Noncompetitive Antagonist Binding Research Articles

Neuroprotective effect of the standardised extract of Bacopa monnieri (BacoMind) in valproic acid model of autism spectrum disorder in rats

Ethnopharmacological relevanceBacopa monnieri (BM) is commonly employed in the Indian traditional system of medicines, i.e. Ayurveda as a memory booster, antioxidant, anti-inflammatory, antipyretic, analgesic, sedative and anti-epileptic for decades. Aim of the studyTo evaluate the neuroprotective effect of Bacopa monnieri (BM) in experimental model of autism spectrum disorder (ASD) in Wistar rats and explore its mechanism of action. Materials and methodsBacoMind, was evaluated for its neuroprotective effect in valproic acid (VPA) model of ASD. For in-vivo study, the pregnant female Wistar rats were divided in two groups; normal control (NC) and VPA group who received single dose of normal saline (0.9%) or 600 mg/kg dose of VPA respectively on gestation day (G.D) 12.5. After the birth, all pups were segregated according to the sex. All the male pups from the dams were divided into six groups: Group 1 (NC, treated with only 0.9% normal saline, group 2 (VPA, treated 600 mg/kg on G.D12.5 and normal saline from post natal day (PND) 23 to 43), group 3 (risperidone 2.5 mg/kg, PND 23 to 43) and groups 4, 5 and 6 (BM 20, 40, 80 mg/kg, PND 23 to 43). All experimental groups were subjected to batteries of behavior parameters (three chamber sociability test, Morris Water Maze, elevated plus maze, open field and rota rod test), biochemical parameters such as oxidative stress (GSH, SOD, Catalase, MDA), inflammatory cytokines (Il-1β, IL-6, IL-10, TNF-α), histopathological examination (cresyl violet staining) of hippocampus (HC) and prefrontal cortex (PFC) regions. Further, the mRNA as well as protein expression of AMPA receptor was evaluated using RT-PCR and western blot respectively to study the mechanism of neuroprotective effect of BM. The in-silico analysis followed evaluating the binding profile of different constituents of BacoMind with AMPA receptor. ResultsThe results of the in-vivo study indicated BM at 80 mg/kg ameliorated abnormal behavioral paradigms such as social deficits, repetitive behavior, learning and memory impairments, and motor coordination exhibited by the VPA model of ASD in rats. Furthermore, BM was found to have a significant anti-oxidant (increasing GSH, SOD, and catalase and decreasing MDA levels) and anti-inflammatory properties (decreasing IL-1β, 6, TNF- α). The histopathological score was also found to be significantly improved by BM in a dose dependent manner in both HC and PFC. In addition to this, the up-regulated mRNA as well as protein expression of AMPA receptor was significantly reduced by 80 mg/kg dose of BM in both HC and PFC. Further, the in-silico analysis of different constituents of BacoMind with AMPA receptor demonstrated that luteolin and apigenin showed good binding to both the competitive antagonist binding site, non-competitive antagonist binding site and allosteric modulator site while Bacosaponin C showed good binding to the non-competitive antagonist binding site. ConclusionThe present study concluded that BM can be a potential candidate for ameliorating the ASD symptoms in rats and acts via modulating the up-regulated AMPA receptor expression.

A photoreactive probe that differentiates the binding sites of noncompetitive GABA receptor antagonists

γ-Aminobutyric acid (GABA) receptors are postsynaptic membrane protein complexes that are important not only in the regulation of the nervous system but also as targets of drugs and insecticides. We synthesized a photoreactive straight-chain noncompetitive antagonist (NCA), 2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl 4-(4-methoxycarbonyl-1-butynyl)benzoate (NMB), to probe the NCA binding site. Our data show that this probe labels the NCA site and demonstrate that the NCA insecticide fipronil binds at a site distinct from that of other NCAs, such as picrotoxinin and 4'-ethynyl-4-n-propylbicycloorthobenzoate. The unique molecule NMB will be useful in identifying the cross-linking site of straight-chain NCAs in GABA receptors and mapping allosteric binding sites. Such studies should provide invaluable information in designing novel NCAs.

GABAA receptor open-state conformation determines non-competitive antagonist binding

The γ-aminobutyric acid (GABA) type A receptor (GABAAR) is one of the most important targets for insecticide action. The human recombinant β3 homomer is the best available model for this binding site and 4-n-[3H]propyl-4′-ethynylbicycloorthobenzoate ([3H]EBOB) is the preferred non-competitive antagonist (NCA) radioligand. The uniquely high sensitivity of the β3 homomer relative to the much-less-active but structurally very-similar β1 homomer provides an ideal comparison to elucidate structural and functional features important for NCA binding. The β1 and β3 subunits were compared using chimeragenesis and mutagenesis and various combinations with the α1 subunit and modulators. Chimera β3/β1 with the β3 subunit extracellular domain and the β1 subunit transmembrane helices retained the high [3H]EBOB binding level of the β3 homomer while chimera β1/β3 with the β1 subunit extracellular domain and the β3 subunit transmembrane helices had low binding activity similar to the β1 homomer. GABA at 3μM stimulated heteromers α1β1 and α1β3 binding levels more than 2-fold by increasing the open probability of the channel. Addition of the α1 subunit rescued the inactive β1/β3 chimera close to wildtype α1β1 activity. EBOB binding was significantly altered by mutations β1S15′N and β3N15′S compared with wildtype β1 and β3, respectively. However, the binding activity of α1β1S15′N was insensitive to GABA and α1β3N15′S was stimulated much less than wildtype α1β3 by GABA. The inhibitory effect of etomidate on NCA binding was reduced more than 5-fold by the mutation β3N15′S. Therefore, the NCA binding site is tightly regulated by the open-state conformation that largely determines GABAA receptor sensitivity.

Molecular determinants of non-competitive antagonist binding to the mouse GPRC6A receptor

GPRC6A displays high sequence homology to the Ca 2+-sensing receptor (CaSR). Here we report that the calcimimetic Calindol and the calcilytic NPS2143 antagonize increases in inositol phosphate elicited by l-ornithine-induced activation of mouse GPRC6A after transient coexpression with Gα qG66D in HEK293 cells. The calcilytic Calhex 231 did not modulate this response. A three-dimensional model of the GPRC6A seven transmembrane domains (TMs) was constructed. It was used to identify seven residues strictly conserved within the CaSR and GPRC6A allosteric binding pockets, and previously demonstrated to interact with calcilytics or calcimimetics. The mutations F666A 3.32, F670A 3.36, W797A 6.48 caused a loss of l-ornithine ability to activate GPRC6A mutants. The F800A 6.51 mutant was not implicated in either Calindol or NPS 2143 recognition. The E816Q 7.39 mutation led to a loss of Calindol antagonist activity but was without effect on NPS2143 inhibitory response. In summary, these data suggest that Calindol is primarily anchored through an H-bond to E816 7.39 in TM7 and highlight important local differences at the level of the CaSR and GPRC6A allosteric binding pockets. We have identified the first antagonists of GPRC6A that could represent new tools to analyze GPRC6A functions and serve as chemical leads for the development of more specific modulators.

Structural model for γ-aminobutyric acid receptor noncompetitive antagonist binding: Widely diverse structures fit the same site

Several major insecticides, including alpha-endosulfan, lindane, and fipronil, and the botanical picrotoxinin are noncompetitive antagonists (NCAs) for the GABA receptor. We showed earlier that human beta(3) homopentameric GABA(A) receptor recognizes all of the important GABAergic insecticides and reproduces the high insecticide sensitivity and structure-activity relationships of the native insect receptor. Despite large structural diversity, the NCAs are proposed to fit a single binding site in the chloride channel lumen lined by five transmembrane 2 segments. This hypothesis is examined with the beta(3) homopentamer by mutagenesis, pore structure studies, NCA binding, and molecular modeling. The 15 amino acids in the cytoplasmic half of the pore were mutated to cysteine, serine, or other residue for 22 mutants overall. Localization of A-1'C, A2'C, T6'C, and L9'C (index numbers for the transmembrane 2 region) in the channel lumen was established by disulfide cross-linking. Binding of two NCA radioligands [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane and [(3)H] 3,3-bis-trifluoromethyl-bicyclo[2,2,1]heptane-2,2-dicarbonitrile was dramatically reduced with 8 of the 15 mutated positions, focusing attention on A2', T6', and L9' as proposed binding sites, consistent with earlier mutagenesis studies. The cytoplasmic half of the beta3 homopentamer pore was modeled as an alpha-helix. The six NCAs listed above plus t-butylbicyclophosphorothionate fit the 2' to 9' pore region forming hydrogen bonds with the T6' hydroxyl and hydrophobic interactions with A2', T6', and L9' alkyl substituents, thereby blocking the channel. Thus, widely diverse NCA structures fit the same GABA receptor beta subunit site with important implications for insecticide cross-resistance and selective toxicity between insects and mammals.

Characterization of the non-competitive antagonist binding site of the NMDA receptor in dark Agouti rats

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [ 3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity ( K d ) of [ 3H]MK-801 for its binding site was 5.5 n M and the binding site density ( B max) was 1.83 pmol/mg protein. Inhibition of [ 3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were −1. A series of eight non-competitive NMDA antagonists inhibited [ 3H]MK-801 binding with the following rank order of affinity ( K i, n M): MK-801 (5.5) > dexoxadrol (21.5) ≥ TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated ( P = 0.0002; r 2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274–281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.

Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives.

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.

Binding modes of noncompetitive AMPA antagonists: a computational approach

The activity of functional AMPA receptors (AMPARs) is modulated by noncompetitive antagonists. So far, no information about the molecular mechanism of action and the localization of the binding pocket(s) is available. We speculated that the leucine/isoleucine/valine binding protein (LIVBP)-like domain of AMPAR, localized at the extracellular N-terminus of the receptor, might be involved in the binding of noncompetitive antagonists and we tested this hypothesis through a computational approach involving the comparison with NMDA and metabotropic glutamate receptors and the generation of a 3D homology model of the LIVBP-like domain of AMPAR. The results suggest that the interdomain cleft of the LIVBP-like domain of AMPAR may contain the noncompetitive antagonist binding pocket.

Allosterically linked noncompetitive antagonist binding sites in the resting nicotinic acetylcholine receptor ion channel

Previous studies have established the presence of overlapping binding sites for the noncompetitive antagonists (NCAs) amobarbital, tetracaine, and 3-trifluoromethyl-3-( m-[ 125 I ]iodophenyl) diazirine ([ 125 I ]TID) within the ion channel of the Torpedo nicotinic acetylcholine receptor (AChR) in the resting state. These well-characterized NCAs and competitive radioligand binding and photolabeling experiments were employed to better characterize the interaction of the dissociative anesthetics ketamine and thienylcycloexylpiperidine (TCP) with the resting AChR. Our experiments yielded what appear to be conflicting results: (i) both ketamine and TCP potentiated [ 125 I ]TID photoincorporation into AChR subunits; and (ii) ketamine and TCP had very little effect on [ 14 C ]amobarbital binding. Nevertheless, (iii) both ketamine and TCP completely displaced [ 3 H ]tetracaine binding ( K is∼20.9 and 2.0 μM, respectively) by a mutually exclusive mechanism. To reconcile these results we propose that, in the resting ion channel, TCP and ketamine bind to a site that is spatially distinct from the TID and barbiturate locus, while tetracaine bridges both binding sites.

Chlorpyrifos, Parathion, and Their Oxons Bind to and Desensitize a Nicotinic Acetylcholine Receptor: Relevance to Their Toxicities

The nicotinic acetylcholine receptor (nAChR) of the electric organ of the electric ray.Torpedosp., the richest source of nAChR, with similar structure and pharmacology to the mammalian skeletal muscle nAChR, carries several binding sites for different ligands. Incubation ofTorpedomembrane-bound nAChRs with the agonist carbamylcholine (Carb) stimulated the binding of [3H]thienyl-cyclohexylpiperidine ([3H]TCP), which binds to the receptor's noncompetitive antagonist binding site in its ionic channel, with high affinity (Kdof 196 nm). The agonist-stimulated binding of [3H]TCP (i.e., binding to activated nAChRs) was inhibited in a concentration-dependent manner by four organophosphate (OP) anticholinesterases, chlorpyrifos oxon (CPO), chlorpyrifos (CPS), parathion (PS), and paraoxon (PO) with IC50 (concentration that inhibits 50% of the effect) values of 5, 150, 200, and 300 μm, respectively. The binding of CPO was totally reversible. The OPs had no effect on equilibrium binding of [α-125I]bungarotoxin ([α-125I]BGT) to the receptor's acetylcholine (ACh)-binding site, but preincubation of the membranes with the OPs increased this site's affinity for Carb. In absence of agonist, 100 μmof the OPs increased the binding of [3H]TCP by two- to fivefold with the following order of decreasing potency: PS > CPO > CPS > PO. The data suggest that in addition to inhibition of acetylcholinesterase, these OPs bind to a site on the nAChR that is different from the sites that bind ACh or TCP and that this binding induces nAChR desensitization. The relevance of this direct action of OPs on nAChRs on their acute toxicities is discussed.

Interaction of d-Tubocurarine Analogs with the Torpedo Nicotinic Acetylcholine Receptor:: METHYLATION AND STEREOISOMERIZATION AFFECT SITE-SELECTIVE COMPETITIVE BINDING AND BINDING TO THE NONCOMPETITIVE SITE

Analogs of d-tubocurarine were used to determine the individual effects of methylation, stereoisomerization, and halogenation of d-tubocurarine on the affinity for each of the two acetylcholine (ACh) binding sites of the Torpedo nicotinic acetylcholine receptor (AChR) and for the noncompetitive antagonist site. Eight analogs were synthesized, including three new compounds: 7'-O-methyl-chondocurarine, 12'-O-methyl-chondocurarine, and 13'-bromo-d-tubocurarine. The two ACh sites differ in their affinities for d-tubocurarine by 400-fold, as shown by inhibition of [3H]ACh binding, whereas the affinity ratio for metocurine, the trimethylated derivative of d-tubocurarine, is reduced to 30 due to a decreased affinity for the high affinity site. Binding analysis of five d-tubocurarine analogs demonstrates that methylation of the phenols alone is responsible for the observed changes in affinity. Substitution with bromine or iodine at the 13'-position affected affinity at both sites with a net increase in site selectivity. Stereoisomers of d-tubocurare had decreased affinity for only the high affinity ACh site. Thus, the ring systems, including the 12'- and 13'-positions and the 1-position stereocenter, appear to be important in discriminating between the two ACh binding sites. Desensitization of the AChR was measured by increased affinity for [3H]phencyclidine. Binding to only the single, high affinity acetylcholine binding site, comprised by the alpha gamma-subunits, was required for partial desensitization of the AChR by d-tubocurarine and its analogs. Stronger desensitization, to the same extent observed in the presence of the agonist carbamylcholine, occurred upon binding by iodonated or brominated d-tubocurarine. Interaction of the analogs at the noncompetitive antagonist site of the AChR was also measured by [3H]phencyclidine binding. The bis-tertiary ammonium analogs of either the d- or l-stereoisomers bound to the noncompetitive antagonist binding site of the AChR with 100-fold higher affinity than the corresponding quaternary ammonium analogs.

The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor.

We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.