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 C-terminal motif of an A. nidulans class 1 histone deacetylase (HDAC) is required for catalytic activity and viability of the fungus. Moreover, this motif seems to play a decisive role for growth and development of other fungal species. Thus, this enzyme/motif may represent a promising target for HDAC-inhibitors acting as antifungal agents.

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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. Furthermore, we demonstrate that a fungus-specific C-terminal region of only a few acidic amino acids is required for both the nuclear localization and catalytic activity of the enzyme in the model organism Aspergillus nidulans. Since strains with single or multiple deletions of other classical HDACs revealed no or only moderate growth deficiencies, it is highly probable that the significant delay of germination and the growth defects observed in strains growing under the HDAC inhibitor trichostatin A are caused primarily by inhibition of catalytic RpdA activity. Indeed, even at low nanomolar concentrations of the inhibitor, the catalytic activity of purified RpdA is considerably diminished. Considering these results, RpdA with its fungus-specific motif represents a promising target for novel HDAC inhibitors that, in addition to their increasing impact as anticancer drugs, might gain in importance as antifungals against life-threatening invasive infections, apart from or in combination with classical antifungal therapy regimes.

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. Molecular modeling studies for a deep binding-mode analysis of test compounds were also performed. The bis-carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI-1) and practically inactive against the lysine methyltransferase SET7/9.

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.

Hydroxyapatite (HAP) is the main inorganic component of hard tissues and shows excellent biocompatibility and osteoconductivity properties. Nanoparticles of HAP can be synthesised by the precipitation method in distilled water. The needle shaped particles are below 100 nm in size with low-crystallinity and high-surfacial activation. Recent studies showed toxic effects of HAP nanoparticles on cancer cells. Other studies focus on the application of HAP nanoparticles as drug and gene delivery system or cell marker. However, to date, the exact internalization pathway of HAP nanoparticles into cells has not been determined. When HAP nanoparticles were added to cell culture medium, the particles immediately became instable and formed agglomerates with a size of about 500-700 nm. Hence, cells seldom encounter single HAP nanoparticles in the environment of cell culture or body fluid. The TEM showed internalized HAP captured by vacuoles in the cytoplasm of the hepatocellular carcinoma cells. The invaginations in the cell membrane before nanoparticle uptake suggested endocytic pathways as internalization mechanism. This study revealed that agglomerated HAP nanoparticles were internalized by cells through the energy-dependent process of clathrin-mediated endocytosis. Depletion of intracellular potassium arrested the formation of coated pit, which inhibited the uptake of HAP.