Fig. 1: Identification and characterization of iBAP-II as a next-generation BAP1 inhibitor.

A The core structure of iBAP compound. B 44 new analogs of iBAP were designed and synthesized based on the core structure of iBAP. The inhibitory effect of all the compounds were determined by Ub-AMC assay. Commercially available iBAP (iBAP: purity >90%) and in-house synthesized iBAP (iBAP’: purity >95%) were used as positive controls, DMSO was used as the negative control, n = 2. C Structures of Analogs #20, #32, and #42. D) Dose-dependent inhibition by iBAP and three analogs from C were determined by Ub-AMC assay, n = 3. E Dose-dependent inhibition of BAP1 and UCHL5 activity by iBAP and Analog #42 by Ub-AMC assay, n = 3. The IC50 for each condition was provided. F The inhibitory effects of different DUBs’ activity by Analog #42 (1 μg/ml) using Ub-AMC assay, n = 3. G NCI-H1963 cells were treated with iBAP-II (10 μM) for 24 h, the protein levels of H2AK119Ub were determined by western blot (upper panel), and quantified by ImageJ (lower panel). Nuclear extracts of SCLC cells were incubated at different temperatures for 5 min in the presence of either DMSO or iBAP-II (1 μg/ml). The protein levels of BAP1 in the soluble fraction were determined by western blot, n = 2 (H) and quantified by ImageJ (I). J Illustrative molecular docking of iBAP and iBAP-II into the three-dimensional X-ray structure of Calypso (the BAP1 ortholog from Drosophila melanogaster, PDB: 6HGC) was carried out using the Discovery Studio (version 4.5) as implemented through the graphical user interface CDOCKER protocol. K The CDOCKER energy and CDOCKER interaction energies for the top ranked binding poses of iBAP/iBAP-II obtained after conducting the molecular docking studies on Calypso (PDB: 6HGC) using the CDOCKER algorithm in Discovery Studio 4.5.