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Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

Glutathione (GSH) is an endogenous tripeptide (Glu-Cys-Gly) and the most abundant endogenous antioxidant. It protects the body against oxidative stress and reactive electrophiles. GSH and its oxidized form glutathione disulfide (GSSG) make up the most important thiol redox buffer in the body, and its homeostasis is critical to many important cellular functions. Dysfunction or disruption of the redox buffer has been implicated in various diseases and the implications make GSH, GSSG, and their related systems valid and effective targets for medicinal chemistry interventions. For example, increased levels of GSH and GSH related systems in cancer have been employed as a basis for anticancer prodrug activation, anticancer drug delivery and anticancer drug development. The enriched GSH transporters in the blood brain barrier has been effectively employed for the design of brain targeting prodrugs and brain drug delivery systems. Further, a glutathione conjugate can serve as a marker for the presence of a reactive electrophile providing valid information for the evaluation of a drug candidate. This review focuses on GSH/GSSG system-based biomedical and pharmaceutical applications with relevant basic and comprehensive background information.

ABSTRACT Purpose To investigate skin penetration of poly (amidoamine) (PAMAM) dendrimers as a function of surface charge and molecular weight in presence and absence of iontophoresis. Methods Dendrimers were labeled with fluoroisothiocynate (FITC); skin penetration of dendrimers was studied using excised porcine skin in-vitro . Skin penetration of FITC-labeled dendrimers was quantified using confocal laser scanning microscope (CLSM). G2-G6 NH 2 , G3.5-COOH and G4-OH dendrimers were used. Results Cationic dendrimers showed higher skin penetration than neutral and anionic dendrimers. Skin penetration of cationic dendrimer increased linearly with increase in treatment time. Iontophoresis enhanced skin penetration of cationic and neutral dendrimers. Increase in current strength and current duration increased skin transport of dendrimers. Passive and iontophoretic skin penetration of cationic dendrimers was inversely related to their molecular weight. Dendrimer penetrated the skin through intercellular lipids and hair follicles. With iontophoresis, dendrimer was also found in localized skin regions. Conclusions The study demonstrates that the physicochemical properties of dendrimers influence their skin transport. Findings can be used to design dendrimer-based nanocarriers for drug delivery to skin.

Thiol molecules play a significant role in cellular structures and functions. These molecules are distributed in cells unevenly at the subcellular level. Disturbance of cellular thiols has been associated with various diseases and disorders. Probes that are able to detect subcellular thiol density in live cells are valuable tools in determining thiols’ roles at the subcellular level. Lysosomes are a subcellular organelle involved in the degradation of macromolecules through the action of proteolytic enzymes. The degradation not only serves as a way to dispose of unwanted macromolecules but also a way to regulate a variety of cellular functions such as autophagy, endocytosis, and phagocytosis to maintain cell homeostasis. A probe that can detect lysosomal thiols in live cells will be useful in unveiling the roles of thiols in lysosomes. Currently, limited probes are available to detect lysosomal thiols in live cells. We would like to report 4,4′-{[7,7′-thiobis(benzo[c][1,2,5]oxadiazole-4,4′-sulfonyl)]bis(oxy))bis(naphthalene-2,7-disulfonicacid) (TBONES) as a thiol-specific fluorogenic agent for lysosomal thiol imaging in live cells through fluorescence microscopy. TBONES exhibits no fluorescence and readily reacts with non-protein thiols to form fluorescent thiol adducts with λex = 400 nm and λem = 540 nm. No reaction was observed when TBONES was mixed with compounds containing nucleophilic functional groups other than thiols such as –OH, –NH2, and –COOH. No reaction was observed either when TBONES was mixed with protein thiols. When incubated with cells, TBONES selectively and effectively imaged lysosomal thiols in live cells. Imaging of lysosomal thiols was confirmed by a co-localization experiment with LysoTracker™ Blue DND-22.

The goal of this study was to determine if depletion of glutathione (GSH) and inhibition of thioredoxin (Trx) reductase (TrxR) activity could enhance radiation responses in human breast cancer stem cells by a mechanism involving thiol-dependent oxidative stress. The following were used to inhibit GSH and Trx metabolism: buthionine sulfoximine (BSO), a GSH synthesis inhibitor; sulfasalazine (SSZ), an inhibitor of xc– cysteine/glutamate antiporter; auranofin (Au), a thioredoxin reductase inhibitor; or 2-AAPA, a GSH-reductase inhibitor. Clonogenic survival, Matrigel assays, flow cytometry cancer stem cell assays (CD44+CD24–ESA+ or ALDH1) and human tumor xenograft models were used to determine the antitumor activity of drug and radiation combinations. Combined inhibition of GSH and Trx metabolism enhanced cancer cell clonogenic killing and radiation responses in human breast and pancreatic cancer cells via a mechanism that could be inhibited by N-acetylcysteine (NAC). Au, BSO and radiation also significantly decreased breast cancer cell migration and invasion in a thiol-dependent manner that could be inhibited by NAC. In addition, pretreating cells with Au sensitized breast cancer stem cell populations to radiation in vitro as determined by CD44+CD24–ESA+ or ALDH1. Combined administration of Au and BSO, given prior to irradiation, significantly increased the survival of mice with human breast cancer xenografts, and decreased the number of ALDH1+ cancer stem cells. These results indicate that combined inhibition of GSH- and Trx-dependent thiol metabolism using pharmacologically relevant agents can enhance responses of human breast cancer stem cells to radiation both in vitro and in vivo.

Inability to achieve therapeutic concentrations of a medication in the brain due to the blood brain barrier (BBB) is the major cause of treatment failure for most brain diseases. The BBB prevents almost 98% of small molecule drugs and almost all large molecule therapeutics from entering the brain. Modifying a drug delivery system with a brain targeting agent has been an effective approach in developing a brain targeting drug delivery system. Most of the brain targeting agents were developed based on a receptor- or carrier-mediated endocytosis process at the BBB. These endocytosis processes are transporting mechanisms for transporting endogenous molecules into the brain. They include those for transporting transferrin, LDL (low density lipoprotein), insulin, etc., with transferrin receptor-mediated endocytosis being the most investigated and successful one for developing a brain targeting agent. The Na + -dependent glutathione transporter is present on the luminal side of the capillary endothelial cells of the brain, kidneys, and small intestine while its presence on the luminal side of the capillary endothelial cells of other organs is very minimal. This organ distribution difference enables the brain, kidneys and small intestines to sequester GSH from the blood circulation to meet the need of these organs for GSH, and provide a solid foundation for developing organ selective agents for these organs in general. This review provides an overview of the GSH transporter and the status of GSH transporter-based brain targeting drug delivery systems with the intention of bringing the field to the attention of a medicinal chemist for his/her expertise in organic synthesis, ligand identification and optimization.

Background Microtubules have been one of the most effective targets for the development of anticancer agents. Cancer cells treated by these agents are characterized by cell arrest at G 2 /M phase. Microtubule-targeting drugs are, therefore, referred to as antimitotic agents. However, the clinical application of the current antimitotic drugs is hampered by emerging drug resistance which is the major cause of cancer treatment failure. The clinical success of antimitotic drugs and emerging drug resistance has prompted a search for new antimitotic agents, especially those with novel mechanisms of action. The aim of this study was to determine whether microtubules can be S- glutathionylated in cancer cells and whether the glutathionylation will lead to microtubule dysfunction and cell growth inhibition. The study will determine whether microtubule S- glutathionylation can be a novel approach for antimitotic agents. Methods 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenyl carbamoylsulfanyl]propionic acid (2-AAPA) was used as a tool to induce microtubule S -glutathionylation. UACC-62 cells, a human melanoma cell line, were used as a cancer cell model. A pull-down assay with glutathione S- transferase (GST)-agarose beads followed by Western blot analysis was employed to confirm microtubule S -glutathionylation. Immunofluorescence microscopy using a mouse monoclonal anti-α-tubulin-FITC was used to study the effect of the S- glutathionylation on microtubule function; mainly polymerization and depolymerization. Flow cytometry was employed to examine the effect of the S- glutathionylation on cell cycle distribution and apoptosis. Cell morphological change was followed through the use of a Zeiss AXIO Observer A1 microscope. Cancer cell growth inhibition by 2-AAPA was investigated with ten human cancer cell lines. Results Our investigation demonstrated that cell morphology was changed and microtubules were S -glutathionylated in the presence of 2-AAPA in UACC-62 cells. Accordingly, microtubules were found depolymerized and cells were arrested at G 2 /M phase. The affected cells were found to undergo apoptosis. Cancer growth inhibition experiments demonstrated that the concentrations of 2-AAPA required to produce the effects on microtubules were compatible to the concentrations producing cancer cell growth inhibition. Conclusions The data from this investigation confirms that microtubule S -glutathionylation leads to microtubule dysfunction and cell growth inhibition and can be a novel approach for developing antimitotic agents.

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY’s reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7′-thiobis(benzo[c][1,2,5]oxadiazole-4,4′-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY’s reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY’s reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY’s reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP

Cancer metastasis is the major cause of cancer mortality. Despite extensive research efforts, effective treatment for cancer metastasis is still lacking. Cancer metastasis involves 4 essential steps: cell detachment, migration, invasion, and adhesion. Detachment is the first and required step for metastasis. Glutathione disulfide (GSSG) is derived from the oxidation of glutathione (GSH), which is present in biological systems in millimolar concentration. Although GSSG is commercially available, the impact of GSSG on cell functions/dysfunctions has not been fully explored due to the fact that GSSG is not cell membrane permeable and a lack of method to specifically increase GSSG in cells. We have developed GSSG liposomes that effectively deliver GSSG to cells. Unexpectedly, cells treated with GSSG liposomes were resistant to detachment by trypsinization. This observation led to the investigation of the antimetastatic effect of GSSG liposomes. Our data demonstrate that GSSG liposomes at 1 mg/mL completely blocked cell detachment and migration, and significantly inhibited cancer cell invasion. Aqueous GSSG showed no such effect, confirming that the effects on cell detachment, migration, and invasion were caused by the intracellular delivery of GSSG. An in vivo experiment with a murine melanoma experimental metastasis model showed that GSSG liposomes prevented melanoma lung metastasis. The unique antimetastatic mechanism through the effects on detachment and migration, and effective in vitro and in vivo metastasis inhibition, warrants further investigation of the GSSG liposomes as a potential treatment for cancer metastasis.

Glutathione disulfide (GSSG) is an endogenous peptide and the oxidized form of glutathione. The impacts of GSSG on cell function/dysfunction remain largely unexplored due to a lack of method to specifically increase intracellular GSSG. We recently developed GSSG liposomes that can specifically increase intracellular GSSG. The increase affected 3 of the 4 essential steps (cell detachment, migration, invasion, and adhesion) of cancer metastasis in vitro and, accordingly, produced a significant inhibition of cancer metastasis in vivo. In this investigation, the effect of GSSG liposomes on cancer growth was investigated with B16-F10 and NCI-H226 cells in vitro and with B16-F10 cells in C57BL/6 mice in vivo. Experiments were conducted to elucidate the effect on cell death through promotion of apoptosis and the effect on the cell cycle. The in vivo results with C57BL/6 mice implanted subcutaneously with B16-F10 cells showed that GSSG liposomes retarded tumor proliferation more effectively than that of dacarbazine, a chemotherapeutic drug for the treatment of melanoma. The GSSG liposomes by intravenous injection (GLS IV) and GSSG liposomes by intratumoral injection (GLS IT) showed a tumor proliferation retardation of 85% ± 5.7% and 90% ± 3.9%, respectively, compared with the phosphate-buffered saline (PBS) control group. The median survival rates for mice treated with PBS, blank liposomes, aqueous GSSG, dacarbazine, GLS IV, and GLS IT were 7, 7, 7.5, 7.75, 11.5, and 16.5 days, respectively. The effective antimetastatic and antigrowth activities warrant further investigation of the GSSG liposomes as a potentially effective therapeutic treatment for cancer.