Determination of binding constants and stoichiometries for platinum anticancer drugs and serum transport proteins by capillary electrophoresis using the Hummel-Dreyer method

Abstract

A CE method has been developed to evidence and quantitatively characterize the interaction between platinum-based antitumor drugs and human serum proteins. This method is a variant of affinity CE modified regarding both experimental setup and data treatment so as to measure the peaks (or vacancies) that correspond to the bound drug when it slowly binds to the protein. Using the formalism of the Hummel-Dreyer method and cisplatin and oxaliplatin as test compounds, a protocol for determining albumin and transferrin binding constants and stoichiometries, including (and distinguished by) 48 hours of incubation of the reaction mixture, was elaborated. Relative affinities of drugs toward different proteins in aqueous solution at physiological pH, chloride concentration, and temperature were compared in terms of overall binding constants and numbers of drug molecules attached to the protein. The results indicate that both platinum drugs bind to albumin more strongly than to transferrin, supporting the concept that the albumin fraction is a major drug supply route for chemotherapeutical needs. From a comparison with the binding parameters measured previously for cisplatin by other methods, conclusions were drawn about the validity of CE as a simple and convenient method for assaying protein-drug reactions with slow kinetics.

Investigation of interaction between human hemoglobin A0 and platinum anticancer drugs by capillary isoelectric focusing with whole column imaging detection

“…Both these aqua species and the parent drug are responsible for the reaction and binding properties of cisplatin. This agrees with previous observations achieved by other methods [20,30].…”

“…ESI-MS was recently shown as an extremely useful tool for studying the cisplatin-protein interactions [19,[21][22][23][24][25]. Also, HPLC [19,24,[26][27][28] and a complementary high-resolution technique with an orthogonal separation mechanism, CE [29][30][31], are growing in importance for characterizing the protein binding for different metal complexes. The potential of resolving intact and proteinbound drug forms appears to be prominent when combined with a metal-specific mode of detection by using inductively coupled plasma MS (ICP-MS) [19,25,[27][28][29].…”

Characterization of the binding sites of the anticancer ruthenium(III) complexes KP1019 and KP1339 on human serum albumin via competition studies

“…In addition to the spectroscopic studies, the reactions of [HSA-Ru(III) complex] with WF and DG were studied by capillary zone electrophoresis (CZE)−UV-vis and ultrafiltration−UV-vis methods (BR could not be studied because of its low aqueous solubility and extinction coefficient). CE has been used to determine chemical equilibria in proteinmetallodrug systems [49,50]. Compared with other separation-based assays such as highperformance liquid chromatography (HPLC), CE possesses distinctive advantages such as shorter analysis time, use of electrolyte compositions compatible with physiological incubation conditions, and no stationary phase involved in separation [51].…”

Capillary electrophoresis hyphenated to inductively coupled plasma‐mass spectrometry: A novel approach for the analysis of anticancer metallodrugs in human serum and plasma

“…It has been applied to investigate the stability of novel compounds but also the interactions of drugs with biomolecules, such as human serum proteins [8][9][10][11][12][13][14][15] and nucleotides [16][17][18][19][20][21], elucidating possible metabolic and reaction pathways of the drug candidates [6,7,16], notably always working under simulated physiological conditions with regard to pH, temperature, aqueous solutions, and/or buffer composition.…”