Substrate Specificity, Processivity, and Kinetic Mechanism of Protein Arginine Methyltransferase 5

Protein arginine methyltransferases (PRMTs) have emerged as attractive therapeutic targets for heart disease and cancers. PRMT5 is a particularly interesting target because it is overexpressed in blood, breast, colon, and stomach cancers and promotes cell survival in the face of DNA damaging agents. As the only known member of the PRMT enzyme family to catalyze the formation of mono- and symmetrically dimethylated arginine residues, PRMT5 is also mechanistically unique. As a part of a program to characterize the mechanisms and regulation of the PRMTs and develop chemical probes targeting these enzymes, we characterized the substrate specificity, processivity, and kinetic mechanism of bacterially expressed <i>Caenorhabditis elegans</i> PRMT5 (cPRMT5). In this report, we demonstrate that distal positively charged residues contribute to substrate binding in a synergistic fashion. Additionally, we show that cPRMT5 catalyzes symmetric dimethylation in a distributive fashion. Finally, the results of initial velocity, product, and dead-end inhibition studies indicate that cPRMT5 uses a rapid equilibrium random mechanism with dead-end EAP and EBQ complexes. In total, these studies will guide PRMT5 inhibitor development and lay the foundation for studying how the activity of this medically relevant enzyme is regulated

Selective small-chemical inhibitors of protein arginine methyltransferase 5 with anti-lung cancer activity

<div><p>Protein arginine methyltransferase 5 (PRMT5) plays critical roles in a wide variety of biological processes, including tumorigenesis. By screening a library of small chemical compounds, we identified eight compounds that selectively inhibit the PRMT5 enzymatic activity, with IC₅₀ values ranging from 0.1 to 6 μM. Molecular docking simulation and site-directed mutagenesis indicated that identified compounds target the substrate-binding site in PRMT5. Treatment of lung cancer cells with identified inhibitors led to inhibition of the symmetrical arginine methylation of SmD3 and histones and the cellular proliferation. Oral administration of the inhibitor demonstrated antitumor activity in a lung tumor xenograft model. Thus, identified PRMT5-specific small-molecule inhibitors would help elucidate the biological roles of PRMT5 and serve as lead compounds for future drug development.</p></div

Chemical structures of identified chemical compounds.

<p>Chemical structures of identified chemical compounds.</p

The C9 analogue has improved PRMT5-inhibitory efficacy and oral drug availability.

<p>A, The chemical structure of the C9 analogue. B, Compound C9a inhibited methylation of SmD3 by PRMT5. The recombinant SmD3 (2 μg) was used as the substrate in the absence (lane 5) or presence of 1, 10 or 100 μM compounds as indicated. Lane 1 shows the product of the reaction without the enzyme. C, C9a has improved efficacy to inhibit lung cancer growth. A549 cells were cultured in the presence of various concentrations of C9 or C9a and growth inhibition was determined by cell counting 2 days post the compound treatment. D, C9a has improved oral drug availability in the mouse. Male BALB/c mice were randomized and administrated C9a by oral gavage and compound concentrations in blood samples were determined.</p

Steady-state kinetic analysis of PRMT5 inhibition by compounds C2 and C5.

<p><b>Double-reciprocal plot of reaction velocities (cpm per minute) versus concentrations of SmD3.</b> Methylation reaction mixtures contained various concentrations of C2 (left) or C5 (right), various concentrations of SmD3, 1.2 μg of PRMT5 and 5 μM AdoMet. The mixtures were incubated for different times and the amounts of methylated products were quantified using DE81 paper and liquid scintillation. Reaction velocity was calculated as H³ incorporation (cpm) into SmD3 per minute.</p

The conserved phenylalanine residue in PRMT5 is critical for inhibition by the five compounds.

<p>A, DS-PAGE of recombinant wild type (WT) and F225M mutant (MT) PRMT5. The gel was stained with Coomassie Brilliant Blue R250. A band corresponding to an unknown bacterial protein is indicated (star). B, The F225M mutation increased the methytransferase activity of PRMT5. <i>In vitro</i> methylation reactions were performed with recombinant wild type (lanes 2–4) or mutant (lanes 5–7) PRMT5 (1.2, 2.4 or 4.8 μg) and SmD3 (2 μg) as the substrate. C, The F225M mutation in PRMT5 decreased inhibition of arginine methylation by the compounds. The reaction mixture contained SmD3 (2 μg); wild type or mutant PRMT5 (1.2 μg); and 1, 10 or 100 μM compounds as indicated. D, The F225M mutation in PRMT5 decreased the IC₅₀ values of the compounds, as determined by quantitation and analysis of methylation assay results.</p

The compound C9a inhibited growth of lung tumor xenografts in nude mice.

<p>A, Lungs derived from mice injected with A549 cells treated with the vehicle or C9a at the dosage of 100 mg/kg for 21 days. B, Mean size of tumors. C, The C9a treatment inhibited histone 2A (H2A) arginine methylation in lung tumor xenografts. Histones were isolated from tumors and analyzed by SDS-PAGE stained with Commassie blue R250 (bottom) or by Western blot with anti-symmetric dimethyl arginine antibody (SYM10) (top).</p

Identified compounds inhibited growth of lung cancer cells.

<p>A, Lung cancer A549 cells were cultured in the presence of various concentrations (μM) of identified compounds and growth inhibition was determined by cell counting at various time points. B, Lung cancer PC14 cells were cultured in the presence of DMSO or various concentrations of identified compounds and growth inhibition was determined by cell counting 2 days post the compound treatment. C, The serum concentrations of compound C9 after oral administration. Mice were sacrificed and blood samples were collected immediately before (0 h) and at 2, 4, 6 and 8h after compound administration.</p

The compounds interact with the substrate-binding region in PRMT5.

<p>Docking of compounds C2, C5, C6 or C9 in PRMT5. The amino acid residues in PRMT5 likely to interact with compound C2, C5, C6 or C9 are indicated in yellow.</p

Identified compounds inhibited cellular targets of PRMT5.

<p><b>A, The cellular permeability of identified compounds.</b> Lung cancer A549 cells were cultured in the presence of various concentrations of compounds for 2h and the compound concentrations in the cell were determined by HPLC chromatography with a C18 column. B, The cellular stability of identified compounds. Lung cancer A549 cells were cultured in the presence of compounds (20 μM) for 2 h and then the medium was removed. Cells were washed with the medium and cultured for additional 5 or 10 h after the compounds were removed. The compound concentrations in the cell were determined by HPLC chromatography with a C18 column. C, Identified compounds inhibited arginine methylation of histones and B-Raf in A549 cells. A549 cells were grown in the presence of DMSO or compound C5, C9 or C11 (20 μM). Histones were purified and analyzed by SDS-PAGE stained with Commassie blue (lanes 1–4, bottom) or by Western blot with anti-symmetric dimethyl arginine antibody (SYM10) (lanes 1–4, top). B-Raf was immunoprecipitated with anti-B-Raf antibody from whole cell lysates and submitted for Western blot analysis with anti-B-Raf (lanes 5 and 6, bottom) or anti-symmetric dimethyl arginine (lanes 5 and 6, top) antibody.</p