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Genetically-Encoded Discovery of Proteolytically Stable Bicyclic Inhibitors of Morphogen NODAL

submitted on 10.02.2021, 20:17 and posted on 11.02.2021, 12:51 by Jeffrey Y. K. Wong, Raja Mukherjee, Olena Bilyk, Jiayuan Miao, Vivian Triana Guzman, Mark Miskoizie, Yu-Shan Lin, Antoine Henninot, John J. Dwyer, Serhii Kharchenko, Anna Iampolska, Dmitriy Volochnyuk, Lynne Postovit, Ratmir Derda
In this manuscript, we developed a Two-fold Symmetric Linchpin (TSL) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where Xi is any amino acids but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-Tky-nu ovarian carcinoma cells with apparent IC50 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control Tky-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (PronaseTM) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.


Ferring Research Institute

NSERC Discovery Grant

The National Institute of General Medical Sciences of the National Institutes of Health

NSERC Accelerator Supplement

CFI New Leader Opportunity

Canadian Institutes of Health Research


Email Address of Submitting Author


University of Alberta



ORCID For Submitting Author


Declaration of Conflict of Interest

Patent application describing this invention was filed by TEC Edmonton in July 2018. R.D. is a shareholder of 48Hour Discovery Inc., the company licensing the technology.

Version Notes

First version