A Cyclic Pentamethinium Salt Induces Cancer Cell Cytotoxicity through Mitochondrial Disintegration and Metabolic Collapse.

Cancer cells preferentially utilize glycolysis for ATP production even in aerobic conditions (the Warburg effect) and adapt mitochondrial processes to their specific needs. Recent studies indicate that altered mitochondrial activities in cancer represent an actionable target for therapy. We previously showed that salt 1-3C, a quinoxaline unit (with cytotoxic activity) incorporated into a meso-substituted pentamethinium salt (with mitochondrial selectivity and fluorescence properties), displayed potent cytotoxic effects in vitro and in vivo, without significant toxic effects to normal tissues. Here, we investigated the cytotoxic mechanism of salt 1-3C compared to its analogue, salt 1-8C, with an extended side carbon chain. Live cell imaging demonstrated that salt 1-3C, but not 1-8C, is rapidly incorporated into mitochondria, correlating with increased cytotoxicity of salt 1-3C. The accumulation in mitochondria led to their fragmentation and loss of function, accompanied by increased autophagy/mitophagy. Salt 1-3C preferentially activated AMP-activated kinase and inhibited mammalian target of rapamycin (mTOR) signaling pathways, sensors of cellular metabolism, but did not induce apoptosis. These data indicate that salt 1-3C cytotoxicity involves mitochondrial perturbation and disintegration, and such compounds are promising candidates for targeting mitochondria as a weak spot of cancer.

Next-generation sequencing of a combinatorial peptide phage library screened against ubiquitin identifies peptide aptamers that can inhibit the in vitro ubiquitin transfer cascade.

Defining dynamic protein-protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.

Kinomics platform using GBM tissue identifies BTK as being associated with higher patient survival.

Better understanding of GBM signalling networks in-vivo would help develop more physiologically relevant ex vivo models to support therapeutic discovery. A "functional proteomics" screen was undertaken to measure the specific activity of a set of protein kinases in a two-step cell-free biochemical assay to define dominant kinase activities to identify potentially novel drug targets that may have been overlooked in studies interrogating GBM-derived cell lines. A dominant kinase activity derived from the tumour tissue, but not patient-derived GBM stem-like cell lines, was Bruton tyrosine kinase (BTK). We demonstrate that BTK is expressed in more than one cell type within GBM tissue; SOX2-positive cells, CD163-positive cells, CD68-positive cells, and an unidentified cell population which is SOX2-negative CD163-negative and/or CD68-negative. The data provide a strategy to better mimic GBM tissue ex vivo by reconstituting more physiologically heterogeneous cell co-culture models including BTK-positive/negative cancer and immune cells. These data also have implications for the design and/or interpretation of emerging clinical trials using BTK inhibitors because BTK expression within GBM tissue was linked to longer patient survival.

An Ultrasensitive Biosensor for Detection of Femtogram Levels of the Cancer Antigen AGR2 Using Monoclonal Antibody Modified Screen-Printed Gold Electrodes.

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

Anticancer pentamethinium salt is a potent photosensitizer inducing mitochondrial disintegration and apoptosis upon red light illumination.

Despite progress in the development and application of novel therapeutic agents, cancer remains a major cause of death worldwide. Therefore, there is a need for new approaches to increase therapeutic options and efficiency. The metabolism of cancer cells differs from that of non-malignant cells and their mitochondria show altered activities that can be utilized as a target for drug development. Salt 1 is a low-molecular weight heterocyclic compound of the polymethine class that accumulates in the mitochondria of cancer cells and selectively disrupts their metabolism. Salt 1 leads to a non-apoptotic form of cell death in vitro that is associated with an autophagic cellular response and eventual metabolic collapse, and inhibits human tumor xenograft growth in vivo without apparent toxicity for normal cells. As a pentamethinium compound, salt 1 exhibits intrinsic fluorescence and is a candidate for photosensitization after excitation by appropriate wavelengths of light. Herein, we report that salt 1 is a potent photosensitizer, which generates a photodynamic effect and provides enhanced cytotoxicity compared to salt 1 without light exposure. Importantly, photosensitization is optimally induced by red light, which is used clinically for photosensitization and penetrates further into tissues than lower wavelengths. Cancer cells treated with non-cytotoxic doses of salt 1 and subsequently exposed to 630 nm light show severely damaged mitochondria, manifested by reduced mitochondrial membrane potential and disintegration of the mitochondrial tubular network. As a consequence, cancer cells lose their proliferative potential and die via apoptosis in the presence of light. These findings indicate that salt 1 is a promising photosensitizer with potential to be combined with 630 nm light to strengthen its efficacy in cancer therapy.

Mitochondrial Processes in Targeted Cancer Therapy.

BACKGROUND: During tumor initiation and progress, cellular functions adapt to the new needs of the transformed cells and mitochondrial processes are also affected. Mitochondria are less extensively used for supplying cells with energy; rather, cancer cells utilize glycolysis to a much greater extent, even under aerobic conditions. Mitochondria produce metabolites required for cellular growth and proliferation. Mutations and alterations in gene expression of citrate cycle enzymes can directly contribute to transformation through the production of oncometabolites. The apoptotic pathway in which mitochondria play a critical role is disrupted in cancer cells, resulting in cells that do not respond to programmed cell death signaling. These differences between mitochondrial processes in healthy and diseased cells suggest they could be used in mitochondria-targeted therapies. To date, many potential molecular targets have been identified, including enzymes, signaling molecules, and membrane transporters. Even though this field has been studied for years, the first drugs, venetoclax and enasidenib, were only approved in the last two years and are the result of two different research approaches. Venetoclax targets the apoptotic pathway and enasidenib targets metabolic processes. The discovery of these two compounds demonstrates that it is possible to develop mitochondria-targeted cancer treatments. PURPOSE: The purpose of this article is to provide an overview of research in the field of mitochondria-targeting therapies for cancer. The main areas of research and the main approaches for treatment development are summarized. Cellular components studied as potential targets for therapy and compounds that are considered exploitable are described, as well as already approved drugs. Key words: neoplasms - molecular targeted therapy - mitochondria - antineoplastic agents - research The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 3. 8. 2018.