Inhibition of recombinant ABAD (unknown origin) assessed as remaining activity at 25 uM using acetoacetyl-CoA as substrate in presence of NADH by spectrophotometry
ABAD Enzymatic Activity Assay: The assay for the inhibition of reduction of S-acetoacetyl-CoA (SAAC) by ABAD was carried out with ABAD (418 ng/ml), SAAC (172 μM), NADH (102 μM), and different concentrations of inhibitors (from 0 to 1000 μM) in 93 mM potassium phosphate buffer (pH 7.3). Before the assay, all the assay components except SAAC were pre-incubated for 5 minutes, and the reaction started with the addition of SAAC into the reaction mix. The reaction was carried out for a total 6 minutes at room temperature under steady-state conditions, and the decrease of NADH absorbance at 340 nm was determined every 10 seconds.
Surface Plasmon Resonance (SPR)Assay: The interactions between compounds and ABAD were performed using the dual flow cell BIACORE 3000 instrument. Surface Plasmon Resonance (SPR) studies were performed on a BIACORE 3000 at 25° C. SPR binding experiments with ABAD were performed in phosphate-buffered saline (PBS, pH 7.4, 0.005% surfactant P20) as the running and the sample buffer. The surface of the sensor chip was first activated with mixtures of N-hydroxysuccinimide (NHS, 115 mg/ml) and N-(3-dimethyl-aminopropyl)-N'-ethyl-carbodiimide-hydrochloride (EDC, 750 mg/ml) for 7 minutes. ABAD was dissolved in PBS buffer (pH 5.0) at a concentration of 10 μg/ml. The protein was immobilized directly and covalently on the hydrophilic carboxymethylated dextran matrix of the CM5 sensor chip (BIACORE) by using the standard primary amine coupling reaction on a CM5 sensor chip according to standard procedures. After the immobilization of the protein, excess activated carboxylic acid groups were quenched with ethanolamine (1 M, pH 8.5). Special care was taken during injection of samples because of carryover effects. Special washing routines were used to clean the system before injection of new samples. In addition, predipping of needles was performed. The sample flow was 40 μl/minute in experiments performed for the determination of the kinetic and equilibrium constants. Regeneration of the surfaces between subsequent binding experiments was achieved by washing the surface extensively (>>1 hour) with buffer solution.
Inhibition of human full length N-terminal GST-tagged 17beta-HSD10 expressed in Escherichia coli BL21 DE3 Codon plus using [6,7-3H]-labeled E2/E2 as substrate and NADH at 1 uM after 30 mins by HPLC based radioflow detection method
Binding affinity to recombinant human HSD17B10 assessed as shift in melting temperature by SYPRO orange dye based differential scanning fluorimetry assay