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

Abstract

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-dtpa), designed for lanthanide-based time resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR2 agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH2, 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH2, 2-at-LIGRL and; 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.

Pathway‐selective antagonism of proteinase activated receptor 2

“…Assays were performed as described (Hoffman et al, 2012). Cells were seeded overnight in a 384-well plate at a density of 2500 cells per well.…”

“…Here, we use a fluorescence-based binding assay (Hoffman et al, 2012) to show that GB88 directly competes with a Eu-tagged peptide agonist of PAR2, namely 2f-LIGRLO-NH2 ( Figure 1A and 1B). CHO cells transfected with human PAR2 were used because a high level of PAR2 expression is required to allow binding measurements.…”

Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3

“…Using europium-labelled peptides for high-throughput screening has previously been highly successful for ligands targeting disease relevant targets, such as the vasopressin receptors, oxytocin receptor and protease activated receptor-2 (PAR 2 ) (Albizu et al 2007;Hoffman et al 2012). Here, we present a site-directed synthesis strategy where the chelating Eu-DTPA is conjugated to the N-terminus of the single-chain R3B1-22R, which results in high yield and purity.…”

Novel agonists & antagonists for protease-activated receptor 2 and C3a receptor

“…The ortho substitution (175) showed better activity than meta (176) or para (177) substitution, suggesting that more space was available for ortho substitution. A phenyl ring at this position may be too hindered and thus confer less potency than the unsubstituted phenyl-piperidine (167).…”

“…176 Diethylenetriaminepentaacetic acid (dtpa) was introduced at the ornithine side chain to give 2f-LIGRLO(dtpa)-NH 2 , and this peptide was then chelated to europium(III). This is a highly sensitive assay and can be used for high-throughput competitive ligand binding, as long as the test ligand binds in the same or overlapping location in PAR2 as this fluorophore.…”