Kainate receptor (IC50 = 7.5 μM); AMPA receptor (IC50 = 11 μM)

In cultured rat hippocampus neurons, GYKI 52466 (0.3-100 μM) suppresses inward currents triggered by AMPA and Kainate receptor[1].

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In whole-cell voltage-clamp recordings from cultured rat hippocampal neurons, the 2,3-benzodiazepine GYKI 52466 was a potent antagonist of kainate- and AMPA-activated currents (IC50 values, 7.5 and 11 microM, respectively), but was inactive against N-methyl-D-aspartate (NMDA) or gamma-aminobutyric acid responses. The block produced by GYKI 52466 occurred in a noncompetitive fashion, was voltage independent, and failed to show use dependence, indicating an allosteric blocking mechanism. In kinetic experiments with kainate as the agonist, the GYKI 52466 binding and unbinding rates were 1.6 x 10(5) M-1 s-1 and 3.2 s-1, respectively. GYKI 52466 also suppressed non-NMDA receptor-mediated spontaneous synaptic currents via a postsynaptic action. Non-competitive AMPA/kainate antagonists such as GYKI 52466 could offer advantages over competitive antagonists in the treatment of glutamate-associated neurological disorders, particularly under conditions in which high levels of the amino acid would render the competitive antagonists relatively ineffective. Moreover, the results demonstrate the existence of a novel recognition site for an atypical benzodiazepine on non-NMDA receptors [1].

In DBA/2 mice, therapy with GYKI-52466 (intraperitoneal injection; 1.76-13.2 mg/kg; once) offers strong anticonvulsant protection against seizures brought on by sound[2].

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The excitatory amino acid antagonists, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline) and GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) that act on non-NMDA receptors, provide potent anticonvulsant protection against AMPA [RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-induced seizures in Swiss mice and against sound-induced seizures in seizure-susceptible DBA/2 mice. Maximal anticonvulsant protection is observed 5-30 min after the i.p. administration of NBQX and 5-15 min after the i.p. administration of GYKI 52466 in DBA/2 mice. The ED50 values for the protection against AMPA-induced seizures by NBQX (30 min, i.p.) and GYKI 52466 (15 min, i.p.) are 23.6 (11.6-48.0) and 18.5 (11.5-29.5) mumol/kg, respectively. The ED50 values at 15 min for the protection against sound-induced seizures in DBA/2 mice are 31.3 (24.9-39.4) mumol/kg (NBQX, i.p.), 37.8 (21.2-67.4) mumol/kg (NBQX, i.v.) and 13.7 (11.5-16.5) mumol/kg (GYKI 52466, i.p.). In DBA/2 mice the therapeutic index (ratio of ED50 values for impaired rotarod performance and anticonvulsant action) is 6.6 for NBQX (15 and 30 min, i.p.) and 2.0 for GYKI 52466 (15 min, i.p.) [2].

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Two-way RM-ANOVA comparing sex and dose showed that GYKI 52466 produced a significant dose-dependent reduction in ethanol reinforced responding [F(4, 44) = 12, p < 0.0001] but there was no effect of sex and no interaction. Planned multiple comparisons showed that GYKI 52466 (5.6 and 10 mg/kg) reduced the number of ethanol reinforced responses as compared to vehicle in both male and female mice (Figure 5B). Analysis of the number of reinforcer earned also showed a significant dose-dependent effect of GYKI 52466 [F(4, 44) = 13, p < 0.0001] that was supported by individual planned comparisons within sex (Figure 5C). Interestingly, RM-ANOVA conducted on ethanol intake (g/kg) found a significant effect of sex [F(1, 11) = 13, p = 0.0041] and GYKI 52466 [F(4, 44) = 9.5, p < 0.0001]. Planned comparisons within sex showed that only GYKI 52466 (10 mg/kg) significantly decreased ethanol intake in both sexes (Figure 5D).

Due to the different baseline levels of self-administration between male and female mice, parameters of ethanol reinforced responding were transformed to percent control to evaluate the relative effects of GYKI 52466 (Figures 5E–G). Visual inspection of the graphs showed a relatively identical degree of change in ethanol self-administration between male and female mice. RM-ANOVA identified significant main effects of GYKI 52466 on ethanol reinforced responses [F(4, 44) = 12.26, p < 0.0001], number of ethanol reinforcers [F(4, 44) = 5.5, p = 0.0011], and ethanol intake [F(4, 44) = 4.7, p = 0.0030] with dose-dependent reductions in total responses and reinforcers, but not intake (Figures 5E-G).

Analysis of the temporal pattern of ethanol reinforced responding following the highest dose of GYKI 52466 was conducted. A two-way RM-ANOVA was performed on raw response totals collected in 5-min bins, with both Time (0–60 min) and GYKI 52466 (0 or 10 mg/kg) as within-subject factors. For males, there was a significant main effect of GYKI 52466 [F(1, 4) = 9.124, p = 0.039] but no effect of Time and no interaction, indicating that response totals were reduced consistently throughout the session. For females, there was a significant effect of GYKI 52466 [F(1, 7) = 39.78, p = 0.0004] and Time [F(11, 77) = 2.876, p = 0.0033] but no interaction, indicating a consistent reduction in responding across time. Ethanol-reinforced response totals are graphically represented in Figure 5H ascumulative distributions to visualize the impact of GYKI 52466 on the ongoing rate of operant behavior, which is critical to assess altered reinforcing function of ethanol.

Finally, to investigate potential nonspecific motor effects of AMPAR inhibition, open-field locomotor activity was measured following GYKI 52466 (0, 5.6, and 10 mg/kg, IP) during separate behavioral tests. A two-way RM ANOVA found no effect on spontaneous locomotor activity during 1 h sessions (Figure 5I). Because the n was low in the male group, a secondary analysis was conducted in which we collapsed sex as a factor and ran a RM one-way ANOVA on the effects of GYKI 52466 on open field locomotor activity. Similarly, we found no effect on 1 h locomotor activity in the open field [4].

Following this, either 60 μl of anti-GluR2 at 3 ng μl−1 for GluA2FRET purification or 60 μl of anti-TARPγ2 at 2.4 ng μl−1 for GluA2-γ2FRET purification in 1× PBS was applied twice through the chamber and incubated for 20 min, followed by washing with 1× PBS. BSA (0.1 mg ml−1) was introduced into the chamber and incubated for 15 min, before washing with 1× PBS. Detergent-solubilized purified proteins were attached to the glass slide using an in situ immunoprecipitation method by applying 50 µl of sample three times through the chamber and incubating for 20 min. Then, 90 µl of oxygen-scavenging solution buffer system (ROXS) was applied inside the chamber containing 1 mM methyl viologen, 1 mM ascorbic acid, 0.01% w/w pyranose oxidase, 0.001% w/v catalase, 3.3% w/w glucose, 1 mM DDM and 0.2 mM CHS in PBS pH 7.4. For the CTZ condition, 1 mM Glu and 100 μM CTZ were introduced into the ROXS. In the GYKI 52466-treated condition, 1 mM Glu and 100 µM GYKI 52466 were introduced into the ROXS.[3]

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Electrophysiology: For electrophysiological measurements of GluA2-γ2EM, which contained eGFP for cell detection, 1 μg of DNA was transfected into HEK293T cells in 3-cm culture dishes using Lipofectamine 2000. Patch-clamp recordings were performed 24–48 h after transfection using fire-polished borosilicate glass. Pipettes with 1–4 MΩ resistance were filled with internal solution: 110 mM CsF, 30 mM CsCl, 4 mM NaCl, 0.5 mM CaCl2, 10 mM HEPES and 5 mM EGTA (adjusted to pH 7.4 with CsOH). The extracellular solution consisted of 150 mM NaCl, 3 mM KCl, 2 mM CaCl2 and 10 mM HEPES adjusted to pH 7.4 with NaOH. External solutions were locally applied to lifted cells or patches using an SF-77B perfusion fast-step. For inhibition concentration–response determination, 100 μM CTZ was preincubated in extracellular buffer for at least 30–60 s, along with the corresponding GYKI 52466 concentration. For channel activation, 1 mM Glu with 100 μM CTZ and the corresponding GYKI 52466 concentration was applied for 500 ms and recordings were allowed to reach equilibrium before obtaining 2–10 sweeps per condition for averaging. The mean of the residual current was obtained using a range between 200 and 500 ms after Glu application and used for inhibition concentration–response analysis. Recordings were performed using an Axopatch 200B amplifier at −60 mV hold potential, acquired at 2 kHz using pCLAMP10 software. Individual patch-clamp traces and the average residual current for IC50 were analyzed using Clampfit 11 software. The inhibition concentration–response results were analyzed using the Levenberg–Marquardt iteration algorithm for a nonlinear curve fit using OriginPro 2023b. The experimental data were fit with the following equation [3].

Animal/Disease Models: Male and female DBA/2 mice tested for sound-induced seizures responses[2]
Doses: 1.76-13.2 mg/kg
Route of Administration: intraperitoneal (ip)injection; 1.76-13.2 mg/kg; once
Experimental Results: Observed Maximal anticonvulsant protection after the ip treatment (5-15 min).

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Effects of GYKI 52466 on operant alcohol self-administration [4]
Male and female mice with a history of alcohol self-administration (n = 16; n = 8/condition) were habituated to injections of 0.9% saline. Injections were given 30 min prior to the start of the operant session with a minimum of 3 operant sessions between injections. Initially, habituation injections were administered until self-administration following injection stabilized (4–6 injections). Once responding was stable, vehicle and 4 doses of GYKI 52466 dihydrochloride (0.3, 1.0, 5.6, and 10.0 mg/kg, i.p.) were administered using a counterbalanced design. A maximum of two injections per week per animal were given so that responding returned to baseline following drug administration.

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Locomotor testing + GYKI 52466 dose effect curve [4]
After operant self-administration, mice were tested for non-specific locomotor effects of GYKI 52466 using computer-controlled open field chambers (27 cm × 27 cm × 20 cm) as previously reported (Riday et al., 2012; Agoglia et al., 2015b; Faccidomo et al., 2015; Stevenson et al., 2019; Faccidomo et al., 2020). X-Y ambulatory movements were recorded with two sets of 16 pulse-modulated infrared photobeams, assessing the mouse’s position every 60 s to quantify distance traveled (cm). Male and female mice with a history of ethanol self-administration were habituated to the open field apparatus for 2 h. One week later, mice were administered the drug and returned to their home cage for 30 min before being placed in the open field apparatus for 1 h. Vehicle and two doses of GYKI 52466 (5.6 and 10.0 mg/kg, IP) were administered in a counter-balanced design, with at least 1 week between tests. Due to technical issues with two of the open field boxes, 2 male mice were excluded from the final analysis. For this reason, a secondary analysis of GYKI 52466, with male and female mice combined, was added to the results.

GYKI 52466 was dissolved in a vehicle of 0.9% NaCl for intraperitoneal (IP) administration in mice at doses of (0.0, 0.3, 1.0, 5.6, and 10 mg/kg).

[1]. GYKI 52466, a 2,3-benzodiazepine, is a highly selective, noncompetitive antagonist of AMPA/kainate receptor responses. Neuron. 1993 Jan;10(1):51-9.

[2]. The anticonvulsant effect of the non-NMDA antagonists, NBQX and GYKI 52466, in mice. Epilepsy Res. 1991 Jul;9(2):92-6.

[3]. Allosteric competition and inhibition in AMPA receptors. Nat Struct Mol Biol. 2024 Nov;31(11):1669-1679.

[4]. Distinct sex differences in ethanol consumption and operant self-administration in C57BL/6J mice with uniform regulation by glutamate AMPAR activity. Front Behav Neurosci. 2025 Jan 22:18:1498201

Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators are therapeutic candidates that inhibit AMPAR activation and can compete with positive modulators to control AMPAR function through unresolved mechanisms. Here we show that allosteric inhibition pushes AMPARs into a distinct state that prevents both activation and positive allosteric modulation. We used cryo-electron microscopy to capture AMPARs bound to glutamate, while a negative allosteric modulator, GYKI 52466, and positive allosteric modulator, cyclothiazide, compete for control of the AMPARs. GYKI 52466 binds in the ion channel collar and inhibits AMPARs by decoupling the ligand-binding domains from the ion channel. The rearrangement of the ligand-binding domains ruptures the cyclothiazide site, preventing positive modulation. Our data provide a framework for understanding allostery of AMPARs and for rational design of therapeutics targeting AMPARs in neurological diseases. [3]

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Introduction: Considering sex as a biological variable (SABV) in preclinical research can enhance understanding of the neurobiology of alcohol use disorder (AUD). However, the behavioral and neural mechanisms underlying sex-specific differences remain unclear. This study aims to elucidate SABV in ethanol (EtOH) consumption by evaluating its reinforcing effects and regulation by glutamate AMPA receptor activity in male and female mice.
Methods: C57BL/6J mice (male and female) were assessed for EtOH intake under continuous and limited access conditions in the home cage. Acute sensitivity to EtOH sedation and blood clearance were evaluated as potential modifying factors. Motivation to consume EtOH was measured using operant self-administration procedures. Sex-specific differences in neural regulation of EtOH reinforcement were examined by testing the effects of a glutamate AMPA receptor antagonist on operant EtOH self-administration.
Results: Female C57BL/6J mice exhibited a time-dependent escalation in EtOH intake under both continuous and limited access conditions. They were less sensitive to EtOH sedation and had lower blood levels post-EtOH administration (4 g/kg) despite similar clearance rates. Females also showed increased operant EtOH self-administration and progressive ratio performance over a 30-day baseline period compared to males. The AMPAR antagonist GYKI 52466 (0-10 mg/kg, IP) dose-dependently reduced EtOH-reinforced lever pressing in both sexes, with no differences in potency or efficacy.
Discussion: These findings confirm that female C57BL/6J mice consume more EtOH than males in home-cage conditions and exhibit reduced acute sedation, potentially contributing to higher EtOH intake. Females demonstrated increased operant EtOH self-administration and motivation, indicating higher reinforcing efficacy. The lack of sex differences in the relative effects of GYKI 52466 suggests that AMPAR activity is equally required for EtOH reinforcement in both sexes.[4]

C17H17CL2N3O2

366.241781949997

365.069

2319722-40-0

GYKI 52466;102771-26-6;GYKI 52466 hydrochloride;192065-56-8

91820611

Yellow to orange solid powder

3

5

1

24

482

0

Cl.Cl.O1COC2=CC3C(C4C=CC(=CC=4)N)=NN=C(C)CC=3C=C12

NDRITPLSHUPVRR-UHFFFAOYSA-N

InChI=1S/C17H15N3O2.2ClH/c1-10-6-12-7-15-16(22-9-21-15)8-14(12)17(20-19-10)11-2-4-13(18)5-3-11;;/h2-5,7-8H,6,9,18H2,1H3;2*1H

4-(8-methyl-9H-[1,3]dioxolo[4,5-h][2,3]benzodiazepin-5-yl)aniline;dihydrochloride

GYKI-52466 DiHCl; GYKI52466; GYKI 52466; GYKI 52466 dihydrochloride; 2319722-40-0; GYKI 52,466 (dihydrochloride); 4-(8-methyl-9H-[1,3]dioxolo[4,5-h][2,3]benzodiazepin-5-yl)aniline;dihydrochloride; 4-(8-Methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzenamine dihydrochloride; SCHEMBL25227675; GYKI-5,2466; GYKI52,466; GYKI 52,466

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~2.78 mg/mL (~7.59 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)