Design, synthesis and antitumor effects of lupeol quaternary phosphonium salt derivatives.

Lupeol is a natural pentacyclic triterpenoid with a wide range of biological activities. To improve the water solubility and targeting of lupeol, in the following study, we synthesized 27 lupeol derivatives in the first series by introducing lipophilic cations with lupeol as the lead compound. Through the screening of different cancer cells, we found that some of the derivatives showed better activity than cisplatin against human non-small cell lung cancer A549 cells, among which compound 6c was found to have an IC50 value of 1.83 µM and a selectivity index of 21.02 (IC50MRC-5/IC50A549) against A549 cells. To further improve the antiproliferative activity of the compounds, we replaced the ester linkage of the linker with a carbamate linkage and synthesized a second series of five lupeol derivatives which were screened for activity, among which compound 14f was found to have an IC50 value of 1.36 µM and a selectivity index of 15.60 (IC50MRC-5/IC50A549) against A549 cells. We further evaluated the bioactivity of compounds 6c and 14f and found that both compounds induced apoptosis in A549 cells, promoted an increase in intracellular reactive oxygen species and decrease in mitochondrial membrane potential, and inhibited the cell cycle in the S phase. Of the compounds, compound 14f showed stronger bioactivity than compound 6c. We then selected compound 14f for molecular-level Western blot evaluation and in vivo evaluation in the zebrafish xenograft A549 tumor cell model. Compound 14f was found to significantly downregulate Bcl-2 protein expression and upregulate Bax, Cyt C, cleaved caspase-9, and cleaved caspase-3 protein expression, and 14f was found to be able to inhibit the proliferation of A549 cells in the zebrafish xenograft model. The above results suggest that compound 14f has great potential in the development of antitumor drugs targeting mitochondria.

Design and synthesis of novel mitochondria-targeted ergosterol peroxide derivatives as potential anti-cancer agents.

Ergosterol peroxide (EP) is a natural steroid compound that has been reported to have significant antitumor activity. However, its poor water solubility and cellular uptake mean that it has weak efficacy against tumor cells. Herein, we designed and synthesized a series of EP derivatives with mitochondrial targeting properties. Of these, compound 15a showed an IC50 value of 0.32 µM against MCF-7 cells, which was 67-fold higher than that of the parental EP (IC50 = 21.46 µM), and was better than cisplatin (IC50 = 4.23 µM), had a selectivity index of 25.28 (IC50MCF-10A/IC50MCF-7). Additionally, compound 15a promoted an increase in intracellular reactive oxygen species levels and a decrease in mitochondrial membrane potential, and blocked the cell cycle in the G0/G1 phase. In a mouse model of breast cancer, 15a showed 89.85 % tumor inhibition at a dose of 20 mg/kg, which is similar to the therapeutic effect of the cisplatin. On the basis of these results, 15a could be considered for further preclinical evaluation for cancer therapy.

Cyanine mitochondrial dye with slightly selective cytotoxicity against A549 cancerous cells.

Delocalized lipophilic cations (DLCs) are known as mitochondria-addressed molecules. Mitochondria targeting may provide opportunities for tumor detection. DLCs may have antioxidant or anticancer properties. In this study, we focused on the toxicity and localization of 2-[(E)-2-(5-fluoro-2-methyl-1H-indol-3-yl)ethenyl]-1,6-dimethylpyridin-1-ium iodide (62E2), which has recently been found as a novel cytotoxic fluorescent compound. The excitation maximum of 62E2 is 452 ± 10 nm and its emission maximum is 579 ± 10 nm. It is accumulated in the cells and stains mitochondria in nanomolar concentrations. 62E2 is cytotoxic and mitotoxic in low micromolar concentrations, and it demonstrates some selectivity of cytotoxicity against A549 cancer cells. The closest analog of 62E2 is F16, which is the fluorescent mitotoxic agent that has been described earlier as a potential anticancer agent. We hope that 62E2 described here is useful in expanding the diversity of cyanine fluorescent mitochondrial dyes and the analysis of their structure-activity relationships.

Mitochondria-targeted delocalized lipophilic cation complexed with human serum albumin for tumor cell imaging and treatment.

Small molecule 5BMF is a novel mitochondria-targeted delocalized lipophilic cation (DLC) with good anti-tumor activity and fluorescence emission suitable for bioimaging. In this study, human serum albumin (HSA) complexed with 5BMF (5BMF@HSA) has been developed to further improve its solubility (from 1.61 to 5.41 mg/mL), and the fluorescent intensity of 5BMF@HSA was improved over 2 times. Nearly 10-fold 5BMF was released from 5BMF@HSA complex in acidic condition when compared with neutral/basic environment. Intracellular distribution of 5BMF was altered by HSA as its signals were observed in lysosomes where free 5BMF barely localized. Both 5BMF@HSA and 5BMF showed selective toxicity toward tumor cells in µM and nM range and effectively inhibited tumor growth in mice model. In summary, 5BMF@HSA shows improved solubility in aqueous buffer and enhanced fluorescence emission, and maintains tumor inhibition capability. It is a promising protein complex for tumor cell imaging and tumor treatment.

Medicinal Chemistry Targeting Mitochondria: From New Vehicles and Pharmacophore Groups to Old Drugs with Mitochondrial Activity.

Interest in tumor cell mitochondria as a pharmacological target has been rekindled in recent years. This attention is due in part to new publications documenting heterogenous characteristics of solid tumors, including anoxic and hypoxic zones that foster cellular populations with differentiating metabolic characteristics. These populations include tumor-initiating or cancer stem cells, which have a strong capacity to adapt to reduced oxygen availability, switching rapidly between glycolysis and oxidative phosphorylation as sources of energy and metabolites. Additionally, this cell subpopulation shows high chemo- and radioresistance and a high capacity for tumor repopulation. Interestingly, it has been shown that inhibiting mitochondrial function in tumor cells affects glycolysis pathways, cell bioenergy, and cell viability. Therefore, mitochondrial inhibition may be a viable strategy for eradicating cancer stem cells. In this context, medicinal chemistry research over the last decade has synthesized and characterized "vehicles" capable of transporting novel or existing pharmacophores to mitochondrial tumor cells, based on mechanisms that exploit the physicochemical properties of the vehicles and the inherent properties of the mitochondria. The pharmacophores, some of which have been isolated from plants and others, which were synthesized in the lab, are diverse in chemical nature. Some of these molecules are active, while others are prodrugs that have been evaluated alone or linked to mitochondria-targeted agents. Finally, researchers have recently described drugs with well-proven safety and efficacy that may exert a mitochondria-specific inhibitory effect in tumor cells through noncanonical mechanisms. The effectiveness of these molecules may be improved by linking them to mitochondrial carrier molecules. These promising pharmacological agents should be evaluated alone and in combination with classic chemotherapeutic drugs in clinical studies.

In vitro and in vivo characterization of anti-malarial acylphenoxazine derivatives prepared from basic blue 3.

BACKGROUND: Basic blue 3 is a promising anti-malarial lead compound based on the π-delocalized lipophilic cation hypothesis. Its derivatives with nitrogen atoms bonded to carbon atoms at the 3- and 7-positions on the phenoxazine ring were previously shown to exert potent antiprotozoal activity against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani parasites in vitro. However, compounds with nitrogen modification at the 10-position on the phenoxazine ring were not evaluated. METHODS: Six acylphenoxazine derivatives (ITT-001 to 006) with nitrogen modification at the 10-position on the phenoxazine ring, which were synthesized from basic blue 3, were characterized and evaluated for anti-malarial activity in vitro with an automated haematology analyzer (XN-30) and light microscopy. Intensity of self-fluorescence was measured using a fluorometer. Localization of basic blue 3 was observed by fluorescence microscopy. Cytotoxicity was evaluated using human cell lines, HEK293T and HepG2 cells. Finally, anti-malarial activity was evaluated in a rodent malaria model. RESULTS: All the six derivatives showed anti-malarial efficacy even against chloroquine-, pyrimethamine-, and artemisinin-resistant field isolates similar to the sensitive strains and isolates in vitro. The efficacy of basic blue 3 was the strongest, followed by that of ITT-001 to 004 and 006, while that of ITT-005 was the weakest. Basic blue 3 showed strong self-fluorescence, whereas ITT derivatives had five- to tenfold lower intensity than that of basic blue 3, which was shown by fluorescence microscopy to be selectively accumulated in the plasmodial cytoplasm. In contrast, ITT-003, 004, and 006 exhibited the lowest cytotoxicity in HEK293T and HepG2 cells in vitro and the highest selectivity between anti-malarial activity and cytotoxicity. The in vivo anti-malarial assay indicated that oral administration of ITT-004 was the most effective against the rodent malaria parasite, Plasmodium berghei NK65 strain. CONCLUSIONS: The six ITT derivatives were effective against chloroquine- and pyrimethamine-resistant strains and artemisinin-resistant field isolates as well as the sensitive ones. Among them, ITT-004, which had high anti-malarial activity and low cytotoxicity in vitro and in vivo, is a promising anti-malarial lead compound.

New TRAP1 and Hsp90 chaperone inhibitors with cationic components: Preliminary studies on mitochondrial targeting.

TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell growth and in inducing apoptotic cell death in colon carcinoma cells.

Mitochondria-Targeted Agents: Mitochondriotropics, Mitochondriotoxics, and Mitocans.

Mitochondria, the powerhouse of the cell, have been known for many years for their central role in the energy metabolism; however, extensive progress has been made and to date substantial evidence demonstrates that mitochondria play a critical role not only in the cell bioenergetics but also in the entire cell metabolome. Mitochondria are also involved in the intracellular redox poise, the regulation of calcium homeostasis, and the generation of reactive oxygen species (ROS), which are crucial for the control of a variety of signaling pathways. Additionally, they are essential for the mitochondrial-mediated apoptosis process. Thus, it is not surprising that disruptions of mitochondrial functions can lead or be associated with human pathologies. Because of diseases like diabetes, Alzheimer, Parkinson's, cancer, and ischemic disease are being increasingly linked to mitochondrial dysfunctions, the interest in mitochondria as a prime pharmacological target has dramatically risen over the last decades and as a consequence a large number of agents, which could potentially impact or modulate mitochondrial functions, are currently under investigation. Based on their site of action, these agents can be classified as mitochondria-targeted and non-mitochondria-targeted agents. As a result of the continuous search for new agents and the design of potential therapeutic agents to treat mitochondrial diseases, terms like mitochondriotropics, mitochondriotoxics, mitocancerotropics, and mitocans have emerged to describe those agents with high affinity to mitochondria that exert a therapeutic or deleterious effect on these organelles. In this chapter, mitochondria-targeted agents and some strategies to deliver agents to and/or into mitochondria will be reviewed.

Research Progress on 18F-Labeled Agents for Imaging of Myocardial Perfusion with Positron Emission Tomography.

Coronary artery disease (CAD) is the leading cause of death in the world. Myocardial perfusion imaging (MPI) plays a significant role in non-invasive diagnosis and prognosis of CAD. However, neither single-photon emission computed tomography nor positron emission tomography clinical MPI agents can absolutely satisfy the demands of clinical practice. In the past decades, tremendous developments happened in the field of 18F-labeled MPI tracers. This review summarizes the current state of 18F-labeled MPI tracers, basic research data of those tracers, and the future direction of MPI tracer research.

Application Prospects of Triphenylphosphine-Based Mitochondria-Targeted Cancer Therapy.

Cancer is one of the leading causes of death and the most important impediments to the efforts to increase life expectancy worldwide. Currently, chemotherapy is the main treatment for cancer, but it is often accompanied by side effects that affect normal tissues and organs. The search for new alternatives to chemotherapy has been a hot research topic in the field of antineoplastic medicine. Drugs targeting diseased tissues or cells can significantly improve the efficacy of drugs. Therefore, organelle-targeted antitumor drugs are being explored, such as mitochondria-targeted antitumor drugs. Mitochondria is the central site of cellular energy production and plays an important role in cell survival and death. Moreover, a large number of studies have shown a close association between mitochondrial metabolism and tumorigenesis and progression, making mitochondria a promising new target for cancer therapy. Combining mitochondrial targeting agents with drug molecules is an effective way of mitochondrial targeting. In addition, hyperpolarized tumor cell membranes and mitochondrial membrane potentially allow selective accumulation of mitochondria-targeted drugs. This enhances the direct killing of tumor cells by drug molecules while minimizing the potential toxicity to normal cells. In this review, we discuss the common pro-mitochondrial agents, the advantages of triphenylphosphine (TPP) in mitochondrial-targeted cancer therapy and systematically summarize various TPP-based mitochondria-targeting anticancer drugs.

Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations.

Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.

Understanding Functional Redundancy and Promiscuity of Multidrug Transporters in E. coli under Lipophilic Cation Stress.

Multidrug transporters (MDTs) are major contributors to microbial drug resistance and are further utilized for improving host phenotypes in biotechnological applications. Therefore, the identification of these MDTs and the understanding of their mechanisms of action in vivo are of great importance. However, their promiscuity and functional redundancy represent a major challenge towards their identification. Here, a multistep tolerance adaptive laboratory evolution (TALE) approach was leveraged to achieve this goal. Specifically, a wild-type E. coli K-12-MG1655 and its cognate knockout individual mutants ΔemrE, ΔtolC, and ΔacrB were evolved separately under increasing concentrations of two lipophilic cations, tetraphenylphosphonium (TPP+), and methyltriphenylphosphonium (MTPP+). The evolved strains showed a significant increase in MIC values of both cations and an apparent cross-cation resistance. Sequencing of all evolved mutants highlighted diverse mutational mechanisms that affect the activity of nine MDTs including acrB, mdtK, mdfA, acrE, emrD, tolC, acrA, mdtL, and mdtP. Besides regulatory mutations, several structural mutations were recognized in the proximal binding domain of acrB and the permeation pathways of both mdtK and mdfA. These details can aid in the rational design of MDT inhibitors to efficiently combat efflux-based drug resistance. Additionally, the TALE approach can be scaled to different microbes and molecules of medical and biotechnological relevance.

Conjugation of Natural Triterpenic Acids with Delocalized Lipophilic Cations: Selective Targeting Cancer Cell Mitochondria.

Currently, a new line of research on mitochondria-targeted anticancer drugs is actively developing in the field of biomedicine and medicinal chemistry. The distinguishing features of this universal target for anticancer agents include presence of mitochondria in the overwhelming majority, if not all types of transformed cells, crucial importance of these cytoplasmic organelles in energy production, regulation of cell death pathways, as well as generation of reactive oxygen species and maintenance of calcium homeostasis. Hence, mitochondriotropic anticancer mitocan agents, acting through mitochondrial destabilization, have good prospects in cancer therapy. Available natural pentacyclic triterpenoids are considered promising scaffolds for development of new mitochondria-targeted anticancer agents. These secondary metabolites affect the mitochondria of tumor cells and initiate formation of reactive oxygen species. The present paper focuses on the latest research outcomes of synthesis and study of cytotoxic activity of conjugates of pentacyclic triterpenoids with some mitochondria-targeted cationic lipophilic molecules and highlights the advantages of applying them as novel mitocan agents compared to their prototype natural triterpenic acids.

Using chemical biology to assess and modulate mitochondria: progress and challenges.

Our understanding of the role of mitochondria in biomedical sciences has expanded considerably over the past decade. In addition to their well-known metabolic roles, mitochondrial are also central to signalling for various processes through the generation of signals such as ROS and metabolites that affect cellular homeostasis, as well as other processes such as cell death and inflammation. Thus, mitochondrial function and dysfunction are central to the health and fate of the cell. Consequently, there is considerable interest in better understanding and assessing the many roles of mitochondria. Furthermore, there is also a growing realization that mitochondrial are a promising drug target in a wide range of pathologies. The application of interdisciplinary approaches at the interface between chemistry and biology are opening up new opportunities to understand mitochondrial function and in assessing the role of the organelle in biology. This work and the experience thus gained are leading to the development of new classes of therapies. Here, we overview the progress that has been made to date on exploring the chemical biology of the organelle and then focus on future challenges and opportunities that face this rapidly developing field.

Re-engineering the mitochondrial genomes in mammalian cells.

Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.

Re-engineering the mitochondrial genomes in mammalian cells / 대한해부학회지

Mitochondria are subcellular organelles composed of two discrete membranes in the cytoplasm of eukaryotic cells. They have long been recognized as the generators of energy for the cell and also have been known to associate with several metabolic pathways that are crucial for cellular function. Mitochondria have their own genome, mitochondrial DNA (mtDNA), that is completely separated and independent from the much larger nuclear genome, and even have their own system for making proteins from the genes in this mtDNA genome. The human mtDNA is a small (~16.5 kb) circular DNA and defects in this genome can cause a wide range of inherited human diseases. Despite of the significant advances in discovering the mtDNA defects, however, there are currently no effective therapies for these clinically devastating diseases due to the lack of technology for introducing specific modifications into the mitochondrial genomes and for generating accurate mtDNA disease models. The ability to engineer the mitochondrial genomes would provide a powerful tool to create mutants with which many crucial experiments can be performed in the basic mammalian mitochondrial genetic studies as well as in the treatment of human mtDNA diseases. In this review we summarize the current approaches associated with the correction of mtDNA mutations in cells and describe our own efforts for introducing engineered mtDNA constructs into the mitochondria of living cells through bacterial conjugation.