This study was designed to develop hypothetical inhibition mechanism of novel UT-B inhibitor and exploit novel compounds with UT-B inhibitory activity and to obtain promising lead compounds. We integrated cell based h...This study was designed to develop hypothetical inhibition mechanism of novel UT-B inhibitor and exploit novel compounds with UT-B inhibitory activity and to obtain promising lead compounds. We integrated cell based high throughput screening and in silico method to identify an undiscovered UT-B inhibitor binding site and proposed the mechanism of UT-B inhibitor in cross-species. We employed high-throughput screening using an erythrocyte os- motic lysis assay and identified 4 compounds PU21, PU168, PU468 and PU474 with UT-B inhibitory activity in vitro from 2319 hits. 16 compounds with UT-B inhibitory activity were screened by erythrocyte osmotic lysis assay from 60 analogues of PU21. PU14, one of 16 compounds exhibited potential inhibition activity in human, rabbit, rat, mouse in vitro and pharmacological diuresis activity in vivo. Based on the physiological data, we built a compu- tational mode of human UT-B by homology modeling. The putative UT-B binding site was identified by structure- based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional differences under inhibitors treated and untreated conditions were simulated by Molecular Dynamics （MD）. The UT-B inhibitor binding site analysis and validation provide structure basses for lead identification and optimization.展开更多
Urea nitrogen secreted from blood to rumen is a crucial factor shaping the symbiotic relationship between host ruminants and their microbial populations.Passage of urea across rumen epithelia is facilitated by urea tr...Urea nitrogen secreted from blood to rumen is a crucial factor shaping the symbiotic relationship between host ruminants and their microbial populations.Passage of urea across rumen epithelia is facilitated by urea transporter B(UT-B),but the long-term regulation of these proteins remains unclear.As ruminal function develops over a period of months,the developing rumen is an excellent model with which to investigate this regulation.Using rumen epithelium samples of calves from birth to 96 d of age,this study performed immunolocalization studies to localize and semi-quantify UT-B protein development.As expected,preliminary experiments confirmed that ruminal monocarboxylate transporter 1(MCT1)short chain fatty acid transporter protein abundance increased with age(P<0.01,n=4).Further investigation revealed that ruminal UT-B was present in the first few weeks of life and initially detected in the basolateral membrane of stratum basale cells.Over the next 2 months,UT-B staining spread to other epithelial layers and semi-quantification indicated that UT-B abundance significantly increased with age(P<0.01,n=4 or 6).These changes were in line with the development of rumen function after the advent of solid feed intake and weaning,exhibiting a similar pattern to both MCT1 transporters and papillae growth.This study therefore confirmed age-dependent changes of in situ ruminal UT-B protein,adding to our understanding of the long-term regulation of ruminal urea transporters.展开更多
The symbiotic relationship between the host and the rumen microbiome plays a crucial role in ruminant physiology.One of the most important processes enabling this relationship is urea nitrogen salvaging(UNS).This proc...The symbiotic relationship between the host and the rumen microbiome plays a crucial role in ruminant physiology.One of the most important processes enabling this relationship is urea nitrogen salvaging(UNS).This process is important for both maintaining ruminant nitrogen balance and supporting production of their major energy supply,bacterially-derived short chain fatty acids(SCFA).The key step in UNS is the trans-epithelial movement of urea across the ruminal wall and this is a highly regulated process.At the molecular level,the key transport route is via the facilitative urea transporter-B2,localized to ruminal papillae epithelial layers.Additional urea transport through aquaporins(AQP),such as AQP3,is now also viewed as important.Long-term regulation of these ruminal urea transport proteins appears to mainly involve dietary fermentable carbohydrates;whereas,transepithelial urea transport is finely regulated by local conditions,such as CO_(2) levels,pH and SCFA concentration.Although the key principles of ruminal urea transport physiology are now understood,there remains much that is unknown regarding the regulatory pathways.One reason for this is the limited number of techniques currently used in many studies in the field.Therefore,future research in this area that combines a greater range of techniques could facilitate improvements to livestock efficiency,and potentially,reductions in the levels of waste nitrogen entering the environment.展开更多
文摘This study was designed to develop hypothetical inhibition mechanism of novel UT-B inhibitor and exploit novel compounds with UT-B inhibitory activity and to obtain promising lead compounds. We integrated cell based high throughput screening and in silico method to identify an undiscovered UT-B inhibitor binding site and proposed the mechanism of UT-B inhibitor in cross-species. We employed high-throughput screening using an erythrocyte os- motic lysis assay and identified 4 compounds PU21, PU168, PU468 and PU474 with UT-B inhibitory activity in vitro from 2319 hits. 16 compounds with UT-B inhibitory activity were screened by erythrocyte osmotic lysis assay from 60 analogues of PU21. PU14, one of 16 compounds exhibited potential inhibition activity in human, rabbit, rat, mouse in vitro and pharmacological diuresis activity in vivo. Based on the physiological data, we built a compu- tational mode of human UT-B by homology modeling. The putative UT-B binding site was identified by structure- based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional differences under inhibitors treated and untreated conditions were simulated by Molecular Dynamics （MD）. The UT-B inhibitor binding site analysis and validation provide structure basses for lead identification and optimization.
基金The authors would like to thank Dr.Carl Ng,Ms.Frances Downey,Dr.Carlotta Sacchi and Dr.Alan Farrell for their technical assistance throughout this study.The authors are also grateful to the scholarship funding(CZ)from The China Scholarship Council and University College Dublin.
文摘Urea nitrogen secreted from blood to rumen is a crucial factor shaping the symbiotic relationship between host ruminants and their microbial populations.Passage of urea across rumen epithelia is facilitated by urea transporter B(UT-B),but the long-term regulation of these proteins remains unclear.As ruminal function develops over a period of months,the developing rumen is an excellent model with which to investigate this regulation.Using rumen epithelium samples of calves from birth to 96 d of age,this study performed immunolocalization studies to localize and semi-quantify UT-B protein development.As expected,preliminary experiments confirmed that ruminal monocarboxylate transporter 1(MCT1)short chain fatty acid transporter protein abundance increased with age(P<0.01,n=4).Further investigation revealed that ruminal UT-B was present in the first few weeks of life and initially detected in the basolateral membrane of stratum basale cells.Over the next 2 months,UT-B staining spread to other epithelial layers and semi-quantification indicated that UT-B abundance significantly increased with age(P<0.01,n=4 or 6).These changes were in line with the development of rumen function after the advent of solid feed intake and weaning,exhibiting a similar pattern to both MCT1 transporters and papillae growth.This study therefore confirmed age-dependent changes of in situ ruminal UT-B protein,adding to our understanding of the long-term regulation of ruminal urea transporters.
基金China Postdoctoral Science Foundation(2021M701532)National Natural Science Foundation of China(32072757).
文摘The symbiotic relationship between the host and the rumen microbiome plays a crucial role in ruminant physiology.One of the most important processes enabling this relationship is urea nitrogen salvaging(UNS).This process is important for both maintaining ruminant nitrogen balance and supporting production of their major energy supply,bacterially-derived short chain fatty acids(SCFA).The key step in UNS is the trans-epithelial movement of urea across the ruminal wall and this is a highly regulated process.At the molecular level,the key transport route is via the facilitative urea transporter-B2,localized to ruminal papillae epithelial layers.Additional urea transport through aquaporins(AQP),such as AQP3,is now also viewed as important.Long-term regulation of these ruminal urea transport proteins appears to mainly involve dietary fermentable carbohydrates;whereas,transepithelial urea transport is finely regulated by local conditions,such as CO_(2) levels,pH and SCFA concentration.Although the key principles of ruminal urea transport physiology are now understood,there remains much that is unknown regarding the regulatory pathways.One reason for this is the limited number of techniques currently used in many studies in the field.Therefore,future research in this area that combines a greater range of techniques could facilitate improvements to livestock efficiency,and potentially,reductions in the levels of waste nitrogen entering the environment.
