The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue.

The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.

Effects of antioxidants on glutathione levels and clinical recovery from the malnutrition syndrome kwashiorkor--a pilot study.

Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many African countries, and repeatedly has been reported to be associated with oxidative stress. The therapy of kwashiorkor is still ineffective. In this pilot study, we tested the hypothesis that oral application of thiol-containing antioxidants increases glutathione status and is beneficial for the clinical recovery of kwashiorkor patients. The longitudinal clinical intervention study was carried out at St Joseph's Hospital, Jirapa, Ghana. Children with severe kwashiorkor were randomly assigned to either a standard treatment (ST) receiving a therapeutic protocol based on the recommendations of the WHO or to one of three study groups receiving in addition 2 x 600 mg reduced glutathione or 2 x 50 mg alpha-lipoic acid or 2 x 100 mg N-acetylcysteine per day. Patients were followed up clinically and biochemically for 20 days and compared with 37 healthy controls. Both glutathione and alpha-lipoic acid supplementation had positive effects on survival. Also, the blood glutathione concentrations correlated positively with survival rates. Furthermore, the initial skin lesions, glutathione and total protein concentrations were found to be strong predictors of survival. The data strongly suggest that a therapy restoring the antioxidative capacity by applying cysteine equivalents in the form of glutathione and/or alpha-lipoic acid is beneficial for biochemical and clinical recovery of kwashiorkor patients.

Human selenoproteins at a glance.

The public perception of selenium has changed significantly over the last decades. Originally mainly known for its high toxicity, it was later recognized as an essential trace element and is now (despite its narrow therapeutic window) almost being marketed as a lifestyle drug. Indeed, some clinical and preclinical studies suggest that selenium supplementation may be beneficial in a large number of clinical conditions. However, its mode of action is unresolved in most of these cases. Selenocysteine - identified as the 21st amino acid used in ribosome-mediated protein synthesis - is incorporated in at least 25 specific, genetically determined human selenoproteins, many of which have only recently been discovered. Restoration of normal selenoprotein levels may be - apart from direct supranutritional effects - one possible explanation for the effects of selenium supplements. In this review we provide a brief but up-to-date overview of what is currently known about these 25 acknowledged human selenoproteins and their synthesis.

Antioxidant status and nitric oxide in the malnutrition syndrome kwashiorkor.

The pathophysiology of kwashiorkor, a severe edematous manifestation of malnutrition, is still poorly understood. The syndrome is, however, known to be associated with alterations in redox metabolism. To further elucidate the role of oxidative stress in kwashiorkor, we carried out a longitudinal study on the major blood antioxidants at the St. Joseph's Hospital, Jirapa, Ghana. All kwashiorkor patients (K) were followed up for 20 d. In comparison with local healthy controls (C), the plasma total antioxidant status was reduced to less than 50% in the patients (C, 0.87 +/- 0.21 mM; K, 0.40 +/- 0.20 mM; p<0.001). Similarly, the major plasma antioxidant albumin (C, 40.9 +/- 2.5 g/L; K, 19.1 +/- 7.4 g/L; p < 0.001) and erythrocyte glutathione (C, 2.39 +/- 0.28 mM; K, 1.01 +/- 0.33; p < 0.001) were decreased, whereas the levels of bilirubin and uric acid were not significantly altered. Nitrite and nitrate were found to be increased by a factor of 2 in kwashiorkor (C, 120 +/- 46 microM; K, 235 +/- 107 microM; p < 0.001). Over the observation period, the trends of albumin and glutathione levels were related to clinical outcome. These concentrations rose in patients who recovered and fell in patients who did not. Our study strongly supports the hypothesis that oxidative and nitrosative stress play a role in the pathophysiology of edematous malnutrition. Prophylactic and therapeutic strategies should aim at the careful correction of the reduced antioxidant status of the patients.

Thioredoxin reductase as a pathophysiological factor and drug target.

Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2-based oxidative stress. Being a selenite-reducing enzyme as well as a selenol-containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti-rheumatic gold-containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nM. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjögren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti-tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance. TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria.

Synthesis of 5,5'-dithiobis(2-nitrobenzamides) as alternative substrates for trypanothione reductase and thioredoxin reductase: a microtiter colorimetric assay for inhibitor screening.

Trypanothione reductases (TR; EC 1.6.4.8) and thioredoxin reductases (TrxR; EC 1.6.4.5.) are enzymes central to cellular thiol metabolism. Trypanosoma cruzi TR (TcTR) is therefore considered as a potential candidate for drug design against trypanosomiasis. Inhibition of human TrxR (hTrxR) is likely to be beneficial in psoriasis, cancer, and autoimmune diseases, while inhibition of a putative TrxR from Plasmodium falciparum (PfTrxR) might prove effective against malaria. The natural substrates of the first two enzymes are very expensive and difficult to obtain; in the case of PfTrxR, the physiological substrate has not yet been identified. We have therefore synthesized and tested three different 5,5'-dithiobis(2-nitrobenzamides) as alternative substrates of the above enzymes. As with 5, 5'-dithiobis(2-nitrobenzoate) (DTNB), which can be reduced by TRs and TrxRs, the new compounds are converted to their corresponding chromophoric thiolates; however, they have much lower Km values and are therefore less likely to interfere with inhibitor testing. Using the new substrates, a novel enzyme assay has been developed which is identical for all three enzymes, can be performed in a microtiter plate, and is amenable to automation. Thus, the assay provides a versatile and inexpensive tool for kinetic studies and high-throughput inhibitor screening.

Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds.

Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 +/- 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of kcat was 3330 +/- 882 min-1, and the Km values were 18 microM for NADPH and 25 microM for Escherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the Ki was 4 nM when measured in the presence of 50 microM thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase.

The 58 kDa mouse selenoprotein is a BCNU-sensitive thioredoxin reductase.

The flavoprotein thioredoxin reductase [EC 1.6.4.5] (NADPH + H+ + thioredoxin-S2 --> NADP+ + thioredoxin-(SH)2) was isolated from mouse Ehrlich ascites tumour (EAT) cells. Like the counterpart from human placenta but unlike the known thioredoxin reductases from non-vertebrate organisms, the mouse enzyme was found to contain 1 equivalent of selenium per subunit of 58 kDa. The K(M) values were 4.5 microM for NADPH, 480 microM for DTNB and 36 microM for Escherichia coli thioredoxin, the turnover number with DTNB being approximately 40 s(-1). As mouse is a standard animal model in cancer and malaria research, thioredoxin reductase and glutathione reductase [EC 1.6.4.2] from EAT cells were compared with each other. While both enzymes in their 2-electron reduced form are targets of the cytostatic drug carmustine (BCNU), no immunologic cross-reactivity between the two mouse disulfide reductases was observed.

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli.

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric flavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M(r) approximately 55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M(r) approximately 35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M(r) of approximately 55,000 [Luthman, M. & Holmgren, A. (1982) Biochemistry 21, 6628-6633; Gasdaska, P. Y., Gasdaska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, 5-9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 6146-6151]. Thus, the evolution of this family is highly unusual. The mechanism of thioredoxin reductase from higher eukaryotes is not known. As reported here, thioredoxin reductase from human placenta reacts with only a single molecule of NADPH, which leads to a stable intermediate similar to that observed in titrations of lipoamide dehydrogenase or glutathione reductase. Titration of thioredoxin reductase from human placenta with dithionite takes place in two spectral phases: formation of a thiolate-flavin charge transfer complex followed by reduction of the flavin, just as with lipoamide dehydrogenase or glutathione reductase. The first phase requires more than one equivalent of dithionite. This suggests that the penultimate selenocysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. Acad. Sci. USA 93, 1006-1011] is in redox communication with the active site disulfide/dithiol. Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.