Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 21;10(8):1770-7.
doi: 10.1021/acschembio.5b00216. Epub 2015 Jun 16.

Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4

Michael Zengerle  1 , Kwok-Ho Chan  1 , Alessio Ciulli  1
Affiliations

Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4

Michael Zengerle et al. ACS Chem Biol. .

Abstract

The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the _targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for _target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis _targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective _targeting of BRD4.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Design, synthesis, and biophysical and biological evaluation of BET bromodomain PROTACs. (a) Chemical structures of BET-bromodomain inhibitors JQ1 and I-BET762 and binders of von Hippel-Lindau protein VHL-1 and VHL-2. (b) Scheme of the synthesis of PROTAC compounds MZ1–3 and cisMZ1; for detailed synthetic procedures see the Supporting Information. (c) Isothermal titration calorimetry data for titration of MZ1 into the individual members of the BET-bromodomain subfamily. Titrations were performed at 30 °C with a protein concentration of 15 μM and ligand concentration of 150 μM (entry 1–6). Titration of MZ1 and cisMZ1 into VBC at 25 °C with identical concentrations (entry 9, 12) and reverse titration of VBC protein (150 μM) into MZ3 (15 μM) at 25 °C (entry 10) were conducted. For ΔS and ΔG values, see the Supporting Information. (d) HeLa cells were treated with either siRNA _targeting individual BET proteins or negative control siRNA 24 h prior to treatment with the compounds MZ1–3, cisMZ1, and JQ1 or vehicle control (0.01% DMSO) for an additional 24 h. Abundance of individual BET protein was analyzed by Western blotting using corresponding specific antibodies accordingly after SDS-PAGE. i, data from ref (8); ii, data from ref (26).
Figure 2
Figure 2
PROTACs induce concentration- and time-dependent selective degradation of BRD4. (a) HeLa cells treated for 24 h with different concentrations of MZ1 (panel I), MZ2 (panel II), and MZ3 (panel III). The bands observed in the BRD4 short isoform lane at a high concentration of each compound are correlated to nonspecific binding. (b) Time dependent treatment over 36 h of HeLa cells with 1 μM (panel I) and 100 nM (panel II) of MZ1. (c) U2OS cells transfected with GFP-BRD4 were treated with either 5 μM of MZ1 or cisMZ1 over a time course of 4 h. BRD4 degradation over time was followed by live fluorescence imaging.
Figure 3
Figure 3
Mechanistic studies on PROTAC biological activity. (a) Time dependent treatment over 36 h of HeLa cells with 1 μM inactive compound cisMZ1. (b) HeLa cells treated with JQ1 or MZ1 at 1 μM in the absence or presence of the proteasome inhibitor MG132. (c) Time dependent treatment over 36 h of HeLa cells with 1 μM MZ1 observing the levels of the von Hippel-Lindau (VHL) protein. (d) HeLa cells treated with 100 μM CoCl2 as a hypoxia control or 0.1, 1, and 10 μM MZ1. (e) BRD4 protein levels were observed (panel I) with single treatment of MZ1 at t = 0 for 4 h and then exchange of media, (panel II) single treatment with MZ1 at t = 0 but no exchange of media, and (panel III) single treatment with 0.01% DMSO for 4 h and then exchange of media.
Figure 4
Figure 4
Selective degradation of BRD4 leads to a differential response between JQ1 and MZ1 on selected genes. mRNA expression profiles of MYC, P21, AREG, FAS, FGFR1, and TYRO3 upon treatment with PROTAC MZ1 and JQ1 were compared. (a) HeLa cells were treated with 100 nM MZ1, VHL-1′, or JQ1 or 0.01% DMSO vehicle control (Veh.) for 24 h. (b) To mimic the protein removal effect, HeLa cells were transfected with siRNA _targeting individual BRD2, BRD3, or BRD4 or negative control siRNA and were harvested after 48 h. Quantitative PCR was performed to analyze relative gene expression level of treated HeLa cells using _target specific primers. Gene expression levels relative to GAPDH were normalized to control treatment. The data shown represent the mean ± SEM (n = 3 technical replicates) of one experiment. Statistical significance compared to the control was determined with two-tailed t tests: *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. = not significant.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gallenkamp D.; Gelato K. A.; Haendler B.; Weinmann H. (2014) Bromodomains and their pharmacological inhibitors. ChemMedChem. 9, 438–464. - PubMed
    1. Belkina A. C.; Denis G. V. (2012) BET domain co-regulators in obesity, inflammation and cancer. Nat. Rev. Cancer 12, 465–477. - PMC - PubMed
    1. Zuber J.; Shi J.; Wang E.; Rappaport A. R.; Herrmann H.; Sison E. A.; Magoon D.; Qi J.; Blatt K.; Wunderlich M.; Taylor M. J.; Johns C.; Chicas A.; Mulloy J. C.; Kogan S. C.; Brown P.; Valent P.; Bradner J. E.; Lowe S. W.; Vakoc C. R. (2011) RNAi screen identifies Brd4 as a therapeutic _target in acute myeloid leukaemia. Nature 478, 524–528. - PMC - PubMed
    1. Baratta M. G.; Schinzel A. C.; Zwang Y.; Bandopadhayay P.; Bowman-Colin C.; Kutt J.; Curtis J.; Piao H.; Wong L. C.; Kung A. L.; Beroukhim R.; Bradner J. E.; Drapkin R.; Hahn W. C.; Liu J. F.; Livingston D. M. (2015) An in-tumor genetic screen reveals that the BET bromodomain protein, BRD4, is a potential therapeutic _target in ovarian carcinoma. Proc. Natl. Acad. Sci. U. S. A. 112, 232–237. - PMC - PubMed
    1. Chung C.-W.; Coste H.; White J. H.; Mirguet O.; Wilde J.; Gosmini R. L.; Delves C.; Magny S. M.; Woodward R.; Hughes S. A.; Boursier E. V.; Flynn H.; Bouillot A. M.; Bamborough P.; Brusq J.-M. G.; Gellibert F. J.; Jones E. J.; Riou A. M.; Homes P.; Martin S. L.; Uings I. J.; Toum J.; Clément C. A.; Boullay A.-B.; Grimley R. L.; Blandel F. M.; Prinjha R. K.; Lee K.; Kirilovsky J.; Nicodeme E. (2011) Discovery and characterization of small molecule inhibitors of the BET family bromodomains. J. Med. Chem. 54, 3827–3838. - PubMed

Publication types

MeSH terms

  NODES
INTERN 1
twitter 2