Figure 1.
Optimization of conditions for high-throughput screening of BSH inhibitors.
(A) Principal of the strategy for HTS screening of BSH inhibitors. (B) Proof of concept experiment to demonstrate the feasibility of the HTS methodology in 96-well plate. Using 200 µl of total reaction volume, BSH inhibitor was added at a final concentration of 0.5 mM in column 1 with 2-fold serial dilution through column 10. KIO3 is used as an example in duplicate rows. The purified BSH was added at final concentration of 8 µg/ml in column wells 1–11. Column 12 served as a negative control with no enzyme and BSH inhibitor added.
Figure 2.
Representative result of high-throughput screening of BSH inhibitors.
(A) Plate layout for screening BSH inhibitors. Pink boxes (columns 3–22) indicate test wells that contain library compounds of interest, BSH, and reaction mix containing substrate. Library compounds were shot into the well bottom using Echo 550/555 and enzyme and reaction mix were added using Multidrop Combi. Controls were added manually to the side wells (columns 1–2 and 23–24): blue boxes indicate activity controls (BSH, reaction mix containing substrate, and solvent DMSO), yellow boxes correspond to inhibition controls (BSH, reaction mix containing substrate, and NaIO3), and white boxes are negative controls with no BSH added but include reaction mix as well as substrate. (B) The HTS results represented by one 384-well plate. Control wells indicate the assay proceeded normally. The wells in columns 3–22 that appeared clear, regardless of alternative color due to compound, and had low absorbance readings were considered hits (putative BSH inhibitors).
Table 1.
Effect of selected HTS hits on BSH activitya.
Figure 3.
Dosing effects of selected BSH inhibitors on BSH activity.
(A) Inhibition of BSH activity by caffeic acid phenethyl ester (CAPE). (B) Inhibition of BSH activity by riboflavin. All assays were performed in triplicate. The procedure is detailed in Materials and Methods.
Figure 4.
Dosing effects of oxytetracycline and roxarsone on BSH activity.
Black bars represent Inhibition of BSH activity by oxytetracyline and white bars by roxarsone. All assays were performed in triplicate. The procedure is detailed in Materials and Methods.
Table 2.
Effect of different classes of antibiotics on BSH activity.