Simple inhibitors of histone deacetylase activity that combine features of short-chain fatty acid and hydroxamic acid inhibitors

Butyric acid and trichostatin A (TSA) are anti-cancer compounds that cause the upregulation of genes involved in differentiation and cell cycle regulation by inhibiting histone deacetylase (HDAC) activity. In this study we have synthesized and evaluated compounds that combine the bioavailability of short-chain fatty acids, like butyric acid, with the bidentate binding ability of TSA. A series of analogs were made to examine the effects of chain length, simple aromatic cap groups, and substituted hydroxamates on the compounds' ability to inhibit rat-liver HDAC using a fluorometric assay. In keeping with previous structure-activity relationships, the most effective inhibitors consisted of longer chains and hydroxamic acid groups. It was found that 5-phenylvaleric hydroxamic acid and 4-benzoylbutyric hydroxamic acid were the most potent inhibitors with IC₅₀'s of 5 μM and 133 μM respectively.     

Synthesis of rigid trichostatin A analogs as HDAC inhibitors

New inhibitors of histone deacetylase (HDAC) have been synthesized and evaluated for their activity toward non small lung cancer cell line H661. Their design is based on indanone (or tetralone) systems leading to trichostatin A (TSA) analogs with limited conformational mobility. Molecular modelization at the AM1 level revealed that the conformations of indane-based analogs and TSA bound to HDAC like protein are similar. The synthesis of these new analogs was achieved by alkylation of an appropriate indanone (or tetralone) to introduce the side chain bearing a terminal ester group, the latter being a precursor of hydroxamic acid and aminobenzamide derivatives. Hydroxamic acids with the TSA side chain were found to be the most active compounds and the presence of the dimethylamino group on the phenyl ring turned out to be essential to achieve low micromolar activities against H661 cancer cells.     

Chlamydocin-hydroxamic acid analogues as histone deacetylase inhibitors

Chlamydocin-hydroxamic acid analogues were designed and synthesized as histone deacetylase (HDAC) inhibitors based on the structure and HDAC inhibitory activity of chlamydocin and trichostatin A. Chlamydocin is a cyclic tetrapeptide containing an epoxyketone moiety in the side chain that makes it an irreversible inhibitor of HDAC. We replaced the epoxyketone moiety of chlamydocin with hydroxamic acid to design potent and reversible inhibitors of HDAC. In addition, a number of amino-cycloalkanecarboxylic acids (Acc) are introduced instead of the simple amino-isobutric acid (Aib) for a variety of the series of chlamydocin analogues. The compounds synthesized were tested for HDAC inhibitory activity and the results showed that many of them are potent inhibitors of HDAC. The replacement of Aib residue of chlamydocin with an aromatic amino acid enhances the in vivo and in vitro inhibitory activity. We have carried out circular dichroism and molecular modeling studies on chlamydocin-hydroxamic acid analogue and compared it with the solution structure of chlamydocin.     

SelSA,selenium analogs of SAHA as potent histone deacetylase inhibitors

Cancer treatment and therapy has moved from conventional chemotherapeutics to more mechanism-based targeted approach. Disturbances in the balance of histone acetyltransferase (HAT) and deacetylase (HDAC) leads to a change in cell morphology, cell cycle, differentiation, and carcinogenesis. In particular, HDAC plays an important role in carcinogenesis and therefore it has been a target for cancer therapy. Structurally diverse group of HDAC inhibitors are known. The broadest class of HDAC inhibitor belongs to hydroxamic acid derivatives that have been shown to inhibit both class I and II HDACs. Suberoylanilide hydroxamic acid (SAHA) and Trichostatin A (TSA), which chelate the zinc ions, fall into this group. In particular, SAHA, second generation HDAC inhibitor, is in several cancer clinical trials including solid tumors and hematological malignancy, advanced refractory leukemia, metastatic head and neck cancers, and advanced cancers. To our knowledge, selenium-containing HDAC inhibitors are not reported in the literature. In order to find novel HDAC inhibitors, two selenium based-compounds modeled after SAHA were synthesized. We have compared two selenium-containing compounds; namely, SelSA-1 and SelSA-2 for their inhibitory HDAC activities against SAHA. Both, SelSA-1 and SelSA-2 were potent HDAC inhibitors; SelSA-2 having IC50 values of 8.9 nM whereas SAHA showed HDAC IC₅₀ values of 196 nM. These results provided novel selenium-containing potent HDAC inhibitors.     

Design and evaluation of 'Linkerless' hydroxamic acids as selective HDAC8 inhibitors

In this report, we describe new HDAC inhibitors designed to exploit a unique sub-pocket in the HDAC8 active site. These compounds were based on inspection of the available HDAC8 crystal structures bound to various inhibitors, which collectively show that the HDAC8 active site is unusually malleable and can accommodate inhibitor structures that are distinct from the canonical 'zinc binding group-linker-cap group' structures of SAHA, TSA, and similar HDAC inhibitors. Some inhibitors based on this new scaffold are >100-fold selective for HDAC8 over other class I and class II HDACs with IC(50) values <1microM against HDAC8. Furthermore, treatment of human cells with the inhibitors described here shows a unique pattern of hyperacetylated proteins compared with the broad-spectrum HDAC inhibitor TSA.     

Effects of suberoylanilide hydroxamic acid and trichostatin A on induction of cytochrome P450 enzymes and benzo[a]pyrene DNA adduct formation in human cells

In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on the metabolism of polycyclic aromatic hydrocarbons (PAH) in human mammary carcinoma derived MCF-7 cells in culture. Benzo[a]pyrene (B[a]P) induces cytochrome P450 (CYP) 1A1, CYP1B1 and other xenobiotic metabolizing enzymes. Results from our study indicated a significant increase in CYP activity in comparison to vehicle control in cells treated with SAHA or TSA as measured by ethoxyresorufin-O-deethylase assay. However, co-treatment with 1.0 microM SAHA and BP, reduced the mRNA levels of CYP1B1 relative to B[a]P alone. When co-treated with 1.0 microM TSA and BP, a reduction in the mRNA levels of both CYP1A1 and CYP1B1 was observed relative to BP alone. We further investigated to ascertain if the differential expression and activity of CYP1A1 and CYP1B1 influenced levels of B[a]P DNA adduct formation. MCF-7 cells co-treated with B[a]P and SAHA or TSA formed DNA adducts, although no significant differences in levels of DNA binding were revealed. These results suggest that while CYP enzyme activity and gene expression were affected by the HDAC inhibitors SAHA and TSA, they had no apparent influence on B[a]P DNA binding.     

Mercaptoamide-based non-hydroxamic acid type histone deacetylase inhibitors

Inhibitors of histone deacetylases (HDAC) are emerging as a promising class of anti-cancer agents. A mercaptoamide functionality was designed as a bidentate zinc chelator and incorporated into the hydroxamic acid based SAHA (1) scaffold in order to identify non-hydroxamate compounds as potential inhibitors of histone deacetylases. Two sets of mercaptoamides 2 and 3 with varying spacer length were synthesized and their HDAC inhibitory activity was evaluated. Low micromolar inhibition was observed for mercaptoamides 2e, 3b, and 3d.     

Histone acetylation-independent effect of histone deacetylase inhibitors on Akt through the reshuffling of protein phosphatase 1 complexes

Despite advances in understanding the role of histone deacetylases (HDACs) in tumorigenesis, the mechanism by which HDAC inhibitors mediate antineoplastic effects remains elusive. Modifications of the histone code alone are not sufficient to account for the antitumor effect of HDAC inhibitors. The present study demonstrates a novel histone acetylation-independent mechanism by which HDAC inhibitors cause Akt dephosphorylation in U87MG glioblastoma and PC-3 prostate cancer cells by disrupting HDAC-protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors examined, trichostatin A (TSA) and HDAC42 exhibit the highest activity in down-regulating phospho-Akt, followed by suberoylanilide hydroxamic acid, whereas MS-275 shows only a marginal effect at 5 microm. This differential potency parallels the respective activities in inducing tubulin acetylation, a non-histone substrate for HDAC6. Evidence indicates that this Akt dephosphorylation is not mediated through deactivation of upstream kinases or activation of downstream phosphatases. However, the effect of TSA on phospho-Akt can be rescued by PP1 inhibition but not that of protein phosphatase 2A. Immunochemical analyses reveal that TSA blocks specific interactions of PP1 with HDACs 1 and 6, resulting in increased PP1-Akt association. Moreover, we used isozyme-specific small interfering RNAs to confirm the role of HDACs 1 and 6 as key mediators in facilitating Akt dephosphorylation. The selective action of HDAC inhibitors on HDAC-PP1 complexes represents the first example of modulating specific PP1 interactions by small molecule agents. From a clinical perspective, identification of this PP1-facilitated dephosphorylation mechanism underscores the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through Akt down-regulation.     

Design, synthesis, and evaluation of cyclic amide/imide-bearing hydroxamic acid derivatives as class-selective histone deacetylase (HDAC) inhibitors

A series of hydroxamic acid derivatives bearing a cyclic amide/imide group as a linker and/or cap structure, prepared during our structural development studies based on thalidomide, showed class-selective potent histone deacetylase (HDAC)-inhibitory activity. Structure-activity relationship studies indicated that the steric character of the substituent introduced at the cyclic amide/imide nitrogen atom, the presence of the amide/imide carbonyl group, the hydroxamic acid structure, the shape of the linking group, and the distance between the zinc-binding hydroxamic acid group and the cap structure are all important for HDAC-inhibitory activity and class selectivity. A representative compound (30w) showed potent p21 promoter activity, comparable with that of trichostatin A (TSA), and its cytostatic activity against cells of the human prostate cell line LNCaP was more potent than that of the well-known HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA).     

Thiol-based SAHA analogues as potent histone deacetylase inhibitors

In order to find novel nonhydroxamate histone deacetylase (HDAC) inhibitors, a series of thiol-based compounds modeled after suberoylanilide hydroxamic acid (SAHA) was synthesized, and their inhibitory effect on HDACs was evaluated. Compound 6, in which the hydroxamic acid of SAHA was replaced by a thiol, was found to be as potent as SAHA, and optimization of this series led to the identification of HDAC inhibitors more potent than SAHA.     

Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition, and binding mode study of SAHA-based non-hydroxamates

In order to find novel non-hydroxamate histone deacetylase (HDAC) inhibitors, a series of compounds modeled after suberoylanilide hydroxamic acid (SAHA) were designed and synthesized as (i) substrate (acetyl lysine) analogues (compounds 3–7), (ii) analogues bearing various functional groups expected to chelate zinc ion (compounds 8–15), and (iii) analogues bearing nucleophilic functional groups which could bind covalently to HDACs (compounds 16–18). In this series, semicarbazide 8b and bromoacetamides 18b,c were found to be potent HDAC inhibitors for non-hydroxamates.     

Structural requirements of HDAC inhibitors: SAHA analogs functionalized adjacent to the hydroxamic acid

Inhibitors of histone deacetylase (HDAC) proteins such as suberoylanilide hydroxamic acid (SAHA) have emerged as effective therapeutic anti-cancer agents. To better understand the structural requirements of HDAC inhibitors, a small molecule library with a variety of substituents attached adjacent to the metal binding hydroxamic acid of SAHA was synthesized. The presence of a substituent adjacent to the hydroxamic acid led to an 800- to 5000-fold decrease in inhibition compared to SAHA. The observed results have implications for drug design, suggesting that HDAC inhibitors with substituents near the metal binding moiety will have inhibitory activities in the micromolar rather than nanomolar range.     

Design,synthesis and biological evaluation of novel hydroxamic acid based histone deacetylase 6 selective inhibitors bearing phenylpyrazol scaffold as surface recognition motif

In recent years, inhibition of HDAC6 became a promising therapeutic strategy for the treatment of cancer and HDAC6 inhibitors were considered to be potent anti-cancer agents. In this work, celecoxib showed moderate degree of HDAC6 inhibition activity and selectivity in preliminary enzyme inhibition activity assay. A series of hydroxamic acid derivatives bearing phenylpyrazol moiety were designed and synthesized as HDAC6 inhibitors. Most compounds showed potent HDAC6 inhibition activity. 11i was the most selective compound against HDAC6 with IC₅₀ values of 0.020 µM and selective factor of 101.1. Structure-activity relationship analysis indicated that locating the linker group at 1′ of pyrazol gave the most selectivity. The most compounds 11i (GI₅₀ = 3.63 μM) exhibited 6-fold more potent than vorinostat in HepG2 cells. Considering of the high selectivity against HDAC6 and anti-proliferation activity, such compounds have potential to be developed as anti-cancer agents.     

Amide-based derivatives of β-alanine hydroxamic acid as histone deacetylase inhibitors: Attenuation of potency through resonance effects

A library of amide-linked derivatives of β-alanine hydroxamic acid were prepared (2-7) and the activity as inhibitors of Zn(II)-containing histone deacetylases (HDACs) determined in vitro against HDAC1 and the anti-proliferative activity determined in BE(2)-C neuroblastoma cells. The IC(50) values of the best-performing compounds (3-7) against HDAC1 ranged between 38 and 84μM. The least potent compound (2) inhibited a maximum of only 40% HDAC1 activity at 250μM. The anti-proliferative activity of 2-7 at 50μM against BE(2)-C neuroblastoma cells ranged between 57.0% and 88.6%. The structural similarity between the potent HDAC inhibitor trichostatin A (TSA, 1; HDAC1, IC(50) 12nM) and the present compounds (2-7) was high at the Zn(II) coordinating hydroxamic acid head group; and in selected compounds (2, 5), at the 4-(dimethylamino)phenyl tail. The significantly reduced potency of 2-7 relative to 1 underscores the rank importance of the linker region as part of the HDAC inhibitor pharmacophore. Molecular modeling of 1-7 using HDAC8 as the template suggested that the conformationally constrained 4'-methyl group of 1 may contribute to HDAC inhibitor potency through a sandwich-like interaction with a hydrophobic region containing F152 and F208; and that the absence of this group in 2-7 may reduce potency. The close proximity of the 5'-carbonyl oxygen atom in 2-7 to the sulfur atom of Met274 in HDAC8 or the corresponding isobutyl group of Leu274 in HDAC1 may attenuate potency through repulsive steric and dipole-dipole forces. In a unique resonance stabilized form of 2, this interaction could manifest as stronger ion-dipole repulsive forces, resulting in a further decrease in potency. This work suggests that resonance structures of HDAC inhibitors could modulate intermolecular interactions with HDAC targets, and potency.