Synthesis and biological evaluation of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides as novel synthetic HDAC inhibitors.

A new series of 2-, 3-, and 4-acylaminocinnamyl-N-hydroxyamides 1-3 have been prepared, and their anti-HDAC (against maize HD2, HD1-B, and HD1-A enzymes) activities have been assessed. Cinnamyl-hydroxyamides bearing acylamino substituents at the C2 position of the benzene ring (compounds 1a-g) showed very low HDAC inhibiting activities, with IC(50) values in the high micromolar range. By shifting the same acylamino groups from C2 to C3 (compounds 2a-g) as well as C4 (compounds 3a-f) position of the benzene ring, a number of highly potent HDAC inhibitors have been obtained. In the anti-HD2 assay 3c (IC(50) = 11 nM) was the most potent compound, being >11600-, 4.5-, and 10-fold more potent than sodium valproate, SAHA, and HC-toxin, respectively, and showing the same activity as trapoxin. HD1-B and HD1-A assays have been performed to screen the inhibitory action of 1-3 against mammalian class I (HD1-B) and class II (HD1-A) HDAC homologous enzymes. From the corresponding IC(50) data, a selectivity ratio has been calculated. In general, compounds 1-3 showed no or little selectivity towards the class II homologue HD1-A, the most selective being 2a with class II selectivity ratio = 4.3. About the inhibitory potency, the 4-(2-naphthoylamino)cinnamyl-N-hydroxyamide 3f showed the highest inhibiting effect against the two enzymes (IC(50-HD1-B) = 36 nM; IC(50-HD1-A) = 42 nM). Selected 2 and 3 compounds will be evaluated to determine their antiproliferative and cyto differentiating activities on HL-60 cells.

An inhibitor-resistant histone deacetylase in the plant pathogenic fungus Cochliobolus carbonum.

We have partially purified and characterized histone deacetylases of the plant pathogenic fungus Cochliobolus carbonum. Depending on growth conditions, this fungus produces HC-toxin, a specific histone deacetylase inhibitor. Purified enzymes were analyzed by immunoblotting, by immunoprecipitation, and for toxin sensitivity. The results demonstrate the existence of at least two distinct histone deacetylase activities. A high molecular weight complex (430,000) is sensitive to HC-toxin and trichostatin A and shows immunoreactivity with an antibody against Cochliobolus HDC2, an enzyme homologous to yeast RPD3. The second activity, a 60,000 molecular weight protein, which is resistant even to high concentrations of well-known deacetylase inhibitors, such as HC-toxin and trichostatin A, is not recognized by antibodies against Cochliobolus HDC1 (homologous to yeast HOS2) or HDC2 and represents a different and/or modified histone deacetylase which is enzymatically active in its monomeric form. This enzyme activity is not present in the related filamentous fungus Aspergillus nidulans. Furthermore, in vivo treatment of Cochliobolus mycelia with trichostatin A and analysis of HDACs during the transition from non-toxin-producing to toxin-producing stages support an HC-toxin-dependent enzyme activity profile.

Histone acetylation: plants and fungi as model systems for the investigation of histone deacetylases.

The basic element of chromatin is the nucleosome. Histones H4, H3, H2A and H2B form the core histone octamer by protein-protein interactions of their folded domains. The free, flexible N-terminal extensions of the histones protrude from the nuclesome; they contain conserved lysines undergoing posttranslational acetylation. Histone acetyltransferases (HATs) transfer the acetyl moiety of acetyl-coenzyme A to the epsilon-amino group; this reaction is reverted by histone deacetylases (HDACs). The dynamic equilibrium of the acetylation/deacetylation reaction varies throughout the genome; some regions in chromatin undergo rapid acetylation/deacetylation, whereas others are fixed in a certain acetylation state without significant changes. In general, chromatin regions engaged in transcription display dynamic acetylation, i.e. HATs and HDACs are recruited to these regions. Higher plants and fungi have considerably contributed to the unraveling of the multiplicity of HDACs; in particular, plants possess HDACs that have so far not been identified in animal cells.

A gene related to yeast HOS2 histone deacetylase affects extracellular depolymerase expression and virulence in a plant pathogenic fungus.

A gene, HDC1, related to the Saccharomyces cerevisiae histone deacetylase (HDAC) gene HOS2, was isolated from the filamentous fungus Cochliobolus carbonum, a pathogen of maize that makes the HDAC inhibitor HC-toxin. Engineered mutants of HDC1 had smaller and less septate conidia and exhibited an approximately 50% reduction in total HDAC activity. Mutants were strongly reduced in virulence as a result of reduced penetration efficiency. Growth of hdc1 mutants in vitro was normal on glucose, slightly decreased on sucrose, and reduced by 30 to 73% on other simple and complex carbohydrates. Extracellular depolymerase activities and expression of the corresponding genes were downregulated in hdc1 mutant strains. Except for altered conidial morphology, the phenotypes of hdc1 mutants were similar to those of C. carbonum strains mutated in ccSNF1 encoding a protein kinase necessary for expression of glucose-repressed genes. These results show that HDC1 has multiple functions in a filamentous fungus and is required for full virulence of C. carbonum on maize.

Comparative analysis of HD2 type histone deacetylases in higher plants.

Zea mays (L.) histone deacetylase HD2 was identified as a new type of histone deacetylase (HDAC) unrelated to the well-known Rpd3p and Hdalp families but with sequence homology to peptidyl-prolyl cis-trans isomerases (PPIases). Here we show that HD2 is a multigene family with highly related members in various plant species. Gene analysis revealed a similar exon/intron structure in Arabidopsis thaliana (L.) Heynh. and Z. mays, and most of the sequences analyzed were demonstrated to possess an intron of the very rare AT-AC type.

Histone deacetylases in replicative senescence: evidence for a senescence-specific form of HDAC-2.

To analyze mechanisms of senescence-associated gene expression, we have investigated histone deacetylases (HDACs) in human fibroblasts undergoing replicative senescence. We found that the overall acetylation pattern of histones does not vary detectably with replicative senescence. By Northern blot and Western blot, we found a significant decrease in the abundance of HDAC-1 in senescent cells. Biochemical analysis of deacetylase activities in extracts from old and young cells revealed a striking difference. While by anion exchange chromatography we found a single peak of activity in extracts from young cells, which coincided with the elution of both HDAC-1 and HDAC-2, in senescent cells a second peak of activity was found. This second peak of activity is associated with HDAC-2 but does not contain HDAC-1. These results suggest that HDAC-2 is present in at least two distinct forms, one of which is specific for senescent cells. Further biochemical characterization of the enzyme activity revealed that addition of nicotinamide adenine dinucleotide (NAD) did not detectably influence the activity of any fraction, suggesting that NAD is not an essential co-factor for the analyzed HDACs from diploid human fibroblasts.

3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides, a new class of synthetic histone deacetylase inhibitors.

Novel 3-(4-aroyl-2-pyrrolyl)-N-hydroxy-2-propenamides are disclosed as a new class of histone deacetylase (HDAC) inhibitors. Three-dimensional structure-based drug design and conformational analyses into the histone deacetylase-like protein (HDLP) catalytic core suggested the synthesis and biological evaluation of compounds 7a-h. Experimental pK(i) values are in good agreement with VALIDATE predicted pK(i) values of new derivatives. All compounds 7a-h show HDAC inhibitory activity in the micromolar range, with 7e as the most potent derivative (IC(50) = 1.9 microM). The influence of the 4'-substituent in the aroyl moiety is not significant for the inhibitory activity, as all compounds 7a-g show IC(50) values between 1.9 and 3.9 microM. Otherwise, the unsaturated chain linking the pyrrole ring to the hydroxamic acid group is clearly important for the anti-HDAC activity, the saturated analogue 7h being 10-fold less active than the unsaturated counterpart 7a.

Sds3 (suppressor of defective silencing 3) is an integral component of the yeast Sin3[middle dot]Rpd3 histone deacetylase complex and is required for histone deacetylase activity.

SDS3 (suppressor of defective silencing 3) was originally identified in a screen for mutations that cause increased silencing of a crippled HMR silencer in a rap1 mutant background. In addition, sds3 mutants have phenotypes very similar to those seen in sin3 and rpd3 mutants, suggesting that it functions in the same genetic pathway. In this manuscript we demonstrate that Sds3p is an integral subunit of a previously identified high molecular weight Rpd3p.Sin3p containing yeast histone deacetylase complex. By analyzing an sds3Delta strain we show that, in the absence of Sds3p, Sin3p can be chromatographically separated from Rpd3p, indicating that Sds3p promotes the integrity of the complex. Moreover, the remaining Rpd3p complex in the sds3Delta strain had little or no histone deacetylase activity. Thus, Sds3p plays important roles in the integrity and catalytic activity of the Rpd3p.Sin3p complex.

First non-radioactive assay for in vitro screening of histone deacetylase inhibitors.

Inhibitors of histone deacetylase (HD) are of great potential as new drugs due to their ability to influence transcriptional regulation and to induce apoptosis or differentiation in cancer cells. So far only radioactive enzyme activity assays or in-vivo assays with subsequent electrophoresis and immunoblotting existed to study the activity of HD and potential inhibitors. To aid in the search of new inhibitors, a non-radioactive screening assay was sought and we have previously succeeded in establishing this for the first time. The assay uses an aminocoumarin derivative of an omega-acetylated lysine as substrate for the enzyme. Here we report full experimental details, the evaluation of other potential substrates, and comparative analysis of various inhibitors. This advantageous method should have an impact on further developments in the field.

Characterization of two putative histone deacetylase genes from Aspergillus nidulans.

In eukaryotic organisms, acetylation of core histones plays a key role in the regulation of transcription. Multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain a dynamic equilibrium of histone acetylation. The latter form a highly conserved protein family in many eukaryotic species. In this paper, we report the cloning and sequencing of two putative histone deacetylase genes (rpdA, hosA) of Aspergillus nidulans, which are the first to be analyzed from filamentous fungi. Hybridization with a chromosome-specific cosmid library of A. nidulans allowed the localization of rpdA to chromosome III and hosA to chromosome II, respectively. PCR analyses and Southern hybridization experiments revealed that no further members of the RPD3 family are present in the genome of the fungus. Although sequence alignment displays significant amino acid similarity to other eukaryotic RPD3-type deacetylases, the deduced RPDA sequence reveals an unusual 200-amino acid extension at the C-terminus. Expression of both genes was determined by RNA blot analysis. Treatment of the cells with trichostatin A (TSA), a potent inhibitor of HDACs, was found to stimulate expression of rpdA of A. nidulans.

RPD3-type histone deacetylases in maize embryos.

Posttranslational core histone acetylation is established and maintained by histone acetyltransferases and deacetylases. Both have been identified as important transcriptional regulators in various eukaryotic systems. In contrast to nonplant systems where only RPD3-related histone deacetylases (HD) have been characterized so far, maize embryos contain three unrelated families of deacetylases (HD1A, HD1B, and HD2). Purification, cDNA cloning, and immunological studies identified the two maize histone deacetylase HD1B forms as close homologues of the RPD3-type deacetylase HDAC1. Unlike the other maize deacetylases, HD1A and nucleolar HD2, HD1B copurified as a complex with a protein related to the retinoblastoma-associated protein, Rbap46. Two HD1B mRNA species could be detected on RNA blots, encoding proteins of 58 kDa (HD1B-I) and 51 kDa (HD1B-II). HD1B-I (zmRpd3) represents the major enzyme form as judged from RNA and immunoblots. Levels of expression of HD1B-I and -II mRNA differ during early embryo germination; HD1B-I mRNA and protein are present during the entire germination pathway, even in the quiescent embryo, whereas HD1B-II expression starts when meristematic cells enter S-phase of the cell cycle. In line with previous results, HD1B exists as soluble and chromatin-bound enzyme forms. In vivo treatment of meristematic tissue with the deacetylase inhibitor HC toxin does not affect the expression of the three maize histone deacetylases, whereas it causes downregulation of histone acetyltransferase B.

Amide analogues of trichostatin A as inhibitors of histone deacetylase and inducers of terminal cell differentiation.

Inhibitors of histone deacetylase (HD) bear great potential as new drugs due to their ability to modulate transcription and to induce apoptosis or differentiation in cancer cells. We have described previously analogues of the complex natural HD inhibitors trapoxin B and trichostatin A with activities in the submicromolar range. Here we report structure-activity relationship analyses of further analogues of trichostatin A with respect to in vitro inhibition of maize HD-2 and their ability to induce terminal cell differentiation in Friend leukemic cells. This is the first report that shows the correlation between HD inhibitory activity and action on cancer cells on a larger series of similar compounds. Only the compounds that inhibit HD induce differentiation and/or exert antiproliferative activities in cell culture. Our studies support the use of in vitro systems as screening tools and provide structure-activity relationships that merit further investigation of this interesting target.

Histone deacetylase 1 can repress transcription by binding to Sp1.

The members of the Sp1 transcription factor family can act as both negative and positive regulators of gene expression. Here we show that Sp1 can be a target for histone deacetylase 1 (HDAC1)-mediated transcriptional repression. The histone deacetylase inhibitor trichostatin A activates the chromosomally integrated murine thymidine kinase promoter in an Sp1-dependent manner. Coimmunoprecipitation experiments with Swiss 3T3 fibroblasts and 293 cells demonstrate that Sp1 and HDAC1 can be part of the same complex. The interaction between Sp1 and HDAC1 is direct and requires the carboxy-terminal domain of Sp1. Previously we have shown that the C terminus of Sp1 is necessary for the interaction with the transcription factor E2F1 (J. Karlseder, H. Rotheneder, and E. Wintersberger, Mol. Cell. Biol. 16:1659-1667, 1996). Coexpression of E2F1 interferes with HDAC1 binding to Sp1 and abolishes Sp1-mediated transcriptional repression. Our results indicate that one component of Sp1-dependent gene regulation involves competition between the transcriptional repressor HDAC1 and the transactivating factor E2F1.

Different types of maize histone deacetylases are distinguished by a highly complex substrate and site specificity.

Enzymes involved in histone acetylation have been identified as important transcriptional regulators. Maize embryos contain three histone deacetylase families: RPD3-type deacetylases (HD1-B), nucleolar phosphoproteins of the HD2 family, and a third form unrelated to RPD3 and HD2 (HD1-A). Here we first report on the specificity of deacetylases for core histones, acetylated histone H4 subspecies, and acetylated H4-lysine residues. HD1-A, HD1-B, and HD2 deacetylate all four core histones, although with different specificity. However, experiments with histones from different sources (hyperacetylated MELC and chicken histones) using antibodies specific for individually acetylated H4-lysine sites indicate that the enzymes recognize highly distinct acetylation patterns. Only RPD3-type deacetylase HD1-B is able to deacetylate the specific H4 di-acetylation pattern (position 12 and 5) introduced by the purified cytoplasmic histone acetyltransferase B after incubation with pure nonacetylated H4 subspecies. HD1-A and HD2 exist as phosphorylated forms. Dephosphorylation has dramatic, but opposite effects; whereas HD2 loses enzymatic activity upon dephosphorylation, HD1-A is activated with a change of specificity against acetylated H4 subspecies. The data suggest that different types of deacetylases interact with different and highly specific acetylation patterns on nucleosomes.

A non-isotopic assay for histone deacetylase activity.

Inhibitors of histone deacetylase (HD) bear great potential as new drugs due to their ability to modulate transcription and to induce apoptosis or differentiation in cancer cells. To study the activity of HD and the effect of potential inhibitors in vitro so far only radio-active assays have existed. For the search of new inhibitors and for the use in HD identification and purification we established a simple, non-radioactive assay that allows screening of large numbers of compounds. The assay is based on an aminocoumarin derivative of an Omega-acetylated lysine as enzyme substrate.

Biochemical methods for analysis of histone deacetylases.

Specific lysine residues in the N-terminal extensions of core histones can be posttranslationally modified by acetylation of the epsilon-amino group. The dynamic equilibrium of core histone acetylation is established and maintained by histone acetyltransferases and deacetylases. Both enzymes exist as multiple enzyme forms. Histone acetyltransferases and deacetylases have recently been identified as transcriptional regulators as well as nucleolar phosphoproteins, and have therefore attracted considerable research interest. Analysis of the functional significance of histone deacetylases for nuclear processes in certain cases demands the separation and biochemical analysis of different members of the histone deacetylase families. We have characterized three different histones deacetylases in maize embryos and subsequently purified these enzymes to homogeneity. Here we describe methods for extraction, enzymatic assay, chromatographic and electrophoretic separation, and purification of deacetylases. A novel one-step procedure for large-scale preparation of individual histones and their acetylated isoforms for the analysis of substrate and site specificity of the enzymes is presented.

The thyroid hormone receptor functions as a ligand-operated developmental switch between proliferation and differentiation of erythroid progenitors.

The avian erythroblastosis virus (AEV) oncoprotein v-ErbA represents a mutated, oncogenic thyroid hormone receptor alpha (c-ErbA/ TRalpha). v-ErbA cooperates with the stem cell factor-activated, endogenous receptor tyrosine kinase c-Kit to induce self-renewal and to arrest differentiation of primary avian erythroblasts, the AEV transformation target cells. In this cooperation, v-ErbA substitutes for endogenous steroid hormone receptor function required for sustained proliferation of non-transformed erythroid progenitors. In this paper, we propose a novel concept of how v-ErbA transforms erythroblasts. Using culture media strictly depleted from thyroid hormone (T3) and retinoids, the ligands for c-ErbA/TRalpha and its co-receptor RXR, we show that overexpressed, unliganded c-ErbA/ TRalpha closely resembles v-ErbA in its activity on primary erythroblasts. In cooperation with ligand-activated c-Kit, c-ErbA/ TRalpha causes steroid-independent, long-term proliferation and tightly blocks differentiation. Activation of c-ErbA/ TRalpha by physiological T3 levels causes the loss of self-renewal capacity and induces synchronous, terminal differentiation under otherwise identical conditions. This T3-induced switch in erythroid progenitor development is correlated with a decrease of c-ErbA-associated histone deacetylase activity. Our results suggest that the crucial role of the mutations activating v-erbA as an oncogene is to 'freeze' c-ErbA/ TRalpha in its non-liganded, repressive conformation and to facilitate its overexpression.

Histone acetyltransferases during the cell cycle and differentiation of Physarum polycephalum.

The dynamic state of histone acetylation is maintained by histone acetyltransferases (HATs) and deacetylases. Cellular fractionation of plasmodia of Physarum polycephalum and partial purification of subcellular fractions by chromatography revealed the existence of a cytoplasmic B-type and four nuclear A-type HATs. The cytoplasmic B-enzyme was highly specific for histone H4, causing di-acetylation of H4 in vitro. The nuclear enzymes (HAT-A1 to HAT-A4) accepted all core histones as substrates, but differed by the preference for certain histone species. Enzymes were analyzed during the naturally synchronous cell cycle of macroplasmodia. Each of the enzymes had its individual cell cycle activity pattern, indicating diverse functions in nuclear metabolism. When growing plasmodia were induced to undergo differentiation into dormant sclerotia, an additional enzyme (HAT-AS) appeared at a late stage of sclerotization which correlated with differentiation-specific histone synthesis and acetylation in the absence of DNA replication. When dormant sclerotia were induced to reenter the cell cycle, a further enzyme form (HAT-AG) appeared during a short time period prior to the first post-germination mitosis. This enzyme had a strong preference for H2B, correlating with the overproportional in vivo acetate incorporation in H2B. Both differentiation-associated HATs were undetectable in growing plasmodia. The results demonstrate that different functions of core histone acetylation are based on multiple enzyme forms that are independently regulated during the cell cycle. Transitions from one developmental stage into another are accompanied by specific enzyme forms. With respect to recent data in the literature it may be assumed that these HAT-forms are subunits of a HAT-complex whose composition changes during the cell cycle and differentiation.

Identification of maize histone deacetylase HD2 as an acidic nucleolar phosphoprotein.

The steady state of histone acetylation is established and maintained by multiple histone acetyltransferases and deacetylases, and this steady state affects chromatin structure and function. The identification of a maize complementary DNA encoding the chromatin-bound deacetylase HD2 is reported. This protein was not homologous to the yeast RPD3 transcriptional regulator. It was expressed throughout embryo germination in correlation with the proliferative activity of cells. Antibodies against recombinant HD2-p39 immunoprecipitated the native enzyme complex, which was composed of phosphorylated p39 subunits. Immunofluorescence microscopy and sequence homologies suggested nucleolar localization. HD2 is an acidic nucleolar phosphoprotein that might regulate ribosomal chromatin structure and function.

Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos.

The dynamic state of core histone acetylation is maintained by histone acetyltransferases and deacetylases. In germinating maize embryos, four nuclear histone deacetylases can be distinguished. From a chromatin fraction prepared at 72 h after start of embryo germination, we have purified the nuclear histone deacetylase HD2 to homogeneity. Using a sequence of chromatographic steps, we achieved the purification of an enzymatically active high molecular weight protein complex with an apparent molecular mass of 400 kDa, as determined by gel filtration chromatography. The purified enzyme was characterized in terms of enzymatic and kinetic properties, and sensitivity to several histone deacetylase inhibitors. In SDS-polyacrylamide gels, HD2 split into three polypeptides of 45, 42, and 39 kDa, suggesting that the native enzyme is a multimer-protein complex. Electrophoresis under nondenaturing conditions in combination with second dimension SDS-gel electrophoresis indicated that all three protein components of the HD2 complex were enzymatically active. Polyclonal antibodies against each of the three polypeptides were raised in rabbits. Each antiserum reacted with all three polypeptides on Western blots, suggesting that p45, p42, and p39 are highly homologous. This homology was confirmed by amino acid sequencing of peptides generated from each of the three HD2 components.