Validation and Invalidation of Chemical Probes for the Human N-myristoyltransferases

摘要

On-target, cell-active chemical probes are of fundamental importance in chemical and cell biology, whereas poorly characterized probes often lead to invalid conclusions. HumanN-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-proteomeN-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis, and cell-cycle assays. We find thatN-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 ofN-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.