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Ion exchange chromatographic studies of several stages of germination of maize embryo indicate the bonding between various forms of histone deacetylase (HD-1A, HD-1B and HD-2) and histone acetyltransferase (HAT-A1 and HAT-A2) enzymes. HD-1B and HD-2 are bound to the chromatin throughout germination, their ratio differing sometimes. Salt extraction leads to the release of HAT-A1 and HAT-A2, which are tightly bound to chromatin. Intranuclear location of these enzymes controls the nuclear processes involved in germination.

Analyzes the association of multiple enzyme forms of histone deactylase (HD) and histone acetyltransferase (HAT) to chromatin using ion exchange chromatography of subcellular fractions from different germination periods. Comparison of binding of HD forms; Comparison of binding of HAT forms; Analysis of nuclear matrix preparations.

We describe 1,3,4-oxadiazole-containing hydroxamates (2) and 2-aminoanilides (3) as histone deacetylase inhibitors. Among them, 2t, 2x, and 3i were the most potent and selective against HDAC1. In U937 leukemia cells, 2t was more potent than SAHA in inducing apoptosis, and 3i displayed cell differentiation with a potency similar to MS-275. In several acute myeloid leukemia (AML) cell lines, as well as in U937 cells in combination with doxorubicin, 3i showed higher antiproliferative effects than SAHA.

A number of new compounds bearing two ortho-bromo- and ortho, ortho-dibromophenol moieties linked through a saturated/unsaturated, linear/(poly)cyclic spacer (compounds 1- 9) were prepared as simplified analogues of AMI-5 (eosin), a recently reported inhibitor of both protein arginine and histone lysine methyltransferases (PRMTs and HKMTs). Such compounds were tested against a panel of PRMTs (RmtA, PRMT1, and CARM1) and against human SET7 (a HKMT), using histone and nonhistone proteins as a substrate. They were also screened against HAT and SIRTs, because they are structurally related to some HAT and/or SIRT modulators. From the inhibitory data, some of tested compounds ( 1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f) were able to inhibit PRMTs, HKMT, HAT, and SIRTs with similar potency, thus behaving as multiple ligands for these epigenetic targets (epi-MLs). When tested on the human leukemia U937 cell line, the epi-MLs induced high apoptosis levels [i.e., 40.7% ( 4l) and 42.6% ( 7b)] and/or massive, dose-dependent cytodifferentiation [i.e., 95.2% ( 1c) and 96.1% ( 4j)], whereas the single-target inhibitors eosin, curcumin, and sirtinol were ineffective or showed a weak effect.

Lysine and arginine methyltransferases participate in the post-translational modification of histones and regulate key cellular functions. So far only one arginine methyltransferase inhibitor discovered by random screening was available. We present the first target-based approach to protein arginine methyltransferase (PRMT) inhibitors. Homology models of human and Aspergillus nidulans PRMT1 were generated from available X-ray structures of rat PRMTs. The NCI diversity set was filtered by a target-based virtual screening to identify PRMT inhibitors. Employing a fungal PRMT for screening and a human enzyme for validation, we have identified seven inhibitors of PRMTs in vitro. Hit validation was achieved for two new inhibitors by antibody mediated detection of histone hypomethylation as well as Western blotting in cancer cells. Functional activity was proven by an observed block of estrogen receptor activation. Thus, valuable chemical tools and potential drug candidates could be identified.

The screening of the inhibition capabilities of dye-like small molecules from a focused library against both human PRMT1 and Aspergillus nidulans RmtA is reported as well as molecular modeling studies (homology modeling, molecular docking, and 3-D QSAR) of the catalytic domain of the PRMT1 fungal homologue RmtA. The good correlation between computational and biological results makes RmtA a reliable tool for screening arginine methyltransferase inhibitors. In addition, the binding mode analyses of tested derivatives reveal the crucial role of two regions, the pocket formed by Ile12, His13, Met16, and Thr49 and the SAM cisteinic binding site subsite. These regions should be taken into account in the design of novel PRMT inhibitors.

A novel series of compounds containing a uracil moiety as the connection unit between a phenyl/phenylalkyl portion and a N-hydroxy-polymethylenealkanamide or -methylenecinnamylamide group (uracil-based hydroxamic acids, UBHAs) was tested against maize histone deacetylases (HDACs) and mouse HDAC1. Compounds with a phenyl/benzyl ring at the uracil-C6 position and bearing 4-5 carbon units as well as a m- or p-methylenecinnamyl moiety as a spacer were the most potent inhibitors. In cell-based human HDAC1 and HDAC4 assays, the two UBHAs tested inhibited the HDAC1 but not HDAC4 immunoprecipitate activity. When tested in human leukemia U937 cells, some UBHAs produced G1 phase arrest of the cell cycle. Moreover, 1j showed high antiproliferative and dose-dependent granulocytic differentiation properties. The tested UBHAs displayed weak p21WAF1/CIP1 induction in U937 cells, and 1d and 1j showed high histone H3 and alpha-tubulin acetylation effects.

Chemical manipulations performed on aroyl-pyrrolyl-hydroxyamides (APHAs) led to (aryloxopropenyl)pyrrolyl hydroxamates 2a-w, and their inhibition against maize HDACs and their class I or class II HDAC selectivity were determined. In particular, from these studies some benzene meta-substituted compounds emerged as highly class II (IIa)-selective HDAC inhibitors, the most selective being the 3-chloro- and 3-fluoro-substituted compounds 2c (SI = 71.4) and2f (SI = 176.4). The replacement of benzene with a 1-naphthyl ring afforded 2s, highly active against the class II homologue HD1-A (IC(50) = 10 nM) but less class II-selective than 2c,f. When tested against human HDAC1 and HDAC4, 2f showed no inhibitory activity against HDAC1 but was able to inhibit HDAC4. Moreover, in human U937 acute myeloid leukaemia cells 2f did not produce any effect on apoptosis, granulocytic differentiation, and the cell cycle, whereas 2s (that retain class I HDAC inhibitory activity) was 2-fold less potent than SAHA used as reference.

We have studied enzymes involved in histone arginine methylation in the filamentous fungus Aspergillus nidulans. Three distinct protein arginine methyltransferases (PRMTs) could be identified, which all exhibit intrinsic histone methyltransferase activity when expressed as glutathione S-transferase (GST) fusion proteins. Two of these proteins, termed RmtA (arginine methyltransferase A) and RmtC, reveal significant sequence homology to the well-characterized human proteins PRMT1 and PRMT5, respectively. Native as well as recombinant RmtA is specific for histone H4 with arginine 3 as the methylation site. Furthermore, methylation of histone H4 by recombinant RmtA affects the acetylation by p300/CBP, supporting an interrelation of histone methylation and acetylation in transcriptional regulation. The second methyltransferase, named RmtB, is only distantly related to human/rat PRMT3 and must be considered as a member of a separate group within the PRMT family. The 61 kDa protein, expressed as a GST fusion protein, exhibits a unique substrate specificity in catalyzing the methylation of histones H4, H3, and H2A. Unlike human PRMT3, the Aspergillus enzyme lacks a Zn-finger domain in the amino-terminal part indicating functional differences of RmtB. Furthermore, phylogenetic analysis indicated that RmtB together with other fungal homologues is a member of a separate group within the PRMT proteins. The existence of in vivo arginine methylation on histones as demonstrated by site-specific antibodies and the high level and specificity of PRMTs for individual core histones in A. nidulans suggests an important role of these enzymes for chromatin modulating activities.

Aroyl-pyrrole-hydroxy-amides (APHAs) are a new class of synthetic HDAC inhibitors recently described by us. Through three different docking procedures we designed, synthesized, and tested two new isomers of APHA lead compound 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1), compounds 3 and 4, characterized by different insertions of benzoyl and propenoylhydroxamate groups onto the pyrrole ring. Biological activities of 3 and 4 were predicted by computational tools up to 617-fold more potent than that of 1 against HDAC1; thus, 3 and 4 were synthesized and tested against both mouse HDAC1 and maize HD2 enzymes. Predictions of biological affinities (K(i) values) of 3 and 4, performed by a VALIDATE model (applied on either SAD or automatic DOCK or Autodock results) and by the Autodock internal scoring function, were in good agreement with experimental activities. Ligand/receptor positive interactions made by 3 and 4 into the catalytic pocket, in addition to those showed by 1, could at least in part account for their higher HDAC1 inhibitory activities. In particular, in mouse HDAC1 inhibitory assay 3 and 4 were 19- and 6-times more potent than 1, respectively, and 3 and 4 antimaize HD2 activities were 16- and 76-times higher than that of 1, 4 being as potent as SAHA in this assay. Compound 4, tested as antiproliferative and cytodifferentiating agent on MEL cells, showed dose-dependent growth inhibition and hemoglobin accumulation effects.

Previous SAR studies (Part 1: Mai, A.; et al. J. Med. Chem. 2003, 46, 512-524) performed on some portions (pyrrole-C4, pyrrole-N1, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1a) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl (1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the present paper, we investigated the effect on anti-HD2 activity of chemical substitutions performed on the pyrrole-C2 ethene chains of 1a-c, which were replaced with methylene, ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl moiety into the 1a chemical structure (IC50(2f) = 0.77 microM; IC50(1a) = 3.8 microM). IC50 values of compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups at the pyrrole-C(4) position a hydrocarbon spacer length ranging from two to five methylenes is well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes) are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives. Particularly, 3a (IC50 = 0.043 microM) showed the same potency as SAHA in inhibiting HD2 in vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was 4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information potentially useful for a future 3D-QSAR study, showed the same (4a,b) or higher (4c,d) HD2 inhibiting activities in comparison with those of the reference drugs. Molecular modeling and docking calculations on the designed compounds performed in parallel with the chemistry work fully supported the synthetic effort and gave insights into the binding mode of the more flexible APHA derivatives (i.e., 3a). Despite the difference of potency between 1b and 3a in the enzyme assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the differentiation assay only at the highest tested dose (48 microM).

Chemical manipulations performed on aroyl pyrrolyl hydroxyamides, a new class of HDAC inhibitors previously reported by us, led to (aryloxopropenyl)pyrrolyl hydroxyamides 3a-g. Such compounds, showing better inhibitory activity against maize HD1-A than HD1-B (two homologues of mammalian class IIa and I HDACs, respectively), are the first class of IIa-selective inhibitors (fold selectivity: 7-78). They could be useful as tools for probing the biology of these enzymes and eventually as new anticancer agents with low toxicity.

Recently we reported a novel series of hydroxamates, called 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides (APHAs), acting as HDAC inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069-2072). Among them, 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide 1 was chosen as lead compound, and its binding mode into the modeled HDAC1 catalytic core together with its histone hyperacetylation, antiproliferative, and cytodifferentiating properties in cell-based assays were investigated (Mai, A.; et al. J. Med. Chem. 2002, 45, 1778-1784). Here we report the results of some chemical manipulations performed on (i) the aroyl portion at the C4-pyrrole position, (ii) the N(1)-pyrrole substituent, and (iii) the hydroxamate moiety of 1 to determine structure-activity relationships and to improve enzyme inhibitory activity of APHAs. In the 1 structure, pyrrole N(1)-substitution with groups larger than methyl gave a reduction in HDAC inhibiting activity, and replacement of hydroxamate function with various non-hydroxamate, metal ion-complexing groups yielded poorly active or totally inactive compounds. On the contrary, proper substitution at the C4-position favorably affected enzyme inhibiting potency, leading to 8 (IC50 = 0.1 micro M) and 9 (IC50 = 1.0 micro M) which were 38- and 3.8-fold more potent than 1 in in vitro anti-HD2 assay. Against mouse HDAC1, 8 showed an IC50 = 0.5 micro M (IC50 of 1 = 4.9 micro M), and also in cell-based assay, 8 was endowed with higher histone hyperacetylating activity than 1, although it was less potent than TSA and SAHA. Such enhancement of inhibitory activity can be explained by the higher flexibility of the pyrrole C4-substituent of 8 which accounts for a considerably better fitting into the HDAC1 pocket and a more favorable enthalpy ligand receptor energy compared to 1. The enhanced fit allows a closer positioning of 8 hydroxamate moiety to the zinc ion. These findings were supported by extensive docking studies (SAD, DOCK, and Autodock) performed on both APHAs and reference drugs (TSA and SAHA).

Inhibitors of histone deacetylases (HDACs) are a new class of anticancer agents that affect gene regulation. We had previously reported the first simple synthetic HDAC inhibitors with in vitro activity at submicromolar concentrations. Here, we present structure-activity data on modifications of a phenylalanine-containing lead compound including amino acid amides as well as variations of the amino acid part. The compounds were tested for inhibition of maize HD-2, rat liver HDAC, and for the induction of terminal cell differentiation and inhibition of proliferation in Friend leukemic cells. In the amide series, in vitro inhibition was potentiated up to 15-fold, but the potential to induce cell differentiation decreased. Interestingly, an HDAC class selectivity was indicated among some of these amides. In the amino acid methyl ester series, a biphenylalanine derivative was identified as a good enzyme inhibitor, which blocks proliferation in the submicromolar range and is also a potent inducer of terminal cell differentiation.

The binding mode of 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides 1a-c, belonging to a recently reported class of synthetic histone deacetylase (HDAC) inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069-2072), into the new modeled HDAC1 catalytic core is presented, and enzyme/inhibitor interactions are discussed. HDAC1 X-ray coordinates were obtained by virtual "mutation" of those of histone deacetylase-like protein, a bacterial HDAC homologue. In in vitro antimaize HD2 as well as antimouse HDAC1 assay, compounds 1a-c showed inhibitory activities in the low micromolar range. Similarly, 1a-c are endowed with anti-HDAC activity in vivo: on mouse A20 cells, 1a-c induced histone hyperacetylation leading to a highly increased acetylation level of H4 as compared to control histones. Results obtained with acid-urea-triton polyacrylamide gel electrophoresis have been confirmed by Western Blot experiments. Finally, compound 1a, chosen as a representative member of this class of HDAC inhibitors, resulted endowed with antiproliferative (45 and 85% cell growth inhibition at 40 and 80 microM, respectively) and cellular differentiation (18 and 21% of benzidine positive cells at the same concentrations) activities in murine erythroleukemic cells.

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.

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.

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.

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.

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.

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.