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.

Tuning the activity of mitochondria-penetrating peptides for delivery or disruption.

Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

Rerouting chlorambucil to mitochondria combats drug deactivation and resistance in cancer cells.

The difficulty of accessing the mitochondrial matrix has limited the targeting of therapeutics to this organelle. Here, we report, to our knowledge, the first successful delivery of an active DNA alkylating agent--chlorambucil--to mitochondria, and describe unexpected features that result from rerouting this drug within the cell. Mitochondrial targeting of this agent dramatically potentiates its activity, and promotes apoptotic cell death in a variety of cancer cell lines and patient samples. This retention of activity is observed even in cells with resistance to chlorambucil or disabled apoptotic triggering.

Peptide-chlorambucil conjugates combat pgp-dependent drug efflux.

Cancer drugs, such as the ovarian cancer drug adriamycin, are effective at slowing disease progression and improving remission rates in patients. However, drug resistance often arises, limiting the activity of these agents in some patients. In particular, efflux pumps, which export drugs out of cells, limit the efficacy of a variety of anticancer agents. While inhibitors to block these pumps currently exist, they are usually not used clinically because they alter other drug properties. Here, we report a novel inhibitor of drug efflux that only reduces pump activity temporarily. This decreases the risk that it will alter drug function and cause nonspecific toxicity. P-glycoprotein efflux pumps are commonly overexpressed by malignant cells and are a major contributing factor to the development of drug resistance. Many therapeutics containing basic nitrogens, hydrophobic character, or aromaticity are efficiently eliminated from cells, and Pgp inhibitors must often be coadministered to limit this process. However, currently available inhibitors often alter the pharmacokinetic profiles of therapeutics or increase off-target toxicity, limiting their clinical utility. Here, we report the development of a novel panel of peptide-chlorambucil conjugates capable of efficiently decreasing efflux of Pgp substrates. These conjugates selectively improve adriamycin toxicity and uptake for short, but not prolonged, periods reducing the risk of altered pharmacokinetics and off-target effects.

The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells.

To identify known drugs with previously unrecognized anticancer activity, we compiled and screened a library of such compounds to identify agents cytotoxic to leukemia cells. From these screens, we identified ivermectin, a derivative of avermectin B1 that is licensed for the treatment of the parasitic infections, strongyloidiasis and onchocerciasis, but is also effective against other worm infestations. As a potential antileukemic agent, ivermectin induced cell death at low micromolar concentrations in acute myeloid leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. Ivermectin also delayed tumor growth in 3 independent mouse models of leukemia at concentrations that appear pharmacologically achievable. As an antiparasitic, ivermectin binds and activates chloride ion channels in nematodes, so we tested the effects of ivermectin on chloride flux in leukemia cells. Ivermectin increased intracellular chloride ion concentrations and cell size in leukemia cells. Chloride influx was accompanied by plasma membrane hyperpolarization, but did not change mitochondrial membrane potential. Ivermectin also increased reactive oxygen species generation that was functionally important for ivermectin-induced cell death. Finally, ivermectin synergized with cytarabine and daunorubicin that also increase reactive oxygen species production. Thus, given its known toxicology and pharmacology, ivermectin could be rapidly advanced into clinical trial for leukemia.

Potential use of cetrimonium bromide as an apoptosis-promoting anticancer agent for head and neck cancer.

A potential therapeutic agent for human head and neck cancer (HNC), cetrimonium bromide (CTAB), was identified through a cell-based phenotype-driven high-throughput screen (HTS) of 2000 biologically active or clinically used compounds, followed by in vitro and in vivo characterization of its antitumor efficacy. The preliminary and secondary screens were performed on FaDu (hypopharyngeal squamous cancer) and GM05757 (primary normal fibroblasts), respectively. Potential hit compounds were further evaluated for their anticancer specificity and efficacy in combination with standard therapeutics on a panel of normal and cancer cell lines. Mechanism of action, in vivo antitumor efficacy, and potential lead compound optimizations were also investigated. In vitro, CTAB interacted additively with gamma radiation and cisplatin, two standard HNC therapeutic agents. CTAB exhibited anticancer cytotoxicity against several HNC cell lines, with minimal effects on normal fibroblasts; a selectivity that exploits cancer-specific metabolic aberrations. The central mode of cytotoxicity was mitochondria-mediated apoptosis via inhibition of H(+)-ATP synthase activity and mitochondrial membrane potential depolarization, which in turn was associated with reduced intracellular ATP levels, caspase activation, elevated sub-G(1) cell population, and chromatin condensation. In vivo, CTAB ablated tumor-forming capacity of FaDu cells and delayed growth of established tumors. Thus, using an HTS approach, CTAB was identified as a potential apoptogenic quaternary ammonium compound possessing in vitro and in vivo efficacy against HNC models.

Recent advances in the use of cell-penetrating peptides for medical and biological applications.

The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.

Mitochondria-penetrating peptides.

Mitochondria are important targets for cancer chemotherapy and other disease treatments. Gaining access to this organelle can be difficult, as the inner membrane is a barrier limiting diffusive transport. A mitochondrial molecular carrier would be a boon to the development of organelle-specific therapeutics. Here, we report a significant advance in the development of mitochondrial transporters-synthetic cell-permeable peptides that are able to enter mitochondria. Efficient uptake of these mitochondria-penetrating peptides (MPPs) is observed in a variety of cell types, and organellar specificity is attained with sequences that possess specific chemical properties. The MPPs identified are cationic, but also lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane. The examination of a panel of MPPs illustrates that mitochondrial localization can be rationally controlled and finely tuned by altering lipophilicity and charge.