GPX4-IN-5

I am somewhat unconvinced by the presented data on this compound as being wholly on-target and therefore useful as a chemical probe. Specifically: - biochemical inhibition data are expressed as %age inhibition at 1uM, and values given would suggest biochemical IC50s in the order of 2uM. For credible tool compound activity, a wider range of more potent analogies would be desirable - these inferred IC50 values are inconsistent with the cell IC50 values obtained, suggesting alternate modes of action - A detailed covalent binding study is not evident, in terms of other targets which are alkylated. This data is critical to understand possible off-targets which may account for the unanticipated and inconsistent cell activity. Whilst the CETSA studies (and others) do point toward interaction of the compound with the stated target, more potent off-target binding cannot be ruled out and may be more responsible for the observed cellular and in vivo activities, particularly given the structural similarity to other compound classes with well documented polypharmacology. - PK studies are incomplete - indications of free drug exposure would be helpful to assess dose vs cellular data, to give some confidence in the in vitro-in vivo experimental correlation Overall, the data are incomplete and do not give a compelling connection from target to observed activity, and as such data generate with these tools may be misleading or consequential, rather than causal. Without further information, I would be hesitant to recommend this derivative as a chemical probe against the suggested target.

C18 demonstrates biologically potent on-target inhibition of GPX4 leading to ferroptosis induction of susceptible cell lines. It is active in vivo in tumour models susceptible to ferroptosis mediated cell death. The ferroptotic phenotype is GPX4 dependent and ameliorated by appropriate ferroptosis antagonists and glutathione rescue (e.g., Fer-1) but not by cell death inhibitors specific for other mechanisms such as apoptosis (e.g., caspase inhibitors). Physico-chemical engagement was confirmed by CETSA for GPX4 and C18 was demonstrated to covalently bind Sec46 of GPX4. However, specific assays to exclude an effect on other GPX enzymes (i.e., GPX1-8) were not performed. As inhibition of these enzymes may not have contributed to phenotypic effects in the cell line tested, the selectivity of C18 for GPX4 over other GPX enzymes is not fully established.

in vivo dose-response not seen above 10 mg/Kg