Synthesis and Biological Validation of Novel Synthetic Histone/Protein Methyltransferase Inhibitors
Antonello MaiSérgio ValenteDonghang ChengAndrea PerroneRino RagnoSilvia SimeoniGianluca SbardellaGerald BroschAngela NebbiosoMariarosaria ConteLucia AltucciMark T. Bedford
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Coding control: Protein arginine methyltransferases (PRMTs) and histone lysine methyltransferases (HKMTs) are epigenetic enzymes involved in regulation of gene expression and cellular processes. A new series of histone/protein methyltransferase inhibitors based on the 1,5-diphenyl-1,4-pentadien-3-one scaffold is reported. The described compounds showed various degrees of selectivity against the tested PRMTs (PRMT1 and CARM1) and HKMT (SET7).Abstract Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM.
HEK 293 cells
Proto-Oncogene Proteins c-myc
Protein methylation
Temozolomide
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Chromatin immunoprecipitation
Histone H4
Immunoprecipitation
Histone Methylation
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Here we report the identification of small molecules that specifically inhibit protein arginine N-methyltransferase (PRMT) activity. PRMTs are a family of proteins that either monomethylate or dimethylate the guanidino nitrogen atoms of arginine side chains. This common post-translational modification is implicated in protein trafficking, signal transduction, and transcriptional regulation. Most methyltransferases use the methyl donor, S-adenosyl-L-methionine (AdoMet), as a cofactor. Current methyltransferase inhibitors display limited specificity, indiscriminately targeting all enzymes that use AdoMet. In this screen we have identified a primary compound, AMI-1, that specifically inhibits arginine, but not lysine, methyltransferase activity in vitro and does not compete for the AdoMet binding site. Furthermore, AMI-1 prevents in vivo arginine methylation of cellular proteins and can modulate nuclear receptor-regulated transcription from estrogen and androgen response elements, thus operating as a brake on certain hormone actions.
Protein methylation
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Histone lysine methyltransferases (KMTs) play an important role in epigenetic gene regulation and have emerged as promising targets for drug discovery. However, the scope and limitation of KMT catalysis on substrates possessing substituted lysine side chains remain insufficiently explored. Here, we identify new unnatural lysine analogues as substrates for human methyltransferases SETD7, SETD8, G9a and GLP. Two synthetic amino acids that possess a subtle modification on the lysine side chain, namely oxygen at the γ position (KO, oxalysine) and nitrogen at the γ position (KN, azalysine) were incorporated into histone peptides and tested as KMTs substrates. Our results demonstrate that these lysine analogues are mono-, di-, and trimethylated to a different extent by trimethyltransferases G9a and GLP. In contrast to monomethyltransferase SETD7, SETD8 exhibits high specificity for both lysine analogues. These findings are important to understand the substrate scope of KMTs and to develop new chemical probes for biomedical applications.
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Protein methylation
Transcription
Posttranslational modification
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The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.
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Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM.
HEK 293 cells
Proto-Oncogene Proteins c-myc
Temozolomide
Druggability
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Protein methylation
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1564 The tumor suppressor p53 is the most commonly mutated gene in human cancer. As a transcription factor, p53 plays an essential role in the induction of cellular processes such as cell cycle arrest, apoptosis, and senescence in response to stress signals. Post-translational modifications and interaction with cofactors are the primary mediators of p53 stabilization and activation upon cellular stresses. Protein arginine methyltransferases (PRMTs) are known to methylate histones and many cellular proteins involved in diverse processes such as DNA repair, transcription, and RNA processing. Here, we showed that inhibition of protein methyltransferases, especially arginine methyltransferases, prevents the induction of p21 and MDM2 as well as the repression of ECT2 in response to DNA damage, in a p53-dependent manner. To identify which arginine methyltransferase is required for p53 transcriptional activity, we generated multiple cell lines in which PRMT1, CARM1, or PRMT5 are inducibly knocked down by the tetracycline-inducible shRNA expression system in MCF7 breast adenocarcinoma cells. We found that PRMT1 and/or CARM1 knockdown has a limited effect on cell proliferation. In addition, PRMT1 and CARM1 deficiency do not have an effect on p53 stabilization and transcriptional activity upon DNA damage or MDM2 inhibition. However, we found that PRMT5 is required for cell proliferation. In addition, we showed that deficiency in PRMT5 leads to cell cycle arrest in G1. Interestingly, we also found that PRMT5 knockdown attenuates p53 stabilization upon DNA damage, leading to a decreased induction of MDM2 and p21. Taken together, we uncovered that the arginine methyltransferase family member PRMT5 has a pro-survival function and also acts as a novel modulator of p53 transcriptional activity.
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