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Optimization of a cyclic peptide inhibitor of Ser/Thr phosphatase PPM1D (Wip1)
Ryo Hayashi1, Kan Tanoue, Stewart R Durell
1Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
PPM1D (PP2Cδ or Wip1) was identified as a wild-type p53-induced Ser/Thr phosphatase that accumulates after DNA damage and classified into the PP2C family. It dephosphorylates and inactivates several proteins critical for cellular stress responses, including p38 MAPK, p53, and ATM. Furthermore, PPM1D is amplified and/or overexpressed in a number of human cancers. Thus, inhibition of its activity could constitute an important new strategy for therapeutic intervention to halt the progression of several different cancers. Previously, we reported the development of a cyclic thioether peptide with low micromolar inhibitory activity toward PPM1D. Here, we describe important improvements in the inhibitory activity of this class of cyclic peptides and also present a binding model based upon the results. We found that specific interaction of an aromatic ring at the X1 position and negative charge at the X5 and X6 positions significantly increased the inhibitory activity of the cyclic peptide, with the optimized molecule having a K(i) of 110 nM. To the best of our knowledge, this represents the highest inhibitory activity reported for an inhibitor of PPM1D. We further developed an inhibitor selective for PPM1D over PPM1A with a K(i) of 2.9 microM. Optimization of the cyclic peptide and mutagenesis experiments suggest that a highly basic loop unique to PPM1D is related to substrate specificity. We propose a new model for the catalytic site of PPM1D and inhibition by the cyclic peptides that will be useful both for the subsequent design of PPM1D inhibitors and for identification of new substrates.
Insights
Researchers developed improved cyclic peptides to inhibit PPM1D (Wip1), a phosphatase implicated in cancer. The optimized peptide shows high inhibitory activity and selectivity, offering a potential new therapeutic strategy for various cancers.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- PPM1D (Wip1) is a phosphatase involved in DNA damage response and cellular stress.
- PPM1D is frequently amplified or overexpressed in human cancers.
- Inhibiting PPM1D activity presents a potential therapeutic strategy for cancer treatment.
Purpose of the Study:
- To improve the inhibitory activity and selectivity of cyclic thioether peptides against PPM1D.
- To develop a binding model for PPM1D inhibitors.
- To investigate the structural basis for PPM1D substrate specificity.
Main Methods:
- Medicinal chemistry for cyclic peptide optimization.
- Enzyme inhibition assays to determine K(i) values.
- Mutagenesis studies to probe substrate specificity.
- Computational modeling to propose a binding site model.
Main Results:
- Optimized cyclic peptides achieved a K(i) of 110 nM against PPM1D.
- A selective inhibitor with a K(i) of 2.9 microM for PPM1D over PPM1A was developed.
- Structural modifications, including aromatic rings and negative charges, enhanced inhibitory activity.
- Mutagenesis suggested a unique basic loop in PPM1D contributes to substrate specificity.
Conclusions:
- Highly potent and selective PPM1D inhibitors were developed.
- A binding model for PPM1D and its inhibitors was proposed.
- Findings will guide future design of PPM1D inhibitors and substrate identification.
