Adriaan P. IJzerman

The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist

The adenosine class of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

The adenosine class of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.

International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and Classification of Adenosine Receptors—An Update

In the 10 years since our previous International Union of Basic and Clinical Pharmacology report on the nomenclature and classification of adenosine receptors, no developments have led to major changes in the recommendations. However, there have been so many other developments that an update is needed. The fact that the structure of one of the adenosine receptors has recently been solved has already led to new ways of in silico screening of ligands. The evidence that adenosine receptors can form homo- and heteromultimers has accumulated, but the functional significance of such complexes remains unclear. The availability of mice with genetic modification of all the adenosine receptors has led to a clarification of the functional roles of adenosine, and to excellent means to study the specificity of drugs. There are also interesting associations between disease and structural variants in one or more of the adenosine receptors. Several new selective agonists and antagonists have become available. They provide improved possibilities for receptor classification. There are also developments hinting at the usefulness of allosteric modulators. Many drugs targeting adenosine receptors are in clinical trials, but the established therapeutic use is still very limited.

Structural basis for allosteric regulation of GPCRs by sodium ions.

GPCR Close-Up Structures of G protein–coupled receptors (GPCRs) determined in the past few years, have provided insight into the function of this important family of membrane proteins. Liu et al. (p. 232) used a protein-engineering strategy to produce a stabilized version of the human A2Aadenosine receptor (A2AAR). The high-resolution structure reveals the position of about 60 internal waters, which suggests an almost continuous channel in the GPCR and can explain the allosteric effects of Na+ on ligand binding and how cholesterol may contribute to GPCR stabilization. A protein-engineering strategy yields a closer look at the receptor-bound water, sodium, and lipid molecules. Pharmacological responses of G protein–coupled receptors (GPCRs) can be fine-tuned by allosteric modulators. Structural studies of such effects have been limited due to the medium resolution of GPCR structures. We reengineered the human A2A adenosine receptor by replacing its third intracellular loop with apocytochrome b562RIL and solved the structure at 1.8 angstrom resolution. The high-resolution structure allowed us to identify 57 ordered water molecules inside the receptor comprising three major clusters. The central cluster harbors a putative sodium ion bound to the highly conserved aspartate residue Asp2.50. Additionally, two cholesterols stabilize the conformation of helix VI, and one of 23 ordered lipids intercalates inside the ligand-binding pocket. These high-resolution details shed light on the potential role of structured water molecules, sodium ions, and lipids/cholesterol in GPCR stabilization and function.

Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase

1 FPL 67156 (6‐N,N‐diethyl‐β,γ‐dibromomethylene‐d‐ATP), is a newly synthesized analogue of ATP. 2 In a rabbit isolated tracheal epithelium preparation, measuring P2U‐purinoceptor‐dependent chloride secretion, FPL 67156 was discovered to potentiate the responses to UTP but not those to ATP‐γ‐S. UTP agonist‐concentration effect (E/[A]) curves were shifted to the left by 5‐fold in the presence of 100 μm FPL 67156. The differential effect of FPL 67156 on UTP and ATP‐γ‐S was hypothesized to be due to the greater susceptibility of UTP to enzymatic dephosphorylation and the ability of FPL 67156 to inhibit this process. 3 FPL 67156 was tested as an ecto‐ATPase inhibitor in a human blood cell assay, measuring [γ32P]‐ATP dephosphorylation. The compound inhibited [γ32P]‐ATP degradation with a pIC50 of 4.6. 4 FPL 67156 was then tested for its effects on ATP and α,β‐methylene‐ATP responses at P2X‐purinoceptors in the rabbit isolated ear artery. In the concentration range 30 μm‐1 mm, the compound potentiated the contractile effects of ATP but not those of α,β‐methylene‐ATP. At 1 mm, FPL 67156 produced a 34‐fold leftward shift of ATP E/[A] curves. 5 The effects of FPL 67156 on ATP E/[A] curves in the rabbit ear artery were analysed using a theoretical model (Furchgott, 1972) describing the action of an enzyme inhibitor on the effects of a metabolically unstable agonist. This analysis provided an estimate of the pK1 for FPL 67156 as an ecto‐ATPase inhibitor of 5.2. 6 Using appropriate assays, FPL 67156 was shown to have weak antagonist effects at P2X‐ and P2T‐purinoceptors (pA2 ≅ 3.3 and 3.5 respectively), and weak agonist effects at P2U‐purinoceptors (p[A50] ≅ 3.5). 7 The degree of potentiation of ATP and UTP effects elicited by FPL 67156 confirms previous results concerning the influence that ecto‐ATPase has on the position of E/[A] curves for metabolically unstable agonists. The magnitude of this influence is predicted to have a major effect on the agonist potency orders currently used to designate purinoceptors. 8 This study indicates FPL 67156 to be a potentially valuable probe in studies on the action of nucleotides and in the classification of purinoceptors.

Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation

G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process.

Drug-target residence time--a case for G protein-coupled receptors.

A vast number of marketed drugs act on G protein‐coupled receptors (GPCRs), the most successful category of drug targets to date. These drugs usually possess high target affinity and selectivity, and such combined features have been the driving force in the early phases of drug discovery. However, attrition has also been high. Many investigational new drugs eventually fail in clinical trials due to a demonstrated lack of efficacy. A retrospective assessment of successfully launched drugs revealed that their beneficial effects in patients may be attributed to their long drug‐target residence times (RTs). Likewise, for some other GPCR drugs short RT could be beneficial to reduce the potential for on‐target side effects. Hence, the compounds’ kinetics behavior might in fact be the guiding principle to obtain a desired and durable effect in vivo. We therefore propose that drug‐target RT should be taken into account as an additional parameter in the lead selection and optimization process. This should ultimately lead to an increased number of candidate drugs moving to the preclinical development phase and on to the market. This review contains examples of the kinetics behavior of GPCR ligands with improved in vivo efficacy and summarizes methods for assessing drug‐target RT.

Pharmacological analysis of ecto-ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2X-purinoceptor ligands

1 Previous studies have shown that suramin and FPL 66301 are competitive antagonists at the P2X‐purinoceptor in the rabbit ear artery. Those studies employed α,β‐methylene ATP, a poorly hydrolysable ATP analogue, as the agonist. In this study these compounds have been tested using ATP as the agonist. 2 Suramin, in the concentration range 30–1000 μm, potentiated the contractile effects of ATP, producing a 3‐fold leftward shift of the ATP E/[A] curves. FPL 66301, in the concentration range 100–1000 μm, produced a significant but small (3‐fold) rightward shift of the ATP curves. These results are in marked contrast with previous studies using α,β‐methylene ATP in which 30‐fold rightward shifts were achieved using the same concentration ranges of suramin and FPL 66301. 3 Suramin and FPL 66301 were tested as ecto‐ATPase inhibitors in a human blood cell assay. Suramin inhibited the enzyme with a pIC50 of 4.3, FPL 66301 with a pIC50 of 3.3. 4 The pharmacological data were analysed using a theoretical model describing the action of a compound with dual enzyme inhibitory and receptor antagonistic properties on the effects of an agonist susceptible to enzymatic degradation. The model was found to fit the data well using the known pKBestimates for suramin and FPL 66301 and similar relative (but not absolute) pK1 estimates to those obtained for the compounds in the enzyme assay. 5 From this analysis it was concluded that the limited shifts of ATP E/[A] curves produced by suramin and FPL 66301 were the result of ‘self‐cancellation’ of the potentiating (enzyme inhibitory) and rightward‐shifting (receptor antagonistic) properties. 6 The analysis also indicated that the presence of ecto‐ATPase activity in the rabbit ear artery preparation has a marked effect on the apparent potency of ATP. The experimental p[A50] was 3.4, whereas the ‘true’ value, that is the value which would be obtained in the absence of ecto‐ATPase activity, was 6.0, some 400‐fold higher. 7 Two conclusions are drawn from this study. Firstly, caution must be exercised in the use of suramin and FPL 66301 as tools for receptor classification. Absence of overt antagonism by these compounds when metabolically unstable agonists are used could lead to erroneous claims for receptor subtypes. Secondly, the agonist potency order currently used to designate P2X‐ purinoceptors may require modification.

Proteochemometric modeling as a tool to design selective compounds and for extrapolating to novel targets

‘Proteochemometric modeling’ is a bioactivity modeling technique founded on the description of both small molecules (the ligands), and proteins (the targets). By combining those two elements of a ligand – target interaction proteochemometrics techniques model the interaction complex or the full ligand – target interaction space, and they are able to quantify the similarity between both ligands and targets simultaneously. Consequently, proteochemometric models or complex based models, can be considered an extension of QSAR models, which are ligand based. As proteochemometric models are able to incorporate target information they outperform conventional QSAR models when extrapolating from the activities of known ligands on known targets to novel targets. Vice versa, proteochemometrics can be used to virtually screen for selective compounds that are solely active on a single member of a subfamily of targets, as well as to select compounds with a desired bioactivity profile – a topic particularly relevant with concepts such as ‘ligand polypharmacology’ in mind. Here we illustrate the concept of proteochemometrics and provide a review of relevant methodological publications in the field. We give an overview of the target families proteochemometrics modeling has previously been applied to, and introduce some novel application areas of the modeling technique. We conclude that proteochemometrics is a promising technique in preclinical drug research that allows merging data sets that were previously considered separately, with the potential to extrapolate more reliably both in ligand as well as target space.

2-Amino-6-furan-2-yl-4-substituted nicotinonitriles as A2A adenosine receptor antagonists.

A 2A adenosine receptor antagonists usually have bi- or tricyclic N aromatic systems with varying substitution patterns to achieve desired receptor affinity and selectivity. Using a pharmacophore model designed by overlap of nonxanthine type of previously known A 2A antagonists, we synthesized a new class of compounds having a 2-amino nicotinonitrile core moiety. From our data, we conclude that the presence of at least one furan group rather than phenyl is beneficial for high affinity on the A 2A adenosine receptor. Compounds 39 (LUF6050) and 44 (LUF6080) of the series had K i values of 1.4 and 1.0 nM, respectively, with reasonable selectivity toward the other adenosine receptor subtypes, A 1, A 2B, and A 3. The high affinity of 44 was corroborated in a cAMP second messenger assay, yielding subnanomolar potency for this compound.