Department of Animal Science, University of Tennessee, Knoxville, Tennessee, United States of America.
PLoS One. 2014 Jan 14;9(1):e85344. doi: 10.1371/journal.pone.0085344. eCollection 2014.
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.
全球范围内限制抗生素生长促进剂（AGP）在动物生产中的使用这一趋势，需要开发有效的替代品来维持食用动物的生产力和可持续性。先前的研究表明，抑制胆盐水解酶（BSH）是一种肠道细菌产生的酶，它对宿主脂肪消化和利用有负面影响，是促进动物生长性能的一种很有前途的方法。为了实现开发新型 AGP 替代品的长期目标，本研究开发了一种快速、方便的高通量筛选（HTS）系统，并成功用于鉴定 BSH 抑制剂。在鸡唾液乳杆菌来源的高纯度 BSH 的辅助下，我们优化了各种筛选条件（例如 BSH 浓度、反应缓冲液 pH 值、孵育温度和时间、底物类型和浓度），并建立了基于沉淀的筛选方法，使用 96 孔或 384 孔微孔板来鉴定 BSH 抑制剂。使用由 2,240 种具有生物活性和结构多样性的化合物组成的小型化合物文库进行了初步 HTS。在 107 个命中化合物中，选择了几种有前途的强效 BSH 抑制剂（例如核黄素和咖啡酸苯乙酯），并通过标准 BSH 活性测定进行了验证。有趣的是，HTS 还鉴定了一组抗生素作为 BSH 抑制剂；特别是，各种四环素类抗生素和罗硝唑，一种广泛使用的 AGP，已被证明对 BSH 具有很强的抑制作用。总的来说，本研究开发了一种高效的 HTS 系统，并鉴定了几种具有替代 AGP 潜力的 BSH 抑制剂。此外，这项研究的结果还提示了 AGP 促进动物生长的一种新作用模式。
