Search

The alkyltriphenylphosphonium (TPP) group is the most widely used vector targeted to mitochondria. Previously, the length of the alkyl linker was varied as well as structural modifications in the TPP phenyl rings to obtain the optimal therapeutic effect of a pharmacophore conjugated with a lipophilic cation. In the present work, we synthesized butyltriphenylphosphonium cations halogenated and methylated in phenyl rings (C

BACKGROUND: Neurological complications in sciatic nerve (SN) after a total hip replacement (THR) are observed in 0.93.2% of cases in patients with arthrosis deformans and age-related morphologic changes in SN. These cause the need for SN evaluation before THR. This research was aimed at the evaluation of the initial SN capacity with electrophysiological findings in patients with arthrosis deformans of the hip. MATERIALS AND METHODS: Electroneuromyography (ENMG) was used to evaluate fibular and tibial nerves M-responses as well as F-waves in 66 patients with dysplastic coxarthrosis and 12 patients with posttraumatic coxarthrosis. The findings were compared to those of the controls. RESULTS: Changes in ENMG findings for fibular nerve in 49 patients with dysplastic coxarthrosis were bilateral and showed significant difference only from the norm. In 19 of 66 cases (27.9%) low M-responses (р 0.02) were found in the side subject to THR. In 87.3% of cases, the signs of a decrease in the conductivity of proximal segments of the tibial nerve were revealed. In patients with posttraumatic coxarthrosis, the significant decrease in ENMG findings from both fibular and tibial nerves was observed in the affected side, they made up just 42-50% of those in the opposite side. Asymptomatic progress of denervation damage in hip and tibia muscles sometimes required needle EMG to fund the signs of motor innervation disorder. A-waves revealed in 65% of patients suggested local damage to one or both portions of SN. CONCLUSION: ENMG findings in patients with dysplastic arthrosis of the hip enabled revealing of the signs of neuropathy before surgeries and decreasing the risk of neurologic post-surgery complications.

An impressive body of evidence has been accumulated now on sound beneficial effects of mitochondrial uncouplers in struggling with the most dangerous pathologies such as cancer, infective diseases, neurodegeneration and obesity. To increase their efficacy while gaining further insight in the mechanism of the uncoupling action has been remaining a challenge. Encouraged by our previous promising results on lipophilic derivatives of 7-hydroxycoumarin-4-acetic acid (UB-4 esters), here, we use a 7-hydroxycoumarin-3-carboxylic acid scaffold to synthesize a new series of 7-hydroxycoumarin (umbelliferone, UB)-derived uncouplers of oxidative phosphorylation - alkyl esters of umbelliferone-3-carboxylic acid (UB-3 esters) with varying carbon chain length. Compared to the UB-4 derivatives, UB-3 esters proved to be stronger uncouplers: the most effective of them caused a pronounced increase in the respiration rate of isolated rat heart mitochondria (RHM) at submicromolar concentrations. Both of these series of UB derivatives exhibited a striking difference between their uncoupling patterns in mitochondria isolated from liver and heart or kidney, namely: a pronounced but transient decrease in membrane potential, followed by its recovery, was observed after the addition of these compounds to isolated rat liver mitochondria (RLM), while the depolarization of RHM and rat kidney mitochondria (RKM) was rather stable under the same conditions. Interestingly, partial reversal of this depolarization in RHM and RKM was caused by carboxyatractyloside, an inhibitor of ATP/ADP translocase, thereby pointing to the involvement of this mitochondrial membrane protein in the uncoupling activity of both UB-3 and UB-4 esters. The fast membrane potential recovery in RLM uncoupled by the addition of the UB esters was apparently associated with hydrolysis of these compounds, catalyzed by mitochondrial aldehyde dehydrogenase (ALDH2), being in high abundance in liver compared to other tissues. Protonophoric properties of the UB derivatives in isolated mitochondria were confirmed by measurements of RHM swelling in the presence of potassium acetate. In model bilayer lipid membranes (liposomes), proton-carrying activity of UB-3 esters was demonstrated by measuring fluorescence response of the pH-dependent dye pyranine. Electrophysiological experiments on identified neurons from Lymnaea stagnalis demonstrated low neurotoxicity of UB-3 esters. Resazurin-based cell viability assay showed low toxicity of UB-3 esters to HEK293 cells and primary human fibroblasts. Thus, the present results enable us to consider UB-3 esters as effective tissue-specific protonophoric mitochondrial uncouplers.

Objective — To investigate the extent of degenerative changes in neurons of spinal ganglion and segmental apparatus in various injuries to sciatic nerve in the experiment on white rats. Material and Methods — The research involved 40 white non-pedigree male rats distributed among four groups. The animals of Group 1 (n=10) underwent the compression of nerve trunks with Mosquito clamp forceps for 15 minutes. In Group 2 (n=10), the animals had their nerve trunks ligated; and in Group 3, they had their nerves completely transected in their middle thirds. The separate group of control animals (n=10) suffered no damage to their sciatic nerves. Spinal cords and spinal ganglia at L4-L6 level were the material for histopathological examination. We calculated the number (percent) of degenerated neurons in spinal cords and spinal ganglia at the affected sides on Day 30, and compared them to those at the intact sides. Results — The number (percent) of degenerated neurons in spinal cord and spinal ganglion, expressed as Me (Q1; Q2), constituted 2.52% (1.92; 2.74) and 3.75% (2.37; 4.74) in Group 1, 9.27% (9.03; 9.94) and 16.74% (16.01; 18.22) in Group 2, 25.59% (24.36; 26.29) and 31.94% (31.44; 33.03) in Group 3, respectively. Depending on the number (percent) of degenerated neurons, we classified three grades of change manifestation: mild (Group 1), medium (Group 2), and severe (Group 3). No degenerated neurons were found in the control animals. Conclusion — The compression, ischemic exposure on the sciatic nerve, and complete anatomical transection of its trunk resulted in Wallerian degeneration, as well as degeneration of segmental apparatus in spinal cord neurons.

A great variety of coumarin-related compounds, both natural and synthetic, being often brightly fluorescent, have shown themselves beneficial in medicine for both therapeutic and imaging purposes. Here, in search for effective uncouplers of oxidative phosphorylation, we synthesized a series of 7-hydroxycoumarin (umbelliferone, UB) derivatives combining rather high membrane affinity with the presence of a hydroxyl group deprotonable at physiological pH - alkyl esters of umbelliferone-4-acetic acid (UB-4 esters) differing in alkyl chain length. Addition of UB-4 esters to isolated rat liver mitochondria (RLM) resulted in their rapid depolarization, unexpectedly followed by membrane potential recovery on a minute time scale. According to TLC and HPLC data, incubation of RLM with UB-4 esters caused their hydrolysis, which led to disappearance of the uncoupling activity (recoupling). Both mitochondrial recoupling and hydrolysis of UB-4 esters were suppressed by inhibitors of mitochondrial aldehyde dehydrogenase (ALDH2), disulfiram and daidzin, thus pointing to the involvement of this enzyme in the recoupling of RLM incubated with UB-4 esters. The protonophoric mechanism of mitochondrial uncoupling by UB-4 esters was proved in experiments with artificial bilayer lipid membranes (BLM): these compounds induced proton-selective electrical current across planar BLM and caused dissipation of pH gradient on liposomes. UB-4 esters showed antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Mycobacterium smegmatis.

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.

Penetrating cations are widely used for the design of bioactive mitochondria-targeted compounds. The introduction of various substituents into the phenyl rings of dodecyltriphenylphosphonium and the measurement of the flip-flop of the synthesized cations by the current relaxation method revealed that methyl groups accelerated significantly the cation penetration through the lipid membrane, depending on the number of groups introduced. However, halogenation slowed down the penetration of the analogues. This result is strictly opposite to the flip-flop acceleration observed for halogenated tetraphenylborate anions. Density functional theory and the polarizable continuum solvent model were used to calculate the solvation energies of methyltriphenylphosphonium and methyltriphenylborate analogues. A good agreement was demonstrated between the difference in the free energy of ion solvation in water and octane and the absolute value of the central free energy barrier estimated from experimental data. Our results reveal that increasing the size of the lipophilic ion can lead to both acceleration and deceleration of the transmembrane flip-flop rate depending on the substituent and sign of the ion. This finding also emphasizes the different nature of ion-water interactions for structurally similar substituted hydrophobic anions and cations.

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 pK(a) value (similar to 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.

We present the study of interaction of fluorescein derivatives, MitoFluo and C 8 -Fl with two types of liposomes modeling cellular and mitochondrial membranes. Polarization anisotropy, fluorescence lifetimes, and rotational diffusion times have been determined from time-resolved polarized fluorescence experiments. The analysis of the experimental results has shown that both fluorescein derivatives interact with liposomes and that MitoFluo embeds in liposomes more effectively than C 8 -Fl.

To clarify the contribution of charge delocalization in a lipophilic ion to the efficacy of its permeation through a lipid membrane, we compared the behavior of alkyl derivatives of triphenylphosphonium, tricyclohexylphosphonium and trihexylphosphonium both in natural and artificial membranes. Exploring accumulation of the lipophilic cations in response to inside-negative membrane potential generation in mitochondria by using an ion-selective electrode revealed similar mitochondrial uptake of butyltricyclohexylphosphonium (C4TCHP) and butyltriphenylphosphonium (C4TPP). Fluorescence correlation spectroscopy also demonstrated similar membrane potential-dependent accumulation of fluorescein derivatives of tricyclohexyldecylphosphonium and decyltriphenylphosphonium in mitochondria. The rate constant of lipophilic cation translocation across the bilayer lipid membrane (BLM), measured by the current relaxation method, moderately increased in the following sequence: trihexyltetradecylphosphonium ([P6,6,6,14])

Appending a lipophylic alkyl chain by ester bond to fluorescein has been previously shown to convert this popular dye into an effective protonophoric uncoupler of oxidative phosphorylation in mitochondria, exhibiting neuro- and nephroprotective effects in murine models. In line with this finding, we here report data on the pronounced depolarizing effect of a series of fluorescein decyl esters on bacterial cells. The binding of the fluorescein derivatives to Bacillus subtilis cells was monitored by fluorescence microscopy and fluorescence correlation spectroscopy (FCS). FCS revealed the energy-dependent accumulation of the fluorescein esters with decyl(triphenyl)- and decyl(tri-p-tolyl)phosphonium cations in the bacterial cells. The latter compound proved to be the most potent in suppressing B. subtilis growth.

Voltage-dependent translocation of a series of cationic rhodamine B derivatives differing in n-alkyl chain length (ethyl, butyl, octyl, dodecyl, octadecyl) from one lipid monolayer to another was studied by measuring electrical current relaxation after a voltage jump on a planar bilayer phosphatidylcholine (PC) membrane. The rate of the translocation decreased in the following series of lipids: diphytanyl-PC > dioleyl-PC > diphytanoyl-PC > dierucoyl-PC. For all the lipids studied, the rate increased with lengthening of the hydrocarbon chain of the rhodamine derivatives, with the increase being most pronounced for the compounds having a short alkyl chain. The results could be well explained by involvement of molecule reorientations in the process of transmembrane flip-flop of the hydrophobic membrane-bound compounds. However, an impact of membrane dipole potential on the translocation rate could not be excluded, because the dipole potential could contribute to the energy barrier for translocation of the compounds located at different depths in the water-membrane interface. Based on the data obtained, a difference in the dipole potential of ester diphytanoyl-PC membranes with respect to ether diphytanyl-PC was estimated to be 108 mV, highlighting the contribution of a layer of oriented carbonyl groups of the lipids to the membrane dipole potential.

Rigid severe post-traumatic thoracic spine deformities result from frequent, recent high-energy trauma in children with an increasing frequency due to a variety of reasons. These types of injuries are commonly followed by spinal cord anatomic injury; therefore, the treatment of these patients warrants special attention from the ethical viewpoint. Generally, the only indication for surgical intervention is spinal dysfunction. Considering this and the patients’ ordinary severe somatic state, surgical trauma should be minimized as much as possible. However, for adequate deformity correction, effective spine stabilization and restoration of liquorodynamics is necessary. Recent studies have reported the successful use of different methods of dorsal interventions (P/VCR) in cases with unstable damages in children. Here, we present the case of a 15-year-old boy who underwent surgical treatment for coarse post-traumatic thoracic spine deformity with chronic fracture-dislocation of Th7 vertebra.

Sterols change the biophysical properties of lipid membranes. Here, we analyzed how sterols affect the activity of widely used antimicrobial membrane-active compounds, sodium dodecyl sulfate (SDS) and benzalkonium chloride (BAC). We also tested a novel benzalkonium-like substance, Kor105. Our data suggest that benzalkonium and Kor105 disturb the ordering of the membrane lipid packaging, and this disturbance is dampened by cholesterol. The disturbance induced by Kor105 is stronger than that induced by BAC because of the higher rigidity of the Kor105 molecule due to a shorter linker between the phenyl group and quaternary nitrogen. On the contrary, individual SDS molecules do not cause the disturbance. Thus, in the tested range of concentrations, SDS&ndash, membrane interaction is not influenced by cholesterol. To study how sterols influence the biological effects of these chemicals, we used yeast strains lacking Lam1&ndash, 4 proteins. These proteins transport sterols from the plasma membrane into the endoplasmic reticulum. We found that the mutants are resistant to BAC and Kor105 but hypersensitive to SDS. Together, our findings show that sterols influence the interaction of SDS versus benzalkonium chloride and Kor105 with the membranes in a completely different manner.

We have propose an original approach for fabrication of flexible antennas for biomedical-related applications. The technology is based on magnetron sputtering and laser ablation methods. The magnetron sputtering method is used to deposit a thin layer of metal (copper) on a flexible substrate (polyimide film). Then laser ablation is utilized to remove excess copper thus forming an antenna pattern on the metal layer. ISM band flexible design with microstrip feeding structure was chosen for testing this technique. Results of numerical simulation are presented. A number of test flexible antennas were fabricated by our original approach. Similar flexible antennas were also fabricated via photolithography process. A comparison of the experimental results of return loss (S11) measurements of antennas with different fabrication approach was carried out. The obtained experimental data is in a good agreement with the numerical modeling results. Proposed technology has significant advantages in cost, speed, and flexibility over photolithography processes commonly utilized for such applications.

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1–2 μM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.

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.

We have proposed and developed an original approach for fabrication flexible antennas for biomedical application. The technology is based on magnetron sputtering and laser ablation methods. The magnetron sputtering method is used to deposit a thin layer of metal (copper) on a flexible (polyimide) substrate. Then laser ablation is utilized to cut an antenna structure from the copper layer. For testing our approach we have chosen a number of examples of flexible antenna scheme with microstrip feeding structure working in ISM bands. A number of test flexible antennas were fabricated. Return loss (S11) of fabricated antennas were measured experimentally and calculated numerically. The obtained experimental results are in good agreement with the numerical ones. The proposed technology has significant advantages in cost, speed and flexibility over lithography processes commonly utilized for such applications.

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.

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.

Background Multiple sclerosis (MS) is one of the most widespread chronic neurological diseases that manifests itself by progressive demyelination in the central nervous system. The study of MS pathogenesis begins with the onset of the relapsing-remitting phase of the disease, which becomes apparent due to microglia activation, neuroinflammation and demyelination/ remyelination in the white matter. The following progressive phase is accompanied by severe neurological symptoms when demyelination and neurodegeneration are spread to both gray and white matter. In this review, we discuss a possible role of mitochondrial reactive oxygen species (mtROS) in MS pathogenesis, mechanisms of mtROS generation and effects of some mitochondria-targeted antioxidants as potential components of MS therapy. Results In the early phase of MS, mtROS stimulate NLRP3 inflammasomes, which is critical for the formation of local inflammatory lesions. Later, mtROS contribute to blood-brain barrier disruption induced by mediators of inflammation, followed by infiltration of leukocytes. ROS generated by leukocytes and activated microglia promote mitochondrial dysfunction and oligodendrocyte cell death. In the progressive phase, neurodegeneration also depends on excessive mtROS generation. Currently, only a few immunomodulatory drugs are approved for treatment of MS. These drugs mainly reduce the number of relapses but do not stop MS progression. Certain dietary and synthetic antioxidants have demonstrated encouraging results in animal models of MS but were ineffective in the completed clinical trials. Conclusion Novel mitochondria-targeted antioxidants could be promising components of combined programs for MS therapy considering that they can be applied at extremely low doses and concurrently demonstrate anti-inflammatory and neuroprotective activities.

The environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD) is generally utilized to monitor dynamic properties of membrane lipids and proteins. Here we studied the behavior of a homologous series of 4- n -alkylamino-substituted NBD derivatives (NBD-C n ; n = 4, 6, 8, 9, 10, 12) in planar lipid bilayers, liposomes and isolated mitochondria. NBD-C 10 induced proton conductivity in planar lipid membranes, while NBD-C 4 was ineffective. The NBD-C n compounds readily provoked proton permeability of neutral liposomes being less effective in negatively charged liposomes. NBD-C n increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Remarkably, the bell-shaped dependence of the uncoupling activity of NBD-C n on the alkyl chain length was found in mitochondria in contrast to the monotonous dependence in liposomes. The effect of NBD-C n on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by mitochondria swelling. Binding of NBD-C n to mitochondria increased with n, as shown by fluorescence correlation spectroscopy. It was concluded that despite a pK a value of the amino group in NBD-C n being about 10, i.e. far from the physiological pH range, the expected hindering of the uncoupling activity could be overcome by inserting the alkyl chain of a certain length.

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.

During protein synthesis the nascent polypeptide chain (NC) extends through the ribosomal exit tunnel (NPET). Also, the large group of macrolide antibiotics binds in the nascent peptide exit tunnel. In some cases interaction of NC with NPET leads to the ribosome stalling, a significant event in regulation of translation. In other cases NC-ribosome interactions lead to pauses in translation that play an important role in cotranslational folding of polypeptides emerging from the ribosome. The precise mechanism of NC recognition in NPET as well as factors that determine NC conformation in the ribosomal tunnel are unknown. A number of derivatives of the macrolide antibiotic 5-O-mycaminosyltylonolide (OMT) containing N-acylated amino acid or peptide residues were synthesized in order to study potential sites of NC-NPET interactions. The target compounds were prepared by conjugation of protected amino acids and peptides with the C23 hydroxyl group of the macrolide. These OMT derivatives showed high although varying abilities to inhibit the firefly luciferase synthesis in vitro. Three glycil-containing derivatives appeared to be strong inhibitors of translation, more potent than parental OMT. Molecular dynamics (MD) simulation of complexes of tylosin, OMT, and some of OMT derivatives with the large ribosomal subunit of E. coli illuminated a plausible reason for the high inhibitory activity of Boc-Gly-OMT. In addition, the MD study detected a new putative site of interaction of the nascent polypeptide chain with the NPET walls.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2-demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria-targeted antioxidants effective in treating a large number of age-related pathologies. This review summarizes new data on the antioxidant and some other properties of membrane-penetrating cationic compounds where 2-demethylplastoquinone substitutes for plastoquinone. It was found that such a substitution significantly increases a window between anti- and prooxidant concentrations of the conjugates. Like the original plastoquinone derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate through model and natural membranes, specifically accumulate in mitochondria in an electrophoretic fashion, and strongly inhibit H2O2-induced apoptosis at pico- and nanomolar concentrations in cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promising mitochondria-targeted drugs of the quinone series.

In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work (Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines (NBD-Cn) were shown to exhibit uncoupling activity. It was concluded that despite a pKa value being about 10, the expected hindering of the uncoupling activity could be overcome by insertion of an alkyl chain. There is evidence in the literature that the introduction of an aryl substituent in the 4-amino NBD group shifts the pKa to neutral values. Here we report the data on the properties of a number of 4-arylamino derivatives of NBD, namely, alkylphenyl-amino-NBD (Cn-phenyl-NBD) with varying alkyl chain Cn. By measuring the electrical current across planar bilayer lipid membrane, the protonophoric activity of Cn-phenyl-NBD at neutral pH grew monotonously from C1- to C6-phenyl-NBD. All of these compounds increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Importantly, the uncoupling action of C6- and C4-phenyl-NBD was partially reversed by glutamate, diethyl pyrocarbonate (DEPC), 6-ketocholestanol, and carboxyatractyloside, thus pointing to the involvement of membrane proteins in the uncoupling activity of Cn-phenyl-NBD in mitochondria. The pronounced recoupling effect of DEPC, an inhibitor of an aspartate–glutamate carrier (AGC), and that of its substrates for the first time highlighted AGC participation in the action of potent uncouplers on mitochondria. C6-phenyl-NBD produced strong antimicrobial effect on Bacillus subtilis, which manifested itself in cell membrane depolarization and suppression of bacterial growth at submicromolar concentrations.

In 2008, using a model of compression brain ischemia, we presented the first evidence that mitochondria-targeted antioxidants of the SkQ family, i.e. SkQR1 [10-(6′-plastoquinonyl)decylrhodamine], have a neuroprotective action. It was shown that intraperitoneal injections of SkQR1 (0.5–1 μmol/kg) 1 day before ischemia significantly decreased the damaged brain area. Later, we studied in more detail the anti-ischemic action of this antioxidant in a model of experimental focal ischemia provoked by unilateral intravascular occlusion of the middle cerebral artery. The neuroprotective action of SkQ family compounds (SkQR1, SkQ1, SkQTR1, SkQT1) was manifested through the decrease in trauma-induced neurological deficit in animals and prevention of amyloid-β-induced impairment of long-term potentiation in rat hippocampal slices. At present, most neurophysiologists suppose that long-term potentiation underlies cellular mechanisms of memory and learning. They consider inhibition of this process by amyloid-β1-42 as an in vitro model of memory disturbance in Alzheimer’s disease. Further development of the above studies revealed that mitochondria-targeted antioxidants could retard accumulation of hyperphosphorylated τ-protein, as well as amyloid-β1-42, and its precursor APP in the brain, which are involved in developing neurodegenerative processes in Alzheimer’s disease.

Background Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. Linking a triphenyl-phosphonium cation to fluorescein through a decyl (C 10 ) spacer yields a fluorescent uncoupler, coined mitoFluo, that selectively accumulates in energized mitochondria (Denisov et al., Chem.Commun. 2014). Methods Proton-transport activity of mitoFluo was tested in liposomes reconstituted with bacteriorhodopsin. To examine the uncoupling action on mitochondria, we monitored mitochondrial membrane potential in parallel with oxygen consumption. Neuro- and nephroprotecting activity was detected by a limb-placing test and a kidney ischemia/reperfusion protocol, respectively. Results We compared mitoFluo properties with those of its newly synthesized analog having a short (butyl) spacer (C 4 -mitoFluo). MitoFluo, but not C 4 -mitoFluo, caused collapse of mitochondrial membrane potential resulting in stimulation of mitochondrial respiration. The dramatic difference in the uncoupling activity of mitoFluo and C 4 -mitoFluo was in line with the difference in their protonophoric activity on a lipid membrane. The accumulation of mitoFluo in mitochondria was more pronounced than that of C 4 -mitoFluo . MitoFluo decreased the rate of ROS production in mitochondria. MitoFluo was effective in preventing consequences of brain trauma in rats: it suppressed trauma-induced brain swelling and reduced a neurological deficit. Besides, mitoFluo attenuated acute kidney injury after ischemia/reperfusion in rats. Conclusions A long alkyl linker was proved mandatory for mitoFluo to be a mitochondria- targeted uncoupler. MitoFluo showed high protective efficacy in certain models of oxidative stress-related diseases. General significance MitoFluo is a candidate for developing therapeutic and fluorescence imaging agents to treat brain and kidney pathologies.

To develop effective mitochondria-targeted antioxidants composed entirely of natural constituents.Novel mitochondria-targeted antioxidants were synthesized containing plant electron carrier and antioxidant plastoquinone conjugated by nonyloxycarbonylmethyl residue with berberine or palmatine, penetrating cations of plant origin. These compounds, SkQBerb and SkQPalm, were tested in model planar phospholipid membranes and micelles, liposomes, isolated mitochondria and living cells.SkQBerb and SkQPalm penetrated across planar bilayer phospholipid membrane in their cationic forms and accumulated in mitochondria isolated or in living human cells in culture. Reduced forms of SkQBerb and SkQPalm as well as C10Berb and C10Palm (SkQBerb and SkQPalm analogs lacking plastoquinol moiety) revealed radical scavenging activity in lipid micelles and liposomes, while oxidized forms were inactive. In isolated mitochondria and in living cells, berberine and palmatine moieties were not reduced, so antioxidant activity of C10Berb and C10Palm was not detected. SkQBerb and SkQPalm inhibited lipid peroxidation in isolated mitochondria at nanomolar concentrations; their prooxidant effect was observed at 1,000 times higher concentrations. In human cell cuture, nanomolar SkQBerb and SkQPalm prevented fragmentation of mitochondria and apoptosis induced by exogenous hydrogen peroxide.This is the first successful attempt to construct mitochondria-targeted antioxidants composed entirely of natural components, namely plastoquinone, nonyl, acetyl and berberine or palmatine residues.

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 β-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.

High negative electric potential inside mitochondria provides a driving force for mitochondria-targeted delivery of cargo molecules linked to hydrophobic penetrating cations. This principle is utilized in construction of mitochondria-targeted antioxidants (MTA) carrying quinone moieties which produce a number of health benefitting effects by protecting cells and organisms from oxidative stress. Here, a series of penetrating cations including MTA were shown to induce the release of the liposome-entrapped carboxyfluorescein anion (CF), but not of glucose or ATP. The ability to induce the leakage of CF from liposomes strongly depended on the number of carbon atoms in alkyl chain ( n ) of alkyltriphenylphosphonium and alkylrhodamine derivatives. In particular, the leakage of CF was maximal at n about 10–12 and substantially decreased at n = 16. Organic anions (palmitate, oleate, laurylsulfate) competed with CF for the penetrating cation-induced efflux. The reduced activity of alkylrhodamines with n = 16 or n = 18 as compared to that with n = 12 was ascribed to a lower rate of partitioning of the former into liposomal membranes, because electrical current relaxation studies on planar bilayer lipid membranes showed rather close translocation rate constants for alkylrhodamines with n = 18 and n = 12. Changes in the alkylrhodamine absorption spectra upon anion addition confirmed direct interaction between alkylrhodamines and the anion. Thus, mitochondria-targeted penetrating cations can serve as carriers of hydrophobic anions across bilayer lipid membranes.

The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273-1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663-668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH2 interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ- being reduced back to SkQH2 by heme bH of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C9 or C13 of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (capital ES, Cyrillic12TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since capital ES, Cyrillic12TPP is specifically accumulated in mitochondria and increases the H+ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by capital ES, Cyrillic12TPP.

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-aminooxy-acetic acid.

A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H + -conducting fatty acid cycling mediated by penetrating cations such as 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C 12 TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δ ψ ) of the Nernstian value (about 60 mV Δ ψ at ΔpH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C 12 TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced ( i ) uncoupling of respiration and phosphorylation, and ( ii ) inhibition of H 2 O 2 formation. In intact yeast cells, C 12 TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization.

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 theβ-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/But strategy and a chlorotrityl anchor group. The cyclization was carried out with benzotriazolyloxytris(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.

We explored the neuroprotective properties of natural plant-derived antioxidants plastoquinone and thymoquinone (2-demethylplastoquinone derivative) modified to be specifically accumulated in mitochondria. The modification was performed through chemical conjugation of the quinones with penetrating cations: Rhodamine 19 or tetraphenylphosphonium. Neuroprotective properties were evaluated in a model of middle cerebral artery occlusion. We demonstrate that the mitochondria-targeted compounds, introduced immediately after reperfusion, possess various neuroprotective potencies as judged by the lower brain damage and higher neurological status. Plastoquinone derivatives conjugated with rhodamine were the most efficient, and the least efficiency was shown by antioxidants conjugated with tetraphenylphosphonium. Antioxidants were administered intraperitoneally or intranasally with the latter demonstrating a high level of penetration into the brain tissue. The therapeutic effects of both ways of administration were similar. Long-term administration of antioxidants in low doses reduced the neurological deficit, but had no effect on the volume of brain damage. At present, cationic decylrhodamine derivatives of plastoquinone appear to be the most promising anti-ischemic mitochondria-targeted drugs of the quinone family. We suggest these antioxidants could be potentially used for a stroke treatment.