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A miniaturized screening assay to discover Pin1 inhibitors as probes of phosphorylation signaling

Wednesday, October 10, 2012 — Poster Session II

Noon – 2:00 p.m

Natcher Conference Center, Building 45

NCATS

CANCER-5

Authors

  • M. I. Davis
  • D. Wei
  • M. Shen
  • D. Auld
  • M. Boxer
  • X. Z. Zhou
  • K. P. Lu
  • A. Simeonov

Abstract

Protein phosphorylation on certain serine or threonine residues preceding a proline (phos.Ser/Thr-Pro) is a major signaling mechanism in diverse cellular processes, and its deregulation contributes to human disease, notably cancer, Alzheimer's disease, and autoimmune disorders. The unique chemical properties of Pro allow for the adoption of two distinct amide bond conformations (cis and trans). Peptidyl-prolyl cis/trans isomerases (PPIases) are evolutionarily conserved enzymes that catalyze cis/trans isomerization of peptidyl-prolyl peptide bonds. Pin1 binds a subset of phos.Ser/Thr-Pro-containing proteins using its protein-targeting WW domain and isomerizes only specific phos.Ser/Thr-Pro bonds, which cannot be effectively catalyzed by other known PPIases. Pin1 has emerged as a novel molecular timer that modulates its multiple targets at various steps of a given cellular process to synergistically control the amplitude and duration of a cellular response or process. A high-throughput screen using a fluorescence polarization competition assay was developed using a HiFluor-488-labeled peptide, which binds to the active site of Pin1 and can be displaced by compounds. The screen encompassed ~400,000 compounds assayed as a concentration-titration series (114 ┬ÁM to 3 nM). Hits were confirmed using a TAMRA-labeled peptide and an orthogonal enzyme activity assay, which identified compounds with IC50s as low as 95 nM.

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