The cognition-enhancing activity of E1R, a novel positive allosteric modulator of sigma-1 receptors.

BACKGROUND AND PURPOSE: Here, we describe the in vitro and in vivo effects of (4R,5S)-2-(5-methyl-2-oxo-4-phenyl-pyrrolidin-1-yl)-acetamide (E1R), a novel positive allosteric modulator of sigma-1 receptors. EXPERIMENTAL APPROACH: E1R was tested for sigma receptor binding activity in a [³H](+)-pentazocine assay, in bradykinin (BK)-induced intracellular Ca²âº concentration ([Ca²âº](i)) assays and in an electrically stimulated rat vas deferens model. E1R's effects on cognitive function were tested using passive avoidance (PA) and Y-maze tests in mice. A selective sigma-1 receptor antagonist (NE-100), was used to study the involvement of the sigma-1 receptor in the effects of E1R. The open-field test was used to detect the effects of E1R on locomotion. KEY RESULTS: Pretreatment with E1R enhanced the selective sigma-1 receptor agonist PRE-084's stimulating effect during a model study employing electrically stimulated rat vasa deferentia and an assay measuring the BK-induced [Ca²âº](i) increase. Pretreatment with E1R facilitated PA retention in a dose-related manner. Furthermore, E1R alleviated the scopolamine-induced cognitive impairment during the PA and Y-maze tests in mice. The in vivo and in vitro effects of E1R were blocked by treatment with the selective sigma-1 receptor antagonist NE-100. E1R did not affect locomotor activity. CONCLUSION AND IMPLICATIONS: E1R is a novel 4,5-disubstituted derivative of piracetam that enhances cognition and demonstrates efficacy against scopolamine-induced cholinergic dysfunction in mice. These effects are attributed to its positive modulatory action on the sigma-1 receptor and this activity may be relevant when developing new drugs for treating cognitive symptoms related to neurodegenerative diseases.

Distinct effects of tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following thrombolytic reperfusion.

BACKGROUND AND PURPOSE: Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. METHODS: The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. RESULTS: Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1ß, TNF-α, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 µg/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. CONCLUSIONS: Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

ARQ-197, an oral small-molecule inhibitor of c-Met for the treatment of solid tumors.

ARQ-197 is an oral, selective c-Met inhibitor under development by ArQule Inc, in partnership with Daiichi Sankyo Co Ltd and Asian licensee Kyowa Hakko Kirin Co Ltd, for the potential treatment of solid tumors, including NSCLC, hepatocellular carcinoma and pancreatic cancer, as well as microphthalmia transcription factor-driven tumors. c-Met, a key cell surface receptor tyrosine kinase involved in diverse regulatory functions, is often aberrantly activated in human cancers. While the precise mechanism of action of ARQ-197 remains undefined, data from preclinical studies have demonstrated that ARQ-197 inhibits c-Met activation in numerous human tumor cell lines and specifically targets c-Met in various cancer types; uniquely, ARQ-197 inhibits c-Met in a non-ATP-competitive manner. Phase I/II clinical trials demonstrated promise in terms of both tolerability and tumor response. Intriguingly, dose-limiting adverse effects were hematological in nature. Combinational trials are also ongoing to take advantage of the signaling crosstalk between c-Met and other oncogenic signaling systems. Prioritization of the clinical development of c-Met inhibitors, such as ARQ-197, among different tumor disease types is a key challenge at present; an improved understanding of the prediction of molecular determinants in tumors with respect to c-Met kinase as the driver oncogenic receptor, and of the prediction of tumor response, is still urgently needed.

Biophysical investigation of the mode of inhibition of tetramic acids, the allosteric inhibitors of undecaprenyl pyrophosphate synthase.

Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.

Pseurotin A and its analogues as inhibitors of immunoglobulin E [correction of immunoglobuline E] production.

A natural product, pseurotin A inhibits IgE production in vitro. Wide variety of chemical modification of pseurotin A was performed. Structure-activity relationship studies of pseurotin analogues elucidated that 10-deoxypseurotin A strongly inhibits IgE production with IC(50) of 0.066 microM. An immunosuppressive activity of another natural product, synerazol was also found.

Potentiation of N-methyl-D-aspartate-induced currents by the nootropic drug nefiracetam in rat cortical neurons.

Nefiracetam is a new pyrrolidone nootropic drug being developed for the treatment of Alzheimer's type and post-stroke vascular-type dementia. In the brain of Alzheimer's disease patients, down-regulation of both cholinergic and glutamatergic systems has been found and is thought to play an important role in impairment of cognition, learning and memory. We have previously shown that the activity of neuronal nicotinic acetylcholine receptors is potently augmented by nefiracetam. The present study was undertaken to elucidate the mechanism of action of nefiracetam on glutamatergic receptors. Currents were recorded from rat cortical neurons in long-term primary culture using the whole-cell patch-clamp technique at a holding potential of -70 mV in Mg2+-free solutions. N-Methyl-D-aspartate (NMDA)-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam resulting in a bell-shaped dose-response curve. The minimum effective nefiracetam concentration was 1 nM, and the maximum potentiation to 170% of the control was produced at 10 nM. Nefiracetam potentiation occurred at high NMDA concentrations that evoked the saturated response, and in a manner independent of NMDA concentrations ranging from 3 to 1,000 microM. Glycine at 3 microM potentiated NMDA currents but this effect was attenuated with an increasing concentration of nefiracetam from 1 to 10,000 nM. 7-Chlorokynurenic acid at 1 microM prevented nefiracetam from potentiating NMDA currents. Nefiracetam at 10 nM shifted the dose-response relationship for the 7-chlorokynurenic acid inhibition of NMDA currents in the direction of higher concentrations. Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid- and kainate-induced currents were not significantly affected by application of 10 nM nefiracetam. It was concluded that nefiracetam potentiated NMDA currents through interactions with the glycine binding site of the NMDA receptor.

Epithelial cell-derived human beta-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway.

Mast cells are known to accumulate at the sites of inflammation in response to chemoattractants generated in the local milieu. Since human beta-defensin-2 (hBD-2) is generated in several epithelial tissues where mast cells are present and because we have recently reported that this human antibacterial peptide induces mast cell degranulation, we thus hypothesized that hBD-2 could be a mast cell chemotaxin. Here we report that hBD-2 directly and specifically induces mast cell migration with an optimal concentration of 3 microg/ml. Checkerboard analysis showed that the migration was more chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125I-labeled hBD-2 revealed that mast cells have at least two classes of receptors, high- and low-affinity receptors, for this peptide. Moreover, the competitive binding assay suggested that hBD-2 is unlikely to utilize CCR6, a functional receptor for hBD-2-mediated dendritic and T cell migration, on mast cells. In addition, treatment of mast cells with G protein inhibitor, pertussis toxin, and phospholipase C inhibitor, U-73122, abolished the cell chemotaxis in response to hBD-2, indicating that the G protein-phospholipase C signaling pathway is involved in hBD-2-induced mast cell activation. Thus, we suggest that hBD-2, which was originally believed to be involved in innate host defense, may participate in the recruitment of mast cells to inflammation foci.

Regulation of IL-15-stimulated TNF-alpha production by rolipram.

Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-alpha. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-alpha release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-alpha production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-alpha. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-alpha from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-alpha release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-alpha. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-alpha from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-alpha from macrophages by inhibiting T cell activation and expression of surface molecules.

CDP840: a novel inhibitor of PDE-4.

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor's activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells.

Although phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a well-characterized precursor for the second messengers inositol 1,4,5-trisphosphate, diacylglycerol [1] and phosphatidylinositol 3,4,5-trisphosphate [2], it also interacts with the actin-binding proteins profilin and gelsolin [3], as well as with many signaling molecules that contain pleckstrin homology (PH) domains [4]. It is conceivable that stimuli received by receptors in the plasma membrane could be sufficiently strong to decrease the PtdIns(4,5)P2 concentration; this decrease could alter the structure of the cortical cytoskeleton and modulate the activity of signaling molecules that have PH domains. Here, we tested this hypothesis by using an in vivo fluorescent indicator for PtdIns(4,5)P2, by tagging the PH domain of phospholipase C delta 1 (PLC-delta 1) with the green fluorescent protein (GFP-PH). When expressed in cells, GFP-PH was found to be enriched at the plasma membrane. Binding studies in vitro and mutant analysis suggested that GFP-PH bound PtdIns(4,5)P2 selectively over other phosphatidylinositol lipids. Strikingly, receptor stimulation induced a transient dissociation of GFP-PH from the plasma membrane, suggesting that the concentration of PtdIns(4,5)P2 in the plasma membrane was effectively lowered. This transient dissociation was blocked by the PLC inhibitor U73122 but was not affected by the phosphoinositide (PI) 3-kinase inhibitor wortmannin, suggesting that it is mostly mediated by PLC and not by PI 3-kinase activation. Overall, our studies show that PtdIns(4,5)P2 can have second messenger functions of its own, by mediating a transient dissociation of proteins anchored in the plasma membrane.

Activation of multiple metabotropic glutamate receptor subtypes prevents NMDA-induced excitotoxicity in rat hippocampal slices.

Metabotropic glutamate receptors (mGluRs) belong to a relative large receptor family consisting of multiple members with important roles in a number of brain functions. We report here that activation of mGluRs prevents the neurotoxic effect induced by N-methyl-D-aspartate (NMDA) in slices from the rat hippocampus. Neuroprotection was elicited when slices were simultaneously exposed to both the selective mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD) and NMDA. Persisting stimulation of mGluRs after the toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD was also evoked by its active isomer, (1S, 3R)-ACPD, and was prevented by the selective mGluR antagonist (+)-alpha-methyl-4-carboxyphenyl-glycine (500 microM), confirming that mGluR activation is involved in the mechanism of action of tACPD. The effect of 100 microM tACPD was reproduced by 100 microM quisqualate, an agonist of mGluR2 and mGluR3 subtypes. No neuroprotection was induced by L-2-amino-4-phosphonobutyrate, a selective agonist for mGluR4, mGluR6, mGluR7 and mGluR8, at 500 microM. Since the NMDA-mediated cell death in hippocampal slices is considered relevant to ischaemia-induced brain injury, these results indicate that mGluRs may be important safety devices used by neurons to decrease their sensitivity to excitotoxic stimuli and increase their chance of survival.

Enhancement of neuronal calcium channel currents by the nootropic agent, nefiracetam (DM-9384), in NG108-15 cells.

Effects of nootropic agents on neuronal calcium channels were studied in NG108-15 cells using the whole-cell patch-clamp technique. Nefiracetam (DM-9384) at a concentration of 1 microM increased a long-lasting component of calcium channel currents two-fold without affecting a transient component. The dose-response relationship yielded a bell-shaped curve with a peak at 1 microM. Similar, but slightly less potent effects were observed by aniracetam. Dibutyryl cyclic AMP (1 mM) also enhanced the currents, which were not further increased by nefiracetam, or vice versa. The currents enhanced by nefiracetam were markedly reduced by nifedipine (10 microM), an 'L-type' calcium channel blocker. Cells treated with pertussis toxin (PTX; 500 ng/ml, > 20 h) to inactivate inhibitory G-proteins were apparently insensitive to nefiracetam. The results suggest that the nootropic agents may enhance the activity of neuronal L-type calcium channels under the regulation of inhibitory G-proteins and possibly, cyclic AMP-dependent processes.

Effects of BMY-21502 on anoxia in mice.

The protective effects of BMY-21502 (1-[[1-[2-(trifluoromethyl)-4-pyrimidinyl]-4-piperidinyl]methyl]-2- pyrrolidinone) against cerebral anoxia were investigated using various models in mice, in comparison with those of other cerebroactive drugs. Oral administration of BMY-21502 (10-100 mg/kg) significantly prolonged the survival time in KCN (2.4 mg/kg, i.v.)-induced anoxia. Oxiracetam and idebenone exerted similar but weak protection at doses above 100 mg/kg, p.o. and only at a dose of 100 mg/kg, p.o., respectively. Significant protection by BMY-21502 against moderate hypobaric hypoxia was observed at doses of 30 and 100 mg/kg, p.o. Idebenone (100 and 300 mg/kg, p.o.) significantly prolonged the survival time of mice in this model, but oxiracetam (30-300 mg/kg, p.o.) did not. Oral administration of all of these drugs (BMY-21502, 3-300 mg/kg; Oxiracetam, 100-1000 mg/kg; Idebenone, 100-1000 mg/kg) failed to increase the number of gasps and the duration of gasping in the decapitated head of mice as a complete ischemic model. The anti-anoxic effect of BMY-21502 in the KCN-anoxia model was blocked by pretreatment with scopolamine. These findings suggest that BMY-21502 has an anti-anoxic action superior to those of the other cerebroactive drugs used, and activation of the CNS cholinergic system is involved as one of the causative mechanisms for the anti-anoxic effect of BMY-21502.

Central and peripheral minocycline suppresses motor activity in rats.

Minocycline (MIN) HCl is a tetracycline derivative previously shown to inhibit agonist-induced accumulation of cyclic adenosine monophosphate (cAMP) in vitro and suppress motor activity and amphetamine-induced hyperactivity in rats following SC injection. The present study examined the effect of IV and intracerebral MIN on baseline activity and amphetamine-induced hyperactivity. IV MIN suppressed both types of activity in doses of 100 and 150 mg/kg. When injected ICV, MIN (50 micrograms/2 microliter) suppressed the increase in rearing elicited by amphetamine but did not affect baseline activity. MIN did not attenuate the behavioral suppression induced by the cAMP phosphodiesterase inhibitor rolipram. MIN apparently has centrally mediated effects on motor activity in rats; however, it is not yet possible to associate MIN's behavioral effects with its ability to inhibit agonist-induced stimulation of cAMP.

The GABAB antagonist, CGP 35348, antagonizes the effects of baclofen, gamma-butyrolactone and HA 966 on rat striatal dopamine synthesis.

The effects of the new GABAB antagonist, CGP 35348 (3-aminopropane-diethoxymethylphosphinic acid), on rat striatal dopamine synthesis and the increases thereof, caused by (-)-baclofen, gamma-butyrolactone (GBL), and HA 966 (3-amino-1-hydroxypyrrolid-2-one), were investigated. CGP 35348 did not alter dopamine synthesis on its own up to the highest dose tested (500 mg/kg i.p.). However, it antagonized the increase elicited by 50 mg/kg s.c. (-)-baclofen at doses above 100 mg/kg i.p.; at 500 mg/kg i.p. this antagonism disappeared within about 6 h of interval between the administration of the compound and (-)-baclofen. CGP 35348 also clearly and significantly attenuated the effects of graded doses of GBL and HA 966 at 500 mg/kg i.p., but was unable to alter the responses elicited by 0.3 mg/kg i.p. haloperidol or 10 mg/kg i.p. tetrabenazine. This indicates that the compound did not generally attenuate increases of dopamine synthesis. It is likely that its GABAB antagonistic properties are responsible for the attenuation of the effect of (-)-baclofen, and our results suggest that this compound is useful for the characterization of the role of GABAB receptors in vivo, e.g. in behaviour. On the other hand, they also suggest the possibility that GBL and HA 966 elicit their effects on dopamine synthesis by means of an interaction with GABAB receptors; a weak in vitro interaction with the latter in radioligand binding experiments has been found for GBL, but not for HA 966.

Effects of nebracetam (WEB 1881 FU), a novel nootropic, as a M1-muscarinic agonist.

We here investigated the effects of nebracetam (WEB 1881 FU 4-aminomethyl-1-benzylpyrrolidin-2-one-hemifumarate), a novel pyrrolidinone nootropic, on the rise of intracellular Ca2+ concentration ([Ca2+]i) in Jurkat cells, a human leukemic T cell line. Nebracetam induced a rise of [Ca2+]i in the medium with 1 mM Ca2+ and without Ca2+ (plus 1 mM EGTA). The nebracetam-induced [Ca2+]i rise was blocked by atropine greater than pirenzepine greater than AF-DX 116. From these results, nebracetam seems to act as an agonist for human M1-muscarinic receptors.