Validation and invalidation of chemical probes for the human N-myristoyltransferases. Wouter W Kallemeijn Gregor A Lueg Monica Faronato Kate Hadavizadeh Andrea Goya Grocin Ok-Ryul Song Michael Howell Dinis P Calado Edward W Tate 10779/crick.11522910.v1 https://crick.figshare.com/articles/journal_contribution/Validation_and_invalidation_of_chemical_probes_for_the_human_N-myristoyltransferases_/11522910 On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as target in cancer and infectious diseases. Here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM), or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies that used off-target compounds. 2020-01-06 17:42:54 2-hydroxymyristic acid D-NMAPPD (B13) IMP-1088 IMP-366 (DDD85646) N-myristoylation N-myristoyltransferases (NMT) Tris-DBA palladium chemical proteomics metabolic tagging sortase A ligation Calado FC001057 HTS CS-ack Tate - sat