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

Bioconjug Chem. 2018 Jun 20;29(6):2100-2106. doi: 10.1021/acs.bioconjchem.8b00315. Epub 2018 Jun 12.

Abstract

MDM2 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Boron Compounds / chemistry*
  • Cell Line, Tumor
  • Click Chemistry
  • Cyclooctanes / analogs & derivatives
  • Cyclooctanes / pharmacology
  • Fluorescent Dyes / chemistry*
  • Humans
  • Molecular Docking Simulation
  • Optical Imaging / methods
  • Protein Binding / drug effects
  • Protein Interaction Maps / drug effects
  • Proto-Oncogene Proteins c-mdm2 / analysis*
  • Proto-Oncogene Proteins c-mdm2 / metabolism*
  • Pyrrolidines / chemistry*
  • Pyrrolidines / pharmacology
  • Tumor Suppressor Protein p53 / metabolism*
  • para-Aminobenzoates / chemistry*
  • para-Aminobenzoates / pharmacology

Substances

  • 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
  • Boron Compounds
  • Cyclooctanes
  • Fluorescent Dyes
  • Pyrrolidines
  • RG7388
  • Tumor Suppressor Protein p53
  • para-Aminobenzoates
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2