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Highly Potent Clickable Probe for Cellular Imaging of MDM2 and Assessing Dynamic Responses to MDM2-p53 Inhibition
journal contribution
posted on 2018-05-31, 00:00 authored by Honorine Lebraud, Richard A. Noble, Nicole Phillips, Keisha Heam, Juan Castro, Yan Zhao, David R. Newell, John Lunec, Stephen R. Wedge, Tom D. HeightmanMDM2
is a key negative regulator of the p53 tumor suppressor. Direct
binding of MDM2 to 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-cyclooctene tagged 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 BODIPY dye enabled
fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells.
RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged
agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO
enables precise imaging of MDM2 in cells and can permit a relative
assessment of target engagement and MDM2-p53 antagonism in vitro.