A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases

Abstract

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.

Figure 1. Design of the type I PRMT inhibitor MS023

Figure 2. Characterization of MS023 in biochemical and biophysical assays

(A) IC₅₀ determination for MS023 against PRMTs. (B) Isothermal titration calorimetry (ITC) was used to assess binding of PRMT6 to MS023 (K_d = 6 nM). (C) Differential scanning fluorimetry (DSF) was used to confirm the binding of PRMT6 to MS023. Experiments were performed for PRMT6 (●) in the absence of any compound as a control (T_m = 52 °C), and in the presence of (○) 100 μM SAM (ΔT_m = 1.6 °C), () 200 μM MS023 (ΔT_m = 20 °C) and (□) 100 μM SAM plus 200 μM MS023 (ΔT_m = 22 °C). The inflection point of each transition curve is considered melting temperature (T_m) and the increase in T_m is an indication of binding. (D) The selectivity of MS023 was determined against a panel of 25 PKMTs and DNMTs and 3 histone lysine demethylases at two compound concentrations of 1 μM and 10 μM. No change was observed in the IC₅₀ values when determined under different peptide (E) and SAM (F) concentrations for PRMT6 indicating a noncompetitive pattern of inhibition.

Figure 3

The X-ray crystal structure of a ternary complex of MS023 (cyan), SAH (green) and PRMT6 (gray). Key interactions shown in yellow dotted lines.

Figure 4. The effect of MS023 and MS094 on inhibiting PRMT1 and PRMT6 in cells

(A) MS023 inhibits PRMT1 methyltransferase activity in MCF7 cells. (B) MS094 does not inhibit PRMT1 methyltransferase activity in MCF7 cells. MCF7 cells were treated with MS023 (A) or MS094 (B) at indicated concentrations for 48 h and H4R3me2a levels were determined by Western blot. The graphs represent nonlinear fits of H4R3me2a signal intensities normalized to total histone H4. The results are MEAN ± SEM of two experiments done in triplicate. (C) MS023 inhibits PRMT6 methyltransferase activity in HEK293 cells. HEK293 cells were transfected with FLAG-tagged PRMT6 or its catalytically inactive mutant V86K/D88A (MUT) and treated with MS023 at indicated concentrations for 20 h. H3R2me2a levels were determined by Western blot. The graphs represent nonlinear fits of H3R2me2a signal intensities normalized to total histone H3. The results are MEAN ± SEM of 3 replicates.

Figure 5. The effect of MS023 on arginine asymmetric dimethylation (Rme2a), symmetric dimethylation (Rme2s) and monomethylation (Rme1) in cells

MS023 reduces cellular levels of Rme2a and concurrently increases cellular levels of Rme1 and Rme2s. MCF7 cells were treated with MS023 at indicated compound concentrations for 48 h. Arginine asymmetric dimethylation, arginine symmetric dimethylation and arginine monomethylation were detected by western blot using indicated pan-arginine antibodies (green). β-actin was used as a loading control (red).

Scheme 1. Synthetic route for MS023

Reagents and conditions: (a) NaH, DMF, THF; (b) Boc₂O, Et₃N, DMAP, DCM, 82% over two steps; (c) DIBALH, DCM, −78 °C, 71%; (d) DMP, DCM, 75%; (e) tert-butyl (2-(methylamino)ethyl)carbamate, NaBH(OAc)₃, DCM, 94%; (f) HCl, MeOH, 45 °C, 87%.