Department of Chemistry, Imperial College London, Molecular Research Science Hub, 80 Wood Lane, London W12 0BZ, UK.
Department of Chemistry, Imperial College London, Molecular Research Science Hub, 80 Wood Lane, London W12 0BZ, UK; The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
Cell Chem Biol. 2019 Jun 20;26(6):892-900.e4. doi: 10.1016/j.chembiol.2019.03.006. Epub 2019 Apr 18.
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.
在化学生物学和细胞生物学中，靶向细胞活性的化学探针具有重要意义，而特征描述不佳的探针往往会导致无效的结论。人类 N-豆蔻酰转移酶（NMT）作为癌症和传染病的靶点，引起了越来越多的关注。在这里，我们使用定量全蛋白质组 N-豆蔻酰化谱分析、生化酶测定、细胞毒性、细胞内蛋白质合成和细胞周期测定等方法，对广泛应用于人类 NMT 抑制剂的五种化合物进行了深入比较。我们发现，2-羟基豆蔻酸（100μM）、D-NMAPPD（30μM）或 Tris-DBA 钯（10μM）对 N-豆蔻酰化没有影响，后两种化合物通过与 NMT 无关的机制引起细胞毒性。相比之下，类药性抑制剂 IMP-366（DDD85646）和 IMP-1088 在 1μM 和 100nM 的范围内，分别在多种细胞系中完全且特异性地抑制了 N-豆蔻酰化。这项研究使我们能够选择合适的靶标探针用于未来的研究，并表明需要重新评估以前使用非靶标化合物的研究。
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