Dissecting the binding interactions of teixobactin with the bacterial cell wall precursor lipid II

The prevalence of life‐threatening, drug‐resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered “resistance‐proof” antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram‐positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram‐positive and Gram‐negative bacteria. Furthermore, we show that when combined with known outer membrane‐disrupting peptides, teixobactin is active against Gram‐negative organisms.</p

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

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