Extended Data Fig. 6: Identification of a PRMT9 inhibitor.
a, Effects of hits (142 from NCI-DTP and 70 from ZINC library) on Molm13 viability. Cells were treated for 4 days with 1 or 5 μM compounds. For each compound, the number represents its library ID. Experiments were triplicated; P values were derived from t tests. b, R493 methylation assay. PABPC1 peptides (G491-T507) were incubated with PRMT9, and SAM. Catalytic activity was assessed by anti-R493 antibody. Synthesized R493me peptide as positive control. c, Catalysis screen of top 20 compounds. PRMT9 protein was pretreated with indicated compound (10 μM), then incubated with PABPC1 peptides and SAM. d, e, Catalytic activity of indicated compounds based on R493 methylation assay (d). Inhibition curves were calculated based on intensity of dots and normalized to DMSO (e). Data is mean ± SD (n = 3 independent experiments). f, g, NSC641396, NSC661221 and NSC645330 share a carbazole ring scaffold. LD2 is designed based on NSC641396 (g). Docking model of NSC641396 in the catalytic pocket (f). h, IC50 analysis of LD2 in B-NHL lines. Cells were treated for 4 days with LD2. Data were mean ± SD (n = 6 independent experiments). i, Substrates of PRMT1 (H4R3me2A, FLT3 R972/R973me), CARM1 (PABPC1 R455/R460me), PRMT5 (H3R8me2S), PRMT7 (HSP70me) were assessed in Molm13 treated 2 days with 2.5 μM LD2 (n = 2 independent experiments). j, Dose-dependent inhibition of PABPC1 R493 methylation level after 2 days of treatment with LD2 in Molm13 cells (n = 1). Cells were treated with a dose titration of 0.5–40 μM LD2 for 48 hr. The inhibition activity of LD2 to PRMT9 (right red curve) was evaluated by calculating the normalized PABPC1 R493 methylation signal. The inhibition activity of LD2 to PRMT5 (right blue curve) was evaluated by calculating the normalized SmB’B’ methylation signal (supplementary Fig. 2). k, Vina docking of LD2 with PRMT9, PRMT5, CARM1 or PRMT7 was performed, and the average docking scores (kcal/mol, with standard deviation) were shown. l, Molecular dynamics simulation analyses of LD2 with PRMT9 or PRMT5. Smaller root-mean-square-fluctuation (RMSF) of ligand (LD2) in the binding pocket of PRMT9 compared with PRMT5 was shown. m, Ctrl and PRMT9 KD Molm13 were treated for 4 days with LD2 (2.5 uM), and viability was assessed. Data were mean ± SD from 4 independent experiments. n, CyTOF of AML MNCs after treatment for 4 days with vehicle or LD2 (2.5 μM). The frequency of CD3 + T cells and CD34+CD45dim AML blasts was noted. o, Relative leukemia (CD34+CD45dim) and T cell frequencies before and after LD2 treatment based on CyTOF from 3 samples. p, Enriched T cells were treated for 4 days with LD2 (2.5 μM) for 4 days, and cell viability was assessed (n = 3 independent experiments). Data were mean ± SD. q, Frequency of PRMT9 high versus low AML samples displaying the Cytotoxic T Lymphocyte (CTL) score high versus low signatures in cohort GSE14468. P value was calculated using two-sided Fisher exact chi-squared test. ‘n’ represents number of patients.
