Development of a histone deacetylase 6 inhibitor and its biological effects

Development of isoform-selective histone deacetylase (HDAC) inhibitors is important in elucidating the function of individual HDAC enzymes and their potential as therapeutic agents. Among the eleven zinc-dependent HDACs in humans, HDAC6 is structurally and functionally unique. Here, we show that a hydroxamic acid-based small-molecule N-hydroxy-4-(2-(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) selectively inhibits HDAC6 catalytic activity in vivo and in vitro. HPOB causes growth inhibition of normal and transformed cells but does not induce cell death. HPOB enhances the effectiveness of DNA-damaging anticancer drugs in transformed cells but not normal cells. HPOB does not block the ubiquitin-binding activity of HDAC6. The HDAC6-selective inhibitor HPOB has therapeutic potential in combination therapy to enhance the potency of anticancer drugs.Histone deacetylase 6 (HDAC6) is unique among the eleven zinc-dependent HDACs in humans. HDAC6 is located in the cytoplasm, and it has two catalytic domains and an ubiquitin-binding domain at the C-terminal region (1–3). This study focused on the development of a HDAC6-selective inhibitor and its biological effects. The substrates of HDAC6 include nonhistone proteins such as α-tubulin, peroxiredoxin (PRX), cortactin, and heat shock protein 90 (Hsp90) but not histones (4–7). HDAC6 plays a key role in the regulation of microtubule dynamics including cell migration and cell–cell interactions. The reversible acetylation of Hsp90, a substrate of HDAC6, modulates its chaperone activity and, accordingly, the stability of survival and antiapoptotic factors, including epidermal growth factor receptor (EGFR), protein kinase AKT, proto-oncogene C-RAF, survivin, and other factors. HDAC6, through its ubiquitin-binding activity and interaction with other partner proteins, plays a role in the degradation of misfolded proteins by binding polyubiquitinated proteins and delivering them to the dynein and motor proteins for transport into aggresomes which are degraded by lysosomes (8–10). Thus, HDAC6 has multiple biological functions through deacetylase-dependent and -independent mechanisms modulating many cellular pathways relevant to normal and tumor cell growth, migration, and death. HDAC6 is an attractive target for potential cancer treatment.There are several previous reports on the development of HDAC6-selective inhibitors (11–15). The most extensively studied is tubacin (16, 17). Tubacin has non–drug-like qualities, high lipophilicity, and difficult synthesis and has proved to be more useful as a research tool rather than as a potential drug (18). We and others (12–15, 19) have developed HDAC6-selective inhibitors whose pharmacokinetics, toxicity, and efficacy make them potentially more useful than tubacin as therapeutic agents. ACY-1215, 2-(Diphenylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)pyrimidine-5-carboxamide, a HDAC6-selective inhibitor, is currently being evaluated in clinical trials (http://clinicaltrials.gov).HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA), consist of three structural domains: a metal-binding domain, a linker domain, and a surface domain (20). The catalytic pocket of HDAC1 is deeper and narrower than the catalytic pocket of HDAC6 (14). To develop HDAC6-selective inhibitors, we synthesized small molecules with bulkier and shorter linker domains than the pan-HDAC inhibitor SAHA (20, 21). A hydroxamic acid-based small-molecule N-hydroxy-4-(2-(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) was synthesized that selectively inhibits HDAC6. We report the effects of this HDAC6-selective inhibitor on normal and transformed cells. Further, we found that selective inhibition of HDAC6 increases the effectiveness of anticancer agents, etoposide, doxorubicin, and SAHA in inducing cell death of transformed cells but not normal cells.