Fig 1: Complex 1a disrupts the VHL–HIF-1a interaction by selectively engaging VHL.a Complex 1a inhibits the interaction of VHL–HIF-1a in cellulo. HEK293 cells were treated with vehicle DMSO (1% for 2 h), 1a (0.3, 1, 3, 6 and 9 µM for 2 h), VH298 (100 µM for 2 h), MG132 (20 µM for 3 h), or FG-4592 (100 µM for 2 h) before lysis. Cell lysates were collected and incubated with VHL antibody overnight at 4 °C. The proteins were immunoprecipitated using agarose beads. The levels of hydroxy-HIF-1a co-precipitated with VHL were detected using a hydroxy-HIF-1a antibody. b Cellular thermal shift assays to monitor cellular target engagement of VHL and HIF-1a. HEK293 cell lysates were treated with 1a (3 µM) at room temperature for 30 min and then heated at different temperatures ranging from 48 to 68 °C for 5 min. The protein samples were collected and detected by Western blotting using either VHL or PHD2 antibodies. c Fluorescence-based protein thermal shift assay of VBC in the presence or absence of VH298 (100 µM) and complex 1a (100 µM). d ITC titration of complex 1a (300 µM) into recombinant VHL protein (30 µM). ITC experiments were carried out in a MicroCal PEAQ-ITC isothermal titration calorimeter (Malvern Panalytical). e A competitive fluorescence polarization binding assay was performed to evaluate displacement of a fluorescent peptide (FAM-DEALAHyp-YIPD) binding to VBC (Kd = 421.50 ± 65.23 nM) by complex 1a. The IC50 of complex 1a (Kd = 2.06 µM) was determined to be 3.79 µM in the presence of 125 nM the fluorescent peptide and 450 nM VBC using a four-parameter logistic equation. f BLI kinetic analysis of the interaction between complex 1a and VBC. VBC was surface-immobilized to Ni-NTA biosensors. BLI sensorgrams showing the binding of complex 1a to surface-immobilized VBC. The Kd values for a 1:1 interaction were calculated from the kinetic fit (Kd = 6.74 ± 0.19 µM) and steady-state fit (Kd = 5.80 ± 0.64 µM), respectively. The Ni-NTA biosensor tips coated with His-tagged VBC were dipped in increasing concentrations of 1a (0.78, 1.56, 3.13, 6.25, and 12.5 µM) to measure the binding affinity of 1a to VBC (kon = 5.22 × 102 M-1 s-1) and subsequently moved to wells containing buffer to measure dissociation rates (koff = 3.52 × 10-2 s-1). Data are expressed as means ± SD (n = 3 independent experiments for figures a and b, samples for figures c and e), P values were calculated using a two-sided t-test. *P < 0.05, **P < 0.01 vs. DMSO group. Source data are provided as a Source Data file.