American Chemical Society
jm901691y_si_001.mpg (1.59 MB)

Frontal Affinity Chromatography−Mass Spectrometry Useful for Characterization of New Ligands for GPR17 Receptor

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posted on 2010-05-13, 00:00 authored by Enrica Calleri, Stefania Ceruti, Gloria Cristalli, Claudia Martini, Caterina Temporini, Chiara Parravicini, Rosaria Volpini, Simona Daniele, Gabriele Caccialanza, Davide Lecca, Catia Lambertucci, Maria Letizia Trincavelli, Gabriella Marucci, Irving W. Wainer, Graziella Ranghino, Piercarlo Fantucci, Maria P. Abbracchio, Gabriella Massolini
The application of frontal affinity chromatography−mass spectrometry (FAC−MS), along with molecular modeling studies, to the screening of potential drug candidates toward the recently deorphanized G-protein-coupled receptor (GPCR) GPR17 is shown. GPR17 is dually activated by uracil nucleotides and cysteinyl-leukotrienes, and is expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a new pharmacological target for acute and chronic neurodegeneration. GPR17 was entrapped on an immobilized artificial membrane (IAM), and this stationary phase was used to screen a library of nucleotide derivatives by FAC−MS to select high affinity ligands. The chromatographic results have been validated with a reference functional assay ([35S]GTPγS binding assay). The receptor nucleotide-binding site was studied by setting up a column where a mutated GPR17 receptor (Arg255Ile) has been immobilized. The chromatographic behavior of the tested nucleotide derivatives together with in silico studies have been used to gain insights into the structure requirement of GPR17 ligands.