No abstract

1 In the present study we have classi®ed the receptor(s) mediating increases in intracellular calcium concentration ([Ca 2+ ] i ) in human washed platelets and compared the pharmacological pro®le obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y 1 -receptor. 2 The P2Y 1 -receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar a nities (pK B of 5.8 and 6.0, respectively). 3 The selective P2Y ADP antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an a nity (pK A ) of 4.9. This value is consistent with its known P2Y 1 -receptor activity. In platelets, AR-C66096 at a concentration (0.1 mM) approximately 100 fold greater than its known P2Y ADP receptor a nity, had no e ect on ADP-induced increases in [Ca 2+ ] i . 4 The ability of adenine nucleotide analogues to elevate [Ca 2+ ] i in the Jurkat cells was also determined. The rank order of agonist potency (p[A] 50 ) was: 2-MeSADP (8.3)42-ClATP (7.8)4ADP (7.5)=2-MeSATP (7.4)4ATPgS (6.5)4ATP (6.2), with ATP appearing to be a partial agonist. 5 The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPgS and ATP were lower in platelets. 6 The operational model of agonism was used to test whether the agonist concentration-e ect pro®les obtained in these two cell types could be explained on the basis of di erences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y 1 -receptor system. 7 The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an a nity value for ATP similar to that obtained previously at P2Y 1 -receptors. 8 In summary, the results of this study indicate that human washed platelets contain P2Y 1 -receptors which mediate increases in [Ca 2+ ] i and that this receptor population is pharmacologically distinct from P2Y ADP -receptors.

By using a pharmacophore model, a geometrical representation of the features necessary for molecules to show a particular biological activity, it is possible to search databases containing the 3D structures of molecules and identify novel compounds which may possess this activity. We describe our experiences of establishing a working 3D database system and its use in rational drug design. By using muscarinic M(3) receptor antagonists as an example, we show that it is possible to identify potent novel lead compounds using this approach. Pharmacophore generation based on the structures of known M(3) receptor antagonists, 3D database searching, and medium-throughput screening were used to identify candidate compounds. Three compounds were chosen to define the pharmacophore: a lung-selective M(3) antagonist patented by Pfizer and two Astra compounds which show affinity at the M(3) receptor. From these, a pharmacophore model was generated, using the program DISCO, and this was used subsequently to search a UNITY 3D database of proprietary compounds; 172 compounds were found to fit the pharmacophore. These compounds were then screened, and 1-[2-(2-(diethylamino)ethoxy)phenyl]-2-phenylethanone (pA(2) 6.67) was identified as the best hit, with N-[2-(piperidin-1-ylmethyl)cycohexyl]-2-propoxybenz amide (pA(2) 4. 83) and phenylcarbamic acid 2-(morpholin-4-ylmethyl)cyclohexyl ester (pA(2) 5.54) demonstrating lower activity. As well as its potency, 1-[2-(2-(diethylamino)ethoxy)phenyl]-2-phenylethanone is a simple structure with limited similarity to existing M(3) receptor antagonists.

fold less potent. AH23848B (30 pM) and AH6809 (1 and 10 gM) caused no significant shift in the location of PGE2 E/[A] curves. 7 These data suggest that the rabbit isolated saphenous vein contains prostanoid, EP-, DP-, IP-and TP-receptors. Based on antagonist affinity information and agonist potency orders, the rabbit saphenous vein contains an inhibitory prostanoid EP-receptor different from that in the rabbit ear artery, but comparable to the recently described EP4-receptor.

Prostaglandin (PG)E 2 has been shown to inhibit mediator release from human alveolar macrophages (AMs), but the prostanoid receptor(s) mediating this response have not yet been documented. To investigate this, the present authors conducted a range of pharmacological and expression-based studies in monocyte-derived macrophages (MDMs) and AMs.MDMs were obtained by in vitro differentiation of monocytes from the peripheral blood of healthy human volunteers. Human AMs were obtained by perfusion of lung tissue from carcinoma resection patients.In MDMs, PGE 2 potently inhibited lipopolysaccharide-induced tumour necrosis factor (TNF)-a release (p[A] 50 8.51¡0.11, maximum inhibition 95.9¡4.8%). In human AMs, PGE 2 also inhibited TNF-a release but the observed concentration-effect curve was very flat and inhibition was incomplete. The shape of the PGE 2 curve in AMs suggested that its effects were mediated by activation of a heterogeneous receptor population. Expression studies combined with the use of various E-prostanoid (EP) receptor agonists and a selective EP 4 -receptor antagonist (Ono-AE2-227) confirmed that the inhibitory effects of PGE 2 in both AMs and MDMs were mediated by activation of EP 4 and EP 2 receptors.These data indicate that both E-prostanoid 4 and E-prostanoid 2 selective agonists may have antiinflammatory properties in lung diseases where macrophages play a role.