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A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6 - PubMed

  • ️Fri Jan 01 2021

A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6

Yudao Shen et al. J Med Chem. 2021.

Abstract

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6-(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2's enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.

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Conflict of interest statement

The authors declare the following competing financial interest(s): R. M. Campbell, M. M. Mader and B. M. Watson were employees of Eli Lilly and Company during the time this research was performed.

Figures

Figure 1.
Figure 1.

The structures and potencies of reported PRMT6 inhibitors

Figure 2.
Figure 2.

Discovery of the PRMT6 chemical probe (R)-2. (A) Structure-activity relationship studies and chiral separation led to the discovery of (R)-2 as a potent PRMT6 inhibitor and its enantiomer, (S)-2, as a negative control. (B) IC50 determination of (R)-2 and (S)-2, n = 3.

Figure 3.
Figure 3.

Selectivity assessment of (R)-2 (SGC6870) at 1 μM (gray) and 10 μM (black) and (S)-2 (SGC6870N) at 1 μM (green) and 10 μM (blue) against 8 PRMTs, 21 protein lysine methyltransferases (PKMTs), 3 DNA methyltransferases (DNMTs), and 1 RNA methyltransferase, n = 3.

Figure 4.
Figure 4.

Mechanism of action. (A) IC50 values were determined at various compound-protein incubation times ranging from 15 to 120 min. The value obtained with no incubation time was used as a control (values are presented in Table S3). (B) Part of the plot in Figure A is magnified for better view of the decrease in the IC50 value at longer incubation times. (C) IC50 values of (R)-2 at increasing concentrations of SAM (up to 30 μM) at fixed concentration of peptide substrate (3 μM of H4 1–24 peptide). (D) IC50 values of (R)-2 at increasing concentration of peptide (up to 8 μM) at fixed concentration of SAM (12 μM).

Figure 5.
Figure 5.

Cocrystal structure of PRMT6 in complex with (R)-2. (A) Cocrystal structure of PRMT6 (tinted blue) in complex with (R)-2 (green), and SAM (orange), (PDB: 6W6D). Dashed yellow lines indicate key hydrogen bonds. (B) Structural alignments of complexes PRMT6-(R)-2-SAM (cyan), PRMT6-MS023-SAH (blue) (PDB: 5E8R) and PRMT6-SAH (magenta) (PDB: 4C05). Movement of loop consisting of nine residues, EWMGYGLLH, indicates that (R)-2 binds to a novel induced allosteric pocket of PRMT6.

Figure 6.
Figure 6.

Inhibition of PRMT6-dependent H3R2 and H4R3 asymmetric di-methylation in cells. HEK293T cells were transfected with Flag-tagged PRMT6 and treated with indicated compounds for 20 h. The flag-tagged PRMT6 catalytically inactive mutant V86K/D88A (mut) was used as a positive control. (A) Western blot representation of the effect of (R)-2 on PRMT6 activity. (B) Western blot representation of the effect of (S)-2 on PRMT6 activity. (C) IC50 determination of (R)-2 and (S)-2 at reducing H3R2me2a. The graph represents nonlinear fit of H3R2me2a fluorescence intensities normalized to intensities of H3, n = 4. (D) IC50 determination of (R)-2 and (S)-2 at reducing H4R3me2a. The graph represents nonlinear fit of H4R3me2a fluorescence intensities normalized to intensities of H4, n = 3.

Figure 7.
Figure 7.

Inhibitory effect of (R)-2 and (S)-2 on cell viability (A) Effect of (R)-2 on MCF-7, PNT2, and HEK293T cell viability, n = 3. (B) Inhibitory effect of (S)-2 on MCF-7, PNT2, and HEK293T cell viability, n = 3.

Scheme 1.
Scheme 1.

Synthesis of Compound (R)-2 and Compound (S)-2a a(a) dichloromethane, −78 °C to rt; (b) HCl, MeOH/H2O, reflux; (c) oxazolidine-2,5-dione, CF3COOH, triethylamine, toluene, 60–85 °C; (d) NaBH3CN, AcOH, MeOH, rt; (e) 5-bromothiophene-2-carboxylic acid, EDCI, HOAt, NMM, DMSO, rt; (f) chiral separation.

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