FP competitive binding assays were performed following the method described previously . All the measurements were taken on a PHERAstar (BMG LABTECH) plate reader installed with a FP filter that sets excitation and emission wavelengths at 485 nm and 520 nM separately. Each well of 384-well plate (Coring 3575) contains 10 nM VCB protein, 5 nM FAM-lableled HIF-1α peptide (FAM-DEALAHypYIPMDDDFQLRSF, "JC9"), and decreasing concentrations of testing compounds (14 concentrations with 2-fold serial dilution starting from 250 µM) in FP assay buffer (100 mM Bis-Tris propane, 100 mM NaCl, 1 mM TCEP, pH 7) with a final DMSO concentration of 5%. The control wells containing the VCB and JC9 with no compound are set as the maximum signals (zero displacement). And the control wells containing JC9 in the absence of protein are set as the minimum signals. Control values were used to obtain the percentage of displacement which was ploted against Log [Compound]. Average IC50 values were determined for each titration using nonlinear regression analysis with GraphPad Prism (v.9.3.1). The Ki values were back-calculated from the Kd of JC9 (1.5 nM -3.4 nM) and the fitted IC50 values, as described previously .