Cystine-knot peptide inhibitors of HTRA1 bind to a cryptic pocket within the active site region.

Cystine-knot peptides (CKPs) are naturally occurring peptides that exhibit exceptional chemical and proteolytic stability. We leveraged the CKP carboxypeptidase A1 inhibitor as a scaffold to construct phage-displayed CKP libraries and subsequently screened these collections against HTRA1, a trimeric serine protease implicated in age-related macular degeneration and osteoarthritis. The initial hits were optimized by using affinity maturation strategies to yield highly selective and potent picomolar inhibitors of HTRA1. Crystal structures, coupled with biochemical studies, reveal that the CKPs do not interact in a substrate-like manner but bind to a cryptic pocket at the S1' site region of HTRA1 and abolish catalysis by stabilizing a non-competent active site conformation. The opening and closing of this cryptic pocket is controlled by the gatekeeper residue V221, and its movement is facilitated by the absence of a constraining disulfide bond that is typically present in trypsin fold serine proteases, thereby explaining the remarkable selectivity of the CKPs. Our findings reveal an intriguing mechanism for modulating the activity of HTRA1, and highlight the utility of CKP-based phage display platforms in uncovering potent and selective inhibitors against challenging therapeutic targets.

A phage-displayed disulfide constrained peptide discovery platform yields novel human plasma protein binders.

Disulfide constrained peptides (DCPs) show great potential as templates for drug discovery. They are characterized by conserved cysteine residues that form intramolecular disulfide bonds. Taking advantage of phage display technology, we designed and generated twenty-six DCP phage libraries with enriched molecular diversity to enable the discovery of ligands against disease-causing proteins of interest. The libraries were designed based on five DCP scaffolds, namely Momordica charantia 1 (Mch1), gurmarin, Asteropsin-A, antimicrobial peptide-1 (AMP-1), and potato carboxypeptidase inhibitor (CPI). We also report optimized workflows for screening and producing synthetic and recombinant DCPs. Examples of novel DCP binders identified against various protein targets are presented, including human IgG Fc, serum albumin, vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF). We identified DCPs against human IgG Fc and serum albumin with sub-micromolar affinity from primary panning campaigns, providing alternative tools for potential half-life extension of peptides and small protein therapeutics. Overall, the molecular diversity of the DCP scaffolds included in the designed libraries, coupled with their distinct biochemical and biophysical properties, enables efficient and robust identification of de novo binders to drug targets of therapeutic relevance.

Disulfide-constrained peptide scaffolds enable a robust peptide-therapeutic discovery platform.

Peptides present an alternative modality to immunoglobulin domains or small molecules for developing therapeutics to either agonize or antagonize cellular pathways associated with diseases. However, peptides often suffer from poor chemical and physical stability, limiting their therapeutic potential. Disulfide-constrained peptides (DCP) are naturally occurring and possess numerous desirable properties, such as high stability, that qualify them as drug-like scaffolds for peptide therapeutics. DCPs contain loop regions protruding from the core of the molecule that are amenable to peptide engineering via direct evolution by use of phage display technology. In this study, we have established a robust platform for the discovery of peptide therapeutics using various DCPs as scaffolds. We created diverse libraries comprising seven different DCP scaffolds, resulting in an overall diversity of 2 x 1011. The effectiveness of this platform for functional hit discovery has been extensively evaluated, demonstrating a hit rate comparable to that of synthetic antibody libraries. By utilizing chemically synthesized and in vitro folded peptides derived from selections of phage displayed DCP libraries, we have successfully generated functional inhibitors targeting the HtrA1 protease. Through affinity maturation strategies, we have transformed initially weak binders against Notch2 with micromolar Kd values to high-affinity ligands in the nanomolar range. This process highlights a viable hit-to-lead progression. Overall, our platform holds significant potential to greatly enhance the discovery of peptide therapeutics.

Potent and selective binders of the E3 ubiquitin ligase ZNRF3 stimulate Wnt signaling and intestinal organoid growth.

Selective and precise activation of signaling transduction cascades is key for cellular reprogramming and tissue regeneration. However, the development of small- or large-molecule agonists for many signaling pathways has remained elusive and is rate limiting to realize the full clinical potential of regenerative medicine. Focusing on the Wnt pathway, here we describe a series of disulfide-constrained peptides (DCPs) that promote Wnt signaling activity by modulating the cell surface levels of ZNRF3, an E3 ubiquitin ligase that controls the abundance of the Wnt receptor complex FZD/LRP at the plasma membrane. Mechanistically, monomeric DCPs induce ZNRF3 ubiquitination, leading to its cell surface clearance, ultimately resulting in FZD stabilization. Furthermore, we engineered multimeric DCPs that induce expansive growth of human intestinal organoids, revealing a dependence between valency and ZNRF3 clearance. Our work highlights a strategy for the development of potent, biologically active Wnt signaling pathway agonists via targeting of ZNRF3.

Synthetic Multivalent Disulfide-Constrained Peptide Agonists Potentiate Wnt1/ß-Catenin Signaling via LRP6 Coreceptor Clustering.

Wnt ligands are critical for tissue homeostasis and form a complex with LRP6 and frizzled coreceptors to initiate Wnt/ß-catenin signaling. Yet, how different Wnts achieve various levels of signaling activation through distinct domains on LRP6 remains elusive. Developing tool ligands that target individual LRP6 domains could help elucidate the mechanism of Wnt signaling regulation and uncover pharmacological approaches for pathway modulation. We employed directed evolution of a disulfide constrained peptide (DCP) to identify molecules that bind to the third ß-propeller domain of LRP6. The DCPs antagonize Wnt3a while sparing Wnt1 signaling. Using PEG linkers with different geometries, we converted the Wnt3a antagonist DCPs to multivalent molecules that potentiated Wnt1 signaling by clustering the LRP6 coreceptor. The mechanism of potentiation is unique as it occurred only in the presence of extracellular secreted Wnt1 ligand. While all DCPs recognized a similar binding interface on LRP6, they displayed different spatial orientations that influenced their cellular activities. Moreover, structural analyses revealed that the DCPs exhibited new folds that were distinct from the parent DCP framework they were evolved from. The multivalent ligand design principles highlighted in this study provide a path for developing peptide agonists that modulate different branches of cellular Wnt signaling.

Directed evolution identifies high-affinity cystine-knot peptide agonists and antagonists of Wnt/ß-catenin signaling.

Developing peptide-based tools to fine-tune growth signaling pathways, in particular molecules with exquisite selectivity and high affinities, opens up opportunities for cellular reprogramming in tissue regeneration. Here, we present a library based on cystine-knot peptides (CKPs) that incorporate multiple loops for randomization and selection via directed evolution. Resulting binders could be assembled into multimeric structures to fine-tune cellular signaling. An example is presented for the Wnt pathway, which plays a key role in the homeostasis and regeneration of tissues such as lung, skin, and intestine. We discovered picomolar affinity CKP agonists of the human LPR6 receptor by exploring the limits of the topological manipulation of LRP6 dimerization. Structural analyses revealed that the agonists bind at the first ß-propeller domain of LRP6, mimicking the natural Wnt inhibitors DKK1 and SOST. However, the CKP agonists exhibit a different mode of action as they amplify the signaling of natural Wnt ligands but do not activate the pathway by themselves. In an alveolosphere organoid model, the CKP agonists induced alveolar stem cell activity. They also stimulated growth in primary human intestinal organoids. The approach described here advances the important frontier of next-generation agonist design and could be applied to other signaling pathways to discover tunable agonist ligands.

Fatty acylation enhances the cellular internalization and cytosolic distribution of a cystine-knot peptide.

Delivering peptides into cells could open up possibilities for targeting intracellular proteins. Although fatty acylation of peptide therapeutics improves their systemic half-life, it remains unclear how it influences their cellular uptake. Here, we demonstrate that a fatty acylated peptide exhibits enhanced cellular internalization and cytosolic distribution compared to the un-acylated version. By using a cystine-knot peptide as a model system, we report an efficient strategy for site-specific conjugation of fatty acids. Peptides modified with fatty acids of different chain lengths entered cells through clathrin-mediated and macropinocytosis pathways. The cellular uptake was mediated by the length of the hydrocarbon chain, with myristic acid conjugates displaying the highest distribution across the cytoplasm including the cytosol, and endomembranes of the ER, Golgi and mitochondria. Our studies demonstrate how fatty acylation improves the cellular uptake of peptides, and lay the groundwork for future development of bioactive peptides with enhanced intracellular distribution.

Discovery of a dual pathway aggregation mechanism for a therapeutic constrained peptide.

Constrained peptides (CPs) have emerged as attractive candidates for drug discovery and development. To fully unlock the therapeutic potential of CPs, it is crucial to understand their physical stability and minimize the formation of aggregates that could induce immune responses. Although amyloid like aggregates have been researched extensively, few studies have focused on aggregates from other peptide scaffolds (e.g., CPs). In this work, a streamlined approach to effectively profile the nature and formation pathway of CP aggregates was demonstrated. Aggregates of various sizes were detected and shown to be amorphous. Though no major changes were found in peptide structure upon aggregation, these aggregates appeared to have mixed natures, consisting of primarily non-covalent aggregates with a low level of covalent species. This co-existence phenomenon was also supported by two kinetic pathways observed in time- and temperature-dependent aggregation studies. Furthermore, a stability study with 8 additional peptide variants exhibited good correlation between aggregation propensity and peptide hydrophobicity. Therefore, a dual aggregation pathway was proposed, with the non-covalent aggregates driven by hydrophobic interactions, whereas the covalent ones formed through disulfide scrambling. Overall, the workflow presented here provides a powerful strategy for comprehensive characterization of peptide aggregates and understanding their mechanisms of formation.

Tracking internal and external ions for constrained peptides leads to enhanced sequence coverage and disulfide bond deciphering.

Top-down characterization of disulfide-rich peptides and proteins presents many challenges due to the constrained and protected amino acid backbone. Typically, chemical reduction is required to reduce the disulfide bonds and/or enzymatic digestion (bottom-up analysis) is utilized to selectively cleave the amino acid sequence prior to mass spectrometry analysis owing to the challenges associated with intact, top-down analysis of these biomolecules. While extravagant top-down characterization techniques such as ultraviolet photodissociation (UVPD) or electron capture dissociation (ECD), have demonstrated the ability to break disulfide bonds in top-down workflows, implementation of these technologies and analysis of the resulting fragmentation spectra is not trivial and often inaccessible to many laboratories and users. In the study presented herein, traditional collision induced dissociation (CID) of disulfide-rich peptides is performed to confirm the disulfide bond connectivity and localize chemical modifications for these synthetic therapeutic peptides. While collisional activation does not fragment the peptide backbone linearly (typical N- and C-terminal fragment ions) within the disulfide-bonded regions, internal and external ions are consistently produced throughout the sequence via secondary fragmentation pathways. In this study, seven disulfide-rich peptides (Peptides A - G) with similar disulfide connectivity but varying amino acid composition were subjected to collisional activation for sequencing and disulfide bond confirmation. While only four linear b- and y-type fragment ions are produced for these peptides, fragmentation throughout the amino acid sequence is observed when searching for internal and external fragment ions. These ions are typically not considered during traditional top-down sequencing experiments due to the computational challenge of having an increased search space for fragment ion identification. Through the identification of reproducible internal and external fragment ions, site-specific modifications can also be localized, such as oxidation on the 18th residue in Peptide A. Ultimately, this observation and identification of internal and external ions simplifies the experimental process and wet-chemistry required to accurately depict the disulfide connectivity and the sequencing of these traditionally challenging biomolecules. Further consideration to these non-traditional fragment ions should be given during top-down intact peptide and protein analysis, especially when non-linear sequences are involved.

Nanolipoprotein particles for co-delivery of cystine-knot peptides and Fab-based therapeutics.

Nanolipoprotein particles (NLPs) have been evaluated as an in vivo delivery vehicle for a variety of molecules of therapeutic interest. However, delivery of peptide-like drugs in combination with therapeutic Fabs has not yet been evaluated. In this study, we describe the development and characterization of cystine-knot peptide (CKP)-containing NLPs and Fab-CKP-NLP conjugates. CKPs were incorporated into NLPs using a self-assembly strategy. The trypsin inhibitor EETI-II, a model CKP, was produced with a C16 fatty acyl chain to enable incorporation of the CKP into the lipid bilayer core during NLP assembly. The CKP-NLP retained trypsin inhibitory function although the overall activity was reduced by ∼5 fold compared to free CKP, which was presumably due to steric hindrance. The NLP platform was also shown to accommodate up to ∼60 CKP molecules. Moreover, the stability of the CKP-NLP was comparable to the NLP control, displaying a relatively short half-life (∼1 h) in 50% serum at 37 °C. Therapeutic Fabs were also loaded onto the CKP-NLP by introducing thiol-reactive lipids that would undergo a covalent reaction with the Fab. Using this strategy, Fab loading could be reliably controlled from 1-50 Fabs per CKP-NLP and was found to be independent of CKP density. Surprisingly, Fab incorporation into CKP-NLPs led to a substantial improvement in NLP stability (half-life > 24 h) at 37 °C; also, there was no reduction in CKP activity in the Fab-CKP-NLP conjugates compared to CKP-NLPs. Altogether, our data demonstrate the potential of NLPs as a promising platform for the targeted or multidrug delivery of peptide-based drug candidates in combination with Fabs.

Lead Optimization Yields High Affinity Frizzled 7-Targeting Peptides That Modulate Clostridium difficile Toxin B Pathogenicity in Epithelial Cells.

Frizzled 7 (FZD7) receptors have been shown to play a central role in intestinal stem cell regeneration and, more recently, in Clostridium difficile pathogenesis. Yet, targeting FZD7 receptors with small ligands has not been explored as an approach to block C. difficile pathogenesis. Here, we report the discovery of high affinity peptides that selectively bind to FZD7 receptors. We describe an integrated approach for lead optimization, utilizing structure-based rational design and directed evolution, to enhance the peptide binding affinity while still maintaining FZD7 receptor selectivity. This work yielded new peptide leads with picomolar binding constants to FZD7 as measured by biophysical methods. The new peptides block the interaction between C. difficile toxin B (TcdB) and FZD receptors and perturb C. difficile pathogenesis in epithelial cells. As such, our findings provide a proof of concept that targeting FZD receptors could be a viable pharmacological approach to protect epithelial cells from TcdB pathogenicity.

Probing Interaction of Lipid-Modified Wnt Protein and Its Receptors by ELISA.

Wnts are lipid-modified proteins that regulate stem cell signaling via Frizzled receptors on the cell surface. Determination of binding interactions between lipid-modified Wnt proteins and their Frizzled receptors has been challenging due to the lack of availability of facile detection methods and technical hurdles associated with generating the relevant reagents. Here we report an enzyme-linked immunosorbent assay to measure the binding of a biotinylated form of lipid-modified Wnt3a to the extracellular cysteine-rich domain of Frizzled receptor. The method described herein is robust and rapid, uses minimum volumes of reagents, and can be conducted in a high-throughput format. Because of these attributes, the method could find utility in drug discovery applications such as characterizing the effect of pharmacological inhibitors on Wnt signaling without the need for sophisticated biophysical instrumentation.

Visualizing the cellular route of entry of a cystine-knot peptide with Xfect transfection reagent by electron microscopy.

Cystine-knot peptides are attractive templates in drug discovery due to a number of features they possess including their 3D conformation, physicochemical stability and synthetic tractability. Yet, their cellular uptake mechanisms remain largely unexplored. Recently, we demonstrated that the cystine-knot peptide EETI-II is internalized into cells and that its cellular uptake could be modulated by using a protein transfection reagent Xfect. However, the mechanism of Xfect-mediated cellular internalization of EETI-II remained unclear. Here, by using high resolution electron microscopy, we observe the formation of EETI-II-positive macropinosomes and clathrin-coated pits at early time points after treatment of cells with EETI-II/Xfect complexes. Internalized EETI-II subsequently accumulates in intracellular Xfect-induced detergent-resistant membrane compartments which appear to lack characteristic endosomal or lysosomal markers. Notably, Xfect enables the uptake of cell impermeable nuclear dyes into similar intracellular compartments that do not seem to deliver the cargo to the cytosol or nucleus. Altogether, our findings reveal mechanistic insights into the cellular uptake route of Xfect, and underscore the need for the development of effective tools to enhance the cytosolic delivery of cystine-knot peptides. Finally, our data illustrate that electron microscopy is a powerful approach for studying endocytic mechanisms of cell-penetrating peptides and their effects on cellular membranes.

Fatty acid recognition in the Frizzled receptor family.

Wnt signaling regulates physiological processes ranging from cell differentiation to bone formation. Dysregulation of Wnt signaling is linked to several human ailments, including colorectal, pancreatic, and breast cancers. As such, modulation of this pathway has been an attractive strategy for therapeutic development of anticancer agents. Since the discovery of Wnt proteins more than 35 years ago, research efforts continue to focus on understanding the biochemistry of their molecular interactions and their biological functions. Wnt is a secreted glycoprotein covalently modified with a cis-unsaturated fatty acyl group at a conserved serine residue, and this modification is required for Wnt secretion and activity. To initiate signaling, Wnt proteins bind to cell-surface Frizzled (FZD) receptors, but the molecular basis for recognition of Wnt's fatty acyl moiety by the extracellular cysteine-rich domain of FZD has become clear only very recently. Here, we review the most recent developments in the field, focusing on structural and biochemical studies of the FZD receptor family and highlighting new insights into their molecular arrangement and mode of regulation by cis-unsaturated fatty acids. Additionally, we examine how other lipid-binding proteins recognize fatty acyl chains on Wnt proteins in the regulation of Wnt secretion and activities. Altogether, this perspective expands our understanding of fatty acid-protein interactions in the FZD system and provides a basis for guiding future research in the field.

A selective peptide inhibitor of Frizzled 7 receptors disrupts intestinal stem cells.

Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.

A Decade of Click Chemistry in Protein Palmitoylation: Impact on Discovery and New Biology.

Protein palmitoylation plays diverse roles in regulating the trafficking, stability, and activity of cellular proteins. The advent of click chemistry has propelled the field of protein palmitoylation forward by providing specific, sensitive, rapid, and easy-to-handle methods for studying protein palmitoylation. This year marks the 10th anniversary since the first click chemistry-based fatty acid probes for detecting protein lipid modifications were reported. The goal of this review is to highlight key biological advancements in the field of protein palmitoylation during the past 10 years. In particular, we discuss the impact of click chemistry on enabling protein palmitoylation proteomics methods, uncovering novel lipid modifications on proteins and elucidating their functions, as well as the development of non-radioactive biochemical and enzymatic assays. In addition, this review provides context for building and exploring new research avenues in protein palmitoylation through the use of clickable fatty acid probes.

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding.

Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt-FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt-FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function.

Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking.

Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.

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.

Fatty acylation of Wnt proteins.

Wnt proteins are critical regulators of signaling networks during embryonic development and in adult tissue homeostasis. The generation of active Wnt proteins requires their regulated secretion into the extracellular space. Once secreted, Wnts signal through the cell surface via receptor binding on Wnt-receiving cells, a mechanism that is prevalent in stem cell and cancer biology. Important to both Wnt secretion and receptor recognition is their post-translational fatty acylation. In this Perspective, we highlight progress in elucidating the biochemistry of Wnt fatty acylation and provide a molecular view on the enzymology of substrate recognition and catalysis, with a focus on the Wnt O-acyltransferase porcupine. Special emphasis is given to Wnt fatty acid biosynthesis, Wnt-porcupine interactions, clinical mutations of porcupine and emerging therapeutics for perturbing Wnt fatty acylation in cancer. Finally, we discuss models for the functional role of the unsaturated fatty acyl chain in mediating lipid-protein interactions and in Wnt trafficking.