J. Med. Chem. 1996, 39, 1549-1554
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Inhibition of Human Glutathione Reductase by 10-Arylisoalloxazines: Crystallographic, Kinetic, and Electrochemical Studies Annette Scho¨nleben-Janas,† Peer Kirsch,‡ Peer R. E. Mittl,§ R. Heiner Schirmer,† and R. Luise Krauth-Siegel*,† Institut fu¨ r Biochemie II der Universita¨ t, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany, Abteilung Organische Chemie, Max-Planck-Institut fu¨ r Medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany, and Institut fu¨ r Organische Chemie und Biochemie der Universita¨ t, Albertstrasse 21, D-79104 Freiburg, Germany Received July 13, 1995X
A series of newly synthesized N10-arylisoalloxazinesssome of which are known to be antimalarial agentsswere studied as inhibitors of human glutathione reductase (GR; NADPH + GSSG + H+ h NADP+ + 2GSH). The flavoenzyme was inhibited with IC50 values between e1 and 100 µM in the presence of 100 µM GSSG and 100 µM NADPH. The isoalloxazines and N3methylisoalloxazines with a 4′-chlorophenyl or a 3′,5′-dichlorophenyl group at N10 were found to be the most promising inhibitors of GR, although even the bulkier 10-naphthyl and -anthryl derivatives were also effective inhibitors. In contrast, at position N3 of the isoalloxazine ring, the size of the substituent was found to strongly influence the inhibitory effect. Introduction of a carboxymethyl group at N3swhich markedly increased the solubility of the derivative in aqueous solutionsscaused a rise in the IC50 values by 1 order of magnitude. 8-Fluoro- and 8-azido-10-arylisoalloxazines were potent inhibitors of GR; consequently position C8 of the benzenoid subnucleus instead of N3 should be considered for introducing substituents. No correlation was observed between the inhibitory strength of several isoalloxazines and their redox potential as measured by cyclovoltammetry. The crystallographic analysis of GR complexed with 10-(4′-chlorophenyl)-3-(carboxymethyl)isoalloxazine and 10-(3′,5′-dichlorophenyl)-3-(carboxymethyl)isoalloxazine, respectively, revealed the presence of one inhibitor molecule bound at the 2-fold axis of the homodimeric protein. This location is consistent with fluorescence titration measurements and enzyme kinetic studies in solution which gave no indication for binding at the substrate sites. Introduction Glutathione reductase (GR, EC 1.6.4.2) is a key enzyme in the cell’s defense mechanisms against oxidative stress. The flavoprotein is responsible for the reducing intracellular milieu by catalyzing the reaction: NADPH + GSSG + H+ h NADP+ + 2GSH.1 From a pharmacological viewpoint, human GR is an attractive target of antimalarial and antitumor drugs.2,3 All requirements for a rational drug development are fulfilled. The three-dimensional structures of human GR4 as well as of complexes with its substrates and several inhibitors have been solved by X-ray diffraction analysis (see ref 3 for a review). In addition, a bacterial strain that overexpresses the recombinant enzyme is available.5 10-(Substituted phenyl)flavins were shown to possess antimalarial activity both in vivo against rodent malaria and in vitro against the human pathogen Plasmodium falciparum.6 Among the most potent compounds, the 10-(4′-chlorophenyl) and 10-[3′-(trifluoromethyl)phenyl] derivatives turned out to be effective inhibitors of GR.7 Here we describe a series of new 10-arylisoalloxazines studied as inhibitors of GR. The poor water solubility of the derivatives was markedly improved by introducing a carboxymethyl group at position N3.8 Molecular interactions between the isoalloxazine derivatives and GR were studied by three different approaches: (i) * Corresponding author. † Institut fu ¨ r Biochemie II der Universita¨t Heidelberg. ‡ Max-Planck-Institut fu ¨ r Medizinische Forschung. § Institut fu ¨ r Organische Chemie und Biochemie der Universita¨t Freiburg. X Abstract published in Advance ACS Abstracts, March 1, 1996.
0022-2623/96/1839-1549$12.00/0
inhibitor constants were determined by enzyme kinetics in solution, (ii) binding to wild-type GR and to an active site Tyr mutant9 were followed by fluorescence measurements, and (iii) X-ray diffraction analysis of crystalline enzyme-inhibitor complexes showed the localization of the inhibitor in the protein structure. Results and Discussion Inhibition of Human GR by Isoalloxazines. Twenty-seven 10-arylisoalloxazines which had been synthesized and characterized as described in ref 8 were studied as inhibitors of human GR. The flavin analogues interact reversibly with the enzyme: inhibition is not time-dependent, and full activity is regained upon dilution or dialysis against an inhibitor-free solution. In order to compare the inhibitory strength among the isoalloxazines, enzyme assays were carried out at physiologic substrate concentrations (100 µM GSSG and 100 µM NADPH) while the inhibitor concentration was varied between 0.5 and 150 µM. The compounds studied can be subdivided on the basis of their substitution pattern (Figure 1). The first group (2a-e) possesses an N3-unsubstituted isoalloxazine ring, the second group (3a-e, 18-20) is N3methylisoalloxazines, the third one (4a-d) is 3-[(ethoxycarbonyl)methyl]isoalloxazines, and the fourth group (5a-d) comprises 3-(carboxymethyl)isoalloxazines (Table 1a). Within each group, the 4′-chloro-, 3′,5′-dichloro-, and 3′-(trifluoromethyl)phenyl derivatives were highly effective inhibitors of human GR. There was no appreciable difference between isoalloxazines 2a-d and 3-methylisoalloxazines 3a-d. The latter compounds are known to be potent antimalarial agents in Plasmo© 1996 American Chemical Society
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Journal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Notes Table 2. Electrochemical Dataa a. 10-Aryl-3-methylisoalloxazines 3a,e, 18, and 19, 10-Aryl-3-(carboxymethyl)isoalloxazines 5a-d, and 10-(4′-Chlorophenyl)-8-fluoro-3-methylisoalloxazine 14
Figure 1. Structures of N10-(halophenyl)-3-substituted isoalloxazines: R ) H in 2a-e, R ) CH3 in 3a-e, R ) (ethoxycarbonyl)methyl in 4a-d, R ) carboxymethyl in 5a-d, X ) 4′-Cl in 2a, 3a, 4a, and 5a, X ) 3′,5′-Cl2 in 2b, 3b, 4b, and 5b, X ) 3′-CF3 in 2c, 3c, 4c, and 5c, X ) 3′,5′-(CF3)2 in 2d, 3d, 4d, and 5d, and X ) 2′,3′,4′,5′,6′-F5 in 2e and 3e.
compd
X substituent
18 3a 3e 5a 5b 5c 5d 14 19
H 4′-Cl F5 4′-Cl 3′,5′-Cl2 3′-CF3 3′,5′-(CF3)2 4′-Cl, 8-fluoro 10-(1-naphthyl)
Table 1. Inhibition of Human GRa
IC50 (µM)
X substituent
2a 2b 2c 2d 2e
10-Phenylisoalloxazines 4′-Cl 3′,5′-Cl2 3′-CF3 3′,5′-(CF3)2 F5
