Dragging Ras Back in the Ring

Lech-Gustav Milroy,† Tom N. Grossmann,‡,§ Sven Hennig,‡ Luc Brunsveld,† and Christian Ottmann*,† †Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Technische Universiteit Eindhoven, Den Dolech 2, 5612 AZ Eindhoven, The Netherlands ‡Chemical Genomics Centre of the Max Planck Society, Otto-Hahn Straße 15, 44227 Dortmund, Germany Department of Chemistry and Chemical Biology, Technical University Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

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Modulators of Protein-Protein Interactions

Protein–protein interactions (PPIs) execute many fundamental cellular functions and have served as prime drug targets over the last two decades. Interfering intracellular PPIs with small molecules has been extremely difficult for larger or flat binding sites, as antibodies cannot cross the cell membrane to reach such target sites. In recent years, peptides smaller size and balance of conformational rigidity and flexibility have made them promising candidates for targeting challenging binding interfaces with satisfactory binding affinity and specificity. Deciphering and characterizing peptide–protein recognition mechanisms is thus central for the invention of peptide-based strategies to interfere with endogenous protein interactions, or improvement of the binding affinity and specificity of existing approaches. Importantly, a variety of computation-aided rational designs for peptide therapeutics have been developed, which aim to deliver comprehensive docking for peptide–protein interaction interfaces. Over 60 peptides have been approved and administrated globally in clinics. Despite this, advances in various docking models are only on the merge of making their contribution to peptide drug development. In this review, we provide (i) a holistic overview of peptide drug development and the fundamental technologies utilized to date, and (ii) an updated review on key developments of computational modeling of peptide–protein interactions (PepPIs) with an aim to assist experimental biologists exploit suitable docking methods to advance peptide interfering strategies against PPIs.

A Comprehensive Review on Current Advances in Peptide Drug Development and Design.

Peptides as a therapeutic modality attract much attention due to their synthetic accessibility, high degree of specific binding, and the ability to target protein surfaces traditionally considered “undruggable”. Unfortunately, at the same time, other pharmacological properties of a generic peptide, such as metabolic stability and cell permeability, are quite poor, which limits the success of de novo discovered biologically active peptides as drug candidates. Here, we review how macrocyclization as well as the incorporation of nonproteogenic amino acids and various conjugation strategies may be utilized to improve on these characteristics to create better drug candidates. We analyze recent progress and remaining challenges in improving individual pharmacological properties of bioactive peptides, and offer our opinion on interfacing these, often conflicting, considerations, to create balanced drug candidates as a potential way to make further progress in this area.

Macrocyclic Peptides as Drug Candidates: Recent Progress and Remaining Challenges

Trp53R172H and kmsg12d cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely "undruggable" through the conventional small-molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure-activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras-binding and cell-penetrating properties. These cell-permeable cyclic peptides inhibit Ras signaling by binding to Ras-GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein-protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

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

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Screening Bicyclic Peptide Libraries for Protein–Protein Interaction Inhibitors: Discovery of a Tumor Necrosis Factor-α Antagonist

Cyclic peptides have great potential as therapeutic agents and research tools. However, their applications against intracellular targets have been limited, because cyclic peptides are generally impermeable to the cell membrane. It was previously shown that fusion of cyclic peptides with a cyclic cell-penetrating peptide resulted in cell-permeable bicyclic peptides that are proteolytically stable and biologically active in cellular assays. In this work, we tested the generality of the bicyclic approach by synthesizing a combinatorial library of 5.7 × 106 bicyclic peptides featuring a degenerate sequence in the first ring and an invariant cell-penetrating peptide in the second ring. Screening of the library against oncoprotein K-Ras G12V followed by hit optimization produced a moderately potent and cell-permeable K-Ras inhibitor, which physically blocks the Ras-effector interactions in vitro, inhibits the signaling events downstream of Ras in cancer cells, and induces apoptosis of the cancer cells. Our appr...

Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

A combinatorial library of 6 × 106 cyclic peptides was synthesized in the one bead-two compound format, with each bead displaying a unique cyclic peptide on its surface and a linear peptide encoding tag in its interior. Screening of the library against K-Ras identified compounds that bound K-Ras with submicromolar affinity and disrupted its interaction with effector proteins.

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Inhibition of Ras-Effector Interaction by Cyclic Peptides.

Cyclic peptides have been a rich source of biologically active molecules. Herein we present a method for the combinatorial synthesis and screening of large one-bead-one-compound (OBOC) libraries of cyclic peptides against biological targets such as proteins. Up to ten million different cyclic peptides are rapidly synthesized on TentaGel microbeads by the split-and-pool synthesis method and subjected to a multistage screening protocol which includes magnetic sorting, on-bead enzyme-linked and fluorescence-based assays, and in-solution binding analysis of cyclic peptides selectively released from single beads by fluorescence anisotropy. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. This method allows a single researcher to synthesize and screen up to ten million cyclic peptides and identify the most active ligand(s) in ~1 month, without the time-consuming and expensive hit resynthesis or the use of any special equipment.

Punit Upadhyaya

Papers

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Screening Bicyclic Peptide Libraries for Protein–Protein Interaction Inhibitors: Discovery of a Tumor Necrosis Factor-α Antagonist

Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides

Inhibition of Ras-Effector Interaction by Cyclic Peptides.

Synthesis and screening of one-bead-one-compound cyclic peptide libraries.