Mitochondria-penetrating Peptides Research Articles

Mitochondrial Probe for Glutathione Depletion Reveals NME3 Essentiality for Mitochondrial Redox Response.

Maintenance of the mitochondrial thiol redox state is essential for cell survival. However, we lack a comprehensive understanding of the redox response to mitochondrial glutathione depletion. We developed a mitochondria-penetrating peptide, mtCDNB, to specifically deplete mitochondrial glutathione. A genome-wide CRISPR/Cas9 screen in tandem with mtCDNB treatment was employed to uncover regulators of the redox response to mitochondrial glutathione depletion. We identified nucleoside diphosphate kinase 3 (NME3) as a regulator of mitochondrial dynamics. We show that NME3 is recruited to the mitochondrial outer membrane when under redox stress. In the absence of NME3, there is impaired mitophagy, which leads to the accumulation of dysfunctional mitochondria. NME3 knockouts depleted of mitochondrial glutathione have increased mitochondrial ROS production, accumulate mtDNA lesions, and present a senescence-associated secretory phenotype. Our findings suggest a novel role for NME3 in selecting mitochondria for degradation through mitophagy under conditions of mitochondrial redox stress.

Preparation of peptide-functionalized affinity materials for the highly specific capture and analysis of mitochondria

Mitochondria perform various metabolic processes that significantly affect cell differentiation, proliferation, signal transduction, and programmed cell death. The disruption of mitochondrial bioenergetic and metabolic functions is closely related to many disorders. The specific isolation and purification of intact, high-purity, and functional mitochondria are central to the understanding of their mechanism of action but remain challenging tasks. In this study, a mitochondrial penetrating peptide (MPP) with the sequence FrFKFrFK(Ac) was used as a mitochondrial recognition motif to construct a peptide-guided affinity separation material. The multiple aromatic phenylalanine (F) residues in this amphiphilic peptide can confer lipophilicity to the mitochondrial membrane, whereas the basic residues (D-arginine and lysine) render the MPP surface positively charged, thereby promoting the binding of negatively charged mitochondria. After the derivatization of the N terminal of MPP with an oligoglycine spacer, the peptide ligands were conjugated to matrix beads (MB) with surface aldehyde functional groups. Peptide functionalization was performed via a condensation reaction between the amino group in the peptide ligand and the aldehyde group on the beads. The generated Schiff bases were reduced, affording stable covalent bonds. The dense and stable functionalization of the beads with the mitochondria-targeting peptides was demonstrated using high performance liquid chromatography (HPLC), zeta potential assay, and scanning electron microscopy (SEM). The immobilization efficiency of the peptide ligands was 1.47 μmol/g, and the surface potential of MB@MPP was 11 mV. MB@MPP was used for the direct isolation of mitochondria after cell homogenization. As observed by SEM, mitochondria with a cross-sectional diameter of 500 nm were efficiently captured on the MB@MPP surface. Because the mitochondrial membrane potential is an important marker of mitochondrial function and the driving force behind the staining of mitochondria with Mito Tracker dyes, the specific binding and separation of fluorescent mitochondria from the cell samples revealed that the proposed MB@MPP-based isolation approach can keep mitochondria intact and retain their functions. Western blot assays were employed to characterize the protein markers of the mitochondria (citrate synthase (CS) and voltage-dependent anion channel protein (VDAC)) and cytoplasmic protein (vinculin), and examine the integrity and purity of the captured mitochondria. The results showed that the lysates released from MB@MPP had high CS and VDAC contents. By contrast, vinculin, which is highly abundant in whole-cell lysates, was barely detected in the lysates from MB@MPP. These results suggest that MB@MPP isolates mitochondria with high affinity, specificity, and antifouling ability by using the targeting peptide as the capture handle. A comparison with a commercial mitochondrial isolation kit demonstrated that MB@MPP can separate mitochondria with higher CS and VDAC abundance and purity. Given the superior separation performance of MB@MPP, the molecular profiles of the isolated mitochondria under stress were subjected to further analysis of their molecular profiles under stress. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established to detect tryptophan (Trp) and riboflavin in the mitochondria. Quantification was performed in multiple-reaction monitoring (MRM) mode. Owing to the high purity of the mitochondria, the Trp and riboflavin contents were determined to be 265 and 0.67 nmol/mg, respectively. The metabolic response of mitochondria to external stimuli was further examined using acadesine, an adenosine 5'-monophosphate (AMP)-activated protein kinase activator with a wide range of metabolic effects, to treat cells. After cell homogenization, MB@MPP was used to separate the mitochondria from the cell samples with and without acadesine treatment, followed by LC-MS/MS analysis. The quantification results demonstrated that acadesine induced a 14% upregulation of Trp content in the mitochondria. By contrast, the riboflavin content decreased to 0.48 nmol/mg, which is 72% of that in untreated mitochondria. The changes in Trp and riboflavin contents could influence their metabolic pathways and, thus, the levels of their metabolites, such as nicotinamide adenine dinucleotide, flavin mononucleotide, and flavin adenine dinucleotide, which are essential coenzymes in mitochondria. Peptide-functionalized affinity microbeads with high affinity and specificity for mitochondria are promising for the efficient isolation of high-quality mitochondria, and offer a useful tool for understanding the complicated functions and dynamics of this unique organelle.

A sojourn on mitochondria targeted drug delivery systems for cancer: Strategies, clinical and future prospects

Mitochondria, often referred to as the powerhouses of the cell, have emerged as promising targets for cancer therapy due to their pivotal roles in cell survival, apoptosis, and energy metabolism. This sojourn emphasizes the significance of mitochondria-targeted drug delivery systems in cancer therapeutics. The unique characteristics of cancer cell mitochondria, such as altered membrane potential and distinct lipid composition, offer an avenue for selective drug targeting. Several strategies have been explored to exploit these features, including the use of lipophilic cations, mitochondria-penetrating peptides, and nanocarriers tailored for mitochondrial delivery. Mitochondria-targeted drug delivery systems have demonstrated enhanced therapeutic efficacy and reduced systemic toxicity in preclinical models. Some of these systems have made a successful transition to clinical trials, illustrating their potential in real-world oncology settings. However, there remain challenges like intracellular barriers, potential off-target effects, and the complexity of tumor heterogeneity that must be addressed to fully harness the potential of mitochondria-targeted drug delivery systems. As research progresses, it is anticipated that innovative approaches and technologies will be developed to improve the specificity and efficacy of mitochondrial targeting, paving the way for more effective and safer cancer treatments in the future. This review serves as a comprehensive guide to the current state of mitochondria-targeted drug delivery systems for cancer, highlighting key strategies, clinical progress, and prospective avenues for future research.

CRISPR Screening in Tandem with Targeted mtDNA Damage Reveals WRNIP1 Essentiality.

A major impediment to the characterization of mtDNA repair mechanisms in comparison to nuclear DNA repair mechanisms is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of an mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox), we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted doxorubicin (Dox) screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentiality of our top hit, WRNIP1─observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

Mitochondrial Dysfunction: Pathophysiology and Mitochondria-Targeted Drug Delivery Approaches.

Mitochondria are implicated in a wide range of functions apart from ATP generation, and, therefore, constitute one of the most important organelles of cell. Since healthy mitochondria are essential for proper cellular functioning and survival, mitochondrial dysfunction may lead to various pathologies. Mitochondria are considered a novel and promising therapeutic target for the diagnosis, treatment, and prevention of various human diseases including metabolic disorders, cancer, and neurodegenerative diseases. For mitochondria-targeted therapy, there is a need to develop an effective drug delivery approach, owing to the mitochondrial special bilayer structure through which therapeutic molecules undergo multiple difficulties in reaching the core. In recent years, various nanoformulations have been designed such as polymeric nanoparticles, liposomes, inorganic nanoparticles conjugate with mitochondriotropic moieties such as mitochondria-penetrating peptides (MPPs), triphenylphosphonium (TPP), dequalinium (DQA), and mitochondrial protein import machinery for overcoming barriers involved in targeting mitochondria. The current approaches used for mitochondria-targeted drug delivery have provided promising ways to overcome the challenges associated with targeted-drug delivery. Herein, we review the research from past years to the current scenario that has identified mitochondrial dysfunction as a major contributor to the pathophysiology of various diseases. Furthermore, we discuss the recent advancements in mitochondria-targeted drug delivery strategies for the pathologies associated with mitochondrial dysfunction.

A new mitochondrial probe combining pyrene and a triphenylphosphonium salt for cellular oxygen and free radical detection via fluorescence lifetime measurements

To improve and diversify the quantification of reactive oxygen species (ROS) in mitochondria of single cells, we connected pyrene derivatives (PB) to a triphenylphosphonium salt (TPP+) as a mitochondrial vector forming PB-TPP+ probes. Two pyrene isomers with the n-butyltriphenylphosphonium moieties attached at their 1- or 2- positions were synthesized and characterized. Using the long fluorescence lifetime of pyrene, it was possible to monitor the variation of cellular free radicals and oxygen and to follow the reversibility of both quenchers in real-time. We compared the behavior of these new probes to the previously published pyrene-probes, functionalized by a mitochondrial-penetrating peptide, allowing their transfer to the mitochondria (Mito-PB) or to the cytosolic membrane for pyrene butyric acid (PBA). The high cellular uptake of the new probes allows cells to be loaded with an initial concentration 40 times lower than that for Mito-PB probes, without inducing perturbations in cell growth. The variation in free radicals and oxygen levels was monitored within cells under different stress conditions through the fluorescence lifetime of the new TPP+-based probes giving comparable results to those obtained for MPP-based probes. However, at a loading concentration as low as 25 nM, our technique allows the detection of increased production of free radicals in the mitochondria in the presence of the TPP+ vector, a warning to the user of this well-known vector.

A Mitochondria-Penetrating Peptide Exerts Potent Anti-Plasmodium Activity and Localizes at Parasites' Mitochondria.

Mitochondria are considered a novel drug target as they play a key role in energy production and programmed cell death of eukaryotic cells. The mitochondria of malaria parasites differ from those of their vertebrate hosts, contributing to the drug selectivity and the development of antimalarial drugs. (Fxr)3, a mitochondria-penetrating peptide or MPP, entered malaria-infected red cells without disrupting the membrane and subsequently killed the blood stage of P. falciparum parasites. The effects were more potent on the late stages than on the younger stages. Confocal microscopy showed that the (Fxr)3 intensely localized at the parasite mitochondria. (Fxr)3 highly affected both the lab-strain, chloroquine-resistant K1, and freshly isolated malaria parasites. (Fxr)3 (1 ng/mL to 10 μg/mL) was rarely toxic towards various mammalian cells, i.e., mouse fibroblasts (L929), human leukocytes and erythrocytes. At a thousand times higher concentration (100 μg/mL) than that of the antimalarial activity, cytotoxicity and hemolytic activity of (Fxr)3 were observed. Compared with the known antimalarial drug, atovaquone, (Fxr)3 exhibited more rapid killing activity. This is the first report on antimalarial activity of (Fxr)3, showing localization at parasites’ mitochondria.

The C-terminal segment of Leishmania major HslU: Toward potential inhibitors of LmHslVU activity

It is urgent to develop less toxic and more efficient treatments for leishmaniases and trypanosomiases. We explore the possibility to target the parasite mitochondrial HslVU protease, which is essential for growth and has no analogue in the human host. For this, we develop compounds potentially inhibiting the complex assembly by mimicking the C-terminal (C-ter) segment of the ATPase HslU. We previously showed that a dodecapeptide derived from Leishmania major HslU C-ter segment (LmC12-U2, Cpd 1) was able to bind to and activate the digestion of a fluorogenic substrate by LmHslV. Here, we present the study of its structure-activity relationships. By replacing each essential residue with related non-proteinogenic residues, we obtained more potent analogues. In particular, a cyclohexylglycine residue at position 11 (cpd 24) allowed a more than three-fold gain in potency while reducing the size of compound 24 from twelve to six residues (cpd 50) without significant loss of potency, opening the way toward short HslU C-ter peptidomimetics as potential inhibitors of HslV proteolytic function. Finally, conjugates constituted of LmC6-U2 analogues and a mitochondrial penetrating peptide were found to penetrate into the promastigote form of L. infantum and to inhibit the parasite growth without showing toxicity toward human THP-1 cells at the same concentration (i.e. 30 μM).

Concurrent impairment of nucleus and mitochondria for synergistic inhibition of cancer metastasis

Cancer metastasis, which increases the mortality in a short period of time, has been considered as the main challenge in tumor treatment. However, tumor growth suppression also should not be ignored in cancer metastasis treatment. Recently, accumulating evidences have suggested that mitochondria play an important role in mitigating caner metastasis. Nucleus, as the repository of genetic information, plays a key role in cell proliferation. However, it remains elusive that the concurrent impairment of nucleus and mitochondria may achieve better anti-tumor and anti-metastatic effects. Here, we designed a mitochondria-penetrating peptide modified doxorubicin (MPP-Dox) loaded N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer conjugates (PM), as well as a nuclear accumulating HPMA copolymer Dox conjugates (PN) by the nuclear tendency of Dox. After co-delivering the two copolymers (abbreviation for PMN), PM promoted cell apoptosis and inhibited tumor metastasis by damaging mitochondria, whereas PN suppressed cell proliferation and promoted apoptosis by destroying nucleus. Importantly, PM and PN complemented each other as expected. The mitochondrial dysfunction and tumor metastasis inhibition of PM was improved by PN, while cell proliferation suppression and apoptosis by nucleus destroying of PN was enhanced by PM. As a result, tumor growth of breast cancer 4T1 cells in vivo was significantly restrained and lung metastasis was potently decreased and almost eradicated, fully reflecting the advantages of organelle targeting combination therapy. As a consequence, our work showed that concurrent impairment of nucleus and mitochondria was feasible and beneficial to metastatic cancer treatment.

Targeted elimination of mutated mitochondrial DNA by a multi-functional conjugate capable of sequence-specific adenine alkylation

Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential to cure mitochondrial diseases, no chemical-based drugs in clinical trials are capable of selective modulation of mtDNA mutations. Here, we construct a class of compounds encompassing pyrrole-imidazole polyamides (PIPs), mitochondria-penetrating peptide, and chlorambucil, an adenine-specific DNA-alkylating reagent. The sequence-selective DNA binding of PIPs allows chlorambucil to alkylate mutant adenine more efficiently than other sites in mtDNA. Invitro DNA alkylation assay shows that our compound 8950A-Chb(Cl/OH) targeting a nonpathogenic point mutation in HeLa S3 cells (m.8950G>A) can specifically alkylate the mutant adenine. Furthermore, the compound reduces the mtDNA possessing the target mutation in cultured HeLa S3 cells. The programmability of PIPs to target different sequences could allow this class of compounds to be developed as designer drugs targeting pathogenic mutations associated with mitochondrial diseases in future studies.

Novel Strategies for Disrupting Cancer-Cell Functions with Mitochondria-Targeted Antitumor Drug-Loaded Nanoformulations.

Any variation in normal cellular function results in mitochondrial dysregulation that occurs in several diseases, including cancer. Such processes as oxidative stress, metabolism, signaling, and biogenesis play significant roles in cancer initiation and progression. Due to their central role in cellular metabolism, mitochondria are favorable therapeutic targets for the prevention and treatment of conditions like neurodegenerative diseases, diabetes, and cancer. Subcellular mitochondria-specific theranostic nanoformulations for simultaneous targeting, drug delivery, and imaging of these organelles are of immense interest in cancer therapy. It is a challenging task to cross multiple barriers to target mitochondria in diseased cells. To overcome these multiple barriers, several mitochondriotropic nanoformulations have been engineered for the transportation of mitochondria-specific drugs. These nanoformulations include liposomes, dendrimers, carbon nanotubes, polymeric nanoparticles (NPs), and inorganic NPs. These nanoformulations are made mitochondriotropic by conjugating them with moieties like dequalinium, Mito-Porter, triphenylphosphonium, and Mitochondria-penetrating peptides. Most of these nanoformulations are meticulously tailored to control their size, charge, shape, mitochondriotropic drug loading, and specific cell-membrane interactions. Recently, some novel mitochondria-selective antitumor compounds known as mitocans have shown high toxicity against cancer cells. These selective compounds form vicious oxidative stress and reactive oxygen species cycles within cancer cells and ultimately push them to cell death. Nanoformulations approved by the FDA and EMA for clinical applications in cancer patients include Doxil, NK105, and Abraxane. The novel use of these NPs still faces tremendous challenges and an immense amount of research is needed to understand the proper mechanisms of cancer progression and control by these NPs. Here in this review, we summarize current advancements and novel strategies of delivering different anticancer therapeutic agents to mitochondria with the help of various nanoformulations.

Sequence-Specific Control of Mitochondrial Gene Transcription Using Programmable Synthetic Gene Switches Called MITO-PIPs.

Mitochondria possess multiple copies of mitochondrial DNA (mtDNA) that encode 37 genes and their transcription and replication get controlled by unique molecular codes different from that in the nuclear DNA. The mtDNA has been gaining increased attention as one of the critical therapeutic targets as mutations in them impair the function of mitochondria and cause mitochondrial diseases like MELAS. In this chapter, we describe artificial control of mitochondrial transcription based on mtDNA sequence information with a new type of compounds termed MITO-PIPs, which encompasses two domains: pyrrole-imidazole polyamide as DNA recognition domain and mitochondrial penetrating peptide as the mitochondria-targeting domain. Because MITO-PIPs are amenable to tunability, they can be expanded as a synthetic strategy to modulate mitochondrial gene(s) on demand.

Photostable NIR emitting ruthenium(II) conjugates; uptake and biological activity in live cells

A photostable Ru(2,2-biquinoline)2(3-(2-pyridyl)-5-(4-carboxyphenyl)-1,2,4-triazolate) (Ru(biq)2(trzbenzCOOH)) complex that exhibits near-infrared (NIR) emission centred at 786 nm is reported. The parent complex was conjugated via amide coupling to a cell-penetrating peptide sequence octa-arginine (R8), and two signal peptide sequences; the nuclear localizing sequence (NLS) VQRKRQKLMP and the mitochondria penetrating peptide (MPP) FrFKFrFK(Ac) (r = D isomer of arginine, Ac = terminal lysine amine acetyl blocked). Notably, none of the peptide conjugates were cell-permeable as chloride salts but efficient and rapid membrane permeation was observed post ion exchange with perchlorate counterion. Also, surprisingly, all three peptide conjugates exhibited potent dark cytotoxicity in both CHO and HeLa cell lines. The peptide conjugates induce cell death through a caspase dependent apoptotic pathway. At the minimum concentration of dye (approx. 15 μM) required for cell imaging, only 20% of the cells were viable after a 24 h incubation period. To overcome cytotoxicity, the parent complex was PEGylated; this dramatically decreased cytotoxicity, where 50% of cells were viable even at 150 μM concentration after 24 h. Confocal luminescence microscopy indicated that all four bioconjugates, peptides in perchlorate form and polyethylene glycol (PEG) in chloride form, were rapidly internalized within the cell. However, interestingly the precise localisation by the signal peptides observed in related complexes was not observed here and the peptide conjugates were unsuitable as luminescent probes for cell microscopy due to their high cell toxicity. The poor targeting of signal peptides in this instance is attributed to the high lipophilicity of the metal centre.

Mitochondria Penetrating Peptide-Conjugated TAMRA for Live-Cell Long-Term Tracking.

Mitochondria are essential targets for treatment of diseases with mitochondrial disorders such as diabetes, cancer, and cardiovascular and neurodegenerative diseases. Mitochondria penetrating peptides (MPPs) are composed of cationic and hydrophobic amino acids that can target and permeate the mitochondrial membrane. Herein, a novel d-argine-phenylalanine-d-argine-phenylalanine-d-argine-phenylalanine-NH2 (rFrFrF) was tagged with a rhodamine-based fluorescent chromophore (TAMRA). This probe (TAMRA-rFrFrF) exhibited advantageous properties for long-term mitochondria tracking as demonstrated by fluorescence microscopy. Cell viability assays and oxygen consumption rates indicate low cytotoxicity and high biocompatibility of the new contrast agent. Colocalization studies suggest that TAMRA-rFrFrF is a promising candidate for continuous mitochondrial tracking for up to 3 days.

Delivery of cell membrane impermeable peptides into living cells by using head-to-tail cyclized mitochondria-penetrating peptides.

A series of cyclic Arg-rich mitochondria-penetrating peptides were prepared with variation in the macrocycle size and the chirality of Arg residues. A cyclic heptapeptide was demonstrated to be an efficient mitochondria-specific delivery vector for delivering membrane impermeable peptides.

Liposomes containing cholesterol and mitochondria-penetrating peptide (MPP) for targeted delivery of antimycin A to A549 cells

•Mitochondria are exclusively employed to produce energy required for the vital metabolic functions of the cell. However, mitochondria also play a key role in mammalian cell death. Dissipation in the mitochondria membrane potential causes cell death. Therefore, in cancer therapy, mitochondria are a novel target. Herein, we developed a nano-formulation of Antimycin A specifically targeted towards mitochondria and lung cancer; A549 cell. The liposomes were prepared using cholesterol and a mitochondria-penetrating peptide (MPP) having a phenylalanine-arginine-phenylalanine-lysine (FRFK) peptide sequence. The FRFK peptide was synthesized using solid phase peptide synthesis (SPPS) and contained cholesterol in the N-terminal end of the phenylalanine (Chol-FRFK). The synthesized material was confirmed using 1H NMR, Fourier transform infrared spectroscopy (FT-IR) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/MS). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and Chol-FRFK, at a molar ratio of 1:1, were used for liposomal (Chol-FRFK/D) formulations; the sizes of the liposomes were confirmed using dynamic light scattering (DLS). Cytotoxicity was evaluated in A549 cells. Cellular uptake and mitochondria targeting were confirmed by flow cytometry and confocal microscopy, respectively. Antimycin A, a hydrophobic and mitochondrial electron transporter inhibitor was encapsulated into the Chol-FRFK/D liposomes. Our results indicate that Chol-FRFK/D liposomes may potentially be used for the nano-formulation of cytotoxic drugs and enhancing their bioavailability in cancer therapy.

Delivery and Release of Small-Molecule Probes in Mitochondria Using Traceless Linkers.

Mitochondria-penetrating peptides (MPPs) are specific targeting vectors for the localization of small molecules to the mitochondrial matrix. Mitochondrial targeting of small molecules has enabled the development of a number of potential therapeutics and chemical probes. However, the need for covalent conjugation of small molecules to MPPs can negatively affect the activity of the appended cargo against its cellular target. Here, we describe cleavable linkers designed for the traceless release of chemical cargo from MPPs following mitochondrial transit. The cleavage kinetics of a number of disulfides were investigated using a fluorescent reporter system in order to optimize linker stability for mitochondrial release. The stability of mono- and disubstituted disulfides was determined to be sufficient during transit through the cytosol while still allowing for release of the cargo within 24 h. This linker system successfully released the compound Luminespib, an HSP90 inhibitor, which was deactivated by direct MPP conjugation. The releasable conjugate regenerated Luminespib activity and induced mitochondrial phenotypes of HSP90 inhibition. This linker may prove useful in expanding the repertoire of small molecules that can be used with mitochondrial targeting vectors.

Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression.

Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs' properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations.

Mitochondria-penetrating peptides conjugated to desferrioxamine as chelators for mitochondrial labile iron.

Desferrioxamine (DFO) is a bacterial siderophore with a high affinity for iron, but low cell penetration. As part of our ongoing project focused on DFO-conjugates, we synthesized, purified, characterized and studied new mtDFOs (DFO conjugated to the Mitochondria Penetrating Peptides TAT49-57, 1A, SS02 and SS20) using a succinic linker. These new conjugates retained their strong iron binding ability and antioxidant capacity. They were relatively non toxic to A2780 cells (IC50 40–100 μM) and had good mitochondrial localization (Rr +0.45 –+0.68) as observed when labeled with carboxy-tetramethylrhodamine (TAMRA) In general, mtDFO caused only modest levels of mitochondrial DNA (mtDNA) damage. DFO-SS02 retained the antioxidant ability of the parent peptide, shown by the inhibition of mitochondrial superoxide formation. None of the compounds displayed cell cycle arrest or enhanced apoptosis. Taken together, these results indicate that mtDFO could be promising compounds for amelioration of the disease symptoms of iron overload in mitochondria.