dda1  (Proteintech)

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , Schematic representation of the FACS reporter system and CRISPR/Cas9 screen. KBM7 iCas9 cells expressing a BRD4-BFP protein stability reporter were transduced with a CRL-focused sgRNA library. After Cas9 induction via doxycycline, cells were treated with DMSO, 10 nM MZ1, 1 nM compound 1 or 1 µM GNE-0011 for 6 hours and sorted based on BRD4-BFP levels. b , KBM7 BRD4-reporter iCas9 CRISPR screen. Average gene-level fold-changes and p-values of BRD4 HIGH and BRD4 LOW cell populations compared to BRD4 MID fraction were calculated using MAGeCK . Essential control genes (BRD4 LOW ) and 20S proteasome subunits, COP9 signalosome subunits and E1 or E2 ubiquitin enzymes (BRD4 HIGH ) inside the scoring window (p-value < 0.01, fold-change > 1.5) are highlighted. c , CRISPR/Cas9 viability screen. HCT-116 cells were transduced with Cas9 and a ubiquitin and NEDD8-system-focused sgRNA library and upon selection treated with compound 1 (58 nM; 4-fold IC 50 ) for 6 days. Average gene-level fold-changes and p-values compared to DMSO treated cells were calculated using MAGeCK. Genes showing > 2-fold enrichment or depletion and p-value < 0.01 are highlighted. d , Flow cytometric screen validation. KBM7 iCas9 BRD4-BFP reporter cells were transduced with the indicated sgRNAs and treated with DMSO, dBET6 (10 nM), GNE-0011 or compound 1 as above and BRD4-BFP levels were quantified. BRD4-BFP/mCherry ratio is normalized to DMSO treated sgAAVS1 cells. e , DCAF16 knockout/rescue. KBM7 iCas9 cells were lentivirally transduced with the indicated DCAF16-targeting or AAVS1 control sgRNAs, as well as a sgRNA resistant DCAF16 cDNA. After knockout of endogenous DCAF16, BRD4 expression levels were assessed via immunoblot. f , Viability assay. KBM7 iCas9 sgAAVS1 control or DCAF16 knockout cells were treated with increasing doses of dBET6, JQ1, GNE-0011 or compound 1 for 72 hours and vell viability was evaluated by CellTiterGlo assay. Dose-response curves fitted using non-linear regression. g , Fluorescence polarization (FP) binary binding assay. FITC-labelled sulfonamide probe (top) was titrated into DCAF15:DDB1ΔBPB:DDA1 or DCAF16:DDB1ΔBPB:DDA1. Data in f - g are shown as mean +/-s.d. (n = 3 technical replicates).

Article Snippet: Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16-DDB1ΔBPB-DDA1, His 6 -BRD4 BD1 , His 10 -BRD4 BD2 , His 10 -BRD4 Tandem , compound 1 and LANCE Eu-W1024 Anti-6xHis donor (PerkinElmer) were prepared in TR-FRET assay buffer (20 mM HEPES pH 7, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20).

Techniques: CRISPR, Expressing, Transduction, Control, Selection, Knock-Out, Western Blot, Viability Assay, Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , MZ1 BRD4-reporter iCas9 CRISPR screen. Average gene-level fold-changes and p-values of BRD4 HIGH and BRD4 LOW cell populations compared to BRD4 MID fraction were calculated using MAGeCK as in . b - g , Immunoblot-based CRISPR/Cas9 screen validation. KBM7 ( b ) or HCT-116 cells ( c - f ) were transduced with sgRNA expressing lentivirus ( b ) or transfected with siRNA pools ( c - f ) targeting the indicated genes and treated with DMSO, compounds 1 or 2 , GNE-0011 or dBET6 for 2 h ( c - f ) or 6 h ( b ) at the indicated concentrations and BET protein levels were analysed via immunoblotting. g , DCAF16 knockout/rescue. KBM7 iCas9 BRD4(S)-BFP reporter cells were transduced with lentivirus expressing AAVS1 or two different DCAF16-targeting sgRNAs as well as an sgRNA-resistant DCAF16 expression vector. After 3 days of dox-induced Cas9 expression, cells were treated with DMSO, GNE-0011 (1 µM), compound 1 (1 nM) or dBET6 (10 nM) for 6 h and BRD4-BFP levels analysed via flow cytometry. Data shown for n = 3 biological replicates, mean +/- s.d.

Article Snippet: Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16-DDB1ΔBPB-DDA1, His 6 -BRD4 BD1 , His 10 -BRD4 BD2 , His 10 -BRD4 Tandem , compound 1 and LANCE Eu-W1024 Anti-6xHis donor (PerkinElmer) were prepared in TR-FRET assay buffer (20 mM HEPES pH 7, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20).

Techniques: CRISPR, Western Blot, Transduction, Expressing, Transfection, Knock-Out, Plasmid Preparation, Flow Cytometry

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , Size exclusion chromatography (SEC) UV chromatogram. DCAF16:DDB1ΔBPB:DDA1 or BRD4 Tandem in the with or without excess compound 1 were run on an S200 10/300 column as in , b. b , Schematic representation of alphaLISA displacement assay in . His-tagged BRD4 Tandem is mixed with a biotinylated-JQ1 probe. Binding of the probe to BRD4 bromodomains in the presence of nickel-chelate acceptor beads and streptavidin-europium donor beads induces proximity of donor and acceptor, energy transfer and light emission (left). Competition of the probe from BRD4 Tandem by compound 1 disrupts donor-acceptor proximity and energy transfer, and the presence of DCAF16 further strengthens compound 1 binding over probe binding (positive cooperativity; α > 1; right). c - f , Protein stability reporter assay. WT or truncated forms of BRD2, BRD3 or BRD4 ( c , e ) fused to mTagBFP were stably expressed in KBM7 cells and protein stability after 6-hour treatment with DMSO, GNE-0011 (1 µM), compound 1 (1 nM) or dBET6 (10 nM) was quantified via flow cytometric evaluation of the mTagBFP/mCherry ratio ( d , f ) as in . BD, bromodomain; NPS, N-terminal phosphorylation sites; BID, basic residue-enriched interaction domain; ET, extraterminal domain; SEED, Serine/Glutamic acid/Aspartic acid-rich region; NLS, Nuclear localization signal. Data shown are from n = 3 biological replicates, mean +/- s.d. g , h , BromoTag degradation. HEK293 cells stably expressing BromoTag-MCM4 were treated for 5 hours with DMSO, BromoTag degrader AGB1 and non-degrader cis-AGB1 , compound 1 , or ‘bumped’ analogue compound 5 ( g ) and BromoTag-MCM4 levels were analysed by immunoblotting ( h ). Data representative of n = 2 replicates.

Article Snippet: Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16-DDB1ΔBPB-DDA1, His 6 -BRD4 BD1 , His 10 -BRD4 BD2 , His 10 -BRD4 Tandem , compound 1 and LANCE Eu-W1024 Anti-6xHis donor (PerkinElmer) were prepared in TR-FRET assay buffer (20 mM HEPES pH 7, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20).

Techniques: Size-exclusion Chromatography, Binding Assay, Reporter Assay, Stable Transfection, Residue, Expressing, Western Blot

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , b , Size exclusion chromatography (SEC) UV chromatograms. DCAF16:DDB1ΔBPB:DDA1 and BRD4 Tandem alone or mixed at a 2:1 molar ratio in the presence of excess compound 1 ( a ), DCAF16:DDB1ΔBPB:DDA1 and BRD4 Tandem mixed at a 1:1 molar ratio in the absence or presence of excess compound 1 ( b ), as well as DCAF16:DDB1ΔBPB:DDA1 mixed with BRD4 BD1 and BRD4 BD2 at a molar ratio of 1:1:1 with excess compound 1 ( b ) were run on an S200 10/300 column. c , alphaLISA displacement assay. Competition of a biotinylated-JQ1 probe by compound 1 with His-BRD4 Tandem or His-BRD4 BD1 in the presence or absence of DCAF16. Data shown are from n = 2 independent experiments each with 3 technical replicates, mean +/- s.d. d , TR-FRET proximity assay. 200 nM His-tagged BRD4 (BD1, BD2 or Tandem), bound to anti-His europium acceptor beads, were incubated with increasing concentrations of Cy5-labelled DCAF16:DDB1ΔBPB:DDA1 complex and 1 µM compound 1 or DMSO. Data shown are from n = 2 independent experiments each with 2 technical replicates, mean +/- s.d. e - h , BRD4 protein stability reporter assay. BRD4(S) as well as BET protein truncations or mutants ( e , g ) fused to mTagBFP were ectopically expressed in KBM7 cells and protein stability after 6-hour treatment with DMSO, GNE-0011 (1 µM), compound 1 (1 nM) or dBET6 (10 nM) was quantified via flow cytometric evaluation of the mTagBFP/mCherry ratio ( f , h ). BD, bromodomain; NPS, N-terminal phosphorylation sites; BID, basic residue-enriched interaction domain; ET, extraterminal domain; SEED, Serine/Glutamic acid/Aspartic acid-rich region. Data shown are from n = 3 biological replicates, mean +/- s.d. i , Structural overlay of BRD2 BD2 , BRD3 BD2 and BRD4 BD2 , PDB codes 3ONI, 2OO1 and 6C7Q, respectively, along with JQ1 in the acetylated lysine binding pocket of BRD2 BD2 . Residue E344 of BRD3, shown in stick representation, is a glycine residue at the equivalent position in BRD2 and BRD4.

Article Snippet: Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16-DDB1ΔBPB-DDA1, His 6 -BRD4 BD1 , His 10 -BRD4 BD2 , His 10 -BRD4 Tandem , compound 1 and LANCE Eu-W1024 Anti-6xHis donor (PerkinElmer) were prepared in TR-FRET assay buffer (20 mM HEPES pH 7, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20).

Techniques: Size-exclusion Chromatography, Proximity Assay, Incubation, Reporter Assay, Residue, Binding Assay

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , Electron density (left) and model (right) for the complex formed between DCAF16 (yellow), DDB1ΔBPB (blue), BRD4 Tandem (BD1 orange, BD2 red) and compound 1 (grey). b , Electron density at the DCAF16:BRD4:compound 1 interface. The JQ1 moiety binds to BD2 while the sulfonamide engages BD1. c , d , Detail views of BRD4 BD1 and BD2 intramolecular interaction surface ( c ) and M442 of BD2 bridging the WPF shelves of BD1 and BD2 ( d ). e , f , Intermolecular interaction surfaces between DCAF16 and BRD4 BD2 ( e ) and BD1 ( f ). G386 of BD2 and N93 of BD1 (spheres) face DCAF16 and solvent, respectively. For b - f , colours as in a . g. Schematic model of the different molecular recognition between intramolecularly bivalent glue as revealed in this work vs. traditional monovalent glues and bivalent PROTACs.

Article Snippet: Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16-DDB1ΔBPB-DDA1, His 6 -BRD4 BD1 , His 10 -BRD4 BD2 , His 10 -BRD4 Tandem , compound 1 and LANCE Eu-W1024 Anti-6xHis donor (PerkinElmer) were prepared in TR-FRET assay buffer (20 mM HEPES pH 7, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20).

Techniques: Solvent

Journal: bioRxiv

Article Title: An intramolecular bivalent degrader glues an intrinsic BRD4-DCAF16 interaction

doi: 10.1101/2023.02.14.528511

Figure Lengend Snippet: a , Superposition of known substrate receptor-DDB1 complexes (cartoon) and DCAF16 density (transparent surface). b , Comparison of binding mode in the acetyl lysine pocket of BRD4 BD1 (orange surface and cartoon) between compound 1 (orange) and known sulfonamide BET inhibitors PFI-1 (left, light blue) and compound 6j (right, grey). The cyano group of compound 1 overlays close to a conserved water molecule found in both crystal structures and many other published BD1 structures. c , FP binary binding assay. Indicated proteins were titrated into 20 nM FITC-sulfonamide probe (from ). Data shown are from n = 3 technical replicates. d , alphaLISA displacement assay. Competition of a biotinylated-JQ1 probe following titration of sulfonamide-containing compounds or JQ1 into His-BRD4 BD1 (left) or His-BRD4 BD2 (right). Data shown are from n = 2 technical replicates. e , Protein stability reporter assay. BRD4 Tandem constructs harbouring either two BD1 or two BD2 domains fused by their endogenous linker and connected to mTagBFP were stably expressed in KBM7 cells and protein stability after 6-hour treatment with DMSO, GNE-0011 (1 µM), compound 1 (1 nM) or dBET6 (10 nM) was quantified via flow cytometric evaluation of the mTagBFP/mCherry ratio as in . Data shown are from n = 3 biological replicates, mean +/- s.d.

Article Snippet: Stock solutions of reaction components including sulfo-Cy5-labelled DCAF16-DDB1ΔBPB-DDA1, His 6 -BRD4 BD1 , His 10 -BRD4 BD2 , His 10 -BRD4 Tandem , compound 1 and LANCE Eu-W1024 Anti-6xHis donor (PerkinElmer) were prepared in TR-FRET assay buffer (20 mM HEPES pH 7, 100 mM NaCl, 1 mM TCEP, 0.05% Tween-20).

Techniques: Comparison, Binding Assay, Titration, Reporter Assay, Construct, Stable Transfection