Belinostat

318, 35

A2780, A2780/cp70 and HCT116 cells are injected s.c. into the right flank of CD1 nu/nu mice.

Belinostat is currently in Phase II clinical trial in patients with relapsed or refractory peripheral T-cell lymphoma.

<=40 mg/kg

27 nM [1], 27 nM [1], 27 nM [1], 27 nM [1], 27 nM [1], 27 nM [1]

Belinostat indicates significant tumor growth delay in A2780 and A2780/cp70 xenograft at a dose of 10mg/kg with no effects on the body weight. [1] Belinostat also induces p21WAF1, HDAC core and cell communication genes in mouse bladder tumors. [2] Belinostat monotherapy induces dose-proportional antitumor effects with TGI of 47% at a dose of 100mg/kg in A2780 xenograft. The combination of Belinostat (100 mg/kg) with carboplatin (40 mg/kg) could delay tumor growth from 18.6 days to 22.5 days. [3] Combining with bortezomib, Belinostat results in great tumor inhibition and gastrointestinal toxicity in mice with bortezomib-resistant UMSCC-11A xenograft. [5]

Histone Deacetylase Activity, Subconfluent cultures are harvested and washed twice in ice cold PBS and pelleted by centrifugation at 200 x g for 5 min. The cell pellet is resuspended in two volumes of lysis buffer [60 mM Tris buffer (pH 7.4) containing 30% glycerol and 450 mM NaCl] and lysed by three freeze (dry ice) thaw (30 C water bath) cycles. Cell debris is removed by centrifugation at 1.2 x 104 g for 5 min, and the supernatant is stored at 80 C. Histone H4 peptide (sequence SGRGKGGKGLGKGGAKRHRK corresponding to the 20 NH2-terminal residues) is acetylated by a recombinant protein containing the hypoxanthine-aminopterin-thymidine domain of p300, using [3H]acetyl CoA as a source of acetate. H4 peptide (100 ug) is mixed 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 x complete protease inhibitors, 50 uL of purified p300, and 1.85 m [3H]acetyl CoA (4.50Ci/mmol) in a final volume of 300 uL and incubated at 30 C for 45 min. The p300 protein is removed by incubation with 20 uL of 50% Ni-agaroase beads for 1 hour at 4 C and centrifugation. The supernatant is applied to a 2 mL Sephadex G15 column, and the flow through is collected. One milliliter of distilled H2O is gently applied, and three drop fractions are collected, this is repeated until 4–5 mL of distilled H2O has been added, and 40 fractions are collected. Three microliters of each fraction are diluted in 2 mL of scintillation fluid and counted in a scintillation counter to identify the fractions containing the labeled peptide. These fractions are pooled, and 1 uL of the combined sample is measured to assess the radioactivity in every peptide batch (3-7x103 cpm/uL). For activity assays, the reaction is carried out in a total volume of 150 uL of buffer [60 mM Tris (pH 7.4) containing 30% glycerol] containing 2 uL of cell extract and, where used, 2 uL of belinostat. The reaction is started by the addition of 2 uL of [3H] labeled subrate (acetylated histone H4 peptide corresponding to the 20 NH2-terminal residues). Samples are incubated at 37 C for 45 min, and the reaction stopped by the addition of HCl and acetic acid (0.72 and 0.12 M final concentrations, respectively). Released [3H]acetate is extracted into 750 uL of ethyl acetate, and samples are centrifuged at 1.2x 104 g for 5 min. The upper phase (600 uL) is transferred to 3 mL of scintillation fluid and counted.

DMSO 64 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

2-Propenamide, N-hydroxy-3-[3-[(phenylamino)sulfonyl]phenyl]-