FB23-2

Considering that the in vitro inhibition of FTO is in the same single digit micromolar range as some of the kinase off-targets, it is important to consider their potential effects in cellular assays. An improved version of FB23-2, Dac51, has since been published by the same authors (Liu et al. Cell Metab. 2021) that is more potent in vitro and in vivo, but has been less comprehensively characterized regarding off-targets.

Although compelling data is presented that FB23 and FB-23-2 are binding to FTO, there are significant data disconnects that call into question whether some of the observed phenotypic effects are correlated with FTO alone. Not only are hydroxamic acids a structural alert, the authors also observed hydrolysis back to FB23 in cells with FB23 reportedly more potent on FTO. Thus, it remains opaque which compound is actually eliciting the response in cells. Additionally, the off-target profiling for FB-23 is incomplete, for example only 2 HDACs were tested when hydroxamic acids are key pharmacophores for the entire family; the kinome profile is quite complete yet the chemical structure does not suggest this would be a concern. Overall, given the modest biochemical potencies, it is difficult to assume that cellular activity only relates to FTO inhibition. In fact many data in the publication with FB23-2 that show target engagement use 20 and sometimes even 50 uM concentration whereas the important data in KO cells strikingly only shows 1 and 2 uM with 2 uM already showing an effect in KO cells. Given the totality of information and considering that no negative control compound is available, the use of FB23-2 as a chemical probe cannot be recommended without great hesitation. Significantly more data and characterization will be needed to further support FB23-2. Given the ambiguity, an in vivo use is not recommended.