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. 2017 May 15;56(21):5738-5743.
doi: 10.1002/anie.201611281. Epub 2017 Apr 18.

Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands

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Free PMC article

Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands

David Remillard et al. Angew Chem Int Ed Engl. .
Free PMC article

Abstract

The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.

Keywords: bromodomain; cancer; drug design; epigenetics; protein degradation.

Figures

Figure 1
Figure 1
Design and characterization of thienopyridinone BRD9-targeted degraders. A) Structures of select BRD9 bromodomain probes. B) Schematic representation of degrader design. C) Vehicle-normalized BRD9(bd) displacement (AlphaScreen quadruplicate means +/− SEM). D) Compound-induced ternary complex formation of recombinant BRD9(bd) and CRBN-DDB1 (AlphaScreen quadruplicate means +/− SEM). E) Cocrystal structure of 3 with BRD9(bd) (PDB 5TWX). F) Docking of (E) into the published CRBN-DDB1 (4CI3).
Figure 2
Figure 2
Performance of thienopyrininone degraders. A) Immunoblot for BRD9 and actin after 4 hour treatment of MOLM-13 with indicated concentrations of 1, 2, and 3. B) Chemical structures of 4 and 5. C) Vehicle-normalized CRBN-DDB1 displacement (AlphaScreen quadruplicate means +/− SEM). D) Immunoblot for BRD9 and actin after 4 hour treatment of MOLM-13 with indicated concentrations of 4 and 5.
Figure 3
Figure 3
Temporal and mechanistic characterization of BRD9 degradation by 5. A) Immunoblot for BRD9 and actin after treatment of MOLM-13 Cells with 100nM 5 for the indicated times. B) Immunoblot for BRD9 and actin after a 4hr pre-treatment of MM.1S cells with vehicle, I-BRD9, Lenalidomide,Carfilzomib*, or MLN-4924, followed by a 2-hour treatment with 5 (100 nM). * Car pretreatment 30 min. C) Immunoblot for BRD9 and actin after 4 hour treatment with 5 at the indicated doses in MM.1Swt or MM.1SCRBN−/− cells.
Figure 4
Figure 4
Napthiridinone degrader 6 (dBRD9) offers improved biochemical and cellular selectivity. A) Selectivity of phage-displayed bromodomain displacement by 5 (Bromoscan). B) Chemical structure of dBRD9. C) Immunoblot of BRD9 and actin after 4hr treatment of MOLM-13 cells with indicated concentrations of dBRD9. D) Selectivity of phage-displayed bromodomain displacement by dBRD9 (Bromoscan). E) Compound-induced ternary complex formation of recombinant BRD9(bd) and CRBN-DDB1 (AlphaScreen quadruplicate means +/− SEM). F) Compound-induced ternary complex formation of recombinant BRD4(1) and CRBN-DDB1 as in (E). G) Immunoblot for BRD7 and actin after 4hr treatment of MOLM-13 cells with indicated concentrations of 5 or dBRD9. H) Immunoblot for BRD4 following treatment as in (G).
Figure 5
Figure 5
dBRD9 selectivity established by whole-cell lysate proteomics. Fold change in relative abundance of 7326 proteins quantified from MOML-13 cells treated for two hours with dBRD9 (100nM) or vehicle (DMSO), versus q-value for quintuplicate replicates.
Figure 6
Figure 6
Impact of BRD9 degradation on cultured human leukemia lines. A) Viability of EOL-1 and MOML-13 cell lines treated for 7 days with the indicated compounds (ATP-Lite quadruplicate means +/− SEM). B) Viability of MOLM-13 AML measured as in (A) following transduction with recombinant BRD9 alleles or vector control.

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