| BackgroundHyperuricemia is a kind of metabolic disease and causes great harm and injury to the human body.However,there is no safe and effective urate-lowering drugs in clinic.URAT1,an organic anion transporter,is responsible for 90%of uric acid reabsorption in the kidney.URAT1 inhibitors are hot spots in the research and development of urate-lowering drugs.URAT1 has no crystal structure at present,and the molecular mechanism of uric acid transport and inhibitors binding of URAT1 remain unknown,which greatly limited the development of selective URAT1 inhibitors.Objective1.To clarify the molecular mechanism of uric acid transport of URAT1,the binding mode and structure-activity relationship of URAT1 inhibitors,and to provide insights for the design of selective URAT1 inhibitors.2.To evaluate the pharmacology and toxicity of the synthesized URAT1 inhibitors,and to obtain safe and effective urate-lowering compounds.Method1.The three-dimensional structure of URAT1 was obtained by homology modeling.Furthermore,molecular dynamic simulation was used to optimize the structure.The binding sites of uric acid and inhibitors with URAT1 were determined by molecular docking,sequence alignment and site-directed mutagenesis.2.HQSAR and topomer CoMFA models were established to analyze the structure-activity relationship of the inhibitors,and molecular docking was used to explore the interactions of compounds with URAT1.Compounds were further modified according to the analysis results.3.The inhibitory effects of the compounds on URAT1,OAT1,GLUT9 and ABCG2 were evaluated by constructing over-expression cell models.Potassium oxonate and hypoxanthine were used to induce hyperuricemia mouse model to evaluate the urate-lowering effects of compounds.The cytotoxicity of the compounds was determined by MTT assay.The liver/kidney toxicity of the compounds was detected by detecting the levels of ALT/AST and CR/BUN in mice.The hERG toxicity of the compounds was evaluated by patch clamp electrophysiology.The hepatotoxicity of benzbromarone analogs was determined by detecting GSH levels.Results1.Molecular mechanism of uric acid transport and inhibitors binding of URAT1Results of molecular dynamics simulation showed that the RMSF values of two loop regions of URAT1(L355-F365 of TMD7 and G483-R487 of TMD11)fluctuated greatly.Mutagenesis and kinetic experiments indicated these two regions are involved in uric acid transport and the structural changes of URAT1.S35,F241,H245,R477,R487,F241 and R487 were identified as the potential binding sites of uric acid and inhibitors.R477 and K393 are highly conserved,and their acid-base and/or charge properties are closely related to uric acid transport of URAT1.2.The contribution of the flexible linker of URAT1 inhibitorsThe results of HQSAR and topomer CoMFA analysis showed that the linkers between the aromatic groups of Verinurad and benzbromarone analogs had positive contributions to the activity.In addition,organic anion groups also played an important role in the inhibitory activity.The docking results indicated that the linkers and anion groups could provide additional hydrogen bond to enhance the binding of compounds with URAT1.3.Pharmacological evaluation of Verinurad and benzbromarone analogs36 verinurad analogs synthesized by introducing different linkers were evaluated for pharmacology and toxicity.Among them,KPH2f exhibited the strongest inhibitory effect on URAT1 with an IC50 value of 0.24 μM.KPH2f exhibited similar urate-lowering and stronger uricosuric effects to Verinurad in hyperuricemia mice.The pharmacokinetic results showed that the half-life of KPH2f was 5.14±1.36 h and the absolute bioavailability of KPH2f was 30.13%±3.24%.The MTT and hERG results indicated that KPH2f has no obvious cytotoxicity and heart toxicity.Results of serum ALT/AST and CR/BUN indicated that KPH2f had no obvious liver and kidney toxicity.24 benzbromarone analogs synthesized by adding anion group were evaluated for pharmacology and safety.Among them,JNS4 exhibited the most strongly URAT1 inhibitory activity with and IC50 of 0.83 μM.JNS4 exhibited stronger selective inhibition effect of URAT1 than benzbromarone.Compared with benzbromarone,JNS4 showed stronger urate-lowering effect in hyperuricemia mouse model.The pharmacokinetic results showed that the absolute bioavailability of JNS4 in rats was 55.28%.The MTT and hERG results indicated that JNS4 had no obvious cytotoxicity and heart toxicity.Results of serum ALT/AST and CR/BUN and HE staining showed that KPH2f had no obvious liver and kidney toxicity.Conclusion1.L355-F365 in TMD7 and G483-R487 in TMD11 of URAT1 are involved in uric acid transport and structural change.2.S35,F241,H245,R477,R487 and F241 are possible binding sites of URAT1 with uric acid and inhibitor.The acid-base(charge properties)of R477 and K393 are essential to uric acid transport.3.The flexible linkers and organic anion groups of URAT1 inhibitors have positive effects on the activity.4.The inhibitory effect of KPH2f on URAT1 was similar to that of Verinurad in vitro.KPH2f exhibited similar urate-lowering activity and pharmacokinetic properties as Verinurad in vivo.KPH2f had no obvious liver,kidney and heart toxicity.5.Compound JNS4 showed a stronger selective inhibition effect on URAT1.Compared with benzbromarone,JNS4 exhibited stronger urate-lowering effect,better pharmacokinetic properties,and no obvious liver,kidney and heart toxicity. |
