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Substrates for the Enzyme Trypanothione Disulfide Reductase

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Substrates for the Enzyme Trypanothione Disulfide Reductase Proc. Nati. Acad. Sci. USA Vol. 85, pp. 5374-5378, August 1988 Biochemistry "Subversive" substrates for the enzyme trypanothione disulfide reductase: Alternative approach to chemotherapy of Chagas disease (Trypanosoma cruzi/leishmaniasis/naphthoquinone/nitrofuran) GRAEME B. HENDERSON*t, PETER ULRICH*, ALAN H. FAIRLAMBt, IAN ROSENBERG§, MIERCIO PEREIRA§, MICHAEL SELA¶1, AND ANTHONY CERAMI* *Laboratory of Medical Biochemistry, The Rockefeller University, New York, NY 10021; *Department of Medical Protozoology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom; §Department of Medicine, New England Medical Center Hospitals, Boston, MA 02111; and $Weizmann Institute of Science, Rehovot, Israel 76100 Contributed by Michael Sela, March 2, 1988 ABSTRACT The trypanosomatid flavoprotein disulfide unusual NADPH-dependent flavoprotein disulfide reductase reductase, trypanothione reductase, is shown to catalyze one- (trypanothione reductase) (8), which maintains trypanothione electron reduction of suitably substituted naphthoquinone and in the dithiol form [Try(SH)2] within the cell. In addition, nitrofuran derivatives. A number of such compounds have trypanosomatids also possess trypanothione-dependent per- been chemically synthesized, and a structure-activity relation- oxidase activity (9, 10). Given that the antioxidant defenses of ship has been established; the enzyme is most active with trypanosomatids are based upon trypanothione, inhibition of compounds that contain basic functional groups in side-chain trypanothione reductase or subversion of its antioxidant role residues. The reduced products are readily reoxidized by within the cell represents an attractive target for the design of molecular oxygen and thus undergo classical enzyme-catalyzed drugs to treat trypanosomatid infections. redox cycling. In addition to their ability to act as substrates for Trypanothione disulfide reductase has been purified from trypanothione reductase, the compounds are also shown to Crithidia fasciculata and T. cruzi (8, 11) and found to have effectively inhibit enzymatic reduction of the enzyme's physi- similar physical and chemical properties to human gluta- ological substrate, trypanothione disulfide. Under aerobic thione reductase [NAD(P)H:oxidized-glutathione oxidore- conditions, trypanothione reductase is not inactivated by these redox-cycling substrates, whereas under anaerobic conditions ductase, EC 1.6.4.2]. In fact, trypanothione and glutathione the nitrofuran compounds cause irreversible inactivation ofthe reductases appear to reduce their respective physiological enzyme. When tested for biological activity against Trypano- disulfide substrates by the same catalytic mechanism. The soma cruzi trypomastigotes, many of the test compounds were parasite and human enzymes do, however, differ in their trypanocidal, and this activity correlated with their relative respective substrate specificities; glutathione disulfide is not ability to act as substrates for trypanothione reductase. The a substrate or inhibitor of trypanothione reductase, and, activity of the enzyme with these redox-cycling derivatives conversely, trypanothione disulfide [Try(S)2] is not a sub- constitutes a subversion of its normal antioxidant role within strate for glutathione reductase. This mutually exclusive the cell. For this reason these compounds may be termed substrate specificity is of key importance in any approach "subversive" substrates for trypanothione reductase. toward the selective inhibition oftrypanothione reductase. In a study on the substrate specificity of trypanothione reduc- There is great need for new and less toxic treatments for tase (12), the enzyme reduced various analogues ofTry(S)2 in human tropical diseases by parasitic trypanosomes [African which the spermidine moiety had been replaced by an sleeping sickness and Chagas disease (South American try- aliphatic side chain that contained at least one amine func- panosomiasis)] and leishmanias (oriental sore and kala-azar). tion. The activity of the enzyme with these analogues most In the case of Chagas disease, which is caused by Trypano- closely reflected the relative ability ofthe compounds to bind soma cruzi, no generally effective chemotherapy currently in the active site and suggested that trypanothione reductase exists for the millions of infected people (1). might possess a binding site for the spermidine moiety of Comparative studies on the metabolism of trypanosoma- Try(S)2. This work led us to propose that suitably substituted tids and their mammalian hosts have pointed to a number of analogues of Try(S)2 might take advantage of this aspect of biochemical differences that might be exploited as targets for the enzyme-substrate interaction to access the catalytic chemotherapy. We have been investigating the biochemical center of trypanothione reductase. basis for the well-documented (2, 3) sensitivity of parasitic Having identified trypanothione reductase as a potential protozoa towards reagents that promote free radical damage target for chemotherapeutic intervention, we developed a in cells. This work has led to the discovery that trypanoso- strategy in which compounds are designed, which effectively matids possess highly unusual antioxidant defense mecha- subvert the physiological function of this enzyme. These nisms (4) based upon the glutathione-spermidine conjugate compounds take advantage of the ability of trypanothione N1,N8-bis(glutathionyl)spermidine, which has been given reductase to catalyze reduction of substances that can un- the trivial name trypanothione (5). dergo redox-cycling processes to produce toxic metabolites In most aerobic organisms, the tripeptide glutathione and of oxygen that can kill T. cruzi trypomastigotes. the glutathione reductase/glutathione peroxidase enzyme In this communication we report the preparation of several couple have key roles in the antioxidant defense process (6). model compounds that appear to be trypanocidal by their In contrast, all species of trypanosomatids examined to date ability to act as redox-cycling or "subversive" substrates for lack classical glutathione reductase and glutathione-dependent trypanothione reductase. peroxidase activities (7). Trypanosomatids possess instead an Abbreviations: Try(SH)2 and Try(S)2, the dithiol and disulfide forms The publication costs of this article were defrayed in part by page charge of trypanothione, respectively; HSVSM, human saphenous vein payment. This article must therefore be hereby marked "advertisement" smooth muscle. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5374 Downloaded by guest on October 1, 2021 Biochemistry: Henderson et al. Proc. Natl. Acad. Sci. USA 85 (1988) 5375 MATERIALS AND METHODS tions used in the assays were treated in this manner. Addi- tions to the cuvette were made with a microsyringe piercing Materials. All reagents and chemicals were of the highest the septum. grade commercially available. Try(S)2 was chemically synthe- Interaction of Trypanothione Reductase with Nitrofuran sized (5), and trypanothione disulfide reductase was purified VIII Under Aerobic and Anaerobic Conditions. Assays were from C. fasciculata as described previously (8). Nifurtimox done in 0.1 M Hepes (pH 7.8) containing 1 mM EDTA, 0.15 and nitrofurazone were obtained from Bayer (Leverkusen, mM NADPH, trypanothione reductase at 500 pmol/ml, and F.R.G.) and Aldrich, respectively. Other compounds (Fig. 1) 10 ,uM nitrofuran VIII. Reactions were started by addition of were chemically synthesized. Details of chemical synthesis enzyme (80 ,1 to a 2-ml assay). Sequential spectra (200-800 (too lengthy for inclusion) are available from the authors on nm) were collected at 20-sec intervals using a Hewlett- request. Packard HP8450A UV/Vis spectrophotometer. At the end of Enzyme Assays and Kinetic Analysis. Trypanothione disul- the experiment, the assay mixtures were dialyzed, and fide reductase activity was assayed spectrophotometrically trypanothione reductase was assayed using 250 gM Try(S)2. by monitoring substrate-dependent oxidation of NADPH at Parasite Cell Culture. Human saphenous vein smooth 340 nm. Alternatively, enzyme activity was monitored by muscle (HSVSM) cells were isolated from outgrowths of to cytochrome c reduction and coupling radical formation explants of unused portions of veins harvested for coronary measuring absorbance changes (E550 = 21 mM-1). Absorb- artery bypass surgery as described (13). The cells were ance changes were monitored on a Varian Cary 219 spectro- Cam- photometer with a thermostated cuvette chamber. Enzyme maintained in 24-well tissue culture plates (Costar, concentration was measured spectrophotometrically using bridge, MA) in a solution of Dulbecco's modified Eagle's medium (DMEM) containing 5.5 mM glucose, 25 mM Hepes, the extinction coefficient E4M = 11.3 mM- (8). Kinetic runs were done at 27°C in 0.1 M 4-(2-hydroxyethyl)-1-piper- and 10% fetal bovine serum (HyClone, Logan, UT). T. cruzi azineethane-sulfonic acid (Hepes) buffer (pH 7.8) containing strain Y trypomastigotes were propagated as described (14), 0.1 mM EDTA and 0.25 mM NADPH. Anaerobic measure- except that HSVSM cells were used in place of bovine aortic ments were performed in rubber-stoppered cuvettes that smooth muscle cells. were flushed repeatedly with helium. All buffers and solu- Trypanocidal Assays. Test compounds were dissolved in 0.3 ml of dimethyl sulfoxide and brought to volume by o 0 dilution
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