A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes.

Novel Pyridine-Based Hydroxamates and 2'-Aminoanilides as Histone Deacetylase Inhibitors: Biochemical Profile and Anticancer Activity.

Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino)phenyl)acrylamide (5 b) previously described by us as a HDAC inhibitor, we prepared four aza-analogues, 6-8, 9 b, as regioisomers containing the pyridine nucleus. Preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide (9 b) as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates (9 a, 9 c-f, and 11 a-f) and 2'-aminoanilides (10 a-f and 12 a-f), related to 9 b, to be tested against HDACs.

RcLS2F - A Novel Fungal Class 1 KDAC Co-repressor Complex in .

The fungal class 1 lysine deacetylase (KDAC) RpdA is a promising target for prevention and treatment of invasive fungal infection. RpdA is essential for survival of the most common air-borne mold pathogen and the model organism . In , RpdA depletion induced production of previously unknown small bioactive substances.

Type I and II PRMTs regulate catabolic as well as detoxifying processes in Aspergillus nidulans.

In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs). In Aspergillus nidulans, three conserved type I and II PRMTs are present that catalyze asymmetric or symmetric dimethylation of arginines.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria.

A Class 1 Histone Deacetylase as Major Regulator of Secondary Metabolite Production in .

An outstanding feature of filamentous fungi is their ability to produce a wide variety of small bioactive molecules that contribute to their survival, fitness, and pathogenicity. The vast collection of these so-called secondary metabolites (SMs) includes molecules that play a role in virulence, protect fungi from environmental damage, act as toxins or antibiotics that harm host tissues, or hinder microbial competitors for food sources. Many of these compounds are used in medical treatment; however, biosynthetic genes for the production of these natural products are arranged in compact clusters that are commonly silent under growth conditions routinely used in laboratories.

A Class 1 Histone Deacetylase with Potential as an Antifungal Target.

Unlabelled: Histone deacetylases (HDACs) remove acetyl moieties from lysine residues at histone tails and nuclear regulatory proteins and thus significantly impact chromatin remodeling and transcriptional regulation in eukaryotes. In recent years, HDACs of filamentous fungi were found to be decisive regulators of genes involved in pathogenicity and the production of important fungal metabolites such as antibiotics and toxins. Here we present proof that one of these enzymes, the class 1 type HDAC RpdA, is of vital importance for the opportunistic human pathogen Aspergillus fumigatus Recombinant expression of inactivated RpdA shows that loss of catalytic activity is responsible for the lethal phenotype of Aspergillus RpdA null mutants.

2-Benzazolyl-4-Piperazin-1-Ylsulfonylbenzenecarbohydroxamic Acids as Novel Selective Histone Deacetylase-6 Inhibitors with Antiproliferative Activity.

We have screened our compound collection in an established cell based assay that measures the derepression of an epigenetically silenced transgene, the locus derepression assay. The screen led to the identification of 4-[4-(1-methylbenzimidazol-2-yl)piperazin-1-yl]sulfonylbenzenecarbohydroxamic acid (9b) as an active which was found to inhibit HDAC1. In initial structure activity relationships study, the 1-methylbenzimidazole ring was replaced by the isosteric heterocycles benzimidazole, benzoxazole, and benzothiazole and the position of the hydroxamic acid substituent on the phenyl ring was varied.

AmcA-a putative mitochondrial ornithine transporter supporting fungal siderophore biosynthesis.

Iron is an essential nutrient required for a wide range of cellular processes. The opportunistic fungal pathogen Aspergillus fumigatus employs low-molecular mass iron-specific chelators, termed siderophores, for uptake, storage and intracellular iron distribution, which play a crucial role in the pathogenicity of this fungus. Siderophore biosynthesis (SB) depends on coordination with the supply of its precursor ornithine, produced mitochondrially from glutamate or cytosolically via hydrolysis of arginine.

1,3,4-Oxadiazole-containing histone deacetylase inhibitors: anticancer activities in cancer cells.

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.

A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog.

The histone deacetylases HDAC1 and HDAC2 are crucial regulators of chromatin structure and gene expression, thereby controlling important developmental processes. In the mouse brain, HDAC1 and HDAC2 exhibit different developmental stage- and lineage-specific expression patterns. To examine the individual contribution of these deacetylases during brain development, we deleted different combinations of Hdac1 and Hdac2 alleles in neural cells.

Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis.

The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue.

Novel cinnamyl hydroxyamides and 2-aminoanilides as histone deacetylase inhibitors: apoptotic induction and cytodifferentiation activity.

Four novel series of cinnamyl-containing histone deacetylase (HDAC) inhibitors 1-4 are described, containing hydroxamate (1 and 3) or 2-aminoanilide (2 and 4) derivatives. When screened against class I (maize HD1-B and human HDAC1) and class II (maize HD1-A and human HDAC4) HDACs, most hydroxamates and 2-aminoanilides displayed potent and selective inhibition toward class I enzymes. Immunoblotting analyses performed in U937 leukemia cells generally revealed high acetyl-H3 and low acetyl-α-tubulin levels.

Novel insights into the functional role of three protein arginine methyltransferases in Aspergillus nidulans.

Protein arginine methylation has been implicated in different cellular processes including transcriptional regulation by the modification of histone proteins. Here we demonstrate significant in vitro activities and multifaceted specificities of Aspergillus protein arginine methyltransferases (PRMTs) and we provide evidence for a role of protein methylation in mechanisms of oxidative stress response. We have isolated all three Aspergillus PRMTs from fungal extracts and could assign significant histone specificity to RmtA and RmtC.

Design, synthesis and biological evaluation of carboxy analogues of arginine methyltransferase inhibitor 1 (AMI-1).

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI-1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis-amidic moiety; 3) the introduction of a N-containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates.

A novel motif in fungal class 1 histone deacetylases is essential for growth and development of Aspergillus.

Acetylation of the N-terminal tails of core histones is an important regulatory mechanism in eukaryotic organisms. In filamentous fungi, little is known about the enzymes that modify histone tails. However, it is increasingly evident that histone deacetylases and histone acetyltransferases are critical factors for the regulation of genes involved in fungal pathogenicity, stress response, and production of secondary metabolites such as antibiotics or fungal toxins.

New pyrrole-based histone deacetylase inhibitors: binding mode, enzyme- and cell-based investigations.

Aroyl-pyrrolyl-hydroxy-amides (APHAs) are a class of synthetic HDAC inhibitors described by us since 2001. Through structure-based drug design, two isomers of the APHA lead compound 1, the 3-(2-benzoyl-1-methyl-1H-pyrrol-4-yl)-N-hydroxy-2-propenamide 2 and the 3-(2-benzoyl-1-methyl-1H-pyrrol-5-yl)-N-hydroxy-2-propenamide 3 (iso-APHAs) were designed, synthesized and tested in murine leukemia cells as antiproliferative and cytodifferentiating agents. To improve their HDAC activity and selectivity, chemical modifications at the benzoyl moieties were investigated and evaluated using three maize histone deacetylases: HD2, HD1-B (class I human HDAC homologue), and HD1-A (class II human HDAC homologue).

epigenetic multiple ligands: mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (sirtuin) inhibitors.

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.

Histone modifications and chromatin dynamics: a focus on filamentous fungi.

The readout of the genetic information of eukaryotic organisms is significantly regulated by modifications of DNA and chromatin proteins. Chromatin alterations induce genome-wide and local changes in gene expression and affect a variety of processes in response to internal and external signals during growth, differentiation, development, in metabolic processes, diseases, and abiotic and biotic stresses. This review aims at summarizing the roles of histone H1 and the acetylation and methylation of histones in filamentous fungi and links this knowledge to the huge body of data from other systems.

Class II-selective histone deacetylase inhibitors. Part 2: alignment-independent GRIND 3-D QSAR, homology and docking studies.

(Aryloxopropenyl)pyrrolyl hydroxamates were recently reported by us as first examples of class II-selective HDAC inhibitors and can be useful tools to probe the biology of such enzymes. Molecular modelling and 3-D QSAR studies have been performed on a series of 25 (aryloxopropenyl)pyrrolyl hydroxamates to gain insights about their activity and selectivity against both maize HD1-B and HD1-A, two enzymes homologous of mammalian class I and class II HDACs, respectively. The studies have been accomplished by calculating alignment-independent descriptors (GRIND descriptors) using the ALMOND software.

Identification of two new synthetic histone deacetylase inhibitors that modulate globin gene expression in erythroid cells from healthy donors and patients with thalassemia.

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold).

A novel arginine methyltransferase inhibitor with cellular activity.

Via virtual screening we identified a thioglycolic amide as an arginine methyltransferase (PRMT) inhibitor and tested it and related compounds against the fungal PRMT RmtA and human PRMT1. Compound RM65 was the most potent druglike inhibitor (IC(50)-PRMT1: 55.4 microM) and showed histone hypomethylation in HepG2 cells.

Novel pyrrole-containing histone deacetylase inhibitors endowed with cytodifferentiation activity.

A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a-i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a-l). Compounds 3a-i and 4a-l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2.

Target-based approach to inhibitors of histone arginine methyltransferases.

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.