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.

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.

Transcriptional perturbation of protein arginine methyltransferase-5 exhibits MTAP-selective oncosuppression

We hypothesized that small molecule transcriptional perturbation could be harnessed to target a cellular dependency involving protein arginine methyltransferase 5 (PRMT5) in the context of methylthioadenosine phosphorylase (MTAP) deletion, seen frequently in malignant pleural mesothelioma (MPM). Here we show, that MTAP deletion is negatively prognostic in MPM. In vitro, the off-patent antibiotic Quinacrine efficiently suppressed PRMT5 transcription, causing chromatin remodelling with reduced global histone H4 symmetrical demethylation. Quinacrine phenocopied PRMT5 RNA interference and small molecule PRMT5 inhibition, reducing clonogenicity in an MTAP-dependent manner. This activity required a functional PRMT5 methyltransferase as MTAP negative cells were rescued by exogenous wild type PRMT5, but not a PRMT5E444Q methyltransferase-dead mutant. We identified c-jun as an essential PRMT5 transcription factor and a probable target for Quinacrine. Our results therefore suggest that small molecule-based transcriptional perturbation of PRMT5 can leverage a mutation-selective vulnerability, that is therapeutically tractable, and has relevance to 9p21 deleted cancers including MPM.