Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist

The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.     

Quantitation of the P2Y(1) receptor with a high affinity radiolabeled antagonist

2-Chloro-N(6)-methyl-(N )-methanocarba-2'-deoxyadenosine-3',5'- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y(1) receptor (P2Y1-R) antagonist (J Med Chem 43:829-842, 2002; Br J Pharmacol 135:2004-2010, 2002). We have taken advantage of the N(6)-methyl substitution in the adenine base to incorporate [(3)H]methylamine into the synthesis of [(3)H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [(3)H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y(2) or P2Y(12) receptors. Equilibrium binding of [(3)H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (K(d) = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R-promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K(d) calculated from independent determinations of the rate constants of association (7.15 x 10(7) M(-1) min(-1)) and dissociation (0.72 min(-1)) of [(3)H]MRS2279 also was in good agreement with the K(d) derived from equilibrium binding studies. Competition binding assays with [(3)H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K(i) values for the P2Y1-R antagonists MRS2279 (K(i) = 13 nM), N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179; K(i) = 84 nM), adenosine-3', 5'-bisphosphate (K(i)=900 nM), and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (K(i) = 6 microM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [(3)H]MRS2279 also bound with high affinity (K(d) = 4-8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [(3)H]MRS2279 bound with high affinity (K(d) = 16 nM) to a binding site on out-dated human platelets (5-35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [(3)H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.     

The histamine H(3) receptor: a target for new drugs

The histamine H3 receptor has been identified in the rat brain as a presynaptic autoreceptor inhibiting histamine synthesis and release. Following its recent cloning, more than fifteen years later, the existence of isoforms, species pharmacological differences and the high constitutive activity of the receptor were established. All these molecular findings have to be taken into account for the optimization of the ligands aiming at a clinical use. They show the interest of antagonists/inverse agonists in the symptomatic treatment of schizophrenia and cognitive and attentional deficits.     

Cangrelor: a novel P2Y12 receptor antagonist

Antiplatelet therapy is critical in the prevention of thrombotic complications of acute coronary syndrome and percutaneous coronary interventions. Current antiplatelet agents (aspirin, clopidogrel and glycoprotein IIb/IIIa antagonists) have demonstrated the capacity to reduce major adverse cardiac events. However, these agents have limitations that compromise their clinical utility. The platelet P2Y₁₂ receptor plays a central role in platelet function and is a focus in the development of antiplatelet therapies. Cangrelor is a potent, competitive inhibitor of the P2Y₁₂ receptor that is administered by intravenous infusion and rapidly achieves near complete inhibition of ADP-induced platelet aggregation. This investigational drug has been studied for use during coronary procedures and the management of patients experiencing acute coronary syndrome and is undergoing evaluation for use in the prevention of perioperative stent thrombosis.     

Inhibition of platelet adhesion to collagen as a new target for antithrombotic drugs

Platelet adhesion to a damaged blood vessel is the initial trigger for arterial hemostasis and thrombosis. Platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between collagen within the damaged vessel wall and the platelet receptor glycoprotein Ib/V/IX (GPIb), an interaction especially important under high shear conditions[1]. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion mediated by the collagen receptors (alpha(2)beta(1), GPVI, ) in addition[2] resulting in platelet activation. This leads to the conformational activation of the platelet alpha(IIb)beta3 receptor, fibrinogen binding and finally to platelet aggregation. Over the past decades, modulation of platelet function has been a strategy for the control of cardiovascular disease. Lately, drugs have been developed that target the fibrinogen receptor alphaIIbbeta3 or the ADP receptor and many of these promising compounds have been tested in clinical trials. However the development of products that interfere with the first step of hemostasis, i.e. the platelet adhesion, has lagged behind. In this review we want to discuss (i) the in vivo studies that were performed with compounds that target proteins involved in different adhesion steps i.e. the VWF-GPIb-axis, the collagen-VWF axis and the collagen-collagen receptor axis and (ii) the possible advantages these putative new drugs could have over the current antiplatelet agents.     

ATP, an agonist at the rat P2Y(4) receptor, is an antagonist at the human P2Y(4) receptor

The nucleotide selectivities of the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor stably expressed in 1321N1 human astrocytoma cells were determined by measuring increases in intracellular [Ca(2+)] under conditions that minimized metabolism, bioconversion, and endogenous nucleotide release. In cells expressing the hP2Y(4) receptor, UTP, GTP, and ITP all increased intracellular [Ca(2+)] with a rank order of potency of UTP (0.55) > GTP (6.59) = ITP (7.38), (EC(50), microM). ATP, CTP, xanthine 5'-triphosphate (XTP), and diadenosine 5',5"'-P(1), P(4)-tetraphosphate (Ap(4)A), all at 100 microM, were inactive at the hP2Y(4) receptor. In cells expressing the rP2Y(4) receptor, all seven nucleotides increased intracellular [Ca(2+)] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0. 51) > Ap(4)A (1.24) approximately ITP (1.82) approximately GTP (2. 28) > CTP (7.24) > XTP (22.9). Because ATP is inactive at the hP2Y(4) receptor, we assessed whether ATP displayed antagonist activity. When coapplied, ATP shifted the concentration-response curve to UTP rightward in a concentration-dependent manner, with no change in the maximal response. A Schild plot derived from these data gave a pA(2) value of 6.15 (K(B) = 708 nM) and a slope near unity. Additionally, CTP and Ap(4)A (each at 100 microM) inhibited the response to an EC(50) concentration of UTP by approximately 40 and approximately 50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap(4)A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y(4) receptor but is a competitive antagonist with moderate potency at the hP2Y(4) receptor.     

Adenosine 5'-O-(1-boranotriphosphate) derivatives as novel P2Y(1) receptor agonists

P2-receptors (P2-Rs) represent important targets for novel drug development. Most ATP analogues proposed as potential drug candidates have shortcomings such as limited receptor-selectivity and limited stability that justify the search for new P2-R agonists. Therefore, a novel series of nucleotides based on the adenosine 5'-O-(1-boranotriphosphate) (ATP-alpha-B) scaffold was developed and tested as P2Y(1)-R agonists. An efficient four-step one-pot synthesis of several ATP-alpha-B analogues from the corresponding nucleosides was developed, as well as a facile method for the separation of the diastereoisomers (A and B isomers) of the chiral products. The potency of the new analogues as P2Y(1)-R agonists was evaluated by the agonist-induced Ca2+ release of HEK 293 cells stably transfected with rat-brain P2Y(1)-R. ATP-alpha-B A isomer was equipotent with ATP (EC50 = 2 x 10(-7) M). However, 2-MeS- and 2-Cl- substitutions on ATP-alpha-B (A isomer) increased the potency of the agonist up to 100-fold, with EC50 values of 4.5 x 10(-9) and 3.6 x 10(-9) M, compared to that of the ATP-alpha-B (A isomer). Diastereoisomers A of all ATP-alpha-B analogues were more potent in inducing Ca2+ release than the corresponding B counterparts, with a 20-fold difference for 2-MeS-ATP-alpha-B analogues. The chemical stability of the new P2Y(1)-R agonists was evaluated by 31P NMR under physiological and gastric-juice pH values at 37 degrees C, with rates of hydrolysis of 2-MeS-ATP-alpha-B of 1.38 x 10(-7) s-1 (t1/2 of 1395 h) and 3.24 x 10(-5) s-1 (t1/2 = 5.9 h), respectively. The enzymatic stability of the new analogues toward spleen NTPDase was evaluated. Most of the new analogues were poor substrates for the NTPDase, with ATP-alpha-B (A isomer) hydrolysis being 5% of the hydrolysis rate of ATP. Diastereoisomers A and B exhibited different stability, with A isomers being significantly more stable, up to 9-fold. Furthermore, A isomers that are potent P2Y(1)-R agonists barely interact with NTPDase, thus exhibiting protein selectivity. Therefore, on the basis of our findings, the new, highly water-soluble, P2Y(1)-R agonists may be considered as potentially promising drug candidates.     

The P2X7 receptor as a therapeutic target

Background: The P2X7 receptor is present in a variety of cell types involved in pain, inflammatory processes and neurodegenerative conditions, thus it may be an appealing target for pharmacological intervention. The extensive use of high-throughput screening (HTS) followed by a hit-to-lead (HtL) program, has prompted a number of firms to identify highly selective and metabolically stable small-molecules possessing activity for both the rat and human P2X₇ receptor, which provide a novel therapeutic approach to the treatment of pain as well as neurodegenerative and inflammatory disorders. Objective: To describe the current status of and potential for development of P2X₇ receptor-antagonists. Methods: A literature review. Results/conclusions: We describe the recent discoveries of novel P2X₇ receptor-selective antagonists, along with their biological activity and therapeutic potential.     

The enteric purinergic P2Y1 receptor

Significant recent discoveries have shown that the P2Y(1) purinergic receptor subtype is expressed in the enteric nervous system and at intestinal neuromuscular junctions. Secretomotor neurons, which release vasoactive intestinal peptide at their junctions with intestinal secretory glands, express the P2Y(1) receptor. Synaptically released ATP acts at these P2Y(1) receptors to stimulate glandular secretion of electrolytes and H(2)O. Motor neurons in the enteric nervous system release ATP as an inhibitory neurotransmitter at neuromuscular junctions in the intestinal circular muscle coat; this action of ATP is mediated by the P2Y(1) receptor. The emerging evidence for significant involvement of P2Y(1) receptors in local enteric neural control and coordination of intestinal secretion and motility suggests that either the receptors themselves or steps in the post-P2Y(1) receptor signal transduction cascade might be potential therapeutic targets.     

(Pro)renin receptor as a new drug target

Over the last few years, the implication of the (pro)renin receptor [(P)RR] in the pathogenesis of end-organ damage has been shown through many different studies. The (P)RR plays a dual role when stimulated by renin or prorenin as it enhances both cell surface production of angiotensin and stimulates angiotensin-independent intracellular signaling cascades. Since Ichihara's group demonstrated activation of prorenin when it was bound to antibodies targeted against a specific region in the renin prosegment, they designed a complementary decapeptide to this region called the handle region to use as a potential (P)RR blocker (PRRB). The effects of systemic administration of the PRRB on the development and progression of different renal, cardiac and ocular pathologies have been observed and have thus proposed the blocker as a potential new treatment for these afflictions. Conversely, the specificity of the PRRB has been questioned as conflicting results have been reported in the literature. A recent study has described a new high affinity binding site for renin and prorenin to the (P)RR called the hinge region. Hence, although there is great promise in the (P)RR potential as a therapeutic target, still much research is required to better identify adequate blockers.     

Molecular cloning of the platelet P2T(AC) ADP receptor: pharmacological comparison with another ADP receptor, the P2Y(1) receptor

Platelet activation plays an essential role in thrombosis. ADP-induced platelet aggregation is mediated by two distinct G protein-coupled ADP receptors, Gq-linked P2Y(1), and Gi-linked P2T(AC), which has not been cloned. The cDNA encoding a novel G protein-coupled receptor, termed HORK3, was isolated. The HORK3 gene and P2Y(1) gene were mapped to chromosome 3q21-q25. HORK3, when transfected in the rat glioma cell subline (C6-15), responded to 2-methylthio-ADP (2MeSADP) (EC(50) = 0.08 nM) and ADP (EC(50) = 42 nM) with inhibition of forskolin-stimulated cAMP accumulation. 2MeSADP (EC(50) = 1.3 nM) and ADP (EC(50) = 18 nM) also induced intracellular calcium mobilization in P2Y(1)-expressing cells. These results show that HORK3 is a Gi/o-coupled receptor and that its natural ligand is ADP. AR-C69931 MX and 2MeSAMP, P2T(AC) antagonists, selectively inhibited 2MeSADP-induced adenylyl cyclase inhibition in HORK3-expressing cells. On the other hand, A3P5PS, a P2Y(1) antagonist, blocked only 2MeSADP-induced calcium mobilization in P2Y(1)-expressing cells. HORK3 mRNA was detected in human platelets and the expression level of HORK3 was equivalent to that of P2Y(1). These observations indicate that HORK3 has the characteristics of the proposed P2T(AC) receptor. We have also determined that [(3)H]2MeSADP binds to cloned HORK3 and P2Y(1). Competition binding experiments revealed a similarity in the rank orders of potency of agonists and the selectivity of antagonists as obtained in the functional assay. These results support the view that P2Y(1) functions as a high-affinity ADP receptor and P2T(AC) as a low-affinity ADP receptor in platelets.     

RNase P: a promising molecular target for the development of new drugs

RNase P is an ubiquitous and essential endonuclease in tRNA biogenesis, which generates the mature 5'-termini of tRNAs. RNase P activities have been identified in all three kingdoms of life (Bacteria, Archaea, Eukarya). Most forms of RNase P are ribonucleoproteins, i.e., they consist of an essential RNA and protein subunits. In bacteria and in some archaea, the catalytic function of this enzyme resides entirely in its RNA subunit, which is one of firstly identified ribozymes. Its high structural and functional diversity among representatives of a vast variety of phylogenetic domains indicates that RNase P could also serve as a molecular target of and a useful screening system for the biological activity of different compounds and give more insight into the molecular mechanisms of their action inside the cell. The emerged information from recent studies on the mechanism and structural idiosyncrasies of RNase P provides a convenient platform for designing specific inhibitors for this ribozyme and potential areas of its application in gene therapy. This review summarises the current information on the effect of several protein synthesis inhibitors, retinoids and arotinoids, vitamin D analogues and anthalin on the activity of RNase P.     

The suramin analogue NF279 is a novel and potent antagonist selective for the P2X(1) receptor

The suramin analogue 8,8'-(carbonylbis(imino-4, 1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)) bis(1,3,5-naphthalenetrisul fonic acid) (NF279) was analysed with respect to its potency and P2X receptor subtype selectivity. Two-electrode voltage-clamp measurements were performed with Xenopus laevis oocytes expressing homomultimeric rat P2X(1), P2X(2), P2X(3) and human P2X(4) receptors. For the fast desensitising P2X(1) and P2X(3) receptors, IC(50) values strongly depended on whether oocytes were pre-incubated with NF279 prior to ATP superfusion or exposed to NF279 simultaneously with ATP. With a 10 s pre-incubation period of NF279, IC(50) values of 19 nM and 1.62 microM were obtained for rat P2X(1) and P2X(3), respectively. Without pre-incubation, IC(50) values amounted to 2 microM and 85.5 microM for P2X(1) and P2X(3), respectively. For the non-desensitising rat P2X(2) receptor NF279 appeared to act as a competitive antagonist with an IC(50) value of 0.76 microM and a K(B) value of 0.36 microM, as derived from Schild analysis. P2X(4) receptors were the least sensitive subtypes for NF279 (IC(50)>300 microM). The antagonism was fully reversible at all P2X subtypes analysed. Our results indicate that NF279 is a potent P2X(1) receptor-selective and reversible antagonist.     

2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist

1. We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y(1) receptor and that modification of the ribose in these analogues is tolerated in the P2Y(1) receptor binding pharmacophore. 2. Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3. MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK(B)=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y(1) receptor (pK(B)=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y(1) receptor since MRS2279 had no effect on activation of the human P2Y(2), P2Y(4), P2Y(6), or P2Y(11) receptors by their cognate agonists. 4. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5. In contrast, the P2Y(1) receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affinity (pK(B)=8.05) similar to that observed at the human P2Y(1) receptor heterologously expressed in 1321N1 cells. 6. Taken together these results illustrate selective high affinity antagonism of the P2Y(1) receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.     

Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family

The cloning of a human G-protein-coupled receptor (GPCR) that specifically responds to UDP-glucose and related sugar-nucleotides has been reported recently. This receptor has important structural similarities to known members of the P2Y receptor family but also shows a distinctly different pharmacological response profile. Here, the IUPHAR Subcommittee for P2Y receptor nomenclature and classification review the current knowledge of this receptor and present their reasons for including this receptor in the P2Y receptor family as the P2Y(14) receptor.     

Substituted benzimidazole derivatives as angiotensin II-AT1 receptor antagonist: a review

The renin angiotensin system (RAS) plays an important role in regulation of blood pressure and fluid-electrolyte homeostasis. The renin-angiotensin system consists of a cascade of enzymatic reactions producing angiotensin II (Ang II). Ang II is a vasoconstrictive peptide hormone that exerts a wide variety of physiological actions on cardiovascular, renal, endocrine and central nervous systems. The RAS can be inhibited at various points to control pathogenesis of hypertension. Renin inhibitors and angiotensin-converting enzyme (ACE) inhibitors were the earliest RAS blocking agents. A relatively new class of compounds known as Ang II receptor antagonists (SARTANs) is developed for the treatment of hypertension. They exert their action by blocking the binding of Ang II on AT(1) receptor. Angiotensin converting enzyme (ACE) inhibitors are associated with incident of side effects such as cough and angioedema while clinical trials with Ang II receptor antagonists have confirmed that these drugs are safe and efficacious for the treatment of hypertension. Based upon the understanding of molecular interaction of Ang II receptor antagonists with AT(1) receptor some of the common structural features have been identified, such as a heterocyclic (nitrogen atom) ring system, an alkyl side chain and an acidic tetrazole group. Research efforts for development of new molecules with similar structural features have led to the discovery of various non-peptidic Ang II receptor antagonists with different substituted heterocyclic such as imidazole (losartan) and benzimidazole (candesartan and telmisartan). In this study we have critically reviewed various benzimidazole substituted compounds as Ang II-AT(1) receptor antagonists and explored other potential clinical uses for this class of compounds.