German Edition: DOI: 10.1002/ange.201603230 Therapeutic Peptides International Edition: DOI: 10.1002/anie.201603230 A Cyclized Helix-Loop-Helix Peptide as a Molecular Scaffold for the Design of Inhibitors of Intracellular Protein–Protein Interactions by Epitope and Arginine Grafting Daisuke Fujiwara, Hidekazu Kitada, Masahiro Oguri, Toshio Nishihara, Masataka Michigami, Kazunori Shiraishi, Eiji Yuba, Ikuhiko Nakase, Haeri Im, Sunhee Cho, JongYoung Joung, Seiji Kodama, Kenji Kono, Sihyun Ham,* and Ikuo Fujii* Abstract: The design of inhibitors of intracellular protein– protein interactions (PPIs) remains a challenge in chemical biology and drug discovery. We propose a cyclized helix-loop- helix (cHLH) peptide as a scaffold for generating cell- permeable PPI inhibitors through bifunctional grafting: epi- tope grafting to provide binding activity, and arginine grafting to endow cell-permeability. To inhibit p53–HDM2 interactions, the p53 epitope was grafted onto the C-terminal helix and six Arg residues were grafted onto another helix. The designed peptide cHLHp53-R showed high inhibitory activity for this interaction, and computational analysis suggested a binding mode for HDM2. Confocal microscopy of cells treated with fluorescently labeled cHLHp53-R revealed cell membrane penetration and cytosolic localization. The peptide inhibited the growth of HCT116 and LnCap cancer cells. This strategy of bifunctional grafting onto a well-structured peptide scaffold could facilitate the generation of inhibitors for intracellular PPIs. The design of inhibitors of intracellular protein–protein interactions (PPIs) remains challenging in chemical biology and drug discovery. [1] To act as an intracellular PPI inhibitor, the inhibitor must exhibit high binding affinity for the target proteins, high resistance to proteolytic degradation, and high stability in the reducing environment found inside cells. In addition, it must be taken up by the cell, penetrate and cross the cell membrane, and be released into the cytosol. One of the methods used to generate such inhibitors is protein grafting on a relatively small protein scaffold. [2] A scaffold capable of comprehensively fulfilling all of these require- ments would enable the design of intracellular PPI inhibitors. Herein, we propose a cyclized helix-loop-helix (cHLH) peptide as a suitable scaffold for the design of intracellular PPI inhibitors by using a bifunctional grafting approach involving “epitope grafting” and “arginine grafting”. [2, 3] Grafting strategies based on protein scaffolds have been developed for the design of novel functional proteins. [2, 3] In epitope grafting, the critical binding residues of proteins, such as the BH3-heilcal region of Bclx2 or the HIV-1-inhibiting epitope, are transferred onto a protein scaffold to provide binding activity. [1] Avian pancreatic polypeptide (aPP), the GCN4 leucine zipper, and Apamin have been used as protein scaffolds onto which epitopes can be grafted. [2] In arginine grafting, polyarginine residues are installed onto the surfaces of GFP and aPP, and these grafted proteins showed cell permeability. [3] In this work, a combination of these two grafting strategies was applied to cHLH to design cell- permeable PPI inhibitors. We have previously designed a helix-loop-helix (HLH) peptide as a scaffold for generating PPI inhibitors. [4] The HLH peptide YT1 consists of two a-helices stabilized by hydro- phobic interactions between leucine residues embedded on the helix–helix interface (Figure 1). The peptide YT1 retains its secondary structure when solvent-exposed amino acids outside the C-terminal helix are randomly substituted with Figure 1. Structures of the cHLHp53-R peptide, its derivatives, and the control peptides. [*] Dr. D. Fujiwara, H. Kitada, M. Oguri, T. Nishihara, Dr. M. Michigami, Dr. K. Shiraishi, Prof. S. Kodama, Prof. I. Fujii Department of Biological Science Graduate School of Science Osaka Prefecture University 1-1, Gakuen-cho, Naka-ku, Osaka 599-8531 (Japan) E-mail: fujii@b.s.osakafu-u.ac.jp Dr. E. Yuba, Prof. K. Kono Department of Applied Chemistry Graduate School of Engineering Osaka Prefecture University 1-1, Gakuen-cho, Naka-ku, Osaka 599-8531 (Japan) Dr. I. Nakase N2RC, Osaka Prefecture University 1–2, Gakuen-cho, Naka-ku, Osaka 599-8570 (Japan) H. Im, S. Cho, Dr. J.Y. Joung, Prof. S. Ham Department of Chemistry, Sookmyung Women’s University Hyochangwongil 52, Yongsanku, Seoul, 140-742 (Korea) E-mail: sihyun@sm.ac.kr Supporting information for this article can be found under: http://dx.doi.org/10.1002/anie.201603230. A ngewandte Chemi e Communications 1 Angew. Chem. Int. Ed. 2016, 55,1–5  2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! Ü Ü