An mRNA Display Approach for Covalent Targeting of a Virulence Factor.

Staphylococcus aureus () is an opportunistic human pathogen that causes over one million deaths around the world each year. We recently identified a family of serine hydrolases termed fluorophosphonate binding hydrolases (Fphs) that play important roles in lipid metabolism and colonization of a host. Because many of these enzymes are only expressed in bacteria, they are valuable targets for diagnostics and therapeutics.

CREMP: Conformer-rotamer ensembles of macrocyclic peptides for machine learning.

Computational and machine learning approaches to model the conformational landscape of macrocyclic peptides have the potential to enable rational design and optimization. However, accurate, fast, and scalable methods for modeling macrocycle geometries remain elusive. Recent deep learning approaches have significantly accelerated protein structure prediction and the generation of small-molecule conformational ensembles, yet similar progress has not been made for macrocyclic peptides due to their unique properties.

Membrane Permeability in a Large Macrocyclic Peptide Driven by a Saddle-Shaped Conformation.

The effort to modulate challenging protein targets has stimulated interest in ligands that are larger and more complex than typical small-molecule drugs. While combinatorial techniques such as mRNA display routinely produce high-affinity macrocyclic peptides against classically undruggable targets, poor membrane permeability has limited their use toward primarily extracellular targets. Understanding the passive membrane permeability of macrocyclic peptides would, in principle, improve our ability to design libraries whose leads can be more readily optimized against intracellular targets.

Hydrogel-Encapsulated Beads Enable Proximity-Driven Encoded Library Synthesis and Screening.

Encoded combinatorial library technologies have dramatically expanded the chemical space for screening but are usually only analyzed by affinity selection binding. It would be highly advantageous to reformat selection outputs to "one-bead-one-compound" solid-phase libraries, unlocking activity-based and cellular screening capabilities. Here, we describe hydrogel-encapsulated magnetic beads that enable such a transformation.

Identification of Macrocyclic Peptide Families from Combinatorial Libraries Containing Noncanonical Amino Acids Using Cheminformatics and Bioinformatics Inspired Clustering.

In the past decade, macrocyclic peptides gained increasing interest as a new therapeutic modality to tackle intracellular and extracellular therapeutic targets that had been previously classified as "undruggable". Several technological advances have made discovering macrocyclic peptides against these targets possible: 1) the inclusion of noncanonical amino acids (NCAAs) into mRNA display, 2) increased availability of next generation sequencing (NGS), and 3) improvements in rapid peptide synthesis platforms. This type of directed-evolution based screening can produce large numbers of potential hit sequences given that DNA sequencing is the functional output of this platform.

Liposomal Permeation Assay for Droplet-Scale Pharmacokinetic Screening.

Combinatorial library screening increasingly explores chemical space beyond the Ro5 (bRo5), which is useful for investigating "undruggable" targets but suffers compromised cellular permeability and therefore bioavailability. Moreover, structure-permeation relationships for bRo5 molecules are unclear partially because high-throughput permeation measurement technology for encoded combinatorial libraries is still nascent. Here, we present a permeation assay that is scalable to combinatorial library screening.

Evaluating Translational Efficiency of Noncanonical Amino Acids to Inform the Design of Druglike Peptide Libraries.

Advances in genetic code reprogramming have allowed the site-specific incorporation of noncanonical functionalities into polypeptides and proteins, providing access to wide swaths of chemical space via translation techniques like mRNA display. Prior efforts have established that the translation machinery can tolerate amino acids with modifications to both the peptide backbone and side chains, greatly broadening the chemical space that can be interrogated in ligand discovery efforts. However, existing methods for confirming the translation yield of new amino acid building blocks for these technologies necessitate multistep workups and, more importantly, are not relevant for measuring translation within the context of a combinatorial library consisting of multiple noncanonical amino acids.

Computational Site Saturation Mutagenesis of Canonical and Non-Canonical Amino Acids to Probe Protein-Peptide Interactions.

Technologies for discovering peptides as potential therapeutics have rapidly advanced in recent years with significant interest from both academic and pharmaceutical labs. These advancements in turn drive the need for new computational tools to design peptides for purposes of advancing lead molecules into the clinic. Here we report the development and application of a new automated tool, AutoRotLib, for parameterizing a diverse set of non-canonical amino acids (NCAAs), N-methyl, or peptoid residues for use with the computational design program Rosetta.

Ribosomal Synthesis of Macrocyclic Peptides with Linear γ- and β-Hydroxy-γ-amino Acids.

Herein, we report the ribosomal elongation of linear γ- and β-hydroxy-γ-amino acids (statines) to expand the nonproteinogenic amino acid repertoire of natural product-like combinatorial peptide libraries. First, we demonstrated the successful ribosomal incorporation of four linear γ-amino acids (γGly, ()-γAla, ()-γNva, and ()-γLeu) into a 10-mer macrocyclic peptide scaffold. Given the promising effects reported for statines on the cell permeability of macrocyclic peptides, we also designed and tested the ribosomal incorporation of six statines derived from Ala and d-val.

Ribosomal Synthesis of Macrocyclic Peptides with β- and β-Homo-Amino Acids for the Development of Natural Product-Like Combinatorial Libraries.

The development of large, natural-product-like, combinatorial macrocyclic peptide libraries is essential in the quest to develop therapeutics for "undruggable" cellular targets. Herein we report the ribosomal synthesis of macrocyclic peptides containing one or more β-homo-amino acids (βhaa) to enable their incorporation into mRNA display-based selection libraries. We confirmed the compatibility of 14 β-homo-amino acids, ()- and ()-stereochemistry, for single incorporation into a macrocyclic peptide with low to high translation efficiency.

Inhibition of Escherichia coli Lipoprotein Diacylglyceryl Transferase Is Insensitive to Resistance Caused by Deletion of Braun's Lipoprotein.

Lipoprotein diacylglyceryl transferase (Lgt) catalyzes the first step in the biogenesis of Gram-negative bacterial lipoproteins which play crucial roles in bacterial growth and pathogenesis. We demonstrate that Lgt depletion in a clinical uropathogenic Escherichia coli strain leads to permeabilization of the outer membrane and increased sensitivity to serum killing and antibiotics. Importantly, we identify G2824 as the first-described Lgt inhibitor that potently inhibits Lgt biochemical activity and is bactericidal against wild-type Acinetobacter baumannii and E.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation.

Targeting the Hippo Pathway and Cancer through the TEAD Family of Transcription Factors.

The Hippo pathway is a critical transcriptional signaling pathway that regulates cell growth, proliferation and organ development. The transcriptional enhanced associate domain (TEAD) protein family consists of four paralogous transcription factors that function to modulate gene expression in response to the Hippo signaling pathway. Transcriptional activation of these proteins occurs upon binding to the co-activator YAP/TAZ whose entry into the nucleus is regulated by Lats1/2 kinase.

Flexibility and Design: Conformational Heterogeneity along the Evolutionary Trajectory of a Redesigned Ubiquitin.

Although protein design has been used to introduce new functions, designed variants generally only function as well as natural proteins after rounds of laboratory evolution. One possibility for this pattern is that designed mutants frequently sample nonfunctional conformations. To test this idea, we exploited advances in multiconformer modeling of room-temperature X-ray data collection on redesigned ubiquitin variants selected for increasing binding affinity to the deubiquitinase USP7.

Palmitoylation of TEAD Transcription Factors Is Required for Their Stability and Function in Hippo Pathway Signaling.

The Hippo signaling pathway is responsible for regulating the function of TEAD family transcription factors in metazoans. TEADs, with their co-activators YAP/TAZ, are critical for controlling cell differentiation and organ size through their transcriptional activation of genes involved in cell growth and proliferation. Dysregulation of the Hippo pathway has been implicated in multiple forms of cancer.

USP30 and parkin homeostatically regulate atypical ubiquitin chains on mitochondria.

Multiple lines of evidence indicate that mitochondrial dysfunction is central to Parkinson's disease. Here we investigate the mechanism by which parkin, an E3 ubiquitin ligase, and USP30, a mitochondrion-localized deubiquitylase, regulate mitophagy. We find that mitochondrial damage stimulates parkin to assemble Lys 6, Lys 11 and Lys 63 chains on mitochondria, and that USP30 is a ubiquitin-specific deubiquitylase with a strong preference for cleaving Lys 6- and Lys 11-linked multimers.

Decoding a chain letter for degradation.

Ubiquitin chains can have distinct signaling outcomes, depending on their conjugation point. In this issue of Structure, Castañeda and colleagues describe a new structure of K11 diubiquitin and investigate its recognition by effectors to target substrates to the proteasome.

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling.

Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin.

The conserved arginine 380 of Hsp90 is not a catalytic residue, but stabilizes the closed conformation required for ATP hydrolysis.

Hsp90, a dimeric ATP-dependent molecular chaperone, is required for the folding and activation of numerous essential substrate "client" proteins including nuclear receptors, cell cycle kinases, and telomerase. Fundamental to its mechanism is an ensemble of dramatically different conformational states that result from nucleotide binding and hydrolysis and distinct sets of interdomain interactions. Previous structural and biochemical work identified a conserved arginine residue (R380 in yeast) in the Hsp90 middle domain (MD) that is required for wild type hydrolysis activity in yeast, and hence proposed to be a catalytic residue.

Mechanistic studies of Sansalvamide A-amide: an allosteric modulator of Hsp90.

Herein we show that San A-amide, a structurally unique molecule, influences a subset of cancer-related pathways involving Hsp90. We show that San A-amide specifically binds to the N-middle domain of Hsp90 allosterically disrupts the binding of proteins thought to interact with the Hsp90 C-terminal domain, while having no effect on an N-terminal domain client protein. This unique mechanism suggests that San A-amide is a potential tool for studying C-terminal binding proteins of Hsp90 as well as a promising lead in the development of new cancer therapeutics.

Intra- and intermonomer interactions are required to synergistically facilitate ATP hydrolysis in Hsp90.

Nucleotide-dependent conformational changes of the constitutively dimeric molecular chaperone Hsp90 are integral to its molecular mechanism. Recent full-length crystal structures (Protein Data Bank codes 2IOQ, 2CG9, AND 2IOP) of Hsp90 homologs reveal large scale quaternary domain rearrangements upon the addition of nucleotides. Although previous work has shown the importance of C-terminal domain dimerization for efficient ATP hydrolysis, which should imply cooperativity, other studies suggest that the two ATPases function independently.