Development of an ELISA-Based HDAC Activity Assay for Characterization of Isoform-Selective Inhibitors

Abstract

Histone deacetylase (HDAC) proteins are promising targets for cancer treatment, with several HDAC inhibitors used clinically as anticancer drugs. Most HDAC inhibitors nonspecifically interact with all or many of the 11 HDAC isoforms. Isoform-selective HDAC inhibitors would be useful tools to dissect the individual functions of HDAC proteins in cancer formation, in addition to potentially displaying effective anticancer properties. We report here a robust HDAC activity assay for screening selective HDAC inhibitors, which is inspired by the traditional enzyme-linked immunosorbent assay (ELISA). The key feature of the ELISA-based HDAC activity assay is use of mammalian cell-derived HDAC isoforms instead of recombinant proteins. Importantly, the assay was validated with several known HDAC inhibitors. The ELISAbased HDAC activity assay will facilitate the characterization of isoform-selective HDAC inhibitors against mammalian cell-derived HDAC proteins, which will enhance HDAC-centered cancer research and provide a foundation for anticancer drug development.

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

“…28 SAHA, as expected, showed no selectivity among HDAC1, 2, 3, and 6. 28 Interestingly, several C5-SAHA analogs displayed more potent inhibition against HDAC6 compared to HDAC1, HDAC2, and HDAC3 (Figure 2).…”

“…28 SAHA, as expected, showed no selectivity among HDAC1, 2, 3, and 6. 28 Interestingly, several C5-SAHA analogs displayed more potent inhibition against HDAC6 compared to HDAC1, HDAC2, and HDAC3 (Figure 2). The analogs that showed the greater difference in potency with HDAC6 versus the other isoforms were C5- n -butyl ( 1b ), C5- n -hexyl ( 1c ), and C5-benzyl ( 1e ).…”

“…The IC 50 values of SAHA as the parent compound were included as well (Table 2). 28 SAHA displayed similar IC 50 values against HDAC1, 2, 3, and 6, with 6- to 27-fold selectivity against HDAC8. 28, 44 Both C5- n -butyl ( 1b ), and C5- n -hexyl ( 1c ) SAHA analogs displayed modest selectivity, with 3- to 5-fold and 5- to 7-fold selectivities for HDAC6 and HDAC8 over HDAC1, 2, and 3 (Tables 2 and S6).…”

“…28 SAHA displayed similar IC 50 values against HDAC1, 2, 3, and 6, with 6- to 27-fold selectivity against HDAC8. 28, 44 Both C5- n -butyl ( 1b ), and C5- n -hexyl ( 1c ) SAHA analogs displayed modest selectivity, with 3- to 5-fold and 5- to 7-fold selectivities for HDAC6 and HDAC8 over HDAC1, 2, and 3 (Tables 2 and S6). In addition, 1b and 1c showed modest reductions in HDAC6 potency (IC 50 values of 320 and 410 nM), but similar potency against HDAC8 (430 and 420 nM) compared to SAHA (Table 2).…”

“…26 SAHA inhibits most of the eleven metal-dependent HDAC proteins, with only a modest selectivity against HDAC8. 27, 28 The non-selectivity of SAHA might explain the side effects observed in clinic, but certainly limits the use of SAHA to study individual HDAC isoforms in cancer biology. 29 In the last four years, several dual HDAC6/HDAC8 selective inhibitors have been developed.…”