Diamidine compounds for selective inhibition of protein arginine methyltransferase 1

Abstract

Protein arginine methylation is a posttranslational modification critical for a variety of biological processes. Misregulation of protein arginine methyltransferases (PRMTs) has been linked to many pathological conditions. Most current PRMT inhibitors display limited specificity and selectivity, indiscriminately targeting many methyltransferase enzymes that use S-adenosyl-l-methionine as a cofactor. Here we report diamidine compounds for specific inhibition of PRMT1, the primary type I enzyme. Docking, molecular dynamics, and MM/PBSA analysis together with biochemical assays were conducted to understand the binding modes of these inhibitors and the molecular basis of selective inhibition for PRMT1. Our data suggest that 2,5-bis(4-amidinophenyl)furan (1, furamidine, DB75), one leading inhibitor, targets the enzyme active site and is primarily competitive with the substrate and noncompetitive toward the cofactor. Furthermore, cellular studies revealed that 1 is cell membrane permeable and effectively inhibits intracellular PRMT1 activity and blocks cell proliferation in leukemia cell lines with different genetic lesions.

Figure 1

Structures of tested amidine compounds.

Figure 2

Kinetic analysis of PRMT1 inhibition by compound 1. (A) and (C) are Michaelis–Menten plots, and (B) and (D) are double-reciprocal plot of initial velocities versus varied concentrations of H4-20 or [³H]SAM. Concentration of 1 was selected at 0 μM (▲), 10 μM (■), 20 μM (●), 30 μM (×), and 40 μM (○). In (A) and (B), the concentration of [³H]AdoMet was fixed at 3 μM, and in (C) and (D), the concentration of H4-20 was fixed at 15 μM.

Figure 3

Competitive binding measurement with fluorescence anisotropy: fluorescence anisotropy (524 nm) of H4-FL and PRMT1 complex at different concentrations of compound 1. The concentrations of H4-FL and PRMT1 were kept constant at 0.2 and 2.0 μM, respectively.

Figure 4

Predicted binding modes of compound 1 in PRMT1 and PRMT5 from docking (AutoDock 4.2) and molecular dynamics simulation (NAMD 2.8). Ligand–residue interaction energies from MM/PBSA energy decomposition for (A) PRMT1 and (B) PRMT5. (C, D) Binding modes of compound 1 with (C) PRMT1 and (D) PRMT5. The best docking pose obtained from AutoDock for 1 in complex with the hPRMT1 homology model (based on 1F3L(44) and 3SMQ(45)) and X-ray hPRMT5 (4GQB) was selected for MD simulation. Dominant structures for the hPRMT1·1 and hPRMT5·1 complexes from the last 20 ns of MD trajectory clustering analysis were used for visualization. PRMT residues engaging the ligand are explicitly shown in ball and stick representation. The protein (in cartoon representation) is colored according to the residue contribution values in the free energy decomposition from red (negative) to blue (positive).

Figure 5

Electrostatic and shape complementarity in diamidine binding to PRTM1 and PRMT5: (A) electrostatic potential surface for the binding pocket of PRMT1 with compound 1; (B) electrostatic potential surface for the binding pocket of PRMT5 with compound 1; (C) shape of the binding cavity of PRMT1 (red) with compound 1 (blue); (D) shape of the binding cavity of PRMT5 (red) with compound 1 (blue). The best docking pose obtained from AutoDock for 1 in complex with the hPRMT1 homology model (based on 1F3L(44) and 3SMQ(45)) and X-ray hPRMT5 (4GQB) was selected for MD simulation. Dominant structures for the hPRMT1·1 and hPRMT5·1 complexes from the last 20 ns of MD trajectory clustering analysis were used for visualization, the same as for Figure 4C,D. The charges of proteins were assigned using PDB2PQR server and electrostatic potential was calculated using APBS. The electrostatic potential varied from −10K_BT/e to +10K_BT/e and was depicted using Chimera in panels A and B from red to blue, respectively. The ligand in panels A and B is color-coded by AM1BCC charge from red (negative) to blue (positive). The surface was visualized in panels C and D using the program VMD.

Figure 6

Compound 1 inhibits proliferation of leukemia cell lines. (A) 1 inhibits GFP-ALY methylation in 293T cells. 20 μM 1 was added to 293T cells for 15 h before harvest. Then the GFP-ALY fusion protein was purified via GFP antibody beads. ASYM24 (Millipore) was used to detect the methylated ALY protein. (B) Compound 1 inhibits leukemic cell growth on day 3. 20 μM 1 was added to the cell culture for 3 days before harvesting for cell viability assay. As a control, the cells were treated with the same amount of DMSO as that added in drug treated samples. The y-axis is the percentage of viable cells in drug treated group by viable cells in control group as denominator. Meg-01 cells and K562 cells have BCR-ABL translocation. HL-60 cells and NB4 cells have PML-RAR α translocation. MOLM13 cells are with MLL-AF9 translocation. HEL cells are with JAK2 V617F mutation. CMK cell, CMY cell, CMS cell, and CHRF cells are with trisomy 21. Jurkat cells derived from T cell leukemia patients had very complicated mutation. (C) Growth curves of CHRF cells. (D) Growth curves of MOLM13 cells. (E) Growth curves of HEL cells.