Kinetics and Mechanism of the Reaction between Serum Albumin and Auranofin (and Its Isopropyl Analogue) in Vitro

Abstract

The first detailed kinetic study of in vitro reactions between serum albumin and the secondgeneration gold drug 3,4,glucopytanosato-S-) gold(I)] and its tri-i-propylphosphine analogue, .iPr3PAuSATg, are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer kinetics.Based on the Penefsky column data, the binding of the Et3PAuSATg to AIbSH (0.600 mM) was complete when gold concentrations were limiting: 0.093, 0.151, and 0.225 mM. The reaction is biphasic. The fast phase is defined by a first order rate constant, (k 2.94 _+ 0.24 x 10 -2 sec"1) and accounts for 95% of the Au(I) bound. This phase is first order with respect to albumin, and zero order with respect to auranofin. A minor, slower step (k 2 2.26 _+ 0.26 x 10 -3 sec"1), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with respect to auranofin. For .iPr3PAuSATg, only the first step was observed, k 1.4 +_ 0.1 x 10 -2 s "1 and is first order in albumin and independent of iPr3PAuSATg concentration. 31p.NMR saturation transfer experiments utilizing the auranofin analogue, .iPr3PAuSATg, under equilibrium conditions with excess .iPr3PAuSATg and ATgSH yielded second order rate constants for both the forward (1.0 x 102 M " sec"1) and the reverse (3.9 x 101 M "1 sec"1) directions. A multi-step mechanism involving a conformationally altered albumin species to which gold binds was developed using the steady-state approximation. The mechanism accounts for the different reaction orders observed under the two set of conditions. A rate determining conformational change on the albumin governs the reaction as monitored by the Penefsky columns. A second order reaction of R3PAuSATg at Cys-34 is observed under the NMR conditions. These results are the first quantitative determination of auranofin-albumin reaction kinetics and the first mechanistic study of the reaction.A novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species can be detected by Penefsky and HummeI-Dreyer gel chromatographic techniques, but not by conventional gel-exclusion chromatography. This complex albumin-auranofin complex is weaklybound and readily dissociates.

Cancer cell death induced by phosphine gold(I) compounds targeting thioredoxin reductase

“…Both deacetylated auranofin and the parent [AuCl(PEt 3 )] complex form the same [(Et 3 P)Au(S-alb)] adduct by displacement of the anion. By studying reactions with auranofin instead of the unstable deacetylated form, under conditions approximating those in vivo, such adduct formation is of the first-order in auranofin with a rate constant of 2.9 ± 0.2 s -1 [6]. These data support the view that auranofin (or its deacetylated metabolite)…”

“…should have a very short lifetime after entering the bloodstream where albumin is present in excessive amount fold) compared to the gold drug [6]. To corroborate this hypothesis, Christodoulou et al [7] reported a conformational change in albumin that accompanies gold binding to cys-34.…”

Anticancer Therapeutics That Target Selenoenzymes: Synthesis, Characterization, in vitro Cytotoxicity, and Thioredoxin Reductase Inhibition of a Series of Gold(I) Complexes Containing Hydrophilic Phosphine Ligands

“…[19,44] Moreover, investigation of the reactions of auranofin with serum albumin showed that, upon gold binding to Cys34, the thioglucose ligand was displaced with the phosphine moiety remaining attached. [45] A similar mechanism of thiolate release may be responsible for the activity of the series of gold(I) compounds described here. Further studies are underway to establish whether TrxR histidines could also form covalent interactions with gold ions and to determine the nature of the protein-bound metallofragments.…”

Novel gold(i) phosphine compounds inhibit HIV-1 enzymes

“…13 Thiol-ligand exchange reactions have been reported by various authors demonstrating the role of ligand exchange of gold compounds in different situations. 12,13,[24][25][26] Ligand exchange reactions with the complexes that have an easily accessible chlorine atom such as 1b, 2b, 3b, 4b, 5, 7 and 8 might therefore have been favoured as all these gold complexes inhibited RT. It is also possible that the gold compounds intercalate in the DNA molecule being formed in the reaction mixture of enzyme and nucleotides, thereby interfering with the reverse transcription reaction catalysed by RT.…”