A-385358 binds to Bcl-XL (Ki = 0.8 ± 0.17 nmol/L) and Bcl-2 (Ki = 67 ± 7 nmol/L) [1].

In fluorescence polarization tests, A-385358 is a selective inhibitor of Bcl-XL with Kis values of 0.80 and 67 nM for Bcl-XL and Bcl-2, respectively. After administering A-385358 to IL-3-deficient FL5.12/Bcl-XL cells for a whole day, the cells died, with an EC50 of 0.47±0.05 μM (n=68). An increase in caspase-3 activity coincides with this impact. The results indicate that Bcl-XL has a stronger affinity than Bcl-2 hydrophobic groove, as evidenced by the 4-fold higher EC50 of A-385358 on IL-3-depleted FL5.12/Bcl-2 cells (1.9±0.1 μM; n=55) in comparison to cytokine-deprived FL5.12/Bcl-XL cells. Additionally, compared to Bcl-2 cells, A-385358 was more successful in inducing the release of cytochrome c from mitochondria isolated from FL5.12/Bcl-XL [1].

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A-385358 efficiently enters FL5.12 cells, with a [cell]/[extracellular] ratio of 51.1 ± 2.7 after 1 hour of incubation at 10 μmol/L. 30% of the cell-associated compound was released upon lysis, indicating the majority is bound to cellular membranes [1].
In NCI-H460 cells, a fluorescent analogue of A-385358 (compound 1) co-localizes with the mitochondrial marker MitoTracker Green FM, as visualized by confocal microscopy [1].
In IL-3-deprived FL5.12/Bcl-XL cells, A-385358 induces cell killing with an EC50 of 0.47 ± 0.05 μmol/L. In IL-3-deprived FL5.12/Bcl-2 cells, the EC50 is 1.9 ± 0.1 μmol/L [1].
In A549 non-small-cell lung cancer cells, A-385358 (10 μmol/L) potentiates the activity of paclitaxel. When paclitaxel is given 24 hours before addition of A-385358, the potentiation factor (EC50 shift) is 15-fold with 48 hours of co-incubation and 26-fold with 72 hours of co-incubation. Under concurrent 48-hour co-incubation, the potentiation factor is 3.5-fold [1].
A-385358 does not alter the kinetics or extent of [3H]paclitaxel uptake or release in A549 cells [1].
A-385358 enhances cell killing by UV-C irradiation in A549 cells by approximately 2-fold. At 1.25 μmol/L A-385358, the viable fraction of A549 cells after 16 mJ/cm² UV-C was reduced to <10% [1].
In various human tumor cell lines, A-385358 enhances the cell killing activity of several chemotherapeutic agents: paclitaxel and doxorubicin in A549 cells (potentiation factor of 13 ± 1 and 3.6 ± 0.7, respectively); etoposide in A549, SN12C, and 786-O cells (potentiation factors of 13 ± 1, 2.3 ± 0.1, and 2.3 ± 0.1, respectively); and cisplatin in SN12C, MiaPaCa-2, NCI-H226, and NCI-H322M cells (potentiation factors of 3.3 ± 0.1, 2.6, 2.4 ± 0.7, and 2.0 ± 0, respectively) [1].
The EC50 for A-385358 in H146 small-cell lung cancer cells is 0.35 ± 0.03 μmol/L under serum-free conditions and 31 ± 6 μmol/L in the presence of 10% human serum [1].
In Molt-4 cells, the EC50 for A-385358 is 0.74 ± 0.23 μmol/L (serum-free) and 32.8 ± 10.3 μmol/L (10% human serum) [1].
In CCRF-CEM cells, the EC50 for A-385358 is 0.21 ± 0.12 μmol/L (serum-free) and >30 μmol/L (10% human serum) [1].
In A549 cells, the EC50 for A-385358 is 16 ± 1 μmol/L (serum-free) and >100 μmol/L (10% human serum) [1].

In comparison to paclitaxel monotherapy, the combination of A-385358 at 100 mg/kg/d and a lower dose of paclitaxel significantly reduced tumor growth (%T/C). In addition, compared to vehicle control, the combination more than 100% accelerated the time it took for tumors to reach 900 mm3 (%ILS). When A-385358 was dosed, maximum efficacy was seen, and after therapy was stopped, tumor growth increased gradually but steadily. In addition to being well tolerated, the combination of 30 mg/kg/d paclitaxel and 75 mg/kg/d A-385358 reduced tumor development by over 80%. At dosages as low as 50 mg/kg/d, notable impacts on tumor development were seen in comparison to paclitaxel monotherapy [1].

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In an early-treatment A549 xenograft model, A-385358 (100 mg/kg/d, i.p., days 7-27) alone provided no significant inhibition of tumor growth but significantly enhanced the activity of paclitaxel (15 or 30 mg/kg/d, i.p., q4d x 3), resulting in a significant reduction in tumor growth (%T/C) and a >100% increase in time for tumors to reach 900 mm³ (%ILS) compared to paclitaxel monotherapy. The combination of A-385358 at 75 mg/kg/d plus paclitaxel at 30 mg/kg/d inhibited tumor growth rate by nearly 80% [1].
In a staged (established tumor) A549 xenograft model (tumors ~240 mm³), the combination of A-385358 (100 mg/kg/d, i.p.) plus paclitaxel (15 mg/kg/d, i.p.) resulted in nearly complete inhibition of tumor growth during the therapy period. The combination of paclitaxel at 30 mg/kg/d plus A-385358 at 75 mg/kg/d led to regression of established tumors (mean tumor size reduced from 400 mm³ to ~200 mm³) [1].
In the LX-1 squamous cell lung carcinoma model, A-385358 (100 mg/kg/d) significantly improved the efficacy of paclitaxel (5 mg/kg/d), resulting in 65% inhibition of tumor growth rate and 30% enhancement of tumor growth delay [1].
In the H146 small-cell lung cancer xenograft model, A-385358 administered at 100 mg/kg/d, i.p., for 21 days showed only modest inhibition of tumor growth (29% TGI) [1].
Immunohistochemical analysis of A549 tumors showed that addition of A-385358 to paclitaxel increased the percentage of MPM-2-positive cells (mitotic arrest) by ~2-fold compared to paclitaxel alone and extended the apoptotic response (caspase-3-positive cells) from 30 hours to at least 36 hours post-treatment [1].

The binding affinities of A-385358 for Bcl-XL and Bcl-2 were determined using fluorescence polarization assays. These assays measure the compound's ability to displace fluorescein-labeled BH3 peptides from the Bcl-2 family proteins. The Ki values were calculated from the competition curves. Under serum-free conditions, the Ki of A-385358 for Bcl-XL was 0.8 ± 0.17 nmol/L (n=5), and for Bcl-2 it was 67 ± 7 nmol/L (n=6) [1].

Cell viability was analyzed using a colorimetric MTS assay. Cells were plated in 96-well plates at 5,000 per well 24 hours before treatment with compound. After various incubation periods, MTS reagent was added and viability was measured [1].
For the UV-C assay, A549 cells were plated at 5,000 per well. The following day, various doses of UV-C irradiation were applied using a Stratalinker, immediately followed by addition of A-385358 [1].
FL5.12 cells stably transfected with either the Bcl-xL or Bcl-2 gene were used. To determine compound-specific killing, transfected cells were cultured in the absence of WEHI 3B medium (source of IL-3) for 24 hours. Cells were then plated at 1x10^5 per well and serial dilutions of A-385358 were added. Cell viability was measured by MTS assay after 24 hours of incubation [1].
For Western blot analysis, 20 μg of total protein were electrophoresed on 4% to 12% Bis/Tris gradient gels and transferred to PVDF membrane. Specific signals were detected with primary antibodies to Bcl-XL or Bcl-2 [1].
For analysis of paclitaxel uptake and efflux, A549 cells (1x10^5) were plated in 96-well plates. [3H] Paclitaxel (5 μmol/L; 0.5 μCi/mL final) was added to all wells, with or without 50 μmol/L A-385358, and incubated for various times. For washout experiments, cells were first exposed to [3H]paclitaxel for 2 hours, washed, and then incubated with fresh medium with or without 50 μmol/L A-385358 [1].

All animal studies were conducted in accordance with internal Institutional Animal Care and Use Committee guidelines [1].
For A549 xenograft studies, 5x10^6 cells mixed with 50% Matrigel were inoculated s.c. into the flank of male scid mice. For early-treatment studies, therapy was initiated on day 7. For established tumor studies, tumors were allowed to grow to ~240 mm³ before treatment [1].
A-385358 was delivered in a vehicle containing 5% Tween 80, 20% propylene glycol, and 75% PBS (pH 3.8). Paclitaxel was formulated according to manufacturer's recommendations. For combination therapy, both drugs were administered i.p., with paclitaxel given several hours before treatment with A-385358 (except for IHC studies where they were given simultaneously) [1].
In the early-treatment A549 model, A-385358 was given once daily at 100 mg/kg/d, i.p., from day 7 to 27. Paclitaxel was given i.p. at 15 or 30 mg/kg/d on a q4d x 3 schedule (days 7, 11, and 15) [1].
In the staged A549 model, A-385358 was administered i.p. once daily at 100 or 75 mg/kg/d on days 16 to 36. Paclitaxel was given i.p. at 15 or 30 mg/kg/d on days 16, 20, and 24 [1].
In the LX-1 model, male CD-1 nude mice were inoculated with a 1:5 dilution of tumor bri in 50% Matrigel. A-385358 (100 mg/kg/d) was administered on days 1-21, and paclitaxel (5 mg/kg/d) on days 1, 5, and 9 [1].
For H146 xenograft studies, A-385358 was administered at 100 mg/kg/d, i.p., for 21 days [1].
For immunohistochemistry, following drug treatments, A549 tumors were collected into Streck Tissue Fixative for 24 hours, processed, embedded in paraffin, and sectioned. MPM-2 antibody (1:350) was used to analyze G2-M arrest, and an antibody against activated caspase-3 (1:400) was used to analyze apoptosis. Secondary antibodies were used at 1:250 [1].

Co-administration of A-385358 plus paclitaxel did not alter either the Cmax (3.0 μg/mL) or the AUC (13 μg·h/mL) of paclitaxel relative to dosing with paclitaxel alone in scid mice [1].
A-385358 does not influence either the uptake or efflux of paclitaxel in A549 cells [1].

A-385358 given once daily at 100 mg/kg/d, i.p., from day 7 to 27 was well tolerated (<5% body weight loss) [1].

[1]. A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo. Cancer Res. 2006 Sep 1;66(17):8731-9.

A-385358 is a small molecule with relative selectivity for binding to Bcl-X versus Bcl-2 (Ki of 0.80 and 67 nmol/L, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane [1].
While ABT-737 is a potent inhibitor of Bcl-2, Bcl-XL, and Bcl-w with broad-spectrum activity, A-385358 is a Bcl-XL-selective inhibitor used to study the biological consequences of inhibiting this specific target [1].
The combination of paclitaxel and A-385358 resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy in A549 tumors [1].
The enantiomer of A-385358, bearing the opposite configuration of the dimethylamino ethyl group, was 20- and 300-fold less active against Bcl-XL and Bcl-2, respectively, and was used as a negative control in cellular assays [1].

C32H41N5O5S2

639.83

639.254

406228-55-5

11556440

White to yellow solid powder

1.3±0.1 g/cm3

1.649

7.6

2

9

12

44

1020

1

CC1(CCN(CC1)C2=CC=C(C=C2)C(=O)NS(=O)(=O)C3=CC(=C(C=C3)N[C@H](CCN(C)C)CSC4=CC=CC=C4)[N+](=O)[O-])C

DWEHITNKTMMZBR-RUZDIDTESA-N

InChI=1S/C32H41N5O5S2/c1-32(2)17-20-36(21-18-32)26-12-10-24(11-13-26)31(38)34-44(41,42)28-14-15-29(30(22-28)37(39)40)33-25(16-19-35(3)4)23-43-27-8-6-5-7-9-27/h5-15,22,25,33H,16-21,23H2,1-4H3,(H,34,38)/t25-/m1/s1

N-[4-[[(2R)-4-(Dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonyl-4-(4,4-dimethylpiperidin-1-yl)benzamide

A 385358 A385358A-385358

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)

DMSO : ~50 mg/mL (~78.15 mM)

Solubility in Formulation 1: 2.5 mg/mL (3.91 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (3.91 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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 (Please use freshly prepared in vivo formulations for optimal results.)