Platinum metallodrug‐protein binding studies by capillary electrophoresis‐inductively coupled plasma‐mass spectrometry: Characterization of interactions between Pt(II) complexes and human serum albumin

Abstract

Characterizing how platinum metallocomplexes bind to human serum albumin (HSA) is essential in evaluating anticancer drug candidates. Using cisplatin as a reference complex, the application of capillary electrophoresis (CE) to reliably assess drug/HSA interactions was validated. Since this complex is small compared to the size of the protein, the binding response could only be recognized when applying CE coupled to a (platinum) metal-specific mode of detection, namely inductively coupled plasma-mass spectrometry (ICP-MS). This coupling allowed for confirmation of a specific affinity of cisplatin and novel Pt complexes to HSA, measurement of the kinetics of binding reactions, and determination of the number of drug molecules attached to the protein. As the cisplatin/HSA molar ratio increased, the reaction rate became faster with a maximum on the kinetic curve appearing at about 50 h of incubation at 20 times excess of cisplatin. The reaction was characterized as a pseudo-first order reaction with the rate constant k = 0.003 min(-1) at 37 degrees C. When incubated with a 20-fold excess of cisplatin, HSA bound up to 10 mol of Pt per mol of the protein. This is indicative for a strong metal-protein coordination occurring at several HSA sites other than the only protein cysteine residue. Structural analogs of cisplatin, bearing aminoalcohol ligands, showed comparable protein binding reactivity and stoichiometry but a common equilibrium was not reached even after one week of incubation. Also apparent was a two-step mechanism of the binding reaction. Results demonstrated the suitability of CE-ICP-MS as a rapid assay for high-throughput studying of drug/HSA interactions.

A Fluorescent Probe for Investigating the Activation of Anticancer Platinum(IV) Prodrugs Based on the Cisplatin Scaffold

“…Elemental-analysis techniques, such as graphite-furnace atomic-absorption spectrometry (GF-AAS) and inductively coupled plasma mass spectrometry (ICP-MS), have also been used extensively to quantitate the Pt distribution in tissue or cellular compartments, but they cannot be applied in cellular imaging. [11] More recently, NanoSIMS was used to map the accumulation of cisplatin and the multinuclear Pt drug candidate TriplatinNC in vitro, but it cannot distinguish between different Pt oxidation states (NanoSIMS = nanoscale secondary ion mass spectrometry). [12] Fluorescence microscopy remains the most effective and accessible method for imaging at the cellular level.…”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[151] Nevertheless, it is worth mentioning that there is still some conflicting information on the affinity of cisplatin for serum proteins, some reports claiming preference for transferrin, [151] others selectivity for albumin [33], as well as on the platination sites on those proteins. [152] Interactions between cisplatin and albumin have been demonstrated to occur, [153] proteins to be characterised in a 24 h period. Via this method, five binding sites were described for cisplatin on HSA, specifically Cys34, Met329, Met548, Tyr150 (or Tyr148) and Asp375 (or Glu376).…”

The application of inductively coupled plasma mass spectrometry in clinical pharmacological oncology research

“…The exact role that Pt binding to proteins such as albumin and transferrin plays in the mechanism of drug action, remains unclear. Interactions with proteins, however, might play an important role in drug efficacy and side effects (Timerbaev et al, 2004). Methionine is important because of its large concentrations and reactivity.…”

“…The bands from the gel were cut out and dissolved in 70% HNO 3 and then heated at 908C before being diluted with water (Carr, Tingle, & McKeage, 2002) or they were extracted with aqua regia (Lustig et al, 1999). The Pt content of the bands was measured by ICP-MS. Timerbaev et al (2004) coupled capillary electrophoresis to ICP-MS using an interface consisting of a microconcentric nebuliser to study the interaction of cisplatin with albumine. A similar method was applied to study the interaction of KP1019 with albumine and transferrin (Polec-Pawlak et al, 2006).…”