Wheat pathogen Zymoseptoria tritici N-myristoyltransferase inhibitors: on-target antifungal activity and an unusual metabolic defense mechanism

Zymoseptoria tritici is the causative agent of Septoria tritici blotch (STB), which costs billions of dollars annually to major wheat-producing countries in terms of both fungicide use and crop loss. Agricultural pathogenic fungi have acquired resistance to most commercially available fungicide classes, and the rate of discovery and development of new fungicides has stalled, demanding new approaches and insights. Here we investigate a potential mechanism of targeting an important wheat pathogen Z. tritici via inhibition of N-myristoyltransferase (NMT). We characterize Z. tritici NMT biochemically for the first time, profile the in vivo Z. tritici myristoylated proteome and identify and validate the first Z. tritici NMT inhibitors. Proteomic investigation of the downstream effects of NMT inhibition identified an unusual and novel mechanism of defense against chemical toxicity in Z. tritici through the application of comparative bioinformatics to deconvolute function from the previously largely unannotated Z. tritici proteome. Research into novel fungicidal modes-of-action is essential to satisfy an urgent unmet need for novel fungicide targets, and we anticipate that this study will serve as a useful proteomics and bioinformatics resource for researchers studying Z. tritici

Discovery of lipid-mediated protein–protein interactions in living cells using metabolic labeling with photoactivatable clickable probes

Protein-protein interactions (PPIs) are essential and pervasive regulatory elements in cell biology. Despite development of a range of techniques to probe PPIs in living systems, there is a dearth of approaches to capture interactions driven by specific post-translational modifications (PTMs). Myristoylation is a lipid PTM added to more than 200 human proteins, where it may regulate membrane localization, stability or activity. Here we report design and synthesis of a panel of novel photocrosslinkable and clickable myristic acid analog probes, and their characterization as efficient substrates for human N -myristoyltransferases NMT1 and NMT2, both biochemically and through X-ray co-crystallography. We demonstrate metabolic incorporation of probes to label NMT substrates in cell culture and in situ intracellular photoactivation to form a covalent crosslink between modified proteins and their interactors, capturing a snapshot of interactions driven by the presence of the lipid PTM. Proteomic analyses revealed both known and multiple novel interactors of a series of myristoylated proteins, including ferroptosis suppressor protein FSP1 and spliceosome-associated RNA helicase DDX46. The concept exemplified by these probes offers an efficient approach for exploring the PTM-specific interactome, which may prove broadly applicable to other PTMs