Discovery of First-in-Class Protein Arginine Methyltransferase 5 (PRMT5) Degraders

Abstract

The aberrant expression of protein arginine methyltransferase 5 (PRMT5) has been associated with multiple cancers. Using the proteolysis targeting chimera technology, we discovered a first-in-class PRMT5 degrader 15 (MS4322). Here, we report the design, synthesis, and characterization of compound 15 and two structurally similar controls 17 (MS4370) and 21 (MS4369), with impaired binding to the von Hippel-Lindau E3 ligase and PRMT5, respectively. Compound 15, but not 17 and 21, effectively reduced the PRMT5 protein level in MCF-7 cells. Our mechanism studies indicate that compound 15 degraded PRMT5 in an E3 ligase- and proteasome-dependent manner. Compound 15 also effectively reduced the PRMT5 protein level and inhibited growth in multiple cancer cell lines. Moreover, compound 15 was highly selective for PRMT5 in a global proteomic study and exhibited good plasma exposure in mice. Collectively, compound 15 and its two controls 17 and 21 are valuable chemical tools for exploring the PRMT5 functions in health and disease.

Figure 1.

Design of PRMT5 putative degraders 9, 10, 11, 12, and 13. (A) Crystal structure of PRMT5:MEP50 (gray) in complex with SAM (magenta) and EPZ015666 (green) (PDB: 4X61). The highlighted oxetane moiety of EPZ015666 is solvent-exposed. Key H-bond interactions between the EPZ015666 and PRMT5 residues are highlighted by red dashed lines. (B) Chemical structures of EPZ015666, compound 3, and the five putative PRMT5 degraders 9–13.

Figure 2.

Effects of compounds 9–13 on reducing the PRMT5 protein level in MCF-7 cells. MCF-7 cells were treated with compounds 9–13 at the indicated concentrations (0.2, 1, and 5 μM) for 6 d. Cell lysates were collected, and the PRMT5 protein levels were detected by western blots. The results are representative of at least two independent experiments.

Figure 3.

Design of the PRMT5 degrader 15 and its two control compounds 17 and 21.

Figure 4.

Effects of compounds 15, 17, and 21 on inhibiting the PRMT5 methyltransferase activity in a radioactive biochemical assay. Substrate: H4 (1–15)-biotin (400 nM). Cofactor: ³H-SAM (1 μM). EPZ015666 was used as a positive control. IC₅₀ determination experiments were performed in duplicate, and the values are presented as mean ± SD.

Figure 5.

Effects of compound 15 on reducing the PRMT5 protein level and inhibiting SDMA in MCF-7 cells. (A,B) Compound 15 concentration-dependently reduced the PRMT5 protein level, whereas compounds 17 and 21 and EPZ015666 did not. MCF-7 cells were treated with the indicated concentration of 15, 17, 21, or EPZ015666 for 6 d. Cell lysates were collected, and the protein levels of PRMT5 were detected by western blotting. The relative strength of the PRMT5 signal on the western blots was measured by densitometry. DC₅₀ and D_max values are presented as mean ± SD from three independent experiments. (C) Compound 15 reduced the PRMT5 protein level in a time-dependent manner. MCF-7 cells were treated with 5 μM of compound 15 for 0, 2, 4, 6, or 8 d. The PRMT5 protein levels were detected by western blotting. (D) Compound 15 and EPZ015666 inhibited SDMA. MCF-7 cells were treated with the indicated concentration of EPZ015666 or compound 15 for 6 d. The SDMA levels were detected by western blotting. Western blot results are representative of at least two independent experiments.

Figure 6.

(A) Compound 15 reduced the PRMT5 protein level in MCF-7 cells in a PRMT5-, E3 ligase VHL-, and proteasome-dependent manner. MCF-7 cells were treated with 5 μM of compound 15 for 7 d. During the last 24 h, the cells were co-treated with 100 μM VH-298, 2 μM MLN4924, 30 μM MG-132, or 30 μM EPZ015666. The protein levels of PRMT5 were detected by western blotting. (B) PRMT5 degradation induced by compound 15 was reversible. MCF-7 cells were treated with 5 μM of compound 15 for 6 d, and compound 15 was then washed out. The PRMT5 protein levels were detected by western blotting at 0, 12, 24, and 48 h post the washout.

Figure 7.

Effects of compounds 15, 17, 21, and EPZ015666 on inhibiting the proliferation of MCF-7 cells. MCF-7 cells were treated with the indicated concentration of compounds 15, 17, 21, or EPZ015666 for 6 d. The relative cell viabilities to the untreated group are shown as mean ± SD (n = 3).

Figure 8.

Global proteomic analysis indicates that compound 15 is a highly selective PRMT5 degrader. MCF-7 cells were treated with compound 15 at 5 μM or DMSO for 5 d and then harvested and lysed for liquid chromatography–MS (LC–MS) analysis. LFQ was used to calculate the peptide intensity; the relative abundance of PRMT5 (LFQ intensity value) between the compound 15-treated and DMSO-treated groups is shown.

Figure 9.

Compound 15 reduced the PRMT5 protein levels in HeLa, A549, A172, and Jurkat cells. The cells were treated with 5 μM of compound 15 for 6 d. The protein levels of PRMT5 were detected by western blotting. The western blotting results are representative of two independent experiments.

Figure 10.

Compound 15 and EPZ015666 inhibited the proliferation of HeLa (A), A549 (B), A172 (C), and Jurkat (D) cells. The cells were treated with 15 or EPZ015666 at the indicated concentrations for 6 d. The relative cell viabilities to the untreated group are shown as mean ± SD (n = 3).

Figure 11.

Plasma concentrations of compound 15 over 12 h, following a single 150 mg/kg IP injection in male Swiss albino mice. The compound concentration shown at each time point is the mean ± SD from three test mice.

Scheme 1.. Synthesis of PRMT5 Putative Degraders 9–13^a

^aReagents and conditions: (a) i-PrOH, reflux; (b) TFA, DCM; (c) EDCI·HCl, HOAt, NMM, 6-chloropyrimidine-4-carboxylic acid, DMSO, 18% over three steps; (d) tert-butyl 3-aminoazetidine-1-carboxylate, NMP, 90%; (e) dicarboxylic acids, EDCI·HCl, HOAt, NMM, DMSO, 54–78%; (f) 3, EDCI·HCl, HOAt, NMM, DMSO, 36–60%.

Scheme 2.. Synthesis of Compounds 15, 17, and 21^a

^aReagents and conditions: (a) EDCI·HCl, HOAt, NMM, DMSO, 50%; (b) 3, EDCI·HCl, HOAt, NMM, DMSO, 35–53%; (c) i-PrOH, reflux; (d) TFA, DCM; (e) EDCI·HCl, HOAt, NMM, 6-chloropyrimidine-4-carboxylic acid, DMSO; (f) tert-butyl 3-aminoazetidine-1-carboxylate, NMP, 13% over four steps; (g) 14, EDCI·HCl, HOAt, NMM, DMSO, 49%.