Species-dependent binding of tocainide analogues to albumin: affinity chromatography and circular dichroism study.
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.
School of Bioscience & Bioengineering, South China University of Technology, Higher Education Mega Center, 510006 Guangzhou, People's Republic of China.
Marco
M
Pistolozzi
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy.
Cecilia
C
Fortugno
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari 'Aldo Moro', Bari, Italy.
Carlo
C
Franchini
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari 'Aldo Moro', Bari, Italy.
Filomena
F
Corbo
Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari 'Aldo Moro', Bari, Italy.
Marilena
M
Muraglia
Centre Interdisciplinaire de Nanoscience de Marseille (CNRS-UMR 7325), Aix-Marseille Université, France.
Myriam
M
Roy
Centre Interdisciplinaire de Nanoscience de Marseille (CNRS-UMR 7325), Aix-Marseille Université, France.
Guy
G
Félix
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy. Electronic address: carlo.bertucci@unibo.it.
Carlo
C
Bertucci
D000418:Albumins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D016677:Tocainide / Q000737:chemistry* / Q000378:metabolism*
D000418:Albumins; D016677:Tocainide
Circular dichroism; HSA; High performance liquid affinity chromatography; Protein binding; RSA; Tocainide analogues