hSMUG1 excises uracil (U) and 5-hydroxymethyluracil (hmU) from DNA to be replaced by the correct base through further enzymes of the base excision repair pathway and is involved in RNA metabolism where Ser26 and Glu35 interact with Dyskerin. We report that hSMUG1 Ser26Arg/Glu35Asp and Pro240Gly excise U but not hmU in single-stranded (ss) DNA. Computer simulations of hSMUG1-complexes of wild-type, Glu35Asp and Pro240Gly with U/hmU in ss/double-stranded (ds) DNA verified interactions previously and recently reported in addition to unravelling new ones. This data provides a qualitative picture of the strength/function of crucial interactions between hSMUG1 and its substrate and suggests a different scheme for their relative strengths, explaining how hSMUG1 Glu35 and Pro240 mutants differently affect excision of U and hmU from ssDNA. Our data suggests that the structural organization of hSMUG1 for the repair of U is more robust than that for hmU in both ss- and dsDNA. Thus, weak or unstable H-bond-type of interactions which may help in positioning the base seem more important for hmU than for U to retain enzyme activity.

Methyl-CpG binding domain 2 (MBD2) is a critical epigenetic regulator that recognizes and binds with high selectivity to methylated CpG dinucleotides, a fundamental epigenetic mark involved in gene regulation and chromatin organization. Therefore, understanding the interactions and conformational dynamics that drive MBD2's high selectivity and strong binding affinity to methylated CpG is crucial to unraveling its regulatory mechanisms. In this study, through extensive classical MD simulations, we investigated the formation of the methylated CpG-MBD2 recognition complex. By positioning MBD2 one base pair downstream of its target mCpG site, we observed its transition to a stable complex at the target site within the microsecond timescale. Significantly, we observe that upon binding to methylated CpG, MBD2 forms two distinct stable complexes, with one state adopting a conformation that agrees well with the X-ray structure of the complex (primary state) and the other exhibiting a distinct binding conformation with lower affinity for methylated CpG (secondary state). Our data indicate that S189 serves as a key macro-switch, where loss of its interaction with the methylcytosine backbone shifts the complex toward the secondary state. This is supported by MD simulations of the S189A mutation, where we observe that the complex adopts a conformation closer to the secondary state. Ultimately, our NMR experiments confirm that the S189A mutation does not alter MBD2's selectivity for methylated CpG, while fluorescence polarization demonstrates a reduction in binding affinity, consistent with our MD simulations. Together, these findings strongly suggest that MBD2 adopts a bistable equilibrium in binding to methylated CpG dinucleotides.

Mitochondria mediate electron transport chain (ETC) to power ATP synthase. In cancer cells, the ETC is disturbed leading to leakage of electrons that ultimately results in generation of H2O2. This difference between cancer and healthy cells can be used for the design of cancer-specific prodrugs as confirmed in several proof-of-concept studies. Unfortunately, the known prodrugs are either insufficiently cancer cell specific or their activation efficacy is low. In this work, we solved this problem by optimizing the conjugate of a drug camptothecin and a H2O2-responsive aminoferrocene to obtain the prodrug C. The multistep reaction of the prodrug C with H2O2 is >20-fold more efficient than that of the parent system. The prodrug is accumulated and activated in mitochondria of cancer cells in a H2O2-dependent fashion. The anticancer efficacy towards human ovarian cancer A2780 and monocytic leukemia THP1 cells is improved from 573 and >3000 nM (for prodrug B) to 142 and 479 nM (for prodrug C) correspondingly. Additionally to the excellent activity, the new prodrug is not toxic in vivo to a range of blood (red and white blood cells, neutrophils, monocytes, platelets) and bone marrow cells (monocytes, B and T cells and others) in contrast to camptothecin.

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.

Lipids are major constituents of food but are also highly relevant substructures of drugs and are increasingly applied for the development of lipid-based drug delivery systems. Lipids are prone to oxidative degradation, thus affecting the quality of food or medicines. Therefore, analytical methods or tools that enable to assess the degree of lipid oxidation are of utmost importance to guarantee food and drug safety. Herein, we report the design, synthesis, and application of the first-in-class fluorogenic triacylglycerols that enable a dynamic monitoring of lipid oxidation via straightforward fluorescence readout. Our fluorogenic triacylglycerols can be used both in an aqueous and lipid-based environment. Further, we showed that the sensitivity of our fluorescent tracers towards oxidation can be tuned by incorporating either saturated or unsaturated acyl chains in their triacylglycerol core structure. With this, we provide a first proof-of-principle for the applicability of fluorescently labelled triacylglycerols as tracers to monitor the dynamics of lipid oxidation, thus paving the way for novel discoveries in the area of lipid analytics.

Carboxypeptidase is a Zn-dependent protease that specifically recognises and hydrolyses peptides with a hydrophobic side chain at the C-terminal residue. According to hydrolysis mechanisms proposed in the literature, catalysis requires a water molecule to be close to the Zn ion so as to be activated as a nucleophile. Among small molecules that resemble the slowly hydrolysed Gly-Tyr peptide, which have been previously designed as inhibitors and characterised structurally, a variant with the terminal amino acid in a D-configuration has been the most effective. Our molecular dynamics simulations of carboxypeptidase complexed with different variants of those inhibitor ligands as well as variants of the Gly-Tyr peptide show that the strength of the inhibitory effect is not related to the binding strength of the ligand. Our data rather support an earlier notion that the inhibition is, at least partially, due to blocking a coordination site at the Zn ion by the ligand coordinating the metal ion in a bidentate fashion.

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.

Recent advances in operating and manipulating DNA have provided unique experimental possibilities in many fields of DNA research, especially in gene therapy. Researchers have deployed many techniques, experimental and theoretical, to study the DNA structure changes due to external perturbation. It is crucial to understand the structural and dynamical changes in the DNA molecules in a confined state to understand and control the self-assembly of DNA confined in a chamber or nano-channel for various applications. In the current manuscript, we extend the work study the effect of confinement on the thermal stability and the structural properties of duplex DNA. The present work is an extension of our previous research works. For our study we have considered a 1 BNA chain that is confined in a cylindrical geometry. How the geometry of the confinement affects the opening and other structural parameters of DNA molecule is the objective of this manuscript. We have used a statistical model(PBD model) and Molecular dynamics simulations for our purpose.

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. It requires for its activation the simultaneous presence of triggers T1 (H2O2) and T2 (pH 8 in mitochondria: Mit). The level of H2O2 in cancer cells is higher than that in normal cells. Thus, T1 discriminates cancer cells from the majority of normal cells excluding neutrophils, known to produce elevated levels of H2O2. T2 discriminates cancer cells from neutrophils, since the former cells have a higher number of Mit. We demonstrated that 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.

Zinc ions are the second most abundant ions found in humans. Their role in proteins can be merely structural but also catalytic, owing to their transition metal character. Modelling their geometric–coordination versatility by empirical force fields is, thus, a challenging task. In this work, we evaluated three popular models, specifically designed to represent zinc ions with regard to their capability of preserving structural integrity. To this end, we performed molecular dynamics simulations of two zinc-containing protein–DNA complexes, which differed in their zinc coordination, i.e., four cysteines or two cysteines and two histidines. The most flexible non-bonded 12-6-4 Lennard–Jones-type model shows a preference for six-fold coordination of the Zn2+-ions in contradiction to the crystal structure. The cationic dummy atom model favours tetrahedral geometry, whereas the bonded extended zinc AMBER force field model, by construction, best preserves the initial geometry of a regular or slightly distorted tetrahedron. Our data renders the extended zinc AMBER force field the best model for structural zinc ions in a given geometry. In more complicated cases, though, more flexible models may be advantageous.

Cytochrome c Oxidase (CcO), a membrane protein of the respiratory chain, pumps protons against an electrochemical gradient by using the energy of oxygen reduction to water. The ("chemical") protons required for this reaction and those pumped are taken up via two distinct channels, named D-channel and K-channel, in a step-wise and highly regulated fashion. In the reductive phase of the catalytic cycle, both channels transport protons so that the pumped proton passes the D-channel before the "chemical" proton has crossed the K-channel. By performing molecular dynamics simulations of CcO in the O→E redox state (after the arrival of the first reducing electron) with various combinations of protonation states of the D- and K-channels, we analysed the effect of protonation on the two channels. In agreement with previous work, the amount of water observed in the D-channel was significantly higher when the terminal residue E286 was not (yet) protonated than when the proton arrived at this end of the D-channel and E286 was neutral. Since a sufficient number of water molecules in the channel is necessary for proton transport, this can be understood as E286 facilitating its own protonation. K-channel hydration shows an even higher dependence on the location of the excess proton in the K-channel. Also in agreement with previous work, the K-channel exhibits a very low hydration level that likely hinders proton transfer when the excess proton is located in the lower part of the K-channel, that is, on the N-side of S365. Once the proton has passed S365 (towards the reaction site, the bi-nuclear centre (BNC)), the amount of water in the K-channel provides hydrogen-bond connectivity that renders proton transfer up to Y288 at the BNC feasible. No significant direct effect of the protonation state of one channel on the hydration level, hydrogen-bond connectivity, or interactions between protein residues in the other channel could be observed, rendering proton conductivity in the two channels independent of each other. Regulation of the order of proton uptake and proton passage in the two channels such that the "chemical" proton leaves its channel last must, therefore, be achieved by other means of communication, such as the location of the reducing electron.

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.

DNA repair proteins are able to discriminate DNA lesions among an abundance of intact DNA with high selectivity. To investigate detectable characteristics of one specific lesion, we compare statistical results from molecular dynamics simulations of two different DNA in water, one with an intact C:G pair and one that contains a U:G mispair, and perform a comparative analysis of the water dynamics around the two. Our data show that in addition to the local DNA conformation, also the surrounding water shell exhibits significant differences that may help mispair discrimination. The chemical groups which account for a U:G mispair to exhibit a wobble conformation instead of the 'proper' Watson-Crick pairing of a C:G pair, that is an oxygen atom (in uracil) instead of an amino group (in cytosine), also order the water molecules around the bases such that they act predominantly as hydrogen-bond donor or acceptor to the uracil or cytosine base, respectively. These changes in water conformation stretch into the second solvation shell, which may be exploited by repair enzymes to achieve lesion detection with high efficiency.

The main objective of the present study is to investigate the molecular structure and DNA binding interaction of the tyrosyl-lysyl-threonine (YKT) tripeptide, which has anticancer, antioxidant and analgesic properties, using various in silico (MD, QM, molecular docking), spectroscopic (UV, FT-IR, FTIR-ATR, Raman, gel electrophoresis) and in vitro (MCF-7 and HeLa cancer cell lines and BEAS-2B cell line) methods. The optimized geometry, vibrational wavenumbers, molecular electrostatic potential (MEP), natural bond orbital (NBO) and HOMO-LUMO (highest occupied molecular orbital- lowest unoccupied molecular orbital) calculations were carried out with Density Functional Theory (DFT) using B3LYP/6-311thornthornG(d,p) basis set to indicate conformational, vibrational and intramolecular charge transfer characteristics. The assignment of all fundamental theoretical vibration wavenumbers was performed using potential energy distribution analysis (PED). DNA is a significant pharmacological target of drugs in several diseases such as cancer. For this reason, molecular docking calculation was used to elucidate the binding and interaction between YKT tripeptide and DNA at the atomic level. Also, the dynamic behaviors of YKT and DNA was examined using MD simulations. Besides, the interaction of YKT with DNA was experimentally examined by UV titration method and agarose gel electrophoresis method. Experimental results showed that YKT was intercalatively and electrostatically bound to CT-DNA (Calf thymus DNA) and cleavage pBR322 DNA in the presence of H2O2. The pharmacokinetic profile of YKT was also obtained. Cytotoxic effect of YKT was evaluated on MCF-7, HeLa and BEAS-2B cell lines. Hence, these studies about YKT tripeptide may pave the way for the development of various cancer drugs.

Cytochrome c oxidase is a membrane protein of the respiratory chain that consumes protons and molecular oxygen to produce water and uses the resulting energy to pump protons across the membrane. Our molecular dynamics simulations with an excess proton located at different positions in one of the proton-conducting channels, the K-channel, show a clear dependence of the number of water molecules inside the channel on the proton position. A higher hydration level facilitates the formation of hydrogen-bonded chains along which proton transfer can occur. However, a sufficiently high hydration level for such proton transport is observed only when the excess proton is located above S365, i.e., the lower third of the channel. From the channel entrance up to this point, proton transport is via water molecules as proton carriers. These hydronium ions move with their surrounding water molecules, up to K362, filling and widening the channel. The conformation of K362 depends on its own protonation state and on the hydration level, suggesting its role to be proton transport from a hydronium ion at the height of K362 to the upper part of the channel via a conformational change. The protonation-dependent conformational dynamics of E101 at the bottom of the channel renders proton transfer via E101 unlikely. Instead, its role is rather that of an amplifier of H96's proton affinity, suggesting H96 as the initial proton acceptor.

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.

Proton transfer reactions are one of the most fundamental processes in biochemistry. We present a simplistic approach for estimating proton transfer probabilities in a membrane protein, cytochrome c oxidase. We combine short molecular dynamics simulations at discrete protonation states with a Monte Carlo approach to exchange between those states. Requesting for a proton transfer the existence of a hydrogen-bonded connection between the two source and target residues of the exchange, restricts the acceptance of transfers to only those in which a proton-relay is possible. Together with an analysis of the hydrogen-bonded connectivity in one of the proton-conducting channels of cytochrome c oxidase, this approach gives insight into the protonation dynamics of the hydrogen-bonded networks. The connectivity and directionality of the networks are coupled to the conformation of an important protein residue in the channel, K362, rendering proton transfer in the entire channel feasible in only one of the two major conformations. Proton transport in the channel can thus be regulated by K362 not only through its possible role as a proton carrier itself, but also by allowing or preventing proton transport via water residues.

EDITORIAL article Front. Chem., 15 September 2021 | https://doi.org/10.3389/fchem.2021.762612

We have used the Cu(i)-catalyzed azide-alkyne Huisgen cycloaddition reaction to obtain two families of bivalent heterodimers where tacrine is connected to an azasugar or iminosugar, respectively, via linkers of variable length. The heterodimers were investigated as cholinesterase inhibitors and it was found that their activity increased with the length of the linker. Two of the heterodimers were significantly stronger acetylcholinesterase inhibitors than the monomeric tacrine. Molecular modelling indicated that the longer heterodimers fitted better into the active gorge of acetylcholinesterase than the shorter counterparts and the former provided more efficient simultaneous interaction with the tryptophan residues in the catalytic anionic binding site (CAS) and the peripheral anionic binding site (PAS).

The vibrational spectrum of the Ala-Leu-Ala-Leu peptide in solution, computed from first-principles simulations, shows a prominent band in the amide I region that is assigned to stretching of carbonyl groups. Close inspection reveals combined but slightly different contributions by the three carbonyl groups of the peptide. The shift in their exact vibrational signature is in agreement with the different probabilities of these groups to form hydrogen bonds with the solvent. The central carbonyl group has a hydrogen bond probability intermediate to the other two groups due to interchanges between different hydrogen-bonded states. Analysis of the interaction energies of individual water molecules with that group shows that shifts in its frequency are directly related to the interactions with the water molecules in the first hydration shell. The interaction strength is well correlated with the hydrogen bond distance and hydrogen bond angle, though there is no perfect match, allowing geometrical criteria for hydrogen bonds to be used as long as the sampling is sufficient to consider averages. The hydrogen bond state of a carbonyl group can therefore serve as an indicator of the solvent's effect on the vibrational frequency.