamics simulation provides input geometries for semiempirical quantum mechanical/molecular mechanical (QM/MM) single-point configuration interaction (CI) calculations, which are used to calculate tryptophan vertical absorption and fluorescence energies and intensities as well as relative FRET rate constants. These rate constants together with the Einstein coefficients for spontaneous emission and an assumed rate for nonradiative deactivation allow us to simulate fluorescence decay curves with and without FRET and for the entire ensemble as well as for individual rotamers. Our results indicate that the classical "rotamer model", used to explain the multiexponential fluorescence-decay curves of time-resolved tryptophan emission spectra, can be extended to systems with FRET acceptors present in the protein matrix but that the interpretation of the fitted lifetimes is different to that usually used.