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

Abstract

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. 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. Arginine methylation is a common posttranslational modification that occurs when methyl groups are transferred to the guanidinyl side chain of arginine residues. The protein arginine methyltransferase (PRMT) family of enzymes are S-adenosylmethionine-dependent enzymes that catalyze this transfer, which can result in significant changes to protein–protein interactions, as well as protein–DNA/RNA interactions. Among the most well-studied substrates for PRMT enzymes are the N-terminal tails of histone proteins. Given the critical role that histones play in the packaging of DNA, it is not surprising that posttranslational modifications to the histones affects the translation of some genes (1Pal S. Sif S. Interplay between chromatin remodelers and protein arginine methyltransferases.J. Cell. Physiol. 2007; 213: 306-315Crossref PubMed Scopus (120) Google Scholar, 2Blanc R.S. Richard S. Arginine methylation: the coming of age.Mol. 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Type I enzymes, a group that includes PRMTs 1, 2, 3, 4, 6, and 8, catalyze the sequential additions of two methyl groups to yield asymmetric dimethylarginine (ADMA) residues. Type II enzymes, which include PRMTs 5 and 9, catalyzed the sequential addition of two methyl groups to yield symmetric dimethylarginine (SDMA) residues. The lone member of the Type III group, PRMT 7, catalyzes the addition of a single methyl group to form monomethylarginine (MMA) residues (6Bedford M.T. Arginine methylation at a glance.J. Cell Sci. 2007; 120: 4243-4246Crossref PubMed Scopus (259) Google Scholar, 7Bedford M.T. Clarke S.G. Protein arginine methylation in mammals: who, what, and why.Mol. Cell. 2009; 33: 1-13Abstract Full Text Full Text PDF PubMed Scopus (1240) Google Scholar, 8Di Lorenzo A. Bedford M.T. Histone arginine methylation.FEBS Lett. 2011; 585: 2024-2031Crossref PubMed Scopus (339) Google Scholar, 9Wolf S.S. 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The conversion of this residue to ADMA is associated with the transcriptional activation of genes under the control of p53 and estrogen receptors, among others (1Pal S. Sif S. Interplay between chromatin remodelers and protein arginine methyltransferases.J. Cell. Physiol. 2007; 213: 306-315Crossref PubMed Scopus (120) Google Scholar, 11Lee Y.H. Stallcup M.R. Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.Mol. Endocrinol. 2009; 23: 425-433Crossref PubMed Scopus (163) Google Scholar). Conversely, the conversion of the same residue to SDMA is associated with transcriptional repression of the same genes (1Pal S. Sif S. Interplay between chromatin remodelers and protein arginine methyltransferases.J. Cell. Physiol. 2007; 213: 306-315Crossref PubMed Scopus (120) Google Scholar, 12Herrmann F. Pably P. Eckerich C. Bedford M.T. Fackelmayer F.O. Human protein arginine methyltransferases in vivo--distinct properties of eight canonical members of the PRMT family.J. Cell Sci. 2009; 122: 667-677Crossref PubMed Scopus (108) Google Scholar, 13Zhao X. Jankovic V. Gural A. Huang G. Pardanani A. Menendez S. et al.Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity.Genes Dev. 2008; 22: 640-653Crossref PubMed Scopus (135) Google Scholar, 14Pal S. Baiocchi R.A. Byrd J.C. Grever M.R. Jacob S.T. Sif S. Low levels of miR-92b/96 induce PRMT5 translation and H3R8/H4R3 methylation in mantle cell lymphoma.EMBO J. 2007; 26: 3558-3569Crossref PubMed Scopus (218) Google Scholar, 15Chen D. Ma H. Hong H. Koh S.S. Huang S.M. Schurter B.T. et al.Regulation of transcription by a protein methyltransferase.Science. 1999; 284: 2174-2177Crossref PubMed Scopus (996) Google Scholar). More specifically, PRMT1 has been shown to catalyze the production of H4R3me2a, and this specific modification is a key switch of the epithelial–mesenchymal transition at the ZEB1 promoter, activating its transcription leading to breast cancer (16Gao Y. Zhao Y. Zhang J. Lu Y. Liu X. Geng P. et al.The dual function of PRMT1 in modulating epithelial-mesenchymal transition and cellular senescence in breast cancer cells through regulation of ZEB1.Sci. Rep. 2016; 6: 1-13PubMed Google Scholar). Further studies have also identified overexpression of PRMT1 in some cancers, including leukemia, prostate, esophageal, lung, bladder, and breast compared to normal cells leading to increased protein levels and promoting cell proliferation (5Yang Y. Bedford M.T. Protein arginine methyltransferases and cancer.Nat. Rev. Cancer. 2013; 13: 37-50Crossref PubMed Scopus (699) Google Scholar, 17Liu L. Sun W. Fan X. Xu Y. Cheng M. Zhang Y. Methylation of C/EBPα by PRMT1 inhibits its tumor-suppressive function in breast cancer.Cancer Res. 2019; 79: 2865-2877Crossref PubMed Scopus (35) Google Scholar, 18Yu Z. Chen T. Hébert J. Li E. Richard S. A mouse PRMT1 null allele defines an essential role for arginine methylation in genome maintenance and cell proliferation.Mol. Cell. Biol. 2009; 29: 2982-2996Crossref PubMed Scopus (140) Google Scholar, 19Zhao Y. Lu Q. Li C. Wang X. Jiang L. Huang L. et al.PRMT1 regulates the tumour-initiating properties of esophageal squamous cell carcinoma through histone H4 arginine methylation coupled with transcriptional activation.Cell Death Dis. 2019; 10: 1-17Crossref Scopus (30) Google Scholar). These and other studies have identified PRMTs, specifically PRMT1, as a key contributor to the proliferation of cancers through various mechanisms including epigenetic-mediated gene expression (20Mathioudaki K. Papadokostopoulou A. Scorilas A. Xynopoulos D. Agnanti N. Talieri M. The PRMT1 gene expression pattern in colon cancer.Br. J. Cancer. 2008; 99: 2094-2099Crossref PubMed Scopus (96) Google Scholar, 21Yoshimatsu M. Toyokawa G. Hayami S. Unoki M. Tsunoda T. Field H.I. et al.Dysregulation of PRMT1 and PRMT6, type I arginine methyltransferases, is involved in various types of human cancers.Int. J. Cancer. 2011; 128: 562-573Crossref PubMed Scopus (228) Google Scholar, 22Baldwin R.M. Morettin A. Paris G. Goulet I. Côté J. Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells.Cell Cycle. 2012; 11: 4597-4612Crossref PubMed Scopus (61) Google Scholar, 23Baldwin R.M. Morettin A. Côté J. Role of PRMTs in cancer: could minor isoforms be leaving a mark?.World J. Biol. Chem. 2014; 5: 115-129PubMed Google Scholar). To date, several PRMT inhibitors have been reported, including some that are isozyme specific. One of the first non-AdoMet–based pan inhibitors for Type I PRMTs was developed in 2004 but only recently has a PRMT inhibitor made it to clinical trials (24Wu Q. Schapira M. Arrowsmith C. Barsyte-Lovejoy D. Protein arginine methylation: from enigmatic functions to therapeutic targeting.Nat. Rev. Drug Discov. 2021; 20: 509-530Crossref PubMed Scopus (58) Google Scholar, 25Jarrold J. Davies C. PRMTs and arginine methylation: cancer's best-kept secret?.Trends Mol. Med. 2019; 25: 993-1009Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Given that PRMTs modify protein substrates, it is not surprising that many peptide-based compounds have been developed to study these enzymes (26Bicker K.L. Obianyo O. Rust H.L. Thompson P.R. A combinatorial approach to characterize the substrate specificity of protein arginine methyltransferase 1.Mol. Biosyst. 2011; 7: 48-51Crossref PubMed Scopus (22) Google Scholar, 27Vhuiyan M. Thomas D. Hossen F. Frankel A. Targeting protein arginine N-methyltransferases with peptide-based inhibitors: opportunities and challenges.Future Med. Chem. 2013; 5: 2199-2206Crossref PubMed Scopus (10) Google Scholar, 28Zhang Y. van Haren M. Martin N. Peptidic transition state analogues as PRMT inhibitors.Methods. 2020; 175: 24-29Crossref PubMed Scopus (5) Google Scholar). One obvious advantage of peptide-based inhibitors is the and the by the which can be substrate we recently the development a peptide-based that for PRMT1 PRMT 5 in S. A. C. The development and of a PRMT1 Med. Chem. 2019; PubMed Scopus Google Scholar). One of peptide-based compounds as in and therapeutics is their by we the of as an to which are of N-substituted glycine as a potential for as are less susceptible to hydrolytic degradation. However, these a of that is to and can be by A. of N-substituted glycine and PubMed Scopus Google Scholar, for the of by Chem. Scopus Google Scholar). specific may among various enzymes, the in and them clinical inhibitors have been developed for enzymes and as their in H. M. J. J. et of for Biol. 2013; 20: Full Text Full Text PDF PubMed Scopus Google Scholar, T. of a inhibitor of the Chem. 2007; PubMed Scopus Google Scholar, M. C. C. van W. of the substrate specificity of by 2020; 10: Scopus Google Scholar, K. of of J. 2020; PubMed Scopus Google Scholar, K. S. in the development of 2020; PubMed Google Scholar). studies found that peptoid-based of a PRMT not by the PRMT these found to the enzyme at S. M. C. Histone are inhibitors of protein arginine methyltransferase 1 J. 2020; PubMed Scopus Google Scholar). This to the development of peptoid-based compounds as inhibitors of Herein, we the development of a peptoid-based inhibitor that inhibits PRMT1 PRMT5 with an in the in studies have shown that this cell and potential in MDA468 breast carcinoma and HCT116 colon carcinoma cell with in normal and nontumorigenic cells. Further this inhibitor induces apoptosis and to and cell death in cancer cells. Histone have been shown to be inhibitors of PRMT1 with some of In this only a single in its which the and can be for the development of this single residue to a the to to the PRMT1 and lead to and PRMT1 a within the but this residue is not in most other PRMT with a a could lead to modification of this we to an with a (Fig. To determine was in to these peptoids, we both an and of the and and by with a of and and to have improved and as compared to the that the to modify PRMT1 S. M. C. Histone are inhibitors of protein arginine methyltransferase 1 J. 2020; PubMed Scopus Google Scholar). To this we an of these compounds for PRMT1 a methyltransferase in addition to their to target PRMT1 PRMT1 was with and resulted in an of (Fig. which is improved the However, resulted in a of with to PRMT1 (Fig. and a in compared to the for PRMT1 of These studies that a N terminus to increased binding and of PRMT1 Obianyo O. Zhang X. Cheng X. Thompson P.R. Protein arginine methyltransferase residues in substrate to the of methylation are for substrate binding and 2007; PubMed Scopus Google Scholar). Furthermore, we to determine the which for PRMT1 The for compounds and with PRMT5 both to be (Fig. 3, and in and improved for PRMT1 The in between PRMT1 and PRMT5 was not surprising but that we can target members of this family to peptoid-based Given the improved and was for To study the anticancer potential of MDA468 breast carcinoma and HCT116 colon carcinoma cells with HepaRG cells in the of various of for and cell was the significant in a on both cancer cell while was in the of the nontumorigenic HepaRG cells (Fig. 4, A and The proliferation was most in MDA468 cells at and at HCT116 to the the effects less with at and at the (Fig. we the specificity of on cancer cells a of cancer and normal cells in the there was a and in cell in both cancer cell lines (Fig. A and In significant in and cells at both and with in a in both MDA468 and HCT116 cancer cells (Fig. and not In nontumorigenic HepaRG and normal human mammary epithelial cells in (Fig. and These that specifically cancer cells and their and potential significant to normal and nontumorigenic cells. the of treatment on the and of the impact of the was on the cells to form and (Fig. 6, The a significant in the of MDA468 and with A of in the of HCT116 was with 5 (Fig. and with treatment in MDA468 (Fig. These the of to of cancer cells. To the mechanism of anticancer we cells with for changes in to the cells and normal in the while several cells in the of apoptosis and of in cells of (Fig. To for was in protein from and cells. was in a and in MDA468 and HCT116 cells and of treatment with (Fig. 7, and In activation of was in HepaRG cells and only at the at (Fig. 7, and these apoptosis as a mechanism of action for the anticancer of In we was also to the treatment with as a of cells of (Fig. we cell in and cells on as a common in cells is a in significant in size of cells in both cancer cell lines while HepaRG (Fig. This in cell size could also be an of (Fig. To we the levels of protein chain a of autophagy, in three cell lines as a to The the was less in the nontumorigenic HepaRG (Fig. the both apoptosis and are by to induce and cell death in cancer cells. PRMT family members catalyze the methylation of arginine residues in a of proteins but have been in to their modification of the N-terminal tails of histone proteins. PRMT have substrate specificity in to these histone PRMT1 and PRMT5 are for of the methylation of on histone H4. not but is for the of methylation found on on histone These and in substrate specificity an to inhibitors either single PRMTs or multiple family based on the N-terminal of these histones have been shown to have and that are to of histones. a of compounds that and to specifically to proteins. have been as to physiological functions and have also been to protein–protein interactions L. Wang N. Zhang W. Cheng X. Z. G. et and 7: PubMed Scopus Google Scholar, Wang W. Ma The and development of inhibitors for cancer 2020; 13: Google Scholar). Furthermore, the of and of production have made compounds as However, of the in peptide-based therapeutics is their poor stability and short due to degradation. The of peptoids, which are the side chain on the of the of the the of Furthermore, these compounds cellular their which also their potential as Y. T. of the cell of and Chem. 2007; PubMed Scopus Google Scholar, J. M. A. K. et to cell in Lett. PubMed Scopus Google Scholar). a of the histone H4 N-terminal recently developed by the residues with monomers, thus the side chain from the to the on the However, the of was a poor PRMT1 substrate with the was a inhibitor of PRMT1 with in the the are for a for inhibitor the PRMT family S. M. C. Histone are inhibitors of protein arginine methyltransferase 1 J. 2020; PubMed Scopus Google Scholar). this was to compounds that have binding and specifically target on this we the side chain of the with a to a and compound. The in of the of the arginine side specifically on the histone H4 N-terminal for specific PRMTs to be by this through of PRMT1 by of a with O. Y.H. Stallcup M.R. et of protein arginine methyltransferase and in and in 11: PubMed Scopus (35) Google Scholar). This was to some peptide-based tool compounds PRMT1, the of we this into the on the the of the into the increased the as compared to the with the in the resulted in in PRMT1 and PRMT5 are for of protein arginine methylation in we the inhibitors PRMT5 to determine between these two on the is for PRMT1 PRMT5 by based on the The result in improved and potential as compared to the However, the of these compounds also and the to the role that the may play in to (Fig. The an to the compared to the was the versus In this the peptoid-based inhibitors and stability while also the as compared with Given the role of PRMTs in as breast and colon we the of in MDA468 and HCT116 cell lines compared to both HepaRG and in a in both cancer cell MDA468 and with the of normal cell lines and This that has both specificity and anticancer This is with the result in PRMT pan inhibitors and by et J. P. M. D. M. et arginine methyltransferase (PRMT) and are in and proliferation in cell J. Mol. Sci. 2021; 22: PubMed Scopus (5) Google a in cell of and cell lines with the of the Given that and are pan PRMT of PRMT1 was by a in the levels of PRMT1 methylated et Y. M. H. G. Li F. et and inhibitor of human type I protein arginine Chem. Biol. 2016; 11: PubMed Scopus Google also of decreased a PRMT type I pan a in cell and a downstream of both PRMT1 and by the of and S. M. C. Histone are inhibitors of protein arginine methyltransferase 1 J. 2020; PubMed Scopus Google Scholar). the PRMT is a and the this specificity to the However, the in specificity of PRMT1 was not in this was in both cancer cell but MDA468 cells less but while HCT116 a but This is by the of cell the and the protein of with MDA468 both the and p53 et A. S. S. S. L. N. et of the type I PRMT with PRMT5 through Cell. 2019; Full Text Full Text PDF PubMed Scopus Google at type I PRMT cell lines and a in proliferation to a These that cell has its In that the of the gene may the of cell lines to PRMT type I inhibitors through of PRMT5 due to the of a PRMT 5 is a gene and can be in cancer cell This a for in to the However, as MDA468 has a this of p53 may to the of The increased in both cancer cell lines compared to the normal cells a role for a apoptosis mechanism of action by This was by the of in cells with the has been as the common of cell death treatment with PRMT pan inhibitors due to the of D. with PRMT inhibitors also cell at the Zhang S. L. Chen X. Zhao Y. L. et methyltransferase inhibitor 1 inhibits cancer by and 2017; PubMed Scopus Google Scholar, P. in Death 2017; PubMed Scopus Google Scholar). Cell studies at cells that treatment with the cells from the cell at the expression of in the three cell and a in expression not However, the of this is to be The studies the of multiple and the in the size specifically in cancer cells with and the increased expression of in three cell that may also play a role in the mechanism of action of is the survival of the cell it or is as a cell are a contributor to cell but studies have also shown can with proteins, the to most and induce However, and have an P. in Death 2017; PubMed Scopus Google Scholar). may be that levels are increased to autophagy, as many have been as substrates for These studies a between apoptosis and of may a in these leading to and cell In their and P. in Death 2017; PubMed Scopus Google that specific including may in the of to the cell from into apoptosis (Fig. The peptoid-based has a potential peptide-based compounds for development into therapeutics this PRMT inhibitor anticancer in MDA468 and HCT116 cell reducing cell viability, growth, and cell Further experimentation its and its mechanism of action are to the specificity of PRMT1 and to the and of the in is essential for its as a The of can be by at the levels of PRMT1 and its products in versus cell These help a of the downstream effects of this compound. be between cells with and a cell with PRMT1 These and will be in the in addition to the mechanism of action by while also to on a of the and the genes being

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