Establishment of a first-in-class PRMT5-specific inhibitor. (A) View of the crystal structure of rPRMT1 (aa 41-353, Protein Data Bank [PDB] ID 1OR8, gray ribbon representation) superimposed on the C-terminal domain of the hPRMT5 model (aa 310-637, green ribbon representation) showing the conserved nature of the PRMT family catalytic domain. The cofactor SAM and substrate arginine residue binding pockets are shown as red and blue regions, respectively. (B) rPRMT3 crystal structure displaying the cocrystallized SAH and arginine residue (purple carbon stick representation). (C) hPRMT5 model showing docked SAH and arginine residue similarly oriented in comparison with the rPRMT3 crystal structure. (D) Close-up view of SAH and arginine (stick representation; purple carbon) docked to the hPRMT5 model (green ribbon and key residues in green carbon stick representation). (E) CMP5 (stick representation; purple carbon) docked to the hPRMT5 model (green ribbon and key residues in green carbon stick representation) showing initial predicted interaction with residues. For clarity, residues covering the catalytic site face are not shown in the figures. (F) Immunofluorescence staining of JeKo cells (MCL cell line) treated with DMSO, CMP5, or CMP6 using antibodies against symmetrically (S) dimethylated S2Me-H4R8 and S2Me-H3R8. DAPI (4,6 diamidino-2-phenylindole) was used to stain nuclei. (G) Chemical structure of selective PRMT5 inhibitor CMP5 and nonreactive control CMP6. (H) Histone methyltransferase assays were performed as described in “Methods” in the presence of DMSO, CMP5 (10-100 μM), or CMP6 (10-100 μM). (I) View of the crystal structure of the C-terminal domain of hPRMT5 (aa 310-637, PDB ID 4GQB, blue ribbon representation) superimposed on model hPRMT5 (green ribbon representation). (J) Close-up catalytic site view of the superposed hPRMT5 crystal structure (blue ribbon and blue carbon stick representation) and model (green ribbon and green carbon stick representation). Cocrystallized SAM analog and substrate arginine residue are shown as yellow carbon stick and line representation, respectively. (K) View of the docked conformation of CMP5 (yellow carbon stick representation) within the active site of the optimized hPRMT5 crystal structure. Interacting amino acid residues are shown in blue stick format. (L) An equal number (0.5 × 10⁶) of normal B cells (resting or activated) or the indicated DLBCL cell lines (Pfeiffer and SUDHL-2) were treated with increasing concentrations of CMP5, and cell viability was determined by annexin V–propidium iodide (PI) staining and flow cytometry at 24 hours. (M) Normal B cells from 3 separate healthy donors were treated with increasing concentrations of CMP5 and cell death was determined by annexin V–PI staining and flow cytometry at 24, 48, and 72 hours. (N-O) A transformed cell line (60A) and an immortalized cell line (D-9) were treated with increasing concentrations of CMP5 and cell death was determined by annexin V–PI staining and flow cytometry at 24 and 48 hours. Data are shown as the percentage of annexin V⁻ PI⁻ cells (live cells) and are normalized to untreated control. *P < .05; **P < .01. Error bars indicate standard error of the mean (SEM).