Publications by authors named "John R Atack"

84 Publications

Inhibition of a tonic inhibitory conductance in mouse hippocampal neurones by negative allosteric modulators of α5 subunit-containing γ-aminobutyric acid type A receptors: implications for treating cognitive deficits.

Br J Anaesth 2021 Mar 30;126(3):674-683. Epub 2020 Dec 30.

Department of Physiology, University of Toronto, Toronto, ON, Canada; Department of Anesthesiology and Pain Medicine, University of Toronto, Toronto, ON, Canada; Department of Anesthesia, Sunnybrook Health Sciences Centre, Toronto, ON, Canada. Electronic address:

Background: Multiple cognitive and psychiatric disorders are associated with an increased tonic inhibitory conductance that is generated by α5 subunit-containing γ-aminobutyric acid type A (α5 GABA) receptors. Negative allosteric modulators that inhibit α5 GABA receptors (α5-NAMs) are being developed as treatments for these disorders. The effects of α5-NAMs have been studied on recombinant GABA receptors expressed in non-neuronal cells; however, no study has compared drug effects on the tonic conductance generated by native GABA receptors in neurones, which was the goal of this study.

Methods: The effects of five α5-NAMs (basmisanil, Ono-160, L-655,708, α5IA, and MRK-016) on tonic current evoked by a low concentration of GABA were studied using whole-cell recordings in cultured mouse hippocampal neurones. Drug effects on current evoked by a saturating concentration of GABA and on miniature inhibitory postsynaptic currents (mIPSCs) were also examined.

Results: The α5-NAMs caused a concentration-dependent decrease in tonic current. The potencies varied as the inhibitory concentration for 50% inhibition (IC) of basmisanil (127 nM) was significantly higher than those of the other compounds (0.4-0.8 nM). In contrast, the maximal efficacies of the drugs were similar (35.5-51.3% inhibition). The α5-NAMs did not modify current evoked by a saturating GABA concentration or mIPSCs.

Conclusions: Basmisanil was markedly less potent than the other α5-NAMs, an unexpected result based on studies of recombinant α5 GABA receptors. Studying the effects of α5 GABA receptor-selective drugs on the tonic inhibitory current in neurones could inform the selection of compounds for future clinical trials.
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http://dx.doi.org/10.1016/j.bja.2020.11.032DOI Listing
March 2021

Crystallization and structure of ebselen bound to Cys141 of human inositol monophosphatase.

Acta Crystallogr F Struct Biol Commun 2020 Oct 15;76(Pt 10):469-476. Epub 2020 Sep 15.

Medicines Discovery Institute, School of Biosciences, Cardiff University, Cardiff CF10 3AT, United Kingdom.

Inositol monophosphatase (IMPase) is inhibited by lithium, which is the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects both in a murine model and in clinical trials. Here, the crystallization and the first structure of human IMPase covalently complexed with ebselen, a 1.47 Å resolution crystal structure (PDB entry 6zk0), are presented. In the complex with human IMPase, ebselen in a ring-opened conformation is covalently attached to Cys141, a residue located away from the active site. IMPase is a dimeric enzyme and in the crystal structure two adjacent dimers share four ebselen molecules, creating a tetramer with approximate 222 symmetry. In the crystal structure presented in this publication, the active site in the tetramer is still accessible, suggesting that ebselen may function as an allosteric inhibitor or may block the binding of partner proteins.
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http://dx.doi.org/10.1107/S2053230X20011310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7531247PMC
October 2020

Conformational flexibility within the small domain of human serine racemase.

Acta Crystallogr F Struct Biol Commun 2020 Feb 3;76(Pt 2):65-73. Epub 2020 Feb 3.

Department of Biochemistry and Biomedicine, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QJ, England.

Serine racemase (SR) is a pyridoxal 5'-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,β-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78-155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded β-sheet of the small domain, including changes in the φ-ψ angles of all three residues in the C-terminal β-strand (residues 149-151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue β-strand (residues 78-83), but in the holoenzyme the β-strand is only four residues (78-81) and His82 has φ-ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target.
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http://dx.doi.org/10.1107/S2053230X20001193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010357PMC
February 2020

Pharmacological characterisation of MDI-222, a novel AMPA receptor positive allosteric modulator with an improved safety profile.

J Psychopharmacol 2020 01 26;34(1):93-102. Epub 2019 Nov 26.

Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Harlow, UK.

Purpose: There is considerable interest in positive allosteric modulators (PAMs) of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) subtype of ionotropic glutamate receptors as therapeutic agents for a range of cognitive and mood disorders. However, the challenge is to increase AMPA receptor (AMPAR) function sufficient to enhance cognitive function but not to the extent that there are mechanism-related pro-convulsant or convulsant side effects. In this present study, we report the preclinical pharmacology data for MDI-222, an AMPAR PAM which enhances cognition but has a much reduced side-effect (i.e. convulsant) liability relative to other molecules of this mechanism.

Methods: The pharmacological effects of MDI-222 were characterised in in vitro and in vivo preclinical electrophysiology, efficacy (cognition), side-effect (pro-convulsant/convulsant), tolerability and toxicity assays.

Results: We demonstrate that MDI-222 is an AMPAR PAM, since it enhanced AMPAR function in vitro at human (hGluA1-4) and rat (rGluA2) homomeric receptors, and potentiated hetero-oligomeric AMPARs in rat neurons. MDI-222 enhanced electrically evoked AMPAR-mediated synaptic transmission in the anaesthetised rat at 10 mg/kg (administered intravenously) and did not significantly lower the seizure threshold in the pro-convulsant maximal electroshock threshold test (MEST) at any dose tested up to a maximum of 30 mg/kg (administered by oral gavage (p.o.)). MDI-222 reversed a delay-induced deficit in novel object recognition (NOR) in rats with a minimum effective dose (MED) of 0.3 mg/kg (p.o.) following acute administration, which was reduced to 0.1 mg/kg following sub-chronic administration, and improved passive avoidance performance in scopolamine-impaired rats with a MED of 10 mg/kg p.o. On the other hand, MDI-222 was not pro-convulsant in the MEST, resulting in a therapeutic window between plasma concentrations that enhanced cognitive performance and those associated with mechanism-related side effects of ⩾1000-fold. Unfortunately, despite the excellent preclinical profile of this compound, further development had to be halted due to non-mechanism-related issues.

Conclusions: We conclude that MDI-222 is an AMPAR PAM which enhances cognitive performance in rats and has a significantly improved safety profile in preclinical species.
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http://dx.doi.org/10.1177/0269881119872198DOI Listing
January 2020

Subtype Selective γ-Aminobutyric Acid Type A Receptor (GABAR) Modulators Acting at the Benzodiazepine Binding Site: An Update.

J Med Chem 2020 04 5;63(7):3425-3446. Epub 2019 Dec 5.

Medicines Discovery Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, U.K.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter within the central nervous system (CNS) with fast, transsynaptic, and modulatory extrasynaptic effects being mediated by the ionotropic GABA type A receptors (GABARs). These receptors are of particular interest because they are the molecular target of a number of pharmacological agents, of which the benzodiazepines (BZDs), such as diazepam, are the best described. The anxiolytic, sedating, and myorelaxant effects of BZDs are mediated by separate populations of GABARs containing either α1, α2, α3, or α5 subunits and the molecular dissection of the pharmacology of BZDs indicates that subtype-selective GABAR modulators will have novel pharmacological profiles. This is best exemplified by α2/α3-GABAR positive allosteric modulators (PAMs) and α5-GABAR negative allosteric modulators (NAMs), which were originally developed as nonsedating anxiolytics and cognition enhancers, respectively. This review aims to summarize the current state of the field of subtype-selective GABAR modulators acting via the BZD binding site and their potential clinical indications.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01312DOI Listing
April 2020

The Molecular Basis for Apolipoprotein E4 as the Major Risk Factor for Late-Onset Alzheimer's Disease.

J Mol Biol 2019 05 30;431(12):2248-2265. Epub 2019 Apr 30.

Sussex Neuroscience, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex BN1 6NN, UK. Electronic address:

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-β amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.
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http://dx.doi.org/10.1016/j.jmb.2019.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556554PMC
May 2019

A Biophysical Approach to the Identification of Novel ApoE Chemical Probes.

Biomolecules 2019 01 29;9(2). Epub 2019 Jan 29.

Medicines Discovery Institute, Cardiff University, Park Place, Cardiff CF10 3AT, UK.

Alzheimer's disease (AD) is the most common type of dementia and, after age, the greatest risk factor for developing AD is the allelic variation of apolipoprotein E (ApoE), with homozygote carriers of the ApoE4 allele having an up to 12-fold greater risk of developing AD than noncarriers. Apolipoprotein E exists as three isoforms that differ in only two amino acid sites, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). These amino acid substitutions are assumed to alter ApoE structure and function, and be responsible for the detrimental effects of ApoE4 via a mechanism that remains unclear. The hypothesis that a structural difference between ApoE4 and ApoE3 (and ApoE2) is the cause of the ApoE4-associated increased risk for AD forms the basis of a therapeutic approach to modulate ApoE4 structure, and we were therefore interested in screening to identify new chemical probes for ApoE4. In this regard, a high-yield protocol was developed for the expression and purification of recombinant full-length ApoE, and three diverse biophysical screening assays were established and characterized; an optical label-free assay (Corning Epic) for hit identification and microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) as orthogonal assays for hit confirmation. The 707 compounds in the National Institute of Health clinical collection were screened for binding to ApoE4, from which six confirmed hits, as well as one analogue, were identified. Although the compounds did not differentiate between ApoE isoforms, these data nevertheless demonstrate the feasibility of using a biophysical approach to identifying compounds that bind to ApoE and that, with further optimization, might differentiate between isoforms to produce a molecule that selectively alters the function of ApoE4.
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http://dx.doi.org/10.3390/biom9020048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406525PMC
January 2019

The X-ray structure of human calbindin-D28K: an improved model.

Acta Crystallogr D Struct Biol 2018 Oct 2;74(Pt 10):1008-1014. Epub 2018 Oct 2.

Medicines Discovery Institute, Cardiff University, Cardiff CF10 3AT, Wales.

Calbindin-D28K is a widely expressed calcium-buffering cytoplasmic protein that is involved in many physiological processes. It has been shown to interact with other proteins, suggesting a role as a calcium sensor. Many of the targets of calbindin-D28K are of therapeutic interest: for example, inositol monophosphatase, the putative target of lithium therapy in bipolar disorder. Presented here is the first crystal structure of human calbindin-D28K. There are significant deviations in the tertiary structure when compared with the NMR structure of rat calbindin-D28K (PDB entry 2g9b), despite 98% sequence identity. Small-angle X-ray scattering (SAXS) indicates that the crystal structure better predicts the properties of calbindin-D28K in solution compared with the NMR structure. Here, the first direct visualization of the calcium-binding properties of calbindin-D28K is presented. Four of the six EF-hands that make up the secondary structure of the protein contain a calcium-binding site. Two distinct conformations of the N-terminal EF-hand calcium-binding site were identified using long-wavelength calcium single-wavelength anomalous dispersion (SAD). This flexible region has previously been recognized as a protein-protein interaction interface. SAXS data collected in both the presence and absence of calcium indicate that there are no large structural differences in the globular structure of calbindin-D28K between the calcium-loaded and unloaded proteins.
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http://dx.doi.org/10.1107/S2059798318011610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173056PMC
October 2018

Co-crystallization of human inositol monophosphatase with the lithium mimetic L-690,330.

Acta Crystallogr D Struct Biol 2018 Oct 2;74(Pt 10):973-978. Epub 2018 Oct 2.

School of Biosciences, Medicines Discovery Institute, Park Place, Cardiff CF10 3AT, Wales.

Lithium, which is still the gold standard in the treatment of bipolar disorder, has been proposed to inhibit inositol monophosphatase (IMPase) and is hypothesized to exert its therapeutic effects by attenuating phosphatidylinositol (PI) cell signalling. Drug-discovery efforts have focused on small-molecule lithium mimetics that would specifically inhibit IMPase without exhibiting the undesired side effects of lithium. L-690,330 is a potent bisphosphonate substrate-based inhibitor developed by Merck Sharp & Dohme. To aid future structure-based inhibitor design, determination of the exact binding mechanism of L-690,330 to IMPase was of interest. Here, the high-resolution X-ray structure of human IMPase in complex with L690,330 and manganese ions determined at 1.39 Å resolution is reported.
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http://dx.doi.org/10.1107/S2059798318010380DOI Listing
October 2018

Evidence That Sedative Effects of Benzodiazepines Involve Unexpected GABA Receptor Subtypes: Quantitative Observation Studies in Rhesus Monkeys.

J Pharmacol Exp Ther 2018 07 2;366(1):145-157. Epub 2018 May 2.

New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts (A.N.D., Z.M., D.M.P., J.K.R.); Department of Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, Mississippi (D.M.P., J.K.R.); Medicines Discovery Institute, Cardiff University, Cardiff, Wales, United Kingdom (J.R.A.); P1Vital, University of Oxford, Warneford Hospital, Headington, Oxford, United Kingdom (G.R.D.); Alzheimer's Research UK, Granta Park, Great Abington, Cambridge, United Kingdom (D.S.R.); Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin (V.V.N.P.B.T., G.L., M.R.S., J.M.C.); and Division of Biochemistry and Molecular Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria (W.S.)

In nonhuman primates we tested a new set of behavioral categories for observable sedative effects using pediatric anesthesiology classifications as a basis. Using quantitative behavioral observation techniques in rhesus monkeys, we examined the effects of alprazolam and diazepam (nonselective benzodiazepines), zolpidem (preferential binding to 1 subunit-containing GABA receptors), HZ-166 (8-ethynyl-6-(2'-pyridine)-4-2,5,10b-triaza-benzo[]azulene-3-carboxylic acid ethyl ester; functionally selective with relatively high intrinsic efficacy for 2 and 3 subunit-containing GABA receptors), MRK-696 [7-cyclobutyl-6-(2-methyl-2-1,2,4-triazol-2-ylmethoxy)-3-(2-flurophenyl)-1,2,4-triazolo(4,3-)pyridazine; no selectivity but partial intrinsic activity], and TPA023B 6,2'-diflouro-5'-[3-(1-hydroxy-1-methylethyl)imidazo[1,2-][1,2,4]triazin-7-yl]biphenyl-2-carbonitrile; partial intrinsic efficacy and selectivity for 2, 3, 5 subunit-containing GABA receptors]. We further examined the role of 1 subunit-containing GABA receptors in benzodiazepine-induced sedative effects by pretreating animals with the 1 subunit-preferring antagonist -carboline-3-carboxylate--butyl ester (CCT). Increasing doses of alprazolam and diazepam resulted in the emergence of observable ataxia, rest/sleep posture, and moderate and deep sedation. In contrast, zolpidem engendered dose-dependent observable ataxia and deep sedation but not rest/sleep posture or moderate sedation, and HZ-166 and TPA023 induced primarily rest/sleep posture. MRK-696 induced rest/sleep posture and observable ataxia. Zolpidem, but no other compounds, significantly increased tactile/oral exploration. The sedative effects engendered by alprazolam, diazepam, and zolpidem generally were attenuated by CCT pretreatments, whereas rest/sleep posture and suppression of tactile/oral exploration were insensitive to CCT administration. These data suggest that 2/3-containing GABA receptor subtypes unexpectedly may mediate a mild form of sedation (rest/sleep posture), whereas 1-containing GABA receptors may play a role in moderate/deep sedation.
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http://dx.doi.org/10.1124/jpet.118.249250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988000PMC
July 2018

Lipophilic nalmefene prodrugs to achieve a one-month sustained release.

J Control Release 2016 06 21;232:196-202. Epub 2016 Apr 21.

Neuroscience, Janssen Pharmaceuticals, Turnhoutseweg 30, B-2340 Beerse, Belgium. Electronic address:

Nalmefene is an opioid antagonist which as a once-a-day tablet formulation has recently been approved for reducing ethanol intake in alcoholic subjects. In order to address the compliance issue in this patient population, a number of potential nalmefene prodrugs were synthesized with the aim of providing a formulation that could provide plasma drug concentrations in the region of 0.5-1.0ng/mL for a one-month period when dosed intramuscular to dogs or minipigs. In an initial series of studies, three different lipophilic nalmefene derivatives were evaluated: the palmitate (C16), the octadecyl glutarate diester (C18-C5) and the decyl carbamate (CB10). They were administered intramuscularly to dogs in a sesame oil solution at a dose of 1mg-eq. nalmefene/kg. The decyl carbamate was released relatively quickly from the oil depot and its carbamate bond was too stable to be used as a prodrug. The other two derivatives delivered a fairly constant level of 0.2-0.3ng nalmefene/mL plasma for one month and since there was no significant difference between these two, the less complex palmitate monoester was chosen to demonstrate that dog plasma nalmefene concentrations were dose-dependent at 1, 5 and 20mg-eq. nalmefene/kg. In a second set of experiments, the effect of the chain length of the fatty acid monoester promoieties was examined. The increasingly lipophilic octanoate (C8), decanoate (C10) and dodecanoate (C12) derivatives were evaluated in dogs and in minipigs, at a dose of 5mg-eq. nalmefene/kg and plasma nalmefene concentrations were measured over a four-week period. The pharmacokinetic profiles were very similar in both species with Cmax decreasing and Tmax increasing with increasing fatty acid chain length and the target plasma concentrations (0.5-1.0ng/mL over a month-long period) were achieved with the dodecanoate (C12) prodrug. These data therefore demonstrate that sustained plasma nalmefene concentrations can be achieved in both dog and minipig using nalmefene prodrugs and that the pharmacokinetic profile of nalmefene can be tuned by varying the length of the alkyl group.
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http://dx.doi.org/10.1016/j.jconrel.2016.04.029DOI Listing
June 2016

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2.

Biochem J 2016 07 20;473(13):1869-79. Epub 2016 Apr 20.

Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer BN1 9RQ, U.K.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5'-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a 'humanized' form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2.
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http://dx.doi.org/10.1042/BCJ20160180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4925160PMC
July 2016

JNJ-40255293, a novel adenosine A2A/A1 antagonist with efficacy in preclinical models of Parkinson's disease.

ACS Chem Neurosci 2014 Oct 24;5(10):1005-19. Epub 2014 Sep 24.

Janssen Research and Development , Welsh and McKean Roads, Spring House, Pennsylvania 19477, United States.

Adenosine A2A antagonists are believed to have therapeutic potential in the treatment of Parkinson's disease (PD). We have characterized the dual adenosine A2A/A1 receptor antagonist JNJ-40255293 (2-amino-8-[2-(4-morpholinyl)ethoxy]-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one). JNJ-40255293 was a high-affinity (7.5 nM) antagonist at the human A2A receptor with 7-fold in vitro selectivity versus the human A1 receptor. A similar A2A:A1 selectivity was seen in vivo (ED50's of 0.21 and 2.1 mg/kg p.o. for occupancy of rat brain A2A and A1 receptors, respectively). The plasma EC50 for occupancy of rat brain A2A receptors was 13 ng/mL. In sleep-wake encephalographic (EEG) studies, JNJ-40255293 dose-dependently enhanced a consolidated waking associated with a subsequent delayed compensatory sleep (minimum effective dose: 0.63 mg/kg p.o.). As measured by microdialysis, JNJ-40255293 did not affect dopamine and noradrenaline release in the prefrontal cortex and the striatum. However, it was able to reverse effects (catalepsy, hypolocomotion, and conditioned avoidance impairment in rats; hypolocomotion in mice) produced by the dopamine D2 antagonist haloperidol. The compound also potentiated the agitation induced by the dopamine agonist apomorphine. JNJ-40255293 also reversed hypolocomotion produced by the dopamine-depleting agent reserpine and potentiated the effects of l-dihydroxyphenylalanine (L-DOPA) in rats with unilateral 6-hydroxydopamine-induced lesions of the nigro-striatal pathway, an animal model of Parkinson's disease. Extrapolating from the rat receptor occupancy dose-response curve, the occupancy required to produce these various effects in rats was generally in the range of 60-90%. The findings support the continued research and development of A2A antagonists as potential treatments for PD.
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http://dx.doi.org/10.1021/cn5001606DOI Listing
October 2014

Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors.

Anal Biochem 2014 Jun 14;454:17-22. Epub 2014 Mar 14.

Translational Drug Discovery Group, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK. Electronic address:

Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.
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http://dx.doi.org/10.1016/j.ab.2014.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007590PMC
June 2014

Pharmacological characterization of JNJ-40068782, a new potent, selective, and systemically active positive allosteric modulator of the mGlu2 receptor and its radioligand [3H]JNJ-40068782.

J Pharmacol Exp Ther 2013 Sep 13;346(3):514-27. Epub 2013 Jun 13.

Janssen Research and Development, Beerse, Belgium.

Modulation of the metabotropic glutamate type 2 (mGlu2) receptor is considered a promising target for the treatment of central nervous system diseases such as schizophrenia. Here, we describe the pharmacological properties of the novel mGlu2 receptor positive allosteric modulator (PAM) 3-cyano-1-cyclopropylmethyl-4-(4-phenyl-piperidin-1-yl)-pyridine-2(1H)-one (JNJ-40068782) and its radioligand [(3)H]JNJ-40068782. In guanosine 5'-O-(3-[(35)S]thio)triphosphate binding, JNJ-40068782 produced a leftward and upward shift in the glutamate concentration-effect curve at human recombinant mGlu2 receptors. The EC50 of JNJ-40068782 for potentiation of an EC20-equivalent concentration of glutamate was 143 nM. Although JNJ-40068782 did not affect binding of the orthosteric antagonist [(3)H]2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY-341495), it did potentiate the binding of the agonist [(3)H](2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV), demonstrating that it can allosterically affect binding at the agonist recognition site. The binding of [(3)H]JNJ-40068782 to human recombinant mGlu2 receptors in Chinese hamster ovary cells and rat brain receptors was saturable with a KD of ∼10 nM. In rat brain, the anatomic distribution of [(3)H]JNJ-40068782 was consistent with mGlu2 expression previously described and was most abundant in cortex and hippocampus. The ability of structurally unrelated PAMs to displace [(3)H]JNJ-40068782 suggests that PAMs may bind to common determinants within the same site. It is noteworthy that agonists also increased the binding affinity of [(3)H]JNJ-40068782. JNJ-40068782 influenced rat sleep-wake organization by decreasing rapid eye movement sleep with a lowest active dose of 3 mg/kg PO. In mice, JNJ-40068782 reversed phencyclidine-induced hyperlocomotion with an ED50 of 5.7 mg/kg s.c. Collectively, the present data demonstrate that JNJ-40068782 has utility in investigating the potential of mGlu2 modulation for the treatment of diseases characterized by disturbed glutamatergic signaling and highlight the value of [(3)H]JNJ-40068782 in exploring allosteric binding.
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http://dx.doi.org/10.1124/jpet.113.204990DOI Listing
September 2013

Reinforcing effects of compounds lacking intrinsic efficacy at α1 subunit-containing GABAA receptor subtypes in midazolam- but not cocaine-experienced rhesus monkeys.

Neuropsychopharmacology 2013 May 27;38(6):1006-14. Epub 2012 Dec 27.

Department of Psychiatry, Division of Neuroscience, New England Primate Research Center, Beth Israel Deaconess Medical Center and Harvard Medical School, MA 01772, USA.

Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors ('α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration.
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http://dx.doi.org/10.1038/npp.2012.265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629390PMC
May 2013

The discovery and synthesis of JNJ 31020028, a small molecule antagonist of the Neuropeptide Y Y₂ receptor.

Bioorg Med Chem Lett 2011 Sep 18;21(18):5552-6. Epub 2011 Jul 18.

Johnson & Johnson Pharmaceutical Research & Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.

A series of small molecules based on a chemotype identified from our compound collection were synthesized and tested for binding affinity (IC(50)) at the human Neuropeptide Y Y(2) receptor (NPY Y(2)). Six of the 23 analogs tested possessed an NPY Y(2) IC(50) ≤ 15 nM. One member of this series, JNJ 31020028, is a selective, high affinity, receptor antagonist existing as a racemic mixture. As such a synthetic route to the desired enantiomer was designed starting from commercially available (S)-(+)-mandelic acid.
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http://dx.doi.org/10.1016/j.bmcl.2011.06.136DOI Listing
September 2011

GABAA receptor alpha2/alpha3 subtype-selective modulators as potential nonsedating anxiolytics.

Authors:
John R Atack

Curr Top Behav Neurosci 2010 ;2:331-60

Department of Neuroscience, Johnson & Johnson Pharmaceutical Research and Development, Building 020, Room 1A6, Turnhoutseweg 30, B-2340, Beerse, Belgium.

Nonselective benzodiazepines exert their pharmacological effects via GABAA receptors containing either an alpha1, alpha2, alpha3, or alpha5 subunit. The use of subtype-selective tool compounds along with transgenic mice has formed the conceptual framework for defining the requirements of subtype-selective compounds with potentially novel pharmacological profiles. More specifically, compounds which allosterically modulate the alpha2 and/or alpha3 subtypes but are devoid of, or have much reduced, effects at the alpha1 subtype are hypothesized to be anxioselective (i.e., anxiolytic but devoid of sedation). Accordingly, three compounds, MRK-409, TPA023 and TPA023B, which selectively potentiated the effects of GABA at the alpha2 and alpha3 compared to alpha1 subtypes were progressed into man. All three compounds behaved as nonsedating anxiolytics in preclinical (rodent and primate) species but, surprisingly, MRK-409 produced sedation in man at relatively low levels of occupancy (< 10%). This sedation liability of MRK-409 in man was attributed to its weak partial agonist efficacy at the alpha1 subtype since both TPA023 and TPA023B lacked any alpha1 efficacy and did not produce overt sedation even at relatively high levels of occupancy (> 50%). The anxiolytic efficacy of TPA023 was evaluated in Generalized Anxiety Disorder and although these clinical trials were terminated early due to preclinical toxicity issues, the combined data from these incomplete studies demonstrated an anxiolytic-like effect of TPA023. This compound also showed a trend to increase cognitive performance in a small group of schizophrenic subjects and is currently under further evaluation of its cognition-enhancing effects in schizophrenia as part of the TURNS initiative. In contrast, the fate of the back-up clinical candidate TPA023B has not been publicly disclosed. At the very least, these data indicate that the pharmacological profile of compounds that differentially modulate specific populations of GABAA receptors is distinct from classical benzodiazepines and should encourage further preclinical and clinical investigation of such compounds, with the caveat that, as exemplified by MRK-409, the preclinical profile might not necessarily translate into man.
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http://dx.doi.org/10.1007/7854_2009_30DOI Listing
March 2011

Contribution of GABA(A) receptors containing α3 subunits to the therapeutic-related and side effects of benzodiazepine-type drugs in monkeys.

Psychopharmacology (Berl) 2011 May 30;215(2):311-9. Epub 2010 Dec 30.

Harvard Medical School, New England Primate Research Center, One Pine Hill Drive, P.O. Box 9102, Southborough, MA 01772-9102, USA.

Rationale: Experimental evidence suggests that the differential behavioral effects of benzodiazepines depend on their relative actions at γ-aminobutyric acid type A (GABA(A)) receptors that contain either an α1, α2, α3, or α5 subunit.

Objectives: The present study was aimed at understanding the role of α3 subunit-containing GABA(A) (α3GABA(A)) receptors by examining the behavioral pharmacology of TP003 (4,2'-difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-a]pyridine-3-yl]biphenyl-2-carbonitrile), which shows functional selectivity for α3GABA(A) receptors.

Methods: First, a conflict procedure was used to assess the anxiolytic-like effects of TP003 and a representative clinically available benzodiazepine. TP003 was also administered before daily periods of sucrose pellet availability to evaluate potential hyperphagic effects. In separate experiments, observable behavioral effects were used to assess the motor and sedative effects of TP003.

Results: Administration of TP003 produced robust anti-conflict effects without the rate-decreasing effects that were observed with the representative benzodiazepine. Unlike the reported effects of benzodiazepines, TP003 did not enhance palatable food consumption. However, increases in observable sleep-associated posture were induced by TP003, as were decreases in some species-typical behaviors (vocalization, locomotion, and environment-directed behaviors). When evaluated for its ability to induce a procumbent posture, TP003 failed to produce an effect.

Conclusions: Based on conflict and observation tests in monkeys, our results suggest that TP003 may have anxiolytic properties but lack ataxic, hyperphagic, and pronounced sedative effects characteristic of classical benzodiazepines. TP003 did induce myorelaxant-like effects and had relatively mild sedative effects. Collectively, these results suggest that α3GABA(A) receptors play an important role in the anxiolytic-like and motor effects of benzodiazepine-type drugs.
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http://dx.doi.org/10.1007/s00213-010-2142-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097109PMC
May 2011

GABAA receptor subtype-selective modulators. I. α2/α3-selective agonists as non-sedating anxiolytics.

Authors:
John R Atack

Curr Top Med Chem 2011 ;11(9):1176-202

Department of Neuroscience, Janssen Pharmaceutical Research and Development, Beerse, Belgium.

The prototypic benzodiazepines, such as diazepam, are not only anxiolytic but also produce sedation. These effects are mediated by GABA(A) receptors containing either an α1, α2, α3 or α5 subunit at which the positive modulatory effects (i.e., agonist efficacy) of benzodiazepines are mediated via a specific benzodiazepine recognition site. Recent molecular genetic and pharmacological data point to α1-containing GABA(A) receptors as the "sedative" and α2- and/or α3-containing receptors as the "anxiolytic" subtype(s). Therefore, at Merck Sharp & Dohme attempts were made to identify subtype-selective compounds that modulate α2/α3 but not α1 receptor function with the prediction that such compounds would be non-sedating anxiolytics. The initial strategy for discovering such "anxioselective" compounds focussed on producing compounds with much higher affinity at the α2/α3 compared to α1 subtypes. The starting point for this approach was the triazolophthalazine series developed from a combination of a screening hit and a literature compound [1]. However, the maximum α3 versus α1 binding selectivity that could be achieved in this series was 12-fold and this was not considered sufficient for an appropriate in vivo pharmacological differentiation compared to non-selective compounds. Nevertheless, within this series compounds demonstrating (albeit to a limited extent) higher agonist efficacy at the α3 versus α1 subtype were also identified. This suggested that it might be possible to synthesize a compound with higher efficacy at the α2 and/or α3 compared to α1 subtypes, ideally with no efficacy at the latter subtype (i.e., a compound with subtype-selective efficacy). By changing the structure from a triazolophthalazine to a triazolopyridazine core, a number of either pharmacological tool compounds (L-838417, MRK-067 and MRK-696) or clinical development candidates (MRK-409 and TPA023) were identified. Encouraged by the success of this approach and the observation that the benzimidazole NS-2710 had a modest degree of α3 versus α1 selectivity efficacy, a structurally-related class of imidazopyridines was also explored. The introduction of an additional nitrogen into the imidazopyridine core gave the imidazopyrimidine series which initially had issues with poor dog pharmacokinetics. However, this was resolved and resulted in the identification of the development candidates MRK-623 and MRK-898. A fluoroimidazopyridine was found to be a bioisostere of the imidazopyrimidine core and in this series the α3-selective tool compound TP003 was identified. The addition of a further nitrogen into the imidazopyrimidine core produced the imidazotriazine series, which yielded the clinical candidate TPA023B. Imidazopyrazinone and imidazotriazinone compounds offered no advantages over their respective imidazopyrimidine and imidazotriazine analogues. Additional pharmacological tool compounds were identified within the pyridine, pyrazolotriazine, pyridazine and pyrazolopyridone series highlighting the general feasibility of GABA(A) receptor subtype selective efficacy as a strategy for developing compounds with novel in vitro and in vivo profiles.
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http://dx.doi.org/10.2174/156802611795371350DOI Listing
August 2011

GABAA receptor subtype-selective modulators. II. α5-selective inverse agonists for cognition enhancement.

Authors:
John R Atack

Curr Top Med Chem 2011 ;11(9):1203-14

Department of Neuroscience, Janssen Pharmaceutical Research and Development, Beerse, Belgium.

Benzodiazepine site agonists (such as diazepam) are well-known to impair cognition. Since benzodiazepines exert their effects via modulation of α1-, α2-, α3- and α5-containing GABA(A) receptors, the cognition-impairing effects of diazepam must be associated with one or several of these subtypes. Of these different subtypes, α5-containing GABA(A) receptors represent an attractive option as the "cognition" subtype based upon the preferential localization of these receptors within the hippocampus and the well-established role of the hippocampus in learning and memory. As a result, it is hypothesized that an inverse agonist selective for the α5 subtype should enhance cognition. For example, L-655708, a partial inverse agonist with 50-100-fold higher affinity for the α5 relative to the α1, α2 and α3 subtypes of GABA(A) receptors, enhanced cognitive performance in rats. Unfortunately, however, pharmacokinetic properties of this compound prevented it being developed further. In order to try achieve binding selectivity in a series structurally distinct from the imidazobenzodiazepines, the group at Merck, Sharp & Dohme commenced studies within the triazolopyridazine series. Although a degree of binding selectivity could be achieved (a maximum of 22-125-fold for α5 versus α1, α2 or α3) this approach was dropped in favour of a strategy to identify compounds with either a combination of selective affinity and selective efficacy or purely selective efficacy. With respect to the former, screening of the Merck chemical collection identified a novel, moderately α5-binding selective thiophene series and further optimization of this series produced MRK-536, which demonstrated a modest α5 binding selectivity (~10-fold) as well as α5-efficacy selectivity. However, the structure-activity relationship within this and the analogous tetralone series proved unpredictable and these series were not pursued further. The success of the selective efficacy approach on the α2/α3-selective agonist project led a similar paradigm being adopted for the α5 project. The starting point for this strategy was the triazolopyridazine 3 which, like MRK-536, possessed a degree of both α5 binding- and efficacy-selectivity. By changing the core from a triazolopyridazine to a triazolophthalazine structure, α5 binding selectivity was lost but with subsequent optimization, compounds with the desired profile (low or antagonist efficacy at the α1, α2 and α3 subtypes and marked inverse agonism at α5-containing receptors) could be achieved, allowing the clinical candidate α5IA as well as the structurally-related pharmacological tool compound α5IA-II to be identified. By appending features of the prototypic α2/α3-selective triazolopyridazine L-838417 (t-butyl and 1,2,4 triazole) along with the isoxazole of α5IA to a pyrazolotriazine core, an additional clinical candidate, MRK-016, was identified. Finally, a degree of α5 efficacy selectivity was achieved the pyridazine series but metabolic instability within this chemotype limited its further optimization. Overall, these studies demonstrate the feasibility of adopting a selective efficacy approach in the identification of α5 selective GABA(A) receptor inverse agonists.
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http://dx.doi.org/10.2174/156802611795371314DOI Listing
August 2011

Pre-clinical characterization of aryloxypyridine amides as histamine H3 receptor antagonists: identification of candidates for clinical development.

Bioorg Med Chem Lett 2010 Jul 16;20(14):4210-4. Epub 2010 May 16.

Johnson & Johnson Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.

The pre-clinical characterization of novel aryloxypyridine amides that are histamine H(3) receptor antagonists is described. These compounds are high affinity histamine H(3) ligands that penetrate the CNS and occupy the histamine H(3) receptor in rat brain. Several compounds were extensively profiled pre-clinically leading to the identification of two compounds suitable for nomination as development candidates.
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http://dx.doi.org/10.1016/j.bmcl.2010.05.041DOI Listing
July 2010

Novel substituted pyrrolidines are high affinity histamine H3 receptor antagonists.

Bioorg Med Chem Lett 2010 May 20;20(9):2755-60. Epub 2010 Mar 20.

Johnson & Johnson Pharmaceutical Research & Development L.L.C., 3210 Merryfield Row, San Diego, CA 92121, United States.

Pre-clinical characterization of novel substituted pyrrolidines that are high affinity histamine H(3) receptor antagonists is described. These compounds efficiently penetrate the CNS and occupy the histamine H(3) receptor in rat brain following oral administration. One compound, (2S,4R)-1-[2-(4-cyclobutyl-[1,4]diazepane-1-carbonyl)-4-(3-fluoro-phenoxy)-pyrrolidin-1-yl]-ethanone, was extensively profiled and shows promise as a potential clinical candidate.
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http://dx.doi.org/10.1016/j.bmcl.2010.03.071DOI Listing
May 2010

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha2 subunit-containing GABAA receptors.

Proc Natl Acad Sci U S A 2010 Feb 19;107(5):2289-94. Epub 2010 Jan 19.

Department of Psychology, University of Sussex, Brighton BN1 9QG, United Kingdom.

Because GABA(A) receptors containing alpha2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine's ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha2-GABA(A) receptors (alpha2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.
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http://dx.doi.org/10.1073/pnas.0910117107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836671PMC
February 2010

In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor.

Psychopharmacology (Berl) 2010 Feb 2;208(2):265-77. Epub 2009 Dec 2.

Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, USA.

Rationale: The lack of potent, selective, brain penetrant Y(2) receptor antagonists has hampered in vivo functional studies of this receptor.

Objective: Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y(2) receptor antagonist.

Methods: The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y(2) receptors in KAN-Ts cells and rat Y(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release.

Results: JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was >100-fold selective versus human Y(1), Y(4), and Y(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y(2) receptor binding sites (approximately 90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake.

Conclusion: These results suggest that Y(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.
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http://dx.doi.org/10.1007/s00213-009-1726-xDOI Listing
February 2010

Discriminative stimulus effects of L-838,417 (7-tert-butyl-3-(2,5-difluoro-phenyl)-6-(2-methyl-2H-[1,2,4]triazol-3-ylmethoxy)-[1,2,4]triazolo[4,3-b]pyridazine): role of GABA(A) receptor subtypes.

Neuropharmacology 2010 Feb 22;58(2):357-64. Epub 2009 Oct 22.

Harvard Medical School, New England Primate Research Center, One Pine Hill Drive, Southborough, MA 01772-9102, USA.

Previous reports suggest that gamma-aminobutyric acid type A (GABA(A)) receptors containing alpha1 subunits may play a pivotal role in mediating the discriminative stimulus effects of benzodiazepines (BZs). L-838,417 (7-tert-Butyl-3-(2,5-difluoro-phenyl)-6-(2-methyl-2H-[1,2,4]triazol-3-ylmethoxy)-[1,2,4]triazolo[4,3-b]pyridazine) is a GABA(A) receptor modulator with intrinsic efficacy in vitro at alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors, and little demonstrable intrinsic efficacy in vitro at alpha1 subunit-containing GABA(A) receptors. The present study evaluated the discriminative stimulus effects of L-838,417 in order to determine the extent to which the alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors contribute to the interoceptive effects of BZ-type drugs. Squirrel monkeys (Saimiri sciureus) were trained to discriminate L-838,417 (0.3 mg/kg, i.v.) from vehicle under a 5-response fixed-ratio schedule of food reinforcement. Under test conditions, L-838,417 administration resulted in dose-dependent increases in drug-lever responding that were antagonized by the BZ-site antagonist, flumazenil. Administration of non-selective BZs, compounds with 10-fold greater affinity for alpha1 subunit-containing GABA(A) receptors compared to alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors, barbiturates and ethanol (which modulate the GABA(A) receptor via a non-BZ site), all resulted in a majority of responses on the L-838,417-paired lever (65-100% drug-lever responding). betaCCT, an antagonist that binds with 20-fold greater affinity for alpha1 subunit-containing GABA(A) receptors relative to alpha2, alpha3, and alpha5-containing GABA(A) receptors, had no significant effect on the discriminative stimulus effects of L-838,417 or the L-838,417-like effects of diazepam or zolpidem. These data suggest that efficacy at alpha2, alpha3, and/or alpha5 subunit-containing GABA(A) receptors likely are sufficient for engendering BZ-like discriminative stimulus effects.
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http://dx.doi.org/10.1016/j.neuropharm.2009.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2813423PMC
February 2010

Reducing abuse liability of GABAA/benzodiazepine ligands via selective partial agonist efficacy at alpha1 and alpha2/3 subtypes.

J Pharmacol Exp Ther 2010 Jan 29;332(1):4-16. Epub 2009 Sep 29.

Division of Behavioral Biology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

Abuse-liability-related effects of subtype-selective GABA(A) modulators were explored relative to the prototypic benzodiazepine lorazepam. 7-Cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine (TPA123) has weak partial agonist efficacy at alpha(1)-, alpha(2)-, alpha(3)-, and alpha(5)-containing GABA(A) receptors, whereas 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) has weaker partial agonist efficacy at alpha(2) and alpha(3) and none at alpha(1) and alpha(5) subtypes. For both compounds, preclinical data suggested efficacy as nonsedating anxiolytics. Self-injection of TPA123 (0.0032-0.1 mg/kg) and TPA023 (0.0032-0.32 mg/kg) was compared with lorazepam (0.01-0.32 mg/kg) in baboons. TPA123 and lorazepam maintained self-injection higher than vehicle at two or more doses in each baboon; peak rate of self-injection of lorazepam was higher than TPA123. Self-injected lorazepam and TPA123 also increased rates of concurrently occurring food-maintained behavior. After the availability of self-administered TPA123 doses ended, an effect consistent with a mild benzodiazepine-like withdrawal syndrome occurred. In contrast with lorazepam and TPA123, TPA023 did not maintain self-administration. Positron emission tomography studies showed that TPA023 produced a dose-dependent inhibition in the binding of [(11)C]flumazenil to the benzodiazepine binding site in the baboon, which was essentially complete (i.e., 100% occupancy) at the highest TPA023 dose (0.32 mg/kg). In a physical dependence study, TPA023 (32 mg/kg/24 h) was delivered as a continuous intragastric drip. Neither flumazenil at 14 days nor stopping TPA023 after 30 to 31 days resulted in the marked withdrawal syndrome characteristic of benzodiazepines in baboons. In the context of other data, elimination of efficacy at the alpha(1) subtype of the GABA/benzodiazepine receptor is not sufficient to eliminate abuse liability but may do so when coupled with reduced alpha(2/3) subtype efficacy.
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http://dx.doi.org/10.1124/jpet.109.158303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2802472PMC
January 2010

Benzodiazepine binding site occupancy by the novel GABAA receptor subtype-selective drug 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) in rats, primates, and humans.

J Pharmacol Exp Ther 2010 Jan 24;332(1):17-25. Epub 2009 Sep 24.

Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, United Kingdom.

The GABA(A) receptor alpha2/alpha3 subtype-selective compound 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023; also known as MK-0777) is a triazolopyridazine that has similar, subnanomolar affinity for the benzodiazepine binding site of alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors and has partial agonist efficacy at the alpha2 and alpha3 but not the alpha1 or alpha5 subtypes. The purpose of the present study was to define the relationship between plasma TPA023 concentrations and benzodiazepine binding site occupancy across species measured using various methods. Thus, occupancy was measured using either in vivo [(3)H]flumazenil binding or [(11)C]flumazenil small-animal positron emission tomography (microPET) in rats, [(123)I]iomazenil gamma-scintigraphy in rhesus monkeys, and [(11)C]flumazenil PET in baboons and humans. For each study, plasma-occupancy curves were derived, and the plasma concentration of TPA023 required to produce 50% occupancy (EC(50)) was calculated. The EC(50) values for rats, rhesus monkeys, and baboons were all similar and ranged from 19 to 30 ng/ml, although in humans, the EC(50) was slightly lower at 9 ng/ml. In humans, a single 2-mg dose of TPA023 produced in the region of 50 to 60% occupancy in the absence of overt sedative-like effects. Considering that nonselective full agonists are associated with sedation at occupancies of less than 30%, these data emphasize the relatively nonsedating nature of TPA023.
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http://dx.doi.org/10.1124/jpet.109.157909DOI Listing
January 2010

Preclinical and clinical pharmacology of the GABAA receptor alpha5 subtype-selective inverse agonist alpha5IA.

Authors:
John R Atack

Pharmacol Ther 2010 Jan 19;125(1):11-26. Epub 2009 Sep 19.

Dept. of In Vivo Neuroscience, The Neuroscience Research Centre, Merck Sharp & Dohme Research Labs., Terlings Park, Eastwick Road, Harlow, Essex, UK.

alpha5IA is a triazolophthalazine that selectively attenuates the effects of GABA at GABA(A) receptors containing an alpha5 subunit. It enhances long-term potentiation in an in vitro model of mouse hippocampal synaptic plasticity, gives good in vivo receptor occupancy and improves cognitive performance in normal rats as measured using the delayed-matching-to-place version of the Morris water maze yet, importantly, it is without anxiogenic or proconvulsant liabilities. The hydroxymethyl isoxazole metabolite, which occurs both in vitro and in vivo, has a very low aqueous solubility (0.6microg/mL) that resulted in renal toxicity (crystal formation) at very high doses in preclinical safety and toxicity studies. Although this precluded it from being dosed to humans over prolonged periods of time, alpha5IA is, nevertheless, well tolerated in young and elderly subjects up to a dose of 6mg in multiple-dose studies and gives a plasma EC(50) for alpha5IA occupancy measured using [(11)C]flumazenil PET of 10ng/mL. The compound was evaluated in experimental studies and although in elderly subjects alpha5IA does not improve performance in a paired-associate learning task (a 4-mg dose actually impairs performance), it is able to reverse the ethanol-induced impairment in performance in healthy young normal volunteers. These data demonstrate that in man an alpha5-selective inverse agonist may be effective at increasing performance under certain conditions. Whether or not such a compound has efficacy in conditions associated with cognitive deficits, such as attention-deficit hyperactivity disorder, Alzheimer's disease or schizophrenia remains to be determined.
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http://dx.doi.org/10.1016/j.pharmthera.2009.09.001DOI Listing
January 2010

In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist.

J Pharmacol Exp Ther 2009 Nov 24;331(2):470-84. Epub 2009 Aug 24.

Merck Sharp & Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, United Kingdom.

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.
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http://dx.doi.org/10.1124/jpet.109.157636DOI Listing
November 2009