Characterization of the recombinant human prostanoid DP receptor and identification of L-644,698, a novel selective DP agonist

Abstract

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D₂ (PGD₂) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3′:5′-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway.

2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (K_i) of 0.9 nM. This K_i value was comparable to the K_i values obtained in this study for ligands that have previously shown high affinity for DP: PGD₂ (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM).

3. L-644,698 was found to be a full agonist with an EC₅₀ value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC₅₀): PGD₂ (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM).

4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP₁ (>25,400 fold), EP₂ (∼300 fold), EP_3-III (∼4100 fold), EP₄ (∼10000 fold), FP (>25,400 fold), IP (>25,400 fold) and TP (>25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described.

5. PGJ₂ and Δ¹²-PGJ₂, two endogenous metabolites of PGD₂, were also tested in this system and shown to be effective agonists with K_i and EC₅₀ values in the nanomolar range for both compounds. In particular, PGJ₂ was equipotent to known DP specific agonists with a K_i value of 0.9 nM and an EC₅₀ value of 1.2 nM.

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