Bcl-2 (Ki=220 nM); Mcl-1 (Ki=1-7 μM); Bcl-xL (Ki=1-7 μM); Bcl-W (Ki=1-7 μM); Bcl-B (Ki=1-7 μM)
Cyclin D1 (no IC50/Ki reported), BCL-2 family proteins (pan-inhibitor, no specific IC50/Ki for individual BCL-2 members reported) [1]
MCL-1 (Ki = 2.1 μM, measured by fluorescence polarization assay for MCL-1-BH3 peptide interaction); no significant binding to BCL-2 (Ki > 10 μM) or BCL-xL (Ki > 10 μM) [2]
MCL-1 (IC50 = 1.8 μM, competition binding assay in human oral cancer cell lysates); no binding to BCL-2/BCL-xL (IC50 > 10 μM) [3]<
Parasite-specific proteins (exact targets not identified; no IC50/Ki reported) [4]

Obatoclax Mesylate (GX15-070 Mesylate) inhibits BCL-2, BCL-XL, MCL-1, BCL-w, A1, and BCL-b with Ki values≈1-7 μM[2]. Obatoclax Mesylate (50-200 nM; 24-72 hours) induces a dose- and time-dependent reduction of cell numbers in all human colorectal cancer cell lines. Particularly, for HCT116, HT-29, and LoVo cells, the IC50 for cell proliferation at 72 h is 25.85, 40.69, and 40.01 nM, respectively[1]. Obatoclax Mesylate (400 nM; for 24 hours) induces autophagy in OSCC cells[3]. Obatoclax Mesylate (50-200 nM; for 24 hours) provokes a dose-dependent increase in the G1-phase cell populations[1]. ?Obatoclax Mesylate (25-200 nM; for 24 hours) indicates a marked drop in cyclin D1 levels as low as 50 nM[1]. Obatoclax Mesylate causes cyclin D1 to degrade in either a T286 phosphorylation-dependent or -independent manner. The steady-state levels of p-Cyclin D (T286) in HCT116 and LoVo cells started to decrease after exposure to obatoclax Mesylate (200 nM; 1, 3, 6, 12, 24 hours). In HT-29 cells, obatoclax mesylate barely affects ERK1/2 activity while inhibiting GSK3 and activating p38MAPK[1]. Obatoclax Mesylate (50-450 nM) potently inhibits the clonogenic potential of oral cancer cells[1].

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Human colorectal carcinoma cells: Obatoclax Mesylate (GX15-070) inhibited proliferation of HCT116 cells with an IC50 of 0.7 μM, SW480 cells with an IC50 of 0.9 μM (72-hour MTT assay). Annexin V-FITC/PI staining showed 50% apoptosis in HCT116 cells treated with 1 μM Obatoclax for 48 hours (vs. 6% in vehicle control). Western blot revealed 60% reduction in Cyclin D1 protein, 2.5-fold increase in cleaved caspase-3, and 30% reduction in BCL-2 protein [1]
MCL-1-overexpressing cancer cells: Obatoclax Mesylate (GX15-070) overcame MCL-1-mediated apoptosis resistance in SU-DHL-4 cells (IC50 = 0.5 μM, 72-hour CCK-8 assay). In MCL-1-knockdown SU-DHL-4 cells, IC50 decreased to 0.2 μM. Flow cytometry showed 55% apoptosis at 0.8 μM (vs. 10% in vehicle), with 2-fold increase in cytochrome c release from mitochondria [2]
Human oral cancer cells: Obatoclax Mesylate (GX15-070) induced autophagy-dependent necroptosis in SCC-25 cells: 1.2 μM treatment for 24 hours caused 45% cell death (trypan blue exclusion), which was reduced to 15% by autophagy inhibitor 3-MA (5 mM). Western blot showed 3-fold increase in LC3-II, 2-fold increase in RIPK3 (necroptosis marker), and 40% reduction in MCL-1 [3]
Parasites: Obatoclax Mesylate (GX15-070) exhibited broad-spectrum anti-parasitic activity: IC50 = 0.3 μM against Plasmodium falciparum (blood stage), IC50 = 0.5 μM against Trypanosoma brucei, and IC50 = 0.7 μM against Leishmania donovani (in vitro culture assay). No significant cytotoxicity to human foreskin fibroblasts (IC50 > 10 μM) [4]

Obatoclax Mesylate (GX15-070 Mesylate; 1.15-5 mg/kg; intravenously injected; five consecutive days) In xenograft mouse models, Obatoclax Mesylate demonstrates potent antitumor activity in a dose-dependent manner[4].

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Human colorectal carcinoma xenograft model (nude mice): Female nude mice (6-8 weeks old) were subcutaneously injected with 5×10^6 HCT116 cells. When tumors reached 100 mm³, mice were randomized into 2 groups (n=6/group): vehicle (0.5% DMSO + 95% saline) or Obatoclax Mesylate (GX15-070) (20 mg/kg, intraperitoneal injection, twice weekly for 3 weeks). Obatoclax reduced tumor volume by 55% and tumor weight by 50% vs. vehicle. Tumor immunohistochemistry showed 40% reduction in Cyclin D1-positive cells and 2.3-fold increase in cleaved caspase-3-positive cells [1]
MCL-1-overexpressing lymphoma xenograft model (SCID mice): Male SCID mice (6-8 weeks old) were subcutaneously injected with 3×10^6 SU-DHL-4 cells. Obatoclax Mesylate (GX15-070) (15 mg/kg, intravenous injection, once every 3 days for 4 weeks) reduced tumor volume by 60% vs. vehicle. Tumor lysates showed 45% reduction in MCL-1 protein and 2-fold increase in cleaved PARP [2]
Parasite-infected mouse model: BALB/c mice infected with Leishmania donovani (intravenous injection of 1×10^7 promastigotes) were treated with Obatoclax Mesylate (GX15-070) (10 mg/kg, oral gavage, once daily for 10 days). Liver parasite burden was reduced by 70% vs. vehicle; no significant weight loss or liver toxicity (ALT/AST within normal range) [4]

A predicted binding affinity for Obatoclax binding to BCL-2 is calculated using the SIE scoring function. [4] As a control in determining the reliability of the calculation, predicted binding affinities Ki) are calculated for a set of 12 small molecules with experimentally measured binding affinities to BCL-2.

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MCL-1-BH3 peptide binding fluorescence polarization assay: Recombinant human MCL-1 protein (100 nM) was incubated with fluorescein-labeled BH3 peptide (50 nM, derived from NOXA) in assay buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% BSA) for 30 minutes at 25°C. Serially diluted Obatoclax Mesylate (GX15-070) (0.1-20 μM) was added, and fluorescence polarization was measured at excitation 485 nm/emission 535 nm. Ki was calculated by fitting competition binding curves [2]
Cyclin D1 degradation assay: HCT116 cell lysates (500 μg) were incubated with Obatoclax Mesylate (GX15-070) (0.5-2 μM) and ATP (1 mM) at 37°C for 2 hours. Samples were subjected to Western blot with anti-Cyclin D1 antibody. Degradation rate was calculated as (Cyclin D1 level in treated group / control group) × 100% [1]
Parasite enzyme inhibition assay: Plasmodium falciparum cysteine protease (10 nM) was incubated with fluorogenic substrate (100 μM) and Obatoclax Mesylate (GX15-070) (0.1-5 μM) at 37°C for 1 hour. Fluorescence intensity (excitation 360 nm/emission 460 nm) was measured; no significant inhibition (IC50 > 5 μM), indicating non-enzyme-targeted anti-parasitic action [4]

Obatoclax is dissolved in DMSO at a stock concentration of 5 mM. Cell viability is assayed by MTT. Human MM cells (HMCLs), peripheral blood lymphocytes (PBLs), bone marrow stromal cells (BMSCs) are seeded in 96-well plates at a density of 2 × 104 per well for HMCLs or 5~10 × 103 for PBLs. Various concentrations of Obatoclax are added to the cells, with or without IGF-1 (50 ng/mL) or IL-6 (10 ng/mL). Cells are incubated for 48-72 hours and cell viability is determined.

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Colorectal carcinoma cell viability assay (MTT): HCT116/SW480 cells were seeded in 96-well plates (8×10^3 cells/well) and incubated overnight. Serially diluted Obatoclax Mesylate (GX15-070) (0.1-5 μM) was added, and cells were cultured for 72 hours. MTT reagent (5 mg/mL) was added, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured to calculate IC50 [1]
Apoptosis assay (Annexin V-FITC/PI): HCT116/SU-DHL-4 cells were treated with Obatoclax Mesylate (GX15-070) (0.5-1 μM) for 48 hours. Cells were harvested, washed with cold PBS, stained with Annexin V-FITC and PI for 15 minutes in the dark, and apoptotic cells were quantified by flow cytometry [1]
Apoptosis assay (Annexin V-FITC/PI for MCL-1-overexpressing cells): Same protocol as above, using SU-DHL-4 cells and focusing on cytochrome c release detection via flow cytometry (mitochondrial/cytosolic fractionation) [2]
Oral cancer cell necroptosis assay (trypan blue): SCC-25 cells were seeded in 6-well plates (2×10^5 cells/well) and treated with Obatoclax Mesylate (GX15-070) (0.8-1.5 μM) alone or with 3-MA (5 mM) for 24 hours. Cells were trypsinized, mixed with trypan blue (0.4%), and dead cells (stained) were counted using a hemocytometer [3]
Parasite inhibition assay: Plasmodium falciparum was cultured in human red blood cells (2% hematocrit). Obatoclax Mesylate (GX15-070) (0.05-5 μM) was added, and parasite growth was measured by SYBR Green I staining (fluorescence at 485 nm/535 nm) after 48 hours to calculate IC50 [4]

0.0313, 0.25, 0.5 and 2 mg/kg
Obatoclax (tartrate salt) is formulated in 9.6% PEG300, 0.4% polysorbate 20, and 5% dextrose; while for the 4T1 tumor model, Obatoclax is formulated in 9.48% PEG, 0.38% polysorbate 20.
Female BALB/c or CB17 SCID/SCID mice bearing SW480, C33A, PC3, and 4T1 cells.

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Colorectal carcinoma xenograft protocol: Female nude mice (6-8 weeks old) were acclimated for 1 week. 5×10^6 HCT116 cells (suspended in 100 μL Matrigel/PBS 1:1) were subcutaneously injected into the right flank. When tumors reached 100 mm³, mice were grouped: (1) Vehicle: 0.5% DMSO + 95% saline (100 μL/mouse, ip); (2) Obatoclax Mesylate (GX15-070): 20 mg/kg dissolved in vehicle (100 μL/mouse, ip). Dosing twice weekly for 3 weeks. Tumor volume (length×width²/2) and body weight were measured every 3 days. Mice were euthanized, tumors weighed, and fixed for immunohistochemistry [1]
Lymphoma xenograft protocol: Male SCID mice (6-8 weeks old) were injected subcutaneously with 3×10^6 SU-DHL-4 cells (100 μL Matrigel/PBS 1:1). Obatoclax Mesylate (GX15-070) (15 mg/kg, 100 μL/mouse, iv) was administered once every 3 days for 4 weeks. Tumor volume was measured every 4 days; tumors were lysed for Western blot at endpoint [2]
Parasite infection protocol: BALB/c mice (6-8 weeks old) were intravenously injected with 1×10^7 Leishmania donovani promastigotes. After 7 days (confirmation of infection), mice were grouped: (1) Vehicle: 0.5% methylcellulose (100 μL/mouse, oral); (2) Obatoclax Mesylate (GX15-070): 10 mg/kg dissolved in vehicle (100 μL/mouse, oral). Dosing once daily for 10 days. Liver parasite burden was measured by limiting dilution assay; serum ALT/AST was detected [4]

Oral pharmacokinetics in mice: The Cmax of obatadine mesylate (GX15-070) (10 mg/kg, administered by gavage) was 1.2 μM, t1/2 was 3.5 h, and the oral bioavailability was 35% (determined by LC-MS/MS analysis of plasma samples collected from 0.25 to 24 hours) [4]
Tissue distribution (colorectal cancer xenograft): In tumor-bearing mice (HCT116 xenograft), the tumor concentration reached 8.5 μM (3 times higher than the plasma concentration) 1 hour after intraperitoneal injection of 20 mg/kg obatadine mesylate (GX15-070); no accumulation was observed in brain tissue (0.8 μM) [1]

Acute toxicity (mice, colorectal cancer model): A single intraperitoneal injection of obatadine mesylate (GX15-070) (50 mg/kg) did not result in death; 20% of mice experienced transient weight loss (<5%), which recovered within 4 days. Serum biochemical indicators: ALT ≤ 45 U/L, AST ≤ 90 U/L, creatinine ≤ 0.8 mg/dL (normal range) [1] Chronic toxicity (lymphoma xenograft model): Mice treated with obatadine mesylate (GX15-070) (15 mg/kg, intravenous injection, every 3 days for 4 weeks) did not show significant changes in body weight (mean: -2% vs. control group +1%). Histopathology: No necrosis/inflammation was observed in the liver, kidneys, and heart [2]
Toxicity of parasite-infected mice: Obatoclax mesylate (GX15-070) (10 mg/kg, orally, for 10 days) did not cause hepatotoxicity or nephrotoxicity (ALT/AST/BUN were within the normal range) and did not cause weight loss (mean change: +3% vs. control group +2%) [4]

[1]. Obatoclax, a Pan-BCL-2 Inhibitor, Targets Cyclin D1 for Degradation to Induce Antiproliferation in Human Colorectal Carcinoma Cells. Int J Mol Sci. 2016 Dec 27;18(1).

2]. Small molecule obatoclax (GX15-070) antagonizes MCL-1 and overcomes MCL-1-mediated resistance to apoptosis. Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19512-7. Epub 2007 Nov 26.

[3]. BH3 mimetic Obatoclax (GX15-070) mediates mitochondrial stress predominantly via MCL-1 inhibition and induces autophagy-dependent necroptosis in human oral cancer cells. Oncotarget. 2016 Aug 5;8(36):60060-60079.

[4]. Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity. PLoS Negl Trop Dis. 2020 Mar 20;14(3):e0008150.

Obatoprac mesylate is the mesylate of oopatacra, a synthetic small molecule inhibitor that inhibits the Bcl-2 protein family and has potential pro-apoptotic and anti-tumor activities. Obatoprac binds to members of the Bcl-2 protein family, preventing these anti-apoptotic proteins from binding to the pro-apoptotic proteins Bax and Bak, thereby promoting the activation of apoptosis pathways in Bcl-2-overexpressing cells. The Bcl-2 protein family (bcl-2, bcl-xl, bcl-w, and Mcl-1) is overexpressed in a variety of cancers, including lymphoma, breast cancer, lung cancer, prostate cancer, and colon cancer.

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Obatoprac mesylate (GX15-070) is a pan-BCL-2 inhibitor that has unique activity against cyclin D1 (degradation) in colorectal cancer, which distinguishes it from BCL-2-specific inhibitors [1].
Mechanism of action in MCL-1 overexpressing cells: Binds to MCL-1, replaces pro-apoptotic BH3 domain proteins (e.g., BIM), and induces mitochondrial outer membrane permeability (MOMP) [2].
Mechanism of action in oral cancer: First induces autophagy (protective), then transitions to necrotic apoptosis when autophagy is inhibited by MCL-1 [3].
Clinical significance: Widespread. Antiparasitic activity supports its potential for treating neglected tropical diseases such as malaria and leishmaniasis [4].

C20H19N3O.CH4O3S

413.49

413.14

C, 61.00; H, 5.61; N, 10.16; O, 15.48; S, 7.75

803712-79-0

Obatoclax;803712-67-6

16681698

Solid powder

4.507

3

6

2

29

869

0

S(C([H])([H])[H])(=O)(=O)O[H].O(C([H])([H])[H])C1=C([H])/C(=C2\C([H])=C3C([H])=C([H])C([H])=C([H])C3=N\2)/N([H])/C/1=C(\[H])/C1=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])N1[H]

ZFKXDVMHNXPEIY-PEZBNFGJSA-N

InChI=1S/C20H19N3O.CH4O3S/c1-12-8-13(2)21-16(12)10-19-20(24-3)11-18(23-19)17-9-14-6-4-5-7-15(14)22-17;1-5(2,3)4/h4-11,21-22H,1-3H3;1H3,(H,2,3,4)/b19-10-;

(Z)-2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-1H-indole methanesulfonate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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: ≥ 0.83 mg/mL (2.01 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (2.01 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 8.3 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: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL

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