ABSTRACT

Basic Information

Abstract Number: 250 - 3
Author Name: Irving W Wainer - National Institutes of Health
Session Title: Chromatography Forum of the Delaware Valley Dal Nogare Award
Event Type: Awards
Event Title: Enantioselective Retention on Cellular Membrane Affinity Chromatography Columns: Using Chiral Separations as a Probe Instead of a Goal

Presider Name:Mary Ellen McNally
Affiliation:DuPont Crop Protection

Date: Monday, March 18, 2013
Start Time: 08:10 AM (Slot #3)
Location: 126A

Abstract Content

Protein-based chiral stationary phases were initially employed in the analytical separation of chiral compounds. In our early studies, we also observed that these columns could be used to probe small molecule-protein interactions and chiral separations became a pharmacological tool rather than an analytical end point. This presentation will discuss the enantioselective interactions of chiral molecules with cellular membrane fragments immobilized on silica supports and on the surface of activated open tubular glass capillaries creating cellular membrane affinity chromatography (CMAC) columns [1]. CMAC columns have been prepared from cell
lines expressing target receptors or transporters or cell line that were stably transfected to express the target protein, CMAC(+) columns. CMAC columns were also constructed using membranes from cell lines in which the expression of the target protein had been reduced using siRNA or from the wild type of the cell line used in the transfections, CMAC(-) columns. Frontal affinity
chromatography (FAC) studies using small molecule ligands were carried out on the CMAC(+) and CMAC(-) columns and the relative difference between the two columns used to characterize the binding of the ligands to the immobilized target protein. When chiral ligands were employed, enantioselective differences in retention were used to differentiate between receptor subtypes and to characterize the effects of single nucleotide polymorphisms (SNPs). This approach will be illustrated using a series of CMAC columns developed using cell lines expressing ATP-drug transporters, Pgp, MRP1, MRP2, BCRP and the human organic cation transporter (hOCT1). In the latter studies, the effects of two SNPs on the binding of chiral substrates were used to create a pharmacophore model of the hOCT1.

References
[1] R. Moaddel, I.W. Wainer, Nature Protocols, 4, 197-205 (2009).
[2] Bhatia P, Moaddel R, Wainer IW. Talanta, 81, 1477-1481 (2010).