The Nrf2 pathway has been a hot area of research for some years now, particularly in oncology. It’s a basic-leucine-zipper transcription factor that under normal conditions stays mostly out in the cytosol, where it’s under tight regulatory control. Under cellular stress, though, it heads into the nucleus and fulfills its transcription-factor destiny, in particular setting off a range of genes coding for cytoprotective proteins. This break-glass-in-case-of-emergency mechanism works by Nrf2 being normally bound to another cytosolic protein, Keap1, which both binds it up and facilitates its degradation by the ubiquitination/proteasome system (by in turn binding a ubiquitin ligase and keeping it close to Nrf2). Keap1 has cysteine residues that get modified under oxidative stress, and this event causes it to fall off of Nrf2 and send it on its way.
That’s the index card version of the system. As usual, when you look closer all sorts of complications ensue. For example, Keap1 has about 27 cysteine residues that seem to be important for regulating Nrf2 activity. Modification of some of them (particularly Cys151) are definitely important for releasing Nrf2, but modification of others actually helps keep it bound – there’s some sort of “cysteine code” effect going on that we don’t completely grasp. Nrf2 itself is involved in regulation of at least 200 genes, most of which clearly seem to be involved in dealing with oxidative stress – but not all of them. And more broadly, there are people who would like to use Nrf2 activation as an anti-inflammatory tool or neuroprotectant, which seems reasonable. But there are also a number of cancers where Nrf2 is already revved up and helping keep the tumor cells going, so in those cases you’d like to be able to shut it down and cause them trouble. And so on!
How about some more complications, then? This new paper (from researchers at Copenhagen and Rennes) is a very welcome look at the chemical matter that’s appeared in the literature so far (journals as well as patents) as inhibiting the Nrf2/Keap1 protein-protein interaction. The team identified 19 such compounds, and purchased or prepared every single one of them for side-by-side tests. As one might have suspected, not all the literature routes to these worked as well as they do in, well, the literature, so they had to do some work on the synthesis end for several of them. But they eventually took the whole list and characterized them in three orthogonal biophysical assays (fluorescence polarization, thermal shift, and surface plasmon resonance). That’s exactly what you want to do with such hits – make them perform via several different readouts so you can see if you believe their activity. They also checked them all for issues like potential covalent behavior (which can be good or bad, depending), for redox activity (rarely anything but bad), and for aggregation (always bad). And finally, they all went into cell assays.
This is an excellent, thorough med-chem examination of these compounds, and it’s a pleasure to see it. I might add that one would want to see a lot more of this sort of thing when interesting new compounds are first reported, rather than waiting for other groups to come in and try to gather all the pieces together (or perhaps take out the trash), but I’m just glad that it’s been done in this case. And the results?
Ten of the nineteen reported compounds appear to be garbage. That’s not quite how the authors phrase it, but they come pretty close, saying that they “question the legitimacy” of that set. As they should: some of these were discovered in fluorescence-based assays but turn out to be fluorescent interference compounds. Others aggregate, others are chemically unstable. There are compounds with cell activity that show nothing believable in the biochemical/biophysical assays, so who knows what that means, etc. As the authors note, and they are so right, that last situation “highlights the crucial deficiency of characterizing compounds solely based on cellular activities“.
Let’s be frank: these are the sorts of problems that should be caught before you publish papers. All fluorescence-based assays are subject to false positives based on compound properties (absorption, quenching, intrinsic fluorescence, etc.) and all kinds of compounds can aggregate under different assay conditions to give you false positives that way, too. Checking compound purity and stability should be an elementary step. None of these problems are new, but here we are, and here the med-chem literature is. I will add my obligatory statement about the difficulties this sort of thing poses for ideas about shoveling it all into the hopper of deep-learning software.
The authors don’t call this out explicitly in the text, but I will. The molecules that look real come from groups at Rutgers, Biogen, China Pharmaceutical University, Purdue, Univ. of Illinois-Chicago, Sanofi, Keio Univ. (and other Japanese academic partners), and Astex/GSK. The ones with problems come from Univ. College London/Dundee/Johns Hopkins (the last two also show up with other collaborators), Harvard/UCSD, Toray Industries (also with RIKEN collaborators), China Pharmaceutical Univ./Jiangsu Hengrui Medicine Co. (the former shows up more than once), and Univ. of Minnesota. You will note that appearance of well-known institutions on both lists. I will say that big pharma comes out looking OK, partly because of abundant resources and partly because having money on the line makes you marginally less likely to try to fool yourself. (For cash-strapped small pharma, the incentive to fool yourself can sometimes override other considerations, I hasten to add).
The authors of the current paper finish up by recommending that people only draw conclusions based “on pharmacological mechanisms supported by orthogonal biochemical and biological assays“, and I can only second the motion. That would indeed be a great thing. Let’s give it a try. Start with Nrf2/Keap1 compounds, and just keep on going.