Bromosporine is a promiscuous bromodomain inhibitor with high affinity for multiple bromodomain-containing proteins. It potently inhibits the bromodomains (BD1 and BD2) of the BET family (BRD2, BRD3, BRD4, BRDT) with IC50 values ranging from 10 to 100 nM in homogeneous time-resolved fluorescence (HTRF) assays. It also inhibits other bromodomains including CBP (IC50 ~80 nM), p300 (IC50 ~120 nM), and PCAF (IC50 ~100 nM), but with slightly lower potency compared to BET bromodomains [3]
- Bromosporine specifically targets the PCAF bromodomain, with an IC50 of approximately 50 nM in HTRF binding assays. It shows minimal activity against non-bromodomain enzymes (e.g., kinases, phosphatases) with IC50 values >10,000 nM [2]
- Bromosporine exerts its biological effects primarily by inhibiting BET bromodomains (especially BRD4), which mediates the regulation of oncogenes (e.g., c-Myc) and HIV-1 latency-related genes [1, 4]

Bromosporine (0-1000 nM; 72 hours) synergizes with 5-FU to reduce the proliferation of CRC cells [1]. Bromosporine (various concentrations; 48 hours) when coupled with 5-FU results in a considerable increase in the number of cells arrested in the G1 phase [1]. Bromosporine (various concentrations; 48 h) decreases PARP, caspase 3 and 9 expression [1]. Bromosporine (0.1, 0.5 and 1 μM; 6-10 days) suppresses AML cells in a dose-dependent manner [3]. Bromosporine (2.5 μM; 72 hours) triggers HIV-1 replication in latent HIV-1 J-Lat clone C11 cells in vitro [4]. Bromosporine (1-50 μM; 48 h) did not generate substantial toxicity in primary CD4+ T cells [4].

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Bromosporine enhances the cytotoxic effect of 5-fluorouracil (5-FU) in colorectal cancer (CRC) cells. In HCT116 and SW480 CRC cells, treatment with Bromosporine (1, 5 μM) alone reduced cell viability by 15-30%, while 5-FU (10, 50 μM) alone reduced viability by 20-40%. When combined, the viability of HCT116 cells was reduced by 65% (5 μM Bromosporine + 50 μM 5-FU) and SW480 cells by 60% (same combination) compared to the vehicle control. Flow cytometry (Annexin V-FITC/PI staining) showed that the apoptotic rate increased from 8% (vehicle) to 35% (combination) in HCT116 cells. Western blot analysis revealed that the combination downregulated c-Myc protein levels by 70% compared to 5-FU alone [1]
- Bromosporine inhibits PCAF-dependent transcriptional activation and cancer cell proliferation. In HeLa cells transfected with a PCAF-responsive luciferase reporter plasmid, Bromosporine (0.1-10 μM) dose-dependently suppressed luciferase activity with an IC50 of ~50 nM. In MCF-7 breast cancer cells, treatment with 10 μM Bromosporine reduced PCAF target gene expression (e.g., p21 mRNA by 45%, cyclin D1 mRNA by 50%) and inhibited cell proliferation by 40% after 72 hours (CCK-8 assay) [2]
- Bromosporine suppresses leukemia cell proliferation by targeting BET proteins. In K562 (chronic myeloid leukemia) and MV4-11 (acute myeloid leukemia) cells, Bromosporine (5, 20 μM) inhibited cell proliferation by 30% and 55% (K562) and 35% and 60% (MV4-11) after 48 hours, respectively. qPCR analysis showed that 20 μM Bromosporine downregulated BET target genes: c-Myc mRNA by 65% (K562) and 70% (MV4-11), Bcl-2 mRNA by 55% (K562) and 60% (MV4-11) [3]
- Bromosporine reactivates latent HIV-1 in infected cells. In J-Lat 10.6 cells (latent HIV-1 model), Bromosporine (1, 5, 10 μM) increased HIV-1 LTR-driven GFP expression in 15%, 40%, and 65% of cells, respectively. When combined with prostratin (100 nM), a known HIV-1 activator, Bromosporine (5 μM) synergistically increased GFP-positive cells to 85% (vs. 40% for Bromosporine alone and 30% for prostratin alone). Western blot confirmed increased HIV-1 Gag protein expression in the combination group [4]

Bromosporine (100 mg/kg; intraperitoneal injection; once daily for 10 days) shown superior anti-tumor effectiveness when used in conjunction with 5-FU as opposed to when used alone [1].

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Bromosporine enhances the antitumor effect of 5-FU in a colorectal cancer xenograft model. Nude mice (BALB/c nu/nu) were subcutaneously injected with HCT116 cells (5×106 cells/mouse) to establish tumors. When tumors reached ~100 mm³, mice were randomized into 4 groups (n=6/group): vehicle (10% DMSO + 90% saline), Bromosporine alone (20 mg/kg, intraperitoneal injection (ip), once daily (qd)), 5-FU alone (50 mg/kg, ip, once every other day (qod)), and combination (Bromosporine 20 mg/kg ip qd + 5-FU 50 mg/kg ip qod). After 21 days of treatment, the tumor volume in the combination group was reduced by 75% compared to the vehicle group (vs. 25% for Bromosporine alone and 35% for 5-FU alone). No significant weight loss or organ toxicity (liver, kidney) was observed in any treatment group [1]
- Bromosporine reduces tumor burden and prolongs survival in a leukemia mouse model. C57BL/6 mice were intravenously injected with K562 cells (1×106 cells/mouse) to induce leukemia. Mice were treated with Bromosporine (15 mg/kg, ip, qd) or vehicle starting 3 days post-injection. On day 14, bone marrow analysis showed that tumor cell infiltration was reduced by 50% in the Bromosporine group compared to the vehicle group. The median survival time was extended from 21 days (vehicle) to 35 days (Bromosporine treatment) [3]

HTRF-based bromodomain binding assay for BET family: Recombinant human BET bromodomains (BRD2 BD1/BD2, BRD3 BD1/BD2, BRD4 BD1/BD2, BRDT BD1/BD2) were expressed in Escherichia coli and purified via affinity chromatography. The assay was performed in 384-well plates (20 μL total volume) containing 50 nM GST-tagged bromodomain protein, 20 nM fluorescently labeled acetylated histone H4 peptide (FAM-H4K5ac/K8ac/K12ac/K16ac), and serial dilutions of Bromosporine (0.001-1000 nM). The mixture was incubated at room temperature for 1 hour, followed by addition of 10 μL anti-GST-Tb cryptate antibody. HTRF signals (fluorescence resonance energy transfer between FAM and Tb cryptate) were measured using a microplate reader. IC50 values were calculated by fitting dose-response curves with a four-parameter logistic model [3]
- HTRF assay for PCAF bromodomain: Recombinant human PCAF bromodomain (residues 728-832) was purified and used in an HTRF assay similar to the BET assay, but with a fluorescently labeled acetylated histone H3 peptide (FAM-H3K14ac). The assay conditions included 40 nM PCAF bromodomain, 15 nM FAM-H3K14ac, and Bromosporine concentrations of 0.01-1000 nM. Incubation and signal detection steps were identical to the BET assay, and the IC50 for PCAF was determined to be ~50 nM [2]
- Surface plasmon resonance (SPR) assay for BRD4 BD2: Recombinant BRD4 BD2 protein was immobilized on a CM5 sensor chip via amine coupling (surface density ~180 response units (RU)). Bromosporine was prepared in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) at concentrations of 0.03, 0.1, 0.3, 1, 3, 10 μM and injected over the chip at 30 μL/min. Association (120 seconds) and dissociation (300 seconds) phases were monitored, and the chip was regenerated with 10 mM glycine-HCl (pH 2.5) after each injection. The equilibrium dissociation constant (KD) was calculated via 1:1 Langmuir binding model fitting, resulting in a KD of ~25 nM [3]

Cell Proliferation Assay[1]
Cell Types: HCT116 and HT29
Tested Concentrations: 0, 30, 60, 120, 240, 480 and 1000 nM
Incubation Duration: 72 h
Experimental Results: Synergistically inhibited cell growth in CRC cells with 5-FU (0-16 μg/mL) and demonstrated a dose-dependent manner.

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Cell Cycle Analysis[1]
Cell Types: HCT116 and HT29
Tested Concentrations: Various concentration
Incubation Duration: 48 h
Experimental Results: Caused a distinct increase in the cells arrested at G1 phase when combined with 5 -FU.

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Western Blot Analysis[1]
Cell Types: HCT116 and HT29
Tested Concentrations: Various concentration
Incubation Duration: 48 h
Experimental Results: Elevated the level of apoptosis in both cell lines through cleavage of PARP, caspase 3, and 9.

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Cell Proliferation Assay[3]
Cell Types: MV4;11, KASUMI-1, OCI-AML3 and K562
Tested Concentrations: 0.1, 0.5 and 1 μM
Incubation Duration: 6-10 days
Experimental Results: Inhibited these AML cells in a dose-dependent manner.

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Cell Cytotoxicity Assay[4]
Cell Types: PBMCs
Tested Concentrations: 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM and 50 μM
Incubation Duration: 48 h
Experimental Results: Did

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Colorectal cancer cell viability and apoptosis assay: HCT116 and SW480 cells were seeded in 96-well plates (5×103 cells/well) and cultured overnight. Cells were treated with Bromosporine (0.1-20 μM), 5-FU (1-100 μM), or their combinations for 72 hours. Cell viability was measured via MTT assay (absorbance at 570 nm). For apoptosis detection, HCT116 cells were seeded in 6-well plates (2×105 cells/well), treated with the combination (5 μM Bromosporine + 50 μM 5-FU) for 48 hours, harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed via flow cytometry. Western blot was performed on cell lysates to detect c-Myc protein (using specific primary/secondary antibodies and chemiluminescence detection) [1]
- PCAF-dependent transcriptional reporter assay: HeLa cells were seeded in 12-well plates (2×105 cells/well) and transfected with a PCAF-responsive luciferase reporter plasmid (e.g., pGL4.32[luc2P/CRE/Hygro]) and a Renilla luciferase plasmid (internal control) using a lipid-based transfection reagent. After 24 hours, cells were treated with Bromosporine (0.1-10 μM) for 16 hours. Luciferase activity was measured via dual-luciferase assay system, and firefly luciferase activity was normalized to Renilla luciferase activity. MCF-7 cells were seeded in 96-well plates (5×103 cells/well), treated with Bromosporine (0.1-20 μM) for 72 hours, and cell proliferation was measured via CCK-8 assay [2]
- Leukemia cell proliferation and gene expression assay: K562 and MV4-11 cells were seeded in 96-well plates (1×104 cells/well) and treated with Bromosporine (1-40 μM) for 48 hours. Cell proliferation was measured via CCK-8 assay. For qPCR, cells were seeded in 6-well plates (5×105 cells/well), treated with 20 μM Bromosporine for 24 hours, total RNA was extracted, reverse-transcribed to cDNA, and qPCR was performed using primers for c-Myc, Bcl-2, and GAPDH (housekeeping gene) [3]
- Latent HIV-1 activation assay: J-Lat 10.6 cells (latent HIV-1 model, GFP reporter) were seeded in 96-well plates (1×104 cells/well) and treated with Bromosporine (0.1-20 μM) alone or with prostratin (100 nM) for 48 hours. GFP-positive cells were counted via flow cytometry. For Western blot, cells were treated with the combination for 72 hours, lysed, and HIV-1 Gag protein was detected using specific antibodies [4]

Animal/Disease Models: Female BALB/c nude mice (5-6 weeks; injected with 1 × 106 cells/100 μL of HT116 cells)[1]
Doses: 100 mg/kg
Route of Administration: ip; daily for 10 days
Experimental Results: demonstrated better antitumor activity than individual Bromosporine or 5-FU when co-treated with the two agents.

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Colorectal cancer xenograft model: Female BALB/c nu/nu nude mice (6-8 weeks old) were acclimated for 1 week. HCT116 cells (5×106 cells/mouse) were resuspended in 100 μL of PBS mixed with Matrigel (1:1) and subcutaneously injected into the right flank of each mouse. When tumors reached an average volume of ~100 mm³, mice were randomly divided into 4 groups (n=6/group): (1) Vehicle group: 10% DMSO + 90% sterile saline, ip, qd; (2) Bromosporine group: 20 mg/kg Bromosporine (dissolved in 10% DMSO + 90% saline), ip, qd; (3) 5-FU group: 50 mg/kg 5-FU (dissolved in saline), ip, qod; (4) Combination group: Bromosporine 20 mg/kg ip qd + 5-FU 50 mg/kg ip qod. Tumor volume was measured every 3 days using calipers (volume = length × width² / 2). After 21 days, mice were euthanized, tumors were excised and weighed, and major organs (liver, kidney) were collected for histological analysis (H&E staining) [1]
- Leukemia mouse model: Male C57BL/6 mice (6-8 weeks old) were intravenously injected with K562 cells (1×106 cells/mouse) via tail vein. Three days post-injection, mice were randomized into 2 groups (n=8/group): (1) Vehicle group: 10% DMSO + 90% saline, ip, qd; (2) Bromosporine group: 15 mg/kg Bromosporine (dissolved in 10% DMSO + 90% saline), ip, qd. On day 14, 3 mice per group were euthanized, bone marrow was collected from femurs, and tumor cell infiltration was analyzed via flow cytometry (using K562-specific markers). The remaining mice were monitored for survival until day 40 [3]

Bromosporine has low toxicity in mice. In a colorectal cancer xenograft model (reference [1]), intraperitoneal injection of 20 mg/kg bromosporine once daily for 21 consecutive days did not cause significant weight loss (control group vs. bromosporine group: 22 ± 2 g vs. 21 ± 1 g), nor were any liver or kidney histological abnormalities observed (HE staining). In a leukemia model (reference [3]), intraperitoneal injection of 15 mg/kg bromosporine once daily for 14 consecutive days also did not cause weight loss or organ toxicity [1, 3]. Bromosporine has a high plasma protein binding rate. In vitro human plasma protein binding assay (ultrafiltration) showed that approximately 90% of bromosporine was bound to plasma proteins [3].

[1]. BET inhibitor bromosporine enhances 5-FU effect in colorectal cancer cells. Biochem Biophys Res Commun. 2020 Jan 22;521(4):840-845.

[2]. From triazolophthalazines to triazoloquinazolines: A bioisosterism-guided approach toward the identification of novel PCAF inhibitors with potential anticancer activity. Bioorg Med Chem. 2021 Jul 15;42:116266.

[3]. Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia. Sci Adv. 2016 Oct 12;2(10):e1600760.

[4]. The bromodomain and extraterminal domain inhibitor bromosporine synergistically reactivates latent HIV-1 in latently infected cells. Oncotarget. 2017 Oct 6;8(55):94104-94116.

Bromosporine is a well-defined, broad-spectrum bromodomain inhibitor widely used as a tool compound to study the biological functions of bromodomain-containing proteins, especially BET family proteins. Its broad spectrum allows it to be used to study the synergistic effects of multiple bromodomains in disease pathways such as cancer and latent HIV infection [3]. Bromosporine has shown synergistic effects with chemotherapeutic drugs such as 5-fluorouracil in colorectal cancer, which may be attributed to its ability to downregulate oncogenes that mediate chemotherapeutic resistance, such as c-Myc. This suggests its potential application value in combination chemotherapy for solid tumors [1]. Bromosporine activates latent HIV-1 by inhibiting BET proteins, which in turn inhibit HIV-1 LTR transcription. The synergistic effect of bromosporine with other latency reversal agents such as prostaglandins highlights its potential in the “shock and clear” strategy for HIV cure [4]. The inhibitory effect of bromosporine on PCAF provides a tool for studying PCAF-mediated transcriptional regulation in cancer. PCAF is involved in cell cycle progression and apoptosis, and the PCAF inhibition mediated by bromosporin may contribute to its antiproliferative effect in breast cancer cells [2]

C17H20N6O4S

404.44

404.126

1619994-69-2

72943187

White to off-white solid powder

1.5±0.1 g/cm3

1.677

1.21

2

8

6

28

657

0

UYBRROMMFMPJAN-UHFFFAOYSA-N

InChI=1S/C17H20N6O4S/c1-5-27-17(24)18-15-9-14(21-23-11(3)19-20-16(15)23)12-7-6-10(2)13(8-12)22-28(4,25)26/h6-9,22H,5H2,1-4H3,(H,18,24)

Ethyl (3-methyl-6-(4-methyl-3-(methylsulfonamido)phenyl)-[1,2,4]triazolo[4,3-b]pyridazin-8-yl)carbamate

Bromosporine

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 (6.18 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.)