Linking 7-Nitrobenzo-2-oxa-1,3-diazole (NBD) to Triphenylphosphonium Yields Mitochondria-Targeted Protonophore and Antibacterial Agent.

Appending lipophilic cations to small molecules has been widely used to produce mitochondria-targeted compounds with specific activities. In this work, we obtained a series of derivatives of the well-known fluorescent dye 7-nitrobenzo-2-oxa-1,3-diazole (NBD). According to the previous data [Denisov et al. (2014) Bioelectrochemistry, 98, 30-38], alkyl derivatives of NBD can uncouple isolated mitochondria at concentration of tens of micromoles despite a high pKa value (~11) of the dissociating group. Here, a number of triphenylphosphonium (TPP) derivatives linked to NBD via hydrocarbon spacers of varying length (C5, C8, C10, and C12) were synthesized (mitoNBD analogues), which accumulated in the mitochondria in an energy-dependent manner. NBD-C10-TPP (C10-mitoNBD) acted as a protonophore in artificial lipid membranes (liposomes) and uncoupled isolated mitochondria at micromolar concentrations, while the derivative with a shorter linker (NBD-C5-TPP, or C5-mitoNBD) exhibited no such activities. In accordance with this data, C10-mitoNBD was significantly more efficient than C5-mitoNBD in suppressing the growth of Bacillus subtilis. C10-mitoNBD and C12-mitoNBD demonstrated the highest antibacterial activity among the investigated analogues. C10-mitoNBD also exhibited the neuroprotective effect in the rat model of traumatic brain injury.

Protonophoric and Photodynamic Effects of Fluorescein Decyl(triphenyl)phosphonium Ester on the Electrical Activity of Pond Snail Neurons.

Uncouplers of oxidative phosphorylation in mitochondria, which have been essential in elucidating the basic principles of cell bioenergetics, have recently attracted a considerable interest as compounds with therapeutic, e.g., neuroprotective, properties. Here, we report the effect of mitofluorescein (mitoFluo), a new protonophoric uncoupler representing a conjugate of fluorescein with decyl(triphenyl)phosphonium, on the electrical activity of neurons from Lymnaea stagnalis. Incubation with mitoFluo in the dark led to a decrease in the absolute value of the resting membrane potential of the neurons and alterations in their spike activity, such as spike broadening, spike amplitude reduction, and increase in the spike frequency. Prolonged incubation at high (tens micromoles) mitoFluo concentrations resulted in complete suppression of neuronal electrical activity. The effect of mitoFluo on the neurons was qualitatively similar to that of the classical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) but manifested itself after much longer incubation and at higher concentrations. The distinctive feature of mitoFluo is its light-induced effect on the electrical activity of neurons. Changes in the parameters of the neuronal activity upon illumination in the presence of mitoFluo were similar to the light-induced effects of the well-known photosensitizer Rose Bengal, although less pronounced. It was suggested that the effects of mitoFluo on the electrical activity of neurons, both as a mitochondrial uncoupler and a photosensitizer, are mediated by the changes in the cytoplasmic calcium concentration.

SkQThy, a novel and promising mitochondria-targeted antioxidant.

Since thymoquinone (2-isopropyl-5-methylbenzoquinone) isolation from Nigella sativa in 1963, various studies have reported on its diverse pharmacological properties. However, despite its versatile healing abilities, clinical trials involving the use of thymoquinone have not been initiated due to its poor bioavailability. Many attempts have been made to improve the therapeutic efficacy of thymoquinone by synthesizing analogs, as well as by developing nanotechnology-based delivery systems. We hypothesized that some of the issues with thymoquinone delivery and bioavailability could be resolved by targeted delivery to mitochondria of thymoquinone derivatives conjugated to the penetrating lipophilic cationic triphenylphosphonium fragment. As mitochondria are the major site of reactive oxygen species generation in the cell, such a membranotropic thymoquinone derivative can act as an efficient antioxidant or prooxidant depending on the concentration used. Based on these theoretical considerations, a novel mitochondria-targeted compound, SkQThy, was synthesized and its effects on rat liver mitochondria and yeast cells were examined. SkQThy was found to exhibit pronounced antioxidant activity in mammalian mitochondria and yeast cells, decreasing hydrogen peroxide production in mitochondria, as well as preventing prooxidant-induced oxidative stress and mitochondrial fragmentation in yeast cells and increasing cell viability. Moreover, SkQThy proved itself to be the most efficient mitochondria-targeted antioxidant within the SkQs family, showing good therapeutic potential.

[Functional state of spinal cord and its radices in patients with thoracic and lumbar spine injuries]. / Élektrofiziologicheskii monitoring funktsional'nogo sostoianiia spinnogo mozga i ego koreshkov u bol'nykh s povrezhdeniiami grudnogo i poiasnichnogo otdelov pozvonochnika.

AIM: To estimate the character of neurophysiological monitoring in patients with thoracic and lumbar spine injuries at different treatment stages. MATERIAL AND METHODS: Thirty-eight patients with non-complicated (22 patients, group 1) and complicated (16 patients, group 2) thoracic and lumbar spine injuries underwent electroneuromyography (ENM) and transcranial magnetic stimulation (TMS). The examination was performed at early (up to 2 weeks) and later (more than 1 month) post-injury periods, before the operation and on the 10th day after decompressing-stabilizing interventions. RESULTS: Before the operation, 71.4% patients of group 1 had ENM-signs of suppressed motor neuron activity in L5 segment of the spinal cord with peroneal nerve axonopathy. The most significant changes in ENM-indexes were observed in medullary channel stenosis of more than 30%. TMS parameters in group 1 were normal while in the 2nd group, EMN and TMS results before the operation demonstrated preserved motor neuron activity at the injury level despite gross neurological symptoms and 100% of medullary channel lumen deficit. In the postoperative period, ENM and TMS revealed no definite negative dynamics in patients of both groups. Patients with locomotor disorders, who underwent surgery at late post-injury periods, showed neurophysiological dynamics on the 10th day postoperatively. Low amplitude motor evoked potentials (kMEP), which were not present before, suggested initial signs of conductibility restoration (in 22% of patients) that proved the effectiveness of decompressive interventions in the long-term post-injury period. CONCLUSION: ENM- and TMS monitoring in patients with complicated and non-complicated injuries of thoracic and lumbar spine allowed revealing the positive influence of decompressing-stabilizing operations conducted both at early and late post-injury periods on the state of spinal cord conductibility and segmental apparatus.

Design, Synthesis, and Some Aspects of the Biological Activity of Mitochondria-Targeted Antioxidants.

This review summarizes for the first time data on the design and synthesis of biologically active compounds of a new generation - mitochondria-targeted antioxidants, which are natural (or synthetic) p-benzoquinones conjugated via a lipophilic linker with (triphenyl)phosphonium or ammonium cations with delocalized charge. It also describes the synthesis of mitochondria-targeted antioxidants - uncouplers of oxidative phosphorylation - based on fluorescent dyes.

Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10 µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified.

Uncoupling and Toxic Action of Alkyltriphenylphosphonium Cations on Mitochondria and the Bacterium Bacillus subtilis as a Function of Alkyl Chain Length.

A series of permeating cations based on alkyl derivatives of triphenylphosphonium (C(n)-TPP(+)) containing linear hydrocarbon chains (butyl, octyl, decyl, and dodecyl) was investigated in systems of isolated mitochondria, bacteria, and liposomes. In contrast to some derivatives (esters) of rhodamine-19, wherein butyl rhodamine possessed the maximum activity, in the case of C(n)-TPP a stimulatory effect on mitochondrial respiration steadily increased with growing length of the alkyl radical. Tetraphenylphosphonium and butyl-TPP(+) at a dose of several hundred micromoles exhibited an uncoupling effect, which might be related to interaction between C(n)-TPP(+) and endogenous fatty acids and induction of their own cyclic transfer, resulting in transport of protons across the mitochondrial membrane. Such a mechanism was investigated by measuring efflux of carboxyfluorescein from liposomes influenced by C(n)-TPP(+). Experiments with bacteria demonstrated that dodecyl-TPP(+), decyl-TPP(+), and octyl-TPP(+) similarly to quinone-containing analog (SkQ1) inhibited growth of the Gram-positive bacterium Bacillus subtilis, wherein the inhibitory effect was upregulated with growing lipophilicity. These cations did not display toxic effect on growth of the Gram-negative bacterium Escherichia coli. It is assumed that the difference in toxic action on various bacterial species might be related to different permeability of bacterial coats for the examined triphenylphosphonium cations.

Modeling Interactions of Erythromycin Derivatives with Ribosomes.

Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3'-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3-oxopropyl group in the 3'-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.

[Modern aspects of pathogenesis of the trauma of the spinal cord and trunks of peripheral nerves].

In pathogenesis of the traumatic disease of the spinal cord, two mechanisms of the injuries of its neuronal apparatus are defined: primary (necrosis) and secondary (apoptosis). In the work a participation of a number of internal causes in the progression of apoptosis in injury of the spinal cord and peripheral nerve trunks, the role of those remains little-studied up to date, is discussed.

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants.

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273-1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.

SkBQ - prooxidant addressed to mitochondria.

Oxidative stress and mitochondrial dysfunction are the key links in the chain of development of pathologies associated with the violation of cellular energy metabolism. Development of mitochondria-addressed compounds highly specific for chemical processes is one of the most promising ways to develop approaches to the treatment of inherited and age-related diseases with mitochondrial etiology. Correlation of structure and chemical activity of the test compounds from a class of lipophilic cations revealed the key role of substituents in the aromatic ring of 1,4-benzoquinones in the manifestation of high antioxidant properties. In this work, it is shown that a synthesized benzoquinone derivative conjugated in position 6 with membrane-penetrating cation of decyltriphenylphosphonium and with substituents at position 2, 3, and 5 (SkBQ) has much lower antioxidant and significantly higher prooxidant activity in comparison with similar derivatives of plasto- and toluquinone SkQ1 and SkQT1 in experiments on isolated mitochondria. At the same time, SkBQ, like SkQ1 and SkQT1, can be reduced by the respiratory chain in the center i of complex III and decrease the mitochondrial membrane potential. In cell cultures of human fibroblasts, it was revealed that SkBQ does not protect cells from apoptosis induced by hydrogen peroxide. Under the same conditions, SkQ1 and SkQT1 exhibit a powerful protective effect. Thus, SkBQ can be seen as a mitochondria-addressed prooxidant. The possibility of using SkBQ as an anticancer drug for the treatment of cancers such as prostate cancer whose cells are sensitive to mitochondrial reactive oxygen species is discussed.

Mitochondrial-targeted plastoquinone derivatives. Effect on senescence and acute age-related pathologies.

Plastoquinone, a very effective electron carrier and antioxidant of chloroplasts, was conjugated with decyltriphenylphosphonium to obtain a cation easily penetrating through membranes. This cation, called SkQ1, is specifically targeted to mitochondria by electrophoresis in the electric field formed by the mitochondrial respiratory chain. The respiratory chain also regenerates reduced SkQ1H(2) from its oxidized form that appears as a result of the antioxidant activity of SkQ1H(2). SkQ1H(2) prevents oxidation of cardiolipin, a mitochondrial phospholipid that is especially sensitive to attack by reactive oxygen species (ROS). In cell cultures, SkQ1 and its analog plastoquinonyl decylrhodamine 19 (SkQR1) arrest H(2)O(2)-induced apoptosis. When tested in vivo, SkQs (i) prolong the lifespan of fungi, crustaceans, insects, fish, and mice, (ii) suppress appearance of a large number of traits typical for age-related senescence (cataract, retinopathies, achromotrichia, osteoporosis, lordokyphosis, decline of the immune system, myeloid shift of blood cells, activation of apoptosis, induction of ß-galactosidase, phosphorylation of H2AX histones, etc.) and (iii) lower tissue damage and save the lives of young animals after treatments resulting in kidney ischemia, rhabdomyolysis, heart attack, arrhythmia, and stroke. We suggest that the SkQs reduce mitochondrial ROS and, as a consequence, inhibit mitochondria-mediated apoptosis, an obligatory step of execution of programs responsible for both senescence and fast "biochemical suicide" of an organism after a severe metabolic crisis.

[Amino acid and peptide derivatives of the tylosin family of macrolide antibiotics modified at the aldehyde group].

Fourteen new functionally active amino acid and peptide derivatives of the antibiotics tylosin, desmycosin, and 5-O-mycaminosyltylonolide were synthesized in order to study the interaction of the growing polypeptide chain with the ribosomal tunnel. The conjugation of various amino acids and peptides with a macrolide aldehyde group was carried out by two methods: direct reductive amination with the isolation of the intermediate Schiff bases or through binding via oxime using the preliminarily obtained derivatives of 2-aminooxyacetic acid.

Ribosomal tunnel and translation regulation.

This review describes the results of recent studies of the ribosomal tunnel (RT), the major function of which is to allow the smooth passage of nascent polypeptides with different sequences from the peptidyl transferase center of the ribosome to the tunnel exit, where the folding of protein molecules begins. The features of structural organization of RT and their role in modulation and stabilization of the nascent chain conformation are discussed. Structural features of macrolide binding sites as well as application of macrolide antibiotics and their derivatives as tools to investigate ligand-tunnel wall interactions are also considered. Several examples of strong and specific interactions of regulatory polypeptides with nucleotide and amino acid residues of RT that lead to ribosome stalling and translational arrest are described in detail. The role of these events in regulation of expression of certain genes is discussed on the basis of recent high-resolution structural studies of nascent chains in the RT.

Molecular mechanisms of transformation of SkQ mitotropic quinones and the search for new approaches to creation of selective free radical traps.

Features of the mechanism of action of positively charged benzoquinone derivatives (SkQ), which are the analogs of coenzyme Q (I), plastoquinone (II), and tocopherol (III), are discussed. It is usually considered that the main target of these compounds is mitochondria, where they accumulate due to the positive charge of the molecule. In the present work, it is shown with model systems that the reduced forms of compounds (I-III) under certain conditions can transform into electrically neutral cyclic zwitterions, which theoretically can escape from the matrix of energized mitochondria against the concentration gradient. A weak uncoupling effect of molecules I-III has been found on mitochondria. Its existence is in agreement with the abovementioned transformation of positively charged hydroquinones of type Ia-IIIa into electrically neutral molecules. The data obtained with model systems suggest that the target of SkQ hydroquinones as free radical traps may be not only mitochondria but also biochemical systems of the cytoplasm. Due to the presence of a large number of reactive oxygen species (ROS)-dependent signal systems in a cell, the functioning of cytoplasmic systems might be disturbed under the action of antioxidants. The problem of selective effect of antioxidants is discussed in detail in the present work, and a functional diagram of selective decrease of the "background level" of ROS based on differences in the intensity of background and "signal" ROS fluxes is considered.

[Synthesis of cyclic analogues of the nerve growth factor loop 4].

Cyclic peptides cyclo(-Gly-Asp-Glu-Lys-), cyclo(-Gly-Gly-Asp-Glu-Lys-) and cyclo(-Gly-Gly-Gly-Asp-Glu-Lys-) were synthesized as models of the beta-turn of nerve growth factor loop 4. The corresponding protected linear precursors were obtained in 52-83% yields by the solid-phase method with the use of the Fmoc/Bu(t) strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxy-tris(dimethylamino)phosphonium (BOP) hexafluorophosphate, N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmetanaminium-N-oxide (HBTU) hexafluorophosphate, and diphenylphosphorylazide (DPPA) at a dilution of 10(-3) M. The distribution of reaction products was studied for each cyclopeptide in dependence on the type of the coupling agent. The use of DPPA was shown to completely inhibit the formation of cyclodimers in the synthesis of five- and six-membered cyclopeptides; however, in the case of a four-membered peptide, an additional tenfold dilution of the reaction mixture was necessary to achieve the effect. The identification of several byproducts during the synthesis showed that the elongation of the polypeptide chain using the BOP reagent can be complicated by substantial racemization and the cleavage of the chlorotrityl anchor group by 0.5% TFA in dichloromethane proceeds with insufficient selectivity and is accompanied by the premature Boc deblocking of the lysine side function.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

[Peptide derivatives of tylosin-related macrolides].

Approaches to the synthesis of model compounds based on the tylosin-related macrolides desmycosin and O-mycaminosyltylonolide were developed using specially designed peptide derivatives of macrolide antibiotics to study the conformation and topography of the nascent peptide chain in the ribosome tunnel. A method for selective bromoacetylation of desmycosin at the hydroxyl group of mycinose was developed, which involves preliminary acetylation of mycaminose. The reaction of the 4"-bromoacetyl derivative of the antibiotic with cesium salts of the dipeptide Boc-Ala-Ala-OH and the hexapeptide MeOTr-Gly-Pro-Gly-Pro-Gly-Pro-OH led to the corresponding peptide derivatives of desmycosin. The protected peptides Boc-Ala-Ala-OH, Boc-Ala-Ala-Phe-OH, and Boc-Gly-Pro-Gly-Pro-Gly-Pro-OH were condensed with the C23-hydroxyl group of O-mycaminosyltylonolide.

Experimental validation of direct long electrostimulation after neurotransplantation of the peripheral nerves.

Effects of direct long-term electric stimulation on the growth of the sciatic nerve regenerating axons through autoneurotransplants were studied in rabbits and an optimal electrostimulation method was developed.

Peptide derivatives of antibiotics tylosin and desmycosin, protein synthesis inhibitors.

Biologically active peptide derivatives of 16-member macrolide antibiotics were synthesized as potential probes for the investigation of nascent peptide chain topography in the ribosomal exit tunnel. The tylosin and desmycosin aldehyde groups at the C6 position of the lactone ring were modified by the aminooxyacetyl-L-alanyl-L-alanine methyl ester.