Bax (IC50 = 250 nM); Bax (IC50 = 144 nM)
BTSA1: High-affinity and selective activator of BAX (BCL-2-associated X protein), binding to the N-terminal activation site (trigger site) of BAX; [1]

BTSA1 has no capacity to directly activate the pro-apoptotic homolog BAK. Recombinant soluble BAX is potently and dose-responsively translocated to the mitochondrial membrane by treatment with BTSA1, which is followed by the induction of BAX oligomerization. Cancer cells are more likely to undergo apoptosis when BAX is activated by BTSA1. With IC50 values ranging between 1 and 4 μM, BTSA1 decreases the viability of all AML cell lines in a dose-dependent manner, with a complete effect occurring within 24 hours of treatment. All five AML cell lines exhibit dose-dependent caspase-3/7 activation[1].

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BAX activation and conformational change: BTSA1 binds specifically to the N-terminal trigger site (α1, α6 regions) of BAX with high affinity. Competitive fluorescence polarization binding assays show it competes with FITC-BIM SAHB A2 for BAX binding, while direct fluorescence polarization binding assays confirm its direct interaction with BAX. ¹⁵N-labeled BAX HSQC analysis reveals significant backbone amide chemical shift changes in residues at the trigger site upon BTSA1 titration, indicating induction of BAX conformational changes. Docking studies show BTSA1 forms hydrophobic interactions with BAX and a key hydrogen bond between its pyrazolone group and BAX K21 residue, mimicking BIM BH3 helix-BAX interactions [1]
- BAX-mediated apoptosis in leukemia cells: BTSA1 induces all steps of the BAX activation pathway, including BAX translocation to membranes, oligomerization, and mitochondrial outer membrane permeabilization (MOMP). In ANTS/DPX liposome assays, BTSA1 (100–400 nM) promotes BAX-mediated membrane permeabilization, which is impaired in BAX K21E mutants. In isolated mouse liver mitochondria, BTSA1 induces BAX mitochondrial translocation, oligomerization, and cytochrome c release in a dose-dependent manner. It effectively promotes apoptosis in human AML cell lines (e.g., NB4, OCI-AML3, THP-1, MOLM-13) and mouse AML cell line WEHI, as evidenced by reduced cell viability, increased caspase 3/7 activity, mitochondrial depolarization (detected by TMRE assay), and cytochrome c release. Western blot analysis confirms BTSA1-induced BAX translocation from the cytosol to mitochondria in NB4 cells [1]
- Selectivity and sensitivity determinants: BTSA1 shows no significant binding to anti-apoptotic BCL-2 family proteins (BCL-XL, MCL-1, BFL-1) in competitive binding assays. It spares non-cancerous cell lines and normal hematopoietic progenitor cells, with minimal toxicity. BAX expression levels and cytosolic monomeric conformation regulate sensitivity to BTSA1: AML cells with higher BAX mRNA/protein levels and monomeric cytosolic BAX (e.g., OCI-AML3, HPB-ALL) are more sensitive, while BAX knockout (KO) MEFs are resistant. Reconstitution of wild-type BAX in BAX KO MEFs restores sensitivity, but BAX mutants with impaired trigger site function do not. BTSA1 synergizes with Venetoclax (BCL-2 inhibitor) in AML cell lines (THP-1, OCI-AML3), enhancing caspase 3/7 activity and reducing cell viability [1]
- Efficacy in patient AML samples: BTSA1 induces apoptosis (detected by Annexin V binding) in primary human AML blasts (n=4) and CD34⁺CD38⁻ AML stem cell-enriched populations (n=4) without affecting healthy CD34⁺CD38⁻ hematopoietic stem and progenitor cells (n=2). AML patient cells (n=542) have significantly higher BAX mRNA expression compared to healthy control cells (n=74) [1]

BTSA1 potently suppresses human acute myeloid leukemia (AML) xenografts and increases host survival without toxicity. The healthy stem cellenriched (LSK) cells, common myeloid progenitors, granulocyte-monocyte progenitors, and megakaryocyte-erythrocyte progenitors are well-tolerated in mice and exhibit no toxic effects. A 10 mg/kg dose of BTSA1 reaches sufficient levels (~15 μM) to cause BAX activation and apoptosis in leukemia cells while also having a significant half-life in mouse plasma (T1/2 = 15 hr) and oral bioavailability (%F = 51). As a result, BTSA1 has excellent pharmacokinetics, is orally bioavailable, significantly inhibits tumor growth in leukemia xenografts by inducing apoptosis, and at therapeutically effective doses exhibits no detectable toxicity in the hematopoietic system or other tissues[1].

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Antitumor efficacy in AML xenografts: In THP-1 human AML xenograft NSG mice, BTSA1 treatment significantly suppresses tumor burden in the liver (reduced human CD45⁺ and CD15⁺ cell infiltration) and prolongs host survival (Kaplan-Meier survival curves, n=7). In MOLM-13 human AML xenografts, BTSA1 reduces human CD45⁺CD15⁺ cell infiltration in the bone marrow and peripheral blood (n=5). Immunohistochemical (IHC) staining of bone marrow sections shows increased cleaved caspase-3-positive cells and TUNEL-positive cells in BTSA1-treated mice, indicating enhanced apoptosis. TMRE staining of bone marrow-derived human leukemia cells confirms mitochondrial depolarization, consistent with BAX-mediated MOMP [1]
- In vivo safety and tolerability: NSG mice treated with BTSA1 (15 mg/kg body weight) for 30 days show no significant changes in body weight compared to vehicle-treated controls. Peripheral blood counts (red blood cells, white blood cells, platelets) remain within normal ranges. Hematoxylin & Eosin (H&E) staining of major organs (liver, spleen, kidney, lung, heart, brain, bone marrow) reveals no histopathological abnormalities, demonstrating no systemic toxicity [1]

Fluorescence polarization binding assays for BAX interaction:
1. Competitive binding assay: Prepare reaction mixtures containing FITC-BIM SAHB A2 (bound to BAX), BTSA1 (serial concentrations), and assay buffer. Incubate at room temperature for a specified time, then measure fluorescence polarization to assess competition between BTSA1 and FITC-BIM SAHB A2 for BAX binding. Perform parallel assays with BCL-XL, MCL-1, and BFL-1 to evaluate selectivity.
2. Direct binding assay: Use fluorescent-labeled BTSA1 (F-BTSA1) and serial concentrations of BAX. Incubate the mixture and measure fluorescence polarization to determine the direct binding affinity of BTSA1 for BAX [1]
- NMR-based BAX conformational change assay:
1. Prepare ¹⁵N-labeled inactive BAX protein and dissolve it in appropriate NMR buffer.
2. Titrate BTSA1 into the ¹⁵N-labeled BAX solution up to a 1:1 molar ratio, collecting ¹H-¹⁵N HSQC spectra at each titration step.
3. Analyze chemical shift changes of BAX backbone amide residues, map significant changes to the BAX structure, and identify the binding site (trigger site) [1]
- Liposome membrane permeabilization assay:
1. Prepare ANTS/DPX-loaded liposomes to mimic mitochondrial outer membranes.
2. Incubate liposomes with 200 nM BAX (or BAX K21E mutant) and serial concentrations of BTSA1 (100–400 nM), with 60 nM tBID as a positive control.
3. Measure fluorescence intensity of ANTS released from liposomes over time to assess BTSA1-induced BAX-mediated membrane permeabilization [1]

AML cells (seeded at 2 × 104 cells/well) are incubated with serial dilutions of BTSA1 or BTSA2 or vehicle (0.15% DMSO) in no FBS media for 2.5 hours before 10% FBS replacement is added to a final volume of 100 l. At 24 hours, cell viability is assessed.

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Cell viability and apoptosis assays:
1. Cell culture: Maintain AML cell lines (NB4, OCI-AML3, THP-1, MOLM-13), mouse AML cell line WEHI, non-cancerous cell lines, MEFs (wild-type, BAX KO, BAK KO), and reconstituted MEFs in appropriate culture media. Isolate primary human AML blasts, CD34⁺CD38⁻ AML cells, and healthy hematopoietic progenitor cells from patient samples and healthy donors.
2. Drug treatment: Seed cells in 96-well plates at appropriate densities, treat with serial concentrations of BTSA1 (alone or in combination with Venetoclax) for 6–24 hours. Set up vehicle control and positive control groups.
3. Viability detection: Use a cell viability assay kit to measure absorbance or luminescence, calculate cell viability, and determine IC50 values.
4. Apoptosis detection: Perform Annexin V binding assays for primary cells and caspase 3/7 activity assays for cell lines, measuring fluorescence or luminescence to quantify apoptosis. Use TMRE staining to assess mitochondrial depolarization via flow cytometry [1]
- Western blot and immunoprecipitation assays:
1. Protein extraction: Lyse treated cells or isolate cytosolic and mitochondrial fractions, extract total proteins using lysis buffer supplemented with protease inhibitors.
2. Western blot: Separate proteins by SDS-PAGE, transfer to membranes, block, and probe with antibodies against BAX, cytochrome c, BCL-2, BCL-XL, MCL-1, Actin (cytosolic loading control), and VDAC (mitochondrial loading control). Detect signals using chemiluminescence.
3. Immunoprecipitation: Incubate cell lysates with BAX antibody or biotin-labeled BTSA1, capture immune complexes with protein A/G beads or streptavidin beads, wash, and perform Western blot to detect BAX-interacting proteins [1]
- Size-exclusion chromatography for BAX monomer detection:
1. Prepare cytosolic extracts from MEF, OCI-AML3, and HPB-ALL cells.
2. Load extracts onto a Superdex 200 HR 10/30 column, elute with buffer, and collect fractions corresponding to different molecular weights.
3. Perform Western blot with BAX antibody on collected fractions to identify monomeric BAX (lower molecular weight fractions) [1]

Formulated in 1% DMSO, 30% PEG-400, 65% D5W (5% dextrose in water), 4% Tween-80; 10 mg/kg; P.O. and I.V.
NOD-SCID IL2Rg null (NSG) mice/ICR (CD-1) male mice, 6-8 weeks old

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AML xenograft mouse models:
1. Animal preparation: Use 6–8-week-old NSG mice, acclimate them to the laboratory environment for 1 week before experiments.
2. Tumor cell inoculation: Inject human AML cells (THP-1 or MOLM-13) into mice via tail vein or intraperitoneal injection to establish xenograft models.
3. Drug preparation and administration: Dissolve BTSA1 in an appropriate vehicle (e.g., DMSO/cremophor/ saline) to a concentration suitable for injection. Administer BTSA1 to mice at a dose of 15 mg/kg body weight via intraperitoneal injection, with a predetermined schedule (e.g., daily for 30 days). Vehicle-treated mice serve as controls.
4. Survival and tumor burden assessment: For THP-1 xenografts, monitor mouse survival daily and generate Kaplan-Meier survival curves. For MOLM-13 and THP-1 xenografts, sacrifice mice at specified time points, collect liver, bone marrow, and peripheral blood samples.
5. Flow cytometry analysis: Stain samples with mouse CD45, human CD45, and human CD15 antibodies, analyze by flow cytometry to quantify human AML cell infiltration.
6. IHC and TMRE assays: Prepare bone marrow sections for cleaved caspase-3 and TUNEL IHC staining to detect apoptosis. Perform TMRE staining on bone marrow-derived human leukemia cells to assess mitochondrial depolarization [1]
- In vivo safety assessment:
1. Animal treatment: Administer BTSA1 (15 mg/kg body weight) or vehicle to NSG mice via intraperitoneal injection daily for 30 days.
2. Physiological monitoring: Measure mouse body weight weekly. At the end of treatment, collect peripheral blood for complete blood count (red blood cells, white blood cells, platelets).
3. Histopathological analysis: Harvest major organs (liver, spleen, kidney, lung, heart, brain, bone marrow), fix in formalin, embed in paraffin, section, and stain with H&E. Examine sections for histopathological abnormalities [1]

In vitro toxicity: BTSA1 showed very low toxicity to non-cancer cell lines and healthy artificial hematopoietic progenitor cells, and no significant decrease in cell viability was observed at effective concentrations for AML cells [1]. In vivo toxicity: BTSA1 (15 mg/kg body weight, 30 days of treatment) did not cause weight loss, hematological abnormalities, or histopathological damage to major organs in NSG mice. BAX KO MEF cells were resistant to BTSA1, confirming that its cytotoxicity was BAX-dependent and selective for cancer cells expressing BAX [1].

[1]. Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia. Cancer Cell. 2017 Oct 9;32(4):490-505.e10.

Mechanism of action: BTSA1 is a pharmacologically optimized BAX activator that binds to the N-terminal trigger site of BAX, inducing conformational changes (exposure of membrane-targeting domains, oligomerization), thereby leading to BAX translocation to mitochondria, mitochondrial outer membrane permeability (MOMP), cytochrome c release, and caspase-dependent apoptosis. Its binding mode mimics the BIM BH3 helix, forming a key hydrogen bond with BAX K21 through hydrophobic interactions [1].
- Therapeutic potential: BTSA1 overcomes apoptosis resistance in acute myeloid leukemia (AML) by directly activating BAX (a key mediator of apoptosis). The efficacy of BTSA1 in AML cell lines, patient samples (including cell populations enriched with stem cells), and xenograft models, along with its high selectivity for cancer cells and lack of systemic toxicity, provides proof of concept for direct activation of BAX as a novel strategy for treating acute myeloid leukemia [1]. Synergistic effect with venetoclax: BTSA1 can synergize with venetoclax (a BCL-2 inhibitor) to enhance the apoptotic response of AML cells by releasing and directly activating BAX. This combination therapy may improve the treatment outcomes for AML patients, especially those with BCL-2 overexpression [1].

C21H14N6OS2

430.51

430.067

C, 58.59; H, 3.28; N, 19.52; O, 3.72; S, 14.89

314761-14-3

3857348

Brown to reddish brown solid powder

1.5±0.1 g/cm3

625.2±48.0 °C at 760 mmHg

331.9±29.6 °C

0.0±1.8 mmHg at 25°C

1.791

3.68

1

8

5

30

689

0

S1C([H])=C(C2C([H])=C([H])C([H])=C([H])C=2[H])N=C1N1C(C(=C(C2C([H])=C([H])C([H])=C([H])C=2[H])N1[H])/N=N/C1=NC([H])=C([H])S1)=O

CTRCXGFSYFTJIW-UHFFFAOYSA-N

InChI=1S/C21H14N6OS2/c28-19-18(24-25-20-22-11-12-29-20)17(15-9-5-2-6-10-15)26-27(19)21-23-16(13-30-21)14-7-3-1-4-8-14/h1-13,26H

5-phenyl-2-(4-phenyl-1,3-thiazol-2-yl)-4-(1,3-thiazol-2-yldiazenyl)-1H-pyrazol-3-one

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)