Extended Data Fig. 7: High-throughput screening identifies a specific NNMT inhibitor.
From: NNMT inhibition in cancer-associated fibroblasts restores antitumour immunity
a, Overview of the methyltransferase (MTase)-GLO assay used for high-throughput screening of compound libraries to identify an NNMT inhibitor (NNMTi). SAM = S-adenosylmethionine, NAM = nicotinamide, 1-MNA = 1-methylnicotinamide, SAH = S-adenosylhomocysteine. b, Cellular thermal shift assay (CETSA). CAOV3 cells were treated with different concentrations of the hit compound and then heated, lysed, and immunoblotted for NNMT. c, The MTase-GLO assay was used to test non-specific inhibition of related MTases (MT) by the hit compound. PNMT = phenylethanolamine N-methyltransferase, HNMT = histamine N-methyltransferase, GNMT = glycine N-methyltransferase, GAMT = guanidinoacetate N-methyltransferase, COMT = catechol O-methyltransferase. d, Blood samples were collected from Nnmt+/+ or Nnmt−/− mice, and relative 1-MNA levels were measured by mass spectrometry (n = 5 per group) (two-tailed Mann-Whitney test). e, CAOV3 cells were treated with different concentrations of the hit compound. After 24 h, 1-MNA concentrations were measured in the cell supernatant by mass spectrometry (n = 5 technical replicates). f, NNMT and substrates were mixed with different concentrations of NNMTi/NNMTi-D, and the abundance of S-adenosylhomocysteine (SAH) was measured by MTase-GLO (n = 3 technical replicates per group). g, HiBiT-NNMT-expressing K562 cells were treated with different concentrations of NNMTi or NNMTi-D and the HiBiT-NNMT bioluminescent signal was measured by cellular thermal shift assay (CETSA) (n = 2 technical replicates). TS = target stabilization. h, CAOV3 cells were treated with different concentrations of NNMTi (n = 2 technical replicates) or NNMTi-D (n = 1 technical replicate). After 24 h, 1-MNA concentration was measured in the cell supernatant by mass spectrometry. i, Inhibition of 659 protein kinases was measured in the presence of 10 μM NNMTi (Supplementary Table 10) (n = 2 technical replicates). The size of the red dots represents the degree of inhibition level of 329 kinases. TK = tyrosine kinase, TKL = tyrosine kinase-like, STE = serine/threonine kinase, CK1 = casein kinase 1, AGC = protein kinase A, G, and C family, CAMK = calcium/calmodulin-dependent protein kinase, CMGC contains CDKs = cyclin-dependent kinases, MAPKs = mitogen-activated protein kinases, GSKs = glycogen synthase kinases, CLKs = Cdc2-like kinases. The illustration was created using CORAL75. j, Indicated enzymes were treated with 10 μM NNMTi and unspecific enzyme inhibition was measured (n = 2 technical replicates). Left panel: Rxn Biology MTase Panel. Right panel: Eurofin Safety Screen. Data are shown as mean ± SEM. One experiment representative of at least three independent experiments is shown for panels f-h.
