Protein kinase C-related kinase targets nuclear localization signals in a subset of class IIa histone deacetylases

Class IIa histone deacetylases (HDACs) -4, -5, -7 and -9 undergo signal-dependent nuclear export upon phosphorylation of conserved serine residues that are targets for 14-3-3 binding. Little is known of other mechanisms for regulating the subcellular distribution of class IIa HDACs. Using a biochemical purification strategy, we identified protein kinase C-related kinase-2 (PRK2) as an HDAC5-interacting protein. PRK2 and the related kinase, PRK1, phosphorylate HDAC5 at a threonine residue (Thr-292) positioned within the nuclear localization signal (NLS) of the protein. HDAC7 and HDAC9 contain analogous sites that are phosphorylated by PRK, while HDAC4 harbors a non-phosphorylatable alanine residue at this position. We provide evidence to suggest that the unique phospho-acceptor cooperates with the 14-3-3 target sites to impair HDAC nuclear import.

Structured summary

MINT-7710106:HDAC5 (uniprotkb:Q9UQL6) physically interacts (MI:0915) with PRK2 (uniprotkb:Q16513) by pull down (MI:0096)     

3,5-diarylazoles as novel and selective inhibitors of protein kinase D

The synthesis and preliminary studies of the SAR of novel 3,5-diarylazole inhibitors of Protein Kinase D (PKD) are reported. Notably, optimized compounds in this class have been found to be active in cellular assays of phosphorylation-dependant HDAC5 nuclear export, orally bioavailable, and highly selective versus a panel of additional putative histone deacetylase (HDAC) kinases. Therefore these compounds could provide attractive tools for the further study of PKD / HDAC5 signaling.     

Inhibition of histone deacetylase activity is a novel function of the antifolate drug methotrexate

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used for treating human cancers, and overexpression of histone deacetylase (HDAC) is usually found in tumors. HDAC inhibitors (HDACi) can reactivate tumor suppressor genes and serve as potential anti-cancer drugs. In this study, we found that MTX shared structural similarity with some HDACi and molecular modeling showed that MTX indeed docks into the active site of HDLP, a bacterial homologue of HDAC. Subsequent in vitro assay demonstrated MTX’s inhibition on HDAC activity in human cancer cells. The global acetylation of histone H3 was also induced by MTX. Moreover, MTX inhibited immunoprecipitated HDAC1/2 activity but not their protein levels. This study provides evidence that MTX inhibits HDAC activity.     

Phenylbutyrate, a histone deacetylase inhibitor, protects against Adriamycin-induced cardiac injury

Cardiac injury is a major complication for oxidative-stress-generating anticancer agents exemplified by Adriamycin (ADR). Recently, several histone deacetylase inhibitors (HDACIs) including phenylbutyrate (PBA) have shown promise in the treatment of cancer with little known toxicity to normal tissues. PBA has been shown to protect against oxidative stress in normal tissues. Here, we examined whether PBA might protect heart against ADR toxicity in a mouse model. The mice were i.p. injected with ADR (20 mg/kg). PBA (400 mg/kg/day) was i.p. injected 1 day before and daily after the ADR injection for 2 days. We found that PBA significantly decreased the ADR-associated elevation of serum lactate dehydrogenase and creatine kinase activities and diminished ADR-induced ultrastructual damages of cardiac tissue by more than 70%. Importantly, PBA completely rescued ADR-caused reduction of cardiac functions exemplified by ejection fraction and fraction shortening, and increased cardiac manganese superoxide dismutase (MnSOD) protein and activity. Our results reveal a previously unrecognized role of HDACIs in protecting against ADR-induced cardiac injury and suggest that PBA may exert its cardioprotective effect, in part, by the increase of MnSOD. Thus, combining HDACIs with ADR could add a new mechanism to fight cancer while simultaneously decrease ADR-induced cardiotoxicity.     

Development of novel ferulic acid derivatives as potent histone deacetylase inhibitors

Histone deacetylase inhibitors (HDACIs) offer a promising strategy for cancer therapy. The discovery of potent ferulic acid-based HDACIs with hydroxamic acid or 2-aminobenzamide group as zinc binding group was reported. The halogeno-acetanilide was introduced as novel surface recognition moiety (SRM). The majority of title compounds displayed potent HDAC inhibitory activity. In particular, FA6 and FA16 exhibited significant enzymatic inhibitory activities, with IC₅₀ values of 3.94 and 2.82 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against a panel of human cancer cells. FA17 displayed promising profile as an antitumor candidate. The results indicated that these ferulic acid derivatives could serve as promising lead compounds for further optimization.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.     

AMP-activated protein kinase regulates beta-catenin transcription via histone deacetylase 5

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; it is inhibited under obese conditions and is activated by exercise and by many anti-diabetic drugs. Emerging evidence also suggests that AMPK regulates cell differentiation, but the underlying mechanisms are unclear. We hypothesized that AMPK regulates cell differentiation via altering β-catenin expression, which involves phosphorylation of class IIa histone deacetylase 5 (HDAC5). In both C3H10T1/2 cells and mouse embryonic fibroblasts (MEFs), AMPK activity was positively correlated with β-catenin content. Chemical inhibition of HDAC5 increased β-catenin mRNA expression. HDAC5 overexpression reduced and HDAC5 knockdown increased H3K9 acetylation and cellular β-catenin content. HDAC5 formed a complex with myocyte enhancer factor-2 to down-regulate β-catenin mRNA expression. AMPK phosphorylated HDAC5, which promoted HDAC5 exportation from the nucleus; mutation of two phosphorylation sites in HDAC5, Ser-259 and -498, abolished the regulatory role of AMPK on β-catenin expression. In conclusion, AMPK promotes β-catenin expression through phosphorylation of HDAC5, which reduces HDAC5 interaction with the β-catenin promoter via myocyte enhancer factor-2. Thus, the data indicate that AMPK regulates cell differentiation and development via cross-talk with the wingless and Int (Wnt)/β-catenin signaling pathway.     

Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression

Modulation of histone acetylation is currently being explored as a therapeutic strategy in treatment of cancer. Specifically, inhibition of histone deacetylase by trichostatin A (TSA) has been shown to prevent tumorigenesis and metastasis. In the present paper we demonstrate that increased histone acetylation by TSA-treated 3T3 cells decreases mRNA as well as zymographic activity of gelatinase A, a matrix metalloproteinase, which is itself, implicated in tumorigenesis and metastasis. Furthermore, TSA inhibits cytochalasin D-induced activation of gelatinase A, but TSA does not affect other members of the gelatinase A activation complex, MT1-MMP and TIMP-2. Thus, TSA is a selective and potent inhibitor of expression and activation of gelatinase A. This finding not only strengthens the rationale for continuing to investigate the therapeutic utility of TSA in cancer, but also, provides evidence that TSA inhibition of gelatinase A expression and activation can be used as a biological marker to monitor and determine end-points of clinical trials involving TSA.     

Search for novel histone deacetylase inhibitors. Part II: Design and synthesis of novel isoferulic acid derivatives

Previously, we described the discovery of potent ferulic acid-based histone deacetylase inhibitors (HDACIs) with halogeno-acetanilide as novel surface recognition moiety (SRM). In order to improve the affinity and activity of these HDACIs, twenty seven isoferulic acid derivatives were described herein. The majority of title compounds displayed potent HDAC inhibitory activity. In particular, IF5 and IF6 exhibited significant enzymatic inhibitory activities, with IC₅₀ values of 0.73 ± 0.08 and 0.57 ± 0.16 μM, respectively. Furthermore, these compounds showed moderate antiproliferative activity against human cancer cells. Especially, IF6 displayed promising profile as an antitumor candidate with IC₅₀ value of 3.91 ± 0.97 μM against HeLa cells. The results indicated that these isoferulic acid derivatives could serve as promising lead compounds for further optimization.     

Design and synthesis of a new generation of substituted purine hydroxamate analogs as histone deacetylase inhibitors

Histone deacetylase inhibitors have been proved to be great potential for the treatment of cancer. Recently, we designed and modified a series of substituted purine hydroxamate analogs as potent HDAC inhibitors based on our previous studies. The target compounds were investigated for their in vitro HDAC inhibitory activities and anti-proliferative activities. Results indicated that these compounds could effectively inhibit HDAC and possess obvious anti-proliferative activity against tumor cells. Promisingly, target compounds 4m and 4n outperformed SAHA in both enzymatic inhibitory activity and cellular anti-proliferative activity assay.     

Design,synthesis and preliminary bioactivity studies of 1,2-dihydrobenzo[d]isothiazol-3-one-1,1-dioxide hydroxamic acid derivatives as novel histone deacetylase inhibitors

Histone deacetylase (HDAC) is a clinically validated target for antitumor therapy. In order to increase HDAC inhibition and efficiency, we developed a novel series of saccharin hydroxamic acids as potent HDAC inhibitors. Among them, compounds 11e, 11m, 11p exhibited similar or better HDACs inhibitory activity compared with the approved drug SAHA. Further biological evaluation indicated that compound 11m had potent antiproliferative activities against MDA-MB-231 and PC-3.     

Design,synthesis, docking studies and biological evaluation of novel chalcone derivatives as potential histone deacetylase inhibitors

A group of novel chalcone derivatives comprising hydroxamic acid or 2-aminobenzamide group as zinc binding groups (ZBG) were synthesized. The structure of the prepared compounds was fully characterized by IR, NMR and elemental microanalyses. Most of the tested compounds displayed strong to moderate HDAC inhibitory activity. Some of these compounds showed potent anti-proliferative activity against human HepG2, MCF-7 and HCT-116 cell lines. In particular, compounds 4a and 4b exhibited significant anti-proliferative activity against the three cell lines compared to SAHA as reference drug and displayed promising profile as anti-tumor candidates. The results indicated that these chalcone derivatives could serve as a promising lead compounds for further optimization as antitumor agents.     

Development and characterization of a CNS-penetrant benzhydryl hydroxamic acid class IIa histone deacetylase inhibitor

We have identified a potent, cell permeable and CNS penetrant class IIa histone deacetylase (HDAC) inhibitor 22, with >500-fold selectivity over class I HDACs (1,2,3) and ～150-fold selectivity over HDAC8 and the class IIb HDAC6 isoform. Dose escalation pharmacokinetic analysis demonstrated that upon oral administration, compound 22 can reach exposure levels in mouse plasma, muscle and brain in excess of cellular class IIa HDAC IC₅₀ levels for ～8?h. Given the interest in aberrant class IIa HDAC function for a number of neurodegenerative, neuromuscular, cardiac and oncology indications, compound 22 (also known as CHDI-390576) provides a selective and potent compound to query the role of class IIa HDAC biology, and the impact of class IIa catalytic site occupancy in vitro and in vivo.     

Utilization of tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinone as a cap moiety in design of novel histone deacetylase inhibitors

A series of novel 5,6,7,8-Tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one derivatives bearing a hydroxamic acid, 2-aminoanilide and hydrazide moieties as zinc-binding group (ZBG) were designed, synthesized and evaluated for the HDAC inhibition activity and antiproliferative activity. Most of the tested compounds displayed strong to moderate HDAC inhibitory activity. Some of these compounds showed potent anti-proliferative activity against human HepG2, MCF-7 and HCT-116 cell lines. In particular, compounds IVa, IVb, IXa and IXb exhibited significant anti-proliferative activity against the three cell lines tested compared to SAHA as a reference. Compound IVb is equipotent inhibitor for HDAC1 and HDAC2 as SAHA. It is evident that the presence of free hydroxamic acid group is essential for Zn binding affinity with maximal activity with a linker of aliphatic 6 carbons. Docking study results revealed that compound IVb could occupy the HDAC2 binding site and had the potential to exhibit antitumor activity through HDAC inhibition, which merits further investigation.     

Coumarin-suberoylanilide hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase inhibitors

Histone deacetylases (HDACs) are intimately involved in epigenetic regulation and, thus, are one of the key therapeutic targets for cancer, and two HDAC inhibitors, namely suberoylanilide hydroxamic acid (SAHA) and romidepsin, have been recently approved for cancer treatment. Because the screening and detailed characterization of HDAC inhibitors has been time-consuming, we synthesized coumarin-SAHA (c-SAHA) as a fluorescent probe for determining the binding affinities (K_d) and the dissociation off-rates (k_off) of the enzyme–inhibitor complexes. The determination of the above parameters relies on the changes in the fluorescence emission intensity (λ_ex = 325 nm, λ_em = 400 nm) of c-SAHA due to its competitive binding against other HDAC inhibitors, and such determination neither requires employment of polarization accessories nor is dependent on the fluorescence energy transfer from the enzyme’s tryptophan residues to the probe. Our highly sensitive and robust analytical protocol presented here is applicable to most of the HDAC isozymes, and it can be easily adopted in a high-throughput mode for screening the HDAC inhibitors as well as for quantitatively determining their K_d and k_off values.     

A gastrin-releasing peptide receptor antagonist stimulates Neuro2a neuroblastoma cell growth: Prevention by a histone deacetylase inhibitor

Gastrin-releasing peptide (GRP) acts as an autocrine growth factor for neuroblastoma and other types of cancer, and its cell-surface receptor, GRPR, is overexpressed in advanced-stage human neuroblastoma. GRPR knockdown and GRPR antagonism inhibit the growth of experimental neuroblastoma. Here we show that a GRPR antagonist promotes rather than inhibits the growth of neuroblastoma cells. The GRPR antagonist, RC-3095, at 0.1 nM inhibited, whereas at 100 nM stimulated proliferation of Neuro2a murine neuroblastoma cells in vitro. The stimulatory effects were prevented by the histone deacetylase inhibitor (HDACi), sodium butyrate (NaB). Expression of GRPR mRNA in Neuro2a cells was analyzed by RT-PCR. These findings provide evidence that a GRPR antagonist can stimulate the growth of cancer cells, and suggest that GRPR might interact with epigenetic mechanisms in regulating neuroblastoma cell growth.     

PKCepsilon is essential for gelsolin expression by histone deacetylase inhibitor apicidin in human cervix cancer cells

Down-regulation of gelsolin expression is associated with cellular transformation and induction of gelsolin exerts antitumorigenic effects. In this study, we show that protein kinase C (PKC) signaling pathway is required for the induction of gelsolin by the histone deacetylase inhibitor apicidin in HeLa cells. Apicidin induces gelsolin mRNA independently of the de novo protein synthesis. Inhibitor study has revealed that the PKC signaling pathway is involved in the gelsolin expression. Furthermore, inhibition of PKCepsilon by either siRNA or dominant-negative mutant completely abrogates the expression of gelsolin by apicidin, indicating that PKCepsilon is the major isoform for this process. In parallel, apicidin induction of gelsolin is antagonized by the inhibition of Sp1 using dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and inhibition of PKC leads to suppression of Sp1 promoter activity. Our results provide mechanistic insights into molecular mechanisms of gelsolin induction by apicidin.