title: Mechanistic studies on a novel, highly potent gold-phosphole inhibitor of human glutathione reductase.
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.
authors.affiliation: Interdisciplinary Research Center, Justus Liebig University, D-35392 Giessen, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Deponte
authors.forename: Sabine
authors.initials: S
authors.lastname: Urig
authors.forename: L David
authors.initials: LD
authors.lastname: Arscott
authors.forename: Karin
authors.initials: K
authors.lastname: Fritz-Wolf
authors.forename: Régis
authors.initials: R
authors.lastname: Réau
authors.forename: Christel
authors.initials: C
authors.lastname: Herold-Mende
authors.forename: Sasa
authors.initials: S
authors.lastname: Koncarevic
authors.forename: Markus
authors.initials: M
authors.lastname: Meyer
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Davioud-Charvet
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Charles H
authors.initials: CH
authors.lastname: Williams
authors.forename: Katja
authors.initials: K
authors.lastname: Becker
mesh_terms: D001665:Binding Sites; D001894:Borohydrides / Q000737:chemistry; D002455:Cell Division / Q000187:drug effects; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005909:Glioblastoma / Q000473:pathology; D005978:Glutathione / Q000378:metabolism; D005980:Glutathione Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D009249:NADP / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
chemical_list: C502744:((1-phenyl-2,5-di-(2-pyridyl)phosphole)gold(I)) chloride; D001894:Borohydrides; D004791:Enzyme Inhibitors; D009942:Organometallic Compounds; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D009249:NADP; C025364:sodium borohydride; D005980:Glutathione Reductase; D005978:Glutathione; D003545:Cysteine