Jean E. Lachowicz

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

ADP Is the Cognate Ligand for the Orphan G Protein-coupled Receptor SP1999

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Gαi have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Gαi-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Gα subunits (Gαq/i). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC50 of 60 nm. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5′-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5′-O-(thiotriphosphate). Thus, SP1999 is a novel, Gαi-linked receptor for ADP.

Phenotypic analysis of dopamine receptor knockout mice; recent insights into the functional specificity of dopamine receptor subtypes.

The functional specificity of dopamine receptor subtypes remains incompletely understood, in part due to the absence of highly selective agonists and antagonists. Phenotypic analysis of dopamine receptor knockout mice has been instrumental in identifying the role of dopamine receptor subtypes in mediating dopamines effects on motor function, cognition, reward, and emotional behaviors. In this article, we provide an update of recent studies in dopamine receptor knockout mice and discuss the limitations and future promise of this approach.

Characterization of the Potent and Highly Selective A2A Receptor Antagonists Preladenant and SCH 412348 [7-[2-[4-2,4-Difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] in Rodent Models of Movement Disorders and Depression

The adenosine A2A receptor has been implicated in the underlying biology of various neurological and psychiatric disorders, including Parkinsons disease (PD) and depression. Preladenant and SCH 412348 [7-[2-[4-2,4-difluorophenyl]-1-piperazinyl]ethyl]-2-(2-furanyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] are potent competitive antagonists of the human A2A receptor (Ki = 1.1 and 0.6 nM, respectively) and have >1000-fold selectivity over all other adenosine receptors, making these compounds the most selective A2A receptor antagonists reported to date. Both compounds attenuate hypolocomotion induced by the A2A receptor agonist CGS-21680 [2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine], suggesting that they inhibit A2A receptor activity in vivo. Their high degree of selectivity and robust in vivo activity make preladenant and SCH 412348 useful tools to investigate the role of the A2A receptor system in animal models of PD and depression. Oral administration of preladenant and SCH 412348 (0.1–1 mg/kg) to rats potentiated 3,4-dihydroxy-l-phenylalanine (l-Dopa)-induced contralateral rotations after 6-hydroxydopamine lesions in the medial forebrain bundle and potently attenuated the cataleptic effects of haloperidol. Preladenant (1 mg/kg) inhibited l-Dopa-induced behavioral sensitization after repeated daily administration, which suggests a reduced risk of the development of dyskinesias. Finally, preladenant and SCH 412348 exhibited antidepressant-like profiles in models of behavioral despair, namely the mouse tail suspension test and the mouse and rat forced swim test. These studies demonstrate that preladenant and SCH 412348 are potent and selective A2A receptor antagonists and provide further evidence of the potential therapeutic benefits of A2A receptor inhibition in PD (with reduced risk of dyskinesias) and depression (one of the primary nonmotor symptoms of PD).

The gerbil elevated plus-maze II: anxiolytic-like effects of selective neurokinin NK1 receptor antagonists.

Neurokinin NK1 receptor antagonists may have therapeutic potential in the treatment of anxiety and depression. Species variants in the NK1 receptor result in reduced affinity of NK1 receptor antagonists at rat and mouse NK1 receptors, making it difficult to test NK1 antagonists in traditional preclinical models of anxiety and depression. Gerbil NK1 receptors are similar in homology to the human NK1 receptor. In a companion article, we described the anxiety-like behavioral profile of gerbils on an adapted elevated plus-maze, and the ability of anxiolytic drugs to produce anti-anxiety effects in the gerbil elevated plus-maze. The aim of the present study was to determine whether oral (p.o.) administration of the NK1 receptor antagonists MK-869, L-742,694, L-733,060, CP-99,994, and CP-122,721 produced anxiolytic-like effects in the gerbil elevated plus-maze. Upon testing, all five NK1 antagonists produced anxiolytic-like effects. MK-869 (0.01–3 mg/kg) was the most potent NK1 antagonist, producing anxiolytic-like effects on percentage of open arm time, percentage of open arm entries, stretch-attend postures, and head dips at 0.03–0.3 mg/kg doses. L-742,694 (1–30 mg/kg) and L-733,060 (1–10 mg/kg) produced anxiolytic-like effects on percentage of open arm time and stretch-attend postures at 3–10 mg/kg doses. CP-99,994 (3–30 mg/kg) only produced an anxiolytic-like effect on stretch-attend postures. CP-122,721 (3–30 mg/kg) produced an anxiolytic-like effect on percentage of open arm time at 30 mg/kg. The order of potency of the NK1 antagonists to increase percentage of open arm time was very similar to their potency to block NK1 agonist-induced foot-tapping. These studies demonstrate that neurokinin NK1 receptor antagonists produce anxiolytic-like effects in a novel gerbil elevated plus-maze, and suggest that this is an appropriate model to test NK1 antagonists for preclinical anxiolytic activity.

Centrally administered hemokinin-1 (HK-1), a neurokinin NK1 receptor agonist, produces substance P-like behavioral effects in mice and gerbils.

Hemokinin-1 (HK-1) is a recently described mouse tachykinin peptide whose biological functions are not fully understood. To date, a unique receptor for HK-1 has not been identified. Recent studies suggest HK-1 may have a role in immunological functions, but there has been little characterization of HK-1s effects in the central nervous system (CNS). In the present studies, we confirm that HK-1 is an endogenous agonist at all of the known tachykinin receptors, and is selective for the NK1 receptor over the NK2 and NK3 subtypes. CHO cells transfected with the human NK1 receptor released intracellular calcium in response to HK-1. In addition, HK-1 competed with substance P (SP) for binding to mouse NK1 and human NK1 receptors. In vivo central administration of HK-1 to gerbils and mice induced foot-tapping and scratching behaviors, respectively, similar to those observed following central administration of SP or the NK1 receptor agonist, GR-73632. Furthermore, these behavioral effects were blocked by the selective NK1 receptor antagonist, MK-869. Finally, a comprehensive expression analysis of HK-1 demonstrated that HK-1 mRNA is much more broadly expressed than previously reported with expression observed in many brain regions. Together these data demonstrate that HK-1 is a functional agonist at NK1 receptors and suggest that HK-1 may function both centrally and peripherally.

Design and synthesis of piperidinyl piperidine analogues as potent and selective M2 muscarinic receptor antagonists.

Identification of a number of highly potent M2 receptor antagonists with >100-fold selectivity against the M1 and M3 receptor subtypes is described. In the rat microdialysis assay, this series of compounds showed pronounced enhancement of brain acetylcholine release after oral administration.

Muscarinic agonists and antagonists in the treatment of Alzheimer's disease☆

Alzheimers disease (AD) is a neurodegenerative disease characterized by cognitive impairment and personality changes. The development of drugs for the treatment of the cognitive deficits of AD has focused on agents which counteract loss in cholinergic activity. Although symptoms of AD have been successfully treated with acetylcholinesterase inhibitors (tacrine, donepezil. rivastigmine, galanthamine), limited success has been achieved with direct M1 agonists, probably due to their lack of selectivity versus other muscarinic receptor subtypes. Muscarinic M2 antagonists have been reported to increase synaptic levels of acetylcholine after oral administration to rats (e.g. BIBN-99, SCH-57790), but their selectivity versus other muscarinic receptor subtypes is modest. Exploration of a series of piperidinylpiperidines has yielded the potent and selective M2 antagonist SCH-217443. This antagonist has excellent bioavailability in rats and dogs and shows activity in a rat model of cognition.

Randomized controlled trials of the D1/D5 antagonist ecopipam for weight loss in obese subjects.

Objective: To evaluate the efficacy and safety of the selective dopamine D1/D5 antagonist ecopipam for the treatment of obesity.

Design and synthesis of ether analogues as potent and selective M2 muscarinic receptor antagonists

Novel, selective M2 muscarinic antagonists, which replace the metabolically labile styrenyl moiety of the prototypical M2 antagonist 1 with an ether linkage, were synthesized. A detailed SAR study in this class of compounds has yielded highly active compounds that showed M2 Ki values of < 1.0 nM and >100-fold selectivity against M1, M3, and M5 receptors.