We're also collaborating with a colleague at Harwell to use neutrons to investigate lipid exchange between membranes. Lots of protein biochemistry and biophysics. Get in touch if that's your idea of a good time!

Could you add me please?

Lucas is now on bluesky at @ungerlucas.bsky.social

This is (bluesky-less) Lucas Unger's first manuscript from his PhD work, and a big team effort involving members of #TeamAQP across the world. We are keen to get feedback on our study, and urge caution to researchers using AER-270 or TGN-020 in their work!

TGN-020 appears to be an allosteric modulator of adenosine receptors, and both compounds modulated mTOR signalling in astrocytes (in opposite directions). These off-target effects mean that existing literature on effects of AER-270 and TGN-020 in in vivo models should be re-evaluated.

We further report a variety of off-target effects of these compounds. AER-270 inhibited carbonic anhydrase and NF-kB signalling via IKKB with downstream effects on AQP4 gene expression.

We conclude that the Xenopus assay is susceptible to false positive hits. Using a proteomics-based target identification method, we found 30 common targets of TGN-020 and AER-270 in Xenopus oocytes which could plausibly explain this.

There are several other examples of “aquaporin-4 inhibitors” in the literature that appear to inhibit aquaporin-4 in the Xenopus assay but not in any other assay system, including acetazolamide, and various antiepileptics and loop diuretics.

We then went back to the “gold standard” Xenopus laevis oocyte swelling assay and found apparent inhibitory effects, consistent with the existing literature.

We tested AER-270 and TGN-020 using recombinant AQP4 protein reconstituted into proteoliposomes and again found no inhibitory effect.

We initially became interested in TGN-020 and AER-270 as positive controls in a screen for novel aquaporin-4 modulators. To our surprise, they were not functional in assays using mammalian cell lines overexpressing aquaporin-4, nor in primary astrocytes.