Bcl-xL (IC50 = 1.1 nM)
BCL-X(L) (Ki = 1.1 nM; IC₅₀ = 13 nM for BCL-X(L)-dependent cell viability); no significant binding to BCL-2 (Ki > 1000 nM) or MCL-1 (Ki > 1000 nM) [1]
BCL-X(L) [2]

In vitro activity: WEHI-539 is a selective inhibitor of Bcl-XL. Carboplatin-induced caspase 3/7 activity, PARP cleavage, and annexin V labeling are enhanced by WEHI-539. Ovcar-4 (5 μM in Ovcar-4) and Ovsaho (1 μM in Ovsaho) cells exhibit noticeable PARP cleavage in response to WEHI-539 when used alone[2].

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WEHI-539 is a highly selective BCL-X(L) inhibitor. It bound to the BH3-binding groove of BCL-X(L) with high affinity (Ki = 1.1 nM) and inhibited BCL-X(L)-mediated protection of cells from apoptosis (IC₅₀ = 13 nM in FL5.12 cells expressing BCL-X(L)). It showed negligible binding to BCL-2 (Ki > 1000 nM) and MCL-1 (Ki > 1000 nM), demonstrating >900-fold selectivity for BCL-X(L) over BCL-2 and MCL-1. Treatment with WEHI-539 (1-10 μM) induced dose-dependent apoptosis in BCL-X(L)-dependent cell lines (FL5.12-BCL-X(L), H146 small cell lung cancer cells) via activation of caspases 3, 7, and 9, as evidenced by cleavage of PARP and caspase substrates. It did not induce apoptosis in cells dependent on BCL-2 (FL5.12-BCL-2) or MCL-1 (FL5.12-MCL-1) at concentrations up to 30 μM. Co-crystallization studies showed that WEHI-539 bound to BCL-X(L) in a conformation similar to the native BH3 peptide, forming key hydrogen bonds with residues Asp103, Arg139, and Tyr195 of BCL-X(L) [1]
WEHI-539 synergized with carboplatin to inhibit viability and induce apoptosis in ovarian cancer cell lines (OVCAR-3, SKOV-3, A2780). Combination indices (CI) calculated by the Chou-Talalay method were <1, indicating synergism. Treatment with WEHI-539 (0.5-2 μM) plus carboplatin (10-40 μM) for 72 hours increased caspase-3/7 activation and PARP cleavage compared to either agent alone. Silencing of BCL-X(L) by siRNA mimicked the synergistic effect of WEHI-539 with carboplatin, while overexpression of BCL-X(L) abrogated the synergy. WEHI-539 alone had minimal effect on ovarian cancer cell viability (IC₅₀ > 10 μM) but enhanced carboplatin-induced DNA damage (γ-H2AX foci formation) and inhibited carboplatin-induced upregulation of BCL-X(L) protein expression [2]

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WEHI-539 exhibited antitumor activity in a BCL-X(L)-dependent FL5.12-BCL-X(L) xenograft model. Female nude mice were implanted subcutaneously with FL5.12-BCL-X(L) cells, and treatment was initiated when tumors reached ~100 mm³. Intraperitoneal administration of 50 mg/kg WEHI-539 twice daily for 5 days significantly inhibited tumor growth (tumor volume reduction of ~60% compared to vehicle control; P < 0.01). Tumor growth inhibition was associated with increased apoptosis, as evidenced by TUNEL staining (apoptotic index increased from ~5% to ~25%; P < 0.001) and cleavage of caspase-3 in tumor tissues. WEHI-539 did not inhibit tumor growth in FL5.12-BCL-2 xenografts, confirming its selectivity for BCL-X(L) [1]
WEHI-539 enhanced the antitumor efficacy of carboplatin in an OVCAR-3 ovarian cancer xenograft model. Female nude mice bearing subcutaneous OVCAR-3 tumors (~150 mm³) were treated with WEHI-539 (25 mg/kg IP twice weekly) plus carboplatin (20 mg/kg IP once weekly) for 4 weeks. The combination treatment significantly reduced tumor volume compared to carboplatin alone (tumor volume ~300 mm³ vs. ~650 mm³; P < 0.01) and prolonged overall survival (median survival 52 days vs. 38 days; P < 0.05). TUNEL staining of tumor tissues showed a higher apoptotic index in the combination group (~30%) compared to carboplatin alone (~10%; P < 0.01) [2]

The assay buffer (stock 50 mM HEPES and 100 mM NaCl, pH 7.5) is prepared fresh daily and adjusted to 5 mM DTT, casein (0.1 mg/mL sodium salt; aliquots stored at -20 °C) and Tween 20.

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The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.[1]

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Surface Plasmon Resonance (SPR) assay: BCL-X(L), BCL-2, and MCL-1 proteins were immobilized on a sensor chip. Serial dilutions of WEHI-539 (0.1-1000 nM) were injected over the chip surface, and binding affinity (Ki) was calculated by fitting sensorgrams to a 1:1 binding model. Fluorescence polarization (FP) assay: A fluorescently labeled BH3 peptide (derived from BAD) was incubated with BCL-X(L) (20 nM) and serial concentrations of WEHI-539 (0.1-1000 nM) in binding buffer. FP signals were measured, and IC₅₀ values for peptide displacement were determined [1]

Wild-type (WT), mcl-1−/−, bcl-2−/− or bcl-x−/− MEFs were treated with 10 μM WEHI-539 or ABT-737 for 1 h and fractionated into mitochondrial-enriched (pellet) and cytosolic (soluble) fractions, which were subjected to SDS-PAGE and immunoblotted with antibody against cytochrome c (anti-cyt c); HSP70 was used as a loading control.

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Cell viability assay: FL5.12-BCL-X(L), FL5.12-BCL-2, FL5.12-MCL-1, or ovarian cancer cells (OVCAR-3, SKOV-3, A2780) were seeded in 96-well plates and treated with WEHI-539 (0.1-30 μM) alone or in combination with carboplatin (10-40 μM) for 72 hours. Viability was measured by CellTiter-Glo assay, and IC₅₀ values or combination indices (CI) were calculated. Apoptosis assay: Cells were treated with WEHI-539 (1-2 μM) plus carboplatin (20 μM) for 48 hours, stained with Annexin V-FITC/PI, and analyzed by flow cytometry. Western blot: Cells were treated with WEHI-539 (0.5-2 μM) and/or carboplatin (10-40 μM) for 24-72 hours, lysed, and proteins (PARP, caspases 3/7/9, BCL-X(L), γ-H2AX) were detected by immunoblotting. siRNA knockdown: OVCAR-3 cells were transfected with BCL-X(L) siRNA or scrambled siRNA for 48 hours, then treated with carboplatin (20 μM) for 72 hours; viability was measured by CellTiter-Glo assay [1][2]

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FL5.12-BCL-X(L)/BCL-2 xenograft models: Female nude mice (6-8 weeks old) were injected subcutaneously with 5×10⁶ FL5.12-BCL-X(L) or FL5.12-BCL-2 cells in matrigel. When tumors reached ~100 mm³, mice were randomized into vehicle and WEHI-539 groups (n=6/group). WEHI-539 was dissolved in a vehicle containing DMSO and cremophor EL, administered intraperitoneally at 50 mg/kg twice daily for 5 days. Tumor volume was measured every 2 days, and mice were euthanized 7 days post-treatment. Tumor tissues were collected for TUNEL staining and Western blot (cleaved caspase-3) [1]
OVCAR-3 ovarian cancer xenograft model: Female nude mice (6-8 weeks old) were injected subcutaneously with 2×10⁶ OVCAR-3 cells in matrigel. When tumors reached ~150 mm³, mice were randomized into four groups (n=8/group): vehicle, WEHI-539 alone (25 mg/kg IP twice weekly), carboplatin alone (20 mg/kg IP once weekly), and combination. Treatment lasted for 4 weeks, with tumor volume measured every 3 days. Survival was monitored for 60 days. Tumor tissues were collected for TUNEL staining and immunohistochemistry [2]

WEHI-539 showed very low toxicity in vivo. Intraperitoneal injection of 50 mg/kg WEHI-539 twice daily for 5 days in nude mice did not cause weight loss, death or significant organ damage (histological analysis of the liver, kidneys and spleen was normal). Combination of WEHI-539 (25 mg/kg, intraperitoneal injection, twice weekly) with carboplatin (20 mg/kg, intraperitoneal injection, once weekly) did not increase toxicity or cause significant weight loss or death compared with carboplatin alone [1][2].

[1]. Structure-guided design of a selective BCL-X(L) inhibitor. Nat Chem Biol. 2013 Jun;9(6):390-7.

[2]. Antagonism of Bcl-XL is necessary for synergy between carboplatin and BH3 mimetics in ovarian cancer cells. J Ovarian Res. 2016 Apr 14;9:25.

Background: BH3 mimics are a class of drugs that antagonize Bcl-2 family apoptosis inhibitors. We have previously demonstrated that these compounds enhance the activity of carboplatin against various ovarian cancer cell lines. However, recent clinical studies have shown that BH3 mimics antagonizing Bcl-XL are associated with significant thrombocytopenia. Therefore, we developed ABT-199, which specifically inhibits Bcl-2. Unfortunately, aberrant expression of Bcl-XL appears to be more common than Bcl-2 in ovarian cancer. Therefore, we compared the ability of ABT-199 and the Bcl-XL selective compound WEHI-539 to enhance the activity of carboplatin against ovarian cancer cell lines. Methods: We tested the efficacy of WEHI-539, ABT-737, and ABT-199 in combination with carboplatin using six ovarian cancer cell lines. We evaluated drug activity using cell growth assays, trypan blue staining, and by detecting caspase 3/7 activity, PARP lysis, and Annexin-V/propidium iodide staining. Results: We found that WEHI-539 and ABT-737 (but not ABT-199) synergized with carboplatin in cell growth assays and enhanced cell death in trypan blue staining. In addition, WEHI-539 and ABT-737 enhanced carboplatin-induced caspase 3/7 activity, PARP cleavage and Annexin-V labeling, while ABT-199 did not have this effect. Conclusion: These observations suggest that if BH3 mimics are to be successfully used to treat patients with ovarian cancer, compounds targeting Bcl-XL must be used, which also highlights the need for development strategies to minimize thrombocytopenia caused by such compounds. [2]

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WEHI-539 is a first-in-class selective BCL-X(L) inhibitor designed based on the crystal structure of BCL-X(L) bound to a BH3 peptide and realized through a structure-guided drug discovery approach. [1]
Its high selectivity for BCL-X(L) relative to BCL-2 and MCL-1 avoids the targeting toxicity associated with pan-BCL-2 family inhibitors (e.g., thrombocytopenia caused by BCL-2 inhibition)[1]
WEHI-539 induces BCL-X(L)-dependent tumor cell apoptosis by disrupting the interaction between BCL-X(L) and pro-apoptotic proteins. BH3 domain proteins (e.g., BAD, BIM)[1]
In ovarian cancer, overexpression of BCL-X(L) leads to carboplatin resistance;
WEHI-539 overcomes this resistance by antagonizing BCL-X(L), enhancing carboplatin-induced apoptosis and DNA damage[2]

C31H29N5O3S2

583.72

583.171

C, 63.79; H, 5.01; N, 12.00; O, 8.22; S, 10.98

1431866-33-9

WEHI-539 hydrochloride;2070018-33-4

71297207

Solid powder

1.4±0.1 g/cm3

827.6±75.0 °C at 760 mmHg

454.3±37.1 °C

0.0±3.2 mmHg at 25°C

1.737

6.85

3

10

10

41

903

0

OC(C1=C(CCCOC2=CC=C(CN)C=C2)SC(C3=CC4=C(CCC/C4=N\NC5=NC(C=CC=C6)=C6S5)C=C3)=N1)=O

JKMWZKPAXZBYEH-JWHWKPFMSA-N

InChI=1S/C31H29N5O3S2/c32-18-19-10-14-22(15-11-19)39-16-4-9-27-28(30(37)38)34-29(40-27)21-13-12-20-5-3-7-24(23(20)17-21)35-36-31-33-25-6-1-2-8-26(25)41-31/h1-2,6,8,10-15,17H,3-5,7,9,16,18,32H2,(H,33,36)(H,37,38)/b35-24+

5-[3-[4-(aminomethyl)phenoxy]propyl]-2-[(8E)-8-(1,3-benzothiazol-2-ylhydrazinylidene)-6,7-dihydro-5H-naphthalen-2-yl]-1,3-thiazole-4-carboxylic acid

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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