David A. Hall

Receptors for ADP on human blood platelets

It is well established that ADP causes aggregation of human blood platelets, and indeed it was the first aggregating agent to be studied, but the ways in which platelets respond to ADP are still relatively obscure. Although it is apparent that increases in intracellular Ca2+ concentrations are of major importance in activating platelets, it is not clearly understood how ADP causes these increases and what other signal transduction mechanisms it uses. It is not even clear whether ADP causes its effects by interacting with only one receptor, or whether multiple receptors for ADP exist on platelets. In this review, Susanna Hourani and David Hall examine some of the conflicting evidence in this field, and draw some tentative conclusions about the number and nature of receptors for ADP on human platelets.

Chemokine Receptor 4 Plays a Key Role in T Cell Recruitment into the Airways of Asthmatic Patients

T lymphocytes of the Th2 type are central orchestrators of airway inflammation in asthma. The mechanisms that regulate their accumulation in the asthmatic airways remains poorly understood. We tested the hypothesis that CCR4, preferentially expressed on T lymphocytes of the Th2 type, plays a critical role in this process. We enumerated by flow cytometry the CCR4-expressing T cells from blood, induced sputum, and biopsy samples of patients with asthma and control subjects. We showed a positive correlation between the numbers of peripheral blood CCR4+ T cells and asthma severity, provided evidence of preferential accumulation of CCR4+ T cells in asthmatic airways, and demonstrated that CCR4+ but not CCR4− cells from patients with asthma produce Th2 cytokines. Explanted airway mucosal biopsy specimens, acquired by bronchoscopy from subjects with asthma, were challenged with allergen and the explant supernatants assayed for T cell chemotactic activity. Allergen-induced ex vivo production of the CCR4 ligand, CCL17 was raised in explants from patients with asthma when compared with healthy controls. Using chemotaxis assays, we showed that the T cell chemotactic activity generated by bronchial explants can be blocked with a selective CCR4 antagonist or by depleting CCR4+ cells from responder cells. These results provide evidence that CCR4 might play a role in allergen-driven Th2 cell accumulation in asthmatic airways. Targeting this chemokine receptor in patients with asthma might reduce Th2 cell-driven airway inflammation; therefore, CCR4 antagonists could be an effective new therapy for asthma. This study also provides wider proof of concept for using tissue explants to study immunomodulatory drugs for asthma.

Effects of the P2-purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5'-diphosphate.

1 The effects of suramin, a trypanocidal drug which has been reported to be a P2‐purinoceptor antagonist on smooth muscle, were investigated in human platelets, where adenosine 5′‐diphosphate (ADP) induces aggregation by acting on a subtype of purinoceptors which has been called P2T. 2 Suramin (100 μm) had no inhibitory effect on ADP‐induced platelet aggregation in plasma, even after 40 min incubation in the presence of bacitracin, a peptidase inhibitor, and did not affect the ability of adenosine 5′‐triphosphate (ATP) (40 μm) to inhibit competitively ADP‐induced aggregation. This lack of effect of suramin on platelets in plasma is probably due to its extensive binding to plasma proteins. 3 In washed platelets, suramin (50–400 μm) acted as an apparently competitive antagonist, causing parallel shifts to the right of the log concentration‐response curve to ADP. No depression of the maximal response to ADP was observed at concentrations of suramin (50–150 μm) for which full log concentration‐response curves to ADP could be obtained, but the slope of the Schild plot was around 2, indicating that this antagonism was not simply competitive. The apparent pA2 value for suramin, taken from this Schild plot, was 4.6. 4 Suramin (200–400 μm) also noncompetitively inhibited aggregation induced by U46619 (a thromboxane receptor agonist) or by 5‐hydroxytryptamine in the presence of adrenaline (100 μm), and caused a depression of the maximal response to these agonists. This nonspecific effect of suramin may explain the high Schild plot slope obtained against ADP. 5 These results provide evidence that the ADP receptor on human platelets is indeed similar to the P2‐purinoceptors responding to adenine nucleotides on smooth muscle and other tissues, and show that suramin cannot distinguish between the proposed subtypes of the P2‐purinoceptors.

Effects of analogues of adenine nucleotides on increases in intracellular calcium mediated by P2T-purinoceptors on human blood platelets

1 By use of a number of analogues of adenine nucleotides, the structure‐activity relationships of the human platelet receptor for adenosine 5′‐diphosphate (ADP) mediating increases in intracellular calcium were investigated, and compared with the known structure‐activity relationships for induction by ADP of platelet aggregation. 2 ADP, 2‐methylthioadenosine 5′‐diphosphate (2‐methylthio‐ADP), adenosine 5′‐O‐(1‐thiodiphosphate) (ADP‐α‐S) and adenosine 5′‐O‐(2‐thiodiphosphate) (ADP‐β‐S) each induced increases in intracellular calcium in a manner similar to their reported ability to induce human platelet aggregation. The effects of these agonists were antagonized by ATP, with a pA2 value in each case consistent with the inhibition by ATP of ADP‐induced aggregation. In the case of ADP, the inhibition by ATP of increases in intracellular calcium was shown to be competitive by Schild analysis. 3 Of the analogues tested as inhibitors of the effect of ADP on intracellular calcium, 2‐chloroadenosine 5′‐triphosphate (2‐chloro‐ATP), adenosine 5′‐O‐(1‐thiotriphosphate) (ATP‐α‐S), P1, P5‐diadenosine pentaphosphate (Ap5A) and adenylyl 5′‐(β,γ‐methylene)diphosphonate (AMPPCP) were apparently competitive antagonists, although only one concentration of each antagonist was used. There was a good correlation between the pA2 values found here for these antagonists including ATP, and their pA2 values reported for inhibition of ADP‐induced aggregation. Adenosine 5′‐(α,β‐methylene)triphosphonate (AMPCPP) and uridine 5′‐triphosphate (UTP) (100 μm) were only very weak inhibitors of the effect of ADP on intracellular calcium, and this is consistent with their weak actions as inhibitors of aggregation. 2‐Methylthioadenosine 5′‐triphosphate (2‐methylthio‐ATP) (50 μm) non‐competitively inhibited the effect of ADP on intracellular calcium, in a very similar way to its inhibition of ADP‐induced aggregation. 4 The good correspondence found for these analogues between their effect on intracellular calcium and on aggregation confirms that there is a causal relationship between these actions of ADP, and that they are mediated by the same receptor on platelets. These findings cast further doubt on the use of the affinity reagent 5′‐fluorosulphonylbenzoyladenosine (FSBA) as an antagonist and label for the ADP receptor, as this compound has been reported to inhibit aggregation but not ADP‐induced increases in intracellular calcium.

A specific CCR3 chemokine receptor antagonist inhibits both early and late phase allergic inflammation in the conjunctiva

Allergic inflammation manifests as one of a number of diseases, including asthma, dermatitis, food allergy, vernal keratoconjunctivitis, and systemic anaphylaxis. Together these diseases affect nearly 25% of the Western world and are a leading health-care problem. The diseases are often biphasic, with an early phase driven primarily by mast cell degranulation and a late phase characterized by leukocyte recruitment. While chemokines are well known to be critical for leukocyte recruitment, their importance in early-phase reactions is poorly defined. We show here that administration of a single oral dose of a high affinity and highly selective CCR3 antagonist ablates both the early and late phase reactions in a mouse model of allergic conjunctivitis. A direct analysis of mast cells in the conjunctiva demonstrates that antagonism of the CCR3 receptor stabilizes the mast cell in vivo, thereby leading to the impaired early phase reaction. The late phase reaction is also strongly inhibited as characterized by both reduced eosinophilia and neutrophilia. These results constitute the first direct evidence that antagonism of CCR3 has clear potential for the treatment of allergic diseases.

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.

Distinct Conformations of the Chemokine Receptor CCR4 with Implications for Its Targeting in Allergy

CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another.

Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4.

Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: A practical guide to assessing functional data.

Stephensons empirical definition of an agonist, as a ligand with binding affinity and intrinsic efficacy (the ability to activate the receptor once bound), underpins classical receptor pharmacology. Quantifying intrinsic efficacy using functional concentration response relationships has always presented an experimental challenge. The requirement for realistic determination of efficacy is emphasised by recent developments in our understanding of G protein coupled receptor (GPCR) agonists, with recognition that some ligands stabilise different active conformations of the receptor, leading to pathway-selective, or biased agonism. Biased ligands have potential as therapeutics with improved selectivity and clinical efficacy, but there are also pitfalls to the identification of pathway selective effects. Here we explore the basics of concentration response curve analysis, beginning with the need to distinguish ligand bias from other influences of the functional system under study. We consider the different approaches that have been used to quantify and compare biased ligands, many of which are based on the Black and Leff operational model of agonism. Some of the practical issues that accompany these analyses are highlighted, with opportunities to improve estimates in future, particularly in the separation of true agonist intrinsic efficacy from the contributions of system dependent coupling efficiency. Such methods are by their nature practical approaches, and all rely on Stephensons separation of affinity and efficacy parameters, which are interdependent at the mechanistic level. Nevertheless, operational analysis methods can be justified by mechanistic models of GPCR activation, and if used wisely are key elements to biased ligand identification.

Synthesis and Structure–Activity Relationships of Indazole Arylsulfonamides as Allosteric CC-Chemokine Receptor 4 (CCR4) Antagonists

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.