Extended Data Fig. 1: Related to Fig. 1.
From: PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase
a, Model of bifunctional degrader molecules (PROTACs) of AURORA-A. b, Ribbon model of AURORA-A and structural formula of alisertib. The structure of AURORA-A bound to MLN8054 (PDB: 2X81)33 identified a solvent-exposed carboxyl group on the compound to which linkers could be attached by amide bonds. c, Degrader and ligand structures and their effects on AURORA-A-NanoLuc target engagement. HEK293 cells transfected with an AURORA-A-NanoLuc fusion construct were incubated with various concentrations of the AURORA-A degrader molecules or their components together with an energy transfer probe for 2 h and luminescence was measured. EC50 values were calculated by assuming a sigmoidal dose–response relationship (four parameters). The graph shows one of the two biological replicates. The graphs display the profile of each compound for the assay as shown in Fig. 1a. d, Bar diagram showing the binding of degrader molecules to AURORA-A. AURORA-A was incubated with degrader molecules and binding was analyzed by thermal shift assays and compared to alisertib. Pomalidomide and VHL-binding moieties were used as controls. Bars represent mean ± s.d. for n = 4 replicates (n = 3 for JB161) e, Immunoblot of AURORA-A. AURORA-A and AURORA-B were depleted in IMR5 cells by siRNA and AURORA-A levels were compared to cells treated with JB170 (1 µM) or alisertib (1 µM) and to control cells (Ctr). f, Immunoblot of AURORA-A. MV4–11 cells were treated with 0.1 µM JB170 for the indicated times and AURORA-A levels were compared to control cells. g, Immunoblot of AURORA-A. MV4–11 cells were treated with 0.1 µM JB158 for the indicated times, and AURORA-A levels were compared to control cells.
