BRD2-BD1 (Kd = 1.6 nM); BRD3-BD1 (Kd = 2.1 nM); BRD4-BD1 (Kd = 1.7 nM); 5 nM (BRDT-BD1), 5.9 nM (BRD2-BD2), 6.2 nM (BRD3-BD2), 6.1 nM (BRD4-BD2), 120 nM (BRDT-BD2), ∼100 nM (CBP), ∼100 nM (EP300)
BRD4 [1]
BET family proteins (structurally distinct BET inhibitor targeting the inducible ZA channel) [1]

With Kds of 1.6, 2.1, 1.7, and 5 nM for BD1, 5.9, 6.2, 6.1, and 120 nM for BRD2, BRD3, and BRD4, respectively, and BRDT, PLX51107 is a strong and selective BET inhibitor. Additionally, PLX51107 interacts with EP300's and CBP's bromodomains (Kd, within a 100 nM range). PLX51107 (0.156-10 μM) prevents primary chronic lymphocytic leukemia (CLL) cells from proliferating when exposed to CpG. In addition, PLX51107 lowers c-MYC levels, increases p21 and IκBα accumulation, and modifies pro- and anti-apoptotic proteins. CLL driver genes are specifically regulated by PLX51107 [1].

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PLX51107 shows dose-dependent inhibition of proliferation in CpG-stimulated primary CLL cells when treated continuously for 72 h or for 4 h followed by washout; its potency is comparable to other BET inhibitors (OTX015, JQ1, iBET762) [1]
In co-culture of CLL patient-derived CD19⁺ cells (n=13) with human Hs27 or murine 9–15c bone marrow-derived stroma, PLX51107 (1, 2, 5 μM) reduces CLL cell viability after 72 h as detected by Annexin-V/PI staining [1]
PLX51107 induces dose-dependent proliferation inhibition in malignant B-cell lines (MEC-1, OCI-LY1) after 72 h treatment (p<0.001 for each cell line), with similar efficacy to other BET inhibitors [1]
Treatment of CpG-stimulated CLL cells with 1 μM PLX51107 for 4 h modulates gene expression (detected by microarray analysis using GeneChip Human Transcriptome Array HTA 2.0), downregulating CLL-relevant genes and enriching canonical pathways related to B-cell receptor signaling and oncogenic circuits [1]
Quantitative real-time PCR and immunoblot analysis confirm that PLX51107 (0.1, 1, 2 μM) downregulates the expression of BTK, IKZF1, cMYC, IKZF3, ZBTB17, and upregulates HEXIM1 in CpG-stimulated CLL cells (24 and 48 h treatment, n=5) [1]
ChIP-seq analysis shows that PLX51107 reduces BRD4 occupancy at enhancer regions of CLL patient-derived cells, alters RNA Pol II occupancy at gene promoters and bodies, and modulates the expression of genes in BRD4-regulated super-enhancers [1]

In the Ba/F3 (mouse IL3-dependent pre-B cell line) splenomegaly mouse model, PLX51107 (2 mg/kg, orally) reduced splenomegaly by 75%, with results resembling those of 25 mg/kg OTX015. When taken orally once daily, PLX51107 (20 mg/kg, qd, po) shows strong antileukemia effects in models of Richter transformation (RT) and aggressive chronic lymphocytic leukemia (CLL) [1].

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In the Ba/F3-induced mouse splenomegaly model, PLX51107 is 10-fold more potent than OTX015 [1]
In MV4-11 tumor xenografts, a single dose of PLX51107 induces transcriptional changes (represented by HEXIM1) that outlast plasma drug levels; pharmacokinetic parameters include AUC₀–₂₄h = 90,100 ng•h/mL (205 μM•hr) and terminal half-life (T₁/₂) = 2.8 h [1]
In Eμ-TCL1 mice with advanced leukemia, oral administration of PLX51107 (20 mg/kg, qd, 8 d) reduces leukemic cells in systemic circulation and spleen, and modulates the protein levels of cMYC, P21, BTK, IKZF1, IKZF3, and TCL1A [1]
In the adoptive transfer model of Eμ-TCL1, PLX51107 (20 mg/kg, qd, oral gavage) administered at leukemia onset significantly prolongs overall survival (median OS: 93 days vs 34 days in vehicle group, p<0.0001), decreases circulating leukemic peripheral blood lymphocytes (PBLs), reduces spleen mass, and depletes lymphocytes in spleen, lung, and blood (confirmed by HE and Ki67 staining) [1]
In C57BL/6 mice engrafted with Eμ-TCL1 leukemic splenocytes, PLX51107 (20 mg/kg, qd, oral gavage) increases median OS to 41 days, which is significantly longer than vehicle (21 days, p=0.024) and ibrutinib (32 days, p=0.049) [1]
In the Eμ-Myc/TCL1 adoptive transfer model of high-grade lymphoma (analogous to Richter’s Transformation), PLX51107 (20 mg/kg, qd, oral gavage) prolongs survival, decreases peripheral WBC counts, reduces CD19/CD5/CD45⁺ PBLs, and lowers spleen and lymph node masses; HE and Ki67 staining shows preserved tissue architecture and depletion of atypical neoplastic lymphocytes [1]

Determine the binding mode of PLX51107 to BRD4-BD1 by analyzing co-crystal structures; the 4-azaindole scaffold-derived compound traverses the ZA channel and forms a salt bridge with Lys91 of BRD4-BD1, inducing a conformational change similar to that caused by the acetylated RelA acK310 peptide [1]
Compare the binding interactions of PLX51107 and OTX015 with BRD4-BD1 to clarify the structural basis of its BET inhibitory activity [1]

ChIP-Seq and Data Processing[1]
Primary CLL cells (1E7 cells per condition) were treated with vehicle (DMSO), or 1μM PLX51107 with or without CpG oligonucleotides (3.2 μM) for 4 h. Cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300–500 bp. See supplementary information for detailed cross-link procedure. Genomic DNA regions of interest were isolated using 4μg antibody against BRD4, H3K27ac and RNA Pol II. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.

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Treat CpG-stimulated primary CLL cells with increasing doses of PLX51107, OTX015, JQ1, or iBET762 for 72 h continuously or 4 h followed by washout; assess cell proliferation to evaluate dose-dependent inhibitory effects [1]
Co-culture CLL patient-derived CD19⁺ cells with bone marrow-derived stroma (Hs27 or 9–15c), treat with PLX51107 (1, 2, 5 μM) for 72 h, and determine cell viability using Annexin-V/PI staining [1]
Treat malignant B-cell lines (MEC-1, OCI-LY1) with gradient concentrations of PLX51107 and other BET inhibitors for 72 h; measure cell proliferation to compare efficacy [1]
Treat CpG-stimulated CLL cells with 1 μM PLX51107 or DMSO for 4 h; perform microarray analysis (GeneChip Human Transcriptome Array HTA 2.0) and quantitative real-time PCR to analyze gene expression changes [1]
Conduct ChIP-seq assays to detect BRD4 and H3K27ac binding, and RNA Pol II occupancy in CLL cells treated with PLX51107; analyze changes in enhancer activity and transcriptional regulation [1]
Perform immunoblot analysis to detect the protein levels of BTK, IKZF1, cMYC, IKZF3, ZBTB17, HEXIM1, etc., in PLX51107-treated CpG-stimulated CLL cells [1]

20 mg/kg; qd, p.o.
Ba/F3 splenomegaly mouse model For engraftment studies, C57BL/6 wild type mice were engrafted with 1E7 cells by tail vein injection of splenocytes derived from Eμ-TCL1 or Eμ-Myc/TCL1 mice with active disease. At the onset of leukemia (Eμ-TCL1: ≥10% CD19/CD5/CD45 positive circulating cells; Eμ-Myc/TCL1: WBC count ≥ 8 and/or ≥ 5% CD19/CD5/CD45 positive circulating cells) mice were randomized to receive treatments as indicated. Vehicle = 10% N-Methyl-2-pyrrolidone (NMP) plus diluent (40% PEG400, 5% TPGS, 5% Poloxamer 407 and 50% water). Mice were sacrificed when meeting early removal criteria (ERC: >20% weight loss, impaired motility, splenomegaly and evident tumor masses), and tissues were collected for further analysis.[1]

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Establish the Ba/F3-induced mouse splenomegaly model; administer PLX51107 and OTX015 to compare their potencies in reducing splenomegaly [1]
Establish MV4-11 tumor xenografts in mice; administer a single dose of PLX51107, collect plasma samples at different time points to determine pharmacokinetic parameters (AUC₀–₂₄h, T₁/₂), and detect HEXIM1 expression in tumors to evaluate transcriptional effects [1]
Use Eμ-TCL1 mice with advanced leukemia; stratify mice by leukemic PBLs and spleen palpation score, then administer PLX51107 (20 mg/kg, qd, oral gavage) for 8 d; collect peripheral blood, spleen, and tissue samples to assess leukemic cell burden and protein expression [1]
Establish the adoptive transfer model of Eμ-TCL1 by transplanting leukemic cells into wild-type mice; randomize mice to vehicle or PLX51107 (20 mg/kg, qd, oral gavage) at leukemia onset, monitor survival and leukemic PBLs for 150 days, and perform HE and Ki67 staining on spleen, lung, and blood samples [1]
Engraft C57BL/6 mice with Eμ-TCL1 leukemic splenocytes; treat mice with PLX51107 (20 mg/kg, qd, oral gavage), ibrutinib, or vehicle at leukemia onset; monitor overall survival to compare therapeutic efficacy [1]
Establish the Eμ-Myc/TCL1 adoptive transfer model of high-grade lymphoma; administer PLX51107 (20 mg/kg, qd, oral gavage) at disease onset (≥5% CD19/CD5/CD45⁺ PBLs and/or increased WBC count); monitor survival, WBC counts, and PBLs, and perform HE and Ki67 staining on spleen and lymph node samples [1]

In MV4-11 tumor xenograft mice, the AUC₀–₂₄h of PLX51107 was 90,100 ng•h/mL (205 μM•hr) and the terminal half-life (T₁/₂) was 2.8 h after a single dose [1].

[1]. BRD4 Profiling Identifies Critical Chronic Lymphocytic Leukemia Oncogenic Circuits and Reveals Sensitivity to PLX51107, a Novel Structurally Distinct BET Inhibitor. Cancer Discov. 2018 Apr;8(4):458-477.

PLX51107 is a potent and selective inhibitor of the brominated domain and terminal extradomain (BET) protein family. PLX51107 is currently being investigated in the clinical trial NCT04022785 (PLX51107 in combination with azacitidine for the treatment of patients with acute myeloid leukemia or myelodysplastic syndrome).
The BRD4 inhibitor PLX51107 is a brominated domain protein 4 (BRD4) inhibitor with potential antitumor activity. After administration, the BRD4 inhibitor PLX51107 binds to the acetylated lysine recognition motif in the brominated domain of the BRD4 protein, thereby preventing BRD4 from binding to acetylated lysine residues on histones. This disrupts chromatin remodeling and gene expression. Inhibition of the expression of certain growth-promoting genes may lead to apoptosis and inhibition of proliferation in BRD4-overexpressing tumor cells. BRD4 is a member of the human brominated domain and terminal extradomain (BET) protein family and is a transcriptional regulator that is overexpressed in some tumor cells and plays an important role in cell proliferation. The bromodomain and terminal extraterminal domain (BET) protein family are key regulators of gene expression in cancer. In this paper, we utilize BRD4 profiling to identify key pathways involved in the pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and enriched near upregulated or newly expressed genes in CLL that have known functions in the pathogenesis and progression of the disease. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a theoretical basis for identifying novel targets for this treatment approach in other types of cancer. Furthermore, we describe PLX51107, a structurally unique BET inhibitor with novel in vitro and in vivo pharmacological properties, demonstrating efficacy comparable to or even surpassing BCR signaling pathway drugs in preclinical models of CLL. The discovery of BRD4's involvement in the core transcriptional program of CLL provides strong theoretical support for the clinical research of PLX51107 as an epigenetic therapy for CLL and the application of BRD4 profiling in other cancers. Significance: To date, research on the function of BRD4 in CLL is lacking. Through integrated genomic, functional, and pharmacological analyses, we revealed the existence of a BRD4-regulated core transcriptional program in CLL and presented a preclinical proof-of-concept study demonstrating that BET inhibition, as an epigenetic approach, can be used to target the BCR signaling pathway in CLL. Cancer Discov; 8(4); 458-77. ©2018 AACR. This article is highlighted in the "Highlights" section, pages 1-10. 371.[1]

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PLX51107 is a novel, structurally unique BET inhibitor whose non-benzodiazepine backbone is derived from PLX5981 (modified 4-azaindole backbone)[1]
It targets the inducible ZA channel of BET family proteins, and its binding mode is different from that of known BET inhibitors (such as OTX015)[1]
PLX51107 regulates the core transcriptional program regulated by BRD4 in CLL, targets key pathways including the B cell receptor signaling pathway, and mimics or surpasses the efficacy of drugs that target the BCR signaling pathway in preclinical CLL models[1]
This drug has shown efficacy in invasive CLL and Richter's transition. PLX51107 has shown potent anti-leukemic activity in preclinical models, supporting its potential as an epigenetic therapy for CLL [1]. The duration of transcriptional changes induced by PLX51107 in tumors exceeds its presence in plasma, indicating sustained pharmacodynamic effects [1].

C26H22N4O3

438.477885723114

438.169

C, 71.22; H, 5.06; N, 12.78; O, 10.95

1627929-55-8

90448953

White to off-white solid powder

4

1

6

5

33

684

1

O1C(C)=C(C(C)=N1)C1=CN=C2C(C3C=CC(C(=O)O)=CC=3)=CN(C2=C1)[C@H](C1C=CC=CN=1)C

AMSUHYUVOVCWTP-INIZCTEOSA-N

InChI=1S/C26H22N4O3/c1-15-24(17(3)33-29-15)20-12-23-25(28-13-20)21(18-7-9-19(10-8-18)26(31)32)14-30(23)16(2)22-6-4-5-11-27-22/h4-14,16H,1-3H3,(H,31,32)/t16-/m0/s1

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)