Glutathione Adducts of Helenalin and 11α,13-Dihydrohelenalin Acetate Inhibit Glutathione S-Transferase from Horse Liver¹

• Abstract
• Introduction
• Materials and Methods
• Chemicals
• Enzyme assay
• Assessment of inhibition kinetics by 2 b
• Glutathione reductase assay
• Glyoxalase I assay
• Results and Discussion
• References

Abstract

The 2-mono- and 2,13-bis-glutathionyl adducts of helenalin and the 2-monoglutathionyl adduct of 11α,13-dihydrohelenalin acetate were previously shown to be formed by spontaneous Michael addition at physiological pH. In living cells, glutathione (GSH) conjugation of many types of electrophilic agents is catalysed by a family of GSH S-transferase enzymes (GST). The capability of a glutathione S-transferase from horse liver to catalyze the reaction of helenalin and other helenanolides with GSH was investigated. The enzyme did not accelerate GSH conjugation of helenalin, 11α,13-dihydrohelenalin, or 2-deacetyl-6-deoxychamissonolide. The GSH-adducts, formed by spontaneous reaction, were found to be inhibitors of this enzyme. Free helenalin, a potent inhibitor of many enzymes containing free sulfhydryl groups, did not show any inhibitory activity on GST. It was thus demonstrated that GSH-adducts of sesquiterpene lactones possess their own specific biological activity. Two further enzymes using GSH as substrate, glutathione reductase and glyoxalase I, were not influenced by free helenalin or its GSH-adducts.

Introduction

Sesquiterpene lactones (STL) are known to exhibit a variety of conspicuous biological activities which, for the major part, are considered to arise from alkylation of free cysteine residues in vital cellular proteins via a Michael-type addition [1]. The question how these natural products may exert this mechanism of action in the presence of normally high concentrations of the low-molecular weight thiol glutathione (γ-glutamylcysteinylglycine, GSH) which should be able to deactivate STL before they reach any vital structures inside the cell, has recently been addressed. It was shown that the spontaneous reaction of helenalolide STL with GSH in aqueous solution proceeds at high rates at physiological pH [2]. A large fraction of STL will thus be transformed to GSH-adducts in a living cell, where GSH is present in considerable concentrations (0.5 - 10 mM [3]). It was, however, found that the reaction is reversible under these conditions [2], so that the fraction of free STL present in the equilibrium is capable of reacting with sulfhydryl groups of more vital protein targets such as enzymes [1] and transcription factors [4]. Living organisms possess an efficient means to detoxify electrophilic agents such as activated aromatic systems, epoxides and others, whose conjugation with the thiol group of GSH is catalysed by a group of GSH S-transferase (GST) enzymes (EC 2.5.1.18) [3], [5] [6] [7] [8]. It was therefore of interest to investigate whether the reaction of STL with GSH is catalysed, and thereby accelerated, by GST and, moreover, the influence of STL and STL-GSH adducts on the activity of GST.

Materials and Methods

Chemicals

For origin of STL, and synthesis and purification of 1 b, 1 c and 2 b see [2], [9]. All other mentioned chemicals and the enzymes were obtained from Sigma Chemicals.

Enzyme assay

The GST-assay was carried out according to [5] [6] [7]. Solutions of CDNB and GSH were prepared in buffer (0.1 M potassium phosphate, pH 6.5). Aliquots of each solution were mixed in 1 cm Quartz cuvettes to give the final specified concentration of each reactand in a final volume of 2.0 ml. A stock solution (0.1 mg protein/ml) of equine hepatic glutathione S-transferase (EC 2.5.1.18) was prepared in the same buffer and 10 μl, corresponding to 1 μg protein, were added to the 2 ml test solution.

Stock solutions of the inhibitors were prepared in water in such a way that the volume to be added in order to give the specified final concentrations was 10 μl. Inhibitors were added to the assay immediately before addition of the enzyme.

The increase in absorbance was monitored with a Beckman DB-G UV-Vis spectrophotometer at λ = 340 nm for 10 min and the instrument reading noted at intervals of 1 min. A solution of the same concentrations of the reactands without addition of enzyme was used for compensation of spontaneous “background reaction” which, however, in a separate control experiment was found to proceed only at an insignificant rate.

The data obtained at different inhibitor concentrations (c_i) were plotted vs. those obtained in the absence of the inhibitor to yield straight lines with slopes representing the relative rate of reaction (V_i/V_o). The reported IC₅₀ values were taken from plots of V_i/V_o vs. - log(c_i) (Fig. [1]). The IC₅₀ data represent arithmetic means from two independent series of experiments for each inhibitor.

Assessment of inhibition kinetics by 2 b

The assays were carried out with the specified concentrations of inhibitor (c_i = 0, 0.5, 1, and 10 μM) and GSH (0.125, 0.25, 0.5, 1 and 2 mM) at constant CDNB and enzyme concentration (1 mM and 0.5 μg/ml, respectively) and the absorption measured 5 min after enzyme addition (compensation: test solutions of the same reactand concentrations, without enzyme). Velocity (V = Δc/min) was calculated under the assumption that the rate of conversion at all substrate concentrations is approximately constant during this time, as had been the case in the time course experiments described above. To determine the type of inhibition and approximate values for K_i and K_i′, 1/V and c(GSH)/V were plotted versus c_i according to [10] (Fig. [3]).

Glutathione reductase assay

Reduction of 0.5 mM glutathione disulfide (GSSG) in the presence of 0.5 mM NADPH-tri-Na catalyzed by 6 μg bovine intestinal glutathione reductase (EC 1.6.4.2, type VII) at pH 7.4 (0.1 M phosphate buffer) was monitored by recording the change in absorption at λ = 340 nm for 20 min. Compounds 1 a, 1 b and 1 c were added at a concentration of 0.1 mM to test for inhibitory activity. No significant difference as compared with the rate of conversion in the absence of these compounds was observed.

Glyoxalase I assay

Conversion of 1 mM GSH and 5 mM methylglyoxal to S-lactoyl glutathione in the presence of ≈ 20 ng glyoxalase I (EC 4.4.1.5, origin: yeast) was monitored at pH 6.5 (0.1 M phosphate buffer) by recording the increase of absorption at λ = 240 nm for 15 min [11]. Addition of 1 a, 1 b and 1 c (0.1 mM) did not result in any detectable inhibition.

Results and Discussion

To the reaction mixtures (pH 6.5) containing 0.1 and 1 mM 11α ,13-dihydrohelenalin acetate (2 a) or 2-deacetyl-6-deoxychamissonolide (3) and 0.1 and 1 mM GSH, a commercially available GST from horse liver (EC 2.5.1.18) was added. The progress of the reaction was monitored by UV spectrophotometry as described previously [2]. Even at high enzyme concentrations (50 and 100 μg/ml ≈ 1 and 2 μM) no changes in the reaction rate as compared with the spontaneous reaction in the absence of GST were observed. Formation of STL-glutathione adducts thus appears to be solely dependent on spontaneous reaciton as described previously [2].

The possibility that STL, which are known to be potent inhibitors of many proteins possessing free sulfhydryl groups [1], [4], might have an inhibitory effect on GST was furthermore investigated. The GST-catalyzed reaction of a substrate used for standard testing of GST activity, 1-chloro-2,4-dinitrobenzene (CDNB) [5] [6] [7] [8], with GSH (1 mM each, enzyme concentration 0.5 μg/ml ≈ 10 nM) at pH 6.5 was inhibited to different extents by 1 a and 2 a and 3 at concentrations of 0.1 and 1 mM (data not shown). At this pH, spontaneous reaction of STL with GSH is known to occur in the solution [2] so that the question arose whether the fraction of free STL or the formed adducts were responsible for the enzyme inhibition. Addition of the isolated 2-mono- (1 b) and 2,13-bis-glutathionyl (1 c) adducts of helenalin (1 a) and of the 2-glutathionyl adduct (2 b) of 11α,13-dihydrohelenalin acetate (2 a), to the assay led to a strong and concentration-dependent inhibition of CDNB conjugation, indicating that the inhibition observed on addition of the free STL was actually due to their GSH-adducts. However, the adducts are known to co-exist in an equilibrium with the free STL at physiological pH [2], so that some free STL released from the adducts might be responsible for this effect. It has been demonstrated that adduct formation of STL at lower pH values occurs only at a drastically diminished rate as compared with the physiological pH range [2]. The effect of STL on the enzymatic conversion of CDNB to its glutathione-S-conjugate should therefore be much weaker at lower pH if the spontaneously formed adducts were responsible for enzyme inhibition, while in the case that the enzyme is inhibited by the free STL, it should be stronger than observed at higher pH. Enzyme catalysis of CDNB-GSH conjugation (conditions as above) was not inhibited by 100 μM 1 a at pH 5.3 during 10 min while a control experiment with 100 μM 1 b showed strong inhibition also at this pH. It could thus unambiguously be shown that the inhibitory effect on GST is caused by the GSH-adducts, and not by the free STL.[]

The concentration dependence of the inhibitory activity of 1 b, 1 c and 2 b on GST was investigated (Fig. [1]). IC₅₀ values were determined by measuring the inhibitory potency of the adducts at different concentrations. At substrate concentrations of c(GSH) = c(CDNB) = 1 mM and an enzyme concentration of 0.5 μg/ml ≈ 10 nM, IC₅₀ values of 15.6 ± 3.8 μM and 37.3 ± 9.3 μM were determined for 1 b and 1 c, respectively. Quite noteworthy, the IC₅₀ value found for 2 b was much lower at 1.09 ± 0.46 μM. Thus, 2 b can be considered a strong GST inhibitor on the scale applied by Mannervik and Danielson who considered as strong inhibitors compounds with IC₅₀ or K_i values ≤ 5 μM [8] [for a list of GST inhibitors, see [8]]. It appears quite straightforward to assume that the GSH-residue(s) of the STL-GSH-adducts give rise to these compounds' affinity to the GSH binding site in the enzyme's catalytic center. It has been demonstrated that GST are subject to product inhibition [6] and that S-alkyl- and arylglutathiones are inhibitors of the enzyme [8]. The inhibitory effect of the STL adducts can thus - although they are not themselves products of enzyme catalysis - be interpreted in such terms.

First experiments on inhibition kinetics were carried out with the strong inhibitor 2 b (0, 0.5, 1 and 10 μM) at various GSH concentrations (0.125, 0.25, 0.5, 1 and 2 mM) and constant CDNB (1 mM). The results (Figs. [2] and [3]) indicate that the inhibition with respect to GSH is non-competitive [mixed inhibition [10]]. K_i and K_i′ were found to lie between 0.5 and 1 μM the former being somewhat lower than the latter (Fig. [3]).

Inhibition by STL-GSH-adducts may be expected to occur also with other enzymes that use GSH as a substrate. The effect of free 1 a and of its GSH-adducts 1 b and 1 c on two further GSH-metabolizing enzymes, glutathione reductase (oxidized glutathione oxidoreductase, EC 1.6.4.2, type VII, origin: bovine intestine) and on glyoxalase I (EC 4.4.1.5, origin: yeast) was therefore investigated. Both enzymes' activities, however, were not influenced by either the free STL or by its adducts (10^-4 M) under the assay conditions (see Materials and Methods), so that effects of STL-GSH-adducts on further GSH-metabolizing enzymes remain to be disclosed.

The inhibitory activity of STL-GSH-adducts on hepatic glutathione S-transferase indicates that such compounds may increase the toxicity of other electrophilic substances whose detoxification requires GST activity. It is known that certain subtypes of hepatic GST account for the “nonselenium peroxidase activity” [3], [8], i.e., they catalyse the reduction of organic hydroperoxides formed in the course of lipid peroxidation, so that inhibition of these enzymes by STL conjugates may lead to increased oxidative stress. It will therefore be of interest to study distinctly the effect of STL-GSH-adducts on different subtypes of GST.

The toxicity of helenalin and further STL is known to be accompanied by a considerable depletion of cellular GSH concentration [12] [13] [14]. As a possible cause, Arrick and coworkers have shown an increased formation of hydrogen peroxide by enhanced autoxidation of free cysteine [14]. It is straightforward to assume a connection with an inhibitory activity on enzymes related to GSH metabolism and it is possible that GSH-adducts play a role in this respect. An inhibition of glutathione reductase as observed with the seco-eudesmanolide vernolepin [14], however, could not be found for helenalin and its GSH-adducts.

The results presented here indicate that not only the free STL, but also their GSH-adducts representing the major fraction of STL molecules under physiological conditions, may contribute to these natural products' toxic activity in vivo.

References

• 1 Picman  A K.. Biological activities of sesquiterpene lactones.  Biochem. Syst. Ecol.. 1986;;  14 255-281
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• 2 Schmidt  T J,, Lyß  G,, Pahl  H L,, Merfort  I.. Helenanolide type sesquiterpene lactones - V. The role of glutathione addition under physiological conditions.  Bioorg. & Med. Chem.. 2000;;  in press
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• 4 Lyß  G,, Knorre  A,, Schmidt  T J,, Pahl  H L,, Merfort  I.. The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-κB by directly targeting p 65.  J. Biol. Chem.. 1998;;  273 33508-33516
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• 5 Habig  W H,, Pabst  M J,, Jakoby  W B.. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation.  J. Biol. Chem.. 1974;;  249 7130-7139
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• 7 Simons  P C,, Vander Jagt  D L.. Purification of glutathione S-transferases from human liver by glutathione-affinity chromatography.  Anal. Biochem.. 1977;;  82 334-341
  CrossrefPubMedSearch in Google Scholar
• 8 Mannervik  B,, Danielson  U H.. Glutathione transferases - structure and catalytic activity.  Crit. Rev. Biochem.. 1988;;  23 283-337
  PubMedSearch in Google Scholar
• 9 Schmidt  T J.. Helenanolide type sesquiterpene lactones. III. Rates and stereochemistry in the reaction of helenalin and related helenanolides with sulfhydryl containing biomolecules.  Bioorg. & Med. Chem.. 1997;;  5 645-653
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• 10 Cornish-Bowden  A.. Fundamentals of Enzyme Kinetics. Butterworths, London; 1979
• 11 Vince  R,, Dalugue  S,, Wadd  W B.. Studies on the inhibition of glyoxalase I by S-substituted glutathiones.  J. Med. Chem.. 1971;;  14 35-37
  CrossrefPubMedSearch in Google Scholar
• 12 Merrill  J C,, Kim  H L,, Safe  S,, Murray  C A,, Hayes  M A.. Role of glutathione in the toxicity of the sesquiterpene lactones hymenoxon and helenalin.  J. Toxicol. Environ. Health. 1988;;  23 159-169
  PubMedSearch in Google Scholar
• 13 Arrick  B A,, Nathan  C F,, Cohn  Z A.. Inhibition of glutathione synthesis augments lysis of murine tumor cells by sulfhydryl-reactive antineoplastics.  J. Clin. Invest.. 1983;;  71 258-267
  PubMedSearch in Google Scholar
• 14 Arrick  B A,, Griffo  W,, Cohn  Z,, Nathan  C.. Hydrogen peroxide from cellular metabolism of cystine. A requirement for lysis of murine tumor cells by vernolepin, a glutathione-depleting antineoplastic.  J. Clin. Invest.. 1985;;  76 567-574
  PubMedSearch in Google Scholar

Dr. Thomas J. Schmidt

Institut für Pharmazeutische Biologie

der Heinrich-Heine-Universität Düsseldorf

Universitätsstraße 1, Geb. 26 23

40225 Düsseldorf

Germany

Email: schmidtt@uni-duesseldorf.de

Phone: +49-211-811-1923

Fax: +49-211-811-4179

References

• 1 Picman  A K.. Biological activities of sesquiterpene lactones.  Biochem. Syst. Ecol.. 1986;;  14 255-281
  PubMedSearch in Google Scholar
• 2 Schmidt  T J,, Lyß  G,, Pahl  H L,, Merfort  I.. Helenanolide type sesquiterpene lactones - V. The role of glutathione addition under physiological conditions.  Bioorg. & Med. Chem.. 2000;;  in press
  PubMedSearch in Google Scholar
• 3 Meister  A,, Anderson  M E.. Glutathione.  Ann. Rev. Biochem.. 1983;;  52 711-760
  PubMedSearch in Google Scholar
• 4 Lyß  G,, Knorre  A,, Schmidt  T J,, Pahl  H L,, Merfort  I.. The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-κB by directly targeting p 65.  J. Biol. Chem.. 1998;;  273 33508-33516
  CrossrefPubMedSearch in Google Scholar
• 5 Habig  W H,, Pabst  M J,, Jakoby  W B.. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation.  J. Biol. Chem.. 1974;;  249 7130-7139
  PubMedSearch in Google Scholar
• 6 Pabst  M J,, Habig  W H,, Jakoby  W B.. Glutathione S-transferase A. A novel kinetic mechanism in which the major reaction pathway depends on substrate concentration.  J. Biol. Chem.. 1974;;  249 7140-7148
  PubMedSearch in Google Scholar
• 7 Simons  P C,, Vander Jagt  D L.. Purification of glutathione S-transferases from human liver by glutathione-affinity chromatography.  Anal. Biochem.. 1977;;  82 334-341
  CrossrefPubMedSearch in Google Scholar
• 8 Mannervik  B,, Danielson  U H.. Glutathione transferases - structure and catalytic activity.  Crit. Rev. Biochem.. 1988;;  23 283-337
  PubMedSearch in Google Scholar
• 9 Schmidt  T J.. Helenanolide type sesquiterpene lactones. III. Rates and stereochemistry in the reaction of helenalin and related helenanolides with sulfhydryl containing biomolecules.  Bioorg. & Med. Chem.. 1997;;  5 645-653
  PubMedSearch in Google Scholar
• 10 Cornish-Bowden  A.. Fundamentals of Enzyme Kinetics. Butterworths, London; 1979
• 11 Vince  R,, Dalugue  S,, Wadd  W B.. Studies on the inhibition of glyoxalase I by S-substituted glutathiones.  J. Med. Chem.. 1971;;  14 35-37
  CrossrefPubMedSearch in Google Scholar
• 12 Merrill  J C,, Kim  H L,, Safe  S,, Murray  C A,, Hayes  M A.. Role of glutathione in the toxicity of the sesquiterpene lactones hymenoxon and helenalin.  J. Toxicol. Environ. Health. 1988;;  23 159-169
  PubMedSearch in Google Scholar
• 13 Arrick  B A,, Nathan  C F,, Cohn  Z A.. Inhibition of glutathione synthesis augments lysis of murine tumor cells by sulfhydryl-reactive antineoplastics.  J. Clin. Invest.. 1983;;  71 258-267
  PubMedSearch in Google Scholar
• 14 Arrick  B A,, Griffo  W,, Cohn  Z,, Nathan  C.. Hydrogen peroxide from cellular metabolism of cystine. A requirement for lysis of murine tumor cells by vernolepin, a glutathione-depleting antineoplastic.  J. Clin. Invest.. 1985;;  76 567-574
  PubMedSearch in Google Scholar

Dr. Thomas J. Schmidt

Institut für Pharmazeutische Biologie

der Heinrich-Heine-Universität Düsseldorf

Universitätsstraße 1, Geb. 26 23

40225 Düsseldorf

Germany

Email: schmidtt@uni-duesseldorf.de

Phone: +49-211-811-1923

Fax: +49-211-811-4179