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 IC50 = 647 μm 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)phenylthiocarbamoylsulfanylpropionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The Ki and kinact of 2-AAPA against yeast GR were determined to be 56 μm 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. 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 IC50 = 647 μm 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)phenylthiocarbamoylsulfanylpropionic acid (2-AAPA) as a novel irreversible GR inhibitor. 2-AAPA was prepared by one-step synthesis from commercially available reagents. The Ki and kinact of 2-AAPA against yeast GR were determined to be 56 μm 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. Thiol redox state (TRS) 2The abbreviations used are: TRS, thiol redox state; GR, glutathione reductase; BCNU, N,N-bis(2-chloroethyl)-N-nitrosourea; 2-AAPA, 2-acetylamino-3-4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanylpropionic acid; HPLC, high pressure liquid chromatography; LC/MS, liquid chromatography/mass spectrometry; MS/MS, tandem mass spectrometry; GST, glutathione S-transferase; GP, glutathione peroxidase; GCS, γ-glutamylcysteine synthetase; GS, glutathione synthetase; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); SOD, superoxide dismutase. has been found to be associated with various essential biochemical processes, such as regulation of protein function, stabilization of protein structures, protection of proteins against irreversible oxidation of critical cysteine residues, and regulation of enzyme functions and transcription (1Biswas S. Chida A.S. Rahman I. Biochem. Pharmacol... 2006; 71: 551-564Google Scholar, 2Ghezzi P. Biochem. Soc. 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However, the toxicity caused by nonspecific interactions, as well as the inhibition of DNA synthesis by BCNU, complicates the use of the compound as a GR inhibitor C. Biochem. Pharmacol... 1991; Scholar, M.A. A. Res... 40: Scholar). We have reported the synthesis, and of a novel of irreversible GR inhibitors T. C. G. J. L. J. Med. Chem... 2005; Scholar). this acid 1) (Fig. was determined to be the most inhibitor with and kinact of and 0.1 min–1, against yeast GR T. C. G. J. L. J. Med. Chem... 2005; Scholar). In an IC50 of μm the However, in to create intracellular thiol oxidative stress, the compound to intracellular GR to its to the cell from this In this 2-acetylamino-3-4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanylpropionic acid a of compound as a novel and irreversible GR inhibitor (Fig. The Ki and kinact of 2-AAPA against yeast GR were with of compound 2-AAPA can intracellular GR and thiol oxidative stress. is that the compound can be prepared by one-step synthesis from commercially available reagents. 2-AAPA can be a useful research tool in intracellular thiol oxidative stress. The synthesis and of the compound as a GR as well as its the intracellular ratio of GSH to GSSG, are of of in was to a of in at with (Fig. was the and the was to for The was in and the was to with The was a with acid and with acid The was by and the was in The the were and 2-AAPA was as a of in The of the was determined high pressure liquid a with a The employed an with a of and the was at was by a of in an of acid in and a of in an of acid in min. The compound was determined to be by was by and mass were with a The high mass was a mass = = and and The of the was not for and GR were as T. C. G. J. L. J. Med. Chem... 2005; Scholar). The and NADPH The was by the of GSSG GR was by the of of NADPH determined at = and of GR was at with 2-AAPA at and in the of and NADPH were and for GR at and 20 as for the GR in the of the inhibitor was as a of of yeast GR by 2-AAPA mm) at by in 0.1 in a with a mass of were at and the GR was determined as for the GR GSSG GR was with 2-AAPA (0.1 mm) at in the or of GSSG mm) in (0.1 were and the GR was determined as for the GR GR GR was with 2-AAPA (0.1 mm) in the or of NADPH were and the GR was determined as for the GR of 2-AAPA with or (0.1 mm) was with mm) or cysteine mm) in (0.1 at were to the 2-AAPA by of chromatography/mass was in the Redox mass in the of at the of at The of a tandem mass to an yeast GR NADPH and 2-AAPA (0.1 mm) in (0.1 was at for At the of of the was a 20 mm) with and with of acid in at a of to the The was a and a from to in at a of = acid in = acid in The from was the mass and a The mass was with a source in a with a mass of mass The was to with a source of a of and a of V. The were and and the of proteins were from the protein in in the of 2-AAPA was also in of 2-AAPA Glutathione and Glutathione of 2-AAPA glutathione S-transferase and glutathione were determined by 2-AAPA (0.1 mm) with or at for min. The enzyme was determined from of the of 2-AAPA GSH 2-AAPA GSH the of 2-AAPA γ-glutamylcysteine and glutathione involved in GSH were determined a reported G. M. C. J. 1990; Scholar) with The the of and in cell by the of glutamylcysteine and GSH, the of and GS, respectively. Because CV-1 is a cell that is in and that GSH and glutamylcysteine N. Biochem. Pharmacol... 1990; 40: Scholar), to use cells, a cancer cell of used in this for the cells were an in mm) with The was at for min. The was and a with a mass of to the protein The protein was and for the of GSH and glutamylcysteine by a of GSH and glutamylcysteine was The protein was with 2-AAPA (0.1 mm) at for min. (0.1 and mm) was the cysteine mm) and acid mm) were to the of glutamylcysteine mm) and mm) were The at for min. The were with 5,5′-dithiobis(2-nitrobenzoic mm) for at B. C. D.P. J. Scholar), by the of acid as the protein was by the of and GSH in the were by was a by a with and with a a with a and a The was at The employed an with and was from to in and to in and at for an min. were The was The was glutamylcysteine and GSH were as the and respectively. was with GSH and of the of 2-AAPA the and the of 2-AAPA 2-AAPA (0.1 mm) was with liver for at The was determined a from The superoxide was determined by 0.1 2-AAPA with Chemical, for at The was determined to the of GR Inhibition in CV-1 growing CV-1 cells were in a in and in a at for for At the of the cells in the were with 2-AAPA (0.1 mm) in a at for 20 min. The was and the cells were with and by The and the cell were and at for min. The was with of with in mm) and with an The was at for 20 at The was and of the was used to GR as for the GR was determined by the of Anal. Biochem... Scholar). of GSH and GSSG in CV-1 of GSH and GSSG in the a reported B. C. D.P. J. Scholar) with The cells were with 2-AAPA in a and as The cell was with and in of The cell was a with a The was with as an and by with The was at for to the of GSH by the was by the of by to The was and of the was to was a mass of GSH and GSSG was of the and of a from the of enzyme at various of The that the enzyme its a of irreversible enzyme The Ki and kinact of 2-AAPA were determined to be 56 μm and 0.1 min–1, the of and R. J. Biol. Chem... Scholar), which the of of inhibition of Fig. against the of inhibitor concentration (Fig. of Ki and kinact the of and R. J. Biol. Chem... Scholar). is a of the of inhibition of Fig. the of inhibitor concentration The Ki and kinact were determined to be 56 μm and 0.1 min–1, the = of that the inhibition was of GR was The enzyme was by 2-AAPA The GR was extensively of the GR by but no of enzyme was (Fig. GSSG of GR against the inhibition at or the were in the and of the 2-AAPA was with GR in the of GSSG, the of 2-AAPA was At GSSG GR from inhibition by 2-AAPA, indicating that the and inhibitor were for the site GR (Fig. of the of NADPH GR the GSSG reduction, NADPH the disulfide and of yeast GR to L.D. Williams 39: Scholar). The GSSG to the at the active site are for the inhibition, in the and of NADPH were Fig. that no inhibition was in the of indicating that the at the active site are involved in the enzyme of 2-AAPA with and 2-AAPA with an or 2-AAPA (0.1 mm) was with mm) in (0.1 at was and 2-AAPA a the other 2-AAPA the with cysteine in min. of was employed to the GR and GR is a homodimeric of However, no was in the The that were to GR and (Fig. was with an report to of yeast GR by in which the to a was and no was M. F. Sci... 44: Scholar). of the mass the and from the that they by an of to a of and Upon the of yeast GR, it was found that there is a at the J. Scholar). that a of amino acids enzyme is not an In the of yeast GR also a of amino acid from the J. Scholar). Therefore, the with in the was from a of amino acid from the as in the yeast GR for the is as GR in of and to GR with and of the GR and GR in a Because a high concentration of GR was for the the of GR used in the for was that in the The 2-AAPA concentration employed was the concentration as that in the The enzyme was in min. However, no GR was by to of the enzyme in the of a high concentration of 2-AAPA Therefore, a concentration of 2-AAPA (0.1 mm) was this 2-AAPA produced GR The of the of the from the are in Fig. with GR inhibition, the to the and were In were and to the and that the of 2-AAPA and the enzyme is for and In other the in the enzyme and the inhibitor 1). the and for the and GR is also with an report that GR was not the yeast GR was with an M. F. Sci... 44: Scholar). of of Inhibition by and were to the of 2-AAPA, as these are involved in glutathione At a 2-AAPA concentration of 0.1 a reduction in was with Inhibition of = was for GP, and inhibition of = was determined for In the for inhibition of GSH by 2-AAPA was determined by the of and in cell with 0.1 The by these glutamylcysteine for and GSH for GS, were by Fig. a from a inhibition was with the glutamylcysteine in the was μm μm = in the the GSH was and μm = in the and respectively. Because 2-AAPA can be used as a tool to oxidative stress, it is important to the GR inhibitor has the other the of 2-AAPA the of other the of and was 2-AAPA at a concentration of 0.1 showed no inhibition of or The was determined to be 0.1 and = in the and respectively. SOD, the enzyme was and = in the and respectively. GR Inhibition and in GSH and GSSG by the 2-AAPA can intracellular GR and modulate intracellular GSH and GSSG, the compound (0.1 mm) was with CV-1 a the GR in the cells was of the indicating that 0.1 2-AAPA the GR in CV-1 of GSH and GSSG by that the inhibition to a 25% decrease in GSH and a 5-fold increase in increase in GSSG and a decrease in GSH an increase in TRS by GR inhibition of GR inhibition, GSH, and GSSG in CV-1 (0.1 0.1 in a GR inhibitors can be valuable tools in TRS-related research and also anticancer and antimalarial agents. We have 2-AAPA as a novel GR inhibitor that is of intracellular GR inhibition and a state of thiol oxidative stress as by a decrease in the ratio of GSH to The compound a and inhibition of an irreversible enzyme inhibitor. The inhibition was by the of the GSSG, indicating that the inhibition at or the The inhibition was also of NADPH the inhibition, that the reduction of the disulfide and to at the active site was for the The inhibition has been used as evidence for the of a an irreversible GR inhibitor and the at the active site M. Keese M.A. Becker K. Busse R. Mülsch A. J. Biol. Chem... 1997; 272: 21767-21773Google Scholar, C. Biochem. Pharmacol... 1991; Scholar, H. S. A. I. K. Krauth-Siegel R.L. J. Med. Chem... 1999; Scholar). inhibition of 2-AAPA are to of its compound T. C. G. J. L. J. Med. Chem... 2005; Scholar), and are also with most other irreversible GR inhibitors (22Schirmer R.H. Krauth-Siegel R.L. Dolphin D. Poulson R. Avramovic O. Glutathione: Chemical, Biochemical, and Medical Aspects. III. John Wiley & Sons, Inc, New York1989: 553-596Google Scholar). Therefore, that 2-AAPA a mechanism as for compound the irreversible inhibition is of the at the active The mechanism was by the from the of the that of 2-AAPA with of GR and that the inhibitor the enzyme or the evidence that the at the active site are for the and that the inhibition at the active site and the of the enzyme and the the most has with of the The is which functional of the enzyme the with the inhibitor. Because are from the of the enzyme with the inhibitor the other functional to be in the of the thiol functional that is to be the is the thiol at the active site because no other in yeast GR are to with the inhibitor to yeast GR the that other such as and not react with these an was by 2-AAPA with in functional groups and as a for the that 2-AAPA not react with a it its with cysteine in min. Therefore, of evidence are with the mechanism that 2-AAPA and the at the active to be that no are to other the at the active site in yeast GR, there are that are to other in GR of other species, such as GR, which cysteine at the The and an to the of GR in the is that the 2-AAPA and the which is a in the enzyme have to the the in the enzyme was to be more to The nature of the was in an to the of was found that the were the of 2-AAPA inhibition is these a mechanism for GR inhibition by 2-AAPA is in Fig. GR is a critical enzyme for maintaining a high ratio of GSH to Inhibition of the enzyme to of GSSG and an increase in intracellular thiol oxidative stress. Therefore, a GR inhibitor will be a valuable research tool in thiol oxidative stress to TRS-related normal and abnormal biochemical processes. various has been the most commonly used GR inhibitor in the literature. the and of the use of as a GR inhibitor. 2-AAPA is more and in intracellular GR and The compound showed minimal inhibition of and GP, indicating that the compound is Additionally, no inhibition of the involved in GSH synthesis or other was of 2-AAPA as a research tool its a one-step from commercially available and in and The compound is employed in this to the of GR inhibition intracellular thiol status as well the also that 2-AAPA can GR in and are to the TRS in