Species-dependent binding of tocainide analogues to albumin: Affinity chromatography and circular dichroism study

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 968; pp. 69 - 78
• Main Authors: Pistolozzi, Marco, Fortugno, Cecilia, Franchini, Carlo, Corbo, Filomena, Muraglia, Marilena, Roy, Myriam, Félix, Guy, Bertucci, Carlo
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.10.2014; Elsevier
• Subjects: Affinity; Albumins - chemistry; Albumins - metabolism; Animals; Binding; Binding sites; Binding Sites - physiology; Chromatography; Chromatography, Affinity - methods; Chromatography, High Pressure Liquid - methods; Circular dichroism; Circular Dichroism - methods; Circularity; Dichroism; High performance liquid affinity chromatography; HSA; Humans; Mathematical models; Protein binding; Protein Binding - physiology; Proteins; Rats; RSA; Tocainide - chemistry; Tocainide - metabolism; Tocainide analogues; RSA; Tocainide analogues; High performance liquid affinity chromatography; HSA; Protein binding; Circular dichroism
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2014.01.007

•We characterize the binding of tocainide analogues to rat and human albumins.•We use HPALC to ranking the analyzed compounds for their binding affinities.•We use HPALC and CD spectroscopy to get information on the binding mechanism.•Drug binding data to albumins from different species show significant differences. A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.