Fig. 6
From: Drugging the intrinsically disordered transactivation domain of androgen receptor
Direct binding of ARTADIs to purified AR-TAD. a Confirmation of AR101-485 isolation by LC-MS and SDS-PAGE. The observed molecular weight (38,500 Da) determined by LC-MS matched the expected theoretical value of the protein construct, with no evidence of cysteine oxidation or other post-translational modifications. A representative SDS-PAGE gel image of the protein used in this analysis is shown in the inset. b Steady-state binding of BU170, BU3-12, and EPI-7386 to AR101-485 measured by MST. The inset compares the calculated apparent dissociation constants KD for compound binding. Data presented as mean ± SEM (n = 3). c SPR sensorgram of BU3-12 binding to His-AR101-485 covalently coupled to an NTA chip. The data fit the kinetic model shown in Fig. 6d. Arrows indicate injection points of BU3-12. The experiment was performed in triplicate, and a representative run is shown. d Reaction scheme of analyte A binding to two transient protein states B1 and B2, applied in the SPR kinetic model. e Calculated kinetic parameters obtained from fitting the SPR kinetic model shown in Fig. 6d. f Component analysis of the fitted SPR model showing accumulation of the second bound state AB2 despite the slower BU3-12 binding to B2 state than to the B1 state. g MS/MS data showing the fragmentation of BU170 modified (top) and BU3-12 modified (middle) GCVPEPGAAVAASK. Fragment ions display localization of the modification on C129. The DMSO control shows only the unmodified form of the peptide (bottom). h A competition binding curve showing inhibition of fluoromone binding to recombinant AR-LBD by dihydrotestosterone (DHT), R1881, bicalutamide (BIC) and enzalutamide (ENZA) which were all positive controls. BU130 and EPI-7386 also competed for the AR-LBD at concentrations similar to those mediating biological activity in the other assays (i.e., see Fig. 1g)
