In-solution enrichment identifies peptide inhibitors of protein–protein interactions


The use of competitive inhibitors to disrupt protein–protein interactions (PPIs) holds great promise for the treatment of disease. However, the discovery of high-affinity inhibitors can be a challenge. Here we report a platform for improving the affinity of peptide-based PPI inhibitors using non-canonical amino acids. The platform utilizes size exclusion-based enrichment from pools of synthetic peptides (1.5–4 kDa) and liquid chromatography-tandem mass spectrometry-based peptide sequencing to identify high-affinity binders to protein targets, without the need for ‘reporter’ or ‘encoding’ tags. Using this approach—which is inherently selective for high-affinity binders—we realized gains in affinity of up to ~100- or ~30-fold for binders to the oncogenic ubiquitin ligase MDM2 or HIV capsid protein C-terminal domain, which inhibit MDM2–p53 interaction or HIV capsid protein C-terminal domain dimerization, respectively. Subsequent macrocyclization of select MDM2 inhibitors rendered them cell permeable and cytotoxic toward cancer cells, demonstrating the utility of the identified compounds as functional PPI inhibitors.

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Fig. 1: Affinity selection platform for the maturation of known peptide binders.
Fig. 2: Affinity selection identifies hotspot residues for MDM2 binding.
Fig. 3: Affinity selection identifies potent variants containing non-canonical amino acids.
Fig. 4: Discovery of potent mirror image knottin derived peptide binders to MDM2.
Fig. 5: Macrocyclic variants were cell penetrating and active against oncogenic cells overexpressing MDM2.

Data availability

The authors declare that all data supporting the findings of this study are available within the manuscript, its Supplementary Information, and Supplementary Notes.

Change history

  • 13 May 2019

    In the version of this article originally published, the peptide sequences of compounds 90, 92 and 93 in Fig. 5b and Supplementary Table 7 contained several errors. In Fig. 5b, position 6 of compound 90 should be Tyr instead of Phe. In both Fig. 5b and Supplementary Table 7, position 9 of compounds 92 and 93 should be Gln instead of Glu. Additionally, the surname of co-author Anupam Bandyopadhyay was incorrectly spelled as Bandyopdhyay. The errors have been corrected in the HTML and PDF versions of the paper and in the Supplementary Information PDF.


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The Bettencourt Schueller Foundation is gratefully acknowledged for postdoctoral support to F.T. The Human Frontier Science Program Organization is thanked for a cross-disciplinary fellowship (LT000745/2014-C) to F.T. This work was supported in part by Servier, the Defense Advanced Research Projects Agency (award no. 023504-001 to B.L.P.), the NIH National Institute of General Medical Sciences (grant no. 5-R01-GM110535 to B.L.P.), a Bristol-Myers Squibb unrestricted grant in Synthetic Organic Chemistry (B.L.P.), and a Novartis Early Career Award (B.L.P). F. Lefoulon (Servier) is thankfully acknowledged for his suggestions and comments on the manuscript. FACS experiments were performed at the MIT Koch Institute Flow Cytometry Core. The authors thank the Biophysical Instrumentation Facility at MIT for providing access to the Octet Bio-Layer Interferometry System (NIH S10OD016326) and D. Pheasant for her technical assistance and B. Dass (Pall Fortebio) for his help with data analysis. We gratefully acknowledge A. Rabideau for peptide synthesis and characterization training, A. Quartararo for help with the Orbitrap LC–MS instrument, and A. Vinogradov and M. Simon for scientific discussions.

Author information

F.T. and B.L.P. conceived the study with input from Z.P.G. F.T. and B.L.P. directed the project and designed experiments. F.T. performed most experiments. Z.P.G. and A.B. contributed to OBOC screen design and experiments, and G.L. contributed to library purification and binder validation. F.T., Z.P.G., and B.L.P. wrote the manuscript. All authors contributed to the analysis, interpretation, and validation of the data.

Correspondence to Fayçal Touti or Bradley L. Pentelute.

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Supplementary information

Supplementary Information

Supplementary Tables 1–7, Supplementary Figures 1–39

Reporting Summary

Supplementary Note 1

Library 1 binder validation traces (Fig. 2 and Supplementary Fig. 13).

Supplementary Note 2

HPSEC and LC–MS traces for model binders; LC–MS characterization of numbered compounds.

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