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. Among the series of hits, the exceptional paradigm of GPM-3, a protein and state-specific bicyclic peptide, is the first chemically identified GAP (GTPase-activating protein) modulator with a high binding affinity for Gαi protein. Computational analyses identified and assessed the structure of the bicyclic peptides, novel ligand-protein interaction sites, and their subsequent impact on the nucleotide binding site. This approach can therefore lead the way for the development of efficient chemical biological probes targeting Gαi protein modulation within a cellular context.

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. This work reveals compounds with mid-micromolar binding affinity (KD = 60 ± 6 µM) to the FSE RNA and supports a binding mode distinct from previously reported FSE binders MTDB and merafloxacin. In addition, compounds are active in in vitro dual-luciferase and in-cell dual-fluorescent-reporter frameshifting assays, highlighting the promise of targeting structured elements of RNAs with druglike compounds to alter expression of viral proteins.

A chemical probe based on the PreQ1 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 PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, 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. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.

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 (KD = 21 nM for KRasG12V-GppNHp) and is highly cell-permeable and metabolically stable (serum t1/2 > 24 h). B4-27 inhibits Ras signaling in vitro and in vivo by blocking Ras from interacting with downstream effector proteins and induces apoptosis of Ras-mutant cancer cells. When administered systemically (i.v.), B4-27 suppressed tumor growth in two different mouse xenograft models at 1-5 mg/kg of daily doses.

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/ß (IKKα/ß)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKKß interaction with low µM IC50 values. Further optimization of one of the hits led to a relatively potent and cell-permeable NEMO inhibitor (IC50 = 1.0 µM), which selectively inhibited canonical NF-κB signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells. The inhibitor provides a useful tool for investigating the biological functions of NEMO/NF-κB and a potential lead for further development of a novel class of anti-inflammatory and anticancer drugs.

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. In this Minireview, we provide a summary of the recent progresses on the synthesis and applications of bicyclic peptides.

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. The resulting bicyclic peptide has greatly enhanced proteolytic stability as well as cell-permeability. Once inside the cell, the disulfide bonds are reduced to produce a linear, biologically active peptide. This strategy was applied to generate a cell-permeable bicyclic peptidyl inhibitor against the NEMO-IKK interaction.

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. Screening of a bicyclic peptide library against tumor necrosis factor-α (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions.