Iain G. Dougall

Further concerns over Cheng-Prusoff analysis

In his article in last months issue of TiPS (14, 89-91), Douglas Craig discussed the application of the Cheng-Prusoff relationship to the analysis of antagonists in functional experiments. Craig described how the translation of this method from biochemistry to pharmacology can be accompanied by errors due to theoretical misunderstanding. He also emphasized the lower level of rigour associated with this means of estimating antagonist affinity constants compared with Schild analysis. In this article, Paul Leff and Iain Dougall extend Craigs arguments by describing some additional ways in which the application of the Cheng-Prusoff method to pharmacological experiments are likely to result in misinterpretation and erroneous estimates of antagonist affinity.

P2Y1‐receptors in human platelets which are pharmacologically distinct from P2YADP‐receptors

In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1‐receptor. The P2Y1‐receptor antagonist, adenosine‐3′‐phosphate‐5′‐phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pKB of 5.8 and 6.0, respectively). The selective P2YADP antagonist, AR‐C66096, exhibited partial agonism in the Jurkat cells with an affinity (pKA) of 4.9. This value is consistent with its known P2Y1‐receptor activity. In platelets, AR‐C66096 at a concentration (0.1 μM) approximately 100 fold greater than its known P2YADP receptor affinity, had no effect on ADP‐induced increases in [Ca2+]i. The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2‐MeSADP (8.3)>2‐ClATP (7.8)>ADP (7.5)=2‐MeSATP (7.4)>ATPγS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist. 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 ATPγS and ATP were lower in platelets. The operational model of agonism was used to test whether the agonist concentration‐effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1‐receptor system. 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 affinity value for ATP similar to that obtained previously at P2Y1‐receptors. In summary, the results of this study indicate that human washed platelets contain P2Y1‐receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2YADP‐receptors.

Agonist-antagonist interactions at angiotensin receptors: application of a two-state receptor model

Interactions between agonists and antagonists at angiotensin receptors are characterized by a number of features: variation of antagonist dynamics between apparent simple competition, insurmountable antagonism and, occasionally, augmentation; the tendency for insurmountable antagonism to be saturable; slow recovery of agonist responses following agonist-induced tachyphylaxis; and the ability of competitive antagonists to accelerate recovery from the latter intervention. Some of these phenomena have also been observed in studies of 5-HT2 receptors where they were attributed to the operation of a two-state model with an allosteric site. In this article, Mark Robertson and colleagues propose that the properties of angiotensin AT1 receptors may be explained by a similar model, but without the need to evoke an allosteric site.

Pharmacological studies on prostanoid receptors in the rabbit isolated saphenous vein: a comparison with the rabbit isolated ear artery

1 In isolated ring preparations of the rabbit saphenous vein, prostaglandin E2 (PGE2) caused well‐defined, stable and concentration‐dependent relaxations of KC1 contracted tissues with a mean potency (p[A50]) of 9.39. 2 The prostanoid EP‐receptor agonists, PGE1, 11‐deoxy PGE1, 16,16‐dimethyl PGE2 and misoprostol were all full agonists in this preparation. The EP2‐receptor selective agonists, butaprost and AH13205, and the EP1/EP3‐receptor selective agonist, sulprostone, also relaxed this tissue but were at least 300 times less potent than PGE2. 3 Prostaglandin D2 (PGD2), the DP‐receptor agonist, BW245C, and the IP‐receptor agonist, cicaprost, caused concentration‐dependent relaxations of the rabbit saphenous vein but were at least 60 times less potent than PGE2. 4 The selective EP4‐receptor antagonist, AH23848B (30 μm), antagonized the PGE2 concentration‐effect (E/[A]) curves yielding a pA2 estimate of 4.96. The EP1/DP‐receptor antagonist, AH6809 (10 μm), had no effect on the location of PGE2 E/[A] curves. 5 The stable thromboxane A2‐mimetic, U46619, elicited concentration‐dependent contractions of the rabbit saphenous vein (p[A50] = 8.01) however, PGE2 and prostaglandin F2α (PGF2α) were unable to produce a contractile response. The response to U46619 was competitively antagonized by the TP‐receptor antagonist, GR32191B, yielding a pKB estimate of 7.08. 6 In the rabbit isolated ear artery, PGE2, misoprostol and AH13205 relaxed tissues pre‐contracted with phenylephrine. PGE2 (p[A50] = 7.04) and misoprostol were equipotent, whereas AH13205 was some 40 fold less potent. AH23848B (30 μm) and AH6809 (1 and 10 μm) 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.

Errors in agonist affinity estimation: do they and should they occur in isolated tissue experiments?

Two plausible theories of agonist action - the isomerization and ternary complex mechanisms - predict that agonist affinity measured using the inactivation method may be subject to overestimation. Moreover, the greater the intrinsic efficacy, the greater the predicted error. But are these predictions correct, and are they borne out by experimental data? Paul Leff and colleagues argue that accurate agonist affinity constants can be measured despite these predictions, but that effort must be made to detect any potential errors that might be anticipated from theory.

Pharmacological classification of α1-adrenoceptors mediating contractions of rabbit isolated ear artery: comparison with rat isolated thoracic aorta

The present study attempted to classify pharmacologically the α1‐adrenoceptor subtype(s) present in two isolated, vascular ring preparations, the rabbit ear artery and rat thoracic aorta. In the ear artery, the agonist effects of phenylephrine were antagonized by 5‐methyl urapidil (pA2=7.90; Schild slope=0.85) and BMY 7378 (pA2=6.11; Schild slope=0.80) but not in a simple competitive manner. The shallow Schild slopes are consistent with the activation of a heterogeneous receptor population. Indeed the 5‐methyl urapidil data set could be fitted to a two‐receptor model yielding a high antagonist affinity (pKBH) estimate of 7.85 and a low affinity (pKBL) estimate of 6.03. The effects of clonidine in the ear artery were competitively antagonised by 5‐methyl urapidil (pKB=7.91) and BMY 7378 (pKB=5.53). These data are consistent with contractions to clonidine being mediated by a single receptor subtype. In the aorta, the effects of phenylephrine were antagonized by 5‐methyl urapidil (pA2=7.95; Schild slope=1.11) and BMY 7378 (pA2=9.08; Schild slope=0.73). Neither data set was consistent with a simple competitive interaction. The BMY 7378 data suggested again, that phenylephrine was acting at a heterogeneous receptor population. Subsequent analysis by the two‐receptor model yielded a high affinity (pKBH) estimate of 8.95 and a low affinity (pKBL) estimate of 7.00. The alkylating agent, chloroethylclonidine (CEC) elicited concentration‐dependent contractions in the ear artery with a potency (p[A]50) of 5.57. Pretreatment of this tissue with CEC (5 μm, 30 min incubation) had no effect on subsequent responses to phenylephrine. In contrast, in the aorta, CEC demonstrated no agonism but pretreatment with this agent (5 μm, 15 min incubation) caused a rightward shift and depression of subsequent phenylephrine concentration‐effect curves. The affinity of clonidine in the rabbit ear artery (pKA=6.17) was found to be significantly different to its affinity in the rat thoracic aorta (pKA=7.12) suggesting that this agonist activates different α1‐adrenoceptor subtypes in the two tissues. These results suggest that heterogeneous populations of α1‐adrenoceptors are present in both tissues. In the ear artery, the profile of antagonist and agonist activity is most consistent with α1A‐adrenoceptors being the predominant receptor subtype. The second receptor population does not appear to correspond to any of the recognized α1‐adrenoceptor subtypes. In the aorta α1D‐adrenoceptors appear to predominate, with α1A‐adrenoceptors being the most likely candidate for the second receptor population.

Differential gene expression analysis in human monocyte-derived macrophages: impact of cigarette smoke on host defence.

Alveolar macrophages have been implicated in the pathophysiology of chronic obstructive pulmonary disease (COPD). In this setting they are routinely exposed to cigarette smoke and a range of pathogens including bacteria and viruses. The gene expression changes that result from these challenges may contribute to the initiation and progression of the disease. Understanding such changes is therefore of great interest and could aid the discovery of novel therapeutics. To study this, we stimulated monocyte-derived macrophages (MDM) from smokers and non-smokers with either cigarette smoke extract (CSE) or bacterially derived lipopolysaccharide (LPS) and profiled global transcriptional changes using Affymetrix arrays. LPS and CSE stimulation elicited markedly different transcriptome profiles with the former agent producing a larger number of significant changes. The CSE evoked changes showed some overlap with those observed when comparing habitual smokers with non-smokers, although the latter changes were generally of a more subtle nature. Detailed pathway analyses indicated that a number of genes involved in host defence were regulated following CSE stimulation and in MDM from smokers. In particular the interferon gamma (IFNgamma)-signalling pathway was significantly down-regulated following CSE stimulation, a finding that was confirmed by RT-PCR analysis. Furthermore, these changes were associated with suppressed release of the IFNgamma-induced chemokines, CXCL10 and CXCL9 from CSE treated MDM. In summary, our data provides evidence that smoking alters key mechanisms of host defence in macrophages. Such changes may explain the increased susceptibility of COPD patients to the lung infections that are associated with exacerbations of this disease.

Substituted indole-1-acetic acids as potent and selective CRTh2 antagonists-discovery of AZD1981.

Novel indole-3-thio-, 3-sulfonyl- and 3-oxy-aryl-1-acetic acids are reported which are potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). Optimization required maintenance of high CRTh2 potency whilst achieving a concomitant reduction in rates of metabolism, removal of cyp p450 inhibition and minimization of aldose reductase and aldehyde reductase activity. High quality compounds suitable for in vivo studies are highlighted, culminating in the discovery of AZD1981 (22).

Biochemical and pharmacological characterization of AZD1981, an orally available selective DP2 antagonist in clinical development for asthma.

The discovery of DP2 as a second receptor for PGD2 has prompted the search for antagonists as potential novel therapies based on the associations between PGD2 and disease. Here we describe the biochemical and pharmacological properties of 4‐(acetylamino)‐3‐[(4‐chlorophenyl)thio]‐2‐methyl‐1H‐indole‐1‐acetic acid (AZD1981), a novel DP2 receptor antagonist.

Switching between agonists and antagonists at CRTh2 in a series of highly potent and selective biaryl phenoxyacetic acids

A novel series of biaryl phenoxyacetic acids was discovered as potent, selective antagonists of the chemoattractant receptor-homologous expressed on Th2 lymphocytes receptor (CRTh2 or DP2). A hit compound 4 was discovered from high throughput screening. Modulation of multiple aryl substituents afforded both agonists and antagonists, with small changes often reversing the mode of action. Understanding the complex SAR allowed design of potent antagonists such as potential candidate 34.