实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

瘤胃细菌脲酶抑制化合物的筛选及评价

  • 张震宇 ,
  • 赵圣国 ,
  • 郑楠 ,
  • 王加启
展开
  • 1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
    2. 中国农业科学院北京畜牧兽医研究所, 农业部奶及奶制品质量安全控制重点实验室, 北京 100193
张震宇(1996-),男,九江武宁人,硕士研究生,研究方向为反刍动物营养与饲料科学。E-mail:zzy123779@126.com

收稿日期: 2020-01-09

  网络出版日期: 2020-06-16

基金资助

国家自然科学基金项目(31430081);现代农业产业技术体系专项(CARS-36);中国农业科学院农业科技创新工程重大产出科研选题(CAAS-ZDXT2019004);中国农业科学院科技创新工程(ASTIP-IAS12)

Screening and Evaluation of Ruminal Bacterial Urease Inhibitors

  • ZHANG Zhenyu ,
  • ZHAO Shengguo ,
  • ZHENG Nan ,
  • WANG Jiaqi
Expand
  • 1. State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Key Laboratory of Quality and Safety Control for Milk and Dairy Products, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2020-01-09

  Online published: 2020-06-16

摘要

本试验以细菌脲酶结构(PDB_ID 4EP8)为靶标,运用分子对接技术虚拟筛选ChemDiv化合物库,结合瘤胃脲酶活性抑制试验,筛选获得1个新型瘤胃脲酶抑制剂——N-(3,4-二甲氧基苄基)-2-{(4S)-1-[2-(1H-吲哚-3-基)乙基]-2,5-二氧杂咪唑啉-4-烃基}乙酰胺,其半抑制浓度(IC50)值为101.4 μmol/L。瘤胃脲酶动力学试验表明,该化合物为混合性脲酶抑制剂。体外微生物厌氧培养试验表明,该化合物能抑制瘤胃微生物氨氮的生成。分子对接表明,该化合物能与脲酶活性中心内及活性中心外柔性片状区域的一些关键的氨基酸残基稳定结合。该化合物对瘤胃脲酶抑制效果较好,可以作为先导化合物进一步优化,为新型脲酶抑制剂的开发提供了基础。

本文引用格式

张震宇 , 赵圣国 , 郑楠 , 王加启 . 瘤胃细菌脲酶抑制化合物的筛选及评价[J]. 动物营养学报, 2020 , 32(6) : 2850 -2860 . DOI: 10.3969/j.issn.1006-267x.2020.06.045

Abstract

In this study, a novel ruminal bacterial urease inhibitor was obtained by targeting bacterial urease structure (PDB_ID 4EP8) with molecular docking based virtual screening to filter through ChemDiv compound database followed by ruminal urease activity test. This compound was N-(3,4-dimethoxybenzyl)-2-{(4S)-1-[2-(1H-indol-3-yl) ethyl]-2,5-dioxoimidazolidin-4-yl} acetamide, and it’s IC50 value was 101.4 μmol/L. Ruminal urease kinetics experiments showed that the compound was a mix-type urease inhibitor. In vitro anaerobic culture experiments demonstrated that the compound could inhibit ruminal bacteria ammonia nitrogen production. Molecular docking illustrated that the compound could combine with some key amino acid residues in the urease active site and the flexible loop region outside the urease active site. This compound has a fair urease inhibition capacity, and it can be used for lead optimization, which laid a groundwork for further development of novel urease inhibitors.

参考文献

[1] PATRA A K,ASCHENBACH J R.Ureases in the gastrointestinal tracts of ruminant and monogastric animals and their implication in urea-N/ammonia metabolism:a review[J].Journal of Advanced Research,2018,13:39-50.
[2] 李晓姣,赵圣国,郑楠,等.细菌脲酶蛋白复合物及其活化机制[J].生物工程学报,2019,35(2):204-215.
[3] ZAMBELLI B,MUSIANI F,BENINI S,et al.Chemistry of Ni2+ in urease:sensing,trafficking,and catalysis[J].Accounts of Chemical Research,2011,44(7):520-530.  
[4] 赵圣国,王加启,卜登攀,等.细菌尿素酶的生化和分子生物学特点[J].微生物学通报,2008,35(7):1146-1152.
[5] MOBLEY H L,ISLAND M D,HAUSINGER R P.Molecular biology of microbial ureases[J].Microbiological Reviews,1995,59(3):451-480.  
[6] MACOMBER L,MINKARA M S,HAUSINGER R P,et al.Reduction of urease activity by interaction with the flap covering the active site[J].Journal of Chemical Information and Modeling,2015,55(2):354-361.  
[7] PEARSON M A,MICHEL L O,HAUSINGER R P,et al.Structures of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease[J].Biochemistry,1997,36(26):8164-8172.  
[8] KRAJEWSKA B.Ureases Ⅰ.Functional,catalytic and kinetic properties:a review[J].Journal of Molecular Catalysis B:Enzymatic,2009,59(1/2/3):9-21.
[9] CHAN C C L,JONES G A.Effect of acetohydroxamic acid on growth and volatile fatty acid production by rumen bacteria[J].Canadian Journal of Microbiology,1973,19(1):27-33.  
[10] ABBASI M A,HASSAN M,AZIZ-UR-REHMAN R,et al.Synthesis,in vitro and in silico studies of novel potent urease inhibitors:N-[4-({5- benzamides[J].Bioorganic & Medicinal Chemistry,2018,26(13):3791-3804.  
[11] ABDEL-BAKY R M,ALI M A,ABUO-RAHMA G E D A A,et al.Inhibition of urease enzyme production and some other virulence factors expression in Proteus mirabilis by n-acetyl cysteine and dipropyl disulphide[M]//DONELLI G.Advances in microbiology.Cham:Springer,2017:99-113.
[12] AHMED M,QADIR M A,HAMEED A,et al.Azomethines,isoxazole,N-substituted pyrazoles and pyrimidine containing curcumin derivatives:urease inhibition and molecular modeling studies[J].Biochemical and Biophysical Research Communications,2017,490(2):434-440.  
[13] AZIZIAN H,NABATI F,SHARIFI A,et al.Large-scale virtual screening for the identification of new Helicobacter pylori urease inhibitor scaffolds[J].Journal of Molecular Modeling,2012,18(7):2917-2927.  
[14] LIPINSKI C A.Drug-like properties and the causes of poor solubility and poor permeability[J].Journal of Pharmacological and Toxicological Methods,2000,44(1):235-249.  
[15] 赵圣国,王加启,刘开朗,等.荷斯坦奶牛瘤胃微生物脲酶的分离与鉴定[J].畜牧兽医学报,2010,41(6):692-696.
[16] WEATHERBURN M W.Phenol-hypochlorite reaction for determination of ammonia[J].Analytical Chemistry,1967,39(8):971-974.  
[17] 王加启.反刍动物营养学研究方法[M].北京:现代教育出版社,2011.
[18] RASHID U,BATOOL I,WADOOD A,et al.Structure based virtual screening-driven identification of monastrol as a potent urease inhibitor[J].Journal of Molecular Graphics and Modelling,2013,43:47-57.
[19] WARKENTIN M,BADEAU R,HOPKINS J B,et al.Spatial distribution of radiation damage to crystalline proteins at 25-300 K[J].Acta Crystallographica Section D:Biological Crystallography,2012,68(9):1108-1117.  
[20] PEARSON M A,KARPLUS P A.Crystal structure of wild-type klebsiella aerogenes urease at 100K[J/OL].Protein Data Bank.2003,[2020-04-05] https://www.rcsb.org/structure/1EJX
[21] ROBERTS B P,Miller ⅢB R,ROITBERG A E,et al.Wide-open flaps are key to urease activity[J].Journal of the American Chemical Society,2012,134(24):9934-9937.  
[22] TAHA M,ISMAIL N H,IMRAN S,et al.Synthesis of potent urease inhibitors based on disulfide scaffold and their molecular docking studies[J].Bioorganic & Medicinal Chemistry,2015,23(22):7211-7218.  
[23] RAHIM F,ALI M,ULLAH S,et al.Development of bis-thiobarbiturates as successful urease inhibitors and their molecular modeling studies[J].Chinese Chemical Letters,2016,27(5):693-697.  
[24] MUHAMMAD M T,KHAN K M,ARSHIA,et al.Syntheses of 4,6-dihydroxypyrimidine diones,their urease inhibition,in vitro,in silico,and kinetic studies[J].Bioorganic Chemistry,2017,75:317-331.
[25] IFTIKHAR F,ALI Y,KIANI F A,et al.Design,synthesis,in vitro evaluation and docking studies on dihydropyrimidine-based urease inhibitors[J].Bioorganic Chemistry,2017,74:53-65.
[26] HAMEED A,AL-RASHIDA M,UROOS M,et al.A patent update on therapeutic applications of urease inhibitors (2012-2018)[J].Expert Opinion on Therapeutic Patents,2019,29(3):181-189.  
[27] SAIFY Z,SULTANA N,KHAN A,et al.(1H-pyrrolo[2,3-b]pyridine)7-azaindole derivatives and their antiurease,phosphodiesterase and β-glucuronidase activity[J].International Journal of Biochemistry Research & Review,2015,8(1):1-12.
[28] NOREEN M,RASOOL N,GULL Y,et al.Synthesis,density functional theory (DFT),urease inhibition and antimicrobial activities of 5-aryl thiophenes bearing sulphonylacetamide moieties[J].Molecules,2015,20(11):19914-19928.  
[29] PERVEZ H,CHOHAN Z H,RAMZAN M,et al.Synthesis and biological evaluation of some new N4-substituted isatin-3-thiosemicarbazones[J].Journal of Enzyme Inhibition and Medicinal Chemistry,2009,24(2):437-446.  
[30] TAHA M,ISMAIL N H,IMRAN S,et al.Hybrid benzothiazole analogs as antiurease agent:synthesis and molecular docking studies[J].Bioorganic Chemistry,2016,66:80-87.
[31] LUDDEN P A,HARMON D L,HUNTINGTON G B,et al.Influence of the novel urease inhibitor N-(n-butyl) thiophosphoric triamide on ruminant nitrogen metabolism:Ⅱ.Ruminal nitrogen metabolism,diet digestibility,and nitrogen balance in lambs[J].Journal of Animal Science,2000,78(1):188-198.  
[32] CARTER E L,FLUGGA N,BOER J L,et al.Interplay of metal ions and urease[J].Metallomics,2009,1(3):207-221.  
[33] PEARSON M A,PARK I S,SCHALLER R A,et al.Kinetic and structural characterization of urease active site variants[J].Biochemistry,2000,39(29):8575-8584.  
[34] 赵圣国.牛瘤胃脲酶基因多样性分析与酶活性调控[D].博士学位论文.北京:中国农业科学院,2012.
文章导航

/