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

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Abstract
The first detailed kinetic analysis and mechanistic interpretation of the reactions between serum albumin and the second-generation gold drug Auranofin [Et3PAuSATg = (triethylphosphine)(2,3,4,6-tetra-O-acetyl-1−β-d-glucopyranosato-S-) gold(I)] and its triisopropylphosphine analogue, iPr3PAuSATg, in vitro are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer methods. Under the Penefsky chromatography conditions with 0.4−0.6 mM albumin and a wide range of Et3PAuSATg concentrations, the reaction is biphasic. The fast phase is apparently first order in albumin with a rate constant [k1 = 3.4 ± 0.3 × 10-2 s-1] that decreases slightly in magnitude and becomes intermediate in order at low gold concentrations, [Et3PAuSATg] < [AlbSH]; it accounts for ∼95% of the Au(I) that binds. A minor, slower step [k2 = 2.3 ± 0.3 × 10-3 s-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, k1 = (1.4 ± 0.1) × 10-2 s-1, and is first order in albumin and independent of the iPr3PAuSATg concentration. 31P-NMR saturation transfer experiments utilizing iPr3PAuSATg, under equilibrium conditions, yielded second-order rate constants for both the forward (1.2 × 102 M-1 s-1) and the reverse (3.9 × 101 M-1 s-1) directions. A multistep mechanism involving a conformationally altered albumin species was developed. Albumin domain IA opens with concomitant Cys-34 rearrangement, allowing facile gold binding and exchange, and then closes. In conjunction with the steady-state approximation, this mechanism accounts for the different reaction orders observed under the two set of conditions. The rate-determining conformational change of albumin governs the reaction as monitored by the Penefsky columns. Rapid second order exchange of R3PAuSATg at the exposed Cys-34 residue is observed under the NMR conditions. The mechanism predicts that under physiological conditions where [Et3PAuSATg] is 10−25 μM, the reaction will be second order and rapid with a rate constant of 8 ± 2 × 102 M-1 s-1. The Penefsky spun columns revealed a previously unreported and novel binding mechanism, association of auranofin in the pocket of albumin−disulfide species, which was confirmed by Hummel−Dreyer gel chromatographic techniques under equilibrium conditions. This albumin−auranofin complex (AlbSSR−Et3PAuSATg) is weakly bound and readily dissociates during conventional gel exclusion chromatography.