Pharmacochemistry of the platelet purinergic receptors

Abstract

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y(1) and P2Y(12) receptors, a P2Y(14) receptor (GPR105) of unknown function, and anti-aggregatory A(2A) and A(2B) adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure-activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y(1), P2Y(12), and P2Y(14) receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y(12) receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y(12) receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y(1) receptor or the P2Y(12) receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A(2A) AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A(2A) agonist CGS21680 and the P2Y(1) receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.

Fig. 1

Nucleotide derivatives that activate P2X and P2Y receptors, with emphasis on agonist ligands for studying these receptors in platelets. a Adenine nucleotides. b Uracil nucleotides. Phosphate derivatives would exist predominantly in an ionized form under physiological conditions

Fig. 2

Thienopyridines as non-nucleotide antagonists of the P2Y₁₂ receptor that require activation in vivo. Enzymatic formation of the active metabolites precedes the formation of the disulfide bond with the target P2Y₁₂ receptor on platelets. CYP=cytochrome P450

Fig. 3

a, b Molecular model of the human P2Y₁ receptor based on the structure of the β₂-adrenergic receptor. The P2Y₁ receptor is shown in complex with a selective antagonist (MRS2500) (b) and a selective agonist (MRS2365) (a). The residues shown are those that, when mutated, lead to a decrease of potency of 20 times or higher. The helices are color coded as TM1 (red), TM2 (orange), TM3 (yellow), TM4 (light green), TM5 (dark green), TM6 (cyan), and TM7 (purple). c, d Molecular model of the human P2Y₁₂ receptor based on the structure of the A_2A adenosine receptor. The P2Y₁₂ receptor is shown in complex with a nonselective agonist (2-MeSADP) (c) and a selective non-nucleotide antagonist (PSB-0739) (d). The helices are color-coded in a progression from TM1 (red) to TM7 (yellow). Two disulfide bridges and a salt bridge give rigidity to the extracellular domains of the P2Y₁ and P2Y₁₂ receptors. In the antagonist-bound state of the P2Y₁ receptor, a salt-bridge between R128 and D204 provides an additional link between TM3 and EL2. Our models suggest that agonist binding causes a disruption of this additional bridge as well as a counter-clockwise rotation (when observed from the extracellular side) of Lys280(6.55). Molecular modeling of these receptors was reported previously [24, 25, 35, 36, 38, 40, 41, 67, 89, 117]

Fig. 4

Nucleotide derivatives that have been useful as antagonists in the study of the P2X and P2Y receptors in platelets. Phosphate derivatives would exist predominantly in an ionized form under physiological conditions

Fig. 5

Non-nucleotides antagonists: Nonselective antagonists and their derivatives and P2X₁ selective antagonists. Phosphate, sulfonate, and carboxylate derivatives would exist predominantly in ionized forms under physiological conditions

Fig. 6

Selective non-nucleotides that have been useful antagonists in the study of P2Y receptors in platelets. See Fig. 2 for thienopyridine antagonists of the P2Y₁₂ receptor. Sulfonate and carboxylate derivatives would exist predominantly in ionized forms under physiological conditions