gfp brd3  (Addgene inc)

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , b , Structure ( a ) and BET protein degradation ( b ) of sulfonamide-based PROTAC DAT389. HeLa cells were treated with increasing concentrations of MZ1 or DAT389 for 16 h and BET protein levels were analysed by immunoblot (n = 1). c , Cytotoxicity of IBG1 and VHL-based PROTAC MZ1. MV4;11 and HCT-116 cells were treated with increasing concentrations of compounds for 24 or 96 h, respectively, and cell viability was assessed via CellTiterGlo assay. Dose-response curves were fitted using non-linear regression. n = 2 biological replicates, mean +/− s.d. d , End-point HiBiT protein degradation. BRD2, BRD3 or BRD4 HiBiT knock-in HEK293 cells were treated with the indicated compounds for 5 h and levels of HiBiT-tagged proteins were quantified via the HiBiT lytic detection system. Dose-response curves were fitted using non-linear regression. n = 3 independent experiments, mean +/− s.d. e , Degradation activities of IBG1. BET protein levels were quantified by immunoblotting after compound treatment in HEK293, HCT-116 WT and DCAF15 KO cells. n = 3 independent experiments, mean +/− s.d. Source data, Supplementary Fig. . f , g , In-cell mechanistic evaluation of IBG1. HCT-116 WT ( f ) or DCAF15 knockdown ( g ) cells were treated for 2 h with E7820 (1 µM) or IBG1 (10 nM) alone, or after 1 h pre-treatment with JQ1 (10 µM), MG132 (50 µM) or MLN4924 (3 µM). Western blot representative of 3 ( f ) or 2 ( g ) independent experiments.

Article Snippet: For the engineering of the fluorescent protein stability reporters, the short isoform of BRD4 ( BRD4 (S)) (Twist Bioscience), BRD2 (Addgene plasmid #65376, a gift from K. Miller ) or BRD3 (Addgene plasmid #65377, a gift from K. Miller ) were cloned into a pRRL lentiviral vector, fused to a 3×V5 tag and mTagBFP, and coupled to mCherry for normalization.

Techniques: Western Blot, Knock-In, Knockdown

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , ITC measurement of DCAF16–DDB1(ΔBPB)–DDA1 binding to pre-incubated BRD4 Tandem –IBG1 complex (1:1.1 molar ratio). Data representative of n = 2 independent experiments. DP, differential power. b , TR-FRET ternary complex-formation assay. Europium-labelled anti-His bound to BRD4 Tandem was incubated with equimolar Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 and increasing concentrations of IBG1 or JQ1. Mean ± s.d. of n = 3 technical replicates. c , TR-FRET complex-stabilization assay. His-tagged BRD4 Tandem - or BRD4 BD1 (200 nM) bound to anti-His–europium was incubated with increasing concentrations of Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 in the presence or absence of 1 µM IBG1. Mean ± s.d. of n = 2 independent experiments, each with 2 technical replicates. The asterisk denotes a datapoint that was excluded from non-linear regression fitting. d , e , UV chromatograms from SEC analysis. DCAF16–DDB1(ΔBPB)–DDA1 and BRD4 Tandem alone or mixed at a 2:1 molar ratio in the presence of excess IBG1 ( d ), DCAF16–DDB1(ΔBPB)–DDA1 and BRD4 Tandem mixed at a 1:1 molar ratio in the absence or presence of excess IBG1 ( e ), or DCAF16–DDB1(ΔBPB)–DDA1 mixed with BRD4 BD1 and BRD4 BD2 at a molar ratio of 1:1:1 with excess IBG1 ( e ) were run on an S200 10/300 column. Data representative of n = 2 independent experiments. mAU, milli-absorbance units. f , BET protein stability reporter assay. Tandem mTagBFP fusions with BRD2, BRD3 or BRD4 bromodomains, isolated BRD4 bromodomains or bromodomain chimeras were expressed in KBM7 cells and protein stability was quantified by FACS following treatment with DMSO, IBG1 (1 nM) or dBET6 (10 nM) for 6 h. Mean ± s.d. of n = 3 independent experiments.

Article Snippet: For the engineering of the fluorescent protein stability reporters, the short isoform of BRD4 ( BRD4 (S)) (Twist Bioscience), BRD2 (Addgene plasmid #65376, a gift from K. Miller ) or BRD3 (Addgene plasmid #65377, a gift from K. Miller ) were cloned into a pRRL lentiviral vector, fused to a 3×V5 tag and mTagBFP, and coupled to mCherry for normalization.

Techniques: Binding Assay, Incubation, Tube Formation Assay, Reporter Assay, Isolation

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Electron density (left) and model (right) of the complex formed between DCAF16, DDB1(ΔBPB), BRD4 Tandem (BD1 and BD2) and IBG1. b , Electron density at the DCAF16–IBG1–BRD4 interface. The JQ1 moiety binds to BD2, and the sulfonamide engages BD1. c , UV chromatograms from SEC analysis. Recombinant BRD4 Tandem was incubated with DMSO, JQ1 or IBG1 at a 1:2 molar ratio and run on an S200 10/300 column. Data representative of n = 2 independent experiments. d , A hydrophobic cage formed by DCAF16 residues C58, L59, Y62 and W181 encloses the JQ1 moiety and linker phenyl ring of IBG1. e , Selectivity-determining residue G386 of BD2 at the interface with DCAF16. Colours in b, d , e as in a . f , FACS reporter assay. KBM7 reporter cells expressing wild-type BRD3, BRD4 or indicated single-point mutant bromodomain tandems were treated with IBG1 (1 nM) or dBET6 (10 nM) for 6 h and BET protein stability was evaluated by FACS. Mean ± s.d. of n = 3 independent experiments. g – j , Structure ( g ) and mechanistic characterization ( h – j ) of the dual-JQ1-containing BET degrader IBG3. h , BRD4 degradation. KBM7 reporter cells expressing BRD4 Tandem were treated for 6 h with increasing concentrations of IBG1, IBG3 or dBET6, and BRD4 protein stability was assessed by FACS. Mean ± s.d. of n = 3 independent experiments. i , TR-FRET ternary complex-formation assay. Anti-His–europium bound to BRD4 Tandem was incubated with equimolar Cy5-labelled DCAF16–DDB1(ΔBPB)–DDA1 and increasing concentrations of IBG1, IBG3 or JQ1. Data for JQ1 and IBG1 as in Fig. . Mean ± s.d. of n = 3 technical replicates. j , ITC measurements of DCAF16–DDB1(ΔBPB)–DDA1 complex binding to pre-incubated BRD4 Tandem –IBG3 (1:1.1 molar ratio). Data representative of n = 2 independent experiments.

Article Snippet: For the engineering of the fluorescent protein stability reporters, the short isoform of BRD4 ( BRD4 (S)) (Twist Bioscience), BRD2 (Addgene plasmid #65376, a gift from K. Miller ) or BRD3 (Addgene plasmid #65377, a gift from K. Miller ) were cloned into a pRRL lentiviral vector, fused to a 3×V5 tag and mTagBFP, and coupled to mCherry for normalization.

Techniques: Recombinant, Incubation, Residue, Reporter Assay, Expressing, Mutagenesis, Tube Formation Assay, Binding Assay

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Structure of double JQ1 containing intramolecular bivalent glue degrader IBG2. b , HiBiT degradation assay. HEK293 HiBiT knock-in cells were treated with IBG1, IBG2 or IBG3 for 5 h and levels of BRD2-, BRD3- and BRD4-HiBiT proteins were quantified via HiBiT lytic detection system. Data, n = 3 independent experiments, mean +/− s.d. c , BET protein degradation specificity. KBM7 cells expressing BRD2 Tandem or BRD3 Tandem dual fluorescence reporters were treated with increasing concentrations of IBG1, IBG3 or dBET6 for 6 h and BET protein levels were quantified via flow cytometry. d , Size exclusion chromatograms of BRD4 Tandem incubated with DMSO, MT1, IBG1 or IBG3. Data for DMSO and IBG1 as in Fig. , data representative of n = 2 independent experiments. e , Bromodomain tandem selectivity. KBM7 cells expressing isolated BRD4 bromodomains or mutated BRD4 Tandem constructs were treated with IBG1 (1 nM), IBG3 (0.1 nM) or dBET6 (10 nM) for 6 h and protein levels were evaluated via flow cytometry. f , BRD4 stability CRISPR screen. KBM7 iCas9 BRD4 dual fluorescence reporter cells expressing a CRL-focused sgRNA library were treated with IBG3 (0.1 nM) for 6 h before flow cytometric cell sorting as in Fig. . 20 S proteasome subunits (blue), COP9 signalosome subunits (cyan) and E1 or E2 ubiquitin enzymes (purple) inside the scoring window (one-sided MAGeCK p-value < 0.01, fold-change > 1.5; dashed lines) are highlighted. g , DCAF16 dependency. BRD4(S) dual fluorescence reporter KBM7 iCas9 cells were lentivirally transduced with a DCAF16 -targeting sgRNA and 3 days post Cas9 induction cells were treated with DMSO, IBG1 (1 nM) or IBG3 (0.1 nM) for 6 h before FACS-based quantification of BRD4 levels. h , Bromodomain arrangement. KBM7 cells expressing dual fluorescence reporters harbouring tandems of either BD1 or BD2 of BRD4 were treated with DMSO, IBG1 (1 nM), IBG3 (0.1 nM) or dBET6 (10 nM) for 6 h and analysed by flow cytometry. i , j , Structures ( i ) and HiBiT-BRD4 degradation activity ( j ) of bivalent BET inhibitors MT1 and MS645 after treatment for 24 h. Data for c , e , g , h , n = 3 independent experiments, mean +/− s.d. Data in j , mean of n = 2 independent experiments.

Article Snippet: For the engineering of the fluorescent protein stability reporters, the short isoform of BRD4 ( BRD4 (S)) (Twist Bioscience), BRD2 (Addgene plasmid #65376, a gift from K. Miller ) or BRD3 (Addgene plasmid #65377, a gift from K. Miller ) were cloned into a pRRL lentiviral vector, fused to a 3×V5 tag and mTagBFP, and coupled to mCherry for normalization.

Techniques: Degradation Assay, Knock-In, Expressing, Fluorescence, Flow Cytometry, Incubation, Isolation, Construct, CRISPR, FACS, Ubiquitin Proteomics, Transduction, Activity Assay

Journal: Nature

Article Title: Targeted protein degradation via intramolecular bivalent glues

doi: 10.1038/s41586-024-07089-6

Figure Lengend Snippet: a , Bromodomain tandem specificity. KBM7 cells expressing bromodomain mutant BRD4 Tandem dual fluorescence reporters were treated with DMSO, IBG4 (100 nM) or dBET6 (10 nM) for 6 h and analysed by flow cytometry. b , NanoBRET bromodomain dimerization assay. Indicated compounds were titrated into HEK293 cells transiently expressing BRD4 Nluc-Tandem-HaloTag . Data, mean of n = 2 independent experiments. c , alphaLISA displacement assay. Increasing concentrations of JQ1, E7820 or the pyrazolo pyrimidine warhead of IBG4 were titrated against His-tagged BRD4 bromodomains and biotinylated JQ1 probe. n = 3 technical replicates, mean +/− s.d. d , BET protein selectivity. Bromodomain tandem BRD2, BRD3 or BRD4 dual fluorescence reporter KBM7 cells were treated with DMSO, IBG4 (100 nM) or dBET6 (10 nM) for 6 h and analysed by flow cytometry. e , Mechanistic FACS reporter assay. KBM7 BRD4 dual fluorescence reporter cells were co-treated with IBG1 (1 nM) or IBG4 (100 nM) and Carfilzomib (1 µM), MLN4924 (1 µM) or TAK243 (0.5 µM) for 6 h and BRD4 levels were analysed via flow cytometry. Data, mean of n = 2 independent experiments. f , DCAF16-independence of IBG4. KBM7 iCas9 WT or DCAF16 knockout cells expressing BRD4(S) dual fluorescence reporter were treated with DMSO, IBG1 (1 nM) or IBG4 (100 nM) for 6 h and BRD4 degradation was assessed via flow cytometry. g , AlphaFold (AlphaFold Monomer v2.0 pipeline) prediction of DCAF11 (red) bound to DDB1 (blue). h , i , Size exclusion chromatograms of different combinations of DCAF11, BRD4 Tandem and IBG4 ( h ), data representative of n = 2 independent experiments, and corresponding peak fractions run on SDS-PAGE ( i ). Data for a , d , f , n = 3 independent experiments, mean +/− s.d.

Article Snippet: For the engineering of the fluorescent protein stability reporters, the short isoform of BRD4 ( BRD4 (S)) (Twist Bioscience), BRD2 (Addgene plasmid #65376, a gift from K. Miller ) or BRD3 (Addgene plasmid #65377, a gift from K. Miller ) were cloned into a pRRL lentiviral vector, fused to a 3×V5 tag and mTagBFP, and coupled to mCherry for normalization.

Techniques: Expressing, Mutagenesis, Fluorescence, Flow Cytometry, Reporter Assay, Knock-Out, SDS Page