Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators.

Noncanonical G protein activation and inactivation, particularly for the Gαi/s protein subfamilies, have long been a focus of chemical research. Combinatorial libraries were already effectively applied to identify modulators of the guanine-nucleotide exchange, as can be exemplified with peptides such as KB-752 and GPM-1c/d, the so-called guanine-nucleotide exchange modulators. In this study, we identified novel bicyclic peptides from a combinatorial library screening that show prominent properties as molecular switch-on/off modulators of Gαi signaling.

Discovery of Small Molecules Targeting the Frameshifting Element RNA in SARS-CoV-2 Viral Genome.

Targeting structured RNA elements in the SARS-CoV-2 viral genome with small molecules is an attractive strategy for pharmacological control over viral replication. In this work, we report the discovery of small molecules that target the frameshifting element (FSE) in the SARS-CoV-2 RNA genome using high-throughput small-molecule microarray (SMM) screening. A new class of aminoquinazoline ligands for the SARS-CoV-2 FSE are synthesized and characterized using multiple orthogonal biophysical assays and structure-activity relationship (SAR) studies.

A chemical probe based on the PreQ metabolite enables transcriptome-wide mapping of binding sites.

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ riboswitch system, assessing sixteen diverse PreQ-derived probes for their ability to selectively modify the class-I PreQ riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch.

Discovery of a Bicyclic Peptidyl Pan-Ras Inhibitor.

The Ras subfamily of small GTPases is mutated in ∼30% of human cancers and represents compelling yet challenging anticancer drug targets owing to their flat protein surface. We previously reported a bicyclic peptidyl inhibitor, cyclorasin B3, which binds selectively to Ras-GTP with modest affinity and blocks its interaction with downstream effector proteins in vitro but lacks cell permeability or biological activity. In this study, optimization of B3 yielded a potent pan-Ras inhibitor, cyclorasin B4-27, which binds selectively to the GTP-bound forms of wild-type and mutant Ras isoforms ( = 21 nM for KRasG12V-GppNHp) and is highly cell-permeable and metabolically stable (serum > 24 h).

Cell-Permeable Bicyclic Peptidyl Inhibitors against NEMO-IκB Kinase Interaction Directly from a Combinatorial Library.

Macrocyclic peptides are capable of binding to flat protein surfaces such as the interfaces of protein-protein interactions with antibody-like affinity and specificity, but generally lack cell permeability in order to access intracellular targets. In this work, we designed and synthesized a large combinatorial library of cell-permeable bicyclic peptides, in which the first ring consisted of randomized peptide sequences for potential binding to a target of interest, while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the IκB kinase α/β (IKKα/β)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKKβ interaction with low μM IC values.

Bicyclic Peptides as Next-Generation Therapeutics.

Bicyclic peptides have greater conformational rigidity and metabolic stability than linear and monocyclic peptides and are capable of binding to challenging drug targets with antibody-like affinity and specificity. Powerful combinatorial library technologies have recently been developed to rapidly synthesize and screen large bicyclic peptide libraries for ligands against enzymes, receptors, and protein-protein interaction targets. Bicyclic peptides have been developed as potential therapeutics against a wide range of diseases, drug targeting agents, imaging/diagnostic probes, and research tools.

Enhancing the Cell Permeability and Metabolic Stability of Peptidyl Drugs by Reversible Bicyclization.

Therapeutic applications of peptides are currently limited by their proteolytic instability and impermeability to the cell membrane. A general, reversible bicyclization strategy is now reported to increase both the proteolytic stability and cell permeability of peptidyl drugs. A peptide drug is fused with a short cell-penetrating motif and converted into a conformationally constrained bicyclic structure through the formation of a pair of disulfide bonds.

Screening bicyclic peptide libraries for protein-protein interaction inhibitors: discovery of a tumor necrosis factor-α antagonist.

Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well-defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions.