| BackgroundGlucose transporter 9(GLUT9)is a glucose-facilitated transporter which exists on the basal membrane of proximal tubule epithelial cells.It is a novel uric acid transporter with high-capacity and low-affinity,which plays an important role in maintaining urac acid balance.in human body,GLUT9 is divided into two subtypes,hGLUT9a and hGLUT9 B,which transport uric acid with the same kinetics.Inhibition of GLUT9 will greatly reduce the re-absorption of uric acid and promote uric acid excretion.GLUT9 is an important target of uric acid excretion,with other uric acid-related transporters to maintain the homeostasis of uric acid in vivoObjectiveConstruct a hGLUT9a three-dimensional model for molecular docking to seek effective key sites and inhibit drugs;To construct the recombinant plasmid of hGLUT9b mutant by point mutation of the predicted active site.Construct a cell model for stable expression of hGLUT9a,to provide the experimental basis for the clinical screening of effective uric acid excretion drugs.A cell model of Chinese hamster ovary(CHO-K1),which can express GLUT9,was established by Liposome transfection to validate the drug action,and an effective GLUT9 inhibitor screening model was constructed to verify the active sites and screened potential inhibitors;To study the uric acid lowering effect of drug in hyperuricemia mice.MethodThis project constructs and optimizes the conformation of uric acid transporters by using homology modeling,the reliability of the constructed structure is analyzed using the topology structure feature,the ERRAT function and the Lagrangian method.Subsequently,the combination of uric acid with hGLUT9a was explored by using molecular docking technique and homologous family sequence alignment,and some drug-receptor binding patterns were analyzed in the research and development phase to find the key sites of hGLUT9a.Constructing the corresponding mutant recombinant carrier of the key sites.A slow viral plasmid containing hGLUT9a gene was constructed and used to infect normal cells,and successfully obtain cell lines stably expressing hGLUT9a through puromycin screening.The expression of hGLUT9a mRNA and protein in cell lines was detected by qPCR and western blot.A hGLUT9a stable cell line screening model was established.Subsequently the CHO-K1 cell model expressing GLUT9 was established by Liposome Transfection.And the screen of effective GLUT9 inhibitor and validation of the effect of several key sites,L303A,F397A,W430A,N433A(for hGLUT9b),on uric acid transport were using patch clamp technique.Hyperuricemic mice induced by potassium oxonate were used to screening and evaluation of active compounds(divided into blank control group,hyperuricemia model group,3170 group,3170-M group,HS062#high or low dose group,benzbromarone group and allopurinol group,determination of uric acid(UA)levels in serum and urine using a test kit).Result:The three-dimensional model of hGLUT9a was successfully constructed,ERRAT and Procheck scores were 94.9495 and 92.7 respectively.By molecular docking with small molecular compound to hGLU9a analog pocket,the binding ability is 3170-M>3170>PB>BM>HS062#>apigenin,in turn.The key sites were identified by molecular docking with uric acid and inhibitor.Several sites L303A,F397A,W430A and N433A of hGULT9b were predicted and successfully mutated.Construct the stable strain CHO-hGLUT9a,the expression of hGLUT9a mRNA was 1.03 million times of the control group.The patch clamp verifies that GLUT9 has an effect on the transport of uric acid in CHO cells with Km value 137.7±37.2μM.The effects of drug 3170(100 μM),3170-M(100μM),HS062#(100 μM),apigenin(100 μM),BM(100 μM)and PB(1mM)on mGLUT9 were verified by electrophysiological model,and the inhibitory rate was 13.7±2.4%、88.7±2.8%、55.7±3.6%、9.5±0.5%,92.1±1.4%,80.9±7.1%respectively,mutant L303A,W430A and N433A can also reduce the effect of hGLUT9b transport of uric acid.At the same time,HS062#and 3170-M significantly reduced the level of uric acid in hyperuricemia mice induced by potassium oxychloride.Among them,HS062#can significantly increase urine uric acid level.ConclusionThe three-dimensional model of hGLUT9a is successfully modeled in this experiment,the binding capacity of different compounds and the related active sites are predicted.The Electrophysiological cell model,which can be used to screen the effective inhibitor of hGLUT9 and to determine several amino acid sites that affect the GLUT9 transport of uric acid.In addition,using the method of lentivirus infection,a cell line that can express hGLUT9a steadily is constructed,which can be used to screen effective hGLUT9a inhibitors and provide an experimental basis for the clinical search of effective uric acid excretion drugs.HS062#and 3170-M can effectively reduce uric acid level in vitro and in vivo,and HS062#and 3170-M had the potential to effectively reduce uric acid compounds. |
