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Chemical Biology

Myristoylation Probes, Rethought

The need for good chemical probes continues, and (sadly) so does the use of crappy ones. That’s what I took away from this recent paper from a multicenter team out of London. They’re looking at commonly used probes for inhibition of N-myristoyltransferase (NMT) enzymes, and it’s one of those good-news/bad-news situations.

N-myristoylation is another one of those funky residue modifications that make the human proteome a lot more versatile than just the count of protein-coding genes would indicate. The enzymes (there are two subtypes) catalyze the conjugation of myristic acid to an N-terminal glycine, and it’s an event that happens with at least 200 different proteins. Some of this takes place right down at the ribosome right at translation, and some of it happens post-translationally: for example, when a protease cleaves an existing protein species to expose a fresh glycine terminus. (Those protein cleavage events are another big reason the protein universe is a lot larger than it appears to be through the lens of sequence information).

Functionally, this modification is particularly involved in pathways to do with T-cell activation and infection, and NMT inhibition has been proposed as an antiviral and antifungal target (there are, for example, viral proteins that use the human N-myristoylation process for their own purposes). Over the years, there have been several compounds reported as NMT inhibitors in such cellular studies, among them 2-hydroxymyristic acid, D-NMAPPD (first reported as a ceramidase inhibitor), and (weirdly) tris-dba-palladium complex. Yep, same stuff you might use for a Suzuki coupling reaction. A group at Imperial College (on this paper as well) has also reported two more recent inhibitors, IMP-366 and IMP-1088.

You may see where this is going. A close look across different cell lines and in greater proteomic and mechanistic detail indicates that only the two IMP compounds are actually NMT inhibitors. The other probe compounds, well. . .there are problems. For starters, 2-hydroxymyristic acid has to be given in rather heroic concentrations to have an effect, and you’re always courting trouble when you go up to hundreds of micromolar anything in cell assays. It seems that its effects in those assays come from some sort of derangement of lipid metabolism and handling rather than NMT inhibition, which means that papers like this one may need some rethinking. Similary, the D-NMAPPD compound is problematic: it is indeed a ceramidase inhibitor, but the current authors could not reproduce its reported inhibition of NMT in either enzyme or cell assays. Instead, it had notable cytotoxicity. (One thing I noticed, though, was that the current paper has a truncated structure for the compound, with a much shorter amide chain than the actual tetradecanamide. I hope that’s just an error in manuscript preparation!) And as for the palladium complex, forget it. In enzyme assays, it shows inhibitory effects at just about the concentration it starts to crystallize out of the buffer, which isn’t good, and in cells it show broad cytotoxicity and no real effects on myristoylation. That was an unlikely candidate from the start, and while it certainly has cellular effects, ascribing those to NMR inhibition doesn’t seem tenable. Pd(dba)3 containing nanoparticles (as reported here) are in the same category.

Meanwhile, the two IMP compounds have reproducible effects against recombinant enzymes, at more lower concentrations, and affect the proteomes of several cell lines in a manner consistent with NMT inhibition. Moreover, there are crystals structures of both complexed with the active site of NMT. That’s not to say that they might not have other effects in cells, but at least you can start by saying that they’re NMT inhibitors, which is something that you can’t say with the other three compounds.

Every time something like this happens, the scientific literature is streaked with results that are probably not what they’re claimed to be. The problem is that this doesn’t get noted. They’re still in Pubmed with no annotation, they’re still cited by other papers, and so on. We have a shortage of good ways to annotate past work with “You know, later on this was shown not to be the case” comments (with PubPeer being the closest thing that comes to mind). This work certainly makes you think that a lot of myristoylation-based conclusions will need to be re-evaluated. But another thing to go back and check will come in a few years’ time – when we see how many papers have continued to use what appear to be invalidated chemical probes, yet again.

11 comments on “Myristoylation Probes, Rethought”

  1. tlp says:

    Looks like scientists need some better system for tracking dependencies than journal citations. Kinda like github for software – so that if the credibility of parent results is questioned the whole fork downstream gets affected instantly. With current pace of publishing we can pretty soon reach the inflection point where it will be easier to rerun known experiment than to dive into literature and try separating wheat from chaff.

    1. Russ says:

      Finally refuting a week in the lab saving an hour in the library?

  2. dearieme says:

    At what point does what does the entropy in the literature outweigh the enthalpy, metaphorically speaking?

  3. drsnowboard says:

    Perhaps your readers can come up with a metal complex that actually is used therapeutically – apart from cytotoxics like the -platins? You know, where the metal complex has been proved to get to the site of action, intact?

    1. Eugene says:

      Would Vitamin B12 to treat Cyanide poisoning and pernicious anemia qualify?

    2. cynical1 says:

      All of the MRI contrast agents are Gadolinium complexes and they are used every day.

    3. Barry says:

      the target is contentious, but Lithium for bipolar disorder certainly gets there (wherever “there” is”

    4. loupgarous says:

      Octreotides are used as carriers for metal ions for both peptide receptor-targeted radiotherapy (Lu-177) and for peptide receptor targeted radiography (Ga-68). In both cases the octreotate delivers the metal ion where it can do the most good.

  4. Phytomig says:

    Probes as we know it don’t really tell us much. Probes now are looking at chemical changes to specific enzymes or groups of enzymes. However, what we need are probes that probe the bulk properties of tissues and cells. Biomaterial probes are what we need, but sadly, all we have is the central dogma and organic chemists :(.

    1. Imaging guy says:

      “we need are probes that probe the bulk properties of tissues and cells”

      Do you mean “probes” that come out of phenotypic screening of cells, tissues cultures and animals?

  5. opensciguy says:

    Derek,, website linked to above, is the beginnings of what you are talking about at the end of your post: an annotation layer for the literature that analyzes agreement between a work’s findings and subsequent papers that cite it. It’s like IF and H-index but with a directional component, not just magnitude.

    A lot of potentially impactful things like this have taken shape over the last few years, including Peter Murray-Rust’s Content Mine, which was originally a pdf-scraping operation to make the literature more machine readable for deep NLP and other analyses.

    Interesting times!

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