Confirmation of the Cardioprotective Effect of MitoGamide in the Diabetic Heart.

PURPOSE: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide. METHODS AND RESULTS: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice. The myocardium of Akita mice had a decreased ATP/ADP ratio, elevated mitochondrial oxidative stress and increased organelle density, compared with that of wild-type mice. MitoGamide, a mitochondria-targeted 1,2-dicarbonyl scavenger, exhibited good stability in vivo, uptake into cells and mitochondria and reactivity with dicarbonyls. Treatment of Akita mice with MitoGamide for 12 weeks significantly improved the E/A ratio compared with the vehicle-treated group. CONCLUSION: Our work confirms that the Akita mouse model of diabetes replicates key clinical features of diabetic HFpEF, including cardiac and mitochondrial dysfunction. Furthermore, in this independent study, MitoGamide treatment improved diastolic function in Akita mice.

Analysis of Memory B-Cell Responses Reveals Suboptimal Dosing Schedule of a Licensed Vaccine.

Current guidance recommends that adolescents receive a 2-dose human papillomavirus (HPV) vaccine, whereas young adults and immunocompromised persons receive 3 doses. We examined secondary responses of vaccine-elicited memory B cells (Bmem) in naive women receiving 3 doses of the quadrivalent HPV vaccine to understand the quality of B-cell memory generated by this highly effective vaccine. Unexpectedly, we observed a lower Bmem response rate and magnitude of Bmem responses to the third dose than to a booster dose administered at month 24. Moreover, high titers of antigen-specific serum antibody at vaccination inversely correlated with Bmem responses. As the purpose of additional doses/boosters is to stimulate Bmem to rapidly boost antibody levels, these results indicate the timing of the third dose is suboptimal and lend support to a 2-dose HPV vaccine for young adults. Our findings also indicate more broadly that multidose vaccine schedules should be rationally determined on the basis of Bmem responses.

ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives.

There is an increasing interest in targeting molecules to the mitochondrial matrix. Many proteins are naturally imported through the translocase complexes found in the outer and inner mitochondrial membranes. One possible means for importing molecules is therefore to use a mitochondrial pre-protein as a vector and assess what forms of molecules can be attached to the pre-protein as cargo. A major difficulty with this approach is to ensure that any chimaeric molecule does indeed access the mitochondrial matrix and does not merely associate with the mitochondrial membranes. We have recently demonstrated that click chemistry can be used both to demonstrate convincingly mitochondrial import of a peptide-peptide nucleic acid conjugate and also to quantify the mitochondrial uptake for specific synthetic conjugates. We now report an adaptation of the synthesis to facilitate simple quantification of multiple molecules and hence to calculate the efficiency of their mitochondrial import.

Desipramine decreases expression of human and murine indoleamine-2,3-dioxygenases.

Abundant evidence connects depression symptomology with immune system activation, stress and subsequently elevated levels of kynurenine. Anti-depressants, such as the tricyclic norepinephrine/serotonin reuptake inhibitor desipramine (Desip), were developed under the premise that increasing extracellular neurotransmitter level was the sole mechanism by which they alleviate depressive symptomologies. However, evidence suggests that anti-depressants have additional actions that contribute to their therapeutic potential. The Kynurenine Pathway produces tryptophan metabolites that modulate neurotransmitter activity. This recognition identified another putative pathway for anti-depressant targeting. Considering a recognized role of the Kynurenine Pathway in depression, we investigated the potential for Desip to alter expression of rate-limiting enzymes of this pathway: indoleamine-2,3-dioxygenases (Ido1 and Ido2). Mice were administered lipopolysaccharide (LPS) or synthetic glucocorticoid dexamethasone (Dex) with Desip to determine if Desip alters indoleamine-dioxygenase (DO) expression in vivo following a modeled immune and stress response. This work was followed by treating murine and human peripheral blood mononuclear cells (PBMCs) with interferon-gamma (IFNγ) and Desip. In vivo: Desip blocked LPS-induced Ido1 expression in hippocampi, astrocytes, microglia and PBMCs and Ido2 expression by PBMCs. Ex vivo: Desip decreased IFNγ-induced Ido1 and Ido2 expression in murine PBMCs. This effect was directly translatable to the human system as Desip decreased IDO1 and IDO2 expression by human PBMCs. These data demonstrate for the first time that an anti-depressant alters expression of Ido1 and Ido2, identifying a possible new mechanism behind anti-depressant activity. Furthermore, we propose the assessment of PBMCs for anti-depressant responsiveness using IDO expression as a biomarker.

A Single Human Papillomavirus Vaccine Dose Improves B Cell Memory in Previously Infected Subjects.

Although licensed human papillomavirus (HPV) vaccines are most efficacious in persons never infected with HPV, they also reduce infection and disease in previously infected subjects, indicating natural immunity is not entirely protective against HPV re-infection. The aim of this exploratory study was to examine the B cell memory elicited by HPV infection and evaluate whether vaccination merely boosts antibody (Ab) levels in previously infected subjects or also improves the quality of B cell memory. Toward this end, the memory B cells (Bmem) of five unvaccinated, HPV-seropositive subjects were isolated and characterized, and subject recall responses to a single HPV vaccine dose were analyzed. Vaccination boosted Ab levels 24- to 930-fold (median 77-fold) and Bmem numbers 3- to 27-fold (median 6-fold). In addition, Abs cloned from naturally elicited Bmem were generally non-neutralizing, whereas all those isolated following vaccination were neutralizing. Moreover, Ab and plasmablast responses indicative of memory recall responses were only observed in two subjects. These results suggest HPV vaccination augments both the magnitude and quality of natural immunity and demonstrate that sexually active persons could also benefit from HPV vaccination. This study may have important public policy implications, especially for the older 'catch-up' group within the vaccine's target population.

Interactions between inflammatory mediators and corticosteroids regulate transcription of genes within the Kynurenine Pathway in the mouse hippocampus.

BACKGROUND: Increased tryptophan metabolism towards the production of kynurenine via indoleamine/tryptophan-2,3-dioxygenases (DOs: Ido1, Ido2, and Tdo2) is strongly associated with the prevalence of major depressive disorder in patients and the induction of depression-like behaviors in animal models. Several studies have suggested that activation of the immune system or elevated corticosteroids drive DO expression; however, mechanisms linking cytokines, corticosteroids, and DOs to psychiatric diseases remain unclear. Various attempts have been made to correlate DO gene expression within the brain to behavior, but disparate results have been obtained. We believe that discrepancies arise as a result of the under-recognized existence of multiple mRNA transcripts for each DO. Unfortunately, there are no reports regarding how the multiple transcripts are distributed or regulated. Here, we used organotypic hippocampal slice cultures (OHSCs) to directly test the ability of inflammatory and stress mediators to differentially regulate DO transcripts. METHODS: OHSCs were treated with pro-inflammatory mediators (interferon-gamma (IFNγ), lipopolysaccharide (LPS), and polyinosine-polycytidylic acid (pI:C)) with or without corticosteroids (dexamethasone (Dex: glucocorticoid receptor (GR) agonist), aldosterone (Aldo: mineralocorticoid receptor (MR) agonist), or corticosterone (Cort: GR/MR agonist)). RESULTS: IFNγ induced Ido1-full length (FL) and Ido1-variant (v) expression, and surprisingly, Dex, Cort, and Aldo interacted with IFNγ to further elevate expression of Ido1, importantly, in a transcript dependent manner. IFNγ, LPS, and pI:C increased expression of Ido2-v1 and Ido2-v3 transcripts, whereas only IFNγ increased expression of Ido2-v2. Overall Ido2 transcripts were relatively unaffected by GR or MR activation. Naïve mouse brain expresses multiple Tdo2 transcripts. Dex and Cort induced expression of only one of the three Tdo2 transcripts (Tdo2-FL) in OHSCs. CONCLUSIONS: These results establish that multiple transcripts for all three DOs are expressed within the mouse hippocampus, under the control of distinct regulatory pathways. These data identify a previously unrecognized interaction between corticosteroid receptor activation and inflammatory signals on DO gene expression, which suggest that corticosteroids act to differentially enhance gene expression of Ido1, Ido2, and Tdo2.

Assessing the Delivery of Molecules to the Mitochondrial Matrix Using Click Chemistry.

Mitochondria are central to health and disease, hence there is considerable interest in developing mitochondria-targeted therapies that require the delivery of peptides or nucleic acid oligomers. However, progress has been impeded by the lack of a measure of mitochondrial import of these molecules. Here, we address this need by quantitatively detecting molecules within the mitochondrial matrix. We used a mitochondria- targeted cyclooctyne (MitoOct) that accumulates several- hundredfold in the matrix, driven by the membrane potential. There, MitoOct reacts through click chemistry with an azide on the target molecule to form a diagnostic product that can be quantified by mass spectrometry. Because the membrane potential-dependent MitoOct concentration in the matrix is essential for conjugation, we can now determine definitively whether a putative mitochondrion-targeted molecule reaches the matrix. This "ClickIn" approach will facilitate development of mitochondria-targeted therapies.

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model.

Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry.

The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo.

Synthesis of triphenylphosphonium vitamin E derivatives as mitochondria-targeted antioxidants.

A series of mitochondria-targeted antioxidants comprising a lipophilic triphenylphosphonium cation attached to the antioxidant chroman moiety of vitamin E by an alkyl linker have been prepared. The synthesis of a series of mitochondria-targeted vitamin E derivatives with a range of alkyl linkers gave compounds of different hydrophobicities. This work will enable the dependence of antioxidant defence on hydrophobicity to be determined in vivo.

A mitochondria-targeted derivative of ascorbate: MitoC.

Mitochondrial oxidative damage contributes to a wide range of pathologies. One therapeutic strategy to treat these disorders is targeting antioxidants to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation. To date only hydrophobic antioxidants have been targeted to mitochondria; however, extending this approach to hydrophilic antioxidants offers new therapeutic and research opportunities. Here we report the development and characterization of MitoC, a mitochondria-targeted version of the hydrophilic antioxidant ascorbate. We show that MitoC can be taken up by mitochondria, despite the polarity and acidity of ascorbate, by using a sufficiently hydrophobic link to the TPP moiety. MitoC reacts with a range of reactive species, and within mitochondria is rapidly recycled back to the active ascorbate moiety by the glutathione and thioredoxin systems. Because of this accumulation and recycling MitoC is an effective antioxidant against mitochondrial lipid peroxidation and also decreases aconitase inactivation by superoxide. These findings show that the incorporation of TPP function can be used to target polar and acidic compounds to mitochondria, opening up the delivery of a wide range of bioactive compounds. Furthermore, MitoC has therapeutic potential as a new mitochondria-targeted antioxidant, and is a useful tool to explore the role(s) of ascorbate within mitochondria.

Multiple binding modes of isothiocyanates that inhibit macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has roles in the innate immune response, and also contributes to inflammatory disease. While the biological properties of MIF are closely linked to protein-protein interactions, MIF also has tautomerase activity. Inhibition of this activity interferes with the interaction of MIF with protein partners e.g. the CD74 receptor, and tautomerase inhibitors show promise in disease models including multiple sclerosis and colitis. Isothiocyanates inhibit MIF tautomerase activity via covalent modification of the N-terminal proline. We systematically explored variants of benzyl and phenethyl isothiocyanates, to define determinants of inhibition. In particular, substitution with hydroxyl, chloro, fluoro and trifluoro moieties at the para and meta positions were evaluated. In assays on treated cells and recombinant protein, the IC50 varied from 250 nM to >100 µM. X-ray crystal structures of selected complexes revealed that two binding modes are accessed by some compounds, perhaps owing to strain in short linkers between the isothiocyanate and aromatic ring. The variety of binding modes confirms the existence of two subsites for inhibitors and establishes a platform for the development of potent inhibitors of MIF that only need to target one of these subsites.

A novel class of mitochondria-targeted soft electrophiles modifies mitochondrial proteins and inhibits mitochondrial metabolism in breast cancer cells through redox mechanisms.

Despite advances in screening and treatment over the past several years, breast cancer remains a leading cause of cancer-related death among women in the United States. A major goal in breast cancer treatment is to develop safe and clinically useful therapeutic agents that will prevent the recurrence of breast cancers after front-line therapeutics have failed. Ideally, these agents would have relatively low toxicity against normal cells, and will specifically inhibit the growth and proliferation of cancer cells. Our group and others have previously demonstrated that breast cancer cells exhibit increased mitochondrial oxygen consumption compared with non-tumorigenic breast epithelial cells. This suggests that it may be possible to deliver redox active compounds to the mitochondria to selectively inhibit cancer cell metabolism. To demonstrate proof-of-principle, a series of mitochondria-targeted soft electrophiles (MTSEs) has been designed which selectively accumulate within the mitochondria of highly energetic breast cancer cells and modify mitochondrial proteins. A prototype MTSE, IBTP, significantly inhibits mitochondrial oxidative phosphorylation, resulting in decreased breast cancer cell proliferation, cell attachment, and migration in vitro. These results suggest MTSEs may represent a novel class of anti-cancer agents that prevent cancer cell growth by modification of specific mitochondrial proteins.

Characteristics of memory B cells elicited by a highly efficacious HPV vaccine in subjects with no pre-existing immunity.

Licensed human papillomavirus (HPV) vaccines provide near complete protection against the types of HPV that most commonly cause anogenital and oropharyngeal cancers (HPV 16 and 18) when administered to individuals naive to these types. These vaccines, like most other prophylactic vaccines, appear to protect by generating antibodies. However, almost nothing is known about the immunological memory that forms following HPV vaccination, which is required for long-term immunity. Here, we have identified and isolated HPV 16-specific memory B cells from female adolescents and young women who received the quadrivalent HPV vaccine in the absence of pre-existing immunity, using fluorescently conjugated HPV 16 pseudoviruses to label antigen receptors on the surface of memory B cells. Antibodies cloned and expressed from these singly sorted HPV 16-pseudovirus labeled memory B cells were predominantly IgG (>IgA>IgM), utilized diverse variable genes, and potently neutralized HPV 16 pseudoviruses in vitro despite possessing only average levels of somatic mutation. These findings suggest that the quadrivalent HPV vaccine provides an excellent model for studying the development of B cell memory; and, in the context of what is known about memory B cells elicited by influenza vaccination/infection, HIV-1 infection, or tetanus toxoid vaccination, indicates that extensive somatic hypermutation is not required to achieve potent vaccine-specific neutralizing antibody responses.

Incorporation of triphenylphosphonium functionality improves the inhibitory properties of phenothiazine derivatives in Mycobacterium tuberculosis.

Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. The need for improved therapies is required to kill different M. tuberculosis populations present during infection and to kill drug resistant strains. Protein complexes associated with energy generation, required for the survival of all M. tuberculosis populations, have shown promise as targets for novel therapies (e.g., phenothiazines that target type II NADH dehydrogenase (NDH-2) in the electron transport chain). However, the low efficacy of these compounds and their off-target effects has made the development of phenothiazines as a therapeutic agent for TB limited. This study reports that a series of alkyltriphenylphosphonium (alkylTPP) cations, a known intracellular delivery functionality, improves the localization and effective concentration of phenothiazines at the mycobacterial membrane. AlkylTPP cations were shown to accumulate at biological membranes in a range of bacteria and lipophilicity was revealed as an important feature of the structure-function relationship. Incorporation of the alkylTPP cationic function significantly increased the concentration and potency of a series of phenothiazine derivatives at the mycobacterial membrane (the site of NDH-2), where the lead compound 3a showed inhibition of M. tuberculosis growth at 0.5µg/mL. Compound 3a was shown to act in a similar manner to that previously published for other active phenothiazines by targeting energetic processes (i.e., NADH oxidation and oxygen consumption), occurring in the mycobacterial membrane. This shows the enormous potential of alkylTPP cations to improve the delivery and therefore efficacy of bioactive agents targeting oxidative phosphorylation in the mycobacterial membrane.

Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration that ultimately results in progressive paralysis and death. Growing evidence indicates that mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in ALS. To further explore the hypothesis that mitochondrial dysfunction and nitroxidative stress contribute to disease pathogenesis at the in vivo level, we assessed whether the mitochondria-targeted antioxidant [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium methane sulfonate (MitoQ) can modify disease progression in the SOD1(G93A) mouse model of ALS. To do this, we administered MitoQ (500 µM) in the drinking water of SOD1(G93A) mice from a time when early symptoms of neurodegeneration become evident at 90 days of age until death. This regime is a clinically plausible scenario and could be more easily translated to patients as this corresponds to initiating treatment of patients after they are first diagnosed with ALS. MitoQ was detected in all tested tissues by liquid chromatography/mass spectrometry after 20 days of administration. MitoQ treatment slowed the decline of mitochondrial function, in both the spinal cord and the quadriceps muscle, as measured by high-resolution respirometry. Importantly, nitroxidative markers and pathological signs in the spinal cord of MitoQ-treated animals were markedly reduced and neuromuscular junctions were recovered associated with a significant increase in hindlimb strength. Finally, MitoQ treatment significantly prolonged the life span of SOD1(G93A) mice. Our results support a role for mitochondrial nitroxidative damage and dysfunction in the pathogenesis of ALS and suggest that mitochondria-targeted antioxidants may be of pharmacological use for ALS treatment.

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes.

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography-tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.

Using exomarkers to assess mitochondrial reactive species in vivo.

BACKGROUND: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. SCOPE OF REVIEW: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I.

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

Re-directing an alkylating agent to mitochondria alters drug target and cell death mechanism.

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.