Structures of synaptic vesicle protein 2A and 2B bound to anticonvulsants.

Epilepsy is a common neurological disorder characterized by abnormal activity of neuronal networks, leading to seizures. The racetam class of anti-seizure medications bind specifically to a membrane protein found in the synaptic vesicles of neurons called synaptic vesicle protein 2 (SV2) A (SV2A). SV2A belongs to an orphan subfamily of the solute carrier 22 organic ion transporter family that also includes SV2B and SV2C. The molecular basis for how anti-seizure medications act on SV2s remains unknown. Here we report cryo-electron microscopy structures of SV2A and SV2B captured in a luminal-occluded conformation complexed with anticonvulsant ligands. The conformation bound by anticonvulsants resembles an inhibited transporter with closed luminal and intracellular gates. Anticonvulsants bind to a highly conserved central site in SV2s. These structures provide blueprints for future drug design and will facilitate future investigations into the biological function of SV2s.

Humanized zebrafish as a tractable tool for in vivo evaluation of pro-myelinating drugs.

Therapies that promote neuroprotection and axonal survival by enhancing myelin regeneration are an unmet need to prevent disability progression in multiple sclerosis. Numerous potentially beneficial compounds have originated from phenotypic screenings but failed in clinical trials. It is apparent that current cell- and animal-based disease models are poor predictors of positive treatment options, arguing for novel experimental approaches. Here we explore the experimental power of humanized zebrafish to foster the identification of pro-remyelination compounds via specific inhibition of GPR17. Using biochemical and imaging techniques, we visualize the expression of zebrafish (zf)-gpr17 during the distinct stages of oligodendrocyte development, thereby demonstrating species-conserved expression between zebrafish and mammals. We also demonstrate species-conserved function of zf-Gpr17 using genetic loss-of-function and rescue techniques. Finally, using GPR17-humanized zebrafish, we provide proof of principle for in vivo analysis of compounds acting via targeted inhibition of human GPR17. We anticipate that GPR17-humanized zebrafish will markedly improve the search for effective pro-myelinating pharmacotherapies.

Connectivity Mapping Using a Novel sv2a Loss-of-Function Zebrafish Epilepsy Model as a Powerful Strategy for Anti-epileptic Drug Discovery.

Synaptic vesicle glycoprotein 2A (SV2A) regulates action potential-dependent neurotransmitter release and is commonly known as the primary binding site of an approved anti-epileptic drug, levetiracetam. Although several rodent knockout models have demonstrated the importance of SV2A for functional neurotransmission, its precise physiological function and role in epilepsy pathophysiology remains to be elucidated. Here, we present a novel sv2a knockout model in zebrafish, a vertebrate with complementary advantages to rodents. We demonstrated that 6 days post fertilization homozygous sv2a-/- mutant zebrafish larvae, but not sv2a +/- and sv2a+/+ larvae, displayed locomotor hyperactivity and spontaneous epileptiform discharges, however, no major brain malformations could be observed. A partial rescue of this epileptiform brain activity could be observed after treatment with two commonly used anti-epileptic drugs, valproic acid and, surprisingly, levetiracetam. This observation indicated that additional targets, besides Sv2a, maybe are involved in the protective effects of levetiracetam against epileptic seizures. Furthermore, a transcriptome analysis provided insights into the neuropathological processes underlying the observed epileptic phenotype. While gene expression profiling revealed only one differentially expressed gene (DEG) between wildtype and sv2a +/- larvae, there were 4386 and 3535 DEGs between wildtype and sv2a-/- , and sv2a +/- and sv2a-/- larvae, respectively. Pathway and gene ontology (GO) enrichment analysis between wildtype and sv2a-/- larvae revealed several pathways and GO terms enriched amongst up- and down-regulated genes, including MAPK signaling, synaptic vesicle cycle, and extracellular matrix organization, all known to be involved in epileptogenesis and epilepsy. Importantly, we used the Connectivity map database to identify compounds with opposing gene signatures compared to the one observed in sv2a-/- larvae, to finally rescue the epileptic phenotype. Two out of three selected compounds rescued electrographic discharges in sv2a-/- larvae, while negative controls did not. Taken together, our results demonstrate that sv2a deficiency leads to increased seizure vulnerability and provide valuable insight into the functional importance of sv2a in the brain in general. Furthermore, we provided evidence that the concept of connectivity mapping represents an attractive and powerful approach in the discovery of novel compounds against epilepsy.

Evaluating Diagnostic Accuracy of Saliva Sampling Methods for Severe Acute Respiratory Syndrome Coronavirus 2 Reveals Differential Sensitivity and Association with Viral Load.

Nasopharyngeal swabs are considered the preferential collection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. Less invasive and simpler alternative sampling procedures, such as saliva collection, are desirable. We compared saliva specimens and nasopharyngeal (NP) swabs with respect to sensitivity in detecting SARS-CoV-2. A nasopharyngeal and two saliva specimens (collected by spitting or oral swabbing) were obtained from >2500 individuals. All samples were tested by RT-qPCR, detecting RNA of SARS-CoV-2. The test sensitivity was compared on the two saliva collections with the nasopharyngeal specimen for all subjects and stratified by symptom status and viral load. Of the 2850 patients for whom all three samples were available, 105 were positive on NP swab, whereas 32 and 23 were also positive on saliva spitting and saliva swabbing samples, respectively. The sensitivity of the RT-qPCR to detect SARS-CoV-2 among NP-positive patients was 30.5% (95% CI, 1.9%-40.2%) for saliva spitting and 21.9% (95% CI, 14.4%-31.0%) for saliva swabbing. However, when focusing on subjects with medium to high viral load, sensitivity on saliva increased substantially: 93.9% (95% CI, 79.8%-99.3%) and 76.9% (95% CI, 56.4%-91.0%) for spitting and swabbing, respectively, regardless of symptomatic status. Our results suggest that saliva cannot readily replace nasopharyngeal sampling for SARS-CoV-2 diagnostics but may enable identification of the most contagious cases with medium to high viral loads.

Pharmacological Profile of the Novel Antiepileptic Drug Candidate Padsevonil: Interactions with Synaptic Vesicle 2 Proteins and the GABAA Receptor.

Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABAA receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil's affinity for SV2A was greater than that of levetiracetam and brivaracetam (pKi 8.5, 5.2, and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil's interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics: dissociation t 1/2 30 minutes from the human protein at 37°C compared with <0.5 minute for levetiracetam and brivaracetam. In addition, its binding was not potentiated by the allosteric modulator UCB1244283. At recombinant GABAA receptors, padsevonil displayed low to moderate affinity (pIC50≤6.1) for the benzodiazepine site, and in electrophysiological studies, its relative efficacy compared with zolpidem (full-agonist reference drug) was 40%, indicating partial agonist properties. In in vivo (mice) receptor occupancy studies, padsevonil exhibited SV2A occupancy at low ED50 (0.2 mg/kg) and benzodiazepine site occupancy at higher doses (ED50 36 mg/kg), supporting in vitro results. Padsevonil's selectivity for its intended targets was confirmed in profiling studies, where it lacked significant effects on a wide variety of ion channels, receptors, transporters, and enzymes. Padsevonil is a first-in-class AED candidate with a unique target profile allowing for presynaptic and postsynaptic activity. SIGNIFICANCE STATEMENT: Padsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both presynaptic and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile: padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B, and C) and micromolar affinity for the benzodiazepine binding site on GABAA receptors. Furthermore, padsevonil showed greater affinity for and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam, and brivaracetam.

Further evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein.

Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. BRV differs from LEV in preclinical studies in that it exhibits a more potent and complete seizure protection across animal models. We reported previously that an allosteric modulator of the SV2A protein had differential effects on BRV compared with LEV, suggesting that they act at different sites or with different conformations of the SV2A protein. If this is the case, then we hypothesized that mutations of specific amino acids in the SV2A protein may have differential effects on BRV and LEV binding by the modulator. Mutation of some amino acids identified previously in the binding site of racetams to the SV2A protein had marked effects on binding of both [3 H]BRV and [3 H]LEV (eg, W300F, F277A, G303A, F658A, Y462A, W666A, I663A, D670A, and V661A). However, 3 amino acids were identified (K694, I273, and S294) in which mutation lost the effect of the modulator on [3 H]LEV binding with no effect on the modulation of [3 H]BRV binding. These results confirm that BRV and LEV bind to the human synaptic vesicle 2A protein at closely related sites but interact with these sites in a different way.

Repurposing HAMI3379 to Block GPR17 and Promote Rodent and Human Oligodendrocyte Differentiation.

Identification of additional uses for existing drugs is a hot topic in drug discovery and a viable alternative to de novo drug development. HAMI3379 is known as an antagonist of the cysteinyl-leukotriene CysLT2 receptor, and was initially developed to treat cardiovascular and inflammatory disorders. In our study we identified HAMI3379 as an antagonist of the orphan G protein-coupled receptor GPR17. HAMI3379 inhibits signaling of recombinant human, rat, and mouse GPR17 across various cellular backgrounds, and of endogenous GPR17 in primary rodent oligodendrocytes. GPR17 blockade by HAMI3379 enhanced maturation of primary rat and mouse oligodendrocytes, but was without effect in oligodendrocytes from GPR17 knockout mice. In human oligodendrocytes prepared from inducible pluripotent stem cells, GPR17 is expressed and its activation impaired oligodendrocyte differentiation. HAMI3379, conversely, efficiently favored human oligodendrocyte differentiation. We propose that HAMI3379 holds promise for pharmacological exploitation of orphan GPR17 to enhance regenerative strategies for the promotion of remyelination in patients.

5-HT7 Receptor Antagonists with an Unprecedented Selectivity Profile.

Selective leads: In this study, we generated a new series of serotonin 5-HT7 receptor antagonists. Their synthesis, structure-activity relationships, and selectivity profiles are reported. This series includes 5-HT7 antagonists with unprecedented high selectivity for the 5-HT7 receptor, setting the stage for lead optimization of drugs acting on a range of neurological targets.

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

BACKGROUND: Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [18 F]AV-1451 uptake in Alzheimer's disease may not be possible. OBJECTIVES: The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. METHODS: [3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. RESULTS: [3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [3 H]AV-1451 for monoamine oxidase A and B enzymes. CONCLUSIONS: High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society.

The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes.

Pairing orphan G protein­coupled receptors (GPCRs) with their cognate endogenous ligands is expected to have a major impact on our understanding of GPCR biology. It follows that the reproducibility of orphan receptor ligand pairs should be of fundamental importance to guide meaningful investigations into the pharmacology and function of individual receptors. GPR17 is an orphan receptor characterized by some as a dualistic uracil nucleotide/cysteinyl leukotriene receptor and by others as inactive toward these stimuli altogether. Whereas regulation of central nervous system myelination by GPR17 is well established, verification of activity of its putative endogenous ligands has proven elusive so far. Herein we report that uracil nucleotides and cysteinyl leukotrienes do not activate human, mouse, or rat GPR17 in various cellular backgrounds, including primary cells, using eight distinct functional assay platforms based on labelfree pathway-unbiased biosensor technologies, as well as canonical second-messenger or biochemical assays. Appraisal of GPR17 activity can neither be accomplished with co-application of both ligand classes, nor with exogenous transfection of partner receptors (nucleotide P2Y12, cysteinyl-leukotriene CysLT1) to reconstitute the elusive pharmacology. Moreover, our study does not support the inhibition of GPR17 by the marketed antiplatelet drugs cangrelor and ticagrelor, previously suggested to antagonize GPR17. Whereas our data do not disagree with a role of GPR17 per se as an orchestrator of central nervous system functions, they challenge the utility of the proposed (ant)agonists as tools to imply direct contribution of GPR17 in complex biologic settings.

Evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2A protein.

OBJECTIVE: Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. However, BRV differs from LEV in that it exhibits more potent and complete seizure suppression in animal models including in amygdala-kindled mice, where BRV afforded nearly complete seizure suppression. This raises the possibility that aside from potency differences, BRV and LEV may interact differently with the SV2A protein, which is not apparent in radioligand-binding competition studies. In this study, we used a recently identified SV2A allosteric modulator, UCB1244283, that appears to induce conformational changes in SV2A, to probe the binding properties of labeled BRV and LEV. METHODS: Radioligand binding studies were carried out using [3 H]BRV and [3 H]LEV. Studies were performed in membranes from both recombinant cells expressing human SV2A protein and human brain tissue. RESULTS: The modulator increased the binding of both radioligands but by different mechanisms. For [3 H]BRV, the increase was driven mainly by an increase in affinity, whereas for [3 H]LEV, the increase was due to an increase in the number of apparent binding sites. Kinetic studies confirmed this differential effect. SIGNIFICANCE: These studies suggest that LEV and BRV may act at different binding sites or interact with different conformational states of the SV2A protein. It is possible that some of the pharmacologic differences between BRV and LEV could be due to different interactions with the SV2A protein.

Characterization of P2X4 receptor agonists and antagonists by calcium influx and radioligand binding studies.

Antagonists for ATP-activated P2X4 ion channel receptors are currently in the focus as novel drug targets, in particular for the treatment of neuropathic and inflammatory pain. We stably expressed the human, rat and mouse P2X4 receptors in 1321N1 astrocytoma cells, which is devoid of functional nucleotide receptors, by retroviral transfection, and established monoclonal cell lines. Calcium flux assay conditions were optimized for high-throughput screening resulting in a Z'-factor of >0.8. The application of ready-to-use frozen cells did not negatively affect the results of the calcium assays, which is of great advantage for the screening of compound libraries. Species differences were observed, the rat P2X4 receptor being particularly insensitive to many ATP derivatives. Membrane preparations of the cell lines showed high levels of specific [35S]ATPγS binding with low nonspecific binding (<5% of total binding), while non-transfected cells were devoid of specific binding sites for the radioligand. Conditions were employed which allow binding studies to be performed at room temperature. While a variety of nucleotide-derived agonists and the antagonist TNP-ATP displaced [35S]ATPγS from its binding site at human P2X4 receptors, the non-nucleotidic antagonists paroxetine and 5-BDBD did not compete with radioligand binding and were therefore characterized as allosteric antagonists. Homology modeling was applied to find an explanation for the observed species differences.

Synaptic Vesicle Glycoprotein 2A Ligands in the Treatment of Epilepsy and Beyond.

The synaptic vesicle glycoprotein SV2A belongs to the major facilitator superfamily (MFS) of transporters and is an integral constituent of synaptic vesicle membranes. SV2A has been demonstrated to be involved in vesicle trafficking and exocytosis, processes crucial for neurotransmission. The anti-seizure drug levetiracetam was the first ligand to target SV2A and displays a broad spectrum of anti-seizure activity in various preclinical models. Several lines of preclinical and clinical evidence, including genetics and protein expression changes, support an important role of SV2A in epilepsy pathophysiology. While the functional consequences of SV2A ligand binding are not fully elucidated, studies suggest that subsequent SV2A conformational changes may contribute to seizure protection. Conversely, the recently discovered negative SV2A modulators, such as UCB0255, counteract the anti-seizure effect of levetiracetam and display procognitive properties in preclinical models. More broadly, dysfunction of SV2A may also be involved in Alzheimer's disease and other types of cognitive impairment, suggesting potential novel therapies for levetiracetam and its congeners. Furthermore, emerging data indicate that there may be important roles for two other SV2 isoforms (SV2B and SV2C) in the pathogenesis of epilepsy, as well as other neurodegenerative diseases. Utilization of recently developed SV2A positron emission tomography ligands will strengthen and reinforce the pharmacological evidence that SV2A is a druggable target, and will provide a better understanding of its role in epilepsy and other neurological diseases, aiding in further defining the full therapeutic potential of SV2A modulation.

[(11)C]UCB-A, a novel PET tracer for synaptic vesicle protein 2A.

INTRODUCTION: Development of a selective and specific high affinity PET tracer, [(11)C]UCB-A, for the in vivo study of SV2A expression in humans. Radiochemistry and preclinical studies in rats and pigs including development of a tracer kinetic model to determine VT. A method for the measurement of percent intact tracer in plasma was developed and the radiation dosimetry was determined in rats. RESULTS: 3-5GBq of [(11)C]UCB-A could be produced with radiochemical purity exceeding 98% with a specific radioactivity of around 65GBq/µmol. In vitro binding showed high selective binding towards SV2A. [(11)C]UCB-A displayed a dose-dependent and reversible binding to SV2A as measured with PET in rats and pigs and the VT could be determined by Logan analysis. The dosimetry was favorable and low enough to allow multiple administrations of [(11)C]UCB-A to healthy volunteers, and the metabolite analysis showed no sign of labeled metabolites in brain. CONCLUSIONS: We have developed the novel PET tracer, [(11)C]UCB-A, that can be used to measure SV2A expression in vivo. The dosimetry allows up to 5 administrations of 400MBq of [(11)C]UCB-A in humans. Apart from measuring drug occupancy, as we have shown, the tracer can potentially be used to compare SV2A expression between individuals because of the rather narrow range of baseline VT values. This will have to be further validated in human studies.

Brivaracetam: Rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment.

Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy continue to experience refractory seizures and adverse events. Achievement of better seizure control and fewer side effects is key to improving quality of life. This review describes the rationale for the discovery and preclinical profile of brivaracetam (BRV), currently under regulatory review as adjunctive therapy for adults with partial-onset seizures. The discovery of BRV was triggered by the novel mechanism of action and atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models. LEV is associated with several mechanisms that may contribute to its antiepileptic properties and adverse effect profile. Early findings observed a moderate affinity for a unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant effects in animal models of epilepsy. This provided a promising molecular target and rationale for identifying selective, high-affinity ligands for LBS with potential for improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A (SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug discovery program resulted in the identification of anticonvulsants, comprising two distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity and differential interaction with SV2A as well as a higher lipophilicity, correlating with more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a greater antiepileptogenic potential than LEV. These properties of BRV highlight its promising potential as an AED that might provide broad-spectrum efficacy, associated with a promising tolerability profile and a fast onset of action. BRV represents the first selective SV2A ligand for epilepsy treatment and may add a significant contribution to the existing armamentarium of AEDs.

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis.

In Vitro and In Vivo Identification of Novel Positive Allosteric Modulators of the Human Dopamine D2 and D3 Receptor.

Agonists at dopamine D2 and D3 receptors are important therapeutic agents in the treatment of Parkinson's disease. Compared with the use of agonists, allosteric potentiators offer potential advantages such as temporal, regional, and phasic potentiation of natural signaling, and that of receptor subtype selectivity. We report the identification of a stereoselective interaction of a benzothiazol racemic compound that acts as a positive allosteric modulator (PAM) of the rat and human dopamine D2 and D3 receptors. The R isomer did not directly stimulate the dopamine D2 receptor but potentiated the effects of dopamine. In contrast the S isomer attenuated the effects of the PAM and the effects of dopamine. In radioligand binding studies, these compounds do not compete for binding of orthosteric ligands, but indeed the R isomer increased the number of high-affinity sites for [(3)H]-dopamine without affecting K(d). We went on to identify a more potent PAM for use in native receptor systems. This compound potentiated the effects of D2/D3 signaling in vitro in electrophysiologic studies on dissociated striatal neurons and in vivo on the effects of L-dopa in the 6OHDA (6-hydroxydopamine) contralateral turning model. These PAMs lacked activity at a wide variety of receptors, lacked PAM activity at related Gi-coupled G protein-coupled receptors, and lacked activity at D1 receptors. However, the PAMs did potentiate [(3)H]-dopamine binding at both D2 and D3 receptors. Together, these studies show that we have identified PAMs of the D2 and D3 receptors both in vitro and in vivo. Such compounds may have utility in the treatment of hypodopaminergic function.

Development of an HTRF Assay for the Detection and Characterization of Inhibitors of Catechol-O-Methyltransferase.

Catechol-O-methyltransferase (COMT) plays an important role in the deactivation of catecholamine neurotransmitters and hormones. Inhibitors of COMT, such as tolcapone and entacapone, are used clinically in the treatment of Parkinson's disease. Discovery of novel inhibitors has been hampered by a lack of suitable assays for high-throughput screening (HTS). Although assays using esculetin have been developed, these are affected by fluorescence, a common property of catechol-type compounds. We have therefore evaluated a new homogenous time-resolved fluorescence (HTRF)-based assay from CisBio (Codolet, France), which measures the production of S-adenosyl-L-homocysteine (SAH). The assay has been run in both HTS and medium-throughput screening (MTS) modes. The assay was established using membranes expressing human membrane-bound COMT and was optimized for protein and time to give an acceptable signal window, good potency for tolcapone, and a high degree of translation between data in fluorescence ratio and data in terms of [SAH] produced. pIC50 values for the hits from the HTS mode were determined in the MTS mode. The assay also proved suitable for kinetic studies such as Km,app determination.

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors.

BACKGROUND AND PURPOSE: Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). EXPERIMENTAL APPROACH: The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. KEY RESULTS: [(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. CONCLUSIONS AND IMPLICATIONS: Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties.

Development of [(3)H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([(3)H]PSB-12150): A Useful Tool for Studying GPR17.

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1H-indole-3-propionic acid (1) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative 8 with tritium gas. The radioligand [(3)H]PSB-12150 (9) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities.