NMDARs are ligand-gated ion channels that cause an influx of Na+ and Ca2+ into postsynaptic neurons. The resulting intracellular Ca2+ transient triggers synaptic plasticity. When prolonged, it may induce excitotoxicity, but it may also activate negative feedback to control the activity of NMDARs. Here, we report that a transient rise in intracellular Ca2+ (Ca2+ challenge) increases the sensitivity of NMDARs but not AMPARs/kainate receptors to the endogenous inhibitory neurosteroid 20-oxo-5β-pregnan-3α-yl 3-sulfate and to its synthetic analogs, such as 20-oxo-5β-pregnan-3α-yl 3-hemipimelate (PAhPim). In cultured hippocampal neurons, 30 μm PAhPim had virtually no effect on NMDAR responses; however, following the Ca2+ challenge, it inhibited the responses by 62%; similarly, the Ca2+ challenge induced a 3.7-fold decrease in the steroid IC50 on recombinant GluN1/GluN2B receptors. The increase in the NMDAR sensitivity to PAhPim was dependent on three cysteines (C849, C854, and C871) located in the carboxy-terminal domain of the GluN2B subunit, previously identified to be palmitoylated (Hayashi et al., 2009). Our experiments suggested that the Ca2+ challenge induced receptor depalmitoylation, and single-channel analysis revealed that this was accompanied by a 55% reduction in the probability of channel opening. Results of in silico modeling indicate that receptor palmitoylation promotes anchoring of the GluN2B subunit carboxy-terminal domain to the plasma membrane and facilitates channel opening. Depalmitoylation-induced changes in the NMDAR pharmacology explain the neuroprotective effect of PAhPim on NMDA-induced excitotoxicity. We propose that palmitoylation-dependent changes in the NMDAR sensitivity to steroids serve as an acute endogenous mechanism that controls NMDAR activity.SIGNIFICANCE STATEMENT There is considerable interest in negative allosteric modulators of NMDARs that could compensate for receptor overactivation by glutamate or de novo gain-of-function mutations in neurodevelopmental disorders. By a combination of electrophysiological, pharmacological, and computational techniques we describe a novel feedback mechanism regulating NMDAR activity. We find that a transient rise in intracellular Ca2+ increases NMDAR sensitivity to inhibitory neurosteroids in a process dependent on GluN2B subunit depalmitoylation. These results improve our understanding of the molecular mechanisms of steroid action at the NMDAR and indeed of the basic properties of this important glutamate-gated ion channel and may aid in the development of therapeutics for treating neurologic and psychiatric diseases related to overactivation of NMDARs without affecting normal physiological functions.
- MeSH
- HEK293 buňky MeSH
- hipokampus fyziologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- lipoylace účinky léků fyziologie MeSH
- neuroprotekce fyziologie MeSH
- potkani Wistar MeSH
- pregnany metabolismus farmakologie MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In mature neurons, postsynaptic N-methyl-D-aspartate receptors (NMDARs) are segregated into two populations, synaptic and extrasynaptic, which differ in localization, function, and associated intracellular cascades. These two pools are connected via lateral diffusion, and receptor exchange between them modulates synaptic NMDAR content. Here, we identify the phosphorylation of the PDZ-ligand of the GluN2B subunit of NMDARs (at S1480) as a critical determinant in dynamically controlling NMDAR synaptic content. We find that phosphorylation of GluN2B at S1480 maintains NMDARs at extrasynaptic membranes as part of a protein complex containing protein phosphatase 1 (PP1). Global activation of NMDARs leads to the activation of PP1, which mediates dephosphorylation of GluN2B at S1480 to promote an increase in synaptic NMDAR content. Thus, PP1-mediated dephosphorylation of the GluN2B PDZ-ligand modulates the synaptic expression of NMDARs in mature neurons in an activity-dependent manner, a process with profound consequences for synaptic and structural plasticity, metaplasticity, and synaptic neurotransmission.
- MeSH
- fosforylace MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- ligandy MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- neurony metabolismus MeSH
- PDZ domény MeSH
- potkani Sprague-Dawley MeSH
- proteinfosfatasa 1 metabolismus MeSH
- receptory N-methyl-D-aspartátu genetika metabolismus MeSH
- synapse metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.
- Publikační typ
- časopisecké články MeSH
Here, we report the synthesis of pregn-5-ene and androst-5-ene dicarboxylic acid esters and explore the structure-activity relationship (SAR) for their modulation of N-methyl-d-aspartate receptors (NMDARs). All compounds were positive modulators of recombinant GluN1/GluN2B receptors (EC50 varying from 1.8 to 151.4 μM and Emax varying from 48% to 452%). Moreover, 10 compounds were found to be more potent GluN1/GluN2B receptor modulators than endogenous pregnenolone sulfate (EC50 = 21.7 μM). The SAR study revealed a relationship between the length of the residues at carbon C-3 of the steroid molecule and the positive modulatory effect at GluN1/GluN2B receptors for various D-ring modifications. A selected compound, 20-oxo-pregnenolone hemiadipate, potentiated native NMDARs to a similar extent as GluN1/GluN2A-D receptors and inhibited AMPARs and GABAAR responses. These results provide a unique opportunity for the development of new steroid based drugs with potential use in the treatment of neuropsychiatric disorders involving hypofunction of NMDARs.
- MeSH
- alosterická regulace MeSH
- HEK293 buňky MeSH
- konformace proteinů MeSH
- lidé MeSH
- modulátory membránového transportu chemie farmakologie MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- pregnenolon farmakologie MeSH
- receptory N-methyl-D-aspartátu antagonisté a inhibitory metabolismus MeSH
- steroidy chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Herein, we report a new class of amide-based inhibitors (1-4) of N-methyl-d-aspartate receptors (NMDARs) that were prepared as analogues of pregnanolone sulfate (PAS) and pregnanolone glutamate (PAG) - the steroidal neuroprotective NMDAR inhibitors. A series of experiments were conducted to evaluate their physicochemical and biological properties: (i) the inhibitory effect of compounds 3 and 4 on NMDARs was significantly improved (IC50=1.0 and 1.4μM, respectively) as compared with endogenous inhibitor - pregnanolone sulfate (IC50=24.6μM) and pregnanolone glutamate (IC50=51.7μM); (ii) physicochemical properties (logP and logD) were calculated; (iii) Caco-2 assay revealed that the permeability properties of compounds 2 and 4 are comparable with pregnanolone glutamate; (iv) compounds 1-4 have minimal or no adverse hepatic effect; (v) compounds 1-4 cross blood-brain-barrier.
- MeSH
- amidy MeSH
- buňky Hep G2 MeSH
- Caco-2 buňky MeSH
- hematoencefalická bariéra účinky léků metabolismus MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- neurotransmiterové látky chemie farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- receptory N-methyl-D-aspartátu antagonisté a inhibitory metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
2. přepracované a doplněné vydání 190 stran : ilustrace, tabulky ; 29 cm
- MeSH
- biologická terapie MeSH
- biotechnologie MeSH
- farmaceutická technologie MeSH
- léčivé přípravky MeSH
- vakcíny MeSH
- Konspekt
- Farmacie. Farmakologie
- NLK Obory
- farmacie a farmakologie
- NLK Publikační typ
- učebnice vysokých škol
N-Methyl-d-aspartate receptors (NMDARs) display a critical role in various diseases of the central nervous system. The activity of NMDARs can be modulated by neurosteroids. Herein, we report a structure-activity relationship study for perhydrophenanthrene analogues possessing a framework that mimics the steroidal ring system. This study comprises the design, synthesis, and assessment of the biological activity of a library of perhydrophenanthrene 2-sulfates and 2-hemisuccinates (1-10). Their ability to modulate NMDAR-induced currents was tested on recombinant GluN1/GluN2B receptors. Our results demonstrate that such structural optimization leads to compounds that are inhibitors of NMDARs. Notably, compound 9 (IC50 = 15.6 μM) was assessed as a more potent inhibitor of NMDAR-induced currents than the known endogenous neurosteroid, pregnanolone sulfate (IC50 = 24.6 μM).
- MeSH
- fenantreny chemická syntéza chemie farmakologie MeSH
- HEK293 buňky MeSH
- kvantová teorie MeSH
- lidé MeSH
- molekulární struktura MeSH
- receptory N-methyl-D-aspartátu antagonisté a inhibitory metabolismus MeSH
- sírany chemie farmakologie MeSH
- sukcináty chemie farmakologie MeSH
- termodynamika MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Many diseases of the nervous system are accompanied by alterations in synaptic functions. Synaptic plasticity mediated by the endogenous cannabinoid system involves the activation of the cannabinoid receptor 1 (CB1R). The principles of CB1R signaling must be understood in detail for its therapeutic exploration. We detected the Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1) as a novel CB1R partner. SGIP1 is functionally linked to clathrin-mediated endocytosis and its overexpression in animals leads to an energy regulation imbalance resulting in obesity. We report that SGIP1 prevents the endocytosis of activated CB1R and that it alters signaling via the CB1R in a biased manner. CB1R mediated G-protein activation is selectively influenced by SGIP1, β-arrestin associated signaling is changed profoundly, most likely as a consequence of the prevention of the receptor's internalization elicited by SGIP1.
- MeSH
- beta arrestin 2 metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- endocytóza účinky léků fyziologie MeSH
- HEK293 buňky MeSH
- lidé MeSH
- MAP kinasový signální systém fyziologie MeSH
- mozek metabolismus MeSH
- myši MeSH
- neurony metabolismus MeSH
- potkani Wistar MeSH
- receptor kanabinoidní CB1 metabolismus MeSH
- Saccharomyces cerevisiae MeSH
- techniky dvojhybridového systému MeSH
- transfekce MeSH
- transportní proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
G-quadruplexes are types of secondary structures of nucleic acids, which are formed in guanine-rich regions. Their existence in vivo was confirmed in promoter regions of some genes and at the end of chromosomes in the telomeric regions. The specific structure of G-quadruplexes is a promising target in cancer treatment. There is a search for small ligands, which could stabilize a specific type of G-quadruplexes. Cancer cells are markedly different from normal cells in the telomeric region: the regions are profoundly shorter. Telomeric regions in cancer cells are prolonged by an enzyme telomerase, while this enzyme is inactive in almost all somatic cells. Stabilization of telomeric G-quadruplex by a specific small ligand induces disruption of a normal protective function of telomers leading to cell damage and its death. This approach could be successful in cancer treatment.
