Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2022 Sep 29;13(10):1621–1627. doi: 10.1021/acsmedchemlett.2c00300

Development of an N-Terminal BRD4 Bromodomain-Targeted Degrader

Anand Divakaran †, Cole R Scholtz ‡, Huda Zahid ‡, Wenwei Lin §, Elizabeth C Griffith §, Richard E Lee §, Taosheng Chen §, Daniel A Harki †,‡,*, William C K Pomerantz ‡,†,*
PMCID: PMC9575167  PMID: 36262390

Abstract

graphic file with name ml2c00300_0005.jpg

Targeted protein degradation is a powerful induced-proximity tool to control cellular protein concentrations using small molecules. However, the design of selective degraders remains empirical. Among bromodomain and extra-terminal (BET) family proteins, BRD4 is the primary therapeutic target over family members BRD2/3/T. Existing strategies for selective BRD4 degradation use pan-BET inhibitors optimized for BRD4:E3 ubiquitin ligase (E3) ternary complex formation, but these result in residual inhibition of undegraded BET-bromodomains by the pan-BET ligand, obscuring BRD4-degradation phenotypes. Using our selective inhibitor of the first BRD4 bromodomain, iBRD4-BD1 (IC50 = 12 nM, 23- to 6200-fold intra-BET selectivity), we developed dBRD4-BD1 to selectively degrade BRD4 (DC50 = 280 nM). Notably, dBRD4-BD1 upregulates BRD2/3, a result not observed with degraders using pan-BET ligands. Designing BRD4 selectivity up front enables analysis of BRD4 biology without wider BET-inhibition and simplifies designing BRD4-selective heterobifunctional molecules, such as degraders with new E3 recruiting ligands or for additional probes beyond degraders.

Keywords: BRD4-BD1 inhibitor, BRD4 degrader, BET domain selectivity, epigenetic reader domain

Bromodomain and extra-terminal (BET) family proteins, BRD2, -3, -4, and testis-specific protein, BRDT, are essential epigenetic regulators of gene expression through molecular recognition of acetylated proteins.1 Of the three ubiquitously expressed BET proteins, BRD4 is accepted as the most disease-relevant target for inflammation and oncology;2−5 but few strategies exist to selectively target native BRD4 function. The tandem bromodomains (BD1 and BD2) are the most ligandable domains of BET proteins, but their selective targeting remains challenging due to a high degree of homology among the eight BET bromodomains.6,7 Although significant advances have been made from pan-BET inhibition to either pan-BD1 or -BD2 inhibition of BET proteins,8,9 isoform selectivity for individual BET bromodomains is only more recently being realized.10,11

As an alternative to inhibition, targeted protein degradation has emerged as a therapeutic modality leveraging event-driven pharmacology over traditional target occupancy models.12 Targeted degraders co-opt cellular proteostasis machinery, inducing proximity between cellular E3 protein-ubiquitin ligases (E3) and proteins of interest, resulting in proteasomal degradation of target proteins. Given their substoichiometric mode of action, modularity in design, and promise of targeting traditionally “undruggable” targets, degraders have been reported for a wide variety of protein classes including BET proteins.13−15 BET-degraders have improved therapeutic efficacy compared to BET-inhibition alone,16 but both face challenges with dose-limiting toxicities including thrombocytopenia attributed in part to targeting BRD2 and BRD3.17 While selective degraders have been developed from promiscuous ligands,18 including selective BRD4-degraders from pan-BET inhibitors (Figure 1),19−21 the design elements for achieving selectivity remains a nontrivial endeavor.

Figure 1.

Figure 1

Open in a new tab

Strategies for selective BRD4 degradation. Previous approaches based on pan-BET ligands optimize linkers for BRD4-E3 ligase binding kinetics and ternary complex formation, whereas this work focuses on BRD4 degradation through selective engagement of its N-terminal bromodomain.

Here we report the design, synthesis, and characterization of a potent, selective BRD4-BD1 inhibitor, iBRD4-BD1 (Figure 1), with 23- to 6200-fold selectivity over ubiquitously expressed BET bromodomains. We use this inhibitor to test the efficacy of selective BRD4 degradation through BRD4-BD1 engagement. Design of our first-generation degrader, dBRD4-BD1, demonstrated sustained BRD4 degradation and unexpectedly increased BRD2 and BRD3 protein levels. This approach is distinct from previous BRD4-selective degraders as the use of a BRD4-BD1 inhibitor avoids reversible bromodomain inhibition of undegraded BET-proteins by a pan-BET ligand (Figure 1).

Wang and co-workers have shown that the expression of BRD4, not BRD2/3, correlates most strongly to sensitivity of BET degraders in lung cancer cells.22 Similar results have been observed in other cancers23−25 supporting BRD4 as the most relevant BET protein to target for degradation.26 We envisioned an approach using a ligand with high BRD4 specificity and affinity to degrade BRD4 in a cell line with high BRD4 sensitivity (e.g., multiple myeloma, MM.1S), but only a few molecules with high selectivity for inhibiting BRD4 exist.27,28 By leveraging selectivity properties and structural information on our BD1-selective inhibitors,29,30 we hypothesized we could degrade BRD4 through an individual bromodomain.31,32

Initially, it remained unclear whether BRD4 was amenable to degradation through BD1. Preferential BD2 engagement of previous degraders,33 coupled with higher chromatin occupancy of BD1 that leaves BD2 unliganded,34 suggested BRD4 degradation likely proceeds through BD2. To test these effects, we performed competition experiments on pan-BET-degrader, MZ1, using pan-BD1 and pan-BD2 selective inhibitors, iBET-BD1 and iBET-BD2.9 In this case, neither BD1 nor BD2 inhibition prevented BRD4 degradation (Figure 2A), demonstrating either bromodomain can provide a suitable handle for degradation despite the preference of MZ1 to bind BRD4-BD2 while forming a ternary complex with VHL.33 The pan-BET inhibitor JQ1, however, was able to prevent degradation of BET proteins (Figure S1). These experiments validated our strategy for domain-targeted BRD4 degradation.

Figure 2.

Figure 2

Open in a new tab

Role of BD1 in BRD4 degradation and biophysical characterization of iBRD4-BD1. (A) Competition of pan-BET degrader, MZ1, with pan-BD1 or pan-BD2 inhibitors, iBET-BD1/BD2. (B) Trisubstituted imidazoles with BET-bromodomain selectivity for BRD4-BD1. (C) Commercial Alphascreen with BET-bromodomains from reaction biology and summary of thermodynamic parameters from BRD4-BD1 isothermal titration calorimetry (ITC) assays. See Figures S2 and S3 for source data, reported as mean values of duplicate and triplicate experiments, respectively. (D) Isothermal dose–response CETSA demonstrating target engagement of BRD4 in MM.1S cells after 1 h of treatment.

In contrast to pan-BET bromodomain inhibitors, 1,4,5-trisubstituted imidazoles such as V and UMN627 from our lab preferentially bind to BD1 of BRD4. These inhibitors achieve moderate selectivity in part by displacing a network of conserved waters from the BD1 acetyl-lysine binding site (Figure 2B).30,35 We hypothesized that a vacant hydrophobic area surrounding a dimethyl-aryl ring could be better occupied by bulky aliphatic groups. From these studies, we found replacement of the 3,5-dimethyl substituents with a 2-methyl-5-isopropyl in iBRD4-BD1 (Figure 1) resulted in an increase in affinity and selectivity within the BET-family (BRD4-BD1 Kd = 45.6 nM by ITC and 23- to 6200-fold BRD4-BD1 selectivity by Alphascreen, respectively; Figure 2C).10 We evaluated the cellular engagement of BRD4 in a cellular thermal shift assay (CETSA) with previously established denaturation conditions.35,36iBRD4-BD1 prevented the denaturation of BRD4 in a dose-dependent manner and showed stabilization of BRD4 at concentrations above 3 nM (Figure 2D).

In cocrystal structures of iBRD4-BD1 analogues, the piperidyl amine was oriented toward solvent (PDB codes 6MH1 and 6WGX). As such, degrader dBRD4-BD1 (Figure 3A) was synthesized via conjugation of iBRD4-BD1 through a PEG linker attached to a 4-hydroxythalidomide analogue. To ensure dBRD4-BD1 forms a ternary complex of BRD4 with CRBN necessary for degradation, we used a TR-FRET assay with His-tagged BRD4 bromodomains and GST-tagged CRBN (Figure 3B).37 dBET1 demonstrates no selectivity between BD1 and BD2 to produce a high TR-FRET signal with both bromodomains. However, dBRD4-BD1 selectively produced a TR-FRET signal with BRD4-BD1 and not BD2, albeit to a lower amplitude than dBET1 (Figure 3C,D). Maximal FRET signals were observed at concentrations of 14 and 4.6 nM, suggesting selective and effective chemically induced dimerization of BRD4-BD1 and CRBN.

Figure 3.

Figure 3

Open in a new tab

Biophysical characterization of dBRD4-BD1. (A) Structure of dBRD4-BD1. (B) Schematic of TR-FRET ternary complexation assay to determine complex formation of His-BRD4-BD1 (C) and His-BRD4-BD2 (D) with GST-CRBN via bivalent molecules. (E) Dose-dependent competition of His-BRD4-BD1 and GST-CRBN complex with 4.6 nM dBRD4-BD1 added. Data are reported as the mean ± SD of three independent trials performed in quadruplicate.

Both dBRD4-BD1 and dBET1 also show a hook-effect at high concentrations, supporting ternary complex formation, and importantly, neither monovalent ligand iBRD4-BD1 nor 4-hydroxythalidomide could assemble ternary complexes in both bromodomains. Further, the ternary complex showed a dose-dependent disassembly upon treatment with ligands for either BRD4-BD1 or CRBN (Figure 3E). Aggregation of dBRD4-BD1 and 4-hydroxythalidomide at concentrations above 10 μM likely resulted in the increasing TR-FRET signal at these concentrations.

Encouraged by the positive biophysical results and insight into domain-selectivity, we assessed the ability to degrade BRD4 through BD1 alone. dBRD4-BD1 demonstrated selective and durable BRD4 degradation (Dmax = 77%, DC50 = 280 nM, Figures 4A and S4), which diminished above concentrations of 5 μM where formation of the productive ternary complex was disfavored due to the hook-effect. Degradation of BRD2 and -3 was not observed; conversely both were upregulated at concentrations where BRD4 was degraded.

Figure 4.

Figure 4

Open in a new tab

BRD4 degradation in MM.1S cells. (A) Representative blots for BRD4 degradation after 24 h treatment; see Figure S4 for quantified densitometry for three biological replicates of blots. (B) NanoBRET degradation assay confirming BRD4 degradation. (C) Time-course study of BRD4 degradation by dBRD4-BD1. (D) Rescue of BRD4 degradation using a pan-BD1 inhibitor, proteasome inhibitor, MG132, and neddylation inhibitor MLN4924, but not pan-BD2 inhibitor, iBET-BD2. Refer to Figure 2A for untreated control from the same gel.

BRD4 degradation was further evaluated using a NanoBRET reporter system expressing HaloTag-CRBN and a full-length BRD4-NanoLuc protein (Figure 4B). In this assay, maximal BRD4 degradation was observed at a concentration of 0.2 μM dBRD4-BD1 based on a loss of lumiscent signal from the NanoLuc BRD4 fusion. BRD4 degradation was rescued with addition of iBRD4-BD1. This assay produced a more pronounced hook-effect than in the corresponding blots from Figure 4A, likely due to the artificial nature of the system. Overall, BRD4 was selectively degraded by dBRD4-BD1 and levels of BRD2 and -3 inversely correlated to BRD4, including at concentrations where the hook-effect was observed. This surprising effect on BRD2 and -3 was not observed with previous BRD4-selective degraders AT1, ZXH-3-26, and KB02-JQ1.19−21 Distinctively, all three use pan-BET ligands that likely occupy bromodomains of BRD2/3 and dampen a cellular feedback response to BRD4 degradation that is unaffected by iBRD4-BD1.

BET inhibition can have a stronger effect on cMyc concentrations relative to BET-degradation.38 Since proliferation of the MM.1S cell-line is heavily cMyc driven39 and BD1 inhibition increases levels of cMyc in some cases,30,40 we compared the antiproliferative effects of our compounds with cMyc expression. dBRD4-BD1 does not downregulate cMyc until near maximal BRD4 degradation is achieved (Figure 4A), likely leading to the lower observed cytotoxicity with dBRD4-BD1 relative to iBRD4-BD1 (Figure S5). Although cMyc is absent at higher dBRD4-BD1 concentrations where the hook-effect is observed, inhibition of BET bromodomains alone at these concentrations, rather than BRD4 degradation, may be sufficient to affect cMyc expression.

Next, we evaluated the effects of dBRD4-BD1 over an extended incubation period to assess weaker degradation targets. A potential benefit of the linker attachment point to iBRD4-BD1 is the effect on kinase binding. Trisubstituted imidazoles are known inhibitors of the p38α kinase, and the piperidine of iBRD4-BD1 analogues (e.g., PDB code 1OUK) occupies a region oriented toward the kinase activation loop. Gratifyingly, p38α levels remain unchanged even after extended treatment periods to suggest p38α is not engaged by dBRD4-BD1 (Figure 4C). This result is consistent with additional kinase profiling performed using this optimized scaffold.10

In contrast to p38α levels in the time-course study, the degradation half-life of BRD4 was 3.3 h with maximal degradation achieved after 8 h (Figure 4C). Similar to our earlier observation, concentrations of BRD2 and BRD3 increased after the 8 and 12 h time points, respectively. A modest recovery in cMyc expression was observed at later time points. Widespread effects have been reported in response to BET-inhibition and degradation.41,42 Upregulation of proteins in response to BRD4 degradation may be broader than our set of evaluated proteins, and cellular toxicity at these later time points may have additional effects.

To verify the mechanism of degradation, we performed competition experiments with domain-selective BET and proteasome inhibitors. iBET-BD1 competitively rescued BRD4 degradation, whereas iBET-BD2 was ineffective (Figure 4D). Additionally, BRD4 degradation was rescued with proteasome and neddylation inhibitors, MG-132 and MLN4924. These results together indicate degradation by dBRD4-BD1 was dependent on BD1 and proceeded via ubiquitination and proteasome-dependent pathways.

In summary, given the disease relevance of BRD4-specific function and to address the lack of tools to selectively target BRD4, we leveraged the unique properties of BRD4- and domain-selective BET inhibitors. iBRD4-BD1 notably has >23-fold selectivity for BRD4-BD1 over other BET bromodomains. Moreover, this is the first instance of selective endogenous BRD4 degradation through an individual bromodomain, which produces divergent effects on BRD2/3 relative to previous BRD4 degraders. While less potent than BET-degraders such as dBET1, linker optimization and improvement in the drug-like properties are anticipated to significantly improve the activity of our first-generation degrader.

Prior to this study, the design of BRD4-selective heterobifunctional ligands relied on optimizing pan-BET ligands for interaction kinetics (e.g., BRD4-BD2 with AT1)19 or optimizing linker geometries for ternary complex formation (e.g., BRD4-BD1 with ZXH-3-26).20 This process is cumbersome, and a major limitation remains in availability of the BET-ligand to bind other BET bromodomains aside from the BRD4-ternary complex, which obscures biological effects and may result in toxicity related to targeting BRD2/3.17 The approach presented here highlights the utility of a BRD4-selective inhibitor in circumventing the need for iterative optimization of linker/complex interactions. While additional SAR and selectivity around the design of iBRD4-BD1 have been disclosed elsewhere,10 BET-selective ligands and this strategy will simplify future isoform selective BET degrader development using new E3-ligase ligands that engender further selectivity and efficacy, and more broadly facilitate the design of additional selective BRD4-targeting heterobifunctional modalities beyond degraders. These new tools will be especially beneficial in the study of domain- and BRD4-specific biology, which will be the focus of our future work.

Acknowledgments

The authors thank Dr. Siva Talluri and Dr. Peter Ycas for their intellectual contributions to this project. Figures were created using GraphPad Prism and BioRender. iBET-BD1 and iBET-BD2 were supplied by the Structural Genomics Consortium under an Open Science Trust Agreement: http://www.thesgc.org/click-trust. The GST-CRBN protein was prepared by the Protein Production Facility at St. Jude Children’s Research Hospital.

Glossary

Abbreviations

BET

bromodomain and extra-terminal

BRD2/3/4/T

bromodomain-containing protein 2/3/4/T

Myc

MYC proto-oncogene, bHLH transcription factor

BD1

N-terminal BET bromodomain

BD2

C-terminal BET bromodomain

E3

protein-ubiquitin ligase

CRBN

cereblon

CETSA

cellular thermal shift assay

ITC

isothermal titration calorimetry

SAR

structure–activity relationship

PEG

polyethylene glycol

DMSO

dimethyl sulfoxide

TR-FRET

time-resolved Förster resonance energy transfer

GST

glutathione S-transferase

TFA

trifluoroacetic acid

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00300.

  • Chemical and biological experimental methods, compound characterization, and supplementary/source data (PDF)

Author Contributions

A.D., D.A.H., and W.C.K.P. conceived project. A.D. designed and synthesized molecules, performed cellular experiments, and wrote the manuscript. H.Z., W.L., and E.C.G. designed and performed biophysical experiments. C.R.S. performed nanoBRET degradation and synthesis experiments. R.E.L., T.C., D.A.H., and W.C.K.P. supervised the study. All authors contributed to the writing and review of the manuscript and have given approval to this final version.

This research was supported by the National Institutes of Health’s National Center for Advancing Translational Sciences, Grant UL1TR002494. A.D. was supported by the UMN Doctoral Dissertation Fellowship and an NIH chemistry–biology interface training grant (Grant T32-GM008700/T32-GM132029-01). H.Z. was supported by UMN IEM Engineering in Medicine Doctoral Fellowship 2020. C.R.S. was supported by the NIH Biotechnology Training Grant NIH T32GM008347. D.A.H. acknowledges funding from the Masonic Cancer Center at the University of Minnesota with resources from Minnesota Masonic Charities. T.C. is supported by National Institute of General Medical Sciences of the National Institutes of Health under Award R35GM118041.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the supporting organizations.

The authors declare no competing financial interest.

Supplementary Material

ml2c00300_si_001.pdf (2.5MB, pdf)

References

  1. Muller S.; Filippakopoulos P.; Knapp S. Bromodomains as Therapeutic Targets. Expert Rev. Mol. Med. 2011, 13, e29 10.1017/S1462399411001992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Nabet B.; Roberts J. M.; Buckley D. L.; Paulk J.; Dastjerdi S.; Yang A.; Leggett A. L.; Erb M. A.; Lawlor M. A.; Souza A.; Scott T. G.; Vittori S.; Perry J. A.; Qi J.; Winter G. E.; Wong K. K.; Gray N. S.; Bradner J. E. The DTAG System for Immediate and Target-Specific Protein Degradation. Nat. Chem. Biol. 2018, 14 (5), 431–441. 10.1038/s41589-018-0021-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Runcie A. C.; Zengerle M.; Chan K. H.; Testa A.; Van Beurden L.; Baud M. G. J.; Epemolu O.; Ellis L. C. J.; Read K. D.; Coulthard V.; Brien A.; Ciulli A. Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition. Chem. Sci. 2018, 9 (9), 2452–2468. 10.1039/C7SC02536J. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Delmore J. E.; Issa G. C.; Lemieux M. E.; Rahl P. B.; Shi J.; Jacobs H. M.; Kastritis E.; Gilpatrick T.; Paranal R. M.; Qi J.; Chesi M.; Schinzel A. C.; McKeown M. R.; Heffernan T. P.; Vakoc C. R.; Bergsagel P. L.; Ghobrial I. M.; Richardson P. G.; Young R. A.; Hahn W. C.; Anderson K. C.; Kung A. L.; Bradner J. E.; Mitsiades C. S. BET Bromodomain Inhibition as a Therapeutic Strategy to Target C-Myc. Cell 2011, 146 (6), 904–917. 10.1016/j.cell.2011.08.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Mertz J. A.; Conery A. R.; Bryant B. M.; Sandy P.; Balasubramanian S.; Mele D. A.; Bergeron L.; Sims R. J. Targeting MYC Dependence in Cancer by Inhibiting BET Bromodomains. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (40), 16669–16674. 10.1073/pnas.1108190108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Filippakopoulos P.; Qi J.; Picaud S.; Shen Y.; Smith W. B.; Fedorov O.; Morse E. M.; Keates T.; Hickman T. T.; Felletar I.; Philpott M.; Munro S.; McKeown M. R.; Wang Y.; Christie A. L.; West N.; Cameron M. J.; Schwartz B.; Heightman T. D.; La Thangue N.; French C. A.; Wiest O.; Kung A. L.; Knapp S.; Bradner J. E. Selective Inhibition of BET Bromodomains. Nature 2010, 468 (7327), 1067–1073. 10.1038/nature09504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Filippakopoulos P.; Picaud S.; Mangos M.; Keates T.; Lambert J. P.; Barsyte-Lovejoy D.; Felletar I.; Volkmer R.; Müller S.; Pawson T.; Gingras A. C.; Arrowsmith C. H.; Knapp S. Histone Recognition and Large-Scale Structural Analysis of the Human Bromodomain Family. Cell 2012, 149 (1), 214–231. 10.1016/j.cell.2012.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Faivre E. J.; McDaniel K. F.; Albert D. H.; Mantena S. R.; Plotnik J. P.; Wilcox D.; Zhang L.; Bui M. H.; Sheppard G. S.; Wang L.; Sehgal V.; Lin X.; Huang X.; Lu X.; Uziel T.; Hessler P.; Lam L. T.; Bellin R. J.; Mehta G.; Fidanze S.; Pratt J. K.; Liu D.; Hasvold L. A.; Sun C.; Panchal S. C.; Nicolette J. J.; Fossey S. L.; Park C. H.; Longenecker K.; Bigelow L.; Torrent M.; Rosenberg S. H.; Kati W. M.; Shen Y. Selective Inhibition of the BD2 Bromodomain of BET Proteins in Prostate Cancer. Nature 2020, 578 (7794), 306–310. 10.1038/s41586-020-1930-8. [DOI] [PubMed] [Google Scholar]
  9. Gilan O.; Rioja I.; Knezevic K.; Bell M. J.; Yeung M. M.; Harker N. R.; Lam E. Y. N.; Chung C.; Bamborough P.; Petretich M.; Urh M.; Atkinson S. J.; Bassil A. K.; Roberts E. J.; Vassiliadis D.; Burr M. L.; Preston A. G. S.; Wellaway C.; Werner T.; Gray J. R.; Michon A. M.; Gobbetti T.; Kumar V.; Soden P. E.; Haynes A.; Vappiani J.; Tough D. F.; Taylor S.; Dawson S. J.; Bantscheff M.; Lindon M.; Drewes G.; Demont E. H.; Daniels D. L.; Grandi P.; Prinjha R. K.; Dawson M. A. Selective Targeting of BD1 and BD2 of the BET Proteins in Cancer and Immunoinflammation. Science 2020, 368 (6489), 387–394. 10.1126/science.aaz8455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cui H.; Divakaran A.; Hoell Z. J.; Ellingson M. O.; Scholtz C. R.; Zahid H.; Johnson J. A.; Griffith E. C.; Gee C. T.; Lee A. L.; Khanal S.; Shi K.; Aihara H.; Shah V. H.; Lee R. E.; Harki D. A.; Pomerantz W. C. K. A Structure-Based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes. J. Med. Chem. 2022, 65 (3), 2342–2360. 10.1021/acs.jmedchem.1c01779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ar B.; Je T.; Jf H. Optimized Use of the Firefly Luciferase Assay as a Reporter Gene in Mammalian Cell Lines. Biotechniques 1988, 7 (10), 1116–1122. [PubMed] [Google Scholar]
  12. Lai A. C.; Crews C. M. Induced Protein Degradation: An Emerging Drug Discovery Paradigm. Nat. Rev. Drug Discovery 2017, 16, 101–114. 10.1038/nrd.2016.211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feral A.; Laconde G.; Amblard M.; Masurier N. PROteolysis TArgetting Chimeras (PROTACs) Strategy Applied to Kinases: Recent Advances. Adv. Ther. 2020, 3 (11), 2000148. 10.1002/adtp.202000148. [DOI] [Google Scholar]
  14. Vogelmann A.; Robaa D.; Sippl W.; Jung M. Proteolysis Targeting Chimeras (PROTACs) for Epigenetics Research. Curr. Opin. Chem. Biol. 2020, 57, 8–16. 10.1016/j.cbpa.2020.01.010. [DOI] [PubMed] [Google Scholar]
  15. Flanagan J. J.; Neklesa T. K. Targeting Nuclear Receptors with PROTAC Degraders. Mol. Cell. Endocrinol. 2019, 493, 110452. 10.1016/j.mce.2019.110452. [DOI] [PubMed] [Google Scholar]
  16. Yang C. Y.; Qin C.; Bai L.; Wang S. Small-Molecule PROTAC Degraders of the Bromodomain and Extra Terminal (BET) Proteins—A Review. Drug Discovery Today: Technol. 2019, 31, 43–51. 10.1016/j.ddtec.2019.04.001. [DOI] [PubMed] [Google Scholar]
  17. Stonestrom A. J.; Hsu S. C.; Jahn K. S.; Huang P.; Keller C. A.; Giardine B. M.; Kadauke S.; Campbell A. E.; Evans P.; Hardison R. C.; Blobel G. A. Functions of BET Proteins in Erythroid Gene Expression. Blood 2015, 125 (18), 2825–2834. 10.1182/blood-2014-10-607309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Bondeson D. P.; Smith B. E.; Burslem G. M.; Buhimschi A. D.; Hines J.; Jaime-Figueroa S.; Wang J.; Hamman B. D.; Ishchenko A.; Crews C. M. Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. Cell Chem. Biol. 2018, 25 (1), 78–87. 10.1016/j.chembiol.2017.09.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gadd M. S.; Testa A.; Lucas X.; Chan K. H.; Chen W.; Lamont D. J.; Zengerle M.; Ciulli A. Structural Basis of PROTAC Cooperative Recognition for Selective Protein Degradation. Nat. Chem. Biol. 2017, 13 (5), 514–521. 10.1038/nchembio.2329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nowak R. P.; Deangelo S. L.; Buckley D.; He Z.; Donovan K. A.; An J.; Safaee N.; Jedrychowski M. P.; Ponthier C. M.; Ishoey M.; Zhang T.; Mancias J. D.; Gray N. S.; Bradner J. E.; Fischer E. S. Plasticity in Binding Confers Selectivity in Ligand-Induced Protein Degradation Article. Nat. Chem. Biol. 2018, 14 (7), 706–714. 10.1038/s41589-018-0055-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zhang X.; Crowley V. M.; Wucherpfennig T. G.; Dix M. M.; Cravatt B. F. Electrophilic PROTACs That Degrade Nuclear Proteins by Engaging DCAF16. Nat. Chem. Biol. 2019, 15 (7), 737–746. 10.1038/s41589-019-0279-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Zong D.; Gu J.; Cavalcante G. C.; Yao W.; Zhang G.; Wang S.; Owonikoko T. K.; He X.; Sun S. Y. BRD4 Levels Determine the Response of Human Lung Cancer Cells to BET Degraders That Potently Induce Apoptosis through Suppression of Mcl-1. Cancer Res. 2020, 80 (11), 2380–2393. 10.1158/0008-5472.CAN-19-3674. [DOI] [PubMed] [Google Scholar]
  23. Wong A. S. L.; Choi G. C. G.; Cui C. H.; Pregernig G.; Milani P.; Adam M.; Perli S. D.; Kazer S. W.; Gaillard A.; Hermann M.; Shalek A. K.; Fraenkel E.; Lu T. K. Multiplexed Barcoded CRISPR-Cas9 Screening Enabled by CombiGEM. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (9), 2544. 10.1073/pnas.1517883113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shi J.; Wang E.; Milazzo J. P.; Wang Z.; Kinney J. B.; Vakoc C. R. Discovery of Cancer Drug Targets by CRISPR-Cas9 Screening of Protein Domains. Nat. Biotechnol. 2015, 33 (6), 661–667. 10.1038/nbt.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Xiang T.; Bai J.-y.; She C.; Yu D.-j.; Zhou X.-z.; Zhao T.-l. Bromodomain Protein BRD4 Promotes Cell Proliferation in Skin Squamous Cell Carcinoma. Cell. Signalling 2018, 42, 106–113. 10.1016/j.cellsig.2017.10.010. [DOI] [PubMed] [Google Scholar]
  26. Wu S.; Jiang Y.; Hong Y.; Chu X.; Zhang Z.; Tao Y.; Fan Z.; Bai Z.; Li X.; Chen Y.; Li Z.; Ding X.; Lv H.; Du X.; Lim S. L.; Zhang Y.; Huang S.; Lu J.; Pan J.; Hu S. BRD4 PROTAC Degrader ARV-825 Inhibits T-Cell Acute Lymphoblastic Leukemia by Targeting “Undruggable” Myc-Pathway Genes. Cancer Cell Int. 2021, 21 (1), 230. 10.1186/s12935-021-01908-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ouyang L.; Zhang L.; Liu J.; Fu L.; Yao D.; Zhao Y.; Zhang S.; Wang G.; He G.; Liu B. Discovery of a Small-Molecule Bromodomain-Containing Protein 4 (BRD4) Inhibitor That Induces AMP-Activated Protein Kinase-Modulated Autophagy-Associated Cell Death in Breast Cancer. J. Med. Chem. 2017, 60 (24), 9990–10012. 10.1021/acs.jmedchem.7b00275. [DOI] [PubMed] [Google Scholar]
  28. Liu Z.; Chen H.; Wang P.; Li Y.; Wold E. A.; Leonard P. G.; Joseph S.; Brasier A. R.; Tian B.; Zhou J. Discovery of Orally Bioavailable Chromone Derivatives as Potent and Selective BRD4 Inhibitors: Scaffold Hopping, Optimization, and Pharmacological Evaluation. J. Med. Chem. 2020, 63 (10), 5242–5256. 10.1021/acs.jmedchem.0c00035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Divakaran A.; Talluri S. K.; Ayoub A. M.; Mishra N.; Cui H.; Widen J. C.; Berndt N.; Zhu J.-Y.; Carlson A. S.; Topczewski J. J.; Schönbrunn E.; Harki D. A.; Pomerantz W. C. K. Molecular Basis for the N-Terminal Bromodomain and Extra Terminal (BET) Family Selectivity of a Dual Kinase-Bromodomain Inhibitor. J. Med. Chem. 2018, 61, 9316–9334. 10.1021/acs.jmedchem.8b01248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Cui H.; Divakaran A.; Pandey A. K.; Johnson J. A.; Zahid H.; Hoell Z. J.; Ellingson M. O.; Shi K.; Aihara H.; Harki D. A.; Pomerantz W. C. K. Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement. Angew. Chemie - Int. Ed. 2021, 60 (3), 1220–1226. 10.1002/anie.202008625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Petretich M.; Demont E. H.; Grandi P. Domain-Selective Targeting of BET Proteins in Cancer and Immunological Diseases. Curr. Opin. Chem. Biol. 2020, 57, 184–193. 10.1016/j.cbpa.2020.02.003. [DOI] [PubMed] [Google Scholar]
  32. Tang P.; Zhang J.; Liu J.; Chiang C. M.; Ouyang L. Targeting Bromodomain and Extraterminal Proteins for Drug Discovery: From Current Progress to Technological Development. J. Med. Chem. 2021, 64 (5), 2419–2435. 10.1021/acs.jmedchem.0c01487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zengerle M.; Chan K. H.; Ciulli A. Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4. ACS Chem. Biol. 2015, 10 (8), 1770–1777. 10.1021/acschembio.5b00216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tyler D. S.; Vappiani J.; Cañeque T.; Lam E. Y. N.; Ward A.; Gilan O.; Chan Y. C.; Hienzsch A.; Rutkowska A.; Werner T.; Wagner A. J.; Lugo D.; Gregory R.; Molina C. R.; Garton N.; Wellaway C. R.; Jackson S.; Macpherson L.; Figueiredo M.; Stolzenburg S.; Bell C. C.; House C.; Dawson S. J.; Hawkins E. D.; Drewes G.; Prinjha R. K.; Rodriguez R.; Grandi P.; Dawson M. A. Click Chemistry Enables Preclinical Evaluation of Targeted Epigenetic Therapies. Science 2017, 356 (6345), 1397–1401. 10.1126/science.aal2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Divakaran A.; Talluri S. K.; Ayoub A. M.; Mishra N. K.; Cui H.; Widen J. C.; Berndt N.; Zhu J. Y.; Carlson A. S.; Topczewski J. J.; Schonbrunn E. K.; Harki D. A.; Pomerantz W. C. K. Molecular Basis for the N-Terminal Bromodomain-and-Extra-Terminal-Family Selectivity of a Dual Kinase-Bromodomain Inhibitor. J. Med. Chem. 2018, 61 (20), 9316–9334. 10.1021/acs.jmedchem.8b01248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Jafari R.; Almqvist H.; Axelsson H.; Ignatushchenko M.; Lundbäck T.; Nordlund P.; Molina D. M. The Cellular Thermal Shift Assay for Evaluating Drug Target Interactions in Cells. Nat. Protoc. 2014, 9 (9), 2100–2122. 10.1038/nprot.2014.138. [DOI] [PubMed] [Google Scholar]
  37. Lin W.; Chen T. General Stepwise Approach to Optimize a TR-FRET Assay for Characterizing the BRD/PROTAC/CRBN Ternary Complex. ACS Pharmacol. Transl. Sci. 2021, 4 (2), 941–952. 10.1021/acsptsci.1c00032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Devaiah B. N.; Mu J.; Akman B.; Uppal S.; Weissman J. D.; Cheng D.; Baranello L.; Nie Z.; Levens D.; Singer D. S. MYC Protein Stability Is Negatively Regulated by BRD4. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (24), 13457–13467. 10.1073/pnas.1919507117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lovén J.; Hoke H. A.; Lin C. Y.; Lau A.; Orlando D. A.; Vakoc C. R.; Bradner J. E.; Lee T. I.; Young R. A. Selective Inhibition of Tumor Oncogenes by Disruption of Super-Enhancers. Cell 2013, 153 (2), 320–334. 10.1016/j.cell.2013.03.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Raux B.; Voitovich Y.; Derviaux C.; Lugari A.; Rebuffet E.; Milhas S.; Priet S.; Roux T.; Trinquet E.; Guillemot J. C.; Knapp S.; Brunel J. M.; Fedorov A. Y.; Collette Y.; Roche P.; Betzi S.; Combes S.; Morelli X. Exploring Selective Inhibition of the First Bromodomain of the Human Bromodomain and Extra-Terminal Domain (BET) Proteins. J. Med. Chem. 2016, 59 (4), 1634–1641. 10.1021/acs.jmedchem.5b01708. [DOI] [PubMed] [Google Scholar]
  41. Bandopadhayay P.; Piccioni F.; O’Rourke R.; Ho P.; Gonzalez E. M.; Buchan G.; Qian K.; Gionet G.; Girard E.; Coxon M.; Rees M. G.; Brenan L.; Dubois F.; Shapira O.; Greenwald N. F.; Pages M.; Balboni Iniguez A.; Paolella B. R.; Meng A.; Sinai C.; Roti G.; Dharia N. V.; Creech A.; Tanenbaum B.; Khadka P.; Tracy A.; Tiv H. L.; Hong A. L.; Coy S.; Rashid R.; Lin J. R.; Cowley G. S.; Lam F. C.; Goodale A.; Lee Y.; Schoolcraft K.; Vazquez F.; Hahn W. C.; Tsherniak A.; Bradner J. E.; Yaffe M. B.; Milde T.; Pfister S. M.; Qi J.; Schenone M.; Carr S. A.; Ligon K. L.; Kieran M. W.; Santagata S.; Olson J. M.; Gokhale P. C.; Jaffe J. D.; Root D. E.; Stegmaier K.; Johannessen C. M.; Beroukhim R. Neuronal Differentiation and Cell-Cycle Programs Mediate Response to BET-Bromodomain Inhibition in MYC-Driven Medulloblastoma. Nat. Commun. 2019, 10 (1), 2400. 10.1038/s41467-019-10307-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Winter G. E.; Mayer A.; Buckley D. L.; Erb M. A.; Roderick J. E.; Vittori S.; Reyes J. M.; di Iulio J.; Souza A.; Ott C. J.; Roberts J. M.; Zeid R.; Scott T. G.; Paulk J.; Lachance K.; Olson C. M.; Dastjerdi S.; Bauer S.; Lin C. Y.; Gray N. S.; Kelliher M. A.; Churchman L. S.; Bradner J. E. BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment. Mol. Cell 2017, 67 (1), 5–18. 10.1016/j.molcel.2017.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml2c00300_si_001.pdf (2.5MB, pdf)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES