Establishment And Evaluation Of Cell Screening Model For URAT1 Inhibitors Based On Fluorescence Method

The incidence of patients with hyperuricemia and gout is gradually increasing,and there are no new therapeutic drugs on the market.Urate transporter 1?URAT1?inhibitors are very promising as a treatment for hyperuricemia and gout,which have been screened for their in vitro activity using the ¹⁴C-labeled uric acid-labeled substrate.On the other hand,fluorescent probes have been widely used in recent years,especially the use of fluorescent substances as substrates for in vitro screening of compounds has become a development trend and a research hotspot.Therefore,based on economic cost,environmental protection,convenient operation and other factors,the development of a fluorescent detection method using fluorescent small molecules as a substrate for in vitro screening of URAT1 inhibitors is one of the urgent needs to accelerate the development of new URAT1 inhibitors.In this paper,we have mainly completed three tasks:1)Established a HEK293T cell line stably expressing h URAT1,and selected HEK293T/h URAT1.3 cells with the highest protein expression content for methodological establishment;2)6-carboxyfluorescein was selected as a substrate by HEK293T/h URAT1.3 cells and HEK293T-WT cells uptake of 4 kinds of small fluorescent molecules,and kinetic studies indicate that URAT1-mediated 6-carboxyfluorescein transport time-dependent and saturable?Km=239.478?M,Vmax=6.211 pmol/well/min?,based on the above parameters,the in vitro evaluation method of URAT1 inhibitor based on fluorescence method using 6-carboxyfluorescein as substrate was determined.Methodological verification using the three URAT1 inhibitors on the market shows that the methodology is feasible;3)The in vitro activity test of 26 compounds was carried out by this method.The inhibitory activities of compound 4(IC₅₀=36.11±6.56?M)and 9(IC₅₀=10.82±5.50?M)were comparable to those of benzbromarone(IC₅₀=14.25±4.96?M)?P>0.5?.At the same time,the inhibitory activity of compound 9 on XOR is comparable to febuxostat?P>0.5?,suggesting that compound 9 may be a dual-target inhibitor.Using fluorescent small molecules as URAT1 substrates for in vitro activity testing can not only achieve low cost and environmental protection,but also perform fast and high-throughput screening,which promote the development of new URAT1 inhibitors,and accelerate the development of anti-hyperuricemia and anti-gout drugs.