Purpose Mitochondrial dysfunction plays a significant role in the death of retinal ganglion cells (RGCs) and their axons in retinal neurodegenerative diseases. We studied the effect of the mitochondrial-targeted HDAP2 (High Density Aromatic Peptide 2) to protect MDBK cells following serum starvation in cultured cells, and in mouse retinal ganglion cells following optic nerve crush.Methods Uptake, toxicity, and efficacy of HDAP2 was studied in cultured cells under conditions of serum starvation. Once preliminary safety and efficacy of the peptide was established in vitro , HDAP2 was administered to mice following unilateral optic nerve crush and the distribution and survival of RBPMS-labeled RGCs was assessed.Results We found that water-soluble HDAP2 localizes to mitochondria and improves the mitochondrial membrane potential, prevents oxidative stress, and reduces death of cells grown in serum-free culture media. In mice treated systemically with biotinylated peptide, we found that HDAP2 labeled all layers of the retina in a pattern similar to the mitochondrial marker Cox IV, and that the peptide was colocalized with retinal ganglion cells and Müller glia. Animals treated with HDAP2 following optic nerve crush showed significantly greater survival of retinal ganglion cells compared to untreated animals at all retinal eccentricities.Conclusion Our results demonstrate that HDAP2 protects cells against serum starvation-induced decreases in mitochondrial membrane potential and leads to significant protection of retinal ganglion cells in the mouse retina following optic nerve crush in vivo. These results suggest that therapeutic agents preserving the mitochondrial membrane potential may help protect neurons following trauma or from neurodegenerative disease.### Competing Interest StatementThe authors have declared no competing interest.

ABSTRACT The mitochondrial membrane potential (ΔΨm) is created by the accumulation of protons on an outer leaflet of the inner mitochondrial membrane and drives the synthesis of most cellular ATP, which is essential for cellular bioenergetics and survival. The ΔΨm also facilitates the electrogenic transport of cations, such as Ca 2+ , and regulates generation of reactive oxygen species, which serves as a powerful bioenergetic and stress-signaling regulator. Proton trapping on the outer leaflet of the inner mitochondrial membrane of mitochondrial cristae could be controlled by cardiolipin when the local pH is above 8. However, there is presently no technology that effectively targets strong bases to cardiolipin. We have developed a novel, high-density aromatic peptide (HDAP2) to preserve a proton gradient-driven potential in mitochondria by increasing proton trapping on cardiolipin (CL). HDAP2-induced formation of cardiolipin-HDAP2 complexes accumulated positive charges at the head of CL. The HDAP2-CL vesicles could accumulate the mitochondrial transmembrane potential probe, Tetramethylrhodamine (TMRM). This potential could be uncoupled with Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and Dinitrophenol (DNP), indicating that an interaction of HDAP2 with CL could support a proton gradient-driven transmembrane potential. We demonstrated that this novel, water-soluble peptide is cell-permeable, targets mitochondria without causing cell toxicity, and promotes cell survival during serum starvation. Importantly, the HDAP2-cardiolipin complex-mediated optimization of the proton gradient was supported by the ability of HDAP2 to prevent CCCP-mediated mitochondrial depolarization in ARPE-19 cells in a dose-dependent manner. Based on its mechanism of action, HDAP2 could promote cellular homeostasis, which would have broad clinical applicability for the prevention, recovery and reversal of many acute and chronic disease conditions, such as neurodegeneration, ischemia– reperfusion injury, and inflammation.

Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.

ABSTRACT The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae . Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals.

Background: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe2+, which inhibits electron transport (ET) and promotes oxygenase activity.Methods: SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy.Results: Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20.Conclusion: Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe2+ that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release.General significance: Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery. (C) 2015 Elsevier B.V. All rights reserved.

SS‐31 is a mitochondria‐targeted therapeutic, currently in Phase 2 clinical trials, which protects cristae structure, prevents swelling of mitochondria and improves cellular respiration. We previously identified the target of SS‐31 as cardiolipin (CL), a phospholipid found only in the inner mitochondrial membrane. Here we utilize DOSY NMR to show that the diffusion rate of SS‐31 is reduced in the presence of CL‐containing bicelles, which is the first kinetic information on the interaction of SS‐31 with CL‐containing membranes. Because mitochondrial swelling involves solute transfer across the membrane, in addition to the changes in membrane morphology, it became important to test for membrane‐perturbing effects of SS‐31, particularly In light of evidence that membrane‐perturbation predicts toxicity for small molecules. We utilize a gramicidin‐based fluorescence assay using CL‐containing liposomes to assess drug‐induced membrane perturbations. We find that SS‐31 has no effect on synthetic membrane systems, even at several log orders above therapeutic concentration. The lack of membrane effects suggest that SS‐31 has no toxicity on the membrane, which is consistent with safety data from Phase 1 trials.

SS‐31 (D‐Arg‐dimethylTyr‐Lys‐Phe‐NH2) is a ROS‐scavenging cardiolipin‐targeting tetrapeptide which is known to protect against oxidative stress‐induced ischemia‐reperfusion (IR) injury. The interaction of CL with cytochrome c (cyt c) is necessary for electron carrier function of cyt c but can also lead to a [CL/cyt c] complex formation, which prevents electron transfer and promotes superoxide and hydrogen peroxide generation. SS‐31 was shown to protect the carrier function of cyt c while preventing oxidative stress and optimizing bioenergetic efficiency. To distinguish between ROS scavenging and inhibition of ROS formation in mitochondrial protection during IR injury, we developed an analog of SS‐31 which lacks the ROS‐scavenging dimethylTyr moiety (SS‐20; Phe‐D‐Arg‐Phe‐Lys‐NH2). We showed that SS‐20 interacts specifically with CL in liposomes and mitochondria. In isolated mitochondria, SS‐20 increases oxygen respiration and reduces hydrogen peroxide formation. Although SS‐20 cannot scavenge ROS, it is as effective as SS‐31 at protecting cristae architecture during ischemia and accelerating ATP recovery upon reperfusion in a model of acute kidney ischemia. Thus we demonstrate that targeting the [CL/cyt c] complex to protect electron transport and prevent ROS formation prevents mitochondrial IR injury without ROS scavenging.

Doxorubicin (DOX) is an effective chemotherapeutic that intercalates DNA and inhibits topoisomerase II in cancer cells. However, clinical use of DOX is limited by its cardiac toxicity, resulting from its ability to bind to cardiolipin (CL) and inhibit mitochondrial function. SS‐31, a mitochondria‐targeted tetrapeptide was shown to prevent DOX‐induced toxicity in cardiac and skeletal muscle, and cultured myocytes. However, the mechanism of protection against DOX by SS‐31 is not well understood. Here, by taking advantage of the intrinsic fluorescence of DOX, we developed assays to measure DOX binding to free CL, CL‐containing liposomes, mitoplasts and mitochondria. We demonstrated that SS‐31 prevents DOX binding in different CL‐containing model systems. Furthermore, we showed that SS‐31 could inhibit DOX binding to endogenous CL and uptake in mitoplasts and mitochondria in a dose dependent manner. Therefore, SS‐31 protects against mitochondrial DOX toxicity by preventing its binding to CL. Targeting CL with SS‐31 has clinical implications in increasing the therapeutic window of DOX and potentially addressing other therapeutics with off‐target side effects associated with CL and mitochondrial toxicity.

Ischemia time during partial nephrectomy is strongly associated with acute and chronic renal injury. ATP depletion during warm ischemia inhibits ATP-dependent processes, resulting in cell swelling, cytoskeletal breakdown, and cell death. The duration of ischemia tolerated by the kidney depends on the amount of ATP that can be produced with residual substrates and oxygen in the tissue to sustain cell function. We previously reported that the rat can tolerate 30-min ischemia quite well but 45-min ischemia results in acute kidney injury and progressive interstitial fibrosis. Here, we report that pretreatment with SS-20 30 min before warm ischemia in the rat increased ischemia tolerance from 30 to 45 min. Histological examination of kidney tissues revealed that SS-20 reduced cytoskeletal breakdown and cell swelling after 45-min ischemia. Electron microscopy showed that SS-20 reduced mitochondrial matrix swelling and preserved cristae membranes, suggesting that SS-20 enhanced mitochondrial ATP synthesis under ischemic conditions. Studies with isolated kidney mitochondria showed dramatic reduction in state 3 respiration and respiratory control ratio after 45-min ischemia, and this was significantly improved by SS-20 treatment. These results suggest that SS-20 increases efficiency of the electron transport chain and improves coupling of oxidative phosphorylation. SS-20 treatment after ischemia also significantly reduced interstitial fibrosis. These new findings reveal that enhancing mitochondrial bioenergetics may be an important target for improving ischemia tolerance, and SS-20 may serve well for minimizing acute kidney injury and chronic kidney disease following surgical procedures such as partial nephrectomy and transplantation.

Cardiolipin (CL) plays a major role in mitochondrial cristae formation and ATP synthesis. Peroxidation of CL during mitochondrial oxidative stress causes cristae remodeling and initiates apoptosis. Cardiolipin peroxidation may occur by free radical lipid oxidation or by peroxidase activity of cyt c. Cyt c peroxidase activity is greatly enhanced when it is in a complex with CL, whereby an acyl chain impales cyt c, exposing the heme Fe to H2O2. Here we report the first mitochondria‐targeted compound that selectively targets CL and inhibits cyt c peroxidase activity. Using a polarity‐sensitive amino acid (aladan), we demonstrate that SS‐31, a tetrapeptide that targets inner mitochondrial membrane, interacts with CL via electrostatic and hydrophobic interactions. SS‐31 inhibits cyt c peroxidase activity induced by CL in a dose‐dependent manner. Circular dichroism indicates that this inhibitory activity is due to the ability of the [CL/SS‐31] complex to protect the heme Fe. This inhibition does not rely on the ability of SS‐31 to scavenge H2O2 as the non‐scavenging analog (SS‐20) also inhibits cyt c peroxidase. Both SS‐31 and SS‐20 inhibits basal and Ca2+‐stimulated cyt c peroxidase activity. Furthermore, we have evidence that SS‐31 and SS‐20 prevent cristae remodeling during ischemia‐reperfusion (IR) and inhibit apoptosis. SS‐31 is the first cyt c peroxidase inhibitor in clinical trial for cardiorenal IR injury

Ischemia causes AKI as a result of ATP depletion, and rapid recovery of ATP on reperfusion is important to minimize tissue damage. ATP recovery is often delayed, however, because ischemia destroys the mitochondrial cristae membranes required for mitochondrial ATP synthesis. The mitochondria-targeted compound SS-31 accelerates ATP recovery after ischemia and reduces AKI, but its mechanism of action remains unclear. Here, we used a polarity-sensitive fluorescent analog of SS-31 to demonstrate that SS-31 binds with high affinity to cardiolipin, an anionic phospholipid expressed on the inner mitochondrial membrane that is required for cristae formation. In addition, the SS-31/cardiolipin complex inhibited cytochrome c peroxidase activity, which catalyzes cardiolipin peroxidation and results in mitochondrial damage during ischemia, by protecting its heme iron. Pretreatment of rats with SS-31 protected cristae membranes during renal ischemia and prevented mitochondrial swelling. Prompt recovery of ATP on reperfusion led to rapid repair of ATP-dependent processes, such as restoration of the actin cytoskeleton and cell polarity. Rapid recovery of ATP also inhibited apoptosis, protected tubular barrier function, and mitigated renal dysfunction. In conclusion, SS-31, which is currently in clinical trials for ischemia-reperfusion injury, protects mitochondrial cristae by interacting with cardiolipin on the inner mitochondrial membrane.

A novel UV-C-light-induced ribozyme activity was discovered within the highly structured 5'-genomic regions of both Hepatitis C Virus (HCV) and the related Classic Swine Fever Virus (CSFV). Cleavage is mediated by exposure to UV-C light but not by exogenous oxygen radicals. It is also very selective, occurring at base positions HCV C-79 and CSFV A(45) in some molecules and at the immediately adjacent 5'-positions HCV U-78 and CSFV U-44 in others. Among other reaction products, the majority of biochemically active products detected contained 3'-phosphate and 5'-phosphate-end groups at the newly generated termini, along with a much lower amount of 3'-hydroxyl end group. While preservation of an E-loop RNA structure in the vicinity of the cleavage site was a requisite for HCV RNA self-cleavage, this was not the case for CSFV RNA. The short size of the reactive domains (similar to 33 nt), which are compatible with primitive RNA motifs, and the lack of sequence homology, indicate that as-yet unidentified UV-activated ribozymes are likely to be found throughout structured RNAs, thereby providing clues to whether early RNA self-cleavage events were mediated by photosensitive RNA structures.

Defects in the mitochondrial electron transport chain (ETC) underlie the pathogenesis of many diseases ranging from metabolic syndrome to heart failure, and Alzheimer's Disease. Optimization of electron transfer (ET) through ETC provides a unifying therapeutic approach to these diverse disorders. SS‐31 is a synthetic tetrapeptide that selectively targets the inner mitochondrial membrane and has been shown to protect mitochondrial integrity, preserve ATP synthesis, and minimize ROS formation in numerous animal models. We now report that that SS‐31 acts by facilitating ET in the ETC. In isolated mitochondria, SS‐31 potentiated O2 consumption and ATP synthesis initiated by complex I or II substrates, or by direct reduction of cyt C. SS‐31 also promoted ET in cyt C‐deficient mitoplasts, suggesting that SS‐31 facilitates ET through cyt C, especially under rate‐limiting conditions. Circular dichroism and Trp fluorescence verified that SS‐31 interacts directly with cyt C. Photoluminescence and cyclic voltammetry established that SS‐31 increases electron capacity of cyt C and promotes electron diffusion through cyt C. We conclude that SS‐31 minimized the energy barrier for ET through cyt C. This concept represents a novel approach for protecting cellular energy and a paradigm shift in the treatment of a multitude of diseases associated with mitochondrial dysfunction.

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-alpha for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-alpha is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.

The aminoglycoside, geneticin (G418), was recently shown to have antiviral activity against bovine viral diarrhea virus (BVDV). Since BVDV, dengue virus (DENV) and yellow fever virus (YFV) all belong to the Flaviviridae family, it seemed possible that a common step in their life cycle might be affected by this aminoglycoside. Here it is shown that geneticin prevented the cytopathic effect (CPE) resulting from DENV-2 infection of BHK cells, in a dose-dependent manner with an 50% effective concentration (EC50) value of 3 ± 0.4 μg/ml. Geneticin had no detectable effect on CPE caused by YFV in BHK cells. Geneticin also inhibited DENV-2 viral yield with an EC50 value of 2 ± 0.1 μg/ml and an EC90 value of 20 ± 2 μg/ml. With a CC50 value of 165 ± 5 μg/ml, the selectivity index of anti-DENV activity of geneticin in BHK cells was established to be 66. Furthermore, 25 μg/ml of geneticin nearly completely blocked plaque formation induced by DENV-2, but not YFV. In addition, geneticin, inhibited DENV-2 viral RNA replication and viral translation. Gentamicin, kanamycin, and the guanidinylated geneticin showed no anti-DENV activity. Neomycin and paromomycin demonstrated weak antiviral activity at high concentrations. Finally, aminoglycoside-3′-phosphotransferase activity of neomycin-resistant gene abolished antiviral activity of geneticin.

It has been proposed that the hepatitis C virus (HCV) internal ribosome entry site (IRES) resides within a locked conformation, owing to annealing of its immediate flanking sequences. In this study, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools showed that this region switches to an open conformation triggered by the liver-specific microRNA, miR-122. This structural transition, observed in vitro, may be the mechanistic basis for the involvement of downstream IRES structural domain VI in translation, as well as providing a role of liver-specific miR-122 in HCV infection. In addition, the induced RNA switching at the 5' untranslated region could ultimately represent a new mechanism of action of micro-RNAs.

Background: Aminoglycoside G418 is commonly used to generate stable replicons for RNA viruses, such as hepatitis C virus, West Nile virus, and bovine viral diarrhoea virus (BVDV). This precludes testing G418's own antiviral activities against those viruses. Here, we report antiviral activity of G418 against BVDV. Methods: Cell viability and virus yield reduction assays were used to investigate antiviral effects of G418 against BVDV. The expression of viral proteins and RNA were determined by western blot and real-time quantitive PCR, respectively. Results: We demonstrated that G418 (50% cytotoxicity concentration of 400 µg/ml) improved cell viability of Madin-Darby bovine kidney cells infected with a cytopathic strain of BVDV (NADL) in a dose-dependent manner with 50% effective concentration of 4 µg/ml. Interestingly, close structural analogues with known properties as translation inhibitors similar to G418 — kanamycin and gentamicin — had no antiviral activity against BVDV. In addition, G418 inhibits virus yield of two different strains of BVDV (NADL and NY-1) without affecting viral RNA replication and translation or viral NS3 protein processing. Conclusion: Our data indicate that antiviral activity of G418 could result from interference with either the assembly or release of active virus, rather than the regulation of viral translation and replication. Thus, we propose the use of chemical analogues of G418 as antiviral therapeutics for treatment of viral diseases associated with the Flaviviridae family, such as hepatitis C virus, dengue virus, yellow fever virus, West Nile virus and others.