Du 640

DEAB inhibition and IC₅₀ curves were assayed spectrophotometrically by monitoring the formation of NADH at 340 nm (molar extinction coefficient of 6220 MT⁻¹ cm⁻¹) on a Beckman DU-640 or Cary 300 Bio UV-vis spectrophotometer using purified recombinant ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH4A1, and ALDH5A1. All assays were performed at 25 °C Following a two minute incubation of enzyme with DEAB and NAD⁺, the reactions were initiated by adding substrate. With the exception of ALDH1L1, ALDH4A1 and ALDH5A1, reactions were. performed in a solution containing 100–200 nM enzyme, 200 μM NAD⁺, 1% DMSO, and 100–200 μM propionaldehyde in 50 mM sodium BES pH 7.5. For ALDH4A1, reactions contained 20 mM propionaldehyde and 1.5 mM NAD⁺. For ALDH5A1, reactions contained 2 mM propionaldehyde and 1.5 mM NAD⁺. For ALDH1L1, reactions contained 500 nM enzyme, 4 mM propionaldehyde and 500 μM NADP⁺. For enzymes that showed inhibition by DEAB, IC₅₀ values for propionaldehyde oxidation were calculated by varying the concentration of DEAB from 0 to 20 μ.M. Higher concentrations of DEAB were not used due to interference at 340 nm. However there was little to no interference at lower DEAB concentrations. Data were fit to the four parameter EC₅₀ equation using SigmaPlot (StatSys v12.3) and the values represent the average of three independent experiments (each n = 3).

Hits from the HTS were ordered from ChemDiv to determine if they had any effect on aldehyde oxidation and whether they were selective for ALDH1A1 compared to ALDH2 and ALDH3A1. Dehydrogenase activity of the three isoenzymes were assayed by monitoring the production of NADH at 340 nm (molar extinction coefficient of 6220 M⁻¹ · cm⁻¹) on a Beckman DU-640 or Cary 300 Bio UV-Vis spectrophotometer for 2 to 3 minutes. For ALDH1A1 and ALDH2, the reaction contained 100–200 nM enzyme, 200 μM NAD⁺, 100 μM propionaldehyde, and 1% DMSO in 50 mM Na⁺ BES, pH 7.5 at room temperature. For ALDH3A1, the reaction contained 20 nM enzyme, 200 μM NAD⁺, 300 μM benzaldehyde, and 1% DMSO in either 100 mM sodium phosphate or 50 mM Na⁺ BES at pH 7.5 at room temperature. For most compounds, 20 μM concentration was used for the selectivity assays. However, due to solubility issues for CM307, 10 μM of compound was used. Following a 2 minute incubation of enzyme, compound, and NAD⁺, the reaction was initiated by adding substrate. For compounds with over 60% inhibition at 20 μM, IC₅₀ values for propionaldehyde oxidation were calculated by varying the concentration of the compounds from 0–200 μM under the same conditions as the selectivity assays. Data were fit to the four parameter EC₅₀ equation using SigmaPlot (StatSys v12.3).