bet inhibitor  (MedChemExpress)

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing how the Parbit expression cassette is used to generate stably expressed Acyl-eCRs in mESCs. RMCE by Cre, followed by a double selection of ganciclovir and puromycin, was applied to generate these constructs at a defined site in the mouse genome. A CAG promoter drives the constitutive expression of the bromodomain of interest, which is fused to a nuclear localization signal (NLS), and an eGFP tag. This construct also fuses a biotin acceptor site to the N-terminus of the protein, which can be biotinylated in vivo by a bacterial BirA ligase. (B) Schematic diagram showing how CBP was endogenously tagged with an eGFP tag. A homology donor construct was generated by cloning a 900 bp upstream and a 1,048 bp (CBP) or 1,068 bp (p300) downstream homology arm flanking a 30 bp flexible GGS linker that was fused to an eGFP tag. This donor construct was co-transfected with a pX330 CRISPR-Cas9 plasmid, which had an sgRNA targeting the C-terminus of the CBP gene. (C-D) Sanger sequencing of genotyping PCR products from the C-terminus of the CBP (C) and p300 (D) loci, confirming the in-frame homologous integration of an eGFP tag. Data shown are from mESC clone #1 for both CBP and p300 tagging. (E) Flow cytometry data showing the eGFP signal from cell lines where either CBP or p300 were endogenously tagged with eGFP. Two clonal replicates for each protein tagging are shown. Cell lines were maintained in culture for more than two weeks to demonstrate stable expression of the eGFP fusion proteins. (F) Western blot of nuclear extracts from cell lines treated with 1 μM dCBP-1 PROTAC for the stated duration. An antibody against GFP was used to probe the eGFP tag on the Acyl-eCR constructs. The CBP_BRD.1x eCR runs at approximately 60 kDa, and the Empty-eGFP construct runs at 33 kDa. Non-specific bands are marked by an asterisk (*). Revert700 Total Protein Stain is used to show equal loading in lanes.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Expressing, Stable Transfection, Selection, Construct, In Vivo, Generated, Cloning, Transfection, CRISPR, Plasmid Preparation, Sequencing, Flow Cytometry, Western Blot, Staining

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing the domain architecture of the Brd4 protein, and how its bromodomains are being used in several combinations to make acyl-eCRs and to determine how the valency of reader domains affects drug perturbations. (B) Immunofluorescence images of mESCs showing the nuclear localization of different valencies of the second bromodomain from BRD4 in the Parbit system (green) and their colocalization with Hoechst (magenta) after drug treatments. All scale bars are 5 µM. Drug treatments were performed at 1 μM concentrations for 24 hours. Bottom panel: Representative pseudocolored images (eGFP signal) depicting the differences in fluorescence intensities in different cell lines. A gradient pseudocolor bar (signal intensity) is shown at the left. (C) Normalized FACS data showing the effects of ARV-825 PROTAC treatment on cells expressing several combinations of bromodomains from BRD4. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Immunofluorescence, Fluorescence, Expressing

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Top: Schematic showing how the competitive binding of small molecule inhibitors versus PROTACs for the binding pocket of Acyl-eCRs can be used to measure the affinity of a small molecule for a bromodomain in cellulo . Inhibitors with higher affinity for a bromodomain, better prevent PROTAC-induced degradation. Bottom : Treatment scheme for competitive binding experiments. Cells were treated with 1 μM inhibitors for 1 hour. Then, varying concentrations of the PROTAC were added in addition to the previously added inhibitor. After 3 hours of treatment, the cell fluorescence was measured via flow cytometry. (B) Competitive binding between ARV-825 and several small molecule inhibitors showing how the inhibitors bind to BRD4(2)_BRD.1x. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of ARV-825 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells. (C) Competitive binding between dCBP-1 and several small molecule inhibitors showing how the inhibitors bind CBP bromodomains in Acyl-eCR constructs versus the endogenous CBP protein. The cells were treated with the indicated inhibitor at a 1 μM concentration for 1 hour. Then, the stated concentration of dCBP-1 PROTAC was added for 3 hours, in addition to the previous concentration of the same inhibitor. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay, Construct

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Phylogenetic tree showing that the panel of Acyl-eCRs comprises representative bromodomains from all classes of bromodomains. All bromodomain protein sequences were obtained from InterPro and aligned with Clustal Omega’s multiple sequence alignment. Bromodomains are classified based on structure & druggability . Black circles represent the bromodomains that are included in the panel of Acyl-eCR cell lines. (B) Normalized FACS data showing the effects of dCBP-1 PROTAC treatments on all Acyl-eCR cell lines. PROTAC was added at a 1 μM concentration for 24 hours of treatment. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Sequencing, Concentration Assay

Journal: bioRxiv

Article Title: A modular toolbox for in cellulo screening of small molecule inhibitors targeting chromatin reader domains

doi: 10.1101/2025.09.06.674632

Figure Lengend Snippet: (A) Schematic showing how nuclei isolation & permeabilization followed by flow cytometry can measure the retention of proteins on chromatin in lieu of high background fluorescence. Nuclei can be harvested and permeabilized from whole cells, and then washed to remove the unbound or weakly bound fraction of the protein of interest. Since the weakly bound fraction of protein is removed, the fraction of protein remaining can be measured at a better signal-to-noise ratio via flow cytometry. (B) Flow cytometry analysis of nuclei harvested from BRD9_BRD.1x or WT cells. N3 gate shows the GFP signal being measured in nuclei, after iBRD9 or control treatments. Treatments in the WT cell line show a change in autofluorescence in the nuclei from the drug treatments. (C) Normalized flow cytometry data showing how Acyl-eCRs with 1x or 2x copies of the BRD9 bromodomain remain bound to chromatin, after iBRD9 treatments. iBRD9 treatments were performed at 1 μM concentration for 24 hours. The percentage represents the GFP signal in treated cells as a ratio of the signal observed in untreated samples of the same cell type, after normalizing for the autofluorescence of the drug treatment in wild-type cells.

Article Snippet: The CBP/p300 bromodomain inhibitor: GNE-049 (MedChemExpress, HY-108435), CBP/p300 PROTAC: dCBP-1 (MedChemExpress, HY-134582), BRD4 bromodomain inhibitor: (+)-JQ-1 (MedChemExpress, HY-13030), BRD4 PROTAC: ARV-825 (MedChemExpress, HY-16954), BRD9 bromodomain inhibitor: iBRD9 (MedChemExpress, HY-18975), and broad-spectrum bromodomain inhibitor: Bromosporine (MedChemExpress, HY-15815) were dissolved in DMSO and then diluted to 1μM in mESC media for 24-hour treatments, unless stated otherwise.

Techniques: Isolation, Flow Cytometry, Fluorescence, Control, Concentration Assay

Journal: Clinical and Experimental Medicine

Article Title: The epigenetic revolution in hematology: from benchside breakthroughs to clinical transformations

doi: 10.1007/s10238-025-01783-z

Figure Lengend Snippet: Summarizes the main types of epigenetic modifications along with their subtypes, associated enzymes, and their roles in gene expression regulation and hematological malignancies . Abbreviation: DNMTs: DNA methyltransferases; KMTs: Lysine methyltransferases; NSD: Nuclear SET domain; PRMTs: Protein arginine methyltransferases; CBP: CREB-binding protein; MBD: methyl-CpG-binding domain proteins; ZBTB: Zinc finger and BTB domain; BET: Bromodomain (BRD) and extra-terminal; IDH: Isocitrate dehydrogenase; KDMs: Lysine demethylases; AID/APOBEC: Activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme complex; HDAC: Histone deacetylase; MOZ: Monocytic leukemic zinc finger; MORF: Monocytic leukemia zinc-finger protein-related factor; TET: Ten-eleven translocation; BL: Burkitt’s lymphoma; DLBCL: Diffuse large B-cell lymphoma; NHL: Non-Hodgkin lymphoma; AML: Acute myeloid leukemia; ALL: Acute lymphoblastic leukemia; CML: Chronic myeloid leukemia; CLL: Chronic lymphocytic leukemia; MCL: Mantle cell lymphoma; MM: Multiple Myeloma; FL: Follicular lymphoma; APL: Acute promyelocytic leukemia; MDS: Myelodysplastic syndromes; PTCL: Peripheral T-cell lymphoma; CTCL: Cutaneous T-cell lymphoma; CMML: Chronic myelomonocytic leukemia; MPN: Myeloproliferative neoplasms; NKTCL: NK/T-cell lymphoma; ALCL: Anaplastic large cell lymphoma; MLL: Mixed lineage leukemia; ATLL: Adult T-cell leukemia/lymphoma; LGL: Large Granular Lymphocyte; AITL: Angioimmunoblastic T-cell lymphoma; ALAL: Acute Leukemia Of Ambiguous Lineage; AMKL: Acute Megakaryoblastic Leukemia.

Article Snippet: Other innovative therapies, including Enhancer of zeste homologue 2 (EZH2) inhibitors and Bromodomain and extra-terminal proteins (BET) inhibitors, are also being studied for their potential to target specific epigenetic alterations associated with hematological malignancies [ ].

Techniques: Gene Expression, Binding Assay, Activation Assay, Histone Deacetylase Assay, Translocation Assay

Journal: Non-coding RNA

Article Title: Bromodomain and Extra-Terminal Family Proteins BRD2, BRD3, and BRD4 Contribute to H19-Dependent Transcriptional Regulation of Cell Adhesion Molecules, Modulating Metastatic Dissemination Program in Prostate Cancer.

doi: 10.3390/ncrna11030033

Figure Lengend Snippet: Figure 3. Effect of several BET family protein inhibitors, BRD2/3/4 or BRD7/9 members, on the gene expression level of E-cadherin and β4 integrin and cell proliferation. E-cadherin (CDH1, (A)), β4 integrin (ITGB4, (B)), Cyclin D1 (CCND1, (C)), Vimentin (VIM, (D)), and c-Myc (E) mRNAs were assessed by qPCR in PC-3-luc (PC-3), 22Rv1-luc (22Rv1), and Du145 in presence or absence of 48 h treatment with BRD2/3/4 family inhibitors, JQ1, dBET6, OTX015 (OTX), or BRD7/9, inhibitor Bi7273, or inactive (R)-(-)-JQ1 (R-JQ1), or DMSO as control. Data, plotted as fold change vs. DMSO, represent the mean ± SEM of 4 independent experiments. * p < 0.05 vs. DMSO. (F) PC-3 cell proliferation was monitored using the IncuCyte live cell analysis system. Cell confluence was calculated from raw data images; the data shown is a representative experiment of 4 biological replicates, and each time point represents mean ± SEM of 4 samples. * p < 0.05 vs. DMSO; # p < 0.05 vs. (R)-JQ1.

Article Snippet: PC-3-luc, 22Rv1-luc, and Du145 cells were treated with the following BET inhibitors and degraders, purchased from MedChemExpress: (S)-(+)-JQ-1 (JQ1, Cat. No.: HY-13030), (R)−(−)−JQ1 (R-JQ1, Cat. No.: HY-13030A), dBET6 (Cat. No.: HY-112588), Bi7273 (Cat. No.: HY-100351), OTX-015 (Cat. No.: HY-15743), and LT052 (Cat. No.: HY-130622).

Techniques: Gene Expression, Control, Cell Analysis