Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a Chemical structures of Hispidulin and TP003. b , c Dose-response analysis of Hispidulin ( b ) and TP003 ( c ). HEK293T cells stably expressing α1(D219N)β2γ2 or α1(G251D)β2γ2 receptors were treated with DMSO vehicle control, Hispidulin (0.05 μM to 25 μM), or TP003 (0.05 μM to 10 μM) for 24h. Cells were lysed, and total proteins were subjected to Western blot analysis. EC 50 values were calculated from the dose-response curve fitting, shown on the bottom panels (n=3). d Time-course analysis of Hispidulin and TP003. HEK293T cells stably expressing α1(D219N)β2γ2 or α1(G251D)β2γ2 receptors were treated with DMSO vehicle control, Hispidulin (10 μM), or TP003 (5 μM) for the indicated time. Cells were lysed, and total proteins were subjected to Western blot analysis. Quantification of the α1 band intensities was shown on the bottom panels (n=3). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. *, p < 0.05; **, p < 0.01. Also see Supplementary Fig. S3 and Fig. S4.

Article Snippet: Hispidulin (catalog#: 2174), CL218872 (catalog#: 1709), ZK93423 (catalog#: 1994), Bretazenil (catalog#: 3568), CGS20625 (catalog#: 2467), and TP003 (catalog#: 4414) were purchased from Tocris Bioscience.

Techniques: Stable Transfection, Expressing, Control, Western Blot

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a-c Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on the surface protein expression of the α1 variants in HEK293T cells stably expressing α1(D219N)β2γ2 ( a ), α1(G251D)β2γ2 ( b ), or α1(P260L)β2γ2 GABA A receptors ( c ) according to surface biotinylation analysis. Na + /K + ATPase serves as a plasma membrane protein loading control. Quantification of the surface α1 band intensities was shown on the bottom panels ( n = 3). d-f Effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on GABA-induced peak current amplitudes in HEK293T cells stably expressing α1(D219N)β2γ2 ( d ), α1(G251D)β2γ2 ( e ), or α1(P260L)β2γ2 GABA A receptors ( f ). Representative whole-cell voltage-clamp recording traces are shown. The holding voltage was set at −60 mV. Application of GABA (100 μM, 3 s) is indicated by the horizontal bar above the current traces. Quantification of the peak currents (I max ) are shown on the bottom panels (n = 4 to 8). pA: pico Ampere; nA: nano Ampere. g-i Dose-response curves of GABA are shown for the evaluation of the effect of Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) on α1(D219N)β2γ2 ( g ), α1(G251D)β2γ2 ( h ), or α1(P260L)β2γ2 GABA A receptors ( i ) (n = 3 to 4). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Hispidulin (catalog#: 2174), CL218872 (catalog#: 1709), ZK93423 (catalog#: 1994), Bretazenil (catalog#: 3568), CGS20625 (catalog#: 2467), and TP003 (catalog#: 4414) were purchased from Tocris Bioscience.

Techniques: Expressing, Stable Transfection, Clinical Proteomics, Membrane, Control

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a,b Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) increases the FRET efficiency between CFP-tagged α1(G251D) subunit and YFP-tagged β2 subunit of GABA A receptors. HEK293T cells were transfected with CFP-tagged α1(G251D) subunit, YFP-tagged β2 subunit, and γ2 subunit; Twenty-four hours post transfection, cells were treated with DMSO, Hispidulin (10 µM) or TP003 (5 µM). Forty-eight hours post transfection, pixel-based FRET was used to calculate the FRET efficiency between α1(G251D)-CFP and β2-YFP. Representative images were shown for the CFP channel (1st columns), YFP channel (2nd columns), and FRET efficiency (3rd columns) ( a ). Scale bar = 20 μm. FRET efficiency was calculated from 30-40 cells from at least three transfections using the ImageJ PixFRET plug-in, shown in b . c Hispidulin (10 µM, 24 h) or TP003 (5 µM, 24 h) treatment increases the ER-to-Golgi trafficking efficiency of α1(G251D) in HEK293T cells stably expressing α1(G251D)β2γ2 GABA A receptors. The Peptide-N-Glycosidase F (PNGase F) enzyme cleavage serves as a control for unglycosylated α1 subunits (lane 7). After endo H digestion, α1 subunits with a molecular weight that is equal to unglycosylated α1 were labeled as endo H-sensitive, whereas those with higher molecular weights were labeled as endo H-resistant. Quantification of the ratio of endo H-resistant α1 / total α1 subunit bands, as a measure of the ER-to-Golgi trafficking efficiency, is shown on the bottom (n=3). d Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the protein levels of selected chaperones and γ2 subunits in HEK293T cells stably expressing α1(G251D)β2γ2 receptors (n=3). e Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the interactions between α1(G251D) and selected chaperones or γ2 in HEK293T cells stably expressing α1(G251D)β2γ2 receptors. Quantification of the ratio of proteins of interest / α1(G251D) post immunoprecipitation is shown in the right panels (n=3). IP: immunoprecipitation. f Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the mRNA levels of genes of interest in HEK293T cells stably expressing α1(G251D)β2γ2 receptors, assessed by quantitative RT-PCR (n=3). Each data point is reported as mean ± SEM. One-way ANOVA followed by post-hoc Tukey test was used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001. Also see Supplementary Table S2 .

Article Snippet: Hispidulin (catalog#: 2174), CL218872 (catalog#: 1709), ZK93423 (catalog#: 1994), Bretazenil (catalog#: 3568), CGS20625 (catalog#: 2467), and TP003 (catalog#: 4414) were purchased from Tocris Bioscience.

Techniques: Transfection, Stable Transfection, Expressing, Control, Molecular Weight, Labeling, Immunoprecipitation, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a HEK293T cells stably expressing α1(G251D)β2γ2 receptors were treated with DMSO, Hispidulin (10 µM), and TP003 (5 µM) for 24 h. Then cycloheximide (100 μg / mL) (CHX), a potent protein synthesis inhibitor, was added to the cell culture media for the indicated time before cell lysis and Western blot analysis. Quantification of the normalized remaining α1 protein band intensity was shown on the right (n = 3). b Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the protein levels of selected ERAD factors in HEK293T cells stably expressing α1(G251D)β2γ2 receptors (n=3). c Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the interactions between α1(G251D) and selected ERAD factors in HEK293T cells stably expressing α1(G251D)β2γ2 receptors. Quantification of the ratio of proteins of interest / α1(G251D) post immunoprecipitation is shown in the right panels (n=3). IP: immunoprecipitation. Each data point is reported as mean ± SEM. ANOVA followed by post-hoc Tukey test was used to evaluate the statistical significance. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Hispidulin (catalog#: 2174), CL218872 (catalog#: 1709), ZK93423 (catalog#: 1994), Bretazenil (catalog#: 3568), CGS20625 (catalog#: 2467), and TP003 (catalog#: 4414) were purchased from Tocris Bioscience.

Techniques: Stable Transfection, Expressing, Cell Culture, Lysis, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: a Schematic of the generation of α1(G215D) knockin in hiPSCs and the transcription factor (TF)-based differentiation into GABAergic neurons. b Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the surface expression of GABA A receptor subunits in hiPSC-derived GABAergic neurons carrying the α1(G215D) variant. Surface GABA A receptors were stained using anti-α1 subunit, anti-β2/β3 subunit, or anti-γ2 subunit antibodies without membrane permeabilization. 50-75 neurons from at least three differentiations were imaged by confocal microscopy for each condition. Scale bar = 20 μm. c Quantification of the fluorescence intensity of the surface GABA A receptor subunits after background correction. d Effect of Hispidulin (10 µM, 24 h) and TP003 (5 µM, 24 h) on the interactions between α1(G251D) and selected ER proteostasis network components. PLA was used to determine the endogenous protein-protein interactions. 35-70 neurons from at least three differentiations were imaged by confocal microscopy for each condition. Scale bar = 20 μm. e Quantification of the PLA puncta number per cell was achieved using the ImageJ Analyze Particles plug-in. Each data point is reported as mean ± SEM. ANOVA followed by post-hoc Tukey test was used to evaluate the statistical significance. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Also see Supplementary Fig. S5 .

Article Snippet: Hispidulin (catalog#: 2174), CL218872 (catalog#: 1709), ZK93423 (catalog#: 1994), Bretazenil (catalog#: 3568), CGS20625 (catalog#: 2467), and TP003 (catalog#: 4414) were purchased from Tocris Bioscience.

Techniques: Knock-In, Expressing, Derivative Assay, Variant Assay, Staining, Membrane, Confocal Microscopy, Fluorescence, Protein-Protein interactions

Journal: bioRxiv

Article Title: Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants

doi: 10.1101/2023.04.18.537383

Figure Lengend Snippet: Hispidulin and TP003 act as pharmacological chaperones to bind GABA A receptor variants to stabilize them in the ER. Pharmacological chaperone treatment enhances the assembly of the variants and their forward trafficking to the Golgi and plasma membrane. The effect of Hispidulin and TP003 is also reflected from the enhanced folding and reduced degradation of the variants. Consistently, the interactions between the variants and pro-folding chaperones (BiP and calnexin) and a trafficking factor (LMAN1) are enhanced, whereas the interactions between the variants and ERAD factors (Grp94 and VCP) are reduced by pharmacological chaperone treatment.

Article Snippet: Hispidulin (catalog#: 2174), CL218872 (catalog#: 1709), ZK93423 (catalog#: 1994), Bretazenil (catalog#: 3568), CGS20625 (catalog#: 2467), and TP003 (catalog#: 4414) were purchased from Tocris Bioscience.

Techniques: Clinical Proteomics, Membrane

Journal: Psychopharmacology

Article Title: Contribution of GABA A Receptors Containing ?3 Subunits to the Therapeutic-Related and Side Effects of Benzodiazepine-Type Drugs in Monkeys

doi: 10.1007/s00213-010-2142-y

Figure Lengend Snippet: Anti-conflict effects of TP003 (a) and alprazolam (b) in rhesus monkeys trained under a multiple schedule of food presentation (non-suppressed responding) and food + shock presentation (suppressed responding). Abscissae, cumulative intravenous dose of drug in mg/kg, i.v. Ordinates, response rate as responses per second. Each data point represents the mean (± S.E.M.) from three or four monkeys. Points above “V” represent data after vehicle administration. Asterisks represent significant differences relative to vehicle (Bonferroni t-tests, p<0.05).

Article Snippet: TP003 was provided by Merck, Sharp, & Dohme Research Laboratories (Harlow, UK), and was prepared in a vehicle of 10% benzyl alcohol, 50% propylene glycol, and 40% sterile water.

Techniques:

Journal: Psychopharmacology

Article Title: Contribution of GABA A Receptors Containing ?3 Subunits to the Therapeutic-Related and Side Effects of Benzodiazepine-Type Drugs in Monkeys

doi: 10.1007/s00213-010-2142-y

Figure Lengend Snippet: Potencies of TP003, alprazolam, HZ-166 and zolpidem to alter suppressed and non-suppressed responding in the conflict procedure

Article Snippet: TP003 was provided by Merck, Sharp, & Dohme Research Laboratories (Harlow, UK), and was prepared in a vehicle of 10% benzyl alcohol, 50% propylene glycol, and 40% sterile water.

Techniques:

Journal: Psychopharmacology

Article Title: Contribution of GABA A Receptors Containing ?3 Subunits to the Therapeutic-Related and Side Effects of Benzodiazepine-Type Drugs in Monkeys

doi: 10.1007/s00213-010-2142-y

Figure Lengend Snippet: Ataxia (a) and resistance (myorelaxation, b) scores of TP003 in squirrel monkeys as determined using quantitative observational procedures. Abscissae, dose of TP003 in mg/kg, i.m. Ordinates, assessment score as described in Materials and Methods section. Each data point represents the median and inter-quartile range from four monkeys. Points above “V” represent data after vehicle administration. Asterisks represent significant differences relative to vehicle (Dunn’s Q statistic, p<0.05).

Article Snippet: TP003 was provided by Merck, Sharp, & Dohme Research Laboratories (Harlow, UK), and was prepared in a vehicle of 10% benzyl alcohol, 50% propylene glycol, and 40% sterile water.

Techniques:

Journal: Psychopharmacology

Article Title: Contribution of GABA A Receptors Containing ?3 Subunits to the Therapeutic-Related and Side Effects of Benzodiazepine-Type Drugs in Monkeys

doi: 10.1007/s00213-010-2142-y

Figure Lengend Snippet: Ataxic- and myorelaxant-like effects of TP003, alprazolam and zolpidem in the observation procedure. Median scores (±interquartile range) are from the maximally effective dose tested (shown in parenthesis).

Article Snippet: TP003 was provided by Merck, Sharp, & Dohme Research Laboratories (Harlow, UK), and was prepared in a vehicle of 10% benzyl alcohol, 50% propylene glycol, and 40% sterile water.

Techniques:

Journal: Psychopharmacology

Article Title: Contribution of GABA A Receptors Containing ?3 Subunits to the Therapeutic-Related and Side Effects of Benzodiazepine-Type Drugs in Monkeys

doi: 10.1007/s00213-010-2142-y

Figure Lengend Snippet: Quantitative behavioral effects of TP003 in squirrel monkeys as determined in the observation procedure. Abscissae, dose of TP003 in mg/kg, i.m. Ordinates, pellet consumption as the mean number of sucrose pellets consumed expressed as percentage of baseline control (a) or frequency score as described in Materials and Methods section (b–f). Each data point represents the mean (± S.E.M.) from five monkeys. Points above “V” represent data after vehicle administration. Asterisks represent significant differences relative to vehicle (Bonferroni t-tests, p<0.05).

Article Snippet: TP003 was provided by Merck, Sharp, & Dohme Research Laboratories (Harlow, UK), and was prepared in a vehicle of 10% benzyl alcohol, 50% propylene glycol, and 40% sterile water.

Techniques:

Journal: Psychopharmacology

Article Title: Contribution of GABA A Receptors Containing ?3 Subunits to the Therapeutic-Related and Side Effects of Benzodiazepine-Type Drugs in Monkeys

doi: 10.1007/s00213-010-2142-y

Figure Lengend Snippet: Effects of TP003, alprazolam and zolpidem on observational measures related to sedation. Frequency scores (±S.E.M.) are from the maximally effective dose tested (shown in parenthesis).

Article Snippet: TP003 was provided by Merck, Sharp, & Dohme Research Laboratories (Harlow, UK), and was prepared in a vehicle of 10% benzyl alcohol, 50% propylene glycol, and 40% sterile water.

Techniques: