Studying Histone Deacetylase Inhibition and Apoptosis Induction of Psammaplin A Monomers with Modified Thiol Group

Abstract

Psammaplin A (PsA) is a bromotyrosine disulfide dimer with histone deacetylase (HDAC) inhibition and acts through reduced monomer PsA-SH. We studied the connection of HDAC inhibition, cell growth inhibition, and apoptosis induction of PsA-SH by modifying the -SH group with deletion (6a) or replacement with hydroxamic acid (10b) or benzamide (12g). PsA-SH inhibits HDAC1/2/3 and 6a loses the HDAC inhibition ability. 10b inhibits HDAC1/2/3/6 while 12g shows selective inhibition of HDAC3. PsA-SH and 10b, but neither 6a nor 12g, induce apoptosis in human leukemia HL-60 cells associated with increased acetylation of Histone H3. PsA-SH and 10b inhibit growth of several solid tumor cell lines in vitro and Lewis lung cancer cell growth in vivo. PsA-SH is a simple scaffold for developing selective HDAC inhibitors and induces apoptosis through inhibiting HDAC1/2.

Figure 1

Chemical structures of Psammaplin A (PsA), Psammaplin A monomer (PsA-SH), and approved HDAC inhibitors.

Scheme 1. Synthesis of PsA Monomer with a Different ZBG

Reagents and conditions: (a) Imidazolidine-2,4-dione, NaHCO₃, H₂O, 120 °C, 10 h, 83–92%; (b) NaOH, N₂, H₂O, 95 °C, 12 h, 65–73%; (c) NaOH, NaHCO₃, HONH₂·HCl, H₂O, r.t., 12 h, 70–84%; (d) HOBt, EDCI, DIEA, DMF, 10 h, 30–47%; (e) DTT, KOH, MeOH, r.t., 30 min; (f) SOCl₂, MeOH, Et₂O, r.t., 3 h, 74–83%; (g) LiAlH₄, THF, r.t., 1 h; (h) HONH₂·H₂O, NaOH, MeOH, r.t., 30 min, 73–88%; (i) NaOH, MeOH, 60 °C, 30 min, 90–95%.

Figure 2

FACS and Western blot analyses of apoptosis induction by PsA and PsA-SH in HL-60 cells. (A) HL-60 cells were treated with PsA and PsA-SH at the indicated concentrations for 24 h, and apoptotic cells were determined by FACS after staining with PI/Annexin-V. (B) Western blot analysis of the protein levels of Ac-H3, H3, Ac-α-tubulin, α-tubulin, PARP, caspase-3, -8, -9, and β-actin of HL-60 cells treated with PsA and PsA-SH for 24 h.

Figure 3

Apoptosis and H3 acetylation levels of HL-60 cells treated with 6a, 9, and 11b. Apoptotic cells were determined by FACS after staining with PI/Annexin-V (A), and the acetylated levels of H3 and α-tubulin were measured with Western blot (B) in HL-60 cells treated with each compound at 20 μM for 24 h.

Figure 4

Apoptosis induction and protein regulation by 10b and Tubacin. Apoptotic cells were determined by FACS after staining with PI/Annexin-V (A), and the protein levels of Ac-H3, Ac-α-tubulin, PARP, caspase-3, -8, -9, and β-actin were determined by Western blotting (B) in HL-60 cells treated with 10b and Tubacin at the indicated concentrations for 24 h.

Figure 5

Apoptosis induction and protein regulation by 12e, 12f, and RGFP966 in HL-60 cells. Apoptotic cells were determined by FACS after staining with PI/Annexin-V (A), and the protein levels of Ac-H3, Ac-α-tubulin, PARP, caspase-3, -8, -9, and β-actin were determined by Western blotting (B) in HL-60 cells treated with 12e, 12f, and RGFP966 for 24 h.

Figure 6

Putative binding patterns of PsA-SH (A) or 12g (B) within HDAC1 (left panel, green) and HDAC3 (right panel, blue) pockets.