2-(2-Cholesteroxyethoxyl)ethyl 3'-S-glutathionylpropionate and its self-assembled micelles for brain delivery: Design, synthesis and evaluation.

The blood-brain barrier (BBB) is a barrier that prevents almost all large and most small exogenous molecules from reaching the brain. The barrier is the major cause of treatment failure for most brain diseases. Extensive efforts have been made to facilitate drug molecules to cross the BBB. One of the approaches is to employ an endogenous ligand or ligand analogue that can enter the brain through its transporter or receptor at the BBB as a brain-targeting agent. Glutathione (GSH) transporters are richly expressed at the BBB with limited presence in other tissues except kidneys. 2-(2-Cholesteroxyethoxyl)ethyl 3'-S-glutathionylpropionate (COXP), formed by connecting GSH with cholesterol through a linker, was designed as a GSH transporter-mediated brain targeting molecule. The amphiphilic nature of COXP enables the molecule to self-assemble to form micelles with a CMC value of 3.9 µM. By using DiR as a fluorescence tracking agent and the whole-body fluorescence imaging technique, the brain distribution of DiR delivered by COXP micelles in mice was 20 folds higher when compared with free DiR. Interestingly, the brain targeting effect was further enhanced by co-administration of GSH. The low CMC value and effective brain targeting make COXP micelles a promising drug delivery system to the brain.

Toxicity of canola-derived glucosinolates in pigs fed resistant starch-based diets.

A study was conducted to determine effects of reducing hindgut pH through dietary inclusion of high-amylose cornstarch (HA-starch) on growth performance, organ weights relative to live body weight (BW), blood thyroid hormone levels, and glucosinolate degradation products of nursery pigs fed cold-pressed canola cake (CPCC). A total of 240 pigs (initial BW: 7.1 kg), which had been weaned at 21 d of age, were housed in 40 pens (6 pigs per pen) and fed 4 diets (10 pens per diet) in a randomized complete block design for 28 d. Four diets were a basal diet with CPCC at 0 or 40%, and with HA-starch at 0 or 40% in a 2 × 2 factorial arrangement. The diets were fed in two phases: Phase 1 from day 0 to 14 and Phase 2 from day 14 to 28 and were formulated to have the same net energy, standardized ileal digestible AA, Ca, and standardized total tract digestible P contents. Dietary inclusion of CPCC and HA-starch was achieved by a partial or complete replacement of corn, soybean meal, and soy protein. At the end of the study, one pig from each pen was euthanized to determine organ weights, blood parameters, hindgut pH, and glucosinolate degradation products. Dietary CPCC reduced (P < 0.05) overall average daily gain (ADG) by 15%; increased (P < 0.05) relative weights of liver and thyroid gland by 27% and 64%, respectively; and reduced (P < 0.05) serum tetraiodothyronine (T4) level from 30.3 to 17.8 ng/mL. Heart, kidney, and gastrointestinal tract weights; serum triiodothyronine level; and hindgut pH of pigs were unaffected by dietary CPCC. Dietary HA-starch reduced (P < 0.05) overall ADG, relative weight of thyroid gland, cecal, and colonic pH; but increased (P < 0.05) relative weight of colon; tended to increase (P = 0.062) serum T4 level. Dietary CPCC and HA-starch interacted (P = 0.024) on relative weight of thyroid gland such that dietary CPCC increased (P < 0.05) weight of thyroid gland for HA-starch-free diet (120 vs. 197 mg/kg of BW) but not for HA-starch-containing diet (104 vs. 130 mg/kg of BW). Dietary CPCC and HA-starch interacted (P = 0.001) on cecal isothiocyanate content such that dietary CPCC increased (P < 0.05) level of isothiocyanates for HA-starch-containing diet but not for HA-starch-free diet. In conclusion, dietary CPCC reduced growth performance, increased liver, size and interfered with thyroid gland functions of pigs. However, the negative effects of dietary CPCC on thyroid gland functions of nursery pigs were alleviated by dietary HA-starch.

The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention.

Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC.

Interprofessional education in pharmacology using high-fidelity simulation.

BACKGROUND: This study examined the feasibility of an interprofessional high-fidelity pharmacology simulation and its impact on pharmacy and nursing students' perceptions of interprofessionalism and pharmacology knowledge. INTERPROFESSIONAL EDUCATION ACTIVITY: Pharmacy and nursing students participated in a pharmacology simulation using a high-fidelity patient simulator. Faculty-facilitated debriefing included discussion of the case and collaboration. To determine the impact of the activity on students' perceptions of interprofessionalism and their ability to apply pharmacology knowledge, surveys were administered to students before and after the simulation. Attitudes Toward Health Care Teams scale (ATHCT) scores improved from 4.55 to 4.72 on a scale of 1-6 (p = 0.005). Almost all (over 90%) of the students stated their pharmacology knowledge and their ability to apply that knowledge improved following the simulation. DISCUSSION: A simulation in pharmacology is feasible and favorably affected students' interprofessionalism and pharmacology knowledge perceptions. IMPLICATIONS: Pharmacology is a core science course required by multiple health professions in early program curricula, making it favorable for incorporation of interprofessional learning experiences. However, reports of high-fidelity interprofessional simulation in pharmacology courses are limited. This manuscript contributes to the literature in the field of interprofessional education by demonstrating that an interprofessional simulation in pharmacology is feasible and can favorably affect students' perceptions of interprofessionalism. This manuscript provides an example of a pharmacology interprofessional simulation that faculty in other programs can use to build similar educational activities.

Salicylic acid metabolites and derivatives inhibit CDK activity: Novel insights into aspirin's chemopreventive effects against colorectal cancer.

Aspirin's potential as a drug continues to be evaluated for the prevention of colorectal cancer (CRC). Although multiple targets for aspirin and its metabolite, salicylic acid, have been identified, no unifying mechanism has been proposed to clearly explain its chemopreventive effects. Our goal here was to investigate the ability of salicylic acid metabolites, known to be generated through cytochrome P450 (CYP450) enzymes, and its derivatives as cyclin dependent kinase (CDK) inhibitors to gain new insights into aspirin's chemopreventive actions. Using in vitro kinase assays, for the first time, we demonstrate that salicylic acid metabolites, 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA), as well as derivatives 2,4-dihydroxybenzoic acid (2,4-DHBA), 2,6-dihydroxybenzoic acid (2,6-DHBA), inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, another derivative, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking studies showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and pre-incubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supported the data obtained from molecular docking studies. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells, or the salicylic acid metabolites generated by gut microflora may significantly contribute to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin's chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of novel CDK inhibitors in cancer prevention and treatment.

In Vitro and In Vivo Antimetastatic Effect of Glutathione Disulfide Liposomes.

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.

In-vitro and in-vivo inhibition of melanoma growth and metastasis by the drug combination of celecoxib and dacarbazine.

Celecoxib has been found to be effective in cancer prevention and treatment. Its combination with other chemotherapeutic agents was reported to produce synergistic/additive effects on various cancers. Dacarbazine (DTIC) is one of the most commonly used drugs in the treatment of metastatic melanoma. This investigation aimed to determine the in-vitro and in-vivo effects of the drug combination of celecoxib and DTIC on melanoma growth and metastasis. Melanoma cells B16-F10 and SK-MEL-28, and female C57BL/6 mice were used for the study. Our in-vitro data showed that significant synergistic effects were obtained when celecoxib was used together with various concentrations of DTIC. A study with B16-F10 cells using flow cytometry analysis showed that the drug combination induced significantly more apoptosis than each drug used individually. Our in-vivo results showed that the drug combination was much more effective than each drug used alone for the inhibition of both melanoma growth and metastasis in the B16-F10+C57BL/6 mouse models. For melanoma growth, the median survival rates for phosphate-buffered saline (PBS) (control), celecoxib (30 mg/kg), DTIC-1 (10 mg/kg), DTIC-2 (positive control, 50 mg/kg), and the drug combination (DTIC 10 mg/kg+celecoxib 30 mg/kg) were 6, 6.5, 7.5, 7.5, and 9 days, respectively. For melanoma metastasis, the average number of metastatic tumors in murine lungs was 53.7±10.7, 31.8±18.6, 21.2±21.7, 7.0±9.0, and 0.8±2.0 for PBS, DTIC-1, celecoxib, the drug combination, and DTIC-2. Our results warrant further investigation of the combination as an effective treatment for melanoma patients.

Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes.

Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H2O2), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases.

Evaluation of a dithiocarbamate derivative as an inhibitor of human glutaredoxin-1.

CONTEXT: Glutaredoxins (GRX) are involved in the regulation of thiol redox state. GRX-1 is a cytosolic enzyme responsible for the catalysis of deglutathionylation of proteins. To date, very few inhibitors of GRX-1 have been reported. OBJECTIVE: The objective of this paper is to report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethyl-sulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as an inhibitor of human GRX-1. MATERIALS AND METHODS: The mechanism of inhibition of GRX-1 was investigated using dialysis, substrate protection, and mass spectrometry. RESULTS: 2-AAPA inhibits GRX-1 in a time and concentration dependent manner. The activity did not return following dialysis indicating that inhibition is irreversible. Results of substrate protection and mass spectrometry indicate that the inhibition is occurring at the active site. The compound also produced GRX inhibition in human ovarian cancer cells. DISCUSSION: 2-AAPA is an irreversible GRX-1 inhibitor with similar or greater potency compared to previously reported inhibitors. CONCLUSION: The inhibition of GRX-1 by 2-AAPA could be used as a tool to study thiol redox state.

Microtubule S-glutathionylation as a potential approach for antimitotic agents.

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 G2/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 G2/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.

Design, synthesis, and biological evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine and its analogs as a novel class of anticancer agents.

N-Acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) was identified as a metabolite of sulofenur. Sulofenur was demonstrated to have broad activity against solid tumors in preclinical studies but exhibited disappointing clinical responses due to its high protein binding related adverse effects. NACC exhibited low protein binding and excellent activity against a sulofenur sensitive human colon cancer cell line. In this study, analogs of NACC were synthesized and evaluated with four human cancer cell lines. Two of the NACC analogs showed excellent activity against two human melanoma cell lines, while NACC remains the most potent of the series. All three compounds were more potent than dacarbazine, which is used extensively in treating melanoma. NACC was shown to induce apoptosis without affecting the cell cycle. Further, NACC exhibited low toxicity against monkey kidney cells. The selective anticancer activity, low toxicity, an unknown yet but unique anticancer mechanism and ready obtainability through synthesis make NACC and its analogs promising anticancer agents.

Increase in thiol oxidative stress via glutathione reductase inhibition as a novel approach to enhance cancer sensitivity to X-ray irradiation.

Depletion of the reduced form of glutathione (GSH) has been extensively studied for its effect on sensitizing cancer to radiation. However, little is known about the effects of thiol oxidative stress created through an increase in glutathione disulfide (GSSG) on cancer sensitivity to radiation. In this study, an increase in GSSG was effectively created using 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), an irreversible glutathione reductase (GR) inhibitor. Our results demonstrate that the GSSG increase significantly enhanced cancer sensitivity to X-ray irradiation in four human cancer cell lines (A431, MCF7, NCI-H226, and OVCAR-3). When cells were pretreated with 2-AAPA followed by X-ray irradiation, the IC(50) values for X-ray irradiation of A431, MCF7, NCI-H226, and OVCAR-3 cells were reduced, from 24.2 +/- 2.8, 42.5 +/- 3.0, 43.0 +/- 3.6, and 27.8+/-3.5 Gy to 6.75 +/- 0.9, 8.1 +/- 1.1, 6.75 +/- 1.0, and 12.1 +/- 1.7 Gy, respectively. The synergistic effects observed from the combination of X-rays plus 2-AAPA were comparable to those from the combination of X-rays plus buthionine sulfoximine, a reference compound known to increase cancer sensitivity to radiation. The synergistic effect was correlated with an increase in cell thiol oxidative stress, which was reflected by a five-to sixfold increase in GSSG and 25% increase in total disulfides. No change in GSH or total thiols was observed as a result of GR inhibition.

Effects of glutathione reductase inhibition on cellular thiol redox state and related systems.

Although inhibition of glutathione reductase (GR) has been demonstrated to cause a decrease in reduced glutathione (GSH) and increase in glutathione disulfide (GSSG), a systematic study of the effects of GR inhibition on thiol redox state and related systems has not been noted. By employing a monkey kidney cell line as the cell model and 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthio carbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a GR inhibitor, an investigation of the effects of GR inhibition on cellular thiol redox state and related systems was conducted. Our study demonstrated that, in addition to a decrease in GSH and increase in GSSG, 2-AAPA increased the ratios of NADH/NAD(+) and NADPH/NADP(+). Significant protein glutathionylation was observed. However, the inhibition did not affect the formation of reactive oxygen species or expression of antioxidant defense enzyme systems [GR, glutathione peroxidase, catalase, and superoxide dismutase] and enzymes involved in GSH biosynthesis [gamma-glutamylcysteine synthetase and glutathione synthetase].

Characterization of a novel dithiocarbamate glutathione reductase inhibitor and its use as a tool to modulate intracellular glutathione.

Thiol redox state (TRS) is an important parameter to reflect intracellular oxidative stress and is associated with various normal and abnormal biochemical processes. Agents that can be used to increase intracellular TRS will be valuable tools in TRS-related research. Glutathione reductase (GR) is a critical enzyme in the homeostasis of TRS. The enzyme catalyzes the reduction of GSSG to GSH to maintain a high GSH:GSSG ratio. Inhibition of the enzyme can be used to increase TRS. Despite the reports of various GR inhibitors, N,N-bis(2-chloroethyl)-N-nitrosourea, an anticancer drug with IC(50) = 647 microm against yeast GR, remains the most commonly used GR inhibitor in the literature. However, the toxicity caused by nonspecific interactions, as well as inhibition of DNA synthesis, complicates the use of N,N-bis(2-chloroethyl)-N-nitrosourea as a GR inhibitor. We report 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The K(i) and k(inact) of 2-AAPA against yeast GR were determined to be 56 microm and 0.1 min(-1), respectively. At the concentration that produced >80% yeast GR inhibition, 2-AAPA showed no inhibition against glutamylcysteine synthetase, glutathione synthetase, catalase, and superoxide dismutase, but minimal inhibition against glutathione S-transferase and glutathione peroxidase. In CV-1 cells, 2-AAPA (0.1 mm) produced 97% GR inhibition, 25% GSH reduction, and a 5-fold increase in GSSG in 20 min. The compound can be a useful tool in TRS-related research.

Determination of thiols and disulfides via HPLC quantification of 5-thio-2-nitrobenzoic acid.

This work presents an assay for total thiols and total disulfides in biological samples via HPLC quantification of 5-thio-2-nitrobenzoic acid (TNB) derived from the reaction of thiols with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent). This method also provides simultaneous quantification of glutathione (GSH) via the measurement of the GSH-DTNB adduct (GSH-TNB). By using 326nm as the detecting wavelength, the HPLC detection limit for TNB and the GSH-TNB adduct was determined to be 15 and 7.5pmol respectively. A recovery study with OVCAR-3 cells revealed that the recovery yields for TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides were 99.4+/-1.2% (n=3), 98.1+/-5.0% (n=3), 95.6+/-0.9% (n=3), and 96.6+/-2.3% (n=3) respectively. The recovery yield for GSH-TNB in the procedures for determining non-protein thiols, protein thiols, non-protein disulfides, and protein disulfides was 99.0+/-0.3% (n=3), 95.1+/-4.9% (n=3), 96.8+/-0.6% (n=3), and 95.1+/-2.9% (n=3) respectively. The reproducibility, expressed as the relative standard deviation for the analyte, for TNB was determined to be 2.8% (n=6) for non-protein thiols, 3.9% (n=6) for protein thiols, 3.6% (n=6) for non-protein disulfides and 4.6% (n=6) for protein disulfides. The reproducibility for GSH-TNB was determined to be 1.6% (n=6) for non-protein thiols and 2.6% (n=6) for non-protein disulfides. By comparing the amount of GSH determined in a biological sample before NaBH(4) reduction with that after the reduction, this method can provide information associated with thiol glutathionylation which would be useful for protein glutathionylation study. This method should be applicable to cellular, subcellular, protein, or other biomatrix samples for thiol and disulfide quantification and will be a useful analytical method in the study of thiol redox state and thiol glutathionylation.

2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)- phenylcarbamoylsulfanyl]propionic acid and its derivatives as a novel class of glutathione reductase inhibitors.

Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione to reduced glutathione. The enzyme is an attractive target for the development of antimalarial agents, agents to decrease malarial drug resistance and anticancer agents. In addition, inhibition of the enzyme has been employed as a tool in research for various purposes. In this paper, we present a rational design of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid and its derivatives as irreversible GR inhibitors. The K(i) and k(inact) values of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid, the most potent derivative of the series, are 88 muM and 0.1 min(-1), respectively. Although the K(i) value of the inhibitor is in the micromolar range, it is more potent than N,N-bis(2-chloroethyl)-N-nitrosourea, which is currently the most commonly employed irreversible GR inhibitor with a reported IC(50) value of 646 microM. Additional attractive features of the inhibitor include its ready availability through a one-step synthesis and good solubility in both organic and aqueous solutions.