Dianne Kowal

Monoacylglycerol lipase activity is a critical modulator of the tone and integrity of the endocannabinoid system

Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.

Procognitive and Neuroprotective Activity of a Novel α7 Nicotinic Acetylcholine Receptor Agonist for Treatment of Neurodegenerative and Cognitive Disorders

The α7 nicotinic acetylcholine receptor (nAChR) is a promising target for treatment of cognitive dysfunction associated with Alzheimers disease and schizophrenia. Here, we report the pharmacological properties of 5-morpholin-4-yl-pentanoic acid (4-pyridin-3-yl-phenyl)-amide [SEN12333 (WAY-317538)], a novel selective agonist of α7 nAChR. SEN12333 shows high affinity for the rat α7 receptor expressed in GH4C1 cells (Ki = 260 nM) and acts as full agonist in functional Ca2+ flux studies (EC50 = 1.6 μM). In whole-cell patch-clamp recordings, SEN12333 activated peak currents and maximal total charges similar to acetylcholine (EC50 = 12 μM). The compound did not show agonist activity at other nicotinic receptors tested and acted as a weak antagonist at α3-containing receptors. SEN12333 treatment (3 mg/kg i.p.) improved episodic memory in a novel object recognition task in rats in conditions of spontaneous forgetting as well as cognitive disruptions induced via glutamatergic [5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); MK-801] or cholinergic (scopolamine) mechanisms. This improvement was blocked by the α7-selective antagonist methyllycaconitine, indicating that it is mediated by α7 activation. SEN12333 also prevented a scopolamine-induced deficit in a passive avoidance task. In models targeting other cognitive domains, including attention and perceptual processing, SEN12333 normalized the apomorphine-induced deficit of prepulse inhibition. Neuroprotection of SEN12333 was demonstrated in quisqualate-lesioned animals in which treatment with SEN12333 (3 mg/kg/day i.p.) resulted in a significant protection of choline acetyltransferase-positive neurons in the lesioned hemisphere. Cumulatively, our results demonstrate that the novel α7 nAChR agonist SEN12333 has procognitive and neuroprotective properties, further demonstrating utility of α7 agonists for treatment of neurodegenerative and cognitive disorders.

The Metabotropic Glutamate Receptor 7 Allosteric Modulator AMN082: A Monoaminergic Agent in Disguise?

Metabotropic glutamate receptor 7 (mGluR7) remains the most elusive of the eight known mGluRs primarily because of the limited availability of tool compounds to interrogate its potential therapeutic utility. The discovery of N,N′-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) as the first orally active, brain-penetrable, mGluR7-selective allosteric agonist by Mitsukawa and colleagues (Proc Natl Acad Sci USA 102:18712–18717, 2005) provides a means to investigate this receptor system directly. AMN082 demonstrates mGluR7 agonist activity in vitro and interestingly has a behavioral profile that supports utility across a broad spectrum of psychiatric disorders including anxiety and depression. The present studies were conducted to extend the in vitro and in vivo characterization of AMN082 by evaluating its pharmacokinetic and metabolite profile. Profiling of AMN082 in rat liver microsomes revealed rapid metabolism (t1/2 < 1 min) to a major metabolite, N-benzhydrylethane-1,2-diamine (Met-1). In vitro selectivity profiling of Met-1 demonstrated physiologically relevant transporter binding affinity at serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) (323, 3020, and 3410 nM, respectively); whereas the parent compound AMN082 had appreciable affinity at NET (1385 nM). AMN082 produced antidepressant-like activity and receptor occupancy at SERT up to 4 h postdose, a time point at which AMN082 is significantly reduced in brain and plasma while the concentration of Met-1 continues to increase in brain. Acute Met-1 administration produced antidepressant-like activity as would be expected from its in vitro profile as a mixed SERT, NET, DAT inhibitor. Taken together, these data suggest that the reported in vivo actions of AMN082 should be interpreted with caution, because they may involve other mechanisms in addition to mGluR7.

Pharmacological comparison of muscarinic ligands: historical versus more recent muscarinic M1-preferring receptor agonists.

In functional assay assessments using the five muscarinic receptor subtypes, a second generation of muscarinic M(1)-preferring receptor agonists [AC-42 (1), AC-260584 (2), 77-LH-28-1 (3) and LY-593039 (4)] was shown to have higher selectivity for muscarinic M(1) over M(3) receptor as compared to historical agonists [talsaclidine (8), sabcomeline (10), xanomeline (11), WAY-132983 (12), cevimeline (9) and NGX-267 (6)]. Another striking difference of these more recent compounds is their affinities for the dopamine D(2) and 5-HT(2B) receptors. Taken together, these results suggest that the newer compounds may have a greater clinical safety profile, especially with regard to muscarinic M(3) receptor-mediated events, than the historical agonists, but their affinities for other receptors may still compromise their use to validate the therapeutic potential of muscarinic M(1) receptor agonists.

Old and New Pharmacology: Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor by the 5-Hydroxytryptamine2B/C Receptor Antagonist SB-206553 (3,5-Dihydro-5-methyl-N-3-pyridinylbenzo[1,2-b:4,5-b′]di pyrrole-1(2H)-carboxamide)

The α7 nicotinic acetylcholine receptor (nAChR) has been implicated in Alzheimers disease and schizophrenia, leading to efforts targeted toward discovering agonists and positive allosteric modulators (PAMs) of this receptor. In a Ca2+ flux fluorometric imaging plate reader assay, SB-206553 (3,5-dihydro-5-methyl -N-3-pyridinylbenzo [1, 2-b:4,5 -b′]-di pyrrole-1(2H)-carboxamide), a compound known as a 5-hydroxytryptamine2B/2C receptor antagonist, produced an 8-fold potentiation of the evoked calcium signal in the presence of an EC20 concentration of nicotine and a corresponding EC50 of 1.5 μM for potentiation of EC20 nicotine responses in GH4C1 cells expressing the α7 receptor. SB-206553 was devoid of direct α7 receptor agonist activity and selective against other nicotinic receptors. Confirmation of the PAM activity of SB-206553 on the α7 nAChR was obtained in patch-clamp electrophysiological experiments in GH4C1 cells, where it failed to evoke any detectable currents when applied alone, yet dramatically potentiated the currents evoked by an EC20 (17 μM) and EC100 (124 μM) of acetylcholine (ACh). Native nicotinic receptors in CA1 stratum radiatum interneurons of rat hippocampal slices could also be activated by ACh (200 μM), an effect that was entirely blocked by the α7-selective antagonist methyllycaconitine (MLA). These ACh currents were potentiated by SB-206553, which increased the area of the current response significantly, resulting in a 40-fold enhancement at 100 μM. In behavioral experiments in rats, SB-206553 reversed an MK-801 (dizocilpine maleate)-induced deficit in the prepulse inhibition of acoustic startle response, an effect attenuated in the presence of MLA. This latter observation provides further evidence in support of the potential therapeutic utility of α7 nAChR PAMs in schizophrenia.

Identification of a series of benzoxazoles as potent 5-HT6 ligands

As part of our continuing efforts to identify therapeutics for CNS diseases such as schizophrenia and Alzheimers disease (AD), we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands as a potential treatment for cognitive dysfunction. Herein we report the identification of a novel series of benzoxazole derivatives as potent 5-HT(6) ligands. The synthesis and detailed SAR of this class of compounds are reported. The compounds have been shown to be full antagonists in a cyclic AMP functional assay.

A [35S]GTPγS binding assessment of metabotropic glutamate receptor standards in Chinese hamster ovary cell lines expressing the human metabotropic receptor subtypes 2 and 4

Abstract The activities of metabotropic glutamate receptor (mGluR) standards were evaluated in the [ 35 S]GTP γ S binding assay and in the forskolin (FSK)-enhanced cyclic AMP assay using Chinese hamster ovary (CHO) cells or homogenates which expressed the human mGluR (hmGluR) subtypes 2 and 4. Though distinct rank orders of activities were determined for the agonists between the cell lines expressing individual hmGluRs, similar rank orders of agonist activities were determined for the standards between assays. O -phospho- l -serine ( l -SOP) and ( S )-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) antagonized agonist EC 90 responses in the cell lines expressing the hmGluR 2 and 4 subtypes, respectively. In addition to its antagonist effect, l -SOP increased the baseline level of cAMP when tested in the absence of agonist. In spite of this anomalous effect, l -SOP was found to be a competitive antagonist in the cAMP assay as well as in the [ 35 S]GTP γ S binding assay with a pA 2 value of 5.2 in both assays. MAP4 was a competitive antagonist of l (+)-2-amino-4-phosphonobutyric acid ( l -AP4)-induced responses in the CHO cell line expressing hmGluR4 with pA 2 values of 4.4 and 4.5 determined in the [ 35 S]GTP γ S binding and cAMP assays, respectively.

Evaluation of FLIPR Calcium 3 Assay Kit—A New No-Wash Fluorescence Calcium Indicator Reagent

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT2C), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT2C) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT2C receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC50 values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compel-ling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format. (Journal of Biomolecular Screening 2003:571-577)

WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate uptake.

The pharmacological profile of a novel glutamate transport inhibitor, WAY‐855 (3‐amino‐tricyclo[2.2.1.02.6]heptane‐1,3‐dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY‐855 inhibited glutamate uptake mediated by all three subtypes in a concentration‐dependent manner, with preferential inhibition of the CNS‐predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 μM, respectively, while EAAT1 activity was inhibited by 50% at 100 μM (IC50 values determined in oocytes were 1.3 μM (EAAT2), 52.5 μM (EAAT3) and 125.9 μM (EAAT1)). Application of WAY‐855 to EAAT‐expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]D‐aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY‐855 inhibited D‐aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY‐855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY‐855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes.

5-Piperazinyl-3-sulfonylindazoles as Potent and Selective 5-Hydroxytryptamine-6 Antagonists

As part of our efforts to develop agents for CNS diseases, we have been focused on the 5-HT(6) receptor in order to identify potent and selective ligands for cognitive enhancement. Herein we report the identification of a novel series of 5-piperazinyl-3-sulfonylindazoles as potent and selective 5-HT(6) antagonists. The synthesis, SAR, and pharmacokinetic and pharmacological activities of some of the compounds including 3-(naphthalen-1-ylsulfonyl)-5-(piperazin-1-yl)-1H-indazole (WAY-255315 or SAM-315) will be described.