Bcl-xL (Ki = 0.01 nM); Bcl-2 (Ki = 80 nM)

A-1155463 disturbs BCL-XL-BIM complexes in cells but not BCL-2-BIM complexes. A-1155463 has no detectable cytotoxicity against BCL-2-dependent RS4;11 cells (EC50>5 mM), but it kills BCL-XL-dependent Molt-4 cells (EC50=70 nM). In BCL-XL-dependent H146 cells, A-1155463 causes the classic signs of apoptosis, as shown by the release of cytochrome c from mitochondria, activation of caspase, and accumulation of caspase-dependent sub-G0-G1 DNA content.

A-1155463 inhibits the growth of the H146 small cell lung cancer xenograft tumor in vivo after several doses and results in a mechanism-based and reversible thrombocytopenia in mice. A rapid and reversible decrease in platelets in SCID-Beige mice after a single IP dose served as evidence of A-1155463's ability to exert in vivo on-target activity. Additionally, A-1155463 treatment provided a modest but statistically significant tumor growth inhibition in SCID-Beige mice bearing tumors. An accompanying manuscript will present thorough mechanistic analyses of A-1155463's activity with appropriate chemotherapy in combination across various tumor types. In nontumor bearing SCID-Beige mice, platelet counts drop significantly after a single 5 mg/kg IP dose of A-1155463 and then return to normal levels within 72 hours. Daily administration of 5 mg/kg IP to SCID-Beige mice for 14 days after they received an inoculation of BCL-XL-dependent H146 tumor cells results in a statistically significant inhibition of tumor growth (maximum tumor growth inhibition = 44%) that is relieved upon stopping the dose.

and selective BCL-XL inhibitor; it binds to BCL-XL with a picomolar affinity (Ki 0.01 nM) while binding to BCL-2 (Ki = 80 nM) and related proteins BCL-W (Ki = 19 nM) and MCL-1 (Ki > 440 nM) is >1000 times weaker.

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Compounds were serially diluted in DMSO starting at 500 M using a Tecan Gemini robot. An intermediate 1:10 dilution in assay buffer was performed using a Tecan Temo and 10 l transferred to a white 384-well low volume Corning #3673 assay plate (2x starting concentration; 10% DMSO). Then 10 l of a protein/probe/antibody mix was added to each well at final concentrations listed in the table shown above. The samples were then incubated for 1 hr at room temperature. For each assay, probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Time resolved fluorescence was measured on the Envision using a 340 nm excitation filter and 520 nm (f-Bak ) and 495 nm (Tb-labeled anti-His antibody) emission filters. Dissociation constants (Ki) were determined using Wang’s equation (Wang Z. An exact mathematical expression for describing competititve binding of two different ligands to a protein molecule. FEBS Lett. 1995, 360,111-114)[2]

A-1155463 is administered to cells in increasing concentrations. Following the manufacturer's instructions, cells are tested for viability using the CellTiter-Glo luminescent cell viability assay after 72 hours. Results are adjusted to reflect untreated cells. With the help of the GraphPad Prism program, EC50 is calculated.

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Cell proliferation and viability assays [1]
Breast cancer cell lines were seeded at 5,000 cells per well in 96-well plates and treated with compound combinations in a 9×3 dose matrix, with navitoclax, venetoclax, and A-1155463 diluted in three-fold steps (20-0.001 μM) and docetaxel at 50, 5.0, or 0.5 nM. Cells were incubated for 72 h before assessing viability. NSCLC cell lines were treated for 72 h with compound combinations in a 5×5 dose matrix and assessed as described previously[1]. Ovarian cancer cell lines were seeded at 10,000 cells per well in 96-well plates and treated with compound combinations in a 9×3 dose matrix for 48 h. Docetaxel was diluted in three-fold steps (10-1.1 nM). Navitoclax, venetoclax, and A-1155463 were diluted in 2-fold steps (20-0.08 μM).[1]

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Colony-Forming Assays[1]
Hematopoietic precursor cells derived from normal human bone marrow (BM) were incubated with various concentrations of navitoclax, venetoclax, or A-1155463 plus or minus 5 nM docetaxel in MethoCult 4230 methylcellulose-based medium supplemented with 30 ng mL-1 recombinant human granulocyte colony stimulating factor (rhGCSF). DMSO was used to make stock solutions of all test compounds and was present at a final concentration of <0.002% in all wells. Frozen BM light density cells from three different lots (BM07B21195, BM0080512A, and BM5H09) were thawed rapidly at 37°C, washed once in 10 mL Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 2% fetal bovine serum (IMDM + 2% FBS), and resuspended in IMDM + 2% FBS. Between 2.4-4.3×104 viable cells were seeded in each well of 6-well plates and incubated at 37°C (5% CO2) in the presence of test compounds for 14-16 days. Colony-forming units comprising at least 30 granulocyte cells were enumerated by a trained technician using light microscopy. Each condition was tested in triplicate to determine mean colony numbers +/- one standard deviation.

Mice and husbandry - SCID and SCID-bg mice were obtained from Charles River. Five, eight, or ten mice were housed per cage. The body weight of the mice upon arrival was 18-20 g. Food and water were available ad libitum. Mice were acclimated to the animal facilities for a period of at least one week prior to commencement of experiments. Animals were tested in the light phase of a 12-h light:12-h dark schedule (lights on at 06.00 hours).[2]

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Formulated in 5% DMSO, 10% EtOH, 20% Cremaphor ELP, and 65% D5W; 5 mg/kg; i.p.

SCID-Beige Mice

[1]. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy. Sci Transl Med.?2015 Mar 18;7(279):279

[2]. Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity. ACS Med Chem Lett. 2014 Aug 26;5(10):1088-93..

[3]. Genomic analysis and selective small molecule inhibition identifies BCL-X(L) as a critical survival factor in a subset of colorectal cancer. Mol Cancer. 2015 Jul 2;14:126.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.[1]

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A-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.[2]

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Defects in programmed cell death, or apoptosis, are a hallmark of cancer. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins, including BCL-2, BCL-X(L), and MCL-1 have been characterized as key survival factors in multiple cancer types. Because cancer types with BCL2 and MCL1 amplification are more prone to inhibition of their respectively encoded proteins, we hypothesized that cancers with a significant frequency of BCL2L1 amplification would have greater dependency on BCL-X(L) for survival. Methods: To identify tumor subtypes that have significant frequency of BCL2L1 amplification, we performed data mining using The Cancer Genome Atlas (TCGA) database. We then assessed the dependency on BCL-X(L) in a panel of cell lines using a selective and potent BCL-X(L) inhibitor, A-1155463, and BCL2L1 siRNA. Mechanistic studies on the role of BCL-X(L) were further undertaken via a variety of genetic manipulations. Results: We identified colorectal cancer as having the highest frequency of BCL2L1 amplification across all tumor types examined. Colorectal cancer cell lines with BCL2L1 copy number >3 were more sensitive to A-1155463. Consistently, cell lines with high expression of BCL-XL and NOXA, a pro-apoptotic protein that antagonizes MCL-1 activity were sensitive to A-1155463. Silencing the expression of BCL-X(L) via siRNA killed the cell lines that were sensitive to A-1155463 while having little effect on lines that were resistant. Furthermore, silencing the expression of MCL-1 in resistant cell lines conferred sensitivity to A-1155463, whereas silencing NOXA abrogated sensitivity. Conclusions: This work demonstrates the utility of characterizing frequent genomic alterations to identify cancer survival genes. In addition, these studies demonstrate the utility of the highly potent and selective compound A-1155463 for investigating the role of BCL-X(L) in mediating the survival of specific tumor types, and indicate that BCL-X(L) inhibition could be an effective treatment for colorectal tumors with high BCL-X(L) and NOXA expression.[3]

C35H32FN5O4S2

669.79

669.187

C, 62.76; H, 4.82; F, 2.84; N, 10.46; O, 9.55; S, 9.57

1235034-55-5

59447577

Off-white to yellow solid

1.5±0.1 g/cm3

1.716

6.61

2

11

11

47

1150

0

S1C(=C(C(=O)O)N=C1N1CC2C(C(NC3=NC4=CC=CC=C4S3)=O)=CC=CC=2CC1)CCCOC1C=CC(C#CCN(C)C)=CC=1F

SOYCFODXNRVBTI-UHFFFAOYSA-N

InChI=1S/C35H32FN5O4S2/c1-40(2)17-6-8-22-14-15-28(26(36)20-22)45-19-7-13-30-31(33(43)44)38-35(47-30)41-18-16-23-9-5-10-24(25(23)21-41)32(42)39-34-37-27-11-3-4-12-29(27)46-34/h3-5,9-12,14-15,20H,7,13,16-19,21H2,1-2H3,(H,43,44)(H,37,39,42)

2-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]-5-[3-[4-[3-(dimethylamino)prop-1-ynyl]-2-fluorophenoxy]propyl]-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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.73 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 25.0 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: ≥ 2.5 mg/mL (3.73 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 25.0 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: ≥ 2.5 mg/mL (3.73 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.)