Protein arginine methyltransferase 5 (PRMT5) is an important epigenetic enzyme involved in cancer development. It carries out symmetric dimethylation on histone proteins, especially on H4R3, utilizing S-adenosyl-L-methionine (SAM) as a cofactor. PRMT5 is overexpressed in a variety of cancers, especially MTAP-deleted cancers, making it a compelling target for anticancer therapeutics. Here, we report an apo structure of the human PRMT5:MEP50 complex determined at a resolution of 3.4 Å by single-particle cryo-EM. The intrinsic flexibility of the loop region (residues 292–329) at the active site of apo-PRMT5:MEP50 suggests a huge conformational change induced by the binding of a nucleoside-containing molecule at the cofactor-binding site. Based on the structural feature of apo-PRMT5:MEP50, we have optimized our carbazole-based lead compound CMP5 into peptide-competitive inhibitors (e.g. 11-1F, 11-2F and 11-9F) that bind to the substrate-binding site in the presence of a nucleoside-containing molecule, such as 5’-methylthioadenosine (MTA), binding at the cofactor-binding site. The enzyme inhibition IC50 of compound 11-1F was determined in MTase-Glo assays at 1.3 μM, and its mechanism of inhibition was confirmed as SAM-uncompetitive and peptide-competitive. The cryo-EM structure of PRMT5:MEP50 bound with MTA and 11-2F (IC50 = 0.9 μM) was determined at a resolution of 3.3 Å. By structure-based drug design and SAR analysis, we have identified compound 11-9F as a potent and selective inhibitor with an IC50 value of 183 nM. Here we describe the design and characterization of 11-9F as a peptide-competitive, MTA- synergistic PRMT5 inhibitor with anti-proliferative effects in several breast cancer cell lines. Protein-inhibitor binding experiments were performed by Surface Plasmon Resonance (SPR) to analyze the binding preference of 11-9F towards apo and SAM/SAH/MTA-bound forms of PRMT5:MEP50 and the binding affinity KD values were determined. 11-9F showed stabilization of PRMT5 in cellular thermal shift (CETSA) target engagement assay and the melting temperature was shifted by ~2 °C. Treatment of 11-9F with or without MTA in breast cancer cell lines led to inhibition of symmetric dimethylation and cell death with IC50 values in the single-digit micromolar range. The in vivo evaluations are currently underway.

Structural and biochemical study of human PRMT5 and its peptide–competitive MTA–synergistic small molecule inhibitors for cancer therapeutics

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.