An Encodable Scaffold for Sequence-Specific Recognition of Duplex RNA.

RNA, unlike DNA, folds into a multitude of secondary and tertiary structures. This structural diversity has impeded the development of ligands that can sequence-specifically target this biomolecule. We sought to develop ligands for double-stranded RNA (dsRNA) segments, which are ubiquitous in RNA tertiary structure. The major groove of double-stranded DNA is sequence-specifically recognized by a range of dimeric helical transcription factors, including the basic leucine zippers (bZIP) and basic helix-loop-helix (bHLH) proteins; however, such simple structural motifs are not prevalent in RNA-binding proteins. We interrogated the high-resolution structures of DNA and RNA to identify requirements for a helix fork motif to occupy dsRNA major grooves akin to dsDNA. Our analysis suggested that the rigidity and angle of approach of dimeric helices in bZIP/bHLH motifs are not ideal for the binding of dsRNA major grooves. This investigation revealed that the replacement of the leucine zipper motifs in bHLH proteins with synthetic crosslinkers would allow recognition of dsRNA. We show that a model bHLH DNA-binding motif does not bind dsRNA but can be reengineered as an RNA ligand. Based on this hypothesis, we rationally designed a miniature synthetic crosslinked helix fork (CHF) as a generalizable proteomimetic scaffold for targeting dsRNA. We evaluated several CHF constructs against a set of RNA and DNA hairpins to probe the specificity of the designed construct. Our studies reveal a new class of proteomimetics as an encodable platform for sequence-specific recognition of dsRNA.

Targeting the interaction of ß-catenin and TCF/LEF transcription factors to inhibit oncogenic Wnt signaling.

The Wnt/ß-catenin signaling pathway is crucially involved in embryonic development, stem cell maintenance and tissue renewal. Hyperactivation of this pathway is associated with the development and progression of various types of cancers. The transcriptional coactivator ß-catenin represents a pivotal component of the pathway and its interaction with transcription factors of the TCF/LEF family is central to pathway activation. Inhibition of this crucial protein-protein interaction via direct targeting of ß-catenin is considered a promising strategy for the inactivation of oncogenic Wnt signaling. This review summarizes advances in the development of Wnt antagonists that have been shown to directly bind ß-catenin.

Protein Cofactor Mimics Disrupt Essential Chaperone Function in Stressed Mycobacteria.

Bacterial DnaK is an ATP-dependent molecular chaperone important for maintaining cellular proteostasis in concert with cofactor proteins. The cofactor DnaJ delivers non-native client proteins to DnaK and activates its ATPase activity, which is required for protein folding. In the bacterial pathogen Mycobacterium tuberculosis, DnaK is assisted by two DnaJs, DnaJ1 and DnaJ2. Functional protein-protein interactions (PPIs) between DnaK and at least one DnaJ are essential for survival of mycobacteria; hence, these PPIs represent untapped antibacterial targets. Here, we synthesize peptide-based mimetics of DnaJ1 and DnaJ2 N-terminal domains as rational inhibitors of DnaK-cofactor interactions. We find that covalently stabilized DnaJ mimetics are capable of disrupting DnaK-cofactor activity in vitro and prevent mycobacterial recovery from proteotoxic stress in vivo, leading to cell death. Since chaperones and cofactors are highly conserved, we anticipate these results will inform the design of other mimetics to modulate chaperone function across cell types.

Design and Synthesis of Crosslinked Helix Dimers as Protein Tertiary Structure Mimics.

Crosslinked helix dimers (CHDs) are synthetic tertiary helical structure motifs designed to modulate interactions of proteins with binding partners. Helix dimers serve as mimics of coiled coils, which are known to be implicated in a multitude of protein complexes. Coiled coils are typically stable in long peptides (>21-28 residues), because sufficient intra- and interstrand contacts are not available in short peptides to coax strand assembly. To engineer conformationally stable CHDs in short sequences, we introduced a covalent linkage in place of an interhelical salt bridge and sculpted the helical interface with optimal hydrophobic packing. CHDs have shown efficacy for the disruption of targeted protein-protein interactions in biochemical, cellular, and animal models. This article describes our optimized approach to design and synthesize parallel and antiparallel helical tertiary structure mimics. Synthesis of CHDs involves conjugation of individual peptide segments, purification of the mono-conjugated strand, and alkylation of the two independent strands to yield crosslinked dimers. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Protocol for bis-triazole CHDs Basic Protocol 2: Protocol for dibenzyl ether CHDs.

A Sos proteomimetic as a pan-Ras inhibitor.

Aberrant Ras signaling is linked to a wide spectrum of hyperproliferative diseases, and components of the signaling pathway, including Ras, have been the subject of intense and ongoing drug discovery efforts. The cellular activity of Ras is modulated by its association with the guanine nucleotide exchange factor Son of sevenless (Sos), and the high-resolution crystal structure of the Ras-Sos complex provides a basis for the rational design of orthosteric Ras ligands. We constructed a synthetic Sos protein mimic that engages the wild-type and oncogenic forms of nucleotide-bound Ras and modulates downstream kinase signaling. The Sos mimic was designed to capture the conformation of the Sos helix-loop-helix motif that makes critical contacts with Ras in its switch region. Chemoproteomic studies illustrate that the proteomimetic engages Ras and other cellular GTPases. The synthetic proteomimetic resists proteolytic degradation and enters cells through macropinocytosis. As such, it is selectively toxic to cancer cells with up-regulated macropinocytosis, including those that feature oncogenic Ras mutations.

Synthetic, structural mimetics of the ß-hairpin flap of HIV-1 protease inhibit enzyme function.

Small-molecule mimetics of the ß-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 µM.

Multi-Facial, Non-Peptidic α-Helix Mimetics.

α-Helices often recognize their target proteins at protein-protein interfaces through more than one recognition face. This review describes the state-of-the-art in the design of non-peptidic α-helix mimetics that reproduce functionality from multiple faces of an α-helix.

α-Helix mimetics: outwards and upwards.

α-Helices are common secondary structural elements forming key parts of the large, generally featureless interfacial regions of many therapeutically-relevant protein-protein interactions (PPIs). The rational design of helix mimetics is an appealing small-molecule strategy for the mediation of aberrant PPIs, however the first generation of scaffolds presented a relatively small number of residues on a single recognition surface. Increasingly, helices involved in PPIs are found to have more complex binding modes, utilizing two or three recognition surfaces, or binding with extended points of contact. To address these unmet needs the design and synthesis of new generations of multi-sided, extended, and supersecondary structures are underway.

4,4'-Unsymmetrically substituted 3,3'-biphenyl alpha helical proteomimetics as potential coactivator binding inhibitors.

A series of unsymmetrically substituted biphenyl compounds was designed as alpha helical proteomimetics with the aim of inhibiting the binding of coactivator proteins to the nuclear hormone receptor coactivator binding domain. These compounds were synthesized in good overall yields in seven steps starting from 2-bromoanisole. The final products were evaluated using cotransfection reporter gene assays and mammalian two-hybrid competitive inhibition assays to demonstrate their effectiveness as competitive binding inhibitors. The results from this study indicate that these proteomimetics possess the ability to inhibit coactivator-receptor interactions, but via a mixed mode of inhibition.