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[Preprint]. 2023 Feb 15:2023.02.14.528208.
doi: 10.1101/2023.02.14.528208.

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Yen-Der Li  1   2   3 , Michelle W Ma  1   4   5 , Muhammad Murtaza Hassan  6 , Moritz Hunkeler  4   5 , Mingxing Teng  7 , Kedar Puvar  4   5 , Ryan Lumpkin  4   5 , Brittany Sandoval  2 , Cyrus Y Jin  4   5 , Scott B Ficarro  4   8 , Michelle Y Wang  4 , Shawn Xu  2 , Brian J Groendyke  4 , Logan H Sigua  4 , Isidoro Tavares  4   8 , Charles Zou  2 , Jonathan M Tsai  2   3   9 , Paul M C Park  2   3 , Hojong Yoon  2   3 , Felix C Majewski  6 , Jarrod A Marto  4   8   9 , Jun Qi  4 , Radosław P Nowak  4   5 , Katherine A Donovan  4   5 , Mikołaj Słabicki  2   3 , Nathanael S Gray  6 , Eric S Fischer  4   5 , Benjamin L Ebert  2   3   10
Affiliations

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders

Yen-Der Li et al. bioRxiv. .

Update in

  • Template-assisted covalent modification underlies activity of covalent molecular glues.
    Li YD, Ma MW, Hassan MM, Hunkeler M, Teng M, Puvar K, Rutter JC, Lumpkin RJ, Sandoval B, Jin CY, Schmoker AM, Ficarro SB, Cheong H, Metivier RJ, Wang MY, Xu S, Byun WS, Groendyke BJ, You I, Sigua LH, Tavares I, Zou C, Tsai JM, Park PMC, Yoon H, Majewski FC, Sperling HT, Marto JA, Qi J, Nowak RP, Donovan KA, Słabicki M, Gray NS, Fischer ES, Ebert BL. Li YD, et al. Nat Chem Biol. 2024 Dec;20(12):1640-1649. doi: 10.1038/s41589-024-01668-4. Epub 2024 Jul 29. Nat Chem Biol. 2024. PMID: 39075252 Free PMC article.

Abstract

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

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Conflict of interest statement

Competing Interests B.L.E. has received research funding from Celgene, Deerfield, Novartis, and Calico and consulting fees from GRAIL. He is a member of the scientific advisory board and shareholder for Neomorph Inc., TenSixteen Bio, Skyhawk Therapeutics, and Exo Therapeutics. E.S.F is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (board of directors). He is an equity holder and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital, and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. N.S.G. is a founder, science advisory board member (SAB) and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GSK, Larkspur (board member), Shenandoah (board member), and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline and Sanofi. M.S. has received research funding from Calico Life Sciences LLC. K.A.D is a consultant to Kronos Bio and Neomorph Inc. J.Q. is an equity holder of Epiphanes, Talus Bioscience, and receives or has received research funding from Novartis. J.A.M. is a founder, equity holder, and advisor to Entact Bio, serves on the SAB of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks and TUO Therapeutics. K.P. is currently employed by Abbvie. B.J.G. is currently employed by Blueprint Medicines.

Figures

Extended Data Figure 1.
Extended Data Figure 1.. Degradation characterization of JQ1-derived compounds.
a. The domain structure of BRD4. b. Schematic of BRD4BD stability reporter. IRES, internal ribosome entry site. c. Flow cytometry analysis of BRD4BD1-eGFP and BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of GNE11 for 16 h (n=3). d. Western blots of BRD4 degradation in K562 cells that were treated with JQ1, TMX1 or GNE11 at 1 μM for increasing time points. e. Flow cytometry analysis of BRD4BD1-eGFP and BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of TMX1 for 16 h (n=3). f. Quantitative whole proteome analysis of K562 cells after treatment with JQ1 at 0.5 μM (n=1) or DMSO (n=3) for 5 h. g. Western blots of BRD4 degradation in K562 cells that were treated with DMSO, TMX1 at 1 μM, GNE11 at 1 μM, MG132 at 10 μM, MLN7243 at 1 μM, and MLN4924 at 1 μM for 16 h.
Extended Data Figure 2.
Extended Data Figure 2.. UPS-targeted BRD4BD2 reporter CRISPR screen for JQ1-derived compounds.
a. Schematic of the CRISPR degradation screen for BRD4BD2 stability. b. UPS-focused CRISPR degradation screen for BRD4BD2-eGFP stability in K562-Cas9 cells treated with GNE11 at 1 μM for 16 h (n=2). c. UPS-focused CRISPR degradation screen for BRD4BD2-eGFP stability in 293T-Cas9 cells treated with TMX1 at 1 μM for 16 h (n=2). d. UPS-focused CRISPR degradation screen for BRD4BD2-eGFP stability in 293T-Cas9 cells treated with GNE11 at 1 μM for 16 h (n=2).
Extended Data Figure 3.
Extended Data Figure 3.. Genome-scale resistance CRISPR screen for JQ1-derived compounds.
a. Schematic of the CRISPR resistance screen. b. Genome-wide CRISPR resistance screen in K562-Cas9 cells treated with GNE11 at 0.1 μM (n=3) or DMSO (n=3) for 14 days. c. Genome-wide CRISPR resistance screen in K562-Cas9 cells treated with JQ1 at 0.1 μM (n=3) or DMSO (n=3) for 14 days. d. Flow cytometry-based competitive growth assay of K562-Cas9 cells expressing BFP- or RFP-tagged sgRNAs against DCAF16 and non-targeting control (NTC) treated with DMSO, JQ1 at 0.1 μM, TMX1 at 0.1 μM, or GNE11 at 0.1 μM for increasing time points (n=3).
Extended Data Figure 4.
Extended Data Figure 4.. Covalent recruitment of DCAF16 to BRD4BD2.
a. Flag immunoprecipitation (IP) followed by mass spectrometry in 293T cells overexpressing BRD4BD2-Flag of cells treated with either MLN4924 plus GNE11 both at 1 μM (n=4), or MLN4924 at 1 μM only (n=4). Fold enrichment and p-values were calculated by comparing GNE11/MLN4924 treated samples to MLN4924 only control samples. b. Schematic of the TR-FRET set-up. Positions of FRET donor (terbium-coupled streptavidin) and acceptor (BODIPY–SpyCatcher) are indicated in the structural model. c. TR-FRET signal for DDB1-DCAF16-BODIPY to BRD4BD1-terbium or BRD4BD2-terbium with increasing concentrations of GNE11 (n=3). d. Intact protein mass spectra of DDB1-DCAF16 co-incubated with GNE11 at 25°C for 16 h, or DDB1-DCAF16 co-incubated with GNE11 and BRD4BD2 at 25°C for 16 h. e. Intact protein mass spectra of DDB1-DCAF16 alone, DDB1-DCAF16 co-incubated with KB02-JQ1 at 4°C for 16 h, or DDB1-DCAF16 co-incubated with KB02-JQ1 and BRD4BD2 at 4°C for 16 h.
Extended Data Figure 5.
Extended Data Figure 5.. Optimized electrophilic warheads increases potency of degraders.
a. Flow cytometry analysis of BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of JQ1, GNE11, TMX1, MMH1 or MMH2 for 16 h (n=3). b. Flow analysis of BRD4BD2-eGFP degradation in K562 cells that were treated with JQ1 at 1 μM, TMX1 at 1 μM, GNE11 at 1 μM, MMH1 at 0.1 μM or MMH2 at 0.1 μM for increasing time points (n=3). c. Flow cytometry analysis of BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of MMH1 or MMH2 for 16 h (n=3). d. TR-FRET signal for DDB1-DCAF16-BODIPY to BRD4BD2-terbium with increasing concentrations of JQ1, GNE11, TMX1, MMH1 or MMH2 (n=3). e. Flow cytometry analysis of BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of MMH1, MMH2, dBET6 or MZ1 for 2 h, 6 h, or 16 h (n=3). f. Western blot of BRD4 degradation in K562 cells pre-treated with MMH1, MMH2, dBET6 or MZ1 at 0.1 μM for 4 h, washed with PBS and resuspended in fresh media or the same drug-treated media for an additional 20 h.
Extended Data Figure 6.
Extended Data Figure 6.. MMH1 and MMH2 conserve the mechanism of action as TMX1 and GNE11.
a. Flow cytometry analysis of BRD4BD1-eGFP and BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of MMH1 for 16 h (n=3). b. Flow cytometry analysis of BRD4BD1-eGFP and BRD4BD2-eGFP degradation in K562 cells that were treated with increasing concentrations of MMH2 for 16 h (n=3). c. Quantitative whole proteome analysis of K562 cells after treatment with MMH1 at 0.1 μM (n=2) or DMSO (n=4) for 5 h. d. Quantitative whole proteome analysis of K562 cells after treatment with MMH2 at 0.1 μM (n=2) or DMSO (n=4) for 5 h. e. Intact protein mass spectra of DDB1-DCAF16 co-incubated with MMH1 at 4°C for 16 h, or DDB1-DCAF16 co-incubated with MMH1 and BRD4BD2 at 4°C for 16 h. f. Intact protein mass spectra of DDB1-DCAF16 co-incubated with MMH2 at 4°C for 16 h, or DDB1-DCAF16 co-incubated with MMH2 and BRD4BD2 at 4°C for 16 h.
Extended Data Figure 7.
Extended Data Figure 7.. Cryo-EM processing workflow of DDB1ΔB-DDA1-DCAF16 in complex with BRD4BD2 and MMH2.
a. Overview of processing workflow for the DDB1ΔB-DDA1-DCAF16-BRD4BD2-MMH2 dataset, from raw micrographs (low pass-filtered to 5 Å) to final maps. All steps performed in cryoSPARC. Particles belonging to colored volumes were taken into the subsequent steps. Maps here and in following panels (unless noted otherwise) are contoured at 0.3 (clusters), 0.743 (initial consensus), 0.6 (final from cryoSPARC), 0.2 (final from deepEMhancer). b. FSC plot. c. Viewing distribution for the final reconstruction. d. Local resolution mapped onto final map. e. Model-to-map FSC, dotted lines indicate FSC=0.5 and FSC=0.143. f. 3D FSC plot and directional resolution histogram.
Extended Data Figure 8.
Extended Data Figure 8.. Map quality of the DCAF16-BRD4BD2-MMH2 interface.
a. Cryo-EM density for DCAF16 containing Cys58 covalently bound to MMH2. Map contoured at 0.251. b. Cryo-EM density for DCAF16. c. Cryo-EM density for BRD4BD2. d. Overlay of MMH2 with JQ1 (PDB: 3ONI, in white). e. Key residues on DCAF16 (in green and blue) and BRD4BD2 (in magenta) close to MMH2.
Extended Data Figure 9.
Extended Data Figure 9.. DCAF16 alanine-scanning reporter screen for BRD4 molecular glue degraders.
a. Schematic of DCAF16 alanine mutagenesis screen for BRD4BD2-eGFP degradation in DCAF16 knockout K562 cells. b. DCAF16 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with GNE11 at 1 μM for 16 h (n=3). c. DCAF16 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with TMX1 at 1 μM for 16 h (n=3). d. DCAF16 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with MMH1 at 0.1 μM for 16 h (n=3). e. DCAF16 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with MMH2 at 0.1 μM for 16 h (n=3).
Extended Data Figure 10.
Extended Data Figure 10.. Cellular validation of representative DCAF16 mutants.
a. Western blots of BRD4 degradation in DCAF16 knockout K562 cells that were transduced with indicated HA-DCAF16 mutants and treated with DMSO or MMH1 at 0.1 μM for 16 h. b. Flag immunoprecipitation (IP) followed by Western blots in the presence of DMSO or MMH1 at 0.1 μM from 293T cells transfected with indicated HA-DCAF16 mutants and BRD4BD2-Flag constructs. c. Western blots of BRD4 degradation in DCAF16 knockout K562 cells that were transduced with indicated HA-DCAF16 mutants and treated with DMSO or MMH2 at 0.1 μM for 16 h. d. Flag immunoprecipitation (IP) followed by Western blots in the presence of DMSO or MMH2 at 0.1 μM from 293T cells transfected with indicated HA-DCAF16 mutants and BRD4BD2-Flag constructs. e. Western blots of BRD4 degradation in DCAF16 knockout K562 cells that were transduced with indicated HA-DCAF16 mutants and treated with DMSO or GNE11 at 1 μM for 16 h. f. Flag immunoprecipitation (IP) followed by Western blots in the presence of DMSO or GNE11 at 1 μM from 293T cells transfected with indicated HA-DCAF16 mutants and BRD4BD2-Flag constructs.
Extended Data Figure 11.
Extended Data Figure 11.. DCAF16 alanine-scanning reporter screen and validation.
a. DCAF16 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with KB02-JQ1 at 10 μM for 16 h (n=3). b. Flow analysis of BRD4BD2-eGFP degradation in DCAF16 knockout K562 cells transduced with indicated HA-DCAF16 mutants and treated with increasing concentrations of KB02-JQ1 for 16 h (n=3). c. TR-FRET signal for DDB1-DCAF16(WT)- or DDB1-DCAF16(C58S)-BODIPY to BRD4BD2-terbium with increasing concentrations of MMH2 (n=3). d. Close up of DCAF16 Ala53 orienting towards the hydrophobic core. e. Close up of DCAF16 Cys177 and Cys179 coordinating a structural zinc ion. f. Intact protein mass spectra of DDB1-DCAF16(WT) co-incubated with MMH2, DDB1-DCAF16(WT) co-incubated with MMH2 and BRD4BD2, DDB1-DCAF16(C58S) co-incubated with MMH2, or DDB1-DCAF16(C58S) co-incubated with MMH2 and BRD4BD2 at 4°C for 16
Extended Data Figure 12.
Extended Data Figure 12.. BRD4BD2 alanine-scanning reporter screen for BRD4 molecular glue degraders.
a. Schematic of alanine mutagenesis degradation screen of the BRD4 BD2 domain in K562 cells. b. BD2 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with GNE11 at 1 μM for 16 h (n=2). c. BD2 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with TMX1 at 1 μM for 16 h (n=2). d. BD2 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with MMH1 at 0.1 μM for 16 h (n=3). e. BD2 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with MMH2 at 0.1 μM for 16 h (n=3).
Extended Data Figure 13.
Extended Data Figure 13.. BRD4BD2 alanine-scanning reporter screen and mechanism of bromodomain selectivity.
a. BD2 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with dBET6 at 1 μM for 16 h (n=2). b. BD2 alanine mutagenesis screen for BRD4BD2-eGFP stability in K562 cells treated with MZ1 at 1 μM for 16 h (n=2). c. Correlation of fold change for two BRD4BD2 alanine mutagenesis screens. The x axis is a degradation screen for BRD4BD2-eGFP in K562 cells upon treatment with TMX1 at 1 μM for 16 h (n=2), and the y axis is another degradation screen for BRD4BD2-eGFP in K562 cells upon treatment with MZ1 at 1 μM for 16 h (n=2). d. Overlay of BRD4BD1 (PDB: 3MXF, in yellow) with BRD4BD2 (in magenta) showing a close-up of residues His437. When substituted for Asp144 in BRD4BD1, there is repulsion between Asp144 and the JQ1 carbonyl. e. Overlay of BRD2BD2 (PDB: 3ONI, in cyan) with BRD4BD2 (in magenta) showing a close-up of residues His437 and the corresponding His433 in BRD2BD2. f. AlphaScreen competitive assay of JQ1, GNE11, and TMX1 to quantify the drug’s inhibition of binding between biotinylated-JQ1 and His-tagged BRD4BD1 or BRD4BD2 (n=4).
Figure 1.
Figure 1.. JQ1-derived compounds degrade BRD4 via DCAF16.
a. Chemical structures of JQ1, GNE11 and TMX1. b. Western blots of BRD4 degradation in K562 cells treated with DMSO or different concentrations of JQ1, TMX1 and GNE11 for 16 h. c. Quantitative whole proteome analysis of K562 cells after treatment with TMX1 at 0.5 μM (n=2) or DMSO (n=3) for 5 h. d. Ubiquitin Proteasome System (UPS)-focused CRISPR degradation screen for BRD4BD2-eGFP stability in K562-Cas9 cells treated with TMX1 at 1 μM for 16 h (n=2). e. Western blots of BRD4 degradation in DCAF16 and non-targeting control (NTC) sgRNA infected K562-Cas9 cells treated with DMSO, TMX1 at 1 μM, GNE11 at 1 μM for 16 h. f. Genome-wide CRISPR resistance screen in K562-Cas9 cells after treatment with TMX1 at 0.1 μM (n=3) or DMSO (n=3) for 14 days. g. Flag immunoprecipitation (IP) followed by mass spectrometry in 293T cells overexpressing BRD4BD2-Flag of cells treated with either MLN4924 plus TMX1 both at 1 μM (n=4), or MLN4924 at 1 μM only (n=4). Fold enrichment and p-values were calculated by comparing TMX1/MLN4924 treated samples to MLN4924 only control samples.
Figure 2.
Figure 2.. Covalent recruitment of DCAF16 to BRD4BD2 and optimized electrophilic warheads.
a. TR-FRET signal for DDB1-DCAF16-BODIPY to BRD4BD1-terbium or BRD4BD2-terbium with increasing concentrations of TMX1 (n=3). b. Intact protein mass spectra of DDB1-DCAF16 alone, DDB1-DCAF16 co-incubated with TMX1 at 4°C for 16 h, or DDB1-DCAF16 co-incubated with TMX1 and BRD4BD2 at 4°C for 16 h. c. Chemical structures of MMH1, MMH2, MMH1-NR, MMH2-NR. d. Western blot of BRD4 degradation in K562 cells that were treated with DMSO or different concentrations of MMH1, MMH2, dBET6, or MZ1 for 6 h. e. TR-FRET signal for DDB1-DCAF16-BODIPY to BRD4BD2-terbium with increasing concentrations of JQ1, MMH1, MMH2, MMH1-NR and MMH2-NR (n=3). f. Western blots of BRD4 degradation in K562 cells that were treated with DMSO or different concentrations of MMH1, MMH1-NR, MMH2, or MMH2-NR for 16 h.
Figure 3.
Figure 3.. BRD4BD2 orients MMH2 for DCAF16 modification.
a. 2.2 Å cryo-EM map of the DDB1ΔB-DDA1-DCAF16-BRD4BD2-MMH2 colored to indicate DDB1BPA (red), DDB1BPC (orange), DDB1CTD (gray), DDA1 (yellow), DCAF16CTD (blue), DCAF16NTD (green), DCAF16HLH (cyan), and BRD4BD2 (magenta). Map shown has been processed with DeepEMhancer b. Cartoon representation of the DDB1-DCAF16 ligase complex bound to BRD4BD2 and MMH2 with same coloring as the cryo-EM map. A sequence scheme for all complex partners is shown at the bottom. c. Cartoon representation of DCAF16 indicating secondary structure elements. d. Close-up of MMH2 covalently modifying DCAF16Cys58 with cryo-EM density around MMH2 shown as mesh.
Figure 4.
Figure 4.. DCAF16Cys58 is targeted by molecular glue degraders.
a. Correlation of fold change for two DCAF16 alanine scans in DCAF16 knockout K562 cells. The x axis is a degradation screen for BRD4BD2-eGFP upon treatment with TMX1 at 1 μM for 16 h (n=3), and the y axis is another degradation screen for BRD4BD2-eGFP upon treatment with KB02-JQ1 at 10 μM for 16 h (n=3). b. Western blots of BRD4 degradation in DCAF16 knockout K562 cells that were transduced with indicated HA-DCAF16 mutants, and treated with DMSO or TMX1 at 1 μM for 16 h. c. Flag immunoprecipitation (IP) followed by Western blots in the presence of DMSO or TMX1 at 1 μM from 293T cells transfected with indicated HA-DCAF16 mutants and BRD4BD2-Flag constructs. d. TR-FRET signal for DDB1-DCAF16(WT)- or DDB1-DCAF16(C58S)-BODIPY to BRD4BD2-terbium with increasing concentrations of TMX1 (n=3). e. Intact protein mass spectra of DDB1-DCAF16(WT) or DDB1-DCAF16(C58S) co-incubated with TMX1 and BRD4BD2 at 4°C for 16 h.
Figure 5.
Figure 5.. BRD4BD2 residues critical for interface conformation confer selectivity.
a. Correlation of fold change for two BRD4BD2 alanine mutagenesis screens. The x axis is a degradation screen for BRD4BD2-eGFP in K562 cells upon treatment with TMX1 at 1 μM for 16 h (n=2), and the y axis is another degradation screen for BRD4BD2-eGFP in K562 cells upon treatment with dBET6 at 1 μM for 16 h (n=2). b. Flow cytometry analysis of K562 cells expressing wild-type or indicated mutant BRD4BD2-eGFP construct and treated with DMSO, GNE11 at 1 μM, TMX1 at 1 μM, MMH1 at 0.1 μM, MMH2 at 0.1 μM, MZ1 at 1 μM or dBET6 at 1 μM for 16 h (n=3). c. Flow cytometry analysis of K562 cells expressing the indicated BRD4BD2-eGFP, BRD4BD1-eGFP mutant construct and treated with increasing concentrations of TMX1 for 16 h (n=3). d. Flag immunoprecipitation (IP) followed by Western blots in the presence of DMSO or TMX1 at 1 μM from 293T cells transfected with HA-DCAF16 and indicated BRD4BD2-Flag, BRD4BD1-Flag mutant constructs. e. Schematic model for the mechanism of action of covalent BRD4 molecular glue degraders.
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