Fig 1: Lead compounds were screened using surface plasmon resonance (SPR) experiments and kinase activity assessment. (A) SDS–PAGE analysis of recombinant BCKDK-His6-tag protein, with a band size of the target protein equal to 47 kDa. (B) Fitting results obtained from surface plasmon resonance (SPR) experiments for the lead compounds. Dashed lines indicate the positions of Kd values. (C) Structure, custom abbreviated name, Enamine ID, and Kd values of each lead compound. In the compound structures, the red group represents the portion that forms hydrogen bonding interactions with key amino acid residues at the binding site, the blue group represents the segment that occupies the hydrophobic cavity of the site, and the black group serves as a linker. (D) The proliferation inhibition rate of 293T cells by PCAB, PPHN, POAB and BT2 at different concentrations from 0.05 μM to 500 μM. (E) Western blotting results after treating 293T cells with the compounds at a concentration of 40 μM for 48 hours. (F) Quantitative results based on the graph in (D), indicating that both compounds PPHN and POAB significantly inhibited the phosphorylation of BCKDHA by BCKDK, with a stronger inhibitory effect compared to that of BT2.