There’s a lot of work in the literature on the TrkB receptor, which responds to brain-derived neutrotrophic factor (BDNF). That name certainly makes the ligand protein sound like a pretty big deal, and so it is: BDNF is involved in a lot of neural development pathways, injuries to nerve tissue, and the like, and given the black-box nature of so many of those processes, it and the other neurotrophin hormones have been the subject of a great deal of research. There’s an oncology angle as well, since fusion proteins involving BDNF and others have been found apparently driving some types of cancer.
That means that there’s potentially room for both agonists and antagonists of the receptor as therapeutic options. Antagonists are being looked at in oncology, and agonists could be of use in Alzheimer’s or other neurodegenerative conditions. But coming up with an agonist of a receptor like this isn’t so easy – the whole class of G-protein-coupled receptors that respond to big protein ligands is notoriously hard to deal with in that fashion, replacing their natural partner with a small molecule, because the binding surfaces are rather large and complex. You’d think, well, fine, I’ll just infuse BDNF right into the brain, because these are desperate conditions that require desperate measures. But every single attempt to use that as a therapy has (to my knowledge, failed) so there’s a lot going on here.
Nonetheless, there are several TrkB agonist compounds reported in the literature, and they’re rather small molecules (mostly flavone derivatives). 7,8-dihydroxyflavone in particular has had a lot of work done on it, in numerous models of disease. It does seem odd that a molecule this size can activate the TrkB receptor, and flavone derivatives in general are known as a class of compounds whose activities are hard to pin down. There are other structural classes reported in this field as well, though, such as LM22A-4 and the classic CNS molecules amitryptaline and deprenyl.
Doubts have been raised about these compounds, although publications on them seem to be continuing at a steady pace. But a new paper throws the gauntlet down. The authors, from Columbia and the Broad Institute, have taken a more detailed look at receptor phosphorylation (which is the big event on activation of this class) and downstream readouts (AKT, ERK1, ERK2, etc.), and have come to the conclusion that none of the reported TrkB agonists are, in fact, TrkB agonists at all. BDNF and neurotrophin-4 light up these assays in the just ways that you’d expect, but not the small molecules. The paper also describes a screen through 40,000 compounds in the Broad’s diversity-oriented synthesis collection, which in the end led to no confirmed hits, either. Finding a small-molecule ligand for this receptor is indeed not an easy task.
So why have these compounds persisted in the literature as long as they have? The authors suggest that the methods involved have obscured things:
We propose that one plausible explanation for the observed discrepancies is methodological in nature. Although Western blotting is a standard core method in life sciences research, it requires many procedural steps, making it impractical to perform multiple repeats for each experimental condition plus controls (47–49). Moreover, densitometry is dependent on the analysis procedure, which may lead to biased results, especially with a low amount of phosphorylation of the protein of interest (50). Specificity of the antibody plays an important role as well (50, 51). Although antibodies recognizing phosphorylated ERK and phosphorylated AKT yielded highly specific staining on blots (fig. S5), we did not find an antibody that was specific for phosphorylated TrkB; the commercially available antibodies tended to recognize additional targets. Complementary methods should be applied to confirm Western blot results. In our view, the described quantitative ELISA provides sufficient throughput for controls and repeats to be used along with the blot to more accurately characterize drug activity. We recommend that Western blot be used as a qualitative or semiquantitative complementary method to the quantitative ELISA assays, as confirmation that ELISA indeed detects the desired phosphorylation reaction. Alternative methods that are independent of antibodies should also be considered, such as methods based on enzymatic activity of reporter proteins as the readout.
When you consider the number of reported results (not just in this field!) that rely on Western blots, this is food for thought. (And once again, antibodies that aren’t as good as they’re supposed to be cause trouble). It’s not a new worry, by any means, but you really do want to keep this in mind when you’re evaluating published work (and keep in mind that these worries are completely separate from ones about outright Western <s>blog</s> blot fakery, which has shown up many times in the last few years). A lot of time and money has been spent on these compounds, and a lot of hypotheses generated which are now going to have to be re-evaluated, to say the least.
7,8-dihydoxyflavone may well have effects in animal models, but they’re apparently independent of anything involving direct activation of TrkB. There could well be indirect TrkB mechanisms (as the authors of this paper take time to point out), involving things that aren’t present in many of the in vitro assays. But that’s not what people have been working on the basis of, and if such mechanisms are indeed in effect, time would have been better spent tracking them down rather that working on a false basis. Let’s see how this paper goes over in the field. . .