Highly Potent Clickable Probe for Cellular Imaging of MDM2 and Assessing Dynamic Responses to MDM2-p53 Inhibition

MDM2is a key negative regulator of the p53 tumor suppressor. Direct binding of MDM2to p53 represses the protein’s transcriptional activity and induces its polyubiquitination, targeting it for degradation by the proteasome. Consequently, small molecule inhibitors that antagonize MDM2-p53 binding, such as RG7388, have progressed into clinical development aiming to reactivate p53 function in TP53 wild-type tumors. Here, we describe the design, synthesis, and biological evaluation of a trans- cyclooctenetagged derivative of RG7388, RG7388-TCO, which showed high cellular potency and specificity for MDM2. The in-cell reaction of RG7388-TCO with a tetrazine-tagged BODIPYdye enabled fluorescence imaging of endogenous MDM2in SJSA-1 and T778 tumor cells. RG7388-TCO was also used to pull down MDM2by reaction with tetrazine-tagged agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO enables precise imaging of MDM2in cells and can permit a relative assessment of target engagement and MDM2-p53 ...