Query String: ALDH2 transgene
- ChEMBL_2236379 (CHEMBL5150275) Inhibition of human ALDH2
- ChEMBL_2112359 (CHEMBL4821209) Inhibition of ALDH2 (unknown origin)
- ChEMBL_489763 (CHEMBL989246) Inhibition of human recombinant ALDH2
- ChEMBL_2113104 (CHEMBL4821954) Agonist activity at human recombinant ALDH2 measured after 1 hr
- ChEMBL_1295311 (CHEMBL3128578) Inhibition of human ALDH2 using propionaldehyde as substrate by Lineweaver-Burk plot analysis
- ChEMBL_1295315 (CHEMBL3128582) Inhibition of human ALDH2 by Lineweaver-Burk plot analysis in presence of NAD+
- ChEMBL_2053026 (CHEMBL4708027) Inhibition of ALDH2 (unknown origin) assessed as NADH formation using acetaldehyde as substrate
- ChEMBL_1295309 (CHEMBL3128576) Competitive inhibition of human ALDH2 using propionaldehyde as substrate by Lineweaver-Burk plot analysis
- ChEMBL_2116259 (CHEMBL4825200) Inhibition of human ALDH2 assessed as NADH formation using propionaldehyde as substrate by spectrophotometry
- ChEMBL_1295313 (CHEMBL3128580) Noncompetitive/mixed type inhibition of human ALDH2 by Lineweaver-Burk plot analysis in presence of NAD+
- ChEMBL_2563843 Inhibition of ALDH2 (unknown origin) incubated for 1 hr by colloidal coomassie staining based LC-MS/MS analysis
- ChEMBL_2349467 Agonist activity at human TLR7 in HEK-Blue hTLR7 cells harboring SEAP reporter transgene incubated for 18 hrs by quanti-blue reagent based assay
- ALDH2 Assay Standard ALDH2 reaction mixtures contained 150 uM formaldehyde, 2.5 mM NAD+, 10 mM MgCl2 and 10 nM recombinant human ALDH2 in 50 mM Hepes buffer, pH 7.4, 0.01% Tween 20 in a final volume of 50 ul using 384-well plates. After 60 min of pre-incubation of compound with ALDH2 and formaldehyde, the reaction was started by adding NAD+ and the reaction mixture was allowed to proceed for 90 minutes. Activity of the enzyme was determined by monitoring NADH formation using Perkin-Elmer Envision Reader with excitation and emission wavelengths set at 340 and 460 nm, respectively.
- ALDH2 Assay Standard ALDH2 reaction mixtures contained 150 uM formaldehyde, 2.5 mM NAD+, 10 mM MgCl2 and 10 nM recombinant human ALDH2 in 50 mM Hepes buffer, pH 7.4, 0.01% Tween 20 in a final volume of 50 ul using 384-well plates. After 60 min of pre-incubation of compound with ALDH2 and formaldehyde, the reaction was started by adding NAD+ and the reaction mixture was allowed to proceed for 90 minutes. Activity of the enzyme was determined by monitoring NADH formation using Perkin-Elmer Envision Reader with excitation and emission wavelengths set at 340 and 460 nm, respectively.
- ChEMBL_1295317 (CHEMBL3130565) Inhibition of human ALDH2 using propionaldehyde as substrate preincubated for 2 mins followed by substrate addition by spectrophotometry in presence of NAD+
- ChEMBL_1473081 (CHEMBL3421191) Inhibition of recombinant human ALDH2 using propionaldehyde as substrate preincubated for 2 mins with NAD+ followed by substrate addition by UV-Vis spectrophotometric analysis
- ChEMBL_1755235 (CHEMBL4189995) Inhibition of human ALDH2 using NAD+/propionaldehyde as substrate/cofactor preincubated for 15 mins followed by substrate/cofactor addition measured for 30 mins by fluorescence assay
- Inhibition Assay Standard ALDH2 reaction mixtures contained 150 uM formaldehyde, 2.5 mM NAD+, 10 mM MgCl2 and 10 nM recombinant human ALDH2 in 50 mM Hepes buffer, pH 7.4, 0.01% Tween 20 in a final volume of 50 ul using 384-well plates. After 60 min of pre-incubation of compound with ALDH2 and formaldehyde, the reaction was started by adding NAD+ and the reaction mixture was allowed to proceed for 90 minutes. Activity of the enzyme was determined by monitoring NADH formation using Perkin-Elmer Envision Reader with excitation and emission wavelengths set at 340 and 460 nm, respectively.
- ChEMBL_2348789 Inhibition of human ALDH2 using acetaldehyde as substrate preincubated with enzyme for 15 mins followed by substrate addition in presence of NAD+ and measured for 5 mins by spectrophotometric analysis
- ChEMBL_2057753 (CHEMBL4712754) Allosteric activation of recombinant human full-length ALDH2 (18 to 517 residues) expressed in Escherichia coli using acetaldehyde as substrate measured after 3 hrs in presence of NAD+ by fluorescence assay
- ChEMBL_1659742 (CHEMBL4009354) Competitive inhibition of full length recombinant human ALDH2 expressed in Escherichia coli assessed as reduction in dehydrogenase activity using propionaldehyde as substrate in presence of varying levels NAD+ by Lineweaver-Burk plot analysis
- ChEMBL_1659745 (CHEMBL4009357) Non-competitive inhibition of full length recombinant human ALDH2 expressed in Escherichia coli assessed as reduction in dehydrogenase activity using varying levels of propionaldehyde as substrate in presence of NAD+ by Lineweaver-Burk plot analysis
- ChEMBL_1659744 (CHEMBL4009356) Mixed-type non-competitive inhibition of full length recombinant human ALDH2 expressed in Escherichia coli assessed as reduction in dehydrogenase activity using propionaldehyde as substrate in presence of varying levels NAD+ by Lineweaver-Burk plot analysis
- ChEMBL_1793466 (CHEMBL4265385) Inhibition of N-terminal His6-tagged recombinant human mitochondrial ALDH2 using propionaldehyde as substrate preincubated for 20 mins to 1 hr followed by substrate addition and measured for 5 mins in presence of NADH by fluorescence assay
- ChEMBL_1659722 (CHEMBL4009334) Inhibition of full length recombinant human ALDH2 expressed in Escherichia coli assessed as reduction in dehydrogenase activity by measuring NAD(P)H level preincubated for 2 mins followed by addition of propionaldehyde as substrate in presence of NAD+ by spectrophotometric method
- Inhibitory Effects Against ALDH1a1 and ALDH2 ALDH Assay Protocols: 5 μl of enzyme (150 nM for ALDH1a1 and 200 nM for ALDH2 in reaction buffer) were delivered to assay wells in corning black, 384 well plate. 5 μl of reaction buffer was delivered to ‘no enzyme’ background control wells. Test compounds were prepared in 100% DMSO by serial dilution in 100× of assay concentration. After adding the test compounds, reaction plate was centrifuged briefly in 1200 rpm, then incubated for 20 min at room temperature, to pre-incubate enzyme and compounds. 5 μl of substrate solution (reaction buffer containing 250 μM Acetaldehyde, 500 μM NAD+ for ALDH1a1 and 100 μM Acetaldehyde, 500 μM NAD+ in the for ALDH2) were then delivered to assay wells. Reaction plate was briefly centrifuged and sealed with a plastic film to limit evaporation. After incubation at room temperature for 60 min, 10 μl of detection reagent (15 μg/ml diaphorase, 30 μM resazurin prepared in the degassed reaction buffer) was added. Reaction plate was briefly centrifuged and incubated for 10 minutes at room temperature in the dark. Fluorescent signal from resorufin was measured by Perkin Elmer Envision at Ex/Em=535/590 nm.
- IC50 Determination To determine the IC50 values for CB29 and its analogues, propionaldehyde was used as the substrate for ALDH1A1 and ALDH2 and benzaldehyde was used as the substrate for ALDH3A1. The assays were performed at various concentrations of inhibitors, ranging from 50 nM to 250 uM, following 1 min preincubation. All reactions were initiated by the addition of the aldehyde substrate.
- ALDH Assay Protocol 5 μl of enzyme (150 nM for ALDH1a1 and 200 nM for ALDH2 in reaction buffer) were delivered to assay wells in coming black, 384 well plate. 5 μl of reaction buffer was delivered to ‘no enzyme’ background control wells. Test compounds were prepared in 100% DMSO by serial dilution in 100× of assay concentration. After adding the test compounds, reaction plate was centrifuged briefly in 1200 rpm, then incubated for 20 min at room temperature, to pre-incubate enzyme and compounds. 5 μl of substrate solution (reaction buffer containing 250 μM Acetaldehyde, 500 μM NAD+ for ALDH1a1 and 100 μM Acetaldehyde, 500 μM NAD+ in the for ALDH2) were then delivered to assay wells. Reaction plate was briefly centrifuged and sealed with a plastic film to limit evaporation. After incubation at room temperature for 60 min, 10 μl of detection reagent (15 μg/ml diaphorase, 30 μM resazurin prepared in the degassed reaction buffer) was added. Reaction plate was briefly centrifuged and incubated for 10 minutes at room temperature in the dark. Fluorescent signal from resorufin was measured by Perkin Elmer Envision at Ex/Em=535/590 nm.
- HEK-Blue TLR7 reporter assay Engineered human embryonic kidney blue cells (HEK-Blue TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
- TLR7 Agonist Activity Assay Engineered human embryonic kidney blue cells (HEK-Blue TLR cells; Invivogen) possessing a human TLR7-secreted embryonic alkaline phosphatase (SEAP) reporter transgene were suspended in a non-selective, culture medium (DMEM high-glucose (Invitrogen), supplemented with 10% fetal bovine serum (Sigma)). HEK-Blue TLR7 cells were added to each well of a 384-well tissue-culture plate (15,000 cells per well) and incubated 16-18 h at 37° C., 5% CO2. Compounds (100 nl) were dispensed into wells containing the HEK-Blue TLR cells and the treated cells were incubated at 37° C., 5% CO2. After 18 h treatment ten microliters of freshly-prepared Quanti-Blue reagent (Invivogen) was added to each well, incubated for 30 min (37° C., 5% CO2) and SEAP levels measured using an Envision plate reader (OD=620 nm). The half maximal effective concentration values (EC50; compound concentration which induced a response halfway between the assay baseline and maximum) were calculated.
- Inhibiton Assay IC50 values were determined for CB29 and its analogs using propionaldehyde as the substrate for ALDH1A1 and ALDH2 or benzaldehyde as the substrate for ALDH3A1. The assays were performed on a Beckman DU-640 spectrophotometer at various concentrations of inhibitors ranging from 50 nM to 250 μM following a 1 minute pre-incubation. There was no pre-incubation time-dependence to the inhibition. All reactions were initiated by the addition of the aldehyde substrate. The inhibition curves were fit to the Logistic four parameter IC50 equation using the SigmaPlot (v11, StatSys). We characterized the mode of inhibition using steady-state kinetics through co-variation of inhibitor and substrate concentrations. The steady state kinetic measurements were performed in 100 mM Na2HPO4 buffer, pH 7.5. The reaction mixture contained 10 nM ALDH3A1, varied benzaldehyde (50 μM-800 μM; fixed NADP+, 1.5 mM) and varied inhibitor concentrations. In all cases−including the control reactions lacking inhibitors, the final reaction mixture contained 2% (v/v) DMSO. The reaction was initiated by addition of substrate and the initial rate of product formation was determined on a Beckman DU-640.