Arginine Methyltransferase Inhibitor Research Articles

Endothelial transcriptome in response to pharmacological methyltransferase inhibition.

The enzymatic activities of protein methyltransferases serve to write covalent modifications on histone and non-histone proteins in the control of gene transcription. Here, we describe gene expression changes in human endothelial cells caused by treatment with methyltransferase inhibitors 7,7'-carbonylbis (azanediyl) bis(4-hydroxynaphthalene-2 -sulfonic acid (AMI-1) and disodium-2-(2,4,5,7- tetrabromo-3-oxido-6-oxoxanthen-9-yl) benzoate trihydrate (AMI-5). Deep sequencing of mRNA indicated robust change on transcription following AMI-5 treatment compared with AMI-1. Functional annotation analysis revealed that both compounds suppress the expression of genes associated with translational regulation, suggesting arginine methylation by protein arginine methyltransferases (PRMTs) could be associated with regulation of this pathway. Interestingly, AMI-5 but not AMI-1 was found to decrease methylation of H3 histones at lysine 4 and down-regulate gene expression associated with interleukin-6 (IL-6) and activator protein-1 (AP-1) signaling pathways. These results imply that inhibition of protein methylation by AMI-1 and AMI-5 can differentially regulate specific pathways with potential to interrupt pathological signaling in the vascular endothelium.

Arginine methylation regulates antibody responses through modulating cell division and isotype switching in B cells

Protein arginine methylation plays crucial roles, including signal transduction, transcriptional control, cell proliferation and/or differentiation. B cells undergo clonal division, isotype switching and differentiate into antibody forming cells following stimulation with Toll-like receptor-ligand, lipopolysaccharide (LPS) and T cell-derived signals, including CD40-ligand (CD40-L) and interleukin 4 (IL-4). Whether protein arginine methylation affects B cell division and/or isotype switching to IgG1 in response to LPS, IL-4, and CD40-L was examined using the arginine methyl transferase inhibitor adenosine-2',3'-dialdehyde (AdOx). Addition of AdOx substantially reduced the number of division cycles of stimulated B cells, whereas cell viability remained intact. Upon stimulation with LPS/IL-4/CD40-L, the proportion of surface IgG1 positive cells in each division cycle was slightly diminished by AdOx. However, the degree of expression of γ1 germ line transcript and activation-induced cytidine deaminase (AID) in response to LPS/IL-4/CD40-L were unaffected by addition of AdOx, suggesting that AdOx influences class switch recombination independent of AID expression through transcriptional control. Taken together, arginine methylation appears to be involved in B cell isotype switching, as well as in clonal expansion of B cells in response to LPS/IL-4/CD40-L.

Selective Inhibitors of Arginine Methyl Transferase 5 (PRMT5) As a Novel Treatment for β-Thalassemia and Sickle Cell Disease.

AbstractAbstract 2129The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing.Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD.To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds.We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined.Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures:No relevant conflicts of interest to declare.

An Allosteric Inhibitor of Protein Arginine Methyltransferase 3

PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.

Upregulated Protein Arginine Methyltransferase 1 by IL-4 Increases Eotaxin-1 Expression in Airway Epithelial Cells and Participates in Antigen-Induced Pulmonary Inflammation in Rats

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

An Allosteric Inhibitor of Protein Arginine Methyltransferase 3

An Allosteric Inhibitor of Protein Arginine Methyltransferase 3

Knock-down of protein L-isoaspartyl O-methyltransferase increases β-amyloid production by decreasing ADAM10 and ADAM17 levels

To examine the role of protein L-isoaspartyl O-methyltransferase (PIMT; EC 2.1.1.77) on the secretion of Aβ peptides. HEK293 APPsw cells were treated with PIMT siRNA or adenosine dialdehyde (AdOX), a broad-spectrum methyltransferase inhibitor. Under the conditions, the level of Aβ secretion and regulatory mechanism by PIMT were examined. Knock-down of PIMT and treatment with AdOX significantly increased Aβ(40) secretion. Reductions in levels of PIMT decreased the secretion of soluble amyloid precursor protein alpha (sAPPα) without altering the total expression of APP or its membrane-bound C83 fragment. However, the levels of the C99 fragment generated by β-secretase were enhanced. Moreover, the decreased secretion of sAPPα resulting from PIMT knock-down seemed to be linked with the suppression of the expression of α-secretase gene products, α-disintegrin and metalloprotease 10 (ADAM10) and ADAM17, as indicated by Western blot analysis. In contrast, ADAM10 was not down-regulated in response to treatment with the protein arginine methyltransferase (PRMT) inhibitor, AMI-1. This study demonstrates a novel role for PIMT, but not PRMT, as a negative regulator of Aβ peptide formation and a potential protective factor in the pathogenesis of AD.

Brief Report: Combined Chemical Treatment Enables Oct4-Induced Reprogramming from Mouse Embryonic Fibroblasts

It has been established that exogenous expression of four transcription factors (Oct4, Klf4, Sox2, and c-Myc) can reprogram mammalian somatic cells to pluripotent states. Further studies demonstrated that such induced pluripotent stem cells (iPSCs) could be generated with fewer exogenous transcription factors, facilitated by endogenous expression of reprogramming factors and/or synthetic small molecules. Here, we reported identification of a new small molecule, a protein arginine methyltransferase inhibitor AMI-5, which enabled Oct4-induced reprogramming of mouse embryonic fibroblasts in combination with transforming growth factor (TGF)-β inhibitor A-83-01. The Oct4-induced iPSCs were shown similar to mouse embryonic stem cells with respect to typical pluripotency criteria. More importantly, they were shown to give rise to liveborn pups through tetraploid complementation assays, demonstrating the high quality of full reprogramming induced by this condition. Furthermore, this study suggests that regulation of protein arginine methylation might be involved in the reprogramming process.

Xenoestrogens regulate the activity of arginine methyltransferases.

Arginine methylation is a common post-translational modification that has been strongly implicated in transcriptional regulation. The arginine methyltransferases (PRMTs) were first reported as transcriptional coactivators for the estrogen and androgen receptors. Compounds that inhibit these enzymes will provide us with valuable tools for dissecting the roles of these enzymes in cells, and will possibly also have therapeutic applications. In order to identify such inhibitors of the PRMTs, we have previously performed a high-throughput screen using a small molecule library. These compounds were named arginine methyltransferase inhibitors (AMIs). The majority of these inhibitors were polyphenols, and one in particular (AMI-18) shared additional features with a group of known xenoestrogens. We, thus, tested a panel of xenoestrogens and found that a number of them possess the ability to inhibit PRMT activity, in vitro. These inhibitors primarily target CARM1, and include licochalcone A, kepone, benzyl 4-hydroxybenzoate, and tamoxifen. We developed a cell-based reporter system for CARM1 activity, and showed that tamoxifen (IC(50) =30 μM) inhibits this PRMT. The ability of these compounds to regulate the activity of transcriptional coactivators may be an unappreciated mechanism of action for xenoestrogens, and might also explain the efficacy of high-dose tamoxifen treatment on estrogen receptor negative cancers.

Signal transducer and activator of transcription 3-mediated regulation of miR-199a-5p links cardiomyocyte and endothelial cell function in the heart: a key role for ubiquitin-conjugating enzymes

Mice with a cardiomyocyte (CM)-restricted knockout of signal transducer and activator of transcription 3 (STAT3-KO) develop spontaneous heart failure. We investigated the impact of STAT3-mediated regulation of microRNAs for pathophysiological alterations in the heart. MicroRNAchip and qRT-PCR analysis revealed elevated cardiac expression of miR-199a in STAT3-KO mice. Lentiviral shRNA-mediated STAT3-knock-down in neonatal rat CMs markedly increased miR-199a promoter activity and miR-199a levels indicative of a suppressive effect of STAT3 on miR-199a transcription. Up-regulated miR-199a in CM by pre-miR-199a transfection (pre-miR-199a-CM) reduced expression of components of the ubiquitin-proteasome system (UPS), i.e. the ubiquitin-conjugating enzymes Ube2g1 (mRNA and protein) and Ube2i (protein). Pre-miR-199a-CM or CM with siRNA-mediated down-regulation of Ube2i and Ube2g1 (siRNA-Ube2i/2g1-CM) displayed massive down-regulation of α- and β-myosin heavy chain expression associated with disrupted sarcomere structures. In addition, protein arginine methyltransferase I (PRMT-I) expression and asymmetric dimethylarginine (ADMA) synthesis were increased in pre-miR-199a-CM or in siRNA-Ube2i/2g1-CM. Increased ADMA in cell culture supernatant (SN) from pre-miR-199a-CM or siRNA-Ube2i/2g1-CM lowered nitric oxide (NO) bioavailability of rat cardiac endothelial cells while lowering ADMA concentration in CM SNs by the PRMT inhibitor arginine methyltransferase inhibitor 1 (AMI-1) (100 µM) improved NO bioavailability. In STAT3-KO hearts Ube2i and Ube2g1 expression were markedly reduced. Human terminal failing hearts harbouring low STAT3 protein levels displayed increased miR-199a levels and decreased Ube2g1 expression. This study identifies a novel pathophysiological circuit in the heart between reduced STAT3 protein levels, increased miR-199a expression, and subsequent impairment of the UPS that disrupts CM sarcomere structure and impairs via the release of ADMA endothelial cell function.

Effects of a novel arginine methyltransferase inhibitor on T‐helper cell cytokine production

The protein arginine methyltransferase (PRMT) family of enzymes catalyzes the transfer of methyl groups from S-adenosylmethionine to the guanidino nitrogen atom of peptidylarginine to form monomethylarginine or dimethylarginine. We created several less polar analogs of the specific PRMT inhibitor arginine methylation inhibitor-1, and one such compound was found to have improved PRMT inhibitory activity over the parent molecule. The newly identified PRMT inhibitor modulated T-helper-cell function and thus may serve as a lead for further inhibitors useful for the treatment of immune-mediated disease.

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.

Toward the development of potent and selective bisubstrate inhibitors of protein arginine methyltransferases

Prototype inhibitors of protein arginine methyltransferases (PRMTs) have been constructed by attaching guanidine functionality via a variable linker to non-reactive amine analogues of the cellular co-factor ( S)-adenosyl methionine (AdoMet). Potent inhibition of PRMT1 (IC 50 of ∼3–6 μM) combined with weak inhibition of the lysine methyltransferase SET7 (∼50% of activity at 100 μM) was observed for two such compounds.

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. 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.

Benzo[ d]imidazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1)—Hit to Lead studies

Hit to Lead optimization and SAR development led to the identification of the potent and selective benzo[ d]imidazole inhibitor ( 17b) of Co-activator Associated Arginine Methyltransferase (CARM1).

Optimization of pyrazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1)

Design, synthesis, and SAR development led to the identification of the potent, novel, and selective pyrazole based inhibitor (7f) of Coactivator Associated Arginine Methyltransferase (CARM1).

Virtual Screening and Biological Characterization of Novel Histone Arginine Methyltransferase PRMT1 Inhibitors

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.

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 μM) 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.

Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function

The HIV-1 nucleocapsid protein (NC) is involved in transfer RNA3 annealing to the primer binding site of viral genomic RNA by means of two basic regions that are similar to the N-terminal portion of the arginine-rich motif (ARM) of Tat. As Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its ARM, we investigated whether NC could also act as a substrate for this enzyme. Arginine methylation of NC was demonstrated in vitro and in vivo, and sites of methylation were determined by mutational analysis. The impact of the arginine methylation of NC was measured in RNA annealing and reverse transcription initiation assays. An arginine methyltransferase inhibitor (AMI)3.4 was tested for its effects on viral infectivity and replication in vivo. NC is a substrate for PRMT6 both in vitro and in vivo. NC possesses arginine dimethylation sites in each of its two basic regions at positions R10 and R32, and methylated NC was less able than wild-type to promote RNA annealing and participate in the initiation of reverse transcription. Exposure of HIV-1-infected MT2 and primary cord blood mononuclear cells to AMI3.4 led to increased viral replication, whereas viral infectivity was not significantly affected in multinuclear-activation galactosidase indicator assays. NC is an in-vivo target of PRMT6, and arginine methylation of NC reduces RNA annealing and the initiation of reverse transcription. These findings may lead to ways of driving HIV-infected cells out of latency with drugs that inhibit PRMT6.

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. 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.