Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response.

Histone acetylation is an essential process in the epigenetic regulation of diverse biological processes, including environmental stress responses in plants. Previously, our research group identified a histone deacetylase (HDAC) inhibitor (HDI) that confers salt tolerance in Arabidopsis (Arabidopsis thaliana). In this study, we demonstrate that class I HDAC (HDA19) and class II HDACs (HDA5/14/15/18) control responses to salt stress through different pathways. The screening of 12 different selective HDIs indicated that seven newly reported HDIs enhance salt tolerance. Genetic analysis, based on a pharmacological study, identified which HDACs function in salinity stress tolerance. In the wild-type Columbia-0 background, hda19 plants exhibit tolerance to high-salinity stress, while hda5/14/15/18 plants exhibit hypersensitivity to salt stress. Transcriptome analysis revealed that the effect of HDA19 deficiency on the response to salinity stress is distinct from that of HDA5/14/15/18 deficiencies. In hda19 plants, the expression levels of stress tolerance-related genes, late embryogenesis abundant proteins that prevent protein aggregation and positive regulators such as ABI5 and NAC019 in abscisic acid signaling, were induced strongly relative to the wild type. Neither of these elements was up-regulated in the hda5/14/15/18 plants. The mutagenesis of HDA19 by genome editing in the hda5/14/15/18 plants enhanced salt tolerance, suggesting that suppression of HDA19 masks the phenotype caused by the suppression of class II HDACs in the salinity stress response. Collectively, our results demonstrate that HDIs that inhibit class I HDACs allow the rescue of plants from salinity stress regardless of their selectivity, and they provide insight into the hierarchal regulation of environmental stress responses through HDAC isoforms.

Ky-2, a Histone Deacetylase Inhibitor, Enhances High-Salinity Stress Tolerance in Arabidopsis thaliana.

Adaptation to environmental stress requires genome-wide changes in gene expression. Histone modifications are involved in gene regulation, but the role of histone modifications under environmental stress is not well understood. To reveal the relationship between histone modification and environmental stress, we assessed the effects of inhibitors of histone modification enzymes during salinity stress. Treatment with Ky-2, a histone deacetylase inhibitor, enhanced high-salinity stress tolerance in Arabidopsis. We confirmed that Ky-2 possessed inhibition activity towards histone deacetylases by immunoblot analysis. To investigate how Ky-2 improved salt stress tolerance, we performed transcriptome and metabolome analysis. These data showed that the expression of salt-responsive genes and salt stress-related metabolites were increased by Ky-2 treatment under salinity stress. A mutant deficient in AtSOS1(Arabidopis thaliana SALT OVERLY SENSITIVE 1), which encodes an Na(+)/H(+)antiporter and was among the up-regulated genes, lost the salinity stress tolerance conferred by Ky-2. We confirmed that acetylation of histone H4 at AtSOS1 was increased by Ky-2 treatment. Moreover, Ky-2 treatment decreased the intracellular Na(+)accumulation under salinity stress, suggesting that enhancement of SOS1-dependent Na(+)efflux contributes to increased high-salinity stress tolerance caused by Ky-2 treatment.

An efficient synthesis of SK-658 and its analogs as potent histone deacetylase inhibitors.

SK-658 is a potent histone deacetylase (HDAC) inhibitor that showed higher activity than SAHA due to the presence of extended hydrophobic group. We designed and synthesized thioester and SS-hybrid bearing SK-658 analogs as HDAC inhibitors. All the compounds were active in nano molar range and showed higher inhibitory activity than SAHA and SK-658. Among these, disulfide compounds showed the highest activity.

A convenient preparation of N (ε)-methyl-L-lysine derivatives and its application to the synthesis of histone tail peptides.

A convenient route is established for the preparation of N (α)-Fmoc-N (ε)-(Boc, methyl)-L-lysine and N (α)-Fmoc-N (ε)-dimethyl-L-lysine as building blocks to be used for the synthesis of methylated peptides. This methodology is based on the use of malonate derivatives and dibromobutane to produce key intermediates, L-2-amino-6-bromohexanoic acid derivatives, which could be modified to the required group at the ε-position. Fmoc-protection is accessible, so these compounds can be used in solution as well as in solid-phase peptide synthesis. Also the peptides containing these methylated lysines have been proved to resist the action of trypsin and lysyl endopeptidase. Thus, this new method could be considered as an improvement of the synthesis of N (ε)-methyl-L-lysine derivatives.

Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases.

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.

Bicyclic tetrapeptides as potent HDAC inhibitors: effect of aliphatic loop position and hydrophobicity on inhibitory activity.

Several histone deacetylase (HDAC) inhibiting bicyclic tetrapeptides have been designed and synthesized through intramolecular ring-closing metathesis (RCM) reaction and peptide cyclization. We designed bicyclic tetrapeptides based on CHAP31, trapoxin B and HC-toxin I. The HDAC inhibitory and p21 promoter assay results showed that the aliphatic loop position as well as the hydrophobicity plays an important role toward the activity of the bicyclic tetrapeptide HDAC inhibitors.

Design and synthesis of CHAP31, trapoxin B and HC-toxin based bicyclic tetrapeptides disulfide as potent histone deacetylase inhibitors.

The naturally occurring cyclic depsipeptide, FK228 inhibits histone deacetylase (HDAC) enzymes after reductive cleavage of intra-molecular disulfide bond. One of the sulfhydryl groups produced in the reduction interacts with zinc atom that involved in the catalytic mechanism of type 1 and 2 HDACs such as HDAC1, HDAC4, and HDAC6. In the present study, we describe the development of CHAP31, trapoxin B and HC-toxin based cyclic tetrapeptides with intra-molecular disulfide bond as HDAC inhibitors. The bicyclic tetrapeptides disulfide showed potent HDAC1 and HDAC4 inhibition and p21 promoting activity.

Design and synthesis of peptide-MCA substrates for a novel assay of histone methyltransferases and their inhibitors.

Histone methyltransferases (HMTs) play an important role in controlling gene expression through site-specific methylation of lysines in core and linker histones within chromatin. As the typical HMTs, G9a and Set7/9 have been intensively studied that G9a is specific to the methylation at H3K9 and H3K27 and represses transcription, while Set7/9 methylates at H3K4. In this report we prepared various peptide-MCAs (4-methylcoumaryl-7-amides) related to histone tail and protein-substrates such as p53 and estrogen receptor-α. The fluorogenic substrates are applied for the assay of HMTs and an inhibitor, for example. The most sensitive and specific MCA-substrates to G9a and Set7/9 are discovered. The peptide-MCAs corresponding to the methylation sequences are promising for screening of HMT inhibitors.

Bicyclic tetrapeptide histone deacetylase inhibitors with methoxymethyl ketone and boronic acid zinc-binding groups.

Histone deacetylase (HDAC) inhibitors are a class of potential therapeutics for the treatment of cancer. Bicyclic tetrapeptides equipped with methoxymethyl ketone and boronic acid as zinc-binding group were designed and synthesized. The inhibitory activities of these compounds were evaluated against HDAC enzymes. The cell-free and cell-based assay data showed that both potency and selectivity changed with the change in zinc-binding group. Boronic acid-based compound showed poor activity whereas methoxymethyl ketone-based compound displayed impressive activity in both cell-free and cell-based conditions.

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor.

OBJECTIVES: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. MATERIALS AND METHODS: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst's staining was used to detect apoptotic cells. RESULTS: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. CONCLUSION: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.

Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos.

Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated α-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and α-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to α-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated α-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including α-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development.

Cyclic tetrapeptides with -SS- bridging between amino acid side chains for potent histone deacetylases' inhibition.

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose -SS- bond is reduced within the cell. Here, cyclic peptides with -SS- bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-L-amino acid-L-amino acid-L-Val-D-Pro-), with an -SS- bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-L-amino acid-D-amino acid-L-Val-D-Pro-), with an -SS- bridge between the first and second amino acids, was also a potent HDAC inhibitor.

Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

Evaluation of functional groups on amino acids in cyclic tetrapeptides in histone deacetylase inhibition.

The naturally occurring cyclic tetrapeptide, chlamydocin, originally isolated from fungus Diheterospora chlamydosphoria, consists of α-aminoisobutyric acid, L-phenylalanine, D-proline and an unusual amino acid (S)-2-amino-8-((S)-oxiran-2-yl)-8-oxooctanoic acid (Aoe) and inhibits the histone deacetylases (HDACs), a class of regulatory enzymes. The epoxyketone moiety of Aoe is the key functional group for inhibition. The cyclic tetrapeptide scaffold is supposed to play important role for effective binding to the surface of enzymes. In place of the epoxyketone group, hydroxamic acid and sulfhydryl group have been applied to design inhibitor ligands to zinc atom in catalytic site of HDACs. In the research for more potent HDAC inhibitors, we replaced the epoxyketone moiety of Aoe with different functional groups and synthesized a series of chlamydocin analogs as HDAC inhibitors. Among the functional groups, methoxymethylketone moiety showed as potent inhibition as the hydroxamic acid. On the contrary, we confirmed that borate, trifruoromethylketone, and 2-aminoanilide are almost inactive in HDAC inhibition.

Peptide-linked porphyrin sensitiser and colloidal Pt or Ir catalyst in the H2 formation reaction.

Porphyrins linking amphiphilic peptides were applied as photosensitisers, assuming that they would interact with the H(+) reduction catalyst, polymer-protected colloidal Pt or Ir. The close orientation of the porphyrin and metal catalyst may facilitate efficient electron transfer. The porphyrin linking a peptide containing glutamic acids (Glu), Ac-Cys(porph)-Glu-Val-Glu-Val-NH(2) (2), was an effective sensitiser for the H(2) generation reaction in the presence of N-benzyldihydronicotinamide and colloidal Pt or Ir in aqueous media under visible light illumination. At pH 5 and 7, 2 was a more efficient photosensitiser than tetrakis(p-carboxyphenyl)porphyrin (TCPP). The efficiency of H(+) reduction catalysts was in the order Ir-pGlu (polyglutamic acid) > Ir-PVP (polyvinylpyrrolidone) > Pt-PVP. Dynamic light scattering and scanning electron microscopy measurements showed that large particles formed when colloidal metal solutions were produced by microwave irradiation. Fluorescence quenching experiments suggested that electron transfer occurred from the photoexcited porphyrin to the colloidal Ir.

Cyclic tetrapeptides with thioacetate tails or intramolecular disulfide bridge as potent inhibitors of histone deacetylases.

Two thioacetate tails were introduced to the chlamydocin- and CHAP31-related cyclic tetrapeptides. An intramolecular disulfide bridge could be formed in the CHAP31-related cyclic peptides. Both the thioacetate-tailed and disulfide-bridged peptides were potent histone deacetylase inhibitors in the presence of sulfhydryl compound. Potent p21 promoter inducing activity was also observed in vivo.

Synthesis, evaluation and molecular modeling of cyclic tetrapeptide histone deacetylase inhibitors as anticancer agents.

Histone deacetylase inhibitors (HDACIs) are a promising class of anticancer agents. To examine whether a slight change in the recognition domain could alter their inhibitory activity, we synthesized a series of cyclo(-L-Am7(S2Py)-Aib-L-Phe(n-Me)-D-Pro)derivatives and evaluated their HDAC inhibitory and anticancer activities. The peptides exhibited potent HDAC inhibitory activity and inhibited three human cancer cell lines with IC50 in the micromolar range. Docking and molecular dynamics simulation were conducted to explore the interaction mechanisms of class I and II HDACs with these inhibitors. It revealed that the zinc ion in the active site coordinated five atoms of HDACs and the sulfur atom of the inhibitor. The metal binding domains of these compounds interacted with HDAC2, and the surface recognition domains of these compounds interacted with HDAC4 through hydrogen bonding. The hydrophobic interactions also provided favorable contributions to stabilize the complexes. The results obtained from this study would be helpful for us to design some novel cyclic tetrapeptides that may act as potent HDACIs.

Real-time imaging of histone H4 hyperacetylation in living cells.

To visualize histone acetylation in living cells, we developed a genetically encoded fluorescent resonance energy transfer (FRET)-based indicator. Response of the indicator reflects changes in the acetylation state of both K5 and K8 in histone H4. Using this acetylation indicator, we were able to monitor the dynamic fluctuation of histone H4 acetylation levels during mitosis, as well as acetylation changes in response to structurally distinct histone deacetylase inhibitors.

Histone deacetylase inhibitors block nuclear factor-κB-dependent transcription by interfering with RNA polymerase II recruitment.

Histone deacetylase inhibitors (HDACi) have been shown to exhibit anti-inflammatory activity, but their mechanism of action is poorly understood. Trichostatin A (TSA) and the cyclic tetrapeptide class inhibitor Ky-2 inhibit both lipopolysaccharide-induced tumor necrosis factor-α (TNF-α) production in rats and TNF-α-induced expression of inflammatory genes in HeLa cells. We assessed the molecular mechanism underlying TSA-induced anti-inflammatory activity by genetically dissecting activation of the nuclear factor-κB (NF-κB) pathway following stimulation with TNF-α. Trichostatin A did not inhibit degradation of IκBα, nuclear translocation and DNA binding of NF-κB; also, the drug did not affect transient expression from exogenous κB-reporter plasmids. However, endogenous expression of inflammatory cytokines such as interleukin-8 (IL-8) was greatly reduced, even in the absence of de novo protein synthesis, suggesting that HDACi directly inhibits NF-κB-induced transcription. Indeed, chromatin immunoprecipitation (ChIP) analysis showed that events related to transcriptional activation of the IL-8 gene region in response to TNF-α, including recruitment of RNA polymerase II (Pol II), were compromised in the presence of TSA. These data indicate that HDAC activity is required for the efficient initiation and/or elongation of inflammatory gene transcription mediated by NF-κB.