An iridium(iii)-based irreversible protein-protein interaction inhibitor of BRD4 as a potent anticancer agent

Abstract

Bromodomain-containing protein 4 (BRD4) has recently emerged as an attractive epigenetic target for anticancer therapy. In this study, an iridium(iii) complex is reported as the first metal-based, irreversible inhibitor of BRD4. Complex 1a is able to antagonize the BRD4-acetylated histone protein-protein interaction (PPI) in vitro, and to bind BRD4 and down-regulate c-myc oncogenic expression in cellulo. Chromatin immunoprecipitation (ChIP) analysis revealed that 1a could modulate the interaction between BRD4 and chromatin in melanoma cells, particular at the MYC promoter. Finally, the complex showed potent activity against melanoma xenografts in an in vivo mouse model. To our knowledge, this is the first report of a Group 9 metal complex inhibiting the PPI of a member of the bromodomain and extraterminal domain (BET) family. We envision that complex 1a may serve as a useful scaffold for the development of more potent epigenetic agents against cancers such as melanoma.

Fig. 1. Chemical structures of the cyclometallated Ir(iii) and Rh(iii) complexes (racemates) used for preliminary screening. Only one enantiomer is shown for clarity; complexes 1 and 17 are OTf salts, and others are PF₆ salts.

Fig. 2. Displacement of a tetra-acetylated H4 peptide from BRD4 by a selection of Ir(iii)/Rh(iii) complexes at 100 μM in a TR-FRET assay. Error bars represent the standard deviations of the results from three independent experiments.

Fig. 3. (a) Chemical structures of the cyclometallated Ir(iii) and Rh(iii) complexes (racemates) used for structure-activity analysis (SAR). (b) Displacement of a tetra-acetylated H4 peptide from BRD4 by complex 1 and analogues 1a–1j at 10 μM in a TR-FRET assay. Error bars represent the standard deviations of the results from three independent experiments.

Fig. 4. Ability of 1a to displace H4AcK4 peptide from (a) BRD4(1) and (b) BRD4(2) in a time-resolved-fluorescent resonance electron transfer (TR-FRET) assay. Binding of H4AcK4 to BRD4(1) was strongly inhibited by 1a, with half-maximum inhibitory concentration (IC₅₀) value of 0.07 μM. Error bars represent the standard deviations of the results from three independent experiments. LC-MS/MS analysis of (c) BRD4(1), (d) BRD4(1) with 1a and (e) 1a only. BRD4(1) and 1a complex were buffered in 10 mM Tris–HCl, pH = 7.5, 500 mM NaCl and incubated at 25 °C for 2 h. The sample was analyzed by positive ion mass spectra.

Fig. 5. Chromatin immunoprecipitation (ChIP) analysis showed that 1a selectively decreased the binding of BRD4 to MYC, Bcl-2 and CDK6, but not housekeeping genes (B2M) in (a) A375 and (b) A2058 cells. Bar graphs represented the mean enrichment relative to input and error bars reflect standard deviation of results derived from biological triplicate experiments. Significantly different at ***p < 0.01. Error bars represent the standard deviations of the results from three independent experiments.

Fig. 6. Immunoblotting analysis of the effect of 1a and (+)-JQ1 treatment in (a) A375 and (b) A2058 cells. Densitometry analysis revealed that 1a inhibited c-myc, Bcl-2, ERK 1/2, p-ERK 1/2 and PARP expression. Dose response analysis of cell viability of complex 1a against (c) A375 cells and (d) A2058 cells. Error bars represent the standard deviations of the results from three independent experiments. Normalized proliferation curves in the colony formation assay for (e) A375 and (f) A2058 cells treated with vehicle or 1a (0.001–10 μM) measured by crystal violet staining. Error bars represent the standard deviations of the results from three independent experiments. (g) The relationship between the IC₅₀ of the binding ability of BRD4(1)/peptide and the log of IC₅₀ of A375 cell viability, and a trend of positive correlation was observed (r = 0.8207, n = 14).

Fig. 7. Anti-proliferative activity of 1a in an in vivo xenograft model of melanoma. (a) Photographs of dissected tumors from the control (vehicle) and treatment (1a, 100 mg kg^–1). (b) Average A375 tumour volume in the control group and treatment group (1a, 100 mg kg^–1). Each group contained six mice and results are reported as the values of the mean ± SEM. (c) Tumour inhibition of A375 xenografts in the treatment group (1a, 100 mg kg^–1) expressed as percentage reduction in tumor volume compared to the control group. The results were analyzed using the Student's t-test. Significantly different at 0.01 < **p < 0.05. (d) Average tumor weight of the vehicle control group versus the treatment group (1a, 100 mg kg^–1). (e) Average body weight of the two groups. Each group contained six mice, and results were reported as the values of the mean ± SEM. The results were analyzed using the Student's t-test. Significantly different at 0.01 < **p < 0.05. (f) Heat map of regulated genes of the ECM pathway and VEGF signaling pathway following treatment with 1a. The color scale in the inset represents the log-fold change of expression compared with untreated control.