Screening And Evaluation Of Novel Rumen Bacterial Urease Inhibitiors

In ruminants,bacterial ureases hydrolyse urea,causing the excessive ammonia producing rate.The producing rate of ammonia is much faster than the volatile fatty acids?carbon scaffolds?.Consequently,the produced ammonia cannot be used timely in the rumen and discharged to the environment.Causing not only decrease in the urea utilization efficiency among ruminant animals,but also inducing ammonia toxication in animals and resulting in environmental pollution.Therefore,reducing bacterial urease activity has research significance in the fields of ruminant nutrition.In this study,the three-dimensional structure of predominant urease was obtained by homology modeling.The screening procedure was constructed by molecular docking based virtual screening technology.Compounds with ruminal bacterial urease inhihitory effect were obtained from compound library and their IC₅₀ values were measured by urease inhibition experiment.Enzyme kinetics,rumen bacteria anaerobic culture,and Caco-2 cell culture were used to evaluate the inhibitory types of compounds to reveal their effects on microbial nitrogen metabolism,compound stability,and cytotoxicity.Molecular docking was also employed to study the binding mode between compounds and urease.The effective fragments of compounds were revealed by receptor-ligand pharmacophore.To obtain the structure of ruminal bacterial urease and screening for candidate compounds with potential urease inhibitory effect,we use the NCBI blastp algorithm to search for urease structures with high similarity and intergrity in the PDB protein database as a template protein.The SWISS-MODEL server was used to construct the three-dimensional structure of the targeted gene of ruminal bacterial urease.SAVES 5.0 was used to analyze the rationality of the rumen urease protein structure.The results showed that the similarity between the target sequence and the template urease protein?PDB?ID 4EP8?was 69.56%.Using this protein as a template to model the rumen urease structure,the GMQE and QMEAN values of the rumen urease protein structure were 0.85 and-0.37,respectively;the ERRAT value was 96.9371,and the Verify 3D value was 89.38%;For amino acids among them,424 amino acid residues?89.6%?fell in the most favorable region,44 amino acid residues?9.3%?fell in the favorable region,and 1 amino acid residue?0.2%?fell in the disallowed region,and The amino acids in the urease active site were in the favorable region.Therefore,the structure of ruminal bacterial urease is reasonable.The ChemDiv compound library was virtual screened using the template urease and ruminal bacterial urease structures as targets respectively.a total of 40 candidate small molecule compounds with potentital rumen urease inhibitory effect were obtained.To evaluate urease inhibitory effect of small molecule compounds,the inhibiton rate of the compounds were determined by ruminal bacterial urese inhibition experiment,and the inhibition type of the compound was evaluated by enzyme kinetics.The results showed that compound 6238-0047 and compound Y041-7877 have higher inhibitory effect on ruminal bacterial urease than other candidate compounds,and the IC50 values were 65.86?mol/L and 101.4?mol/L respectively.Under different concentrations of compound 6238-0047 and compound Y041-7877,the ratio of the enzyme kinetic curve parameter Vmax/Km changes.Therefore,both compounds are mixed-type urease inhibitor.To further evaluate small molecule compounds.Rumen bacteria anaerobic culture and Caco-2 cell culture were used to evaluate the ability of compounds to inhibit ammonia nitrogen production and urea degradation of rumen bacteria,the stability of compounds,and the adaptation of rumen bacteria to the compounds and cytotoxicity of the compound.The results showed that compound 6238-0047 and compound Y041-7877 could inhibit the production of ammonia nitrogen in the anaerobic culture throughout the six generations of culturing with no microbial adaptation.Compound 6238-0047 can inhibit the degradation of urea in the anaerobic culture of rumen bacteria.The 24 h degradation rate of compound 6238-0047 in the first and sixth generations was less than that of the standard inhibitor acetohydroxmic acid,indicating that compound 6238-0047 is more stable.At the same time,the cytotoxicity evaluation using Caco-2 as a cell model showed that it had no cytotoxicity at a concentration of 100?mol/L.To obtain the binding mode of small molecule compounds with urease structures and the effective urease inhibitory chemical structures.Compound 6238-0047 and compound Y041-7877 were docked with corresponding urease structures respectively.The effective inhibitory structures of compounds6238-0047 and Y041-7877 were analyzed by"receptor-ligand-pharmacophore".The results indicated that compound 6238-0047 forms hydrogen bonds with Arg335 and Asp220 respectively,and the carboxyl group of the compounds also forms two hydrogen bonds with Ala362 and Asp359.In addition,the pyrrole ring structure in the middle of compound 6238-0047 can form a Pi-Pi stacking force with the His319 of the ruminal bacterial urease protein active site and interact with the Cys318 in the flexible loop region outside the rumen urease active site with Pi-Alkyl Force.The methoxybenzene structure of compound 6238-0047 can also form a Pi-Pi stacking force with the His319.Compound Y041-7877 can form a hydrogen bond with His246 and His321,and its benzene ring structure can form a Pi-Anion force with Asp221 and Arg336.In addition,the heterocyclic structure at the tail of compound Y041-7877 can form Pi-Sigma and Pi-Pi stacked forces with His320 and Phe332.Pharmacophore analysis revealed that compound 6238-0047 has two hydrogen bond acceptors?HBA?,one hydrophobic group?HY?,two hydrogen bond donors?HBD?,and one aromatic ring?RA?.Compound Y041-7877 has two hydrogen bond acceptors?HBA?,one hydrophobic group?HY?,one hydrogen bond donor?HBD?,and two aromatic rings?RA?.The diversity of ureolytic bacterial community in the rumen of ruminant animals and the structural differences between ureases of different bacteria origin makes the nitrogen utilization process of ruminal microbiota a particularly hard process to regulate.Using the ruminal predominant urease gene as a target.The homology modeling was used to obtain the three dementional structure of the target sequence,and a series of screening and evaluation models were constructed based on molecular docking against the ruminal bacterial urease structure.Small molecule compound 6238-0047 and compound Y041-7877 were screened from the ChemDiv compound library with effective inhibition capacity on ruminal microbiota nitrogen metabolism.These compounds could serve as leads for further optimization.This study serve as a paradigm for regulating the ruminal microbial activity and a technical approach to reduce urea degradation rate in the rumen to improve the utilization efficiency and save the dietary protein resources for the ruminant animals.