Mechanistic Studies on a Novel, Highly Potent Gold-Phosphole Inhibitor of Human Glutathione Reductase

David P. Ballou,Sabine Urig,Markus Meyer,Régis Réau,Marcel Deponte,L. David Arscott,Katja Becker,Karin Fritz-Wolf,Charles H. Williams,Elisabeth Davioud-Charvet,Christel Herold-Mende,Sasa Koncarevic

doi:10.1074/jbc.m412519200

Abstract

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols. In this work, the inhibition of human GR by a novel gold-phosphole inhibitor (GoPI) has been studied in vitro. Two modes of inhibition are observed, reversible inhibition that is competitive with GSSG followed by irreversible inhibition. When approximately 1 nm GoPI is incubated with NADPH-reduced GR (1.4 nm) the enzyme becomes 50% inhibited. This appears to be the most potent stable inhibitor of human GR to date. Analyzing the monophasic oxidative half-reaction of reduced GR with GSSG at pH 6.9 revealed a K(d)((app)) for GSSG of 63 microm, and a k((obs)max) of 106 s(-1) at 4 degrees C. The reversible inhibition by the gold-phosphole complex [{1-phenyl-2,5-di(2-pyridyl)phosphole}AuCl] involves formation of a complex at the GSSG-binding site of GR (K(d) = 0.46 microm) followed by nucleophilic attack of an active site cysteine residue that leads to covalent modification and complete inactivation of the enzyme. Data from titration spectra, molecular modeling, stopped-flow, and steady-state kinetics support this theory. In addition, covalent binding of the inhibitor to human GR was demonstrated by mass spectrometry. The extraordinary properties of the compound and its derivatives might be exploited for cell biological studies or medical applications, e.g. as an anti-tumor or antiparasitic drug. Preliminary experiments with glioblastoma cells cultured in vitro indicate an anti-proliferative effect of the inhibitor in the lower micromolar range.

Highlights

The homodimeric flavoprotein glutathione reductase (GR) is a central player of cellular redox metabolism, connecting NADPH to the large pool of redox-active thiols
We propose a mechanism for the reaction of gold-phosphole inhibitor (GoPI) with hGR based on studies using mass spectrometry, titrations, stopped-flow kinetics, and steady-state kinetics
The reaction was initiated by mixing equal volumes of substrate and NaBH4-reduced enzyme in the stopped-flow instrument: in a series of control experiments the first syringe was loaded with 28.1 ␮M hGR(EH2), and the second syringe was loaded with GR buffer containing 0, 30, 60, 120, or 600 ␮M GSSG

Summary

EXPERIMENTAL PROCEDURES

Materials—Recombinant hGR was produced as described [2]. The protein concentration of purified hGR was determined using ⑀463 nm(␭max) ϭ 11.3 mMϪ1 cmϪ1 for Eox (FAD-containing subunit, see Ref. 3). After various intervals (0.5–10 min) of incubation, the enzymatic activity was measured by adding 1 mM GSSG to the mixture and compared with that of an untreated control Based on this second approach a second-order rate constant of the reaction between enzyme and inhibitor was determined from the equation, d[E]/dt ϭ k[E][I] [22]. The reaction was initiated by mixing equal volumes of substrate and NaBH4-reduced enzyme (preincubated with or without GoPI) in the stopped-flow instrument: in a series of control experiments the first syringe was loaded with 28.1 ␮M hGR(EH2), and the second syringe was loaded with GR buffer containing 0, 30, 60, 120, or 600 ␮M GSSG. GoPI was added to the enzyme-containing solution at an excess over hGR (ϳ2.4 eq, ϳ3% DMF, v/v) and mixed with 600 ␮M GSSG. All values are means of at least two independent experiments (each comprising 8 replicas)

RESULTS

Reduced enzyme was preincubated prior to mixing with or without

GSSG kobsa kobsb

DISCUSSION