A novel method for screening antihyperuricemic drugs by combining aptamer sensor array, exonuclease III-DNA walker and linear discriminant analysis

The lack of a cell-based screening method limits urate-lowering drug development. A novel method combining aptamer sensor array (ASA), exonuclease III (Exo III)- powered 3D DNA walker (DW), and linear discriminant analysis (LDA) was developed for detecting uric acid (UA) in cell lysates, referred to as ASA–Exo III-DW–LDA. Three aptamers (Apts) with different affinities for UA and its structurally similar compound, xanthine (Xan), were used to design the ASA. The combination of ASA and Exo III-DW enabled the detection of UA at the picomolar level, whereas LDA was employed to differentiate UA signals from the mixed signals of UA and Xan. Significantly, Pearson correlation analysis revealed a strong correlation between our method and the ¹⁴C radioactive labeling method for urate anion exchanger 1 (URAT1) inhibitors, with r = 0.9880 for lesinurad and r = 0.9777 for benzbromarone. Using our method, kaempferol was identified as a promising hit compound for inhibiting the URAT1, because of its low half-maximal inhibitory concentration (IC₅₀) (18.96 μM) low toxicity in mouse renal tubular epithelial cells (mTECs), and significant uratelowering effect in hyperuricemic mice at 5 mg/kg. Overall, this method is sensitive, cost-effective and safe, offering a novel strategy for routine urate-lowering drug screening in standard laboratories.