CHARACTERIZATION OF THE RECOMBINANT HUMAN PROSTANOID DP RECEPTOR AND IDENTIFICATION OF L-644,698, A NOVEL SELECTIVE DP AGONIST

1 A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D-2 (PGD(2)) receptor (hDP) has b een characterized with respect to radioligand binding and signal trans duction properties by use of prostanoids and prostanoid analogues. Rad ioligand 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'-cycli c monophosphate (cyclic AMP) generation in response to ligand challeng e 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 hi gh 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 l igands that have previously shown high affinity for DP: PGD(2) (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 ge nerating cyclic AMP following activation of hDP. L-644,698 is, therefo re, comparable to those agonists with known efficacy at the DP recepto r (EC50): PGD(2) (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 (similar to 300 fold), EP3-III (similar to 4100 fold), EP4 (simi lar to 10000 fold), FP (>25,400 fold), IP (>25,400 fold) and TP (>25,4 00 fold). L-644,698 is, therefore, one of the most selective DP agonis ts as yet described. 5 PGJ(2) and Delta(12)-PGJ(2), two endogenous met abolites of PGD(2), were also tested in this system and shown to be ef fective agonists with K-i and EC50 values in the nanomolar range for b oth compounds. In particular, PGJ(2) was equipotent to known DP specif ic agonists with a K-i value of 0.9 nM and an EC50 value of 1.2 nM.