Discovery of bile salt hydrolase inhibitors using an efficient high-throughput screening system

Abstract

The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth.

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).

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.