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

Abstract

In this manuscript, we developed a Two-fold Symmetric Linchpin (<b>TSL</b>) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. <b>TSL</b> 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 SX<sub>1</sub>CX<sub>2</sub>X<sub>3</sub>X<sub>4</sub>X<sub>5</sub>X<sub>6</sub>X<sub>7</sub>C sequences, where X<i><sub>i</sub></i> is any amino acids but Cys, were converted to a library of bicyclic <b>TSL</b>-[<u>S</u>]X<sub>1</sub><u>[C]</u>X<sub>2</sub>X<sub>3</sub>X<sub>4</sub>X<sub>5</sub>X<sub>6</sub>X<sub>7</sub>[<u>C]</u> 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 <b>TSL</b>-[<u>S</u>]Y<u>[C]</u>KRAHKN[<u>C]</u> 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. <b>TSL</b>-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 (Pronase<sup>TM</sup>) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. <b>TSL</b>-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.

Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulationin-vivo

“…Late-stage modification of peptides and genetically-encoded (GE) libraries of peptides by cross-linkers (linchpins) is one of the common approaches to incorporate beneficial attributes to their properties. 49 Alkylation of cysteine residues in peptides via an SN2 reaction using bi-or tri-dentate alkyl halides has been use for cyclization of peptides, incorporation of unnatural fragments into the resulting macrocycles, [50][51][52] and late-stage modification of phage-and mRNA-displayed libraries to yield billion-scale GE libraries. Peptide cyclization via SNAr reaction with perfluoroarenes popularized by the Pentelute group forms alkyl-aryl thioethers; 43 other classes reactions have been developed to form aryl [53][54][55] , alkenyl and alkynyl thioethers 56 in unprotected peptides.…”

Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulation in-vivo.

“…All the analyses were performed by next generation sequencing (NGS) of phage DNA as previously reported. 50,52,70 Preparation of Illumina sequencing samples Similar to previous reports, 50,52,70 phage eluted from the target was subjected to PCR ampli cation (see SI for PCR protocols) to append Illumina multiplexing barcodes, and sequencing adapters to randomized library regions. All PCR products were quanti ed by 2% (w/v) agarose gel in Tris-Borate-EDTA buffer at 100 volts for ~35 min using a low molecular weight DNA ladder as standard (NEB, cat# N3233S).…”

“…(linchpins) is one of the common approaches to incorporate bene cial attributes to their properties. 49 Alkylation of cysteine residues in peptides via an S N 2 reaction using bi-or tri-dentate alkyl halides has been use for cyclization of peptides, incorporation of unnatural fragments into the resulting macrocycles, [50][51][52] and late-stage modi cation of phage-and mRNA-displayed libraries to yield billionscale GE libraries. Peptide cyclization via S N Ar reaction with per uoroarenes popularized by the Pentelute group forms alkyl-aryl thioethers; 43 other classes reactions have been developed to form aryl [53][54][55] , alkenyl and alkynyl thioethers 56 in unprotected peptides.…”

Suprastapled Peptides: Hybridization-Enhanced Peptide Ligation and Enforced α-Helical Conformation for Affinity Selection of Combinatorial Libraries

“…Following the pioneering work establishing the utility of stapled peptides, [9][10][11][12] there has been continued interest to advance the stapling chemistry with a particular emphasis on methodologies (for recent examples, see refs [13][14][15][16][17][18][19][20][21] ) that can be applied to unprotected peptides that can be embraced in genetically encoded libraries. [22][23][24][25] Herein, we report a supramolecular approach to stapling the α-helical conformation leveraged on the hybridization of short peptide nucleic acid [26][27] (PNA) sequences. Besides being efficacious in stabilizing the helical conformation, this approach also enables a simple means to generate libraries by templating native chemical ligation of two fragments.…”