Reevaluation of bromodomain ligands targeting BAZ2A

Abstract

BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands.

Keywords: BAZ2 bromodomain; prostate cancer; protein crystallography; small molecule inhibitors.

FIGURE 1

Effect of GSK2801 and BAZ2‐ICR on PCa cells. Each point represents the mean ± SD of 3–5 independent replicates, depending on the cell lines and the treatment. For each replicate, three wells/conditions were seeded. Lines are obtained using the linear regression analysis of Graphpad Prism software. The treatment was considered to have an effect when slopes for treated conditions were statistically different from the untreated. ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (a), (b) GSK2801, but not BAZ2‐ICR, reduces 2D growth of PC3 (AR‐negative) and 22Rv1 (AR‐positive) cells. (c), (d) The BRD9 inhibitor BI9564 shows limited activity only on 22Rv1 cells. (e), (f) Cotreatment with BAZ2‐ICR and BI9564 does not reproduce the effects observed with GSK2801.

FIGURE 2

Effect of tested inhibitors on PCa spheroids. GSK2801 (25 μM) and BAZ2‐ICR (25 μM) similarly affect the growth of 22Rv1 cells in a 3D setting, while only GSK2801 is effective on PC3 cells. Co‐treatment with BAZ2‐ICR (25 μM) and BI9564 (20 μM) does not cause a different effect compared to treatment with BI9564 alone, nor reproduces the GSK2801 effects. Lines are obtained using the segmented linear regression analysis of the Graphpad Prism software. The growth rate of the spheroids changes at days 6 and 9 for PC3 and 22Rv1, respectively. The treatment was considered to have an effect when the slopes obtained from the lines of the treated condition were statistically different from the one of the untreated. ns = p > 0.05, *p < 0.05, ****p < 0.0001. The comparison was also made between BI9564 alone and co‐treatment with BI9564 and BAZ2‐ICR; slopes were not found to be statistically different for either cell line. For each replicate, 5/6 spheroids/conditions were seeded. Doubling time for the two cell lines is significantly different, about 24 and 50 h for PC3 and 22Rv1 cells, respectively.

FIGURE 3

Crystallographic structures of BAZ2 chemical probes bound to BAZ2A. (a) Binding pose of BAZ2‐ICR (ivory) to BAZ2A (orange) superposed to BAZ2‐ICR (light green) bound to BAZ2B (green, PDB 4XUB). (b) GSK2801 (gold) in complex with BAZ2A (yellow) compared to the same inhibitor (turquoise) bound to BAZ2B (cyan, PDB 4RVR); the different conformations assumed by the ZA loop are circled in purple. Residues numbering refers to BAZ2A; substituted residues in BAZ2B are reported according to the structure color. Exit vectors for PROTACs generation are indicated with red arrows.

FIGURE 4

Crystallographic structures of TP‐238, GSK4027 and UP39 in complex with the BAZ2A bromodomain. Residues numbering refers to BAZ2A; substituted residues in BPTF, GCN5 and CBP are reported accordingly to the structure color. (a) Comparison of TP‐238 binding pose in BAZ2A (pink, TP‐238 in magenta) and BPTF (dark green, TP‐238 in green, PDB 7KDZ). ITC titration identifies limited but favorable enthalpic and entropic contributions (blank titration is shown as Figure S6). (b) GSK4027 binds to BAZ2A bromodomain (ivory, GSK4027 in white) similarly to BPTF (light green, GSK4027 in dark green, PDB 7K6R) and GCN5 (dark cyan, GSK4027 in cyan, PDB 5MLJ) bromodomains. The favorable enthalpic contribution is partially counteracted by the opposite entropic effect. (c) The largest divergence for compound UP39 bound to BAZ2A (white, UP39 in gray) or CBP (aquamarine, UP39 in cyan, PDB 5ENG) is the different orientation of the central methylbenzoate ring caused by the substitution of CBP Leu1109 with BAZ2A Trp1816.