MTA-cooperative PRMT5 inhibitors from cofactor-directed DNA-encoded library screens

Abstract

Methylthioadenosine phosphorylase (MTAP) gene deletions are frequent in human cancers. Loss of MTAP leads to significantly increased cellular levels of methylthioadenosine (MTA), a cellular metabolite and specific inhibitor of the cell-essential enzyme Protein Arginine Methyltransferase-5 (PRMT5). Using a cofactor-directed screening strategy and DNA-encoded libraries, we identify a class of PRMT5 inhibitors that cooperatively inhibit PRMT5 in the presence of MTA. An optimized inhibitor, AM-9934, selectively inhibits PRMT5 in MTAP-deleted cells and in transplanted tumors while sparing MTAP-expressing counterparts, leading to specific suppression of viability in MTAP-deleted cells. Structural studies show that AM-9934 occupies the arginine substrate pocket of MTA-bound PRMT5. This study introduces a broadly applicable method for directed DNA-encoded library screening toward a desired mechanistic outcome and highlights MTA-selective PRMT5 inhibition as an attractive therapeutic strategy with a potentially broad therapeutic index in patients with MTAP-deleted cancers.

Keywords: DNA-encoded library screens; PRMT5; cancer.

Fig. 1.

Identification and validation of an MTA-selective PRMT5 inhibitor. (A) Screening strategy. (B and C) Box scatter plots illustrating enriched compounds as a function of their individual building blocks (axes) and relative enrichment (CPM, sequence counts per million reads, dot size). Screens shown are DEL91 (B) and DEL123 (C) in the presence of MTA (Left panels) or SIN (Right panels). (D) Representative hit compound AM-9959 and structure of EPZ015666 and AM-9934. The arrow denotes point of DNA attachment in the oligonucleotide-conjugated AM-9959 derivative. (E) Fold enrichment of oligonucleotide-conjugated compounds from single-round selections in the presence of cofactors as indicated.

Fig. 2.

Validation of MTA co-operativity. (A) Thermal shift assay showing stabilization of PRMT5:MEP50 complex as a function of compound and cofactor addition. (B) Inhibition of PRMT5 methyltransferase activity +/− addition of MTA. (C) Fluorescence polarization (FP) assay demonstrating binding of FITC-AM-9934 to PRMT5 after 300 min incubation without or with cofactor in the presence of indicated concentrations of PRMT5:MEP50 complex. Inset: K_D value of FITC-labeled AM-9934 derivative binding to PRMT5:MEP50 with or without the indicated cofactor as derived from Lineweaver–Burk analysis. (D) Microscale Thermophoresis analysis of AM-9934 binding to PRMT5 in the presence of indicated cofactors. (E) IC₅₀ values of AM-9934 or EPZ015666 against indicated methyltransferase activity. In cases where the IC₅₀ was not reached at 10 µM of compound, this is indicated as >10 µM on the secondary X-axis.

Fig. 3.

Cellular activity of AM-9934. (A) Inhibition of cellular SDMA in HCT116 isogenic cell pair. (B) Inhibition of cell viability in HCT116 isogenic cell pair. (C) Cell viability IC₅₀s across a panel of MTAP WT (Blue) and MTAP^−/− (Red) cell lines. Insets report fold differences in isogenic cell pairs, P-values for WT (Blue) and MTAP^−/− by ANOVA.

Fig. 4.

Substantiation of the AM-9934 mechanism of action. (A) Design of mouse pharmacodynamic model. Contralateral wt/MTAP^−/− tumors were treated once daily for 4 d as indicated before tumor harvesting for SDMA quantification. (B) Tumor SDMA levels (left y-axis) and blood/tumor compound concentrations (right y-axis) 4 h post–last dose of AM-9934 (Dark blue bars) or GSK3326595 (Light blue bars). (C) Overall view of AM-9934 in complex with PRMT5 and MTA with secondary structure renditions of MEP50 (warm pink), PRMT5 catalytic domain (green), and the TIM barrel domain (violet), and the loop linking these two domains (orange). Ligands are in stick representation and their carbon atoms are color-coded for AM-9934 (pink) and MTA (cyan). (D) Zoom of the substrate/cosubstrate pockets of MTA- and AM-9934-bound PRMT5 with the protein depicted in secondary structure, with key residues depicted in stick representation. The arginine substrate from the superposition of PRMT5:MEP50 in complex with MTA and H4 peptide (PDB: 5FA5) onto the complex has also been rendered (ball and stick). Carbon atoms are color-coded for protein residues (light gray), MTA (cyan), Arginine substrate (violet), and AM-9934 (pink).