• Authors: McKinney D. et al.
  • Year: 2021
  • Journal: J Med Chem 64 11148-11168
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi


Expi293 cells were cultured in a 1:1 mixture of Expi293:Freestyle media. At a cell density of ∼2.5 × 106 cells/mL, 250 mL of cells was transfected using 200 μL FectoPRO (Polyplus Transfection) reagent mixed with 100 μg of PRMT5 and 100 μg HA-tagged WDR77 plasmids. One day after transfection, 3 mM valproic acid and 0.4% w/v glucose were added to the culture. Two days after transfection, cells were split into multiple flasks with 30 mL cells each and treated with compound or DMSO at a final 0.2% DMSO concentration. Cell sample was collected after 6 h, pelleted, and washed 3 times with ice-cold PBS. Additional aliquots were analyzed for viability and cell number using a Vi-Cell XR Cell Counter, and all samples were determined to have >98% viability based on trypan blue exclusion. Each sample was lysed in buffer 50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP, 1% v/v Tween-20, and 2 mM reduced GSH. The PRMT5:WDR77 complex was immunoprecipitated by anti-HA agarose resin and then eluted by 50 μM 3X-HA peptide. Each eluate was measured by intact mass LC-MS to determine the percentage of complex with compound adduct.


PRMT5 and its substrate adaptor proteins (SAPs), pICln and Riok1, are synthetic lethal dependencies in MTAP-deleted cancer cells. SAPs share a conserved PRMT5 binding motif (PBM) which mediates binding to a surface of PRMT5 distal to the catalytic site. This interaction is required for methylation of several PRMT5 substrates, including histone and spliceosome complexes. We screened for small molecule inhibitors of the PRMT5-PBM interaction and validated a compound series which binds to the PRMT5-PBM interface and directly inhibits binding of SAPs. Mode of action studies revealed the formation of a covalent bond between a halogenated pyridazinone group and cysteine 278 of PRMT5. Optimization of the starting hit produced a lead compound, BRD0639, which engages the target in cells, disrupts PRMT5-RIOK1 complexes, and reduces substrate methylation. BRD0639 is a first-in-class PBM-competitive inhibitor that can support studies of PBM-dependent PRMT5 activities and the development of novel PRMT5 inhibitors that selectively target these functions.