Publications by authors named "Ingo Bauer"

21 Publications

  • Page 1 of 1

Arginine Auxotrophy Affects Siderophore Biosynthesis and Attenuates Virulence of .

Genes (Basel) 2020 04 15;11(4). Epub 2020 Apr 15.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.

is an opportunistic human pathogen mainly infecting immunocompromised patients. The aim of this study was to characterize the role of arginine biosynthesis in virulence of via genetic inactivation of two key arginine biosynthetic enzymes, the bifunctional acetylglutamate synthase/ornithine acetyltransferase (/AFUA_5G08120) and the ornithine carbamoyltransferase (/AFUA_4G07190). Arginine biosynthesis is intimately linked to the biosynthesis of ornithine, a precursor for siderophore production that has previously been shown to be essential for virulence in . ArgJ is of particular interest as it is the only arginine biosynthetic enzyme lacking mammalian homologs. Inactivation of either ArgJ or ArgB resulted in arginine auxotrophy. Lack of ArgJ, which is essential for mitochondrial ornithine biosynthesis, significantly decreased siderophore production during limited arginine supply with glutamine as nitrogen source, but not with arginine as sole nitrogen source. In contrast, siderophore production reached wild-type levels under both growth conditions in ArgB null strains. These data indicate that siderophore biosynthesis is mainly fueled by mitochondrial ornithine production during limited arginine availability, but by cytosolic ornithine production during high arginine availability via cytosolic arginine hydrolysis. Lack of ArgJ or ArgB attenuated virulence of in the insect model and in murine models for invasive aspergillosis, indicating limited arginine availability in the investigated host niches.
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http://dx.doi.org/10.3390/genes11040423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7231135PMC
April 2020

Multiplex Genetic Engineering Exploiting Pyrimidine Salvage Pathway-Based Endogenous Counterselectable Markers.

mBio 2020 04 7;11(2). Epub 2020 Apr 7.

Institute of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to , we validated the applicability of this novel toolbox in and Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering. This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.
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http://dx.doi.org/10.1128/mBio.00230-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157766PMC
April 2020

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

Front Microbiol 2020 4;11:43. Epub 2020 Feb 4.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

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. As known from yeasts and mammals, class 1 KDACs act as components of multimeric protein complexes, which previously was indicated also for . Composition of these complexes, however, remained obscure. In this study, we used tandem affinity purification to characterize different RpdA complexes and their composition in . In addition to known class 1 KDAC interactors, we identified a novel RpdA complex, which was termed RcLS2F. It contains ScrC, previously described as suppressor of the transcription factor CrzA, as well as the uncharacterized protein FscA. We show that recruitment of FscA depends on ScrC and we provide clear evidence that Δ suppression by ScrC depletion is due to a lack of transcriptional repression caused by loss of the novel RcLS2F complex. Moreover, RcLS2F is essential for sexual development and engaged in an autoregulatory feed-back loop.
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http://dx.doi.org/10.3389/fmicb.2020.00043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010864PMC
February 2020

The Lysine Deacetylase RpdA Is Essential for Virulence in .

Front Microbiol 2019 4;10:2773. Epub 2019 Dec 4.

Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria.

Current suboptimal treatment options of invasive fungal infections and emerging resistance of the corresponding pathogens urge the need for alternative therapy strategies and require the identification of novel antifungal targets. is the most common airborne opportunistic mold pathogen causing invasive and often fatal disease. Establishing a novel conditional gene expression system, we demonstrate that downregulation of the class 1 lysine deacetylase (KDAC) RpdA leads to avirulence of in a murine model for pulmonary aspergillosis. The promoter used has previously been shown to allow xylose-induced gene expression in different molds. Here, we demonstrate for the first time that this promoter also allows tuning of gene activity by supplying xylose in the drinking water of mice. In the absence of xylose, an strain expressing under control of the promoter, , was avirulent and lung histology showed significantly less fungal growth. With xylose, however, displayed full virulence demonstrating that xylose was taken up by the mouse, transported to the site of fungal infection and caused induction . These results demonstrate that (i) RpdA is a promising target for novel antifungal therapies and (ii) the expression system is a powerful new tool for gene silencing in .
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http://dx.doi.org/10.3389/fmicb.2019.02773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905131PMC
December 2019

The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in .

Virulence 2019 12;10(1):925-934

Institute of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine . Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in , the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in and . Here, we demonstrate that in inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.
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http://dx.doi.org/10.1080/21505594.2019.1682760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844326PMC
December 2019

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

Fungal Genet Biol 2019 08 28;129:86-100. Epub 2019 May 28.

Division of Molecular Biology, Medical University of Innsbruck, 6020 Innsbruck, Austria. Electronic address:

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. We generated a double type I mutant (ΔrmtA/rmtB) and a combined type I and type II mutant (ΔrmtB/rmtC) to perform genome-wide comparison of their effects on gene expression, but also to monitor putative overlapping activities and reciprocal regulations of type I and type II PRMTs in Aspergillus. Our study demonstrates, that rmtA and rmtC as type I and type II representatives act together as repressors of proteins that are secreted into the extracellular region as the majority of up-regulated genes are mainly involved in catabolic pathways that constitute the secretome of Aspergillus. In addition to a strong up-regulation of secretory genes we found a significant enrichment of down-regulated genes involved in processes related to oxidation-reduction, transmembrane transport and secondary metabolite biosynthesis. Strikingly, nearly 50% of down-regulated genes in both double mutants correspond to redox reaction/oxidoreductase processes, a remarkable finding in light of our recently observed oxidative stress phenotypes of ΔrmtA and ΔrmtC. Finally, analysis of nuclear and cytoplasmic extracts for mono-methylated proteins revealed the presence of both, common and specific substrates of RmtA and RmtC. Thus, our data indicate that type I and II PRMTs in Aspergillus seem to co-regulate the same biological processes but also specifically affect other pathways in a non-redundant fashion.
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http://dx.doi.org/10.1016/j.fgb.2019.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6837890PMC
August 2019

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

J Vis Exp 2019 05 1(147). Epub 2019 May 1.

Division of Molecular Biology, Biocenter, Medical University of Innsbruck.

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. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.
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http://dx.doi.org/10.3791/59527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7319801PMC
May 2019

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Genes (Basel) 2018 Dec 7;9(12). Epub 2018 Dec 7.

Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, 30100 Murcia, Spain.

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of , one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the promoter P. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.
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http://dx.doi.org/10.3390/genes9120613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315630PMC
December 2018

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

Front Microbiol 2018 19;9:2212. Epub 2018 Sep 19.

Division of Molecular Biology, Medical University of Innsbruck, Innsbruck, Austria.

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. Consequently, a wide arsenal of yet unknown fungal metabolites is waiting to be discovered. Here, we describe the effects of deletion of , one of four classical histone deacetylase (HDAC) genes in ; we show that HosA acts as a major regulator of SMs in with converse regulatory effects depending on the metabolite gene cluster examined. Co-inhibition of all classical enzymes by the pan HDAC inhibitor trichostatin A and the analysis of HDAC double mutants indicate that HosA is able to override known regulatory effects of other HDACs such as the class 2 type enzyme HdaA. Chromatin immunoprecipitation analysis revealed a direct correlation between deletion, the acetylation status of H4 and the regulation of SM cluster genes, whereas H3 hyper-acetylation could not be detected in all the upregulated SM clusters examined. Our data suggest that HosA has inductive effects on SM production in addition to its classical role as a repressor via deacetylation of histones. Moreover, a genome wide transcriptome analysis revealed that in addition to SMs, expression of several other important protein categories such as enzymes of the carbohydrate metabolism or proteins involved in disease, virulence, and defense are significantly affected by the deletion of HosA.
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http://dx.doi.org/10.3389/fmicb.2018.02212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156440PMC
September 2018

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

mBio 2016 11 1;7(6). Epub 2016 Nov 1.

Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innsbruck, Austria

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.

Importance: This paper reports on the fungal histone deacetylase RpdA and its importance for the viability of the fungal pathogen Aspergillus fumigatus and other filamentous fungi, a finding that is without precedent in other eukaryotic pathogens. Our data clearly indicate that loss of RpdA activity, as well as depletion of the enzyme in the nucleus, results in lethality of the corresponding Aspergillus mutants. Interestingly, both catalytic activity and proper cellular localization depend on the presence of an acidic motif within the C terminus of RpdA-type enzymes of filamentous fungi that is missing from the homologous proteins of yeasts and higher eukaryotes. The pivotal role, together with the fungus-specific features, turns RpdA into a promising antifungal target of histone deacetylase inhibitors, a class of molecules that is successfully used for the treatment of certain types of cancer. Indeed, some of these inhibitors significantly delay the germination and growth of different filamentous fungi via inhibition of RpdA. Upcoming analyses of clinically approved and novel inhibitors will elucidate their therapeutic potential as new agents for the therapy of invasive fungal infections-an interesting aspect in light of the rising resistance of fungal pathogens to conventional therapies.
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http://dx.doi.org/10.1128/mBio.00831-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5090035PMC
November 2016

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

Fungal Genet Biol 2010 Jun 23;47(6):551-61. Epub 2010 Mar 23.

Division of Molecular Biology, Biocenter-Innsbruck Medical University, Innsbruck, Austria.

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. In addition, both enzymes were able to methylate several non-histone proteins in chromatographic fractions. For endogenous RmtB a remarkable change in its substrate specificity compared to the recombinant enzyme form could be obtained. Phenotypic analysis of mutant strains revealed that growth of DeltarmtA and DeltarmtC strains was significantly reduced under conditions of oxidative stress. Moreover, mycelia of DeltarmtC mutants showed a significant retardation of growth under elevated temperatures.
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http://dx.doi.org/10.1016/j.fgb.2010.03.006DOI Listing
June 2010

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

ChemMedChem 2010 Mar;5(3):398-414

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano SA, Italy.

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.
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http://dx.doi.org/10.1002/cmdc.200900459DOI Listing
March 2010

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

Mol Biol Cell 2010 Jan 25;21(2):345-53. Epub 2009 Nov 25.

Division of Molecular Biology, Biocenter, Innsbruck Medical University, A-6020 Innsbruck, Austria.

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. Here, we show that depletion of RpdA, an RPD3-type histone deacetylase of Aspergillus nidulans, leads to a pronounced reduction of growth and sporulation of the fungus. We demonstrate that a so far unnoticed motif in the C terminus of fungal RpdA histone deacetylases is required for the catalytic activity of the enzyme and consequently is essential for the viability of A. nidulans. Moreover, we provide evidence that this motif is also crucial for the survival of other, if not all, filamentous fungi, including pathogens such as Aspergillus fumigatus or Cochliobolus carbonum. Thus, the extended C terminus of RpdA-type enzymes represents a promising target for fungal-specific histone deacetylase-inhibitors that may have potential as novel antifungal compounds with medical and agricultural applications.
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http://dx.doi.org/10.1091/mbc.e09-08-0750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808227PMC
January 2010

Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors.

ChemMedChem 2009 Jan;4(1):69-77

Universität Halle-Wittenberg, Department of Pharmaceutical Chemistry, Wolfgang-Langenbeckstrasse 4, 06120 Halle/Saale, Germany.

Lysine and arginine methyltransferases participate in the posttranslational modification of histones and regulate key cellular functions. Protein arginine methyltransferase 1 (PRMT1) has been identified as an essential component of mixed lineage leukemia (MLL) oncogenic complexes, revealing its potential as a novel therapeutic target in human cancer. The first potent arginine methyltransferase inhibitors were recently discovered by random- and target-based screening approaches. Herein we report virtual and biological screening for novel inhibitors of PRMT1. Structure-based virtual screening (VS) of the Chembridge database composed of 328 000 molecules was performed with a combination of ligand- and target-based in silico approaches. Nine inhibitors were identified from the top-scored docking solutions; these were experimentally tested using human PRMT1 and an antibody-based assay with a time-resolved fluorescence readout. Among several aromatic amines, an aliphatic amine and an amide were also found to be active in the micromolar range.
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http://dx.doi.org/10.1002/cmdc.200800301DOI Listing
January 2009

Effect of hydroxyapatite nanoparticles on the ultrastructure and function of hepatocellular carcinoma cells in vitro.

Biomed Mater 2006 Mar 15;1(1):38-41. Epub 2006 Mar 15.

Center of Biomedical Material and Engineering, Wuhan University of Technology, Wuhan 430070, People's Republic of China.

The interaction of Bel-7402 hepatocellular carcinoma cells (HCC) as a single cell suspension with hydroxyapatite (HAP) nanoparticles was investigated. It was observed by an inverted microscope that the cells were still homogeneously distributed in the culture medium after 24 h. A TEM analysis showed that the HAP nanoparticles attached to the Bel-7402 cells were finally swallowed by the cells after 4 h, and induced ultrastructural changes of the cells after 4 days. A MTT assay and cell count test for the HAP nanoparticles of various concentrations from 0.14 to 0.56 mmol L(-1) showed that the HAP nanoparticles at a concentration of 0.56 mmol L(-1) induced the strongest effect on the inhibition of Bel-7402 cell proliferation and induced a dramatic decline in cell numbers. Proliferation of Bel-7402 was inhibited by more than 70%, compared to the control. A cell cycle analysis revealed that HAP can arrest Bel-7402 cells at the G1 phase with increasing effect over time. These findings demonstrated that HAP can enter into HCC very easily, change their ultrastructure, and evidently suppress their proliferation.
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http://dx.doi.org/10.1088/1748-6041/1/1/006DOI Listing
March 2006

Internalization of hydroxyapatite nanoparticles in liver cancer cells.

J Mater Sci Mater Med 2008 Mar 15;19(3):1091-5. Epub 2007 Aug 15.

Biomedical Materials and Engineering Research Center, Wuhan University of Technology, Luoshi Road 122#, Wuhan 430070, P.R. China.

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.
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http://dx.doi.org/10.1007/s10856-007-3124-4DOI Listing
March 2008

A novel arginine methyltransferase inhibitor with cellular activity.

Bioorg Med Chem Lett 2007 Aug 3;17(15):4150-3. Epub 2007 Jun 3.

Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Germany.

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. Docking studies proposed binding at the substrate and SAM cofactor binding pocket. It may serve as a lead for further PRMT inhibitors useful for the treatment for hormone dependent cancers.
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http://dx.doi.org/10.1016/j.bmcl.2007.05.088DOI Listing
August 2007

Target-based approach to inhibitors of histone arginine methyltransferases.

J Med Chem 2007 May 14;50(10):2319-25. Epub 2007 Apr 14.

Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.

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.
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http://dx.doi.org/10.1021/jm061250eDOI Listing
May 2007

Small molecule inhibitors of histone arginine methyltransferases: homology modeling, molecular docking, binding mode analysis, and biological evaluations.

J Med Chem 2007 Mar 27;50(6):1241-53. Epub 2007 Feb 27.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Università degli Studi di Roma La Sapienza, P.le Aldo Moro 5, I-00185 Roma.

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.
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http://dx.doi.org/10.1021/jm061213nDOI Listing
March 2007

Sonochemical preparation of hydroxyapatite nanoparticles stabilized by glycosaminoglycans.

Ultrason Sonochem 2007 Mar 10;14(3):286-90. Epub 2006 Aug 10.

Biomedical Materials and Engineering Center, Wuhan University of Technology, Wuhan, PR China.

Stable hydroxyapatite (HAP) nanoparticles system was synthesized from Ca(H(2)PO(4))(2) aqueous solution and saturated Ca(OH)(2) aqueous solution by an improved precipitation method. This method was reformed through using ultrasound irradiation as assistant technology due to its unique chemical reaction effects and adding glycosaminoglycans (GAGs) as regulation additive due to its strong interaction with HAP. The products were characterized by Malvern Zetasizer 3000HS Analysis system, TEM and ED. The size distribution and zeta potential of HAP nanoparticles were influenced by the concentration of GAGs. With the GAGs concentration of 0.35g/L, the better excellent HAP nanoparticle system could be obtained with the number-averaged particle size of 22.2nm in 84.5% area and 54.6nm in 15.5% area between 18.1nm and 117.4nm and the zeta potential of -60.9mV. In the presence of GAGs, the particle size and size distribution are little sensitive to the ultrasound irradiation (UI) time. With the increasing of UI time from 0.5h to 3h and 5h, the particle size increased a little and the crystallinity was improved. GAGs inhibited HAP crystal growth and stabilized HAP nanoparticles. Based on the TEM observation and size distribution determination of HAP nanoparticles, the possible formation mechanism of HAP nanoparticles stabilized by GAGs under UI was discussed.
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http://dx.doi.org/10.1016/j.ultsonch.2006.06.002DOI Listing
March 2007

Histone methyltransferases in Aspergillus nidulans: evidence for a novel enzyme with a unique substrate specificity.

Biochemistry 2004 Aug;43(33):10834-43

Department of Molecular Biology, Innsbruck Medical University, A-6020 Innsbruck, Austria.

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.
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http://dx.doi.org/10.1021/bi049626iDOI Listing
August 2004
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