Cryo-EM Structure-guided Selection of Computed Ligand Poses to Enhance Potency in MTA-synergic Inhibition of Human Protein Arginine Methyltransferase 5

The potential of using cryo-electron microscopic (cryo-EM) structures of 2.5-4.0 Å resolutions for structure-based drug design was proposed recently, but is yet to be materialized. Here we show that a 3.1 Å cryo-EM structure of protein arginine methyltransferase 5 (PRMT5) is sufficient to guide the selection of computed poses of a bound inhibitor and its redesign for much higher potency. PRMT5 is an oncogenic target and its multiple inhibitors are in clinical trials for various cancer types. However, all these PRMT5 inhibitors manifest negative cooperativity with a metabolic co-factor analog --- 2-methylthioadenosine (MTA), which is accumulated substantially in cancer patients carrying defective MTA phosphorylase (MTAP). To achieve MTA-synergetic inhibition, we obtained a pharmacophore from virtual screen and synthesized a specific inhibitor (11-2F). Cryo-EM structures of the 11-2F/MTA-bound human PRMT5: MEP50 complex and its apo form together showed that the inhibitor binding in the catalytic pocket causes a shift of the cofactor-binding site by 1.5 – 2.0 Å, disfavoring cofactor-binding and resulting in positive cooperativity between 11-2F and MTA. Coarse-grained and full-atomistic MD simulations of the ligands in their binding pockets were performed to compare computed poses of 11-2F and its redesigned analogs. Three new analogs were predicted to have much better potency. One of them, after synthesis, was ~4 fold more efficient in PRMT5 inhibition in the presence of MTA than 11-2F itself. Computational analysis also suggests strong subtype specificity of 11-2F among PRMTs. These data demonstrate the feasibility of using cryo-EM structures of near-atomic resolutions and computational analysis of ligand poses for better small molecule therapeutics.

Structure, Activity and Function of the PRMT2 Protein Arginine Methyltransferase

PRMT2 belongs to the protein arginine methyltransferase (PRMT) family, which catalyzes the arginine methylation of target proteins. As a type I enzyme, PRMT2 produces asymmetric dimethyl arginine and has been shown to have weak methyltransferase activity on histone substrates in vitro, suggesting that its authentic substrates have not yet been found. PRMT2 contains the canonical PRMT methylation core and a unique Src homology 3 domain. Studies have demonstrated its clear implication in many different cellular processes. PRMT2 acts as a coactivator of several nuclear hormone receptors and is known to interact with a multitude of splicing-related proteins. Furthermore, PRMT2 is aberrantly expressed in several cancer types, including breast cancer and glioblastoma. These reports highlight the crucial role played by PRMT2 and the need for a better characterization of its activity and cellular functions.

Protein Arginine Methyltransferase 5 Promotes the Migration of AML Cells by Regulating the Expression of Leukocyte Immunoglobulin-Like Receptor B4

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults with poor prognosis. Especially for AML-M5 type, due to the strong cell migration ability, the possibility of extramedullary invasion is large and widespread, which leads to poor therapeutic effect. Previous studies have found that protein arginine methyltransferase 5 (PRMT5) could promote the proliferation and differentiation of leukemic cells in AML, but its regulation on the invasive ability of AML cells remains unclear. This study was designed to explore the role of PRMT5 in regulating the invasion of AML cells and to investigate the mechanisms. Patient samples were collected for detection of PRMT5 expression level. AML cells were used for exploring the function of PRMT5. The results of clinical samples showed that the expression of PRMT5 was significantly increased in newly diagnosed and recurrent AML patients, and the expression of leukocyte immunoglobulin-like receptor B4 (LILRB4) was positively correlated with the level of PRMT5. In the cell experiment in vitro, we found that when PRMT5 was knocked down, the invasion, migration, and adhesion capacities of MV-4-11 cells and THP-1 cells were decreased, and the mRNA and protein levels of LILRB4 were also decreased. Moreover, we screened related signaling pathways and found that PRMT5 affected the expression of downstream LILRB4 by activating mTOR pathway, which in turn enhanced the invasive ability of AML cells. Taken together, PRMT5 plays an important role in the invasion of AML, which acts via regulating the expression of LILRB4. PRMT5 could act as a potential therapeutic candidate for AML.

Structure, Activity and Function of the Protein Arginine Methyltransferase 6

Members of the protein arginine methyltransferase (PRMT) family methylate the arginine residue(s) of several proteins and regulate a broad spectrum of cellular functions. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that asymmetrically dimethylates the arginine residues of numerous substrate proteins. PRMT6 introduces asymmetric dimethylation modification in the histone 3 at arginine 2 (H3R2me2a) and facilitates epigenetic regulation of global gene expression. In addition to histones, PRMT6 methylates a wide range of cellular proteins and regulates their functions. Here, we discuss (i) the biochemical aspects of enzyme kinetics, (ii) the structural features of PRMT6 and (iii) the diverse functional outcomes of PRMT6 mediated arginine methylation. Finally, we highlight how dysregulation of PRMT6 is implicated in various types of cancers and response to viral infections.