Dataset Information

Validation and invalidation of chemical probes for the human N-myristoyltransferases.

ABSTRACT: On-target, cell-active chemical probes are of fundamental importance in both chemical and cell biology, whereas the application of poorly-characterised probes often leads to invalid conclusions. Human N-myristoyltransferase (NMT) has attracted increasing interest as a 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 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 which used off-target compounds.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Wouter Kallemeijn

LAB HEAD: Edward W. Tate

PROVIDER: PXD012722 | Pride | 2019-11-14

REPOSITORIES: Pride

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Publications

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

Kallemeijn Wouter W WW Lueg Gregor A GA Faronato Monica M Hadavizadeh Kate K Goya Grocin Andrea A Song Ok-Ryul OR Howell Michael M Calado Dinis P DP Tate Edward W EW

Cell chemical biology 20190418 6

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, ...[more]

PMID: 31006618

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Project description:Rhinovirus causes the common cold and drives exacerbation in asthma and COPD, leading to substantial morbidity and healthcare cost. The single positive strand RNA genome of this non-enveloped virus is translated into a single polyprotein that is processed by virus 3C protease to form three structural capsid proteins VP0, VP1 and VP3, and other proteins required for viral life cycle completion. Capsid proteins assemble into the 5S capsid protomer, five of which form a 14S pentamer, and 12 pentamers then assemble to form the 150S icosahedral provirion into which the viral genome is incorporated. During the final step of viral maturation, VP0 is cleaved into VP2 and VP4. VP4 is encoded at the N-terminus of the viral polyprotein, and in many Picornaviruses is N-myristoylated by host cell N-myristoyltransferase (NMT). We have explored the impact of IMP-1088 (the first sub-nanomolar IC50 dual inhibitor of human N-myristoyl transferases HsNMT1 and HsNMT2) on HRV capsid myristoylation in cells. Global identification of proteins for which myristoylation is selectively inhibited by IMP-1088 was performed by quantitative chemical proteomic analysis of YnMyr-tagged proteins in HeLa cells infected with RV16 treated with 50 nM IMP-1088 or vehicle (DMSO). Proteins were subjected to ligation to AzRB, an azide capture reagent bearing a trypsin-cleavable linker and biotin dual label followed by affinity enrichment on Neutravidin beads, on-bead digestion with trypsin, nanoLC-MS/MS analysis of tryptic peptides, and data processing by label-free protein quantification (LFQ) in MaxQuant. 60 N-myristoylated proteins were identified, including the RV16 polyprotein which responded to IMP-1088 inhibition, with no significant change in protein abundances across the whole proteome in the presence of inhibitor. The majority of viral capsid peptides identified in the pull-down mapped to the VP0 domain of the polyprotein, and the N-terminal YnMyr-tagged peptide was readily identified by MS/MS. These data provide the first direct evidence for HRV capsid myristoylation in human cells, and demonstrate that this lipid modification is highly susceptible to inhibition by a selective NMT inhibitor.

Dataset Information

Validation and invalidation of chemical probes for the human N-myristoyltransferases.

Publications

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

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