Immobilized P2X2 purinergic receptor stationary phase for chromatographic determination of pharmacological properties and drug screening
Trujillo, C.A.; Majumder, P.; Gonzalez, F.A.; Moaddel, R.; Ulrich, H.
Universidade de São Paulo, Instituto de Química - Departamento de Bioquímica, São Paulo, Brazil
The biological effects of extracellular purine nucleotides acting through P2 receptors have been studied in many cell and tissue types, and ATP is now recognized as an important messenger molecule in cell-cell communication in the central nervous system and has been indicated to be involved in brain development. Thus, this signaling system, through P2X receptors, opens new opportunities for therapy and screening of potential drugs. Our objective is to explore the pharmacological properties of this target, and establish a new methodology for ligand screening for the P2X2 receptor, developed by the combinatorial RNA library approach SELEX (Systematic Evolution of Ligands by EXponential enrichment).
For receptor immobilization, membranes of 1321N1 cells, stably transfected with P2X2 receptors, were resuspended in 2% cholate detergent. Dried IAM (Immobilized Artificial Membrane) particles were added to the solution containing cholate-protein. The mixture of IAM-cholate-P2X2 receptor containing protein was dialyzed, centrifuged, followed by collection of the solid phase and packing into a FPLC column. The equilibrium binding and competition between the purinergic antagonists, suramin and TNP-ATP, and ATP was determined by a chromatographic assay using 32P alpha ATP as a radioligand to verify specific binding of these compounds, and whether suramin or TNP-ATP and 32P alpha ATP compete for the same binding site. Our data, using immobilized P2X2 receptor in the stationary phase of the column, indicate that suramin does not compete with ATP for the ligand binding site and TNP-ATP is a competitive antagonist, confirming previous studies [Trujillo et al. Biochemistry 45, 224-33, 2006]. In addition, we demonstrate that this assay can be used as tool for SELEX of P2X2 receptor-binding aptamers.
These results show that the development of receptor-coupled stationary phases for liquid chromatography offers a number of unique advantages. The receptor can be immobilized in a stable format, and therefore, be reused over an extended period of time. Besides producing rapid and reproducible results, receptor columns facilitate exploration of ligand-receptor interactions and screening of combinatorial pools of possible ligands. Chromatographic retention times could be used to sort a mixture of compounds according to their relative binding affinities for the isolation and identification of lead drug candidates from complex biological systems.
Financial support: FAPESP and CNPq.
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