BDBM50080153 2-(1-(aminomethyl)cyclohexyl)acetic acid Neurontin (1-Aminomethyl-cyclohexyl)-acetic acid CHEMBL940 [1-(AMINOMETHYL)CYCLOHEXYL]ACETIC ACID GABAPENTIN GOE-3450 CI-945
- Burgos-Lepley, CE; Thompson, LR; Kneen, CO; Osborne, SA; Bryans, JS; Capiris, T; Suman-Chauhan, N; Dooley, DJ; Donovan, CM; Field, MJ; Vartanian, MG; Kinsora, JJ; Lotarski, SM; El-Kattan, A; Walters, K; Cherukury, M; Taylor, CP; Wustrow, DJ; Schwarz, JB Carboxylate bioisosteres of gabapentin. Bioorg Med Chem Lett 16: 2333-6 (2006)
- ChEMBL_348348 (CHEMBL866241) Displacement of [3H]gabapentin from alpha-2delta calcium channel in pig brain membrane
- ChEMBL_348644 (CHEMBL866258) Displacement of [3H]gabapentin from alpha-2delta calcium channel in pig brain membrane
- ChEMBL_332923 (CHEMBL854074) [3H]Gabapentin binding in human A710 membrane expressing alpha-2delta subunit of calcium channel
- ChEMBL_605315 (CHEMBL1071340) Displacement of [3H]gabapentin from calcium channel alpha2delta in pig cerebral cortex membrane after 30 mins
- ChEMBL_605546 (CHEMBL1067300) Displacement of [3H]gabapentin from calcium channel alpha2delta in pig cerebral cortex membrane after 30 mins
- ChEBML_32526 Binding affinity towards alpha2-delta subunit of a voltage gated calcium channel using [3H]gabapentin in human brain membrane (A710 membrane)
- ChEBML_32533 Binding affinity for huamn alpha2-delta1 subunit of voltage gated calcium channel over-expressed in A710 cell membranes using [3H]-gabapentin
- ChEMBL_2059156 (CHEMBL4714157) Displacement of [3H]-gabapentin from human Cav alpha2delta2 expressed in HEK293 cell membranes incubated for 60 mins by scintillation counting method
- ChEMBL_2059155 (CHEMBL4714156) Displacement of [3H]-gabapentin from human Cav alpha2delta1 expressed in CHO-K1 cell membranes incubated for 60 mins by scintillation counting method
- ChEMBL_2116664 (CHEMBL4825605) Displacement of [3H]-gabapentin from human Cav alpha2delta1 expressed in CHO-K1 cell membranes incubated for 60 mins by scintillation counting method
- ChEMBL_1579725 (CHEMBL3811918) Inhibition of human LAT1 expressed in HEK cells assessed as reduction in uptake of [3H]-gabapentin after 3 mins by scintillation counting based cis-inhibition assay
- ChEMBL_2063643 (CHEMBL4718896) Inhibition of human LAT1 expressed in HEK293-T-Rex cells assessed as inhibition of [3H]-gabapentin uptake by scintillation counting cis-inhibition assay relative to BCH inhibition (RV = 100%)
- ChEMBL_1885219 (CHEMBL4386801) Cis-inhibition of human LAT1 expressed in TREx HEK293 cells assessed as inhibition of [3H]-gabapentin uptake preincubated for 3 mins at 37 degC followed by washing with choline buffer and measured after 3 hrs by scintillation counting analysis
- ChEMBL_1777837 (CHEMBL4234829) Cis-inhibition of human LAT1 expressed in TREx HEK293 cells at 200 uM assessed as inhibition of [3H]-gabapentin uptake preincubated for 3 mins at 37 degC followed by washing with choline buffer and measured after 3 hrs by scintillation counting analysis relative to BCH
- [3H]-Gabapentin Binding Assay A test compound (20 μL) diluted to 5 times of the final concentration with Binding buffer, [3H]-gabapentin diluted to 100 nmol/L with binding buffer [20 μL (final concentration 20 nmol/L)], and the rat cerebral cortex membrane fraction (60 μL; 12 μg membrane fraction) obtained in the (1) above were added to each well of a 96-well round-bottom plate. After being sufficiently mixed, these were allowed to react at room temperature for 1 hour. After the reaction, the reaction sample was suction filtered using a filter plate with 50 μL/well of 0.3 vol % polyethyleneimine added, and a cell harvester. The filter was then washed with ice-cooled Wash buffer (100 mmol/L NaCl, 0.1 w/v % BSA). After being washed, the filter plate was dried, and a scintillation cocktail (MicroScint-20, purchased from PerkinElmer) was added (50 μL/well), and the radioactivity on the filter was measured. The radioactivity obtained in the absence of the test compound was measured as total binding amount, and the radioactivity obtained by addition of an unlabeled gabapentin (final concentration 100 mmol/L) as a test compound was measured as non-specific binding amount.
- Binding Assay Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured in five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
- Binding Assay Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured at five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4. Filter plates were dried at 60° C. for 1 h and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
- Human alpha2delta-1 Subunit of Cav2.2 Calcium Channel Assay Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4. NSB (non specific binding) was measured by adding 10 μM pregabalin. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4. Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading. Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
- Binding Assay α2δ-1: Binding Assay to Human α2δ-1 Subunit of Cav2.2 Calcium Channel.Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4.Filter plates were dried at 60° C. for 1 hour and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
- Binding Assay This section describes a scintillation proximity assay (SPA) to measure [3H] gabapentin ([3H]GBP) binding to membranes containing α2δ-1 and its use for profiling compounds (Calvo et al (2012) J. Biomol. Screen. 17:1041-1049).Human Cav1.2/β3/α2δ-1 Calcium Channel Membranes (Chantest) were thawed on ice, aliquotted and stored at −80° C. for subsequent use. Membranes were diluted to 200 μg/ml (3 μg/well final assay concentration (FAC)) in assay buffer (10 mM HEPES (Sigma), (pH7.4)). The [3H]GBP (Perkin Elmer) stock solution was stored at −20° C. [3H]GBP was diluted to 40 nM (10 nM FAC) in assay buffer. SPA beads (Perkin Elmer) were re-suspended at 100 mg/ml in 10 mM HEPES (pH 7.4). Beads were diluted to 40 mg/ml (0.6 mg/well FAC) in assay buffer. Nonspecific binding (NSB) was generated using an excess of pregabalin (Tocris). Pregabalin was solubilized in Milli-Q H2O at 10 mM. 10 mM pregabalin was diluted to 400 μM (100 μM FAC) in assay buffer.Compounds were diluted to 100 μM then half log diluted. These were then diluted 1:100 in assay buffer to a 4× assay concentration (1 μM FAC top dilution).SPA beads 15 μl; membranes 15 μl; pregabalin or assay buffer/test compound 15 μl and [3H]GBP 15 μl were added to a white 96 well isoplate, (Perkin Elmer). The assay plate was sealed and mixed for 10 s on a plate shaker then placed into a plate rack and slotted into the reader stacker. The plate was incubated overnight (20 hours) then read on a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter at ambient room temperature (RT).