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Purification of Ubiquitinated p53 Proteins from Mammalian Cells
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Purification of Ubiquitinated p53 Proteins from Mammalian Cells

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

Honorine Lebraud1, Richard A Noble2, Nicole Phillips2

  • 1Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge CB4 0QA , United Kingdom.

Bioconjugate Chemistry
|June 1, 2018
PubMed

Insights

Researchers developed RG7388-TCO, a novel molecule targeting MDM2, a key regulator of the p53 tumor suppressor. This tool allows for precise imaging and assessment of MDM2 engagement in cancer cells.

Area of Science:

  • Oncology
  • Molecular Biology
  • Chemical Biology

Background:

  • MDM2 is a critical negative regulator of the p53 tumor suppressor protein.
  • MDM2 inhibition is a therapeutic strategy for TP53 wild-type tumors.
  • RG7388 is a small molecule inhibitor of MDM2-p53 binding in clinical development.

Purpose of the Study:

  • To design, synthesize, and evaluate RG7388-TCO, a trans-cyclooctene tagged derivative of RG7388.
  • To utilize RG7388-TCO for imaging endogenous MDM2 in cancer cells.
  • To assess RG7388-TCO's utility in evaluating MDM2-p53 antagonism.

Main Methods:

  • Synthesis of RG7388-TCO, a functionalized MDM2 inhibitor.
  • Bioorthogonal chemistry: click reaction between trans-cyclooctene (TCO) and tetrazine.
  • Fluorescence imaging of endogenous MDM2 using tetrazine-BODIPY dye.
  • Affinity pull-down assay using tetrazine-agarose beads.

Main Results:

  • RG7388-TCO demonstrated high cellular potency and specificity for MDM2.
  • Successful in-cell fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells.
  • RG7388-TCO enabled pull-down of MDM2 from cell lysates.
  • The compound facilitates assessment of target engagement and MDM2-p53 antagonism.

Conclusions:

  • RG7388-TCO is a valuable chemical biology tool for MDM2 research.
  • This derivative enables precise visualization and quantification of MDM2 activity in vitro.
  • RG7388-TCO supports the development of MDM2-targeted cancer therapies.

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