Insights into the equilibrium structure and translocation mechanism of TP1, a spontaneous membrane-translocating peptide.

Crossing the cellular membrane is one of the main barriers during drug discovery; many potential drugs are rejected for their inability to integrate into the intracell fluid. Although many solutions have been proposed to overcome this barrier, arguably the most promising solution is the use of cell-penetrating peptides. Recently, an array of hydrophobic penetrating peptides was discovered via high throughput screening which proved to be able to cross the membrane passively, and although these peptides proved to be effective at penetrating the cell, the details behind the underlying mechanism of this process remain unknown. In this study, we developed a method to find the equilibrium structure at the transmembrane domain of TP1, a hydrophobic penetrating peptide. In this method, we selectively deuterium-label amino acids in the peptidic chain, and employ results of [Formula: see text]H-NMR spectroscopy to find a molecular dynamics simulation of the peptide that reproduces the experimental results. Effectively finding the equilibrium orientation and dynamics of the peptide in the membrane. We employed this equilibrium structure to simulate the entire translocation mechanism and found that after the peptide reaches its equilibrium structure, it must undergo a two-step mechanism in order to completely translocate the membrane, each step involving the flip-flop of each arginine residue in the peptide. This leads us to conclude that the RLLR motif is essential for the translocating activity of the peptide.

l-DOPA modulates the kinetics but not the thermodynamic equilibrium of TTA+ amphiphiles forming lyotropic nematic liquid crystals.

Lyotropic liquid crystals (LLCs) are mixtures of amphiphile molecules usually studied as mimetic of biological membrane. The equilibrium dynamics of tetradecyltrimethyl ammonium cation (TTA+) molecules forming nematic LLCs (LNLCs) is guided by a dive-in mechanism where TTA+ molecules spontaneously leave and re-enter the bicelle. Of note, this dynamic behavior could be exploited to produce drug nano-delivery systems based on LNLCs. Therefore, the understanding of the effect of pharmaceutically interesting molecules in the dynamics of the dive-in mechanism should be crucial for drug delivery applications. In this work, we studied the effects of l-DOPA in the equilibrium dynamics of TTA+ bicelles forming LNLCs, employing a transdisciplinary approach based on 2H-NMR together with molecular modeling and molecular dynamics simulations. Our data suggest that l-DOPA perturbs the kinetic of the dive-in mechanism but not the thermodynamics of this process. As whole, our results provide fundamental insights on the mechanisms by which l-DOPA govern the equilibrium of LNLCs bicelles.

Assessing Parameter Suitability for the Strength Evaluation of Intramolecular Resonance Assisted Hydrogen Bonding in o-Carbonyl Hydroquinones.

Intramolecular hydrogen bond (IMHB) interactions have attracted considerable attention due to their central role in molecular structure, chemical reactivity, and interactions of biologically active molecules. Precise correlations of the strength of IMHB's with experimental parameters are a key goal in order to model compounds for drug discovery. In this work, we carry out an experimental (NMR) and theoretical (DFT) study of the IMHB in a series of structurally similar o-carbonyl hydroquinones. Geometrical parameters, as well as Natural Bond Orbital (NBO) and Quantum Theory of Atoms in Molecules (QTAIM) parameters for IMHB were compared with experimental NMR data. Three DFT functionals were employed to calculated theoretical parameters: B3LYP, M06-2X, and ωB97XD. O…H distance is the most suitable geometrical parameter to distinguish among similar IMHBs. Second order stabilization energies ΔEij(2) from NBO analysis and hydrogen bond energy (EHB) obtained from QTAIM analysis also properly distinguishes the order in strength of the studied IMHB. ΔEij(2) from NBO give values for the IMHB below 30 kcal/mol, while EHB from QTAIM analysis give values above 30 kcal/mol. In all cases, the calculated parameters using ωB97XD give the best correlations with experimental ¹H-NMR chemical shifts for the IMHB, with R² values around 0.89. Although the results show that these parameters correctly reflect the strength of the IMHB, when the weakest one is removed from the analysis, arguing experimental considerations, correlations improve significantly to values around 0.95 for R².

Experimental and Theoretical Reduction Potentials of Some Biologically Active ortho-Carbonyl para-Quinones.

The rational design of quinones with specific redox properties is an issue of great interest because of their applications in pharmaceutical and material sciences. In this work, the electrochemical behavior of a series of four p-quinones was studied experimentally and theoretically. The first and second one-electron reduction potentials of the quinones were determined using cyclic voltammetry and correlated with those calculated by density functional theory (DFT) using three different functionals, BHandHLYP, M06-2x and PBE0. The differences among the experimental reduction potentials were explained in terms of structural effects on the stabilities of the formed species. DFT calculations accurately reproduced the first one-electron experimental reduction potentials with R² higher than 0.94. The BHandHLYP functional presented the best fit to the experimental values (R² = 0.957), followed by M06-2x (R² = 0.947) and PBE0 (R² = 0.942).

A Study about Regioisomeric Hydroquinones with Multiple Intramolecular Hydrogen Bonding.

A theoretical exploration about hydrogen bonding in a series of synthetic regioisomeric antitumor tricyclic hydroquinones is presented. The stabilization energy for the intramolecular hydrogen bond (IHB) formation in four structurally different situations were evaluated: (a) IHB between the proton of a phenolic hydroxyl group and an ortho-carbonyl group (forming a six-membered ring); (b) between the oxygen atom of a phenolic hydroxyl group and the proton of an hydroxyalkyl group (seven membered ring); (c) between the proton of a phenolic hydroxyl group with the oxygen atom of the hydroxyl group of a hydroxyalkyl moiety (seven-membered ring); and (d) between the proton of a phenolic hydroxyl group and an oxygen atom directly bonded to the aromatic ring in ortho position (five-membered ring). A conformational analysis for the rotation around the hydroxyalkyl substituent is also performed. It is observed that there is a correspondence between the conformational energies and the IHB. The strongest intramolecular hydrogen bonds are those involving a phenolic proton and a carbonyl oxygen atom, forming a six-membered ring, and the weakest are those involving a phenolic proton with the oxygen atom of the chromenone, forming five-membered rings. Additionally, the synthesis and structural assignment of two pairs of regioisomeric hydroquinones, by 2D-NMR experiments, are reported. These results can be useful in the design of biologically-active molecules.

A Computational Study of Structure and Reactivity of N-Substitued-4-Piperidones Curcumin Analogues and Their Radical Anions.

In this work, a computational study of a series of N-substitued-4-piperidones curcumin analogues is presented. The molecular structure of the neutral molecules and their radical anions, as well as their reactivity, are investigated. N-substituents include methyl and benzyl groups, while substituents on the aromatic rings cover electron-donor and electron-acceptor groups. Substitutions at the nitrogen atom do not significantly affect the geometry and frontier molecular orbitals (FMO) energies of these molecules. On the other hand, substituents on the aromatic rings modify the distribution of FMO. In addition, they influence the capability of these molecules to attach an additional electron, which was studied through adiabatic (AEA) and vertical electron affinities (VEA), as well as vertical detachment energy (VDE). To study electrophilic properties of these structures, local reactivity indices, such as Fukui (f⁺) and Parr (P⁺) functions, were calculated, and show the influence of the aromatic rings substituents on the reactivity of α,ß-unsaturated ketones towards nucleophilic attack. This study has potential implications for the design of curcumin analogues based on a 4-piperidone core with desired reactivity.

Determinants of Anti-Cancer Effect of Mitochondrial Electron Transport Chain Inhibitors: Bioenergetic Profile and Metabolic Flexibility of Cancer Cells.

Recent evidence highlights that energy requirements of cancer cells vary greatly from normal cells and they exhibit different metabolic phenotypes with variable participation of both glycolysis and oxidative phosphorylation (OXPHOS). Interestingly, mitochondrial electron transport chain (ETC) has been identified as an essential component in bioenergetics, biosynthesis and redox control during proliferation and metastasis of cancer cells. This dependence converts ETC of cancer cells in a promising target to design small molecules with anti-cancer actions. Several small molecules have been described as ETC inhibitors with different consequences on mitochondrial bioenergetics, viability and proliferation of cancer cells, when the substrate availability is controlled to favor either the glycolytic or OXPHOS pathway. These ETC inhibitors can be grouped as 1) inhibitors of a respiratory complex (e.g. rotenoids, vanilloids, alkaloids, biguanides and polyphenols), 2) inhibitors of several respiratory complexes (e.g. capsaicin, ME-344 and epigallocatechin-3 gallate) and 3) inhibitors of ETC activity (e.g. elesclomol and VLX600). Although pharmacological ETC inhibition may produce cell death and a decrease of proliferation of cancer cells, factors such as degree of inhibition of ETC activity by small molecules, bioenergetic profile and metabolic flexibility of different cancer types or subpopulations of cells in a particular cancer type, can affect the impact of the anti-cancer actions. Particularly interesting are the adaptive mechanisms induced by ETC inhibition, such as induction of glutamine-dependent reductive carboxylation, which may offer a strategy to sensitize cancer cells to inhibitors of glutamine metabolism.

Protonation Sites, Tandem Mass Spectrometry and Computational Calculations of o-Carbonyl Carbazolequinone Derivatives.

A series of a new type of tetracyclic carbazolequinones incorporating a carbonyl group at the ortho position relative to the quinone moiety was synthesized and analyzed by tandem electrospray ionization mass spectrometry (ESI/MS-MS), using Collision-Induced Dissociation (CID) to dissociate the protonated species. Theoretical parameters such as molecular electrostatic potential (MEP), local Fukui functions and local Parr function for electrophilic attack as well as proton affinity (PA) and gas phase basicity (GB), were used to explain the preferred protonation sites. Transition states of some main fragmentation routes were obtained and the energies calculated at density functional theory (DFT) B3LYP level were compared with the obtained by ab initio quadratic configuration interaction with single and double excitation (QCISD). The results are in accordance with the observed distribution of ions. The nature of the substituents in the aromatic ring has a notable impact on the fragmentation routes of the molecules.

Small structural changes on a hydroquinone scaffold determine the complex I inhibition or uncoupling of tumoral oxidative phosphorylation.

Mitochondria participate in several distinctiveness of cancer cell, being a promising target for the design of anti-cancer compounds. Previously, we described that ortho-carbonyl hydroquinone scaffold 14 inhibits the complex I-dependent respiration with selective anti-proliferative effect on mouse mammary adenocarcinoma TA3/Ha cancer cells; however, the structural requirements of this hydroquinone scaffold to affect the oxidative phosphorylation (OXPHOS) of cancer cells have not been studied in detail. Here, we characterize the mitochondrial metabolism of TA3/Ha cancer cells, which exhibit a high oxidative metabolism, and evaluate the effect of small structural changes of the hydroquinone scaffold 14 on the respiration of this cell line. Our results indicate that these structural changes modify the effect on OXPHOS, obtaining compounds with three alternative actions: inhibitors of complex I-dependent respiration, uncoupler of OXPHOS and compounds with both actions. To confirm this, the effect of a bicyclic hydroquinone (9) was evaluated in isolated mitochondria. Hydroquinone 9 increased mitochondrial respiration in state 4o without effects on the ADP-stimulated respiration (state 3ADP), decreasing the complexes I and II-dependent respiratory control ratio. The effect on mitochondrial respiration was reversed by 6-ketocholestanol addition, indicating that this hydroquinone is a protonophoric uncoupling agent. In intact TA3/Ha cells, hydroquinone 9 caused mitochondrial depolarization, decreasing intracellular ATP and NAD(P)H levels and GSH/GSSG ratio, and slightly increasing the ROS levels. Moreover, it exhibited selective NAD(P)H availability-dependent anti-proliferative effect on cancer cells. Therefore, our results indicate that the ortho-carbonyl hydroquinone scaffold offers the possibility to design compounds with specific actions on OXPHOS of cancer cells.

Heterocyclic Curcumin Derivatives of Pharmacological Interest: Recent Progress.

Curcumin, a natural yellow polyphenol, is isolated from the herb Curcuma longa L. (turmeric), a member of the ginger family. It has been extensively studied due to their multiple pharmacological properties. Nevertheless, curcumin has disadvantages such as poor water solubility, poor bioavailability and rapid metabolism, which has prompted the search for analogues that overcome these shortcomings while maintaining or improving their good pharmacological properties. Among the main curcumin analogues that have been developed, the heterocyclic curcuminoids show a high interest. In this review, we describe recent progress in the synthesis and pharmacological properties of new heterocyclic curcumin derivatives. The most recent developments in anti-cancer, anti-Alzheimer, anti-bacterial and anti-oxidants heterocyclic curcumin derivatives are covered.

Intramolecular hydrogen bond in biologically active o-carbonyl hydroquinones.

Intramolecular hydrogen bonds (IHBs) play a central role in the molecular structure, chemical reactivity and interactions of biologically active molecules. Here, we study the IHBs of seven related o-carbonyl hydroquinones and one structurally-related aromatic lactone, some of which have shown anticancer and antioxidant activity. Experimental NMR data were correlated with theoretical calculations at the DFT and ab initio levels. Natural bond orbital (NBO) and molecular electrostatic potential (MEP) calculations were used to study the electronic characteristics of these IHB. As expected, our results show that NBO calculations are better than MEP to describe the strength of the IHBs. NBO energies (∆Eij(2)) show that the main contributions to energy stabilization correspond to LP-->σ* interactions for IHBs, O1…O2-H2 and the delocalization LP-->π* for O2-C2=Cα(ß). For the O1…O2-H2 interaction, the values of ∆Eij(2) can be attributed to the difference in the overlap ability between orbitals i and j (Fij), instead of the energy difference between them. The large energy for the LP O2-->π* C2=Cα(ß) interaction in the compounds 9-Hydroxy-5-oxo-4,8, 8-trimethyl-l,9(8H)-anthracenecarbolactone (VIII) and 9,10-dihydroxy-4,4-dimethylanthracen-1(4H)-one (VII) (55.49 and 60.70 kcal/mol, respectively) when compared with the remaining molecules (all less than 50 kcal/mol), suggests that the IHBs in VIII and VII are strongly resonance assisted.

Thermodynamics and 2H NMR study on the insertion of small quinones into a discotic nematic lyotropic liquid crystal.

A detailed description of the distribution, interaction, and dynamics of molecules with biological activity dissolved in a hydrophobic bilayer, a simple model of a biological membrane, provides valuable information for a better understanding of drug functioning, which can be very useful in drug design. Here we present an (2)H NMR and molecular dynamics study on the insertion, distribution, interactions, and thermodynamics of two biologically active molecules, 9,10-dihydroxy-4,4-dimethyl-1,4,5,8-tetrahydroanthracen-1-one (HQ), with anticancer activity, and 4,4-dimethyl-1,4,5,8,9,10-hexahydroanthracen-1,9,10-trione (Q) a fungicide, dissolved in a nematic discotic lyotropic liquid crystal (ndllc) composed of sodium dodecylsulphate (SDS), decanol (DecOH) and Na2 SO4 in water. (2)H NMR quadrupole splittings (ΔνQ ) and longitudinal relaxation times (T1) from HQ-d6, Q-d4, DecOH-α-d2, partially deuterated water, and SDS-d25 were measured and several molecular dynamics trajectories were also calculated. In particular, ΔG, ΔH, and ΔS profiles for the process of both molecules crossing the bilayer were estimated. It was evidenced that the insertion of both molecules into the aggregate is a spontaneous process, and the molecules are mainly distributed in the internal side of the interface. Addition of HQ or Q decreased the mobility of all aggregate components, but this effect was more pronounced for HQ. The rotational correlation time of Q allowed an estimate of 5.3 cP for the microviscosity inside the ndllc aggregate, in the order of previously measured values in similar environments. Both guest molecules display similar free-energy profiles for the process of crossing the bilayer, with a calculated barrier height of 25 and 36 kJ mol(-1) for HQ and Q, respectively.

Mitochondria: a promising target for anticancer alkaloids.

A great number of alkaloids exhibit high potential in cancer research. Some of them are anticancer drugs with well-defined clinical uses, exerting their action on microtubules dynamics or DNA replication and topology. On the other hand, mitochondria have been recognized as an essential organelle in the establishment of tumor characteristics, especially the resistance to cell death, high proliferative capacity and adaptation to unfavorable cellular environment. Interestingly, many alkaloids exert their anticancer activities affecting selectively some functions of the tumor mitochondria by 1) modulating OXPHOS and ADP/ATP transport, 2) increasing ROS levels and mitochondrial potential dissipation by crosstalk between endoplasmic reticulum (ER) and mitochondria, 3) inducing mitochondria-dependent apoptosis and autophagy, 4) inhibiting mitochondrial metabolic pathways and 5) by alteration of the morphology and biogenesis of this organelle. These antecedents show the relevance of developing research about the effects of alkaloids on functions controlled by tumor mitochondria, offering an attractive target for the design of new alkaloid derivatives, considering organelle- specific delivery strategies. This review describes mitochondria as a central component in the anticancer action of a set of alkaloids, in a way to illustrate the importance of this organelle in medicinal chemistry.

NMR assignment in regioisomeric hydroquinones.

A set of regioisomeric pairs of tricyclic hydroquinones, analogues of antitumor 9,10-dihydroxy-4,4-dimethyl-5,8-dihydroanthracen-1(4H)-one (1) and other derivatives, were synthesized and their regiochemistry and NMR spectra assigned by using (1)H-detected one-bond (C-H) HMQC and long-range C-H HMBC, in good agreement with theoretical O3LYP/Alhrichs-pVTZ calculations. The 5-hydroxymethyl derivatives (11, 15, 19) showed a (3)J(H, H) coupling constant of methylene protons evidencing the presence of a seven-membered intramolecular hydrogen bonded ring, not observed for the 8-hydroxymethyl isomers.

Counterion and composition effects on discotic nematic lyotropic liquid crystals II. Ion exchange and molecular dynamics.

The static fluorescence quenching of pyrene by bromide, at the interface of mixed TTAC/TTAB discotic nematic lyotropic liquid crystals, allowed an estimation of the equilibrium constant for the exchange of chloride by bromide. The affinity of the interface for bromide is much higher than for chloride (K(Br-/Cl-) = 13.2). For a molecular level understanding of the experimental results of this and the preceding paper, 20 ns molecular dynamics (MD) simulations were calculated for samples with TTAB/TTAC molar percent ratios 100/0 (A), 50/50 (B) and 0/100 (C). The increment in the concentration of chloride induces a wider distribution of ammonium headgroups along the axis normal to the bilayer surface, increasing the width of the interface. The charge density profile of simulation B shows that the concentration of bromide is higher than the concentration of chloride in the vicinity the ammonium headgroups. The short range contribution to the electrostatic energy from the ammonium-ammonium repulsion is 291.7 kJ/mol for TTAC and 195.6 kJ/mol for TTAB, and the short range ammonium-halide interaction is -6166 kJ/mol for TTAC and -6607 kJ/mol for TTAB, from simulations A and C, respectively. These results are in agreement with a more neutralized TTAB interface. Consistently, the electric dipole moments of water are significantly more aligned with the larger electric field of the TTAB interface.

Counterion and composition effects on discotic nematic lyotropic liquid crystals I. Size and order.

Counterion and composition effects on the size and interface dynamics of discotic nematic lyotropic liquid crystals made of tetradecyltrimethylammonium halide (TTAX)-decanol (DeOH)-water-NaX, with X = Cl(-) and Br(-), were investigated using NMR and fluorescence spectroscopies. The dynamics of the interface was examined by measuring deuterium quadrupole splittings from HDO (0.1% D(2)O in H(2)O) and 1,1-dideuterodecanol (20% 1,1-dideuterodecanol in DeOH) in 27 samples of each liquid crystal. Aggregation numbers, N(D), from 15 samples of each mesophase were obtained using the fluorescence of pyrene quenched by hexadecylpyridinium chloride. N(D) of TTAB and TTAC are about 230+/-30 and 300+/-20, respectively. N(D) of TTAC increases with increasing concentration of all mesophase components, whereas TTAB shows no correlation between size and composition. The dimension of these aggregates prevents the occurrence of undulations, previously observed in lamellar phases. The quadrupole splitting of decanol-d(2) in TTAC is about 5 kHz smaller than in TTAB, and the splitting of HDO is observed only in TTAB. All results are consistent with a more dynamic TTAC interface. The TTAC aggregate should be more dissociated from counterions and the excess ammonium-ammonium electrostatic repulsions contribute to increase the mobility of the interface components.

Effects of 9,10-dihydroxy-4,4-dimethyl-5,8-dihydro-1(4H)-anthracenone derivatives on tumor cell respiration.

A series of tricyclic hydroquinones, incorporating a carbonyl group in the ortho position relative to the phenol function, were tested as inhibitors of oxygen uptake against the TA3 mouse carcinoma cell line and its multidrug-resistant variant TA3-MTX-R. The title compound, which proved to be the most active one, also exhibited low micromolar dose-dependent growth inhibition of the human tumor U937 cell line (human monocytic leukemia). A tentative structure-activity relationship is proposed for these substances. A comparison between the cytotoxicities of the title compound and 4,4-dimethyl-5,8-dihydroxynaphthalene-1-one, with their activities as inhibitors of oxygen uptake by the TA3-MTX-R cell line, is presented. Also, the inhibition of oxygen uptake by 6-(4-methylpent-3-enyl)-1,4-naphthoquinone was determined and compared with its reported cytotoxicity toward P-388 (murine lymphocytic leukemia), A-549 (human lung carcinoma), HT-29 (human colon carcinoma), and MEL-28 (human melanoma) cells. The inhibition of oxygen uptake by TA3-MTX-R cells is useful as a quick test for preliminary screening of possible anticancer activity.

(2Z)-3-(5-Hydroxy-4-oxo-4H-chromen-3-yl)acrylonitrile.

The title compound, C12H7NO3, consists of a chromone moiety substituted in position 3 with an acrylonitrile group in a Z configuration. The two planar groups are twisted with respect to one another. The only significant hydrogen bond in the structure is an intramolecular O-H...O bond. pi-pi contacts connecting aromatic groups and C-H...O intermolecular weak interactions lead to a supramolecular layer arrangement.