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. Here, we investigate the permeabilities of over 200 macrocyclic 10-mers using the thioether cyclization motif commonly found in mRNA display macrocycle libraries. We identified the optimal lipophilicity range for achieving permeability in thioether-cyclized 10-mer cyclic peptide-peptoid hybrid scaffolds and showed that permeability could be maintained upon extensive permutation in the backbone. In one case, changing a single amino acid from d-Pro to d-NMe-Ala, representing the loss of a single methylene group in the side chain, resulted in a highly permeable scaffold in which the low-dielectric conformation shifted from the canonical cross-beta geometry of the parent compounds into a novel saddle-shaped fold in which all four backbone NH groups were sequestered from the solvent. This work provides an example by which pre-existing physicochemical knowledge of a scaffold can benefit the design of macrocyclic peptide mRNA display libraries, pointing toward an approach for biasing libraries toward permeability by design. Moreover, the compounds described herein are a further demonstration that geometrically diverse, highly permeable scaffolds exist well beyond conventional drug-like chemical space.

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. Bulk emulsion polymerization of polyacrylamide hydrogel shells around magnetic microbeads yielded uniform particles (7 ± 2 µm diameter) that are compatible with diverse in-gel functionalization (amine, alkyne, oligonucleotides) and transformations associated with DNA-encoded library synthesis (acylation, enzymatic DNA ligation). In a case study of reformatting mRNA display libraries, transcription from DNA-templated magnetic beads encapsulated in gel particles colocalized both RNA synthesis via hybridization with copolymerized complementary DNA and translation via puromycin labeling. Two control epitope templates (V5, HA) were successfully enriched (50- and 99-fold, respectively) from an NNK5 library bead screen via FACS. Proximity-driven library synthesis in concert with magnetic sample manipulation provides a plausible means for reformatting encoded combinatorial libraries at scale.

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. The current standard for selecting hit peptides from these selections for downstream follow-up relies on the frequency counting and sorting of unique peptide sequences which can result in the generation of false negatives due to technical reasons including low translation efficiency or other experimental factors. To overcome our inability to detect weakly enriched peptide sequences among our large data sets, we wanted to develop a clustering method that would enable the identification of peptide families. Unfortunately, utilizing traditional clustering algorithms, such as ClustalW, is not possible for this technology due to the incorporation of NCAAs in these libraries. Therefore, we developed a new atomistic clustering method with a Pairwise Aligned Peptide (PAP) chemical similarity metric to perform sequence alignments and identify macrocyclic peptide families. With this method, low enriched peptides, including isolated sequences (singletons), can now be clustered into families providing a comprehensive analysis of NGS data resulting from macrocycle discovery selections. Additionally, upon identification of a hit peptide with the desired activity, this clustering algorithm can be used to identify derivatives from the initial data set for structure-activity relationship (SAR) analysis without requiring additional selection experiments.

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. A liposomal fluorogenic azide probe transduces permeation of alkyne-labeled molecules into small unilamellar vesicles via copper-catalyzed azide-alkyne cycloaddition. Control alkynes (e.g., propargylamine, various alkyne-labeled PEGs) benchmarked the assay. Cell-permeable macrocyclic peptides, exemplary bRo5 molecules, were alkyne labeled and shown to retain permeability. The assay was miniaturized to microfluidic droplets with high assay quality (Z' ≥ 0.5), demonstrating excellent discrimination of photocleaved known membrane-permeable and -impermeable model library beads. Droplet-scale permeation screening will enable pharmacokinetic mapping of bRo5 libraries to build predictive models.

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 in vitro 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. In this study, we developed a luminescence-based assay to rapidly assess the relative translation yield of any noncanonical amino acid in real time. Among the 59 amino acids tested here, we found that many translate with high efficiency, but translational yield is not necessarily correlated to whether the amino acid is proteinogenic or has high tRNA acylation efficiency. Interestingly, we found that single-template translation data can inform the library-scale translation yield and that shorter peptide libraries are more tolerant of lower-efficiency amino acid monomers. Together our data show that the luminescence-based assay described herein is an essential tool in evaluating new building blocks and codon table designs within mRNA display toward the goal of developing druglike peptide-based libraries for drug discovery campaigns.

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. In addition, we developed a protocol for designing thioether-cyclized macrocycles within Rosetta, due to their common application in mRNA display using the RaPID platform. To evaluate the utility of these new computational tools, we screened a library of canonical and NCAAs on both a linear peptide and a thioether macrocycle, allowing us to quickly identify mutations that affect peptide binding and subsequently measure our results against previously published data. We anticipate in silico screening of peptides against a diverse chemical space will be a fundamental component for peptide design and optimization, as more amino acids can be explored in a single in silico screen than an in vitro screen. As such, these tools will enable maturation of peptide affinity for protein targets of interest and optimization of peptide pharmacokinetics for therapeutic applications.

An Integrated Strategy for Assessing the Metabolic Stability and Biotransformation of Macrocyclic Peptides in Drug Discovery toward Oral Delivery.

Macrocyclic peptides (MCPs) are an emerging class of promising drug modalities that can be used to interrogate hard-to-drug ("undruggable") targets. However, their poor intestinal stability is one of the major liabilities or obstacles for oral drug delivery. We therefore investigated the metabolic stability and biotransformation of MCPs via a systematic approach and established an integrated in vitro assay strategy to facilitate MCP drug discovery, with a focus on oral delivery liabilities. A group of diverse MCPs were incubated with representative matrices, including simulated intestinal fluid with pancreatin (SIFP), human enterocytes, liver S9 fractions, liver lysosomes, plasma, and recombinant enzymes. The results revealed that the stability and biotransformation of MCPs varied, with the major metabolic pathways identified in different matrices. Under the given conditions, the selected MCPs generally showed better stability in plasma compared to that in SIFP. Our data suggest that pancreatic enzymes act as the primary metabolic barrier for the oral delivery of MCPs, mainly through hydrolysis of their backbone amide bonds. Whereas in enterocytes, multiple metabolic pathways appeared to be involved and resulted in metabolic reactions such as oxidation and reduction in addition to hydrolysis. Further studies suggested that lysosomal peptidase cathepsin B could be a major enzyme responsible for the cleavage of side-chain amide bonds in lysosomes. Collectively, we developed and implemented an integrated assay for assessing the metabolic stability and biotransformation of MCPs for compound screening in the discovery stage toward oral delivery. The proposed question-driven assay cascade can provide biotransformation insights that help to guide and facilitate lead candidate selection and optimization.

Ribosomal Synthesis of Macrocyclic Peptides with Linear γ4- and ß-Hydroxy-γ4-amino Acids.

Herein, we report the ribosomal elongation of linear γ4- and ß-hydroxy-γ4-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 γ4-amino acids (γ4Gly, (S)-γ4Ala, (S)-γ4Nva, and (R)-γ4Leu) 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. Four Ala-derived statines were successfully incorporated into peptides, and γ4SAla3R-OH (GP2) showed a similar efficiency of incorporation to that of (S)-ß2hAla and l-Ala. These new building blocks might confer the important pharmacological properties of protease resistance and membrane permeability to macrocyclic peptides and expand the diversity of future combinatorial peptide libraries that can be translated by the ribosome.

Ribosomal Synthesis of Macrocyclic Peptides with ß2- and ß2,3-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 ß2-homo-amino acids (ß2haa) to enable their incorporation into mRNA display-based selection libraries. We confirmed the compatibility of 14 ß2-homo-amino acids, (S)- and (R)-stereochemistry, for single incorporation into a macrocyclic peptide with low to high translation efficiency. Interestingly, N-methylation of the backbone amide of ß2haa prevented the incorporation of this amino acid subclass by the ribosome. Additionally, we designed and incorporated several α,ß-disubstituted ß2,3-homo-amino acids (ß2,3haa) with different R-groups on the α- and ß-carbons of the same amino acid. Incorporation of these ß2,3haa enables increased diversity in a single position of a macrocyclic peptide without significantly increasing the overall molecular weight, which is an important consideration for passive cell permeability. We also successfully incorporated multiple (S)-ß2hAla into a single macrocycle with other non-proteinogenic amino acids, confirming that this class of ß-amino acid is suitable for development of large scale macrocyclic peptide libraries.

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 in vitro and is bactericidal against wild-type Acinetobacter baumannii and E. coli strains. While deletion of a gene encoding a major outer membrane lipoprotein, lpp, leads to rescue of bacterial growth after genetic depletion or pharmacologic inhibition of the downstream type II signal peptidase, LspA, no such rescue of growth is detected after Lgt depletion or treatment with G2824. Inhibition of Lgt does not lead to significant accumulation of peptidoglycan-linked Lpp in the inner membrane. Our data validate Lgt as a novel antibacterial target and suggest that, unlike downstream steps in lipoprotein biosynthesis and transport, inhibition of Lgt may not be sensitive to one of the most common resistance mechanisms that invalidate inhibitors of bacterial lipoprotein biosynthesis and transport. IMPORTANCE As the emerging threat of multidrug-resistant (MDR) bacteria continues to increase, no new classes of antibiotics have been discovered in the last 50 years. While previous attempts to inhibit the lipoprotein biosynthetic (LspA) or transport (LolCDE) pathways have been made, most efforts have been hindered by the emergence of a common mechanism leading to resistance, namely, the deletion of the gene encoding a major Gram-negative outer membrane lipoprotein lpp. Our unexpected finding that inhibition of Lgt is not susceptible to lpp deletion-mediated resistance uncovers the complexity of bacterial lipoprotein biogenesis and the corresponding enzymes involved in this essential outer membrane biogenesis pathway and potentially points to new antibacterial targets in this pathway.

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. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

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. In recent years, it has become apparent that the dysregulation and/or overexpression of Hippo pathway effectors is implicated in a wide range of cancers, including prostate, gastric and liver cancer. A large body of work has been dedicated to understanding the therapeutic potential of modulating the phosphorylation and localization of YAP/TAZ. However, YAP/TAZ are considered to be natively unfolded and may be intractable as drug targets. Therefore, TEAD proteins present themselves as an excellent therapeutic target for intervention of the Hippo pathway. This review summarizes the functional role of TEAD proteins in cancer and assesses the therapeutic potential of antagonizing TEAD function in vivo.

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. Initial core mutations disrupt natural packing and lead to increased flexibility. Additional, experimentally selected mutations quenched conformational heterogeneity through new stabilizing interactions. Stabilizing interactions, such as cation-pi stacking and ordered waters, which are not included in standard protein design energy functions, can create specific interactions that have long-range effects on flexibility across the protein. Our results suggest that increasing flexibility may be a useful strategy to escape local minima during initial directed evolution and protein design steps when creating new functions.

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. Here, we identify a novel form of regulation of TEAD family proteins. We show that human TEADs are palmitoylated at a universally conserved cysteine, and report the crystal structures of the human TEAD2 and TEAD3 YAP-binding domains in their palmitoylated forms. These structures show a palmitate bound within a highly conserved hydrophobic cavity at each protein's core. Our findings also demonstrate that this modification is required for proper TEAD folding and stability, indicating a potential new avenue for pharmacologically regulating the Hippo pathway through the modulation of TEAD palmitoylation.

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. Using mass spectrometry, we show that recombinant USP30 preferentially removes these linkage types from intact ubiquitylated mitochondria and counteracts parkin-mediated ubiquitin chain formation in cells. These results, combined with a series of chimaera and localization studies, afford insights into the mechanism by which a balance of ubiquitylation and deubiquitylation regulates mitochondrial homeostasis, and suggest a general mechanism for organelle autophagy.

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. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.

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. As part of our investigations on the origins of species-specific differences in Hsp90 conformational dynamics we probed the role of this MD arginine in bacterial, yeast, and human Hsp90s using a combination of structural and functional approaches. While the R380A mutation compromised ATPase activity in all three homologs, the impact on ATPase activity was both variable and much more modest (2-7 fold) than the mutation of an active site glutamate (40 fold) known to be required for hydrolysis. Single particle electron microscopy and small-angle X-ray scattering revealed that, for all Hsp90s, mutation of this arginine abrogated the ability to form the closed "ATP" conformational state in response to AMPPNP binding. Taken together with previous mutagenesis data exploring intra- and intermonomer interactions, these new data suggest that R380 does not directly participate in the hydrolysis reaction as a catalytic residue, but instead acts as an ATP-sensor to stabilize an NTD-MD conformation required for efficient ATP hydrolysis.

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. Using the crystal structures as a guide, we examined the role of intra- and intermonomer interactions in stabilizing the ATPase activity of a single active site within an intact dimer. This was accomplished by creating heterodimers that allow us to differentially mutate each monomer, probing the context in which particular residues are important for ATP hydrolysis. Although the ATPase activity of each monomer can function independently, we found that the activity of one monomer could be inhibited by the mutation of hydrophobic residues on the trans N-terminal domain (opposite monomer). Furthermore, these trans interactions are synergistically mediated by a loop on the cis middle domain. This loop contains hydrophobic residues as well as a critical arginine that provides a direct linkage to the gamma-phosphate of bound ATP. Small angle x-ray scattering demonstrates that deleterious mutations block domain closure in the presence of AMPPNP (5'-adenylyl-beta,gamma-imidodiphosphate), providing a direct linkage between structural changes and functional consequences. Together, these data indicate that both the cis monomer and the trans monomer and the intradomain and interdomain interactions cooperatively stabilize the active conformation of each active site and help explain the importance of dimer formation.