the most consumed psychoactive substance worldwide, may
have beneficial effects on Parkinson’s disease (PD) therapy.
The mechanism by which caffeine contributes to its antiparkinsonian
effects by acting as either an adenosine A2A receptor (A2AR) neutral antagonist or an inverse agonist is unresolved.
Here we show that caffeine is an A2AR inverse agonist in
cell-based functional studies and in experimental parkinsonism. Thus,
we observed that caffeine triggers a distinct mode, opposite to A2AR agonist, of the receptor’s activation switch leading
to suppression of its spontaneous activity. These inverse agonist-related
effects were also determined in the striatum of a mouse model of PD,
correlating well with increased caffeine-mediated motor effects. Overall,
caffeine A2AR inverse agonism may be behind some of the
well-known physiological effects of this substance both in health
and disease. This information might have a critical mechanistic impact
for PD pharmacotherapeutic design.
The origins of the Laboratory of Bioorganic Chemistry, NIDDK, NIH can be traced to events that occurred in the early 20th century. From its beginning to the present, as the laboratory evolved through several organizational changes, many important historical contributions to organic chemistry and biochemistry were made. For example, its early precursor, the Division of Chemistry of the Hygienic Laboratory, was assigned the responsibility of safeguarding public health by analyzing environmental and other chemical risks. This review will trace important developments from the early twentieth century to the present. The topics covered in this review include a historical synopsis, early work on receptors, carbohydrates, heterocycles and nucleotides, with specific emphasis on frog skin alkaloids, the NIH shift (a transfer of an aromatic hydrogen atom to a neighboring ring position during ring hydroxylation, important in the biochemical processing of aromatic substrates), the methionine-specific cleavage of proteins using cyanogen bromide (used commercially and in peptide research) as well as other fundamental contributions. Ongoing research in medicinal chemistry, natural products, biochemistry, vaccines and pharmacology, some leading to clinical applications, will be discussed.
The adenosinergic system operates
through G protein-coupled adenosine
receptors, which have become promising therapeutic targets for a wide
range of pathological conditions. However, the ubiquity of adenosine
receptors and the eventual lack of selectivity of adenosine-based
drugs have frequently diminished their therapeutic potential. Accordingly,
here we aimed to develop a new generation of light-switchable adenosine
receptor ligands that change their intrinsic activity upon irradiation,
thus allowing the spatiotemporal control of receptor functioning (i.e.,
receptor activation/inactivation dependent on location and timing).
Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective
adenosine receptor agonist, nucleoside derivative MRS5543 containing
an aryl diazo linkage on the N6 substituent, which in the
dark (relaxed isomer) behaved as a full adenosine A3 receptor
(A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue
light (460 nm), it remained a full A3R agonist but became
an A2AR antagonist. Interestingly, molecular modeling suggested
that structural differences encountered within the third extracellular
loop of each receptor could modulate the intrinsic, receptor subtype-dependent,
activity. Overall, the development of adenosine receptor ligands with
photoswitchable activity expands the pharmacological toolbox in support
of research and possibly opens new pharmacotherapeutic opportunities.
The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells.
Adenosine receptors (ARs) are members
of the G protein-coupled
receptor (GPCR) superfamily and have shown much promise as therapeutic
targets. We have used an agonist-bound A2AAR X-ray crystallographic
structure to design a chemically reactive agonist for site-specific
chemical modification of the receptor. To further explore and chemically
engineer its binding cavity, a 2-nitrophenyl active ester was attached
through an elongated chain at adenine C2 position. This general structure
was designed for irreversible transfer of a terminal acyl group to
a nucleophilic amino group on the A2AAR. Preincubation
with several O-acyl derivatives prevented radioligand
binding that was not regenerated upon extensive washing. In silico
receptor docking suggested two lysine residues (second extracellular
loop) as potential target sites for an O-acetyl derivative
(MRS5854, 3a), and site-directed mutagenesis indicated
that K153 but not K150 is essential. Similarly, a butyl azide for
click reaction was incorporated in the active ester moiety (3b). These promising results indicate a stable, covalent modification
of the receptor by several reactive adenosine derivatives, which could
be chemical tools for future imaging, structural probing, and drug
discovery. Thus, structure-based ligand design has guided the site-specific
modification of a GPCR.
G protein-coupled receptor; nucleoside; adenosine
receptor; covalent modification; affinity labeling
ligand display system can be used to dissect the
multivalent effects of ligand binding to a membrane receptor. An antagonist
of the A2A adenosine receptor, a G-protein-coupled receptor
that is a drug target for neurodegenerative conditions, was displayed
in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics
was developed to interpret the binding data, suggesting the importance
of receptor dimers. Using this model, extended multivalent arrangements
of ligands were constructed with progressive improvements in binding
to A2A. The results highlight the ability to use a highly
controllable multivalent approach to determine optimal ligand valency
and spacing that can be subsequently optimized for binding to a membrane
receptor. Models explaining the multivalent binding data are also
Binding assays and assays of activation of adenylate cyclase with the agonists 5′-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.
2-Arylethynyl-(N)-methanocarba adenosine 5′-methyluronamides containing rigid N6-(trans-2-phenylcyclopropyl) and 2-phenylethynyl groups were synthesized as agonists for probing structural features of the A3 adenosine receptor (AR). Radioligand binding confirmed A3AR selectivity and N6-1S,2R stereoselectivity for one diastereomeric pair. The environment of receptor-bound, conformationally constrained N6 groups was explored by docking to an A3AR homology model, indicating specific hydrophobic interactions with the second extracellular loop able to modulate the affinity profile. 2-Pyridylethynyl derivative 18 was administered orally in mice to reduce chronic neuropathic pain in the chronic constriction injury model.
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylate cyclase
Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A3AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of β-D-apio-D-furano- and α-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N6 of β-D-apio-D-furanoadenosine afforded an A3AR antagonist (10c, Ki = 0.98 μM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki = 3.07 μM). The structural basis for this difference was examined by docking to an A3AR model; the antagonist lacked a crucial interaction with Thr94.
G protein-coupled receptor; apionucleosides; Adenosine A3 receptor
G protein-coupled A2B adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A2BAR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A2BAR. In cAMP accumulation assays, both 5′-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5′-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A2BAR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A2BAR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative ‘operational model’ characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N6-Substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A2BAR antagonist in MIN-6 mouse pancreatic β cells expressing low A2BAR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target.
GPCR; adenosine receptor; purines; cyclic AMP; calcium; arrestin
The conventional route to alkoxyamine hydrochloride derivatives is by reaction of alkyl bromides with N-hydroxyphthalimide or N-hydroxysuccinimide followed by addition of hydrazine and HCl. Transformation of an alkyl bromide to the corresponding alkoxyamine hydrochloride can be accomplished more rapidly in high yield and without using hazardous hydrazine by reaction of (Boc)2NOH (N,N’-di-tert-butoxycarbonylhydroxylamine) and alkyl bromide followed by addition of HCl. Alkoxyamine hydrochlorides are powerful reagents in organic synthesis that can be used to synthesize alkoxyimino derivatives after condensation with a ketone or aldehyde.
alkoxyamine; alkyl bromide; O-alkylation
thermodynamics of ligand–receptor interactions at the
surface of living cells represents a fundamental aspect of G protein-coupled
receptor (GPCR) biology; thus, its detailed elucidation constitutes
a challenge for modern pharmacology. Interestingly, fluorescent ligands
have been developed for a variety of GPCRs in order to monitor ligand–receptor
binding in living cells. Accordingly, new methodological strategies
derived from noninvasive fluorescence-based approaches, especially
fluorescence resonance energy transfer (FRET), have been successfully
developed to characterize ligand–receptor interactions. Importantly,
these technologies are supplanting more hazardous and expensive radioactive
binding assays. In addition, FRET-based tools have also become extremely
powerful approaches for visualizing receptor–receptor interactions
(i.e., GPCR oligomerization) in living cells. Thus, by means of the
synthesis of compatible fluorescent ligands these novel techniques
can be implemented to demonstrate the existence of GPCR oligomerization
not only in heterologous systems but also in native tissues. Finally,
there is no doubt that these methodologies would also be relevant
in drug discovery in order to develop new high-throughput screening
approaches or to identify new therapeutic targets. Overall, herein,
we provide a thorough assessment of all technical and biological aspects,
including strengths and weaknesses, of these fluorescence-based methodologies
when applied to the study of GPCR biology at the plasma membrane of
John W. Daly was engaged in groundbreaking basic research for nearly 50 years at NIH in Bethesda, Maryland. A primary focus of his research included the discovery, structure elucidation, synthesis and pharmacology of alkaloids and other biologically active natural products. However, he earned further acclaim in other areas that included the investigation of the structure-activity relationships for agonists/antagonists at adenosine, adrenergic, histamine, serotonin, and acetylcholine receptors. In addition he was a pioneer in studies of the modulation and functional relationships for systems involving calcium, cyclic nucleotides, ion channels and phospholipids and in the mechanism of actions of caffeine and other xanthines.
Adenosine receptors (ARs) and P2Y receptors for purine and pyrimidine nucleotides have widespread distribution and regulate countless physiological processes. Various synthetic ligands are in clinical trials for treatment of inflammatory diseases, pain, cancer, thrombosis, ischemia, and other conditions. The methanocarba (bicyclo[3.1.0]hexane) ring system as a rigid substitution for ribose, which maintains either a North (N) or South (S) conformation, tends to preserve or enhance the potency and/or selectivity for certain receptor subtypes. This review summarizes recent developments in the synthetic approaches to these biologically important nucleoside and nucleotide analogues.
4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we probed two positions (N4 and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y6R.
5'-Ether derivatives of the potent adenosine agonist N6-cyclopentyladenosine (CPA) were designed as “caged” ligands for the activation of A1-adenosine receptors following in situ photolysis. The synthesis involved a 2',3'-diol protection scheme using the acid labile ethoxymethynyl group. Generation of CPA was demonstrated chromatographically and in a bioassay measuring the inhibition of synaptic potentials in the rat hippocampus.
Homologated analogues 3a and 3b of potent and selective A3 adenosine receptor ligands, IB-MECA and dimethyl-IB-MECA were synthesized from commercially available 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose (4) via Co2(CO)8-catalyzed siloxymethylation as a key step. Unfortunately, homologated analogues 3a and 3b did not show significant binding affinities at three subtypes of adenosine receptors, indicating that free rotation, resulting from homologation, induced unfavorable interactions in the binding site of the receptor maybe due to the presence of many conformations.
Co2(CO)8-catalyzed siloxymethylation; A3 adenosine receptor; Homologation
John Daly played an important role in defining adenosine receptors as an important target for drug discovery. His systematic work characterized the effects of adenosine analogues on cyclic AMP in the brain that were antagonized by methylxanthines. He also played a decisive role in establishing these receptors as bona fide biochemical entities and contributed to the discovery of receptor heterogeneity. This brief review will cover some of his important early discoveries in the pharmacology and medicinal chemistry of adenosine receptors.
Adenosine analogues modified at the 5′-position as uronamides and/or as N6-benzyl derivatives were synthesized. These derivatives were examined for affinity in radioligand binding assays at the newly discovered rat brain A3 adenosine receptor and at rat brain A1 and A2a receptors. 5′-Uronamide substituents favored A3 selectivity in the order N-methyl > N-ethyl ∞ unsubstituted carboxamide > N-cyclopropyl. 5′-(N-Methylcarboxamido)-N6-benzyladenosine was 37–56-fold more selective for A3 receptors. Potency at A3 receptors was enhanced upon substitution of the benzyl substituent with nitro and other groups. 5′-N-Methyluronamides and N6-(3-substituted-benzyl)adenosines are optimal for potency and selectivity at A3 receptors. A series of 3-(halobenzyl)-5′-N-ethyluronamide derivatives showed the order of potency at A1 and A2a receptors of I ~ Br > Cl > F. At A3 receptors the 3-F derivative was weaker than the other halo derivatives. 5′-N-Methyl-N6-(3-iodobenzyl)adenosine displayed a Ki value of 1.1 nM at A3 receptors and selectivity versus A1 and A2a receptors of 50-fold. A series of methoxybenzyl derivatives showed that a 4-methoxy group best favored A3 selectivity. A 4-sulfobenzyl derivative was a specific ligand at A3 receptors of moderate potency. An aryl amino derivative was prepared as a probe for radioiodination and receptor cross-linking.
Adenosine has been found to be cardioprotective during episodes of cardiac ischemia/reperfusion through activation of the A1 and possibly A3 receptors. Therefore, we have investigated whether activation of these receptors can protect also against apoptotic death induced by angiotensin II (Ang II) in neonatal rat cardiomyocyte cultures. Exposure to Ang II (10 nM) resulted in a 3-fold increase in programmed cell death (p < 0.05). Pretreatment with the A1 adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1 μM), abolished the effects of Ang II on programmed cardiomyocyte death. Moreover, exposure of cells to the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) before pretreatment with CCPA, prevented the protective effect of the latter. Pretreatment with the A3 adenosine receptor agonist N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA, 0.1 μM), led to a partial decrease in apoptotic rate induced by Ang II. Exposure of myocytes to Ang II caused an immediate increase in the concentration of intracellular free Ca2+ that lasted 40–60 sec. Pretreatment of cells with CCPA or IB-MECA did not block Ang II-induced Ca2+ elevation. In conclusion, activation of adenosine A1 receptors can protect the cardiac cells from apoptosis induced by Ang II, while activation of the adenosine A3 receptors confers partial cardioprotection.
A1 and A3 adenosine receptors; angiotensin II; apoptosis; cardiomyocytes; Feulgen and TUNEL stainings
Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonists, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coil, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5–770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6–8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coli P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.
We have previously found that uridine 5′-triphosphate (UTP) significantly reduced cardiomyocyte death induced by hypoxia via activating P2Y2 receptors. To explore the effect of UTP following myocardial infarction (MI) in vivo we studied four groups: sham with or without LAD ligation, injected with UTP (0.44 µg/kg i.v.) 30 min before MI, and UTP injection (4.4 µg/kg i.v.) 24 h prior to MI. Left ventricular end diastolic area (LVEDA), end systolic area (LVESA) fractional shortening (FS), and changes in posterior wall (PW) thickness were performed by echocardiography before and 24 h after MI. In addition, we measured different biochemical markers of damage and infarct size using Evans blue and TTC staining. The increase in LVEDA and LVESA of the treated animals was significantly smaller when compared to the MI rats (p < 0.01). Concomitantly, FS was higher in groups pretreated with UTP 30 min or 24 h (56 ± 14.3 and 36.7 ± 8.2%, p < 0.01, respectively). Ratio of infarct size to area at risk was smaller in the UTP pretreated hearts than MI rats (22.9 ± 6.6, 23.1 ± 9.1%, versus 45.4 ± 7.6%, respectively, p < 0.001). Troponin T and ATP measurements, demonstrated reduced myocardial damage. Using Rhod-2-AM loaded cardiomyocytes, we found that UTP reduced mitochondrial calcium levels following hypoxia. In conclusion, early or late UTP preconditioning is effective, demonstrating reduced infarct size and superior myocardial function. The resulting cardioprotection following UTP treatment post ischemia demonstrates a reduction in mitochondrial calcium overload, which can explain the beneficial effect of UTP.
Heart protection; Ischemia; P2Y receptors
In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-γ-S and UDP-β-S are selective agonists for P2Y2/P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7.
Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface—the Gq-coupled P2Y1 subtype and the Gi-coupled P2Y12 subtype—are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methano-carba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y1 receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y12 receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X1 receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although α,β-methylene–adenosine triphosphate (ATP) is the classic agonist for the P2X1 receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X1 receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y1 receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.
G protein–coupled receptors; P2Y receptors; agonist; antagonist; mutant