Silvia Paoletta

Structure of the human P2Y12 receptor in complex with an antithrombotic drug

P2Y receptors (P2YRs), a family of purinergic G-protein-coupled receptors (GPCRs), are activated by extracellular nucleotides. There are a total of eight distinct functional P2YRs expressed in human, which are subdivided into P2Y1-like receptors and P2Y12-like receptors. Their ligands are generally charged molecules with relatively low bioavailability and stability in vivo, which limits our understanding of this receptor family. P2Y12R regulates platelet activation and thrombus formation, and several antithrombotic drugs targeting P2Y12R—including the prodrugs clopidogrel (Plavix) and prasugrel (Effient) that are metabolized and bind covalently, and the nucleoside analogue ticagrelor (Brilinta) that acts directly on the receptor—have been approved for the prevention of stroke and myocardial infarction. However, limitations of these drugs (for example, a very long half-life of clopidogrel action and a characteristic adverse effect profile of ticagrelor) suggest that there is an unfulfilled medical need for developing a new generation of P2Y12R inhibitors. Here we report the 2.6 Å resolution crystal structure of human P2Y12R in complex with a non-nucleotide reversible antagonist, AZD1283. The structure reveals a distinct straight conformation of helix V, which sets P2Y12R apart from all other known class A GPCR structures. With AZD1283 bound, the highly conserved disulphide bridge in GPCRs between helix III and extracellular loop 2 is not observed and appears to be dynamic. Along with the details of the AZD1283-binding site, analysis of the extracellular interface reveals an adjacent ligand-binding region and suggests that both pockets could be required for dinucleotide binding. The structure provides essential insights for the development of improved P2Y12R ligands and allosteric modulators as drug candidates.

Agonist-bound structure of the human P2Y12 receptor

The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, is one of the most prominent clinical drug targets for inhibition of platelet aggregation. Although mutagenesis and modelling studies of the P2Y12R provided useful insights into ligand binding, the agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here we report the structures of the human P2Y12R in complex with the full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. These structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283), reveal striking conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions. Further analysis of these changes provides insight into a distinct ligand binding landscape in the δ-group of class A G-protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing questions surrounding P2Y12R–agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example, to our knowledge, of a GPCR in which agonist access to the binding pocket requires large-scale rearrangements in the highly malleable extracellular region, the structural and docking studies will therefore provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.

Two disparate ligand-binding sites in the human P2Y1 receptor.

In response to adenosine 5′-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1Rs unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.

2-Phenylpyrazolo[4,3-d]pyrimidin-7-one as a New Scaffold To Obtain Potent and Selective Human A3 Adenosine Receptor Antagonists: New Insights into the Receptor-Antagonist Recognition

A molecular simplification approach of previously reported 2-arylpyrazolo[3,4-c]quinolin-4-ones was applied to design 2-arylpyrazolo[4,3-d]pyrimidin-7-one derivatives as new human A(3) adenosine receptor antagonists. Substituents with different lipophilicity and steric hindrance were introduced at the 5-position of the bicyclic scaffold (R(5) = H, Me, Et, Ph, CH(2)Ph) and on the 2-phenyl ring (OMe, Me). Most of the synthesized derivatives were highly potent hA(3) adenosine receptor antagonists, the best being the 2-(4-methoxyphenyl)pyrazolo[4,3-d]pyrimidin-7-one (K(i) = 1.2 nM). The new compounds were also highly selective, being completely devoid of affinity toward hA(1), hA(2A), and hA(2B) adenosine receptors. On the basis of the recently published human A(2A) receptor crystallographic information, we propose a novel receptor-driven hypothesis to explain both A(3) AR affinity and A(3) versus A(2A) selectivity profiles of these new antagonists.

Nucleotides Acting at P2Y Receptors: Connecting Structure and Function.

Eight G protein–coupled P2Y receptor (P2YR) subtypes are important physiologic mediators. The human P2YRs are fully activated by ATP (P2Y2 and P2Y11), ADP (P2Y1, P2Y12, and P2Y13), UTP (P2Y2 and P2Y4), UDP (P2Y6 and P2Y14), and UDP glucose (P2Y14). Their structural elucidation is progressing rapidly. The X-ray structures of three ligand complexes of the Gi-coupled P2Y12R and two of the Gq-coupled P2Y1Rs were recently determined and will be especially useful in structure-based ligand design at two P2YR subfamilies. These high-resolution structures, which display unusual binding site features, complement mutagenesis studies for probing ligand recognition and activation. The structural requirements for nucleotide agonist recognition at P2YRs are relatively permissive with respect to the length of the phosphate moiety, but less so with respect to base recognition. Nucleotide-like antagonists and partial agonists are also known for P2Y1, P2Y2, P2Y4, and P2Y12Rs. Each P2YR subtype has the ability to be activated by structurally bifunctional agonists, such as dinucleotides, typically, dinucleoside triphosphates or tetraphosphates, and nucleoside polyphosphate sugars (e.g., UDP glucose) as well as the more conventional mononucleotide agonists. A range of dinucleoside polyphosphates, from triphosphates to higher homologs, occurs naturally. Earlier modeling predictions of the P2YRs were not very accurate, but recent findings have provided much detailed structural insight into this receptor family to aid in the rational design of new drugs.

The significance of 2-furyl ring substitution with a 2-(para-substituted) aryl group in a new series of pyrazolo-triazolo-pyrimidines as potent and highly selective hA3 adenosine receptors antagonists: new insights into structure-affinity relationship and receptor-antagonist recognition

Among the heterocyclic structures identified as potent human A(3) (hA(3)) adenosine receptors antagonists, we have demonstrated that the new pyrazolo-triazolo-pyrimidines, bearing an aryl group in replacement of the C(2)-furyl ring, not only confer a good pharmacological profile (with significantly enhanced selectivity against other adenosine receptor subytpes) but also overcome the metabolic transformation of the furan ring into toxic intermediates. All the synthesized [2-(para-substituted) phenyl]-pyrazolo-triazolo-pyrimidines showed affinity at the hA(3) receptor in the low nanomolar range. The most potent derivative of the series presented better affinity and excellent selectivity (compound 31, K(i) hA(3) = 0.108 nM; hA(1)/hA(3) = 5200; hA(2A)/hA(3) = 7200), in comparison to the C(2)-furyl counterpart. A receptor-driven molecular modeling investigation, based on a recently proposed model of A(3) receptor derived from the crystallographic structure of human A(2A) receptor, has been carried out in order to support the experimental binding data and to justify the enhanced selectivity against the other receptor subtypes.

Discovery of novel A3 adenosine receptor ligands based on chromone scaffold.

A project focused on the discovery of new chemical entities (NCEs) as AR ligands that incorporate a benzo-γ-pyrone [(4H)-1-benzopyran-4-one] substructure has been developed. Accordingly, two series of novel chromone carboxamides placed at positions C2 (compounds 2-13) and C3 (compounds 15-26) of the γ-pyrone ring were synthesized using chromone carboxylic acids (compounds 1 or 14) as starting materials. From this study and on the basis of the obtained structure-activity relationships it was concluded that the chromone carboxamide scaffold represent a novel class of AR ligands. The most remarkable chromones were compounds 21 and 26 that present a better affinity for A3AR (Ki = 3680 nM and Ki = 3750 nM, respectively). Receptor-driven molecular modeling studies provide information on the binding/selectivity data of the chromone. The data so far acquired are instrumental for future optimization of chromone carboxamide as a selective A3AR antagonist.

The identification of the 2-phenylphthalazin-1(2H)-one scaffold as a new decorable core skeleton for the design of potent and selective human A3 adenosine receptor antagonists.

Following a molecular simplification approach, we have identified the 2-phenylphthalazin-1(2H)-one (PHTZ) ring system as a new decorable core skeleton for the design of novel hA(3) adenosine receptor (AR) antagonists. Interest for this new series was driven by the structural similarity between the PHTZ skeleton and both the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (TQX) and the 4-carboxamido-quinazoline (QZ) scaffolds extensively investigated in our previously reported studies. Our attention was focused at position 4 of the phthalazine nucleus where different amido and ureido moieties were introduced (compounds 2-20). Some of the new PHTZ compounds showed high hA(3) AR affinity and selectivity, the 2,5-dimethoxyphenylphthalazin-1(2H)-one 18 being the most potent and selective hA(3) AR antagonist among this series (K(i) = 0.776 nM; hA(1)/hA(3) and hA(2A)/hA(3) > 12000). Molecular docking studies on the PHTZ derivatives revealed for these compounds a binding mode similar to that of the previously reported TQX and QZ series, as was expected from the simplification approach.

Rational design of sulfonated A3 adenosine receptor-selective nucleosides as pharmacological tools to study chronic neuropathic pain.

(N)-Methanocarba(bicyclo[3.1.0]hexane)adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g., blood-brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N(6)-p-sulfophenylethyl substituent would determine higher hA3AR vs mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N(6)-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki(hA3AR) = 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered ip reduced mouse chronic neuropathic pain that was ascribed to either A3AR or A1/A3AR using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosines CNS vs peripheral actions.

In vivo phenotypic screening for treating chronic neuropathic pain: modification of C2-arylethynyl group of conformationally constrained A3 adenosine receptor agonists.

(N)-Methanocarba adenosine 5′-methyluronamides containing 2-arylethynyl groups were synthesized as A3 adenosine receptor (AR) agonists and screened in vivo (po) for reduction of neuropathic pain. A small N6-methyl group maintained binding affinity, with human > mouse A3AR and MW < 500 and other favorable physicochemical properties. Emax (maximal efficacy in a mouse chronic constriction injury pain model) of previously characterized A3AR agonist, 2-(3,4-difluorophenylethynyl)-N6-(3-chlorobenzyl) derivative 6a, MRS5698, was surpassed. More efficacious analogues (in vivo) contained the following C2-arylethynyl groups: pyrazin-2-yl 23 (binding Ki, hA3AR, nM 1.8), fur-2-yl 27 (0.6), thien-2-yl 32 (0.6) and its 5-chloro 33, MRS5980 (0.7) and 5-bromo 34 (0.4) equivalents, and physiologically unstable ferrocene 36, MRS5979 (2.7). 33 and 36 displayed particularly long in vivo duration (>3 h). Selected analogues were docked to an A3AR homology model to explore the environment of receptor-bound C2 and N6 groups. Various analogues bound with μM affinity at off-target biogenic amine (M2, 5HT2A, β3, 5HT2B, 5HT2C, and α2C) or other receptors. Thus, we have expanded the structural range of orally active A3AR agonists for chronic pain treatment.