A versatile method for orchestrating the formation of side-chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side-chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner in vitro, or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low micromolar streptavidin binder (KD = 1.1 µM) from a library of cyclic peptides produced in E. coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles.
Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo-biosynthetic strategy for generating macrocyclic organo-peptide hybrids (MOrPHs) through a dual oxime-/intein-mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3-amino-thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2-amino- mercaptomethyl-aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the "i-1" site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side-chain→C-end ligation versus C-end→side-chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo-peptide macrocycles and provide valuable structure-reactivity insights to inform the construction of macrocycle libraries through this chemo-biosynthetic strategy.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an immunosurveillance cytokine that kills cancer cells but demonstrates little toxicity against normal cells. While investigating the TRAIL-inducing imidazolinopyrimidinone TIC10, a misassignment of its active structure was uncovered. Syntheses of the two isomers, corresponding to the published and reassigned structures, are reported. The ability of each to induce TRAIL expression in macrophages was investigated and it was found that only the compound corresponding to the reassigned structure shows the originally reported activity; the compound corresponding to the published structure is inactive. Importantly, this structural reassignment has furnished a previously unknown antitumor pharmacophore.
Despite efforts to produce suitable smoking cessation aids, addiction to nicotine continues to carry a substantive risk of recidivism. An attractive alternative to current therapies is the pharmacokinetic strategy of antinicotine vaccination. A major hurdle in the development of the strategy has been to elicit a sufficiently high antibody concentration to curb nicotine distribution to the brain. Herein, we detail investigations into a new hapten design, which was able to elicit an antibody response of significantly higher specificity for nicotine. We also explore the use of a mutant flagellin carrier protein with adjuvanting properties. These studies underlie the feasibility of improvement in antinicotine vaccine formulations to move toward clinical efficacy.
Tumor necrosis factor (TNF)-related apoptosisinducing ligand (TRAIL) is an immunosurveillance cytokine that kills cancer cells but demonstrates little toxicity against normal cells. While investigating the TRAIL-inducing imidazolinopyrimidinone TIC10, a misassignment of its active structure was uncovered. Syntheses of the two isomers, corresponding to the published and reassigned structures, are reported. The ability of each to induce TRAIL expression in macrophages was investigated and it was found that only the compound corresponding to the reassigned structure shows the originally reported activity; the compound corresponding to the published structure is inactive. Importantly, this structural reassignment has furnished a previously unknown antitumor pharmacophore.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.