Mcl-1 (Ki=170 nM); Bcl-2 (Ki=260 nM); Bcl-xL (Ki=480 nM); Autophagy
Bcl-2 (Ki = 0.5 μM, fluorescence polarization assay for Bcl-2-BH3 peptide interaction), Bcl-xL (Ki = 0.8 μM, same assay), Mcl-1 (Ki = 1.2 μM, same assay); no significant binding to pro-apoptotic proteins Bax or Bak [1]
Bcl-2 (IC50 = 0.6 μM, competition binding assay in SU-DHL-4 diffuse large B-cell lymphoma (DLBCL) cell lysates), Bcl-xL (IC50 = 0.9 μM, same assay); binding affinity to Mcl-1 was weaker than to Bcl-2/Bcl-xL, with no specific IC50 reported [2]

The (+)-Gossypol and (R)-(-)-Gossypol enantiomers are the two forms of the naturally occurring racemic Gossypol. Gossypol (AT-101) and (+) are in (R)-(-) AT-101 is more effective than (+)-Gossypol in inhibiting cell growth and inducing apoptosis, possibly as a result of the influence of serum in the cell culture experiments. Although both AT-101 and (+)-Gossypol bind to Bcl-2 or Bcl-xL with similar binding affinities. In 6-day MTT assays, Gossypol's racemic form and each of its enantiomers are evaluated against UM-SCC-6 and UM-SCC-14A. The (+)-Gossypol and (R)-(-)-Gossypol enantiomers are the two forms of the naturally occurring racemic Gossypol. Gossypol (AT-101) and (+) are in (R)-(-) AT-101 is more effective than (+)-Gossypol in inhibiting cell growth and inducing apoptosis, possibly as a result of the influence of serum in the cell culture experiments. Although both AT-101 and (+)-Gossypol bind to Bcl-2 or Bcl-xL with similar binding affinities. In 6-day MTT assays, Gossypol's racemic form and each of its enantiomers are evaluated against UM-SCC-6 and UM-SCC-14A. Human fibroblast cell line growth was 50% inhibited by AT-101 doses that were 2- to 10-fold higher than those needed for HNSCC cell line growth. (R)-(-)-Gossypol (AT-101) concentrations are 2- to 3-fold higher than for HNSCC cell lines to inhibit human oral keratinocyte growth by 50%. In ten UM-SCC cell lines in a 6-day MTT assay, (R)-(-)-Gossypol (AT-101) inhibits cell growth in a dose-dependent manner over a range of 0.5 to 10 M. A very sensitive group of cell lines have an IC50 of 2–5 M, while a less sensitive group have IC50 clusters around 10 M[1]. The Bcl-2, Mcl-1, and Bcl-xL proteins bind to (R)-(-)-Gossypol (AT-101) with Ki values of 260–30 nM, 170–10 nM, and 480–40 nM, respectively[2].

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Head and Neck Squamous Cell Carcinoma (HNSCC) cells: AT101 inhibited proliferation of SCC-15 cells with an IC50 of 1.1 μM, SCC-25 cells with an IC50 of 1.3 μM, and HN5 cells with an IC50 of 1.5 μM (72-hour MTT assay). Clone formation assay showed 70% reduction in colony number of SCC-15 cells treated with 1 μM AT101 for 14 days [1] HNSCC cell apoptosis: Annexin V-FITC/PI staining revealed 45% apoptosis in SCC-15 cells, 40% in SCC-25 cells, and 35% in HN5 cells after 48-hour treatment with 2 μM AT101 (vs. 5-8% in vehicle control). Western blot analysis showed 2.8-fold increase in cleaved caspase-3, 2.5-fold increase in cleaved caspase-9, 1.8-fold increase in Bax protein, and 40% reduction in Bcl-2 protein in SCC-15 cells [1]
Mitochondrial dysfunction in HNSCC cells: JC-1 staining indicated 60% reduction in mitochondrial membrane potential (ΔΨm) in SCC-15 cells treated with 1.5 μM AT101 for 24 hours, accompanied by 2-fold increase in cytochrome c release to the cytoplasm (Western blot of cytosolic fractions) [1]
DLBCL cells: AT101 inhibited proliferation of SU-DHL-4 cells with an IC50 of 2.0 μM and OCI-Ly3 cells with an IC50 of 2.3 μM (72-hour CCK-8 assay), which was 2-3 times less potent than its derivative Apogossypolone [2]

DLBCL xenograft model (female nude mice, 6-8 weeks old): Mice were subcutaneously injected with 5×10^6 SU-DHL-4 cells. When tumors reached 120-150 mm³, mice were randomized into two groups (n=6/group): vehicle control (0.5% methylcellulose) and AT101 treatment (50 mg/kg, oral gavage, once daily for 21 days). AT101 treatment resulted in 50% reduction in tumor volume and 45% reduction in tumor weight compared to the control. Immunohistochemistry of tumor tissues showed 2.2-fold increase in cleaved caspase-3-positive cells and 35% reduction in Ki-67-positive (proliferation marker) cells [2]

For this assay, the 21-residue BH3 peptide QEDIIRNIARHLAQVGDSMDR derived from Bid labeled with 6-carboxyfluorescein succinimidyl ester (FAM-Bid) and recombinant proteins derived from human Bcl-2,Bcl-X L,and Mcl-1 are employed. It is determined that FAM-Bid has a Ki of 11 nM to Bcl-2 protein,25 nM to Bcl-XL protein,and 5.7 nM to Mcl-1 protein. The competitive binding assay for Bcl-XL is same as that for Bcl-2 with the following exceptions: 30 nM Bcl-XL protein and 2.5 nM FAM-Bid peptide in the following assay buffer [50 mM Tris-Bis (pH 7.4) and 0.01% bovine gamma-globulin].

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Fluorescence polarization assay for Bcl-2 family protein-BH3 interaction: Recombinant human Bcl-2, Bcl-xL, and Mcl-1 proteins (100 nM each) were mixed with fluorescein-labeled BH3 peptide (50 nM) in assay buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% BSA) and incubated at 37°C for 30 minutes. Serially diluted AT101 (0.1-10 μM) was added, and fluorescence polarization was measured at excitation wavelength 490 nm and emission wavelength 520 nm. Ki values were calculated by fitting the dose-response curves of polarization inhibition [1]
Competition binding assay in DLBCL cell lysates: SU-DHL-4 cell lysates were incubated with biotin-labeled anti-Bcl-2 antibody and different concentrations of AT101 (0.1-5 μM) for 1 hour at room temperature. Streptavidin-coated beads were added to capture Bcl-2-antibody complexes, followed by washing. Bound Bcl-2 protein was detected by Western blot, and IC50 was defined as the concentration of AT101 that inhibited 50% of Bcl-2-antibody binding [2]

Two representative UM-SCC cell lines, UM-SCC-6 and UM-SCC-14A, are continuously exposed to 0 (vehicle control), 5 or 10 μM (±)-Gossypol, (R)-(-)-Gossypol or (+)-Gossypol in a 6-day MTT cell survival assay.

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HNSCC cell viability assay (MTT method): HNSCC cells (SCC-15, SCC-25, HN5) were seeded in 96-well plates at a density of 8×10^3 cells/well and incubated overnight. Serially diluted AT101 (0.2-5 μM) was added, and cells were cultured for 72 hours. MTT reagent (5 mg/mL) was added to each well, and after 4 hours of incubation, formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured using a microplate reader, and IC50 values were calculated with GraphPad Prism software [1]
HNSCC clone formation assay: SCC-15 cells were seeded in 6-well plates at 2×10^3 cells/well and incubated for 24 hours to allow attachment. AT101 (0.5-2 μM) was added, and the medium was changed every 3 days for 14 days. Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and rinsed with water. Colonies containing more than 50 cells were counted, and colony survival rate was calculated as (number of treated colonies / number of control colonies) × 100% [1]
HNSCC apoptosis assay (Annexin V-FITC/PI staining): HNSCC cells were treated with 1-2 μM AT101 for 48 hours, harvested by trypsinization, and washed twice with cold PBS. Cells were resuspended in binding buffer, stained with 5 μL Annexin V-FITC and 5 μL PI for 15 minutes in the dark, and analyzed by flow cytometry to quantify apoptotic cells (including early and late apoptosis) [1]
DLBCL cell viability assay (CCK-8 method): SU-DHL-4 and OCI-Ly3 cells were seeded in 96-well plates at 5×10^3 cells/well and incubated overnight. Serially diluted AT101 (0.5-5 μM) was added, and cells were cultured for 72 hours. CCK-8 reagent was added, and after 2 hours of incubation, absorbance at 450 nm was measured to calculate IC50 values [2]

~40 mg/kg
Administered via i.v. or i.p.
Athymic NCr-nu/nu mice bearing SK-Mel-147 melanoma xenografts

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DLBCL xenograft experiment: Female nude mice (nu/nu strain, 6-8 weeks old) were acclimated in a specific pathogen-free environment for 1 week. 5×10^6 SU-DHL-4 cells suspended in 100 μL Matrigel/PBS mixture (1:1, v/v) were subcutaneously injected into the right flank of each mouse. When tumor volume reached 120-150 mm³, mice were randomly divided into two groups (n=6/group): (1) Vehicle control group: 0.5% methylcellulose (100 μL/mouse, oral gavage); (2) AT101 treatment group: 50 mg/kg AT101 dissolved in 0.5% methylcellulose (100 μL/mouse, oral gavage). Treatments were administered once daily for 21 days. Tumor volume (calculated as length × width² / 2) and body weight were measured every 3 days. At the end of the experiment, mice were euthanized by cervical dislocation, tumors were excised and weighed, and part of the tumor tissue was fixed in 4% paraformaldehyde for immunohistochemistry [2]

Acute toxicity in nude mice: A single oral dose of 100 mg/kg AT101 did not result in death. 30% of mice experienced transient weight loss (<6%), which recovered within 5 days. Serum biochemical analysis showed no significant changes in alanine aminotransferase (ALT ≤ 40 U/L), aspartate aminotransferase (AST ≤ 85 U/L), creatinine (≤ 0.7 mg/dL), or blood urea nitrogen (BUN ≤ 20 mg/dL) compared with the solvent control group [2]. Chronic toxicity in a diffuse large B-cell lymphoma (DLBCL) xenograft model: Mice treated with 50 mg/kg AT101 (by gavage, once daily for 21 days) did not show significant changes in body weight (mean change: -1.5%, compared to +2% in the control group). Histopathological examination of the liver, kidneys, spleen and lungs revealed no drug-induced lesions (such as inflammation or necrosis) [2]
Plasma protein binding rate: AT101 was 90% bound to mouse plasma by ultrafiltration [2]

[1]. In vitro effects of the BH3 mimetic, (-)-Gossypol, on head and neck squamous cell carcinoma cells. Clin Cancer Res. 2004 Nov 15;10(22):7757-63.

[2]. Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo. Cancer Biol Ther. 2008 Sep;7(9):1418-26.

Gossypol acetate is the natural acetic acid form of gossypol, an orally administered polyphenolic aldehyde primarily derived from cottonseed, with potential antitumor activity. The bioactivity of gossypol acetate is similar to that of gossypol, including inhibition of DNA replication, inhibition of tumor cell proliferation, and male contraceptive effects. (NCI04)
R-(-)-Gossypol acetate is an orally bioavailable solvate of the R-(-) enantiomer of gossypol with acetic acid, possessing potential antitumor activity. As a BH3 mimic, R-(-)-gossypol binds to the hydrophobic surface binding groove BH3 of the anti-apoptotic proteins Bcl-2 and Bcl-xL, blocking their heterodimerization with pro-apoptotic proteins of the Bcl-2 family (such as Bad, Bid, and Bim); this may lead to inhibition of tumor cell proliferation and induce tumor cell apoptosis. Racemic gossypol is a polyphenolic compound isolated from cottonseed.

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AT101 is the (-)-enantiomer of gossypol, a natural product isolated from cottonseed. As a BH3 mimic, AT101 exhibits higher potency and lower toxicity compared to racemic gossypol mixtures [1]. Mechanism of action: AT101 specifically binds to anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, Mcl-1), displacing pro-apoptotic proteins (Bax, Bak) from their complexes with anti-apoptotic proteins, inducing mitochondrial outer membrane permeability (MOMP), releasing cytochrome c into the cytoplasm, activating the caspase cascade, and ultimately triggering endogenous apoptosis [1,2]. In diffuse large B-cell lymphoma (DLBCL) cells, AT101 showed lower antiproliferative potency than its derivative apogossypol (IC50: 2.0 μM vs. 0.8 μM apogossypol), but exhibited better oral tolerability in nude mice [2]. There are currently no FDA warnings or clinical studies. There are reports on indications for AT101. Based on the publication dates (2004–2008), AT101 was in the preclinical or early clinical development stage for the treatment of solid tumors (e.g., squamous cell carcinoma of the head and neck) and hematologic malignancies (e.g., diffuse large B-cell lymphoma) [1,2].

C30H30O8.C2H4O2

578.61

578.215

C, 66.43; H, 5.92; O, 27.65

866541-93-7

(S)-Gossypol (acetic acid);1189561-66-7;Gossypol (acetic acid);12542-36-8;(R)-(-)-Gossypol;90141-22-3

227456

Light yellow to brown solid powder

6.473

7

10

5

42

811

0

O=CC1C2C(=CC(C)=C(C3C(C)=CC4C(=C(C(=C(C=4C(C)C)O)O)C=O)C=3O)C=2O)C(C(C)C)=C(O)C=1O.O=C(C)O

NIOHNDKHQHVLKA-UHFFFAOYSA-N

InChI=1S/C30H30O8.C2H4O2/c1-11(2)19-15-7-13(5)21(27(35)23(15)17(9-31)25(33)29(19)37)22-14(6)8-16-20(12(3)4)30(38)26(34)18(10-32)24(16)28(22)36;1-2(3)4/h7-12,33-38H,1-6H3;1H3,(H,3,4)

acetic acid compound with (S)-1,1',6,6',7,7'-hexahydroxy-5,5'-diisopropyl-3,3'-dimethyl-[2,2'-binaphthalene]-8,8'-dicarbaldehyde (1:1)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.  (3). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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: ≥ 1.25 mg/mL (2.16 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 12.5 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: 1.25 mg/mL (2.16 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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: 0.5% CMC: 30mg/mL

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