Enzymatic and Nonenzymatic Synthesis of Glutathione Conjugates: Application to the Understanding of a Parasite's Defense System and Alternative to the Discovery of Potent Glutathione S-Transfer
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- 作者：Wei-Jen Lo ; Yu-Ching Chiou ; Yu-Ting Hsu ; Wing See Lam ; Ming-Yun Chang ; Shu-Chuan Jao ; Wen-Shan Li
- 刊名：Bioconjugate Chemistry
- 出版年：2007
- 出版时间：January 2007
- 年：2007
- 卷：18
- 期：1
- 页码：109 - 120
- 全文大小：354K
- 年卷期：v.18,no.1(January 2007)
- ISSN：1520-4812
文摘
A primary pathway for metabolism of electrophilic compounds in Schistosoma japonicum involves glutathioneS-transferase (SjGST)-catalyzed formation of glutathione (GSH) conjugates. As part of a program aimed at gaininga better understanding of the defense system of parasites, a series of aromatic halides (1-8), aliphatic halides (9,10), epoxides (11-20), 
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-unsaturated esters (21, 22), and ,-unsaturated amides (23, 24) were prepared, andtheir participation in glutathione conjugate formation was evaluated. Products from enzymatic and nonenzymaticreactions of these substances with glutathione were characterized and quantified by using reverse-phase high-performance liquid chromatography (HPLC), NMR, and fast atom bombardment mass spectrometry (FAB-MS)analysis. Mechanisms for formation of specific mono(glutathionyl) or bis(glutathionyl) conjugates are proposed.Although the results of this effort indicate that SjGST does not catalyze addition or substitution reactions of 1, 3,4, 7-9, 11-13, 15-17, 19-21, and 24, they demonstrate that 2, 5, 6, 14, 18, and 23 undergo efficient enzyme-catalyzed conjugation reactions. The kcat values for SjGST with 23 and 18 are about 886-fold and 14-fold,respectively, larger than that for 5. This observation suggests that 23 is a good substrate in comparison to otherelectrophiles. Furthermore, the initially formed conjugation product, 23a, is also a substrate for SjGST in a processthat forms the bis(glutathionyl) conjugate 23b. Products arising by enzymatic and nonenzymatic pathways aregenerated under the conditions of SjGST-activated GSH conjugation. Interestingly, production of nonenzymaticGSH conjugates with electrophilic substrates often overwhelms the activity of the enzyme. The nonenzymaticGSH conjugates, 9a-11a, 16a, 21a, and 22a, are inhibitors of SjGST with respective IC50 values of 1.95, 75.5,0.96, 19.0, 152, and 0.36 
M, and they display moderate inhibitory activities against human GSTA2. Directevidence has been gained for substrate inhibition by 10 toward SjGST and GSTA2 that is more potent than thatof its GSH conjugate 10a. The significance of this work is found in the development of a convenient NMR-basedtechnique that can be used to characterize glutathione conjugates derived from small molecule libraries as part ofefforts aimed at uncovering specific potent SjGST and GSTA2 inhibitors. This method has potential in applicationsto the identification of novel inhibitors of other GST targets that are of chemotherapeutic interest.
,-unsaturated esters (21, 22), and ,-unsaturated amides (23, 24) were prepared, andtheir participation in glutathione conjugate formation was evaluated. Products from enzymatic and nonenzymaticreactions of these substances with glutathione were characterized and quantified by using reverse-phase high-performance liquid chromatography (HPLC), NMR, and fast atom bombardment mass spectrometry (FAB-MS)analysis. Mechanisms for formation of specific mono(glutathionyl) or bis(glutathionyl) conjugates are proposed.Although the results of this effort indicate that SjGST does not catalyze addition or substitution reactions of 1, 3,4, 7-9, 11-13, 15-17, 19-21, and 24, they demonstrate that 2, 5, 6, 14, 18, and 23 undergo efficient enzyme-catalyzed conjugation reactions. The kcat values for SjGST with 23 and 18 are about 886-fold and 14-fold,respectively, larger than that for 5. This observation suggests that 23 is a good substrate in comparison to otherelectrophiles. Furthermore, the initially formed conjugation product, 23a, is also a substrate for SjGST in a processthat forms the bis(glutathionyl) conjugate 23b. Products arising by enzymatic and nonenzymatic pathways aregenerated under the conditions of SjGST-activated GSH conjugation. Interestingly, production of nonenzymaticGSH conjugates with electrophilic substrates often overwhelms the activity of the enzyme. The nonenzymaticGSH conjugates, 9a-11a, 16a, 21a, and 22a, are inhibitors of SjGST with respective IC50 values of 1.95, 75.5,0.96, 19.0, 152, and 0.36 M, and they display moderate inhibitory activities against human GSTA2. Directevidence has been gained for substrate inhibition by 10 toward SjGST and GSTA2 that is more potent than thatof its GSH conjugate 10a. The significance of this work is found in the development of a convenient NMR-basedtechnique that can be used to characterize glutathione conjugates derived from small molecule libraries as part ofefforts aimed at uncovering specific potent SjGST and GSTA2 inhibitors. This method has potential in applicationsto the identification of novel inhibitors of other GST targets that are of chemotherapeutic interest.