Reviews pubs.acs.org/acschemicalbiology

Molecular Vehicles for Mitochondrial Chemical Biology and Drug Delivery Sae Rin Jean,† David V. Tulumello,‡ Simon P. Wisnovsky,‡ Eric K. Lei,‡ Mark P. Pereira,‡ and Shana O. Kelley*,†,‡,§ †

Department of Chemistry, Faculty of Arts and Science, ‡Department of Biochemistry, Faculty of Medicine, §Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy,University of Toronto, Toronto, Ontario, Canada ABSTRACT: The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. An understanding of mitochondrial involvement in various human diseases has generated an appreciable amount of interest in exploring this organelle as a potential drug target. As a result, a number of strategies to probe and combat mitochondria-associated diseases have emerged. Access to mitochondria-specific delivery vectors has allowed the study of biological processes within this intracellular compartment with a heightened level of specificity. In this review, we summarize the features of existing delivery vectors developed for targeting probes and therapeutics to this highly impermeable organelle. We also discuss the major applications of mitochondrial targeting of bioactive molecules, which include the detection and treatment of oxidative damage, combating bacterial infections, and the development of new therapeutic approaches for cancer. Future directions include the assessment of the therapeutic benefit achieved by mitochondrial targeting for treatment of disease in vivo. In addition, the availability of mitochondria-specific chemical probes will allow the elucidation of the details of biological processes that occur within this cellular compartment.

The mitochondrion is a vital organelle in eukaryotic cells that serves as the powerhouse for generating cellular energy via oxidative phosphorylation and also plays a key role in major cell death pathways.1−3 Mitochondria also have crucial secondary functions in a number of essential cellular processes such as modulating calcium homeostasis, the tricarboxylic acid and urea cycles, fatty acid oxidation, amino acid metabolism, and redox signaling.1,3,4 The mitochondrion’s structure therefore has evolved to provide tight regulation of these cellular processes. This organelle is comprised of two membranes: a porous outer mitochondrial membrane (OMM) and within it, a highly invaginated inner mitochondrial membrane (IMM).4 The IMM is unique in that it is highly dense due to the abundance of saturated phospholipids and also has a high protein to lipid ratio (3:1) compared to the plasma membrane (1:1).1,4 Additionally, the mitochondrion has a high membrane potential (Δψm of approximately −180 mV) compared to the plasma membrane (Δψp of approximately −60 mV).4 These features allow the mitochondrion to effectively exclude a wide range of ions and molecules, and special transport machinery is typically required to penetrate the IMM.5 The mitochondrion must be able to preclude even small ions such as protons from regaining access through the IMM after being transported into the intermembrane space (IMS).5 This high exclusivity is required to maintain the proton gradient that is necessary for oxidative phosphorylation.5 Residing within the mitochondrial matrix, which is enclosed by the IMM, is the mitochondrial DNA (mtDNA).4 This 16.6 © 2014 American Chemical Society

kb genome encodes for the mitochondrion’s translational machinery as well as 13 subunits of electron transport proteins.6 There has been evidence that mutations in mtDNA are the cause of many of the mitochondrial diseases that occur at a frequency of 1 in 5000 of the general population.7 Typically, point mutations and deletions in mtDNA lead to reduced oxidative phosphorylation and ATP production, increased heat dissipation, and generation of reactive oxygen species (ROS).7,8 These mutations are particularly problematic for organs that have high energy demands such as the heart, brain, and skeletal muscle, and can cause lactic acidosis and neurological disorders.9,10 Thus, maintaining the integrity of mtDNA is critical to regulating essential cellular processes and preventing mitochondria-associated diseases. Moreover, the replication and repair of mtDNA are significantly less well-characterized relative to the analogous processes occurring in the nucleus, and it will be important to elucidate the details of these pathways. The mitochondrion is a major regulator of cell death pathways and is involved in the activation and propagation of apoptosis, necroptosis, and necrosis.8 In healthy cells, various activators of pro-apoptotic proteases and nucleases are under inhibitory control via sequestration within the IMS.2 During Received: November 1, 2013 Accepted: January 11, 2014 Published: January 12, 2014 323

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Figure 1. Structure of the mitochondrion and its therapeutic targets. The mitochondrion possesses a double membrane comprised of the OMM and IMM, the latter of which maintains a highly negative inward potential. The densely packed, hydrophobic IMM (left panel) introduces a high exclusivity of ions and molecules that can be contained within the matrix. A number of mitochondria-specific targets have been explored for disease detection and treatment, such as mtDNA, HSP90, ROS, transcription and translational machinery, and MOMP.

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cell death, proteases are activated upon release of these molecules through mitochondrial outer membrane permeabilization (MOMP).2 This leads to deterioration of vital components of the cell, thereby systematically eliminating it. Dysregulation of these processes has been implicated in cancer progression, where the inhibition of cell death mechanisms allows for increased tumor cell proliferation.8 The mitochondrion therefore plays a major role in both cell survival and death, providing the energy necessary for running essential cellular processes while also directing the cell death machinery. An increasing number of diseases are linked to mitochondrial dysfunction. Organelle-specific probes have been developed to detect and measure molecular changes that occur in mitochondria of healthy versus diseased cells.1,11,12 Targeting antioxidants to mitochondria can minimize oxidative damage and can be an effective approach in treating ROS related diseases.13,14 Moreover, recognizing the mitochondrion’s role in cancer initiation and progression, potential mitochondriatargeted chemotherapeutics have surfaced. There is a host of mitochondrial targets within the organelle that have specific roles in cell death or cancer progression.8 Thus, mitochondrial targeting potentially presents an opportunity to eliminate chemotherapy-refractory cancer cells by acting directly on the cell death machinery. Lastly, another intriguing feature of mitochondria is that their structure and functions in mammalian cells can be traced back to their bacterial origin through evolution.15 Given the similarities shared between mitochondria and bacteria, mitochondrial delivery strategies have been applied to antimicrobials for the treatment of bacterial infections and have been proven to enhance therapeutic efficacy.16 Despite all of the interesting features of the mitochondrion that make it a desirable target for probes and therapeutics, its double-membrane structure makes it a difficult organelle to penetrate (Figure 1). Traversing the barriers protecting mitochondria using organelle-specific targeting delivery vectors is a key step that will enable a more thorough characterization of mitochondrial biology and the exploration of this organelle as a potential drug target. Here, we review delivery strategies and recent successes of mitochondria-targeted therapeutics and probes.

STRATEGIES FOR TARGETING MITOCHONDRIA

Engineering an efficient mitochondria-targeting cell permeable vector is a challenge due to the mitochondrion’s structure that results in impermeability against a wide range of molecules. Nature’s solution to penetrating this barrier relies on the attachment of peptide sequences to nuclear-encoded mitochondrial proteins that are imported into mitochondria through the TIM/TOM translocation complex.5 This process is typically directed by a cleavable mitochondria targeting signal (MTS), composed of a long stretch (20−40) of hydrophobic and cationic residues.5 One strategy to selectively target bioactive molecules to mitochondria is to harness this import machinery by appending this type of directing sequence onto biological molecules of interest. This approach has been successful in directing a variety of proteins to mitochondria in order to study a wide range of biological phenomena.17−19 Using this strategy to externally deliver bioactive molecules is limited, however, due to the length of the polypeptide sequence as well as reliance on additional cellular uptake systems such as cationic polymers or liposomes.11 The development of delivery vectors that do not rely on mitochondrial import machinery has been made possible by a greater understanding of mitochondrial structure and chemical features of molecules that selectively localize to this organelle.20 From these findings, two generalized requirements for mitochondrial localization are apparent: delocalized positive charge and lipophilicity.20 The cationic character is needed for driving the uptake of the delivery vectors through the cellular and mitochondrial membrane, both of which have a negative membrane potential. One example of a vector that makes use of positive charge to drive its mitochondrial uptake is the gamitrinib (geldanamycin mitochondrial matrix inhibitors) scaffold, where the therapeutically active portion of the molecule, geldanamycin, was conjugated to short repeating units of cyclic guanidinium moieties that were used for targeting.21 The delocalization of the positive charge reduces the activation energy associated with desolvating the ion that must occur prior to its uptake through the hydrophobic IMM.22,23 The positive charge is spread over a large surface area in order to increase its ionic radius, effectively reducing the interaction with surrounding water molecules.24 These features allow such molecules to partition across the IMM driven by its large negative potential dictated by the Nernst equation.1 This results in a 100−500× concentration of mitochondria-targeted 324

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Table 1. Summary of Mitochondria-Targeting Vectors

detail although their highly hydrophobic nature and β-turn motif has been suggested.34 By incorporating delocalized cationic charge as well as lipophilicity, several mitochondria-targeting scaffolds have been developed that are capable of delivering a wide variety of cargo, including both neutral and charged (positive or negative) small molecules.35,36 A selection of the most versatile and widely used of these mitochondrial delivery vectors are summarized in Table 1 and described in greater detail below. For a more exhaustive list of mitochondria-targeted small molecules, refer to the following recent reviews: refs 1, 25, 37, and 38. Delocalized Lipophilic Cations. The most notable example of a delocalized lipophilic cation that has been used for mitochondrial targeting is the triphenylphosphonium (TPP) cation.39 This small targeting vector contains a single positive charge that is resonance-stabilized over three phenyl groups.39 In addition to the delocalized positive charge, the large hydrophobic surface area of TPP allows favorable interaction with the lipid bilayer of the IMM.40 This permits the uptake of TPP into the mitochondrial matrix, where it is freely soluble or adsorbed on the inner leaflet of IMM depending on the hydrophobicity of the cargo.4,40 A wide variety of small molecules have been conjugated to TPPs including natural and synthetic antioxidants and probes (see Table 1 for a partial list).41−45 The conjugation of these molecules to TPPs typically occur through a simple SN2 reaction.40 TPPs can be designed to include a reversible tag that is only cleaved by a mitochondria-specific enzyme in order to release the chemically unaltered cargo.46 The small size of TPPs limits its cargo to low molecular weight compounds.47 High molecular weight cargos may potentially affect the subcellular localization, thus multiple TPPs may be required to drive their uptake into mitochondria.48 Interestingly, the wide use of TPPs have been extended to in vivo applications as recent studies have shown that TPP conjugates generally have

conjugates in the matrix. While a partial membrane depolarization and mild ETC uncoupling results from the translocation of cations across the IMM, these effects are largely reversible.25 Furthermore, alterations in mitochondrial bioenergetics, such as a decrease in basal respiration rate, are only evident upon the addition of high concentrations of targeting vectors.26 It should also be noted that the efficacy and uptake of these targeting agents are dependent on the tissue type as there is a large variability in mitochondrial biogenesis and turnover rates.27 Additionally, mitochondria-targeted small molecules that do not contain a tissue-targeting moiety tend to preferentially accumulate within metabolically active cells such as those belonging to the heart and brain.4 Passage of cations across the hydrophobic IMM is facilitated by introducing lipophilicity to the molecule. For instance, nitrooxy-doxorubicin was designed to include a nitric oxide releasing phenyl group that effectively increases its hydrophobicity compared to its parent compound doxorubicin.28 This modification shifts its intracellular localization from the nucleus to membrane-rich organelles such as mitochondria and endoplasmic reticulum.28 Similarly, short peptide sequences have also been adapted for either nuclear or mitochondrial localization by modifying their hydrophobicity.29 Both hydrophobicity and positive charge have been combined in a series of tetrapeptides that are comprised of alternating aromatic and cationic amino acids, known as Schiller−Szeto (SS) peptides, which selectively localize to mitochondria.30 As their cellular uptake is likely an energy-independent direct translocation, they are only partially sensitive to mitochondrial membrane potential and localized primarily in the IMM and are not delivered to the mitochondrial matrix.31 A fragment of the antibiotic peptide gramicidin S (GS) has also been employed to aid in the direction of ROS scavengers to mitochondria.32,33 The physicochemical features that impart mitochondrial localization of this fragment have not been explored in great 325

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low toxicity and a high level of uptake in vivo with some even having the ability to cross the blood-brain barrier (BBB).49 Liposomal and Nanoparticle Systems. Delivery of molecules to mitochondria has also been achieved through encapsulation in liposomes and nanoparticles (NPs). The structure of these systems can be modified for the intracellular delivery of different cargos and has proven to be particularly useful in delivering high MW cargo, improving in vivo pharmacokinetics of a variety of drugs.50 These systems have been adapted for mitochondrial delivery by appending positively charged targeting moieties such as TPP on the outer surface or within its structure.50 Some other notable examples of targeting molecules include dequalinium (dicationic units in DQAsomes) and octaarginine in MITOPorter.50,51 The uptake of liposomes is mediated by macropinocytosis and relies on endosomal escape to interact with the OMM of mitochondria.50 In the past, liposomes have been successful in delivering various cargos ranging in size such as paclitaxel, sclareol, and DNA.50,52−54 Some limitations of this method include complex synthesis, immune response, and toxicity in vivo, although there has been recent improvements to liposomal systems.55,56 More recently, mitochondria-targeted polymeric NP systems have been developed to deliver bioactive cargo such as lonidamine, α-tocopheryl succinate, curcumin, 2,4-dinitrophenol, and zinc phthalocyanine (ZnPc).57,58 In general, NPs are comprised of a biodegradable polymer system (e.g., PLGA-b-PEG) with TPP molecules as the mitochondriatargeting moiety.57 These NPs have been optimized for efficient endosomal escape, mitochondrial uptake, and stability, while minimizing immunologic effect in cellulo.57 Future work is required to elucidate the in vivo efficacy and toxicity of the NP system. Peptidic Delivery Vectors. Peptide vectors have also been proven to provide robust carriers for mitochondrial cargo delivery. Mitochondria-penetrating peptides have been created by combining synthetic and natural residues that are either cationic (e.g., arginine) or hydrophobic (e.g., cyclohexylalanine).59,60 This requisite cationic and hydrophobic composition has also been created though the modification of a polyproline scaffold to contain leucine and arginine derivatives.61−63 As with CPPs, these segments are easily made using solid phase peptide synthesis, and facile conjugation to a cargo is possible through a peptide bond formation.35 The simplistic peptide synthesis method provides tunability to this type of delivery vector that is useful in targeting a chemically diverse range of cargo to mitochondria.35 The modular nature of these peptide delivery vectors also provides a convenient platform to systematically investigate the physiochemical requirements for uptake into mitochondria. The positive charge of these peptides has been found to provide the electrostatic driving force responsible for their translocation utilizing the negative inward potential of both the cellular and mitochondrial membranes.59,64 Overall, cellular uptake of these peptides is highly dependent on the individual potential across both membranes, while mitochondrial localization relies primarily on the mitochondrial membrane potential.59,61 These observations are consistent with a direct, energy-dependent translocation mechanism such as that observed for arginine-rich CPPs.65 At lower peptide concentrations, an endocytic mechanism may also contribute to the uptake of certain peptide vectors.61,63 A systematic study of the sequence determinates of mitochondrial localization of these peptides revealed that a critical lipophilicity threshold must be

exceeded to enable partitioning into the highly hydrophobic IMM.59 Although the positive charge is not delocalized across the entire molecule, this is overcome by alternating cationic and lipophilic moieties, which distributes the positive charge evenly across the entire vector.35 Moreover, increased delocalization of charged residues further promotes mitochondrial localization, at the expense of reducing overall cellular uptake.22 These peptides have been used to deliver a variety of cargo including peptide sequences and charged small molecules that would otherwise be excluded from mitochondria.35 By using solely synthetic and D-amino acids, these sequences can also be made resistant to proteolytic degradation and demonstrate excellent serum stability.66 The MPPs are nontoxic at concentrations up to 10 to 100-fold higher than required for mitochondrial localization, although it should be noted that further increases in hydrophobicity and cationic charge can lead to mitochondrial dysfunction.64 The tunability of these peptides also offers the opportunity to incorporate features that allow for specific organ or tumor targeting for in vivo applications. In summary, a small and easily adaptable peptidic delivery vector can be an ideal scaffold in mitochondrial targeting of different types of small molecules.

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APPLICATIONS OF MITOCHONDRIA-TARGETING VECTORS Probes of Mitochondrial Status, Structure, and Function. The evaluation of mitochondrial structure, function, and biochemical processes are key issues to be addressed in the field of mitochondrial chemical biology, and their study requires organelle-specific delivery vectors. The mitochondriaspecific probes that have been developed thus far include those used for imaging mitochondrial morphology (Mitotracker Dyes, rhodamine) and those that are responsive toward mitochondrial membrane potential (JC-1 and JC-9), and those that indicate the total cellular mitochondrial mass through cardiolipin binding (nonylacridine orange).1 The introduction of mitochondrial-targeting vectors, however, has expanded this field to include probes that were previously limited to the cytoplasm or extracellular environment. One of most pertinent factors in mitochondrial homeostasis is levels of ROS.9 With the development of mitochondriaspecific agents for measuring (e.g., mitoSOX)67 and creating (e.g., Thiazole Orange-FrFK)29 reactive oxygen species, it is now possible to study the cause and effect of ROS within this organelle in a live cell system, eliminating the need to use isolated mitochondrial preparations. Recently, mitochondrial targeting technologies have advanced the field and allowed for the measurement of hydrogen peroxide (H2O2) in vivo. Through the use of a mitochondrial targeted redox probe, MitoB, the quantification of H2O2 in living drosophila is possible.68 Targeted probes have also been modified to investigate specific suborganelle locations, such as probing the antioxidant load in the IMM.69 Using versatile mitochondrial delivery strategies such as TPP, MPPs, and rhodamine derivatives, a number of probes have been developed to assess several key aspects of mitochondrial biology.1,29 Furthermore, these delivery strategies have allowed for greater spatial and temporal control of probe utilization. In one example, through the use of a photocleavable linker, probe molecules can be activated within selected mitochondria allowing for investigation of the effects of a depolarization event without perturbing overall cellular health.70 This allows for the study of a single mitochondrion or a small group of 326

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shown to significantly decrease peroxynitrite (ONO2−) production in irradiated cells, while a targeted nitrate oxide synthase agonist [2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT)] nearly completely abolished the formation of this damaging free radical.82 The ability of targeted antioxidants to reduce ROS generated during cellular stress has also been extended to provide transient protection during neuronal83 or cardiac13 ischemia. Aside from antioxidants, protection from ischemia has also been achieved using targeted therapies that reversibly inhibit oxidative respiration by generation of nitric oxide and nitrosylation of thiol containing proteins that are involved in oxidative phosphorylation.84,85 Similarly, protection from ischemia may also be achieved through the inhibition of the MOMP.45 All of the successful examples of mitochondriatargeted antioxidants described here indicate that organellar targeting can be a promising approach for treatment of the oxidative stress related diseases. Rerouting Antimicrobials to Maximize Therapeutic Benefit. Cellular targeting of antimicrobials using delivery vectors is an advancing field of research that provides novel strategies in the treatment of pathogenic infections. In particular, this approach provides a unique strategy to effectively target intracellular pathogens. This specific type of bacteria presents a challenging target for antimicrobial treatment due to the additional barrier to drug access provided by the mammalian cell membrane.86 A considerable amount of research has been put forth in developing methods of targeting these pathogens, utilizing strategies such as NP mediated antimicrobial delivery systems87 and receptor mediated targeting.88 However, the use of mitochondrial targeting scaffolds for the delivery of antimicrobials provides a method to overcome the cellular barrier in addition to a number of other beneficial properties such as reduced host toxicity.16 Given the evolutionary origin shared between bacteria and mitochondria, the strong negative cellular potential generated by metabolically active bacteria allows for effective targeting by cationic delivery agents typically used for mitochondrial delivery (Figure 2).15 These targeting properties have been successfully integrated with an antimicrobial peptide resulting in effective clearance of intracellular Salmonella typhimurium and Brucella abortus89 while MPPs have been used to successfully target intracellular Listeria monocytogenes.90 Conjugation of methotrexate, an inhibitor of human and bacterial dihydrofolate reductase, to a MPP resulted in effective delivery of the active molecule to intracellular listeria.90 Modification of the chemical properties of these vectors allows for modulation and optimization of partitioning between mitochondria and bacteria, a property important when considering the diversity of bacterial membrane structure and potential, and their life cycle dynamics.90 This provides greater control over the subcellular distribution without modifying the structure of the appended drug, which may affect target interactions. In addition, conjugation of methotrexate to a MPP vector was found to enhance antimicrobial activity through increased uptake in a variety of clinically relevant extracellular bacteria compared to the parent compound.16 In human cells, the sequestration of methotrexate into mitochondria prevented its interaction with cytosolic targets, thereby limiting human cell toxicity. Taken together, mitochondrial targeting of antimicrobials presents a unique strategy to broaden the spectrum of bacteria that can be treated and to increase the therapeutic window of existing and newly discovered antimicrobials.

mitochondria rather than all mitochondria within the cell, an important issue for the study of the redox state of proteins within the mitochondrion. Fluorescent probes have been developed to investigate thioredoxin redox states (MitoNaph71) within the organelle, and the development of mitochondria-specific tools that covalently tag reduced thiols with iodinated TPP (IBTP(4-iodobutyltriphenylphosphonium iodide)),72 or iodoacetamide functionalized TPP (IAM-TPP, [5-(2-iodo-acetylamino-pentyl]-triphenyl-phosophonium mesylate),73 allowed for proteomic study of mitochondrial protein redox state when mitochondrial function was perturbed.72,74 The development of these types of mitochondria-specific proteomic and metabolic tools will aid in further understanding the careful balance of processes within mitochondria of healthy and diseased cells. Protection from Oxidative Stress. One of the most widely studied applications of mitochondrial targeting is the protection of the organelle from oxidative stress. Many mitochondria-related pathologies stem from oxidative damage.9 Mitochondrial dysfunction can lead to the production of partially or completely unreduced oxygen that provides fuel for the production of ROS, which in turn, damages lipids and proteins within mitochondria.4,9,11 Oxidative damage is propagated when ROS cause mutations in mtDNA, leading to further mitochondrial dysfunction, which ultimately damages the rest of the cell.4 For instance, mitochondrial ROS production has been linked to aging and neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.3,9 Among the most widely employed application of mitochondrial delivery vectors is for the protection of mitochondria in such diseases through targeting antioxidants to scavenge ROS within the organelle thereby preventing or reversing oxidative damage.4,11 The aforementioned mitochondria-targeting vectors have been employed to successfully deliver a range of natural antioxidants (e.g., vitamin E,43 coenzyme Q,13,75 plastoquinone,41,76 and lipoic acid44), non-natural antioxidants [e.g., (2,2,6,6-tetramethylpiperidin-1-yl)oxidanyl (TEMPO),34 and dimethyltyrosine (DMT)30,61], and catalytic enzyme mimetics (e.g., peroxidase77 and superoxide dismutase78 mimics). These conjugates have been found to reduce oxidative stress through various means, resulting in increased cell survival under a variety of experimental conditions. For example, the conjugated form of coenzyme Q (MitoQ) does not directly prevent the production of superoxide radical (O2−) but instead limits mitochondrial dysfunction through reduction of lipid peroxidation.75 Other targeted antioxidants, such as the SS-peptides that contain DMT in their sequence, are capable of directly scavenging H2O2 and preventing lipid oxidation in vivo.30 Furthermore, many of these antioxidants have been shown to exhibit ROS scavenging behavior in hydrophobic environments such as lipid bilayers, which are often essential, as their activity relies on IMM localization.79 The reduction of ROS has also been achieved by the targeting of mild membrane potential uncoupling agents to limit ROS production. 80 These uncoupling agents have also been proposed to combat obesity.81 The ability of these ROS scavengers to reduce superoxide production has also been shown to prevent apoptosis in a variety of model systems.34,76 Evasion of apoptosis allows these molecules to mitigate radiation damage to mitochondria and thereby serve as radioprotectants. For example, the effects of mitochondrial delivery of a free radical scavenger, TEMPO, was 327

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can enhance the anticancer activity of current chemotherapeutics. Mitochondrial Targeting of DNA Damaging Agents to Increase Specificity and Efficacy. Mitochondria, similar to nuclei, possess functionally important genetic material and therefore should, in principle, be sensitive to DNA-damaging agents. Moreover, mtDNA has a substantially higher mutation rate compared to nuclear DNA (nDNA).95 MPPs provide an ideal platform for the targeting of structurally distinct anticancer compounds to the mitochondrial matrix, where mtDNA resides. To date, several MPP-DNA damaging drug conjugates have been developed. These include conjugates of chlorambucil (mtCbl), doxorubicin (mtDox), and a platinum chemotherapeutic based on cisplatin (mtPt).96−98 All of these agents act through induction of different types of nDNA damage. Chlorambucil and cisplatin form covalent bifunctional cross-links with nuclear DNA, whereas doxorubicin induces double-stranded DNA breaks by stalling topoisomerase II during DNA replication. Recent studies have indicated that mitochondria-targeted chlorambucil, doxorubicin, and platinum all produce mtDNA damage.96−98 Interestingly, mitochondrial dysfunction, as indicated by ROS generation96,97 and reduction in the mitochondrial membrane potential,96 is also observed following treatment with these agents. These mitochondria-targeted conjugates all demonstrate cytotoxicity against a host of cancer cell models, typically in the low micromolar range.96−98 Delivery of these agents to the mitochondrial matrix dramatically changes their drug properties, and in some cases alters their efficacy. For example, although chlorambucil has long been approved for use against chronic lymphocytic leukemia, in recent years it has been supplanted by fludarabine as the front line treatment for this disease, due to chlorambucil’s relatively low efficacy.99 However, mtCbl shows a ∼100-fold increase in potency against cancer cell models.96 More significantly, mtCbl was also shown to selectively kill leukemic cells while having little effect on healthy white blood cells.96 This selectivity originates from the elevated membrane potential observed in such cancers, which drives increased mitochondrial accumulation of this toxic agent.96 Interestingly, mtCbl is active against xenograft cancer models in vivo and is well tolerated by animals.66 This finding was significant, as one concern in targeting mitochondria for anticancer therapy is the potential for introducing new doselimiting toxicities to certain cell types. Neuronal cells, for example, are particularly sensitive to mitochondrial dysfunction and damage owing to their nearly exclusive reliance on oxidative metabolism for bioenergetic function.9 Nervous system related toxicity was not observed in mtCbl treated animals, however, perhaps reflecting an inability of the MPP vector used to penetrate the BBB.66 More study is needed to evaluate the full effects of mitochondria-targeted drugs on neuronal cells, particularly in peripheral nervous tissue not protected by the BBB. A recent study showed that the mechanism of cytotoxicity of mitochondria-targeted anticancer drugs can diverge considerably from the parent compound. Both mtDox and mtPt produce apoptotic cell death,97,98 while mtCbl causes rapid cell necrosis.66 In the case of mtPt, cell death has been functionally linked to mtDNA damage.97 For mtCbl, however, activity is likely mediated by alkylation of other macromolecules such as mitochondrial proteins rather than mtDNA.66 These differences in activity of mitochondria-targeted anticancer agents are

Figure 2. Delivery of untargeted and mitochondria-targeted antimicrobials to intracellular bacteria. (A) Treatment of intracellular pathogens with untargeted antimicrobials may result in low efficacy due to poor mammalian cell permeability. (B) Human cell toxicity can result if there are homologous cytoplasmic targets in the mammalian cell. (C) Mitochondrial targeting of antimicrobials allows for effective penetration of mammalian cell membrane. The negative membrane potential of bacteria drives the uptake of the mitochondria-targeted antimicrobial, resulting in increased accumulation compared to the untargeted compound (bolded arrow). Additionally, mitochondrial sequestration of antimicrobials also reduces host cell toxicity by preventing interaction with homologous targets.

Targeting Mitochondria for Anticancer Therapy. In recent years, a greater understanding of the association of mitochondrial dysfunction with cancer has led to a surge of interest in exploring the mitochondrion as a target for anticancer therapy.8 One area of research focuses on exploiting metabolic differences in mitochondria between cancerous and normal cell types to develop more selective anticancer drugs. Acute myeloid leukemia (AML) cells, for example, are known to display abnormally elevated levels of mitochondrial biogenesis and oxygen consumption.91,92 Hyperactive mitochondrial metabolism renders AML cells highly sensitive to inhibitors of mitochondrial translation such as the antibacterial drug tigecycline.91 Mitochondrial delivery of therapeutics can also improve specificity for cancer cells by acting on targets that only exist in mitochondria of cancer cells. Mitochondrial HSP90, for example, is a heat shock protein essential for protein folding that is overexpressed in many cancer subtypes, where it is essential for cancer cell survival and proliferation due to its role in suppressing the pro-apoptotic pathways that exist in mitochondria.21,93 A class of HSP90 inhibitors (gamitrinibs) induces rapid mitochondrial membrane permeabilization and cell death in cancer cells while leaving normal cells unaffected.21,93 Enhancing anticancer activity through mitochondrial localization has also been demonstrated for the proapoptotic peptides, D-(KLAKLAK)2, where improved mitochondrial localization correlated to increased efficacy.94 In recent years, rerouting of existing chemotherapeutics to mitochondria has been exploited as a promising way to enhance the drug’s anticancer activity. This novel approach demonstrates many of the principles that highlight the effectiveness of targeted anticancer therapies. Using examples of this approach, we will outline the major features specific to mitochondria that 328

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Figure 3. Evasion of tumor resistance mechanisms through mitochondrial delivery of DNA damaging agents. Cancer cells often acquire resistance in order to counteract the therapeutic action of DNA-damaging anticancer agents through a variety of mechanisms along this pathway. Untargeted DNA damaging agents must accumulate in the cytoplasm and subsequently enter the nucleus. At this stage, the overexpression of drug efflux pumps on the plasma membrane can severely reduce the intracellular accumulation of anticancer drugs.100 Following the entry into the nucleus, DNAdamaging agents must then generate DNA damage. For some drugs, this process may be dependent upon the presence of nuclear enzymes, such as topoisomerases, the downregulation of which may also lead to resistance.105 When DNA damage does occur, cancer cells often upregulate DNA repair pathways that limit the drug’s cytotoxic effects.102 Finally, the signaling pathways leading from DNA damage to cell death can be negated by the overexpression of antiapoptotic proteins such as Bcl2 or downregulation of pro-apoptotic proteins.104 Mitochondria-targeted drugs are able to evade the aforementioned resistance mechanisms through rapid localization into mitochondria to induce mtDNA damage, directly leading to cell death.

these platform-based approaches provide an excellent opportunity for developing therapeutics that have activity within the mitochondrion for the treatment of numerous disorders that are associated with this organelle (e.g., neurodegenerative diseases, metabolic disorders, diabetes, and cancer). As the idea of drug repurposing through organelle targeting is relatively new, the development of many of these therapeutics is still in its infancy. Currently the antioxidant MitoQ, constructed on the TPP platform, has progressed through two separate phase II studies and established long-term safety for the drug.106 The compound was also found to provide protection of the liver in hepatitis C patients demonstrating clinical safety and efficacy of the mitochondria-targeted compound.106 Many of the compounds developed using the other main platforms for mitochondrial delivery (e.g., MPPs, DQAsomes, NPs), however, require further preclinical evaluation. Lastly, incorporation of organ-specific targeting properties in the delivery vector could be considered to further enhance the efficacy of these therapeutics. From the studies outlined in this review, it is clear that mitochondrial targeting shows considerable promise for the development of new classes of drugs. Two of the most prevalent challenges in chemotherapy are (i) resistance to the therapeutic agent and (ii) the production of undesirable secondary effects. As described in previous sections, organellar targeting appears to successfully overcome many common cellular resistance mechanisms to cancer chemotherapeutics.97,98 Perhaps even more intriguing, is to determine if the shift in cellular targets (or added specificity for targets) for these therapeutics facilitated by organellar targeting alters or abolishes their secondary effects. For instance, treatment with doxorubicin and platinum compound are complicated through cardiotoxicity and peripheral nerve toxicity, respectively.107,108 Even if the intended target of a drug is not within the mitochondrion, many compounds have demonstrated interactions with this organelle that can lead to secondary toxicities limiting the effectiveness of the drug. Through organellar targeting, and selection of a single target within the cell, specific cellular interactions can be paired with the phenotypes observed. These studies will shed light on the effects that

an interesting subject for future research, and may be useful for studying the effects of different types of targets in mitochondria. Delivery of DNA-Damaging Agents to Mitochondria for Evasion of Tumor Resistance. One of the most promising aspects of mitochondrial targeting of anticancer drugs has been the circumvention of acquired tumor resistance that ultimately diminishes the effectiveness of existing anticancer agents over the course of the treatment.100 Evasion of resistance is primarily achieved through sequestration of the drug into mitochondria, where it is unexposed to many of the acquired resistance mechanisms that exist in the nucleus and cytoplasm (Figure 3). For instance, many clinically observed cancer resistance phenotypes, including doxorubicin and cisplatin resistance, are proposed to arise from overexpression of Pgp efflux pumps in the plasma membrane.100 As these pumps act only on cytoplasmic substrates, the mitochondrial sequestration of doxorubicin enabled the drug to be highly active against Pgp overexpressing cells, showing no sensitivity to drug efflux.66 A similar evasion of Pgp resistance was also observed for mtCbl, indicating that this is a universal evasion mechanism that arises from mitochondria-targeting.101 Resistance to cisplatin may also develop through overexpression of nucleotide excision repair (NER) proteins that act to excise Pt-DNA adducts from the nuclear genome.102 Targeting a platinum drug to mitochondria, which completely lacks the NER pathway,103 restores activity against a platinum-resistant cell line.97 In the case of chlorambucil, drug resistance in leukemia is most often mediated by the inactivation of apoptotic triggering through the overexpression of Bcl2 family of antiapoptotic proteins.104 mtCbl, however, kills such chlorambucil-resistant leukemic cells by activating cell death from within mitochondria.96 Many of these evasion mechanisms should also apply to other mitochondria-specific anticancer agents. Future studies are required to assess the activity of these novel compounds against resistant cancers in vivo. Conclusions and Future Perspectives. The development of mitochondrial delivery vectors has provided a unique opportunity to create tools for the study and manipulation of an important organelle. In addition to providing tools that allow mitochondrial chemical biology to be probed systematically, 329

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altering the target of the compounds has on increasing the effectiveness of the therapeutic and ablating organ-associated toxicities and can guide future generations of chemotherapeutics.

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AUTHOR INFORMATION

Corresponding Author

*(S.O.K.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Work in the Kelley Laboratories is supported by the Canadian Institute of Health. (CIHR). D.V.T. and S.P.W. are scholars from the Terry Fox Foundation Strategic Training Initiative for Excellence in Radiation Research for the 21st Century at CIHR. E.K.L. holds a Master’s graduate award from CIHR.

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(101) Fonseca, S. B., and Kelley, S. O. (2011) Peptide-chlorambucil conjugates combat pgp-dependent drug efflux. ACS Med. Chem. Lett. 2, 419−423. (102) Martin, L. P., Hamilton, T. C., and Schilder, R. J. (2008) Platinum resistance: The role of DNA repair pathways. Clin. Cancer Res. 14, 1291−1295. (103) LeDoux, S. P., Wilson, G. L., Beecham, E. J., Stevnsner, T., Wassermann, K., and Bohr, V. A. (1992) Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells. Carcinogenesis 13, 1967−1973. (104) Tzifi, F., Economopoulou, C., Gourgiotis, D., Ardavanis, A., Papageorgiou, S., and Scorilas, A. (2012) The role of bcl2 family of apoptosis regulator proteins in acute and chronic leukemias. Adv. Hematol. 2012, 524308. (105) Pommier, Y., Leo, E., Zhang, H., and Marchand, C. (2010) DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem. Biol. 17, 421−433. (106) Gane, E. J., Weilert, F., Orr, D. W., Keogh, G. F., Gibson, M., Lockhart, M. M., Frampton, C. M., Taylor, K. M., Smith, R. A., and Murphy, M. P. (2010) The mitochondria-targeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients. Liver Int. 30, 1019−1026. (107) Zhang, S., Liu, X., Bawa-Khalfe, T., Lu, L. S., Lyu, Y. L., Liu, L. F., and Yeh, E. T. (2012) Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat. Med. 18, 1639−1642. (108) Screnci, D., McKeage, M. J., Galettis, P., Hambley, T. W., Palmer, B. D., and Baguley, B. C. (2000) Relationships between hydrophobicity, reactivity, accumulation, and peripheral nerve toxicity of a series of platinum drugs. Br. J. Cancer 82, 966−972. (109) Boddapati, S. V., D’Souza, G. G., Erdogan, S., Torchilin, V. P., and Weissig, V. (2008) Organelle-targeted nanocarriers: Specific delivery of liposomal ceramide to mitochondria enhances its cytotoxicity in vitro and in vivo. Nano Lett. 8, 2559−2563. (110) Dikalova, A. E., Bikineyeva, A. T., Budzyn, K., Nazarewicz, R. R., McCann, L., Lewis, W., Harrison, D. G., and Dikalov, S. I. (2010) Therapeutic targeting of mitochondrial superoxide in hypertension. Circ. Res. 107, 106−116. (111) Lei, W., Xie, J., Hou, Y., Jiang, G., Zhang, H., Wang, P., Wang, X., and Zhang, B. (2010) Mitochondria-targeting properties and photodynamic activities of porphyrin derivatives bearing cationic pendant. J. Photochem. Photobiol. B 98, 167−171. (112) Filipovska, A., Eccles, M. R., Smith, R. A., and Murphy, M. P. (2004) Delivery of antisense peptide nucleic acids (pnas) to the cytosol by disulphide conjugation to a lipophilic cation. FEBS Lett. 556, 180−186. (113) Sikora, A., Zielonka, J., Adamus, J., Debski, D., DybalaDefratyka, A., Michalowski, B., Joseph, J., Hartley, R. C., Murphy, M. P., and Kalyanaraman, B. (2013) Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: Identification of diagnostic marker products and biological implications. Chem. Res. Toxicol. 26, 856−867. (114) Ji, J., Kline, A. E., Amoscato, A., Samhan-Arias, A. K., Sparvero, L. J., Tyurin, V. A., Tyurina, Y. Y., Fink, B., Manole, M. D., Puccio, A. M., Okonkwo, D. O., Cheng, J. P., Alexander, H., Clark, R. S. B., Kochanek, P. M., Wipf, P., Kagan, V. E., and Bayir, H. (2012) Lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of brain injury. Nat. Neurosci. 15, 1407−1413.

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dx.doi.org/10.1021/cb400821p | ACS Chem. Biol. 2014, 9, 323−333