There are quite a few histone deacetylase inhibitors out there, from research tools to FDA-approved drugs. Those inhibit the enzymes that remove the acetyl epigenetic markers from histone proteins – but what about inhibitors of the enzymes that put them on? Those are histone acetyltransferases (HATs), and they’ve naturally been the subject of a lot of work as well. There’s a whole list of reported compounds (although nothing’s made it through the clinic yet), but how useful are they?
Not very, according to this new paper. The authors (a multicenter team from Brigham & Women’s Hospital, the University of Minnesota, the SGC, NCI, Monash, and Nanjing) investigate 23 reported HAT inhibitors, and right off the bat find that 15 of them are active in the ALARM-NMR assay. That’s a screen for thiol reactivity – you expose a solution of the La antigen protein to your compound of interest, and look for specific changes in its conformation when one or both of its sensitive Cys residues gets alkylated. The readout is “This compound can react with proteins”, and of course it’s up to you to decide what to do about that. If you’re using it as an in vitro tool compound, you’ll want to check the specific proteins you’re using – maybe things are still OK. But if you’re going into cells (or worse, in vivo), this throws suspicion on your phenotypic readouts, because there are so many other things that could be happening. You can do a whole proteome screen for reactivity, if you have the time, equipment, and money, but even that’s not going to pick up everything. In this case, the team went on to check reactivity with small-molecule biological thiols like GSH and CoA, and many of them formed adducts with these as well. (As a side note, the profile in these assays compared to the ALARM NMR data don’t completely overlap, in either direction, and the paper has a discussion of how to interpret such situations – if you’re dealing with potentially reactive screening hits, it’s definitely worth a read).
The data get worse. Those latter experiments were monitored by LC/MS, and that showed that six of the reported compounds are not even stable in buffer, but degrade to various by-products under standard assay conditions. Twelve out of the 23 also showed detergent sensitivity in a standard aggregation assay test as well. All of these problems are going to show up no matter what sort of assay readout you use, but it should also be noted that several compounds showed potential fluorescence problems as well (which is always something that has to be checked with any such assay; sometimes half of your “hits” are merely fluorescent interference compounds).
I’ve never worked in the HAT area myself, and I hadn’t paid that much attention to the reported chemical matter. Looking at the compounds, I’m not the least bit surprised that many of them are reactive or otherwise problematic, though: it’s a very unappealing collection (which even includes our old friend curcumin). How you’re supposed to make sense of cellular assays with such compounds is a mystery, and in fact, the paper shows that the great majority of reported HAT compounds cause nonspecific effects in cell lines. When your tool compound is indistinguishable from a stink bomb like rottlerin, you have problems: you may think that you’ve asked your compound to tell you all about some specific pathway, but in reality, you asked it to just go in and mess your cells up, any old way.
. . .our data show none of the reported HAT inhibitors contain a substantial number of attributes associated with high-quality chemical probes such as target potency and selectivity, tractable mechanism of target modulation, and meaningful target-based cellular activity12. Histone acetylation is a complex biological phenomenon, and we argue the use of suboptimal compounds to probe for HAT cellular functions leads to tenuous, and even false, scientific conclusions that cannot fully account for the contributions of off-target activity, promiscuous reactivity, and/or nonspecific cytotoxicity to cellular, biochemical, or phenotypic readouts.
. . .The cell-based data indicate nonspecific assay interference compounds can disrupt cell proliferation, decrease cellular histone acetylation levels, and even perturb HAT levels. These findings suggest that test compounds have the potential to be misinterpreted as useful HAT inhibitors if the appropriate mechanistic, selectivity, and analytical experiments are not performed.
Absolutely. Garbage in, garbage out is the law, and wishful thinking does nothing to abrogate it. As the paper shows, these compounds appear in hundreds of papers (and hundreds of review articles), so just reading the literature would give you the impression that there are plenty of useful HAT compounds out there, and that we’re figuring out what they do in cells. Not so. These results need to be regarded with skepticism until proven by other means. Looking at the chemical suppliers would give you the impression that you can order up useful compounds. Not so – the authors of this paper end up by being unable to recommend any of the 23 compounds they profile. There are, they note, HAT compounds being reported in industrial patents that look to be better, and further profiling of these by different labs may well validate them as useful tools. But what’s in the open literature now is not cutting it.