Protein Arginine Methyltransferase Family Research Articles

Genome-wide identification and expression analysis of protein arginine methyltransferase and JmjC domain-containing family in apple

Histone methylation is an important type of histone modification that regulates gene expression in plants. In this study, we identified 14 arginine methylation-related genes (Protein Arginine Methyltransferase, MdPRMT) and 32 demethylation-related genes (JmjC Domain-Containing Family, MdJMJ) in apple. Furthermore, we investigated the phylogenetic relationship, chromosome distribution, gene structure, motif analysis, promoter sequence analysis, and expression patterns of MdPRMT and MdJMJ genes. Homology analysis showed a high degree of conservation and homology between PRMT and JMJ genes in Arabidopsis and apple. We identified the types of duplicated genes in the MdJMJ and MdPRMT gene families, found a large number of whole-genome duplicates (WGD) gene pairs and a small number of tandem duplicates (TD) pairs, transposed duplication (TRD) gene pairs as well as proximal duplicates (PD) pairs, and discussed the possible evolutionary pathways of the gene families from the perspective of duplicated genes. Homology analysis showed a high degree of conservation and homology between PRMT and JMJ genes in Arabidopsis and apple. In addition, the promoter regions of MdPRMT and MdJMJ contain numerous cis-acting elements involved in plant growth and development, hormone response, and stress responses. Based on the transcriptional profiles of MdPRMT and MdJMJ in different tissues and developmental stages, it was found that MdPRMT and MdJMJ may play multiple roles in apple growth and development, for example, MdJMJ21 may be involved in the regulation of apple endosperm formation. MdPRMT and MdJMJ exhibit different expression patterns in response to hormone signaling in apple, MdJMJ3, MdJMJ18, MdJMJ30, MdPRMT2, MdPRMT13, and MdPRMT14 may play roles in apple response to drought stress, while the expression of MdJMJ13, MdPRMT3, MdPRMT4, and MdPRMT6 is affected by cold stress. Our study provides a foundation for determining the functional roles of MdPRMT and MdJMJ genes in apple.

A CARM1 Inhibitor Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

CARM1, belonging to the protein arginine methyltransferase (PRMT) family, is intricately associated with the progression of cancer and is viewed as a promising target for both cancer diagnosis and therapy. However, the number of specific and potent CARM1 inhibitors is limited. We herein discovered a CARM1 inhibitor, iCARM1, that showed better specificity and activity toward CARM1 compared to the known CARM1 inhibitors, EZM2302 and TP-064. Similar to CARM1 knockdown, iCARM1 suppressed the expression of oncogenic estrogen/ERα-target genes, whereas activated type I interferon (IFN) and IFN-induced genes (ISGs) in breast cancer cells. Consequently, iCARM1 potently suppressed breast cancer cell growth both in vitro and in vivo. The combination of iCARM1 with either endocrine therapy drugs or etoposide demonstrated synergistic effects in inhibiting the growth of breast tumors. In summary, targeting CARM1 by iCARM1 effectively suppresses breast tumor growth, offering a promising therapeutic approach for managing breast cancers in clinical settings.

Discovery of a first-in-class degrader for the protein arginine methyltransferase 6 (PRMT6)

PRMT6 is a member of the protein arginine methyltransferase family, which participates in a variety of physical processes and plays an important role in the occurrence and development of tumors. Using small molecules to design and synthesize targeted protein degraders is a new strategy for drug development. Here, we report the first-in-class degrader SKLB-0124 for PRMT6 based on the hydrophobic tagging (HyT) method.Importantly, SKLB-0124 induced proteasome dependent degradation of PRMT6 and significantly inhibited the proliferation of HCC827 and MDA-MB-435 cells. Moreover, SKLB-0124 effectively induced apoptosis and cell cycle arrest in these two cell lines. Our data clarified that SKLB-0124 is a promising selective PRMT6 degrader for cancer therapy which is worthy of further evaluation.

Identification of protein methyltransferases 5 associated with ferroptosis and immune cell infiltration of head and neck squamous cell carcinoma.

Head and neck tumors are malignant tumors that appear in the head and neck. Although much progress has been made in the treatment of head and neck tumors, many challenges remain. The prognosis of some advanced cases remains poor and survival and quality of life after treatment face certain limitations. Therefore, further research into the pathogenesis and treatment options for head and neck tumors is important in order to improve the prognosis and quality of life of patients. The Protein Arginine Methyltransferase (PRMT) family is a class of enzymes that are responsible for adding methyl groups to arginine residues in proteins. PRMT family members play important roles in regulating many cellular processes, such as transcriptional regulation, signaling, and cell cycle regulation. Recent studies have shown that the PRMT family also plays an important function in tumorigenesis and development. Here, we found that PRMT family members are significantly overexpressed in head and neck tumors and that PRMT5 may serve as an independent prognostic factor in head and neck tumors. We found that PRMT5-regulated differential genes were significantly enriched in tumor-associated signaling pathways such as IL-17 and p53. And we also found that the expression of PRMT5 in head and neck tumors was significantly correlated with immune cell infiltration, m6A as well as the expression of ferroptosis-related genes, and drug sensitivity. These results suggest that PRMT may play an important role in the development of head and neck tumors.

Abstract 3226: PRMT1 is a critical dependency in clear cell renal cell carcinoma with roles in post- transcriptional regulation and DNA damage response

Abstract The majority of sporadic renal cell carcinoma (RCC) cases present as the clear cell subtype (ccRCC) and are associated with the biallelic inactivation of the von Hippel-Lindau (VHL) gene, leading to constitutive activation of the cellular hypoxia response and deleterious alterations to gene expression, metabolism, and growth. Despite the near ubiquity of VHL loss, evidence suggests that it alone is insufficient to drive tumor establishment. Additional loss of function (LOF) mutations in epigenetic regulatory proteins including PBRM1, SETD2 and BAP1 have been identified as tumor drivers. The frequency and penetrance of these mutations in epigenetic regulators implicates a vulnerability in ccRCC that may be exploited to develop new therapies. To explore this vulnerability, the Ailles lab completed a screen of the Structural Genomics Consortium’s (SGC) epiprobe library - a panel of inhibitors targeting an array of epigenetic regulators in patient derived ccRCC models. Among the several promising targets identified, a particularly favorable inhibition profile was noted for MS023, an inhibitor of the type I protein arginine methyltransferase family (PRMT1/3/4/6 and 8). PRMTs transfer methyl groups to nuclear histones and cytoplasmic targets, influencing gene expression, mRNA processing and viability. This growth inhibition was corroborated using an additional type I PRMT compound, GSK3368715. CRISPR-mediated deletion of type I PRMT targets indicated that PRMT1 is the main enzyme responsible for growth inhibition in our ccRCC models. Furthermore, genetic depletion of PRMT1 via shRNA phenocopied pharmacological inhibition and proliferative growth was restored in the presence of MS023 by overexpressing PRMT1. Finally, MS023 treatment of mice bearing ccRCC cell line and patient derived xenografts led to significant inhibition of tumor growth in vivo. Transcriptomic profiling of chemical treatment and PRMT1 genetic depletion demonstrated that PRMT1 inhibition triggers a significant down regulation of genes related to cell cycle progression and DNA damage. ccRCC cells treated with MS023 accumulated significant levels of DNA damage before undergoing cell death, with several DDR-related proteins being down-regulated. Proximity ligation and proteomic analysis (Bio-ID) revealed that PRMT1’s interactors skew heavily towards RNA metabolism regulators and widespread alterations in mRNA processing were noted with MS023 treatment. Evidence continues to mount that PRMT1 is implicated in the biology of cancer, but to our knowledge, no thorough investigations have been performed in the context of ccRCC. Our data suggests that inhibition of PRMT1 inhibits processing of mRNA transcripts, compromises DDR mechanisms, leading to an accumulation of double stranded breaks, cytostasis and cell death. Thus PRMT1 represents a viable therapeutic target in ccRCC. Citation Format: Joseph Walton, Karen Arevalo, Angel SN Ng, Jalna Meens, Christina Karamboulas, Panagiotis Prinos, Brian Raught, Eric Chen, Cheryl Arrowsmith, Laurie Ailles. PRMT1 is a critical dependency in clear cell renal cell carcinoma with roles in post- transcriptional regulation and DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3226.

PRMT1 Integrates Immune Microenvironment and Fatty Acid Metabolism Response in Progression of Hepatocellular Carcinoma.

Protein arginine methyltransferase (PRMT) family members have important roles in cancer processes. However, its functions in the regulation of cancer immunotherapy of hepatocellular carcinoma (HCC) are incompletely understood. This study aimed to investigate the roles of PRMT1 in HCC. Single-cell RNA sequencing (scRNA-seq) and clinicopathological data were obtained and used to explore the diagnostic and prognostic value, cellular functions and roles in immune microenvironment regulation of PRMT1 in HCC. The functions of PRMT1 were explored using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), as well as gene set enrichment analysis (GSEA). TIMER and CIBERSORT were used to analyze the relationships between PRMT1 expression and immune cell infiltration. The STRING database was used to construct a protein-protein interaction (PPI) network. PRMT1 was aberrantly expressed in HCC, which high expression was associated with tumor progression, worse overall survival (OS) and disease-free survival (DFS) of patients with HCC. PRMT1 was also associated with immune cell infiltration. Moreover, it was specifically expressed in immune cells, including exhausted CD8 T cells, B cells, and mono/macro cells in patients with immunotherapy. The expression of immune checkpoints was significantly increased in the high-PRMT1 expression groups of HCC patients. Regarding biological mechanisms, cell viability, migration and invasion, and the expression of genes related to fatty acid metabolism were suppressed in PRMT1 knockdown HCC cells. Moreover, genes co-expressed with PRMT1 were involved in the fatty acid metabolic process and enriched in fatty and drug-induced liver disease. Taken together, these results indicate that PRMT1 might exert its oncogenic effects via immune microenvironment regulation and fatty acid metabolism in HCC. Our finding will provide a foundation for further studies and indicate a potential clinical therapeutic target for liver cancer.

The emerging roles of protein arginine methyltransferases in antiviral innate immune signaling pathways.

The Protein Arginine Methyltransferases (PRMTs) family is involved in various biological processes, including gene transcription, pre-mRNA splicing, mRNA translation, and protein stability. Recently, mounting evidence has shown that PRMTs also play critical roles in regulating the host antiviral immune response, either in an enzymatic activity dependent or independent manner. This review aims to provide an overview of the recent findings regarding the function and regulatory mechanisms of PRMTs in the antiviral response. These findings have the potential to aid in the discovery and design of novel therapeutic strategies for viral infections.

PRMT1 accelerates cell proliferation, migration, and tumor growth by upregulating ZEB1/H4R3me2as in thyroid carcinoma.

Thyroid carcinoma (TC) represents the most prevalent malignancy of the endocrine system. Protein arginine methyltransferase 1 (PRMT1) is a critical member of the protein arginine methyltransferase family in mammals and is involved in multiple biological processes. This study aimed to investigate the function of PRMT1 in TC. In the present study, human TC cell lines (8505C, CAL62, and BCPAP) and a normal human thyroid cell line Nthy‑ori 3‑1 were employed. Small interfering RNA targeting PRMT1 was used to knock down PRMT1 expression in 8505C cells, and PRMT1 overexpression plasmids were transfected into BCPAP cells. Cell viability was assessed using a CCK‑8 and colony formation assays. Apoptosis was measured using flow cytometry and TUNEL assays. Cell migration was assessed using wound healing and Transwell assays. Reverse transcription‑quantitative PCR was used to determine the mRNA expression levels of PRMT1. Western blotting was used to detect the protein expression levels of PRMT1, E‑cadherin, vimentin, H4R3me2as, and zinc‑finger E homeobox‑binding 1 (ZEB1). Notably, PRMT1 expression was elevated in TC (P<0.01). PRMT1 knockdown inhibited TC cell proliferation and migration and concurrently enhanced migration. Furthermore, PRMT1 knockdown suppressed tumor growth and metastasis in a mouse model of TC. PRMT1 downregulation increased E‑cadherin expression and decreased the expression of vimentin, H4R3me2as, and ZEB1 in the TC cells and the mouse model of TC. Conversely, PRMT1 overexpression had the opposite effect on TC malignant characteristics (P<0.05). These findings suggest that PRMT1 knockdown inhibited TC malignancy by downregulating H4R3me2as/ZEB1, thereby highlighting novel therapeutic targets and diagnostic markers for the management of TC.

Research progress on small-molecule inhibitors of protein arginine methyltransferase 5 (PRMT5) for treating cancer.

The protein arginine methyltransferase family includes nine members, with PRMT5 being the major type II arginine methyltransferase. PRMT5 is upregulated in a variety of tumors and promotes tumorigenesis and tumor cell proliferation and metastasis, making it a potential tumor therapy target. Recently, PRMT5 inhibitor research and development have become hotspots in the tumor therapy field. We classified and summarized PRMT5 inhibitors according to different binding mechanisms. We mainly analyzed the structure, biological activity, and binding interactions of PRMT5 inhibitors with the PRMT5 enzyme. At present, many PRMT5 inhibitors with various mechanisms of action have been reported, including substrate-competitive inhibitors, SAM-competitive inhibitors, dual substrate-/SAM-competitive inhibitors, allosteric inhibitors, PRMT5 degraders, MTA-cooperative PRMT5 inhibitors and PPI inhibitors. These inhibitors are beneficial to the treatment of tumors. Some drugs are being used in clinical trials. PRMT5 inhibitors have broad application prospects in tumor therapy.

Discovery of 2,4-diphenyl-substituted thiazole derivatives as PRMT1 inhibitors and investigation of their anti-cervical cancer effects

Cervical cancer is one of the most common cancers that affects middle-aged women and the discovery of new drugs to aid clinical management is needed. As an important member of the protein arginine methyltransferases (PRMTs) family, PRMT1 catalyzes the methylation of protein arginine, which can influence multiple biological processes of cancer cells, such as activating epithelial-mesenchymal transformation (EMT) and acquiring resistance to apoptosis. Therefore, PRMT1 can be considered as a potential drug target for cervical cancer. In the current study, a new sub-binding pocket was discovered by molecular modeling, and by introducing a third substitute on the thiazole group to occupy this pocket, a series of compounds were designed and synthesized as potential PRMT1 inhibitors. Of these, two compounds (ZJG51 and ZJG58) exhibited significant inhibitory activities against PRMT1 without significantly inhibiting PRMT5. Both ZJG51 and ZJG58 displayed potent inhibitory effects on the proliferation of four cancer-derived cell lines and ZJG51 exerted relative selectivity against the cervical cancer cell line, HeLa. Further studies showed that ZJG51 inhibited migration and induce the apoptosis of HeLa cells. Mechanistically, ZJG51 significantly regulated PRMT1 related proteins, and indicated that the induction of apoptosis and inhibition of migration by ZJG51 may involve the activation of Caspase 9 and the inhibition of EMT, respectively. Molecular dynamic simulation and free energy calculation showed that ZJG51 can bind to PRMT1 stably and the binding mode was predicted. These data indicated that introducing the third substitute on the five-membered ring could be a future direction for structure-based optimization of PRMT1 inhibitors, and ZJG51 could be an important lead compound to inform the design of more potent inhibitors.

Effectors and effects of arginine methylation.

Arginine methylation is a ubiquitous and relatively stable post-translational modification (PTM) that occurs in three types: monomethylarginine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Methylarginine marks are catalyzed by members of the protein arginine methyltransferases (PRMTs) family of enzymes. Substrates for arginine methylation are found in most cellular compartments, with RNA-binding proteins forming the majority of PRMT targets. Arginine methylation often occurs in intrinsically disordered regions of proteins, which impacts biological processes like protein-protein interactions and phase separation, to modulate gene transcription, mRNA splicing and signal transduction. With regards to protein-protein interactions, the major 'readers' of methylarginine marks are Tudor domain-containing proteins, although additional domain types and unique protein folds have also recently been identified as methylarginine readers. Here, we will assess the current 'state-of-the-art' in the arginine methylation reader field. We will focus on the biological functions of the Tudor domain-containing methylarginine readers and address other domains and complexes that sense methylarginine marks.

Classification molecular subtypes of hepatocellular carcinoma based on PRMT-related genes.

Background: Recent studies highlighted the functional role of protein arginine methyltransferases (PRMTs) catalyzing the methylation of protein arginine in malignant progression of various tumors. Stratification the subtypes of hepatocellular carcinoma (HCC) is fundamental for exploring effective treatment strategies. Here, we aim to conduct a comprehensive analysis of PRMTs with bioinformatic tools to identify novel biomarkers for HCC subtypes classification and prognosis prediction, which may be potential ideal targets for therapeutic intervention. Methods: The expression profiling of PRMTs in HCC tissues was evaluated based on the data of TCGA-LIHC cohort, and further validated in HCC TMA cohort and HCC cell lines. HCC was systematically classified based on PRMT family related genes. Subsequently, the differentially expressed genes (DEGs) between molecular subtypes were identified, and prognostic risk model were constructed using least absolute shrinkage and selection operator (LASSO) and Cox regression analysis to evaluate the prognosis, gene mutation, clinical features, immunophenotype, immunotherapeutic effect and antineoplastic drug sensitivity of HCC. Results: PRMTs expression was markedly altered both in HCC tissues and HCC cell lines. Three molecular subtypes with distinct immunophenotype were generated. 11 PRMT-related genes were enrolled to establish prognostic model, which presented with high accuracy in predicting the prognosis of two risk groups in the training, validation, and immunotherapy cohort, respectively. Additionally, the two risk groups showed significant difference in immunotherapeutic efficacy. Further, the sensitivity of 72 anticancer drugs was identified using prognostic risk model. Conclusion: In summary, our findings stratified HCC into three subtypes based on the PRMT-related genes. The prognostic model established in this work provide novel insights into the exploration of related therapeutic approaches in treating HCC.

Histone H4K20 monomethylation enables recombinant nucleosome methylation by PRMT1 in vitro

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to specific arginine residues of histones and nonhistone proteins. There are nine members in the PRMT family (PRMT1 to PRMT9), and PRMT1 is a dominant member catalyzing majority of arginine methylation in the cell. However, none of the PRMTs is active with recombinant nucleosome as substrate in vitro. Here, we report the discovery of the first in class novel crosstalk between histone H4 lysine 20 (H4K20) monomethylation on nucleosome by SETD8 and histone H4 arginine 3 (H4R3) methylation by PRMT1 in vitro. Full kinetic characterization and mass spectrometry analysis indicated that PRMT1 is only active with recombinant nucleosomes monomethylated at H4K20 by SETD8. These data suggests that the level of activity of PRMT1 could potentially be regulated selectively by SETD8 in various pathways, providing a new approach for discovery of selective regulators of PRMT1 activity.

Chicken PRMT1 promotes infectious bursal disease virus replication via suppressing IFN-β production

The protein arginine methyltransferase (PRMT) family, such as PRMT1, regulates the arginine methylation of various substrates. Many studies have examined the role of PRMT1 in mammals, however, it is still unknown how PRMT1 works in chickens. To investigate the effect of chicken PRMT1 (chPRMT1) on regulating IFN-β production and IBDV replication, chPRMT1 knock out DF-1 cells were constructed in this study. First, we found that chPRMT1 was widely expressed in a variety of chicken tissues and that it was distributed in the cytoplasm and nucleus of DF-1 cells. Additionally, IFN-β activation was inhibited by chPRMT1 at the step of chMAVS. In addition, chPRMT1 knock out DF-1 cells were constructed using CRISPR-Cas9 technique. The morphology and viability of chPRMT1 knock out DF-1 cells were similar with the wild-type cells. In addition, the IFN-β as well as interferon stimulate genes activation induced by chMAVS in PRMT1 knock out DF-1 cells were significantly higher than that in WT cells. Furthermore, ectopic expression of chPRMT1 significantly supports IBDV replication. We also found that the ability of IBDV replication in PRMT1 knock out DF-1 cells was remarkably lower than that of in WT cells, suggesting that PRMT1 negatively regulate IBDV replication via suppressing IFN-β production. In conclusion, the PRMT1 knock out DF-1 cells were constructed, which was further used to demonstrate an inhibitory role of chPRMT1 in IFN-β production, and a contributor of chPRMT1 in IBDV replication.

Design, synthesis and evaluation of antitumor activity of selective PRMT6 inhibitors

PRMT6 is a member of the protein arginine methyltransferase family, which is involved in a variety of physiological processes and plays an important role in the occurrence and development of tumors. Due to the high homology of type Ⅰ PRMTs and the two close binding sites of the SAM pocket and the substrate pocket, selective PRMT6 inhibitors have rarely been reported. In this study, a series of (5-phenylpyridin-3-yl)methanamine derivatives were designed and synthesized, which could form hydrogen bonding interactions with the unique Glu49 of PRMT6, thereby improving the selectivity of the compounds for PRMT6. Among them, a25 had the best activity and selectivity, with more than 25-fold selectivity for PRMT1/8 and more than 50-fold selectivity for PRMT3/4/5/7, which was superior to these reported SAM competitive and substrate competitive PRMT6 inhibitors. Importantly, a25 could significantly inhibit the proliferation of various tumor cells and effectively induce apoptosis of cancer cells. Our data clarified that a25 is a promising selective PRMT6 inhibitor for cancer therapy which is worthy of further evaluation.

Arginine Methylation of the PGC-1α C-Terminus Is Temperature-Dependent.

We set out to determine whether the C-terminus (amino acids 481-798) of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α, UniProt Q9UBK2), a regulatory metabolic protein involved in mitochondrial biogenesis, and respiration, is an arginine methyltransferase substrate. Arginine methylation by protein arginine methyltransferases (PRMTs) alters protein function and thus contributes to various cellular processes. In addition to confirming methylation of the C-terminus by PRMT1 as described in the literature, we have identified methylation by another member of the PRMT family, PRMT7. We performed in vitro methylation reactions using recombinant mammalian PRMT7 and PRMT1 at 37, 30, 21, 18, and 4 °C. Various fragments of PGC-1α corresponding to the C-terminus were used as substrates, and the methylation reactions were analyzed by fluorography and mass spectrometry to determine the extent of methylation throughout the substrates, the location of the methylated PGC-1α arginine residues, and finally, whether temperature affects the deposition of methyl groups. We also employed two prediction programs, PRmePRed and MePred-RF, to search for putative methyltransferase sites. Methylation reactions show that arginine residues R548 and R753 in PGC-1α are methylated at or below 30 °C by PRMT7, while methylation by PRMT1 was detected at these same residues at 30 °C. Computational approaches yielded additional putative methylarginine sites, indicating that since PGC-1α is an intrinsically disordered protein, additional methylated arginine residues have yet to be experimentally verified. We conclude that temperature affects the extent of arginine methylation, with more methylation by PRMT7 occurring below physiological temperature, uncovering an additional control point for PGC-1α.

PRMT3 drives glioblastoma progression by enhancing HIF1A and glycolytic metabolism

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, but the mechanisms underlying tumor growth and progression remain unclear. The protein arginine methyltransferases (PRMTs) regulate a variety of biological processes, however, their roles in GBM growth and progression are not fully understood. In this study, our functional analysis of gene expression networks revealed that among the PRMT family expression of PRMT3 was most significantly enriched in both GBM and low-grade gliomas. Higher PRMT3 expression predicted poorer overall survival rate in patients with gliomas. Knockdown of PRMT3 markedly reduced the proliferation and migration of GBM cell lines and patient-derived glioblastoma stem cells (GSC) in cell culture, while its over-expression increased the proliferative capacity of GSC cells by promoting cell cycle progression. Consistently, stable PRMT3 knockdown strongly inhibited tumor growth in xenograft mouse models, along with a significant decrease in cell proliferation as well as an increase in apoptosis. We further found that PRMT3 reprogrammed metabolic pathways to promote GSC growth via increasing glycolysis and its critical transcriptional regulator HIF1α. In addition, pharmacological inhibition of PRMT3 with a PRMT3-specific inhibitor SGC707 impaired the growth of GBM cells. Thus, our study demonstrates that PRMT3 promotes GBM progression by enhancing HIF1A-mediated glycolysis and metabolic rewiring, presenting a point of metabolic vulnerability for therapeutic targeting in malignant gliomas.

Conjugates of adenosine mimetics and arginine-rich peptides serve as inhibitors and fluorescent probes but not as long-lifetime photoluminescent probes for protein arginine methyltransferases.

The conjugates of an adenosine mimetic and oligo-l-arginine or oligo-d-arginine (ARCs) were initially designed in our research group as inhibitors and photoluminescent probes targeting basophilic protein kinases. Here, we explored a panel of ARCs and their fluorescent derivatives in biochemical assays with members of the protein arginine methyltransferase (PRMT) family, focusing specifically on PRMT1. In the binding/displacement assay with detection of fluorescence anisotropy, we found that ARCs and arginine-rich peptides could serve as high-affinity ligands for PRMT1, whereas the equilibrium dissociation constant values depended dramatically on the number of arginine residues within the compounds. The fluorescently labeled probe ARC-1081 was displaced from its complex with PRMT1 by both S-adenosyl-l-methionine (SAM) and S-adenosyl-l-homocysteine (SAH), indicating binding of the adenosine mimetic of ARCs to the SAM/SAH-binding site within PRMT1. The ARCs that had previously shown microsecond-lifetime photoluminescence in complex with protein kinases did not feature such property in complex with PRMT1, demonstrating the selectivity of the time-resolved readout format. When tested against a panel of PRMT family members in single-dose inhibition experiments, a micromolar concentration of ARC-902 was required for the inhibition of PRMT1 and PRMT7. Overall, our results suggest that the compounds containing multiple arginine residues (including the well-known cell-penetrating peptides) are likely to inhibit PRMT and thus interfere with the epigenetic modification status in complex biological systems, which should be taken into consideration during interpretation of the experimental data.

A peptoid-based inhibitor of protein arginine methyltransferase 1 (PRMT1) induces apoptosis and autophagy in cancer cells

Protein arginine methyltransferases (PRMTs) are S-adenosylmethionine-dependent enzymes that transfer a methyl group to arginine residues within proteins, most notably histones. The nine characterized PRMT family members are divided into three types depending on the resulting methylated product: asymmetric dimethylarginine (Type I PRMT), symmetric dimethylarginine (Type II PRMT), or monomethylated arginine (Type III PRMT). In some cancers, the resulting product can lead to either increased or decreased transcription of cancer-related genes, suggesting PRMT family members may be valid therapeutic targets. Traditionally, peptide-based compounds have been employed to target this family of enzymes, which has resulted in multiple tool and lead compounds being developed. However, peptide-based therapeutics suffer from poor stability and short half-lives, as proteases can render them useless by hydrolytic degradation. Conversely, peptoids, which are peptide-mimetics composed of N-substituted glycine monomers, are less susceptible to hydrolysis, resulting in improved stability and longer half-lives. Herein, we report the development of a bioavailable, peptoid-based PRMT1 inhibitor that induces cell death in MDA468 and HCT116 cancer cell lines while not exhibiting any significant impact on nontumorigenic HepaRG or normal human mammary epithelial cells. Furthermore, the inhibitor described herein appears to induce both apoptosis and autophagy, suggesting it may be a less toxic cytostatic agent. In conclusion, we propose this peptoid-based inhibitor has significant anticancer and therapeutic potential by reducing cell viability, growth, and size in breast and colon cancer. Further experimentation will help determine the mechanism of action and downstream effects of this compound.

A Direct Assay for Measuring the Activity and Inhibition of Coactivator-Associated Arginine Methyltransferase 1.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a member of the family of protein arginine methyltransferases. CARM1 catalyzes methyl group transfer from the cofactor S-adenosyl-l-methionine (AdoMet) to both histone and nonhistone protein substrates. CARM1 is involved in a range of cellular processes, mainly involving RNA transcription and gene regulation. As the aberrant expression of CARM1 has been linked to tumorigenesis, the enzyme is a potential therapeutic target, leading to the development of inhibitors and tool compounds engaging with CARM1. To evaluate the effects of these compounds on the activity of CARM1, sensitive and specific analytical methods are needed. While different methods are currently available to assess the activity of methyltransferases, these assays mainly focus on either the measurement of the cofactor product S-adenosyl-l-homocysteine (AdoHcy) or employ radioactive or expensive reagents, each with their own advantages and limitations. To complement the tools currently available for the analysis of CARM1 activity, we here describe the development of a convenient assay employing peptide substrates derived from poly(A)-binding protein 1 (PABP1). This operationally straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach allows for the direct detection of substrate methylation with minimal workup. The method was validated, and its value in characterizing CARM1 activity and inhibition was demonstrated through a comparative analysis involving a set of established small molecules and peptide-based CARM1 inhibitors.