BDBM327847 US9663479, Pregabalin
(S)-3-(aminomethyl)-5-methylhexanoic acid (S)-3-Aminomethyl-5-methyl-hexanoic acid PREGABALIN CHEMBL1059 BDBM50164279 CI-1008 Lyrica PD-144723
- 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).
- 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).