Anti-malarial effect of histone deacetylation inhibitors and mammalian tumour cytodifferentiating agents

The histones of Plasmodium falciparum represent a potential new target for anti-malarial compounds. A naturally occurring compound, apicidin, has recently been shown to inhibit the in vitro growth of P. falciparum. Apicidin was shown to hyperacetylate histones, suggesting that its mode of action is through histone deacetylase inhibition. We have tested the ability of known histone deacetylase inhibitors, mammalian tumour suppressor compounds, and cytodifferentiating agents to inhibit the in vitro growth of a drug sensitive and resistant strain of P. falciparum. Seven of the tested compounds had microM IC50 values, and trichostatin A, a histone deacetylation inhibitor and cytodifferentiating agent, was active at low nM concentrations. One compound, suberic acid bisdimethylamide, which selectively arrests tumour cells as opposed to normal mammalian cells, had an in vivo cytostatic effect against the acute murine malaria Plasmodium berghei, and one round of treatment with the compound failed to select for resistant mutations. These results suggest a promising role for histone deacetylase inhibitors and cytodifferentiating agents as antimalarial drug candidates.     

Substituted N-(2-aminophenyl)-benzamides, (E)-N-(2-aminophenyl)-acrylamides and their analogues: novel classes of histone deacetylase inhibitors

Inhibition of histone deacetylases (HDACs) is emerging as a new strategy in human cancer therapy. Novel 2-aminophenyl benzamides and acrylamides, that can inhibit human HDAC enzymes and induce hyperacetylation of histones in human cancer cells, have been designed and synthesized. These compounds selectively inhibit proliferation and cause cell cycle arrest in various human cancer cells but not in normal cells. The growth inhibition of 2-aminophenyl benzamides and acrylamides against human cancer cells in vitro is reversible and is dependent on the induction of histone acetylation. Compounds of this class can significantly reduce tumor growth in human tumor xenograft models.     

Haspin: a newly discovered regulator of mitotic chromosome behavior

The haspins are divergent members of the eukaryotic protein kinase family that are conserved in many eukaryotic lineages including animals, fungi, and plants. Recently-solved crystal structures confirm that the kinase domain of human haspin has unusual structural features that stabilize a catalytically active conformation and create a distinctive substrate binding site. Haspin localizes predominantly to chromosomes and phosphorylates histone H3 at threonine-3 during mitosis, particularly at inner centromeres. This suggests that haspin directly regulates chromosome behavior by modifying histones, although it is likely that additional substrates will be identified in the future. Depletion of haspin by RNA interference in human cell lines causes premature loss of centromeric cohesin from chromosomes in mitosis and failure of metaphase chromosome alignment, leading to activation of the spindle assembly checkpoint and mitotic arrest. Haspin overexpression stabilizes chromosome arm cohesion. Haspin, therefore, appears to be required for protection of cohesion at mitotic centromeres. Saccharomyces cerevisiae homologues of haspin, Alk1 and Alk2, are also implicated in regulation of mitosis. In mammals, haspin is expressed at high levels in the testis, particularly in round spermatids, so it seems likely that haspin has an additional role in post-meiotic spermatogenesis. Haspin is currently the subject of a number of drug discovery efforts, and the future use of haspin inhibitors should provide new insight into the cellular functions of these kinases and help determine the utility of, for example, targeting haspin for cancer therapy.     

Structure-activity relationship study of beta-carboline derivatives as haspin kinase inhibitors

Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure-activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented.     

Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.     

Haspin inhibition delays cell cycle progression through interphase in cancer cells

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.     

Synthesis and biological testing of novel pyridoisothiazolones as histone acetyltransferase inhibitors

We present a combination of database screening, synthesis and in vitro testing to identify novel histone acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors. Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for new anticancer agents directed against HATs as new targets have been identified.     

第二代 mTOR 抑制剂的抗肿瘤研究进展

哺乳动物雷帕霉素靶蛋白(mTOR)是 PI3K/Akt/mTOR 等多种信号通路的下游分子,在细胞增殖、分化、转移和存活中发挥 重要作用,已成为癌症治疗的一个重要靶标。传统的 mTOR 抑制剂主要是雷帕霉素及其衍生物,能特异性抑制 mTORC1,但在部分癌 症临床治疗中未达到预期疗效,且易产生耐药性。第二代 mTOR 抑制剂即双重或多重 mTOR 抑制剂能与 mTOR 的催化位点竞争 ATP, 高度选择性地抑制 mTORC1 和 mTORC2,比单靶点 mTOR 抑制剂具有更大的治疗优势。此外,某些天然来源产物也具有对 mTOR 的 抑制作用,且毒性、副作用更小。综述近几年有关 mTOR 及其抑制剂在抗肿瘤方面的研究进展。     

Histone deacetylase modulators provided by Mother Nature

Protein acetylation status results from a balance between histone acetyltransferase and histone deacetylase (HDAC) activities. Alteration of this balance leads to a disruption of cellular integrity and participates in the development of numerous diseases, including cancer. Therefore, modulation of these activities appears to be a promising approach for anticancer therapy. Histone deacetylase inhibitors (HDACi) are epigenetically active drugs that induce the hyperacetylation of lysine residues within histone and non-histone proteins, thus affecting gene expression and cellular processes such as protein–protein interactions, protein stability, DNA binding and protein sub-cellular localization. Therefore, HDACi are promising anti-tumor agents as they may affect the cell cycle, inhibit proliferation, stimulate differentiation and induce apoptotic cell death. Over the last 30 years, numerous synthetic and natural products, including a broad range of dietary compounds, have been identified as HDACi. This review focuses on molecules from natural origins modulating HDAC activities and presenting promising anticancer activities.     

A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells

Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.     

Inhibitors of histone deacetylase as antitumor agents: A critical review

Histone deacetylase (EC 3.5.1.98 – HDAC) is an amidohydrolase involved in deacetylating the histone lysine residues for chromatin remodeling and thus plays a vital role in the epigenetic regulation of gene expression. Due to its aberrant activity and over expression in several forms of cancer, HDAC is considered as a potential anticancer drug target. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as cell survival, differentiation and apoptosis in tumor cells and thus exhibit anticancer activity. Till date, four drugs, namely Vorinostat (SAHA), Romidepsin (FK-228), Belinostat (PXD-101) and Panobinostat (LBH-589) have been granted FDA approval for cancer and several HDAC inhibitors are currently in various phases of clinical trials, either as monotherapy and/or in combination with existing/novel anticancer agents. Regardless of this, today scientific efforts have fortified the quest for newer and novel HDAC inhibitors that show isoform selectivity. This review focuses on the chemistry of the molecules of two classes of HDAC inhibitors, namely short chain fatty acids and hydroxamic acids, investigated so far as novel therapeutic agents for cancer.     

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.     

Histone carbonylation occurs in proliferating cells

Chromatin is a dynamic structure formed mainly by DNA and histones, and chemical modifications on these elements regulate its compaction. Histone posttranslational modifications (PTMs) have a direct impact on chromatin conformation, controlling important cellular events such as cell proliferation and differentiation. Redox-related posttranslational modifications may have important effects on chromatin structure and function, offering a new intriguing area of research termed "redox epigenetics." Little is known about histone carbonylation, a PTM that may be related to modifications in the cellular redox environment. The aim of our study was to determine the carbonylation of the various histones during cell proliferation, a moment in cell life during which important redox changes take place. Here, we describe changes in histone carbonylation during cell proliferation in NIH3T3 fibroblasts. In addition, we have studied the variations of poly(ADP-ribosyl)ation and phospho-H2AX at the same time, because both modifications are related to DNA damage responses. High levels of carbonylation on specific histones (H1, H1(0), and H3.1 dimers) were found when cells were in an active phase of DNA synthesis. The modification decreased when nuclear proteasome activity was activated. However, these results did not correlate completely with poly(ADP-ribosyl)ation and phospho-H2AX levels. Therefore, histone carbonylation may represent a specific event during cell proliferation. We describe a new methodology named oxy-2D-TAU Western blot that allowed us to separate and analyze the carbonylation patterns of the histone variants. In addition we offer a new role for histone carbonylation and its implication in redox epigenetics. Our results suggest that histone carbonylation is involved in histone detoxification during DNA synthesis.     

Differentiation of eosinophilic leukemia EoL-1 cells into eosinophils induced by histone deacetylase inhibitors

EoL-1 cells differentiate into eosinophils in the presence of n-butyrate, but the mechanism has remained to be elucidated. Because n-butyrate can inhibit histone deacetylases, we hypothesized that the inhibition of histone deacetylases induces the differentiation of EoL-1 cells into eosinophils. In this study, using n-butyrate and two other histone deacetylase inhibitors, apicidin and trichostatin A, we have analyzed the relationship between the inhibition of histone deacetylases and the differentiation into eosinophils in EoL-1 cells. It was demonstrated that apicidin and n-butyrate induced a continuous acetylation of histones H4 and H3, inhibited the proliferation of EoL-1 cells without attenuating the level of FIP1L1-PDGFRA mRNA, and induced the expression of markers for mature eosinophils such as integrin beta7, CCR1, and CCR3 on EoL-1 cells, while trichostatin A evoked a transient acetylation of histones and induced no differentiation into eosinophils. These findings suggest that the continuous inhibition of histone deacetylases in EoL-1 cells induces the differentiation into mature eosinophils.     

Biotinyl-methyl 4-(amidomethyl)benzoate is a competitive inhibitor of human biotinidase

Posttranslational modification of histones by biotinylation can be catalyzed by both biotinidase (BTD) and holocarboxylase synthetase. Biotinylation of histones is an important epigenetic mechanism to regulate gene expression, DNA repair, and chromatin remodeling. The role of BTD in histone biotinylation is somewhat ambiguous, given that BTD also catalyzes removal of the biotin tag from histones. Here, we sought to develop BTD inhibitors for future studies of the role of BTD in altering chromatin structure. We adopted an existing colorimetric BTD assay for use in a novel 96-well plate format to permit high-throughput screening of potential inhibitors. Biotin analogs were chemically synthesized and tested for their ability to inhibit human BTD. Seven of these compounds inhibited BTD by 26–80%. Biotinyl-methyl 4-(amidomethyl)benzoate had the largest effect on BTD, causing an 80% inhibition at 1 mM concentration. Enzyme kinetics studies were conducted to determine V_max, K_m and K_i for the seven inhibitors; kinetics were consistent with the hypothesis that biotinyl-methyl 4-(amidomethyl)benzoate and the other compounds acted by competitive inhibition of BTD. Finally, biotinyl-methyl 4-(amidomethyl)benzoate did not affect biotin transport in human cells, suggesting specificity in regard to biotin-related processes.     

Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications.

The modification patterns of histones present in various forms of intracellular simian virus 40 nucleoprotein complexes were analyzed by acetic acid-urea-polyacrylamide gel electrophoresis. The results showed that different viral nucleoprotein complexes contain different histone patterns. Simian virus 40 chromatin, which contains the activities for the synthesis of viral RNA and DNA, exhibits a histone modification pattern similar to that of the host chromatin. However, virion assembly intermediates and mature virions contain highly modified histones. Pulse-chase experiments with [3H]lysine showed that the newly incorporated histones in the virion assembly intermediates were already highly modified. The majority of in vivo acetylation activity of histones occurred on the 70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate. These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into the mature virion: (i) modification of histones, (ii) accumulation of capsid protein around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion assembly is discussed.