Chemical Instability and Promiscuity of Arylmethylidenepyrazolinone-Based MDMX Inhibitors

[Image: see text] Targeting the protein–protein interaction between p53 and MDM2/MDMX (MDM4) represents an attractive anticancer strategy for the treatment of p53-competent tumors. Several selective and potent MDM2 inhibitors have been developed and entered the clinic; however, the repertoire of MDMX antagonists is still limited. The arylmethylidenepyrazolinone SJ-172550 has been reported as a selective MDMX antagonist; yet, uncertainties about its mechanism of action have raised doubts about its use as a chemical probe. Here, we show that, in addition to its unclear mode of action, SJ-172550 is unstable in aqueous buffers, giving rise to side products of unknown biological activity. Using an SJ-172550-derived affinity probe, we observed promiscuous binding to cellular proteins whereas cellular thermal shift assays did not reveal a stabilizing effect on MDMX. Overall, our results raise further questions about the interpretation of data using SJ-172550 and related compounds to investigate cellular phenotypes.