HDAC ( IC50 = 27 nM )

Belinostat, at a dose of 10 mg/kg with no effect on body weight, shows significant tumor growth delay in A2780 and A2780/cp70 xenograft.[1] In mouse bladder tumors, belinostat also induces the expression of CDM, HDAC core, and p21WAF1. With a dose of 100 mg/kg, belinastat monotherapy produces dose-proportional antitumor effects in an A2780 xenograft, with a TGI of 47%. Tumor growth could be delayed from 18.6 days to 22.5 days by combining belimostat (100 mg/kg) with carboplatin (40 mg/kg).[3] Belinostat, when combined with bortezomib, causes significant tumor inhibition and gastrointestinal toxicity in mice bearing a xenograft of the bortezomib-resistant UMSCC-11A.[5]

Pelletization of subconfluent cultures is achieved by centrifugation at 200 × g for 5 minutes after they are harvested and twice washed in ice cold PBS. The lyse is performed using three freeze (dry ice) thaw (30 °C water bath) cycles after the cell pellet has been resuspended in two volumes of lysis buffer [60 mM Tris buffer (pH 7.4) containing 30% glycerol and 450 mM NaCl]. The supernatant is kept at -80 °C after cell debris is extracted using centrifugation at 1.2 × 10⁴ g for 5 minutes. Histone H4 peptide (sequence SGRGKGGKGLGKGGAKRHRK, which corresponds to the 20 NH2-terminal residues) is acetylated by a recombinant protein that uses [³H]acetyl CoA as an acetate source and contains the hypoxanthine-aminopterin-thymidine domain of p300. H4 peptide (100 μg) is combined with hypoxanthine-aminopterin-thymidine buffer (50 mM Tris HCl pH 8.0, 5% glycerol, 50 mM KCl, and 0.1 mM EDTA), 1 mM DTT, 1 mM 4-(2-aminoethyl) benzenesulfonylfluoride, 1 × complete protease inhibitors, 50 μL of purified p300, and 1.85 m [³H]acetyl CoA (4.50Ci/mmol) in a final volume of 300 μL. The mixture is then incubated at 30 °C for 45 minutes. Centrifugation and an hour at 4 °C are used to extract the p300 protein from 20 μL of 50% Ni-agaroase beads. After applying the supernatant to a 2 mL Sephadex G15 column, the flow through is gathered. After adding one milliliter of distilled H₂O gently and collecting three drop fractions, this process is repeated until four to five milliliters of distilled H₂O are added and approximately forty fractions are collected. The fractions containing the labeled peptide are identified by diluting three microliters of each fraction in two milliliters of scintillation fluid and counting the results in a scintillation counter. These fractions are combined, and a 1 μL sample is measured to determine the radioactivity in each batch of peptides (3-7×10³ cpm/μL). The reaction for activity assays is conducted in a total volume of 150 μL of buffer [60 mM Tris (pH 7.4) containing 30% glycerol] with 2 μL of cell extract and, if applicable, 2 μL of belinostat added. 20 NH₂-terminal residues' worth of acetylated histone H4 peptide, or 2 μL of [³H] labeled substrate, is added to initiate the reaction. Samples are incubated for 45 minutes at 37 °C, after which the addition of acetic acid and HCl (0.72 and 0.12 M final concentrations, respectively) stops the reaction. Samples are centrifuged at 1.2× 10⁴ g for 5 minutes after released [³H]acetate is extracted into 750 μL of ethyl acetate. After being transferred to 3 milliliters of scintillation fluid, the upper phase (600 μL) is counted.

After seeding tumor cell lines at a density of 8 × 10⁴ cells/25 cm2 flask in 5 mL of medium, they are incubated for 48 hours. For a full day, cells are subjected to varying concentrations of belinostat (0.016 to 10 μM). After the medium is gone, each flask receives 1 mL of trypsin/EDTA. Following their detachment, the cells are resuspended in 1 mL of medium, and the number of cells from the untreated control flask is recorded. Depending on the cell line, three cells are diluted and plated into 6-cm Petri dishes per flask, with a density of 0.5-2× 10³ cells/dish. The drug-treated flasks' cells are diluted and plated similarly to the control flasks. Dishes are incubated at 37 °C for ten to fifteen days. After the cells are fixed in methanol, stained with crystal violet, and rinsed with PBS, colonies containing 50 or more cells are counted. The belinostat concentration needed to lower the number of colonies to 50% of the untreated control cells is known as the IC50, which is used to express sensitivity.

[1]. Mol Cancer Ther . 2003 Aug;2(8):721-8.

[2]. J Transl Med . 2007 Oct 12:5:49.

[3]. Mol Cancer Ther . 2006 Aug;5(8):2086-95.

[4]. J Biol Chem . 2011 Sep 2;286(35):30937-30948.

[5]. Mol Cancer Ther . 2007 Jan;6(1):37-50.

C15H14N2O4S

318.35

318.07

C, 56.59; H, 4.43; N, 8.80; O, 20.10; S, 10.07.

414864-00-9

414864-00-9; 866323-14-0

White solid powder

C1=CC=C(C=C1)NS(=O)(=O)C2=CC=CC(=C2)/C=C/C(=O)NO

NCNRHFGMJRPRSK-MDZDMXLPSA-N

InChI=1S/C15H14N2O4S/c18-15(16-19)10-9-12-5-4-8-14(11-12)22(20,21)17-13-6-2-1-3-7-13/h1-11,17,19H,(H,16,18)/b10-9+

(E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide

NSC726630; NSC-726630; PX-105684; PXD 101; PXD101; PXD-101; PX105684; PX 105684; NSC-726630; Trade name: Beleodaq

2934.99.03.00

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)