Synthesis and Validation of Substrates for PRMT1 using Plate-Based Screening Assay

PRMT (Protein Arginine Methyltransferase) is a mammalian enzyme that catalyzes methylation of arginine residues in a polypeptide chain. PRMT is categorized as 3 different types. The methylation can occur as asymmetric dimethylation (ADMA, PRMT 1, 2, 3, 4, 6, and 8), symmetric dimethylation (SDMA, PRMT 7, 5 and 9) or monomethylation (MMA, PRMT 7), Type I, II and III respectively. PRMT1 generates ADMA on arginine residues of the Histone H4 N-terminal tail, which can lead to transcription of cancer-related genes. Alternatively, PRMT5 can modify the same arginine residue to produce SDMA, which represses the development of those same cancer-related genes. A better understanding of the substrate specificity of these enzymes can assist in the development of novel isozyme-specific pharmaceuticals.1 To identify these differences, we synthesized a 96-well plate of peptides based on the Histone H4 N-terminal tail, screened them against PRMT1 using a screening method previously developed in the Knuckley lab.2 This medium-throughput screen identified 7 “hit” peptide sequences and consensus sequences based on the “hit” peptides were synthesized by solid-phase peptide synthesis. Each of these consensus sequences varied at the N-terminus, while retaining the more distal positive charges of H4-16 peptide. The peptides were validated using a MTase-GloTM Methyltransferase Assay to determine if they were indeed substrates for PRMT1. The kinetic values indicate their efficiency as PRMT1 substrates and further investigations are being conducted to identify the differences in the substrate specificity regarding PRMT4 and PRMT5. These continued efforts will help us gain a better understanding of the role PRMT isozymes play in the onset of cancer, while assisting in the design of novel pharmaceuticals to battle this disease

Investigating the Substrate Specificity of PRMT1 using a Plate Based Screening Method

Enzymes are biological catalysts that speed up the rate of a reaction by lowering the activation energy and converting substrate (reactant) to product more expeditiously. Enzymes have a high degree of specificity and will only catalyze selective reactions by targeting particular substrates. In mammals, there exists a family of 11 enzymes, PRMT’s (Protein Arginine Methyl transferase), that target protein arginine for post translational methylation on the guanidino nitrogen of the residue. They occur naturally as Type I, II and III and can administer asymmetric di-methylation (ADMA), symmetric dimethylation (SDMA) and monomethylation, respectively. The focal point of this project is to determine the substrate specificity of PRMT1 (Type I), PRMT 4, (Type I) and PRMT5 (Type II) on arginine residues in histone tails. Histones are eukaryotic proteins. The “tails” of the histones (polypeptide chains) are projected out from the protein and are often targets of various post translational modifications (PTM’s). Overexpression of PRMT 1 and PRMT 4 results in increased enzyme activity leading to ADMA on the histones arginine’s. This modification recruit’s transcription factors to the histone tails and induces the transcription of cancerous genes (colon, breast, prostate cancer, etc.). Meanwhile, PRMT 5 is repressive of these developments by leading to SDMA on the arginine, which blocks the enzyme activity and recruitment of these same transcription factors.  By synthesizing the histone H4 tail in this experiment and varying different regions using high-throughput libraries, a screening of PRMT1, 4 and 5 can be completed

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