Journal: The Journal of Biological Chemistry
Article Title: Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation
doi: 10.1016/j.jbc.2022.102469
Figure Lengend Snippet: High-throughput screen (HTS) for the discovery of chemical compounds modulating human zDHHC2 autoacylation activity: TR-FRET-based assay. A , experimental design of the HTS. HA-tagged zDHHC2 enzyme expressed at the surface of lipoparticles was incubated for 15 min at room temperature (RT) with NBD-tagged palmitoyl-CoA and an anti-HA antibody coupled to terbium (Tb) cryptate. Autoacylation of zDHHC2 on its catalytic cysteine brings closer the FRET donor Tb cryptate bound to the anti-HA antibody to the NBD fluorophore coupled to the palmitate and triggers FRET that can be followed over time (time-resolved FRET [TR-FRET]). B , TR-FRET was measured with increasing concentrations of NBD-palmitoyl-CoA (NBD-C16:0-CoA) and either WT zDHHC2 ( left panel ) or catalytically dead zDHHC2 C157S ( right panel ) over a period of 35 min. C , experimental conditions of the HTS screen. About 350,000 compounds were incubated individually in 1536-well plates at a concentration of 4.2 μM with zDHHC2 lipoparticles (42 μg/ml stock) for a few minutes before the addition of a mix containing the Tb cryptate–tagged anti-HA antibody (1:200 dilution) and 1 μM of NBD-palmitoyl-CoA (NBD-C16:0-CoA). TR-FRET was measured after a 15 min incubation at room temperature on a Biotek Synergy Neo apparatus. Results are given as a ratio between the FRET signal measured over a background baseline. D , histogram representation of ONO library screen showing the percentage of activity of zDHHC2 (as measured by TR-FRET) in the presence of each compound. The graph represents the number of compounds that are either activating (0 to 100) or inhibiting (0 to −100) zDHHC2 activity. HA, hemagglutinin; NBD, nitrobenzoxadiazole; TR-FRET, time-resolved FRET; zDHHC, zinc finger DHHC domain–containing protein.
Article Snippet: Palmitoyl-CoA-NBD (catalog no.: 810705) was purchased from Avanti Polar Lipids.
Techniques: High Throughput Screening Assay, Activity Assay, Incubation, Concentration Assay
![High-throughput screen (HTS) for the discovery of chemical compounds modulating human zDHHC2 autoacylation activity: TR-FRET-based assay. A , experimental design of the HTS. HA-tagged zDHHC2 enzyme expressed at the surface of lipoparticles was incubated for 15 min at room temperature (RT) with <t>NBD-tagged</t> <t>palmitoyl-CoA</t> and an anti-HA antibody coupled to terbium (Tb) cryptate. Autoacylation of zDHHC2 on its catalytic cysteine brings closer the FRET donor Tb cryptate bound to the anti-HA antibody to the NBD fluorophore coupled to the palmitate and triggers FRET that can be followed over time (time-resolved FRET [TR-FRET]). B , TR-FRET was measured with increasing concentrations of NBD-palmitoyl-CoA (NBD-C16:0-CoA) and either WT zDHHC2 ( left panel ) or catalytically dead zDHHC2 C157S ( right panel ) over a period of 35 min. C , experimental conditions of the HTS screen. About 350,000 compounds were incubated individually in 1536-well plates at a concentration of 4.2 μM with zDHHC2 lipoparticles (42 μg/ml stock) for a few minutes before the addition of a mix containing the Tb cryptate–tagged anti-HA antibody (1:200 dilution) and 1 μM of NBD-palmitoyl-CoA (NBD-C16:0-CoA). TR-FRET was measured after a 15 min incubation at room temperature on a Biotek Synergy Neo apparatus. Results are given as a ratio between the FRET signal measured over a background baseline. D , histogram representation of ONO library screen showing the percentage of activity of zDHHC2 (as measured by TR-FRET) in the presence of each compound. The graph represents the number of compounds that are either activating (0 to 100) or inhibiting (0 to −100) zDHHC2 activity. HA, hemagglutinin; NBD, nitrobenzoxadiazole; TR-FRET, time-resolved FRET; zDHHC, zinc finger DHHC domain–containing protein.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8053/pmc09558053/pmc09558053__gr1.jpg)
