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. 2010 Jun 25;285(26):19947-58.
doi: 10.1074/jbc.M110.111286. Epub 2010 Apr 19.

Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-methyl-D-aspartate Receptors With Fast Off-Rate by Novel Multifunctional Dimer Bis(propyl)-Cognitin

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Pathologically Activated Neuroprotection via Uncompetitive Blockade of N-methyl-D-aspartate Receptors With Fast Off-Rate by Novel Multifunctional Dimer Bis(propyl)-Cognitin

Jialie Luo et al. J Biol Chem. .
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Abstract

Uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonists with fast off-rate (UFO) may represent promising drug candidates for various neurodegenerative disorders. In this study, we report that bis(propyl)-cognitin, a novel dimeric acetylcholinesterase inhibitor and gamma-aminobutyric acid subtype A receptor antagonist, is such an antagonist of NMDA receptors. In cultured rat hippocampal neurons, we demonstrated that bis(propyl)-cognitin voltage-dependently, selectively, and moderately inhibited NMDA-activated currents. The inhibitory effects of bis(propyl)-cognitin increased with the rise in NMDA and glycine concentrations. Kinetics analysis showed that the inhibition was of fast onset and offset with an off-rate time constant of 1.9 s. Molecular docking simulations showed moderate hydrophobic interaction between bis(propyl)-cognitin and the MK-801 binding region in the ion channel pore of the NMDA receptor. Bis(propyl)-cognitin was further found to compete with [(3)H]MK-801 with a K(i) value of 0.27 mum, and the mutation of NR1(N616R) significantly reduced its inhibitory potency. Under glutamate-mediated pathological conditions, bis(propyl)-cognitin, in contrast to bis(heptyl)-cognitin, prevented excitotoxicity with increasing effectiveness against escalating levels of glutamate and much more effectively protected against middle cerebral artery occlusion-induced brain damage than did memantine. More interestingly, under NMDA receptor-mediated physiological conditions, bis(propyl)-cognitin enhanced long-term potentiation in hippocampal slices, whereas MK-801 reduced and memantine did not alter this process. These results suggest that bis(propyl)-cognitin is a UFO antagonist of NMDA receptors with moderate affinity, which may provide a pathologically activated therapy for various neurodegenerative disorders associated with NMDA receptor dysregulation.

Figures

FIGURE 1.
FIGURE 1.
Tether length differential potency and voltage dependence of bis(n)-cognitins. A, the graph shows the inhibitory effects of memantine, tacrine, and bis(n)-cognitins (B2C, B3C, B5C, B7C, and B10C) on 30 μm NMDA-activated currents at a holding potential of −50 mV in cultured rat hippocampal neurons. Each point represents the mean of percentage inhibition from 5–7 neurons. The IC50 values are indicated in the graph. Inset, molecular structure of bis(propyl)-cognitin. B, voltage dependence of tacrine and bis(n)-cognitin blockade of NMDA-activated currents is analyzed by plotting the mean of percentage inhibition against holding potentials.
FIGURE 2.
FIGURE 2.
Selective, agonist- and use-dependent effects of bis(propyl)-cognitin inhibition. A, current traces show the effect of 10 μm bis(propyl)-cognitin (B3C) on 30 μm AMPA- or kainic acid (KA)-activated currents. Each row of traces is recorded from the same neuron and repeated in five different neurons. Bis(propyl)-cognitin is co-applied with AMPA or kainic acid for the time course indicated above each trace. B, representative current traces recorded from the same neuron show three different modes of drug application: co-application of bis(propyl)-cognitin (0.3 μm) and NMDA (30 μm), co-application made after the onset of the response to NMDA, and sequential application of NMDA following bis(propyl)-cognitin preapplication. Note the different effects of bis(propyl)-cognitin under these different conditions. A similar result was obtained in four different neurons. C, representative current traces (top) and graph (bottom, n = 8) show the control NMDA response and the cumulative inhibition incurred by consecutive co-application of NMDA and 1 μm bis(propyl)-cognitin, followed by the response to NMDA alone for recovery. Each response, normalized to the first NMDA-activated current, is activated at intervals of 1 min. The solid bars indicate the application of NMDA and/or bis(propyl)-cognitin.
FIGURE 3.
FIGURE 3.
Bis(propyl)-cognitin is an uncompetitive inhibitor of NMDA receptors. A, NMDA dose-response curves in the absence (○) and presence (●) of 0.3 μm bis(propyl)-cognitin (B3C). The steady state response for each concentration of NMDA in the absence or presence of bis(propyl)-cognitin is normalized to the response produced by 30 μm NMDA in the absence of bis(propyl)-cognitin (marked with an asterisk). The responses are the means ± S.E. (error bars) from 9–15 neurons at each concentration of NMDA. Inset, plot showing the percentage inhibition of B3C and d(-)-2-amino-5-phosphonopentanoic acid (AP5) against NMDA concentrations ranging from 3 to 300 μm. B, dose-response relationships for bis(propyl)-cognitin inhibition of INMDA obtained under the conditions of 0.1 μm (●) and 10 μm (■) glycine. The curves were fitted using the logistic equation under “Experimental Procedures.”
FIGURE 4.
FIGURE 4.
Onset and offset time constants of bis(propyl)-cognitin inhibition. A, traces show currents activated by 30 μm NMDA and its inhibition by 1 μm bis(propyl)-cognitin applied after the activation of NMDA receptors. The periods of drug application are indicated by the solid bar above the trace. White dotted lines drawn through the current trace (black part) are the theoretical fit of onset (τon) and offset (τoff) of bis(propyl)-cognitin inhibition using single exponential functions. B, graph plotting average τon (■) and τoff (●) values against bis(propyl)-cognitin concentration shows the concentration dependence of onset and offset kinetics. Error bars, S.E.
FIGURE 5.
FIGURE 5.
Acting site of bis(propyl)-cognitin on the NMDA receptor. A, a close-up view of the low energy pose of bis(propyl)-cognitin in the NMDA receptor channel pore generated by molecular docking. The NMDA receptor is depicted in ribbon form, bis(propyl)-cognitin (tan) is depicted as a stick model, and the surface of the ligand pocket is shown as transparent gray skin. The homology model of the ion channel of NMDA receptor is constructed based on the x-ray crystal structure of the potassium channel KcsA (Protein Data Bank code 1BL8). B, graph shows the inhibition of [3H]MK-801 binding to rat in rat cerebellar cortex membrane preparations by bis(propyl)-cognitin (B3C). Membranes from rat cerebellar cortex were incubated with [3H]MK-801 (4 nm) and bis(propyl)-cognitin at gradually increasing concentrations. The data, expressed as percentage of control, are means according to three independent experiments. The Ki value was calculated from the corresponding IC50 Value according to the equation, Ki = IC50/(1 + C/Kd), where C is the concentration of radioligand and Kd is the dissociation constant obtained from the saturation experiment (12 nm). C, representative current traces and plots show the concentration-response relationships for bis(propyl)-cognitin inhibition of currents mediated by recombinant NR1/NR2A (wild type (WT)) and NR1(N616R)/NR2A receptors expressed in HEK293T cells. Error bars, S.E.
FIGURE 6.
FIGURE 6.
Neuroprotection of bis(propyl)-cognitin in vitro. A, CGNs, at 8 days in vitro, were preincubated with bis(propyl)-cognitin (B3C) at different concentrations as indicated for 2 h and then exposed to 75 μm glutamate for another 24 h. Cell viability is measured using an MTT assay. All of the data, expressed as percentage of untreated control, are the means ± S.E. (error bars) of three separate experiments. Inset, plots show dose-response relationships for bis(propyl)-cognitin and memantine protection. B, CGNs, at 8 days in vitro, were preincubated with 3 μm bis(propyl)-cognitin (B3C) or bis(heptyl)-cognitin (B7C) for 2 h and then exposed to different concentrations of glutamate as indicated for another 24 h. The cell survival after the treatment with glutamate or glutamate following bis(propyl)-cognitin or bis(heptyl)-cognitin preincubation is expressed as percentage of untreated control.
FIGURE 7.
FIGURE 7.
Neuroprotective activity of bis(propyl)-cognitin in vivo. A, representative 2,3,5-triphenyltetrazolium chloride-stained coronal brain sections from animals of the sham group (left column), vehicle-treated control group (middle column), and bis(propyl)-cognitin (B3C) (0.65 μmol/kg)-treated group (right column), respectively. The white area represents the area of infarction in the brains of stroke rats. Drug or saline was intraperitoneally administered 15 min after the onset of MCAO, and rats were decapitated 24 h later. B, infarct volume for seven sequential coronal sections from anterior to posterior in rats receiving saline (Control) or bis(propyl)-cognitin (0.65 μmol/kg) 15 min after the induction of ischemia. C, total infarct volume (left) and neurological deficit scores (right) at 24 h after reperfusion for saline-, bis(propyl)-cognitin (0.19 and 0.65 μmol/kg)-, and memantine (93 μmol/kg)-treated rats, respectively. *, p < 0.05; **, p < 0.01 compared with control. Error bars, S.E.
FIGURE 8.
FIGURE 8.
Bis(propyl)-cognitin enhances LTP. A, the graph shows induction of LTP in control (●) and in the presence of bis(propyl)-cognitin (B3C), perfused for 60 min prior to HFS (○). B, statistical analyses of the effect of bis(propyl)-cognitin, memantine, and MK-801 on LTP. The agents were perfused over the slices for 60 min prior to HFS, and LTP induction was recorded. All data are the means ± S.E. of EPSPs at 60 min after HFS (n = 5). *, p < 0.05; **, p < 0.01 versus LTP control.

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