The structural requirements of histone deacetylase inhibitors: SAHA analogs modified at the C5 position display dual HDAC6/8 selectivity

Abstract

Histone deacetylase (HDAC) proteins have emerged as important targets for anti-cancer drugs, with four small molecules approved for use in the clinic. Suberoylanilide hydroxamic acid (Vorinostat, SAHA) was the first FDA-approved HDAC inhibitor for cancer treatment. However, SAHA inhibits most of the eleven HDAC isoforms. To understand the structural requirements of HDAC inhibitor selectivity and develop isoform selective HDAC inhibitors, SAHA analogs modified in the linker at the C5 position were synthesized and tested for potency and selectivity. C5-modified SAHA analogs displayed dual selectivity to HDAC6 and HDAC8 over HDAC 1, 2, and 3, with only a modest reduction in potency. These findings are consistent with prior work showing that modification of the linker region of SAHA can alter isoform selectivity. The observed HDAC6/8 selectivity of C5-modified SAHA analogs provide guidance toward development of isoform selective HDAC inhibitors and more effective anti-cancer drugs.

Keywords: HDAC inhibitors; HDAC6/8 selective inhibitor; Histone deacetylase; Isoform selectivity; Vorinostat.

Figure 1

Chemical structures of the FDA-approved drugs SAHA, Belinostat, and Panobinistat, along with the C5-modified SAHA analogs reported here.

Figure 2

In vitro isoform selectivity screening of C5-modified SAHA analogs (1a–e) against HDAC1, HDAC2, HDAC3, and HDAC6 using the ELISA-based HDAC activity assay. Analogs 1a–e were tested at 0.025, 0.25, 1.25, 0.125, and 1.25 µM final concentrations, respectively. SAHA was tested at 1 µM concentration in a previous report using the same assay procedure. Mean percent deacetylase activities from a minimum of two independent trials with standard errors were plotted (Table S2).

Figure 3

Western blots analysis of acetyl-lysine 9 of histone H3, (AcH3) and acetyl-lysine 40 of α-tubulin (AcTub) after treatment with SAHA or C5-benzyl SAHA 1e. U937 cells were treated with DMSO (1%), SAHA (5 µM), or increasing concentrations of C5-benzyl SAHA (1e) analog (10–40 µM), before lysis, SDS-PAGE separation, transfer to a PVDF membrane, and western analysis with AcH3 or AcTub antibodies. GAPDH levels in the samples were also probed as a gel load control. A DMSO control sample was included for comparison to inhibitor-treated samples. Repetitive trials are shown in Figures S56.

Figure 4

Cytotoxicity screening of SAHA and C5-modified SAHA analogs 1b, 1c, and 1e, at 1 and 10 µM concentrations using an MTT assay with the Jurkat cells. Mean percent cell viability from a minimum of three independent trials with standard errors were plotted (Table S7).

Scheme 1

Synthesis of C5-modified SAHA analogs (1a–e)