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Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor.

ABSTRACT: 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 ([(35)S]GTPgammaS 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.

SUBMITTER: Calleri E

PROVIDER: S-EPMC6201305 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor.

Calleri Enrica E Ceruti Stefania S Cristalli Gloria G Martini Claudia C Temporini Caterina C Parravicini Chiara C Volpini Rosaria R Daniele Simona S Caccialanza Gabriele G Lecca Davide D Lambertucci Catia C Trincavelli Maria Letizia ML Marucci Gabriella G Wainer Irving W IW Ranghino Graziella G Fantucci Piercarlo P Abbracchio Maria P MP Massolini Gabriella G

Journal of medicinal chemistry 20100501 9

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 neurodegene ...[more]

PMID: 20394377

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Project description:Antibodies facilitate targeted cell killing by engaging with immune cells such as natural killer cells through weak binding interactions with Fcγ receptors on the cell surface. Here, we evaluate the binding affinity of the receptor FcγRIIIa V158 (CD16a) for several therapeutic antibody classes, isoforms, and Fc-fusion proteins using an immobilized receptor affinity liquid chromatography (LC) approach coupled with online mass spectrometry (MS) detection. Aglycosylated FcγRIIIa was used in the affinity chromatography and compared with published affinities using glycosylated receptors. Affinity LC-MS differentiated the IgG1 antibodies primarily according to their Fc glycosylation patterns, with highly galactosylated species having greater affinity for the immobilized receptors and thus eluting later from the column (M5< G0F < G0 afucosylated ≅ G1F < G2F). Sialylated species bound weaker to their asialylated counterparts as reported previously. High mannose glycoforms bound weaker than G0F, contrary to previously published studies using glycosylated receptors. Also, increased receptor binding affinity associated with afucosylated antibodies was not observed with the aglycosylated FcγRIIIa. This apparent difference from previous findings highlighted the importance of the glycans on the receptors for mediating stronger binding interactions. Characterization of temperature-stressed samples by LC-MS peptide mapping revealed over 200 chemical and post-translational modifications, but only the Fc glycans, deamidation of EU N325, and an unknown modification to either proline or cysteine residues of the hinge region were found to have a statistically significant impact on binding.Abbreviations: Antibody-dependent cell-mediated cytotoxicity (ADCC), chimeric antigen receptor (CAR), Chinese hamster ovary (CHO), dithiothreitol (DTT), electrospray ionization (ESI), hydrogen-deuterium exchange (HDX), filter aided-sample preparation (FASP), Fcγ receptor (FcγR), fragment crystallizable (Fc), high-pressure liquid chromatography (HPLC), immunoglobulin G (IgG), liquid chromatography (LC), monoclonal antibody (mAb), mass spectrometry (MS), natural killer (NK), N-glycolylneuraminic acid (NGNA), N-acetylneuraminic acid (NANA), principal component analysis (PCA), surface plasmon resonance (SPR), trifluoroacetic acid (TFA), and extracted mass chromatogram (XMC).

Dataset Information

Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor.

Publications

Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor.

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