Lanthanide Labeling of a Potent Protease Activated Receptor-2 Agonist for Time-Resolved Fluorescence Analysis

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• 作者：Justin Hoffman ; Andrea N. Flynn ; Dipti V. Tillu ; Zhenyu Zhang ; Renata Patek ; Theodore J. Price ; Josef Vagner ; Scott Boitano
• 刊名：Bioconjugate Chemistry
• 出版年：2012
• 出版时间：October 17, 2012
• 年：2012
• 卷：23
• 期：10
• 页码：2098-2104
• 全文大小：348K
• 年卷期：v.23,no.10(October 17, 2012)
• ISSN：1520-4812

文摘

Protease activated receptor-2 (PAR₂) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR₂ can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR₂ to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR₂ peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH₂ (2-f-LIGRLO鈥揹tpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO鈥揹tpa is a potent and specific PAR₂ agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO鈥揹tpa can be utilized in an affinity binding assay to evaluate the ligand鈥搑eceptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH₂, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH₂, 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH₂, 6-an-LIGRL) and PAR₂. A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH₂, 3-ia-LIGRL) that does not activate PAR₂ signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR₂ binding. In summary, we have developed a europium-containing PAR₂ agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR₂ ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR₂ ligands.