Comparing Cell Penetration of Biotherapeutics across Human Cell Lines.

A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

Computational Prediction of Cyclic Peptide Structural Ensembles and Application to the Design of Keap1 Binders.

The Nrf2 transcription factor is a master regulator of the cellular response to oxidative stress, and Keap1 is its primary negative regulator. Activating Nrf2 by inhibiting the Nrf2-Keap1 protein-protein interaction has shown promise for treating cancer and inflammatory diseases. A loop derived from Nrf2 has been shown to inhibit Keap1 selectively, especially when cyclized, but there are no reliable design methods for predicting an optimal macrocyclization strategy. In this work, we employed all-atom, explicit-solvent molecular dynamics simulations with enhanced sampling methods to predict the relative degree of preorganization for a series of peptides cyclized with a set of bis-thioether "staples". We then correlated these predictions to experimentally measured binding affinities for Keap1 and crystal structures of the cyclic peptides bound to Keap1. This work showcases a computational method for designing cyclic peptides by simulating and comparing their entire solution-phase ensembles, providing key insights into designing cyclic peptides as selective inhibitors of protein-protein interactions.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy.

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Stapled ß-Hairpins Featuring 4-Mercaptoproline.

Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel ß-strands, and then we incorporated that staple within a ß-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein-protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein-protein interactions.

Directed evolution of cyclic peptides for inhibition of autophagy.

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B-two essential components of the autophagosome maturation machinery-with mid-nanomolar affinities and disrupted protein-protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

HaloTag Forms an Intramolecular Disulfide.

HaloTag is a modified haloalkane dehalogenase used for many applications in chemical biology including protein purification, cell-based imaging, and cytosolic penetration assays. While working with purified, recombinant HaloTag protein, we discovered that HaloTag forms an internal disulfide bond under oxidizing conditions. In this work, we describe this internal disulfide formation and the conditions under which it occurs, and we identify the relevant cysteine residues. Further, we develop a mutant version of HaloTag, HaloTag8, that maintains activity while avoiding internal disulfide formation altogether. While there is no evidence that HaloTag is prone to disulfide formation in intracellular environments, researchers using recombinant HaloTag, HaloTag expressed on the cell surface, or HaloTag in the extracellular space might consider using HaloTag8 to avoid intramolecular disulfide formation.

A cell-penetrant lactam-stapled peptide for targeting eIF4E protein-protein interactions.

Eukaryotic translation initiation factor 4E (eIF4E) has emerged as a promising cancer therapeutic target due to its role in the initiation of cap-dependent translation, a process that is accelerated during tumorigenesis. To regulate the initiation of cap-dependent translation, eIF4E participates in protein-protein interactions (PPI) with binding partners, 4E-BP1 and eIF4G, which act as an inhibitor and stimulator of translation, respectively. As both of these proteins interact with eIF4E by utilizing a short, α-helical stretch of amino acids, our laboratory has been working to develop helical mimetics of these proteins, in particular 4E-BP1, to inhibit eIF4E PPIs. Herein, we describe our continued efforts in this area and report the development and characterization of a cell-penetrant lactam stapled peptide for targeting cellular eIF4E.

Quantitative measurement of cytosolic penetration using the chloroalkane penetration assay.

A major barrier for drug development is ensuring molecules can access intracellular targets. This is especially true for biomolecules, which are notoriously difficult to deliver to the cytosol. Many current methods for measuring the internalization of therapeutic biomolecules are largely indirect and qualitative, and they do not offer information about subcellular localization. We recently reported a new assay, called the ChloroAlkane Penetration Assay (CAPA), that addresses some of the drawbacks of existing methods. CAPA is high-throughput, quantitative, and compartment-specific, and can be used to monitor cytosolic penetration over time and under a variety of culture conditions. We have used CAPA to investigate the cytosolic localization of peptides, proteins, and oligonucleotides. In this chapter, we discuss the materials, protocols, and troubleshooting necessary to perform CAPA and appropriately analyze the data. We end with a discussion about the applications and limitations of CAPA, and we speculate on the potential of the assay and its variations.

Cytosolic delivery of peptidic STAT3 SH2 domain inhibitors.

The signal transducer and activator of transcription 3 (STAT3) protein is constitutively activated in several cancers. STAT3 activity can be blocked by inhibiting its Src Homology 2 (SH2) domain, but phosphotyrosine and its isosteres have poor bioavailability. In this work, we develop peptide-based inhibitors of STAT3-SH2 by combining chemical strategies that have proven effective for targeting other SH2 domains. These strategies include a STAT3-specific selectivity sequence, non-hydrolyzable phosphotyrosine isosteres, and a high-efficiency cell-penetrating peptide. Peptides that combined these three strategies had substantial biological stability and cytosolic delivery, as measured using highly quantitative cell-based assays. However, these peptides did not inhibit STAT3 activity in cells. By comparing in vitro binding affinity, cell penetration, and proteolytic stability, this work explores the delicate balance of factors that contribute to biological activity for peptidic inhibitors of STAT3.

Stapled Peptide Inhibitors of Autophagy Adapter LC3B.

A growing body of evidence suggests that autophagy inhibition enhances the effectiveness of chemotherapy, especially in difficult-to-treat cancers. Existing autophagy inhibitors are primarily lysosomotropic agents. More specific autophagy inhibitors are highly sought-after. The microtubule-associated protein 1A/1B light chain 3B protein, LC3B, is an adapter protein that mediates key protein-protein interactions at several points in autophagy pathways. In this work, we used a known peptide ligand as a starting point to develop improved LC3B inhibitors. We obtained structure-activity relationships that quantify the binding contributions of peptide termini, individual charged residues, and hydrophobic interactions. Based on these data, we used artificial amino acids and diversity-oriented stapling to improve affinity and resistance to biological degradation, while maintaining or improving LC3B affinity and selectivity. These peptides represent the highest-affinity LC3B-selective ligands reported to date, and they will be useful tools for further elucidation of LC3B's role in autophagy and in cancer.

Cellular Uptake and Cytosolic Delivery of a Cyclic Cystine Knot Scaffold.

Cyclotides are macrocyclic peptides with exceptionally stable structures and have been reported to penetrate cells, making them promising scaffolds for the delivery of inhibitory peptides to target intracellular proteins. However, their cellular uptake and cytosolic localization have been poorly understood until now, which has limited their therapeutic potential. In this study, the recently developed chloroalkane penetration assay was combined with established assays to characterize the cellular uptake and cytosolic delivery of the prototypic cyclotide, kalata B1. We show that kalata B1 enters the cytosol at low efficiency. A structure-activity study of residues in loop 6 showed that some modifications, such as increasing cationic residue content, did not affect delivery efficiency, whereas others, including introducing a single hydrophobic amino acid, did significantly improve cytosolic delivery. Our results provide a foundation for the further development of a structurally unique class of scaffolds for the delivery of therapeutic cargoes into cells.

Phosphotyrosine isosteres: past, present and future.

Tyrosine phosphorylation is a critical component of signal transduction for multicellular organisms, particularly for pathways that regulate cell proliferation and differentiation. While tyrosine kinase inhibitors have become FDA-approved drugs, inhibitors of the other important components of these signaling pathways have been harder to develop. Specifically, direct phosphotyrosine (pTyr) isosteres have been aggressively pursued as inhibitors of Src homology 2 (SH2) domains and protein tyrosine phosphatases (PTPs). Medicinal chemists have produced many classes of peptide and small molecule inhibitors that mimic pTyr. However, balancing affinity with selectivity and cell penetration has made this an extremely difficult space for developing successful clinical candidates. This review will provide a comprehensive picture of the field of pTyr isosteres, from early beginnings to the current state and trajectory. We will also highlight the major protein targets of these medicinal chemistry efforts, the major classes of peptide and small molecule inhibitors that have been developed, and the handful of compounds which have been tested in clinical trials.

Yeast can accommodate phosphotyrosine: v-Src toxicity in yeast arises from a single disrupted pathway.

Tyrosine phosphorylation is a key biochemical signal that controls growth and differentiation in multicellular organisms. Saccharomyces cerevisiae and nearly all other unicellular eukaryotes lack intact phosphotyrosine signaling pathways. However, many of these organisms have primitive phosphotyrosine-binding proteins and tyrosine phosphatases, leading to the assumption that the major barrier for emergence of phosphotyrosine signaling was the negative consequences of promiscuous tyrosine kinase activity. In this work, we reveal that the classic oncogene v-Src, which phosphorylates many dozens of proteins in yeast, is toxic because it disrupts a specific spore wall remodeling pathway. Using genetic selections, we find that expression of a specific cyclic peptide, or overexpression of SMK1, a MAP kinase that controls spore wall assembly, both lead to robust growth despite a continuous high level of phosphotyrosine in the yeast proteome. Thus, minimal genetic manipulations allow yeast to tolerate high levels of phosphotyrosine. These results indicate that the introduction of tyrosine kinases within single-celled organisms may not have been a major obstacle to the evolution of phosphotyrosine signaling.

Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1.

Recycling of receptors from the endosomal recycling compartment to the plasma membrane is a critical cellular process, and recycling is particularly important for maintaining invasiveness in solid tumors. In this work, we continue our efforts to inhibit EHD1, a critical adaptor protein involved in receptor recycling. We applied a diversity-oriented macrocyclization approach to produce cyclic peptides with varied conformations, but that each contain a motif that binds to the EH domain of EHD1. Screening these uncovered several new inhibitors for EHD1's EH domain, the most potent of which bound with a Kd of 3.1µM. Several of the most potent inhibitors were tested in a cellular assay that measures extent of vesicle recycling. Inhibiting EHD1 could potentially slow cancer invasiveness and metastasis, and these cyclic peptides represent the most potent inhibitors of EHD1 to date.

Diversity-Oriented Stapling Yields Intrinsically Cell-Penetrant Inducers of Autophagy.

Autophagy is an essential pathway by which cellular and foreign material are degraded and recycled in eukaryotic cells. Induction of autophagy is a promising approach for treating diverse human diseases, including neurodegenerative disorders and infectious diseases. Here, we report the use of a diversity-oriented stapling approach to produce autophagy-inducing peptides that are intrinsically cell-penetrant. These peptides induce autophagy at micromolar concentrations in vitro, have aggregate-clearing activity in a cellular model of Huntington's disease, and induce autophagy in vivo. Unexpectedly, the solution structure of the most potent stapled peptide, DD5-o, revealed an α-helical conformation in methanol, stabilized by an unusual (i,i+3) staple which cross-links two d-amino acids. We also developed a novel assay for cell penetration that reports exclusively on cytosolic access and used it to quantitatively compare the cell penetration of DD5-o and other autophagy-inducing peptides. These new, cell-penetrant autophagy inducers and their molecular details are critical advances in the effort to understand and control autophagy. More broadly, diversity-oriented stapling may provide a promising alternative to polycationic sequences as a means for rendering peptides more cell-penetrant.

A bicyclic peptide scaffold promotes phosphotyrosine mimicry and cellular uptake.

While peptides are promising as probes and therapeutics, targeting intracellular proteins will require greater understanding of highly structured, cell-internalized scaffolds. We recently reported BC1, an 11-residue bicyclic peptide that inhibits the Src homology 2 (SH2) domain of growth factor receptor-bound protein 2 (Grb2). In this work, we describe the unique structural and cell uptake properties of BC1 and similar cyclic and bicyclic scaffolds. These constrained scaffolds are taken up by mammalian cells despite their net neutral or negative charges, while unconstrained analogs are not. The mechanism of uptake is shown to be energy-dependent and endocytic, but distinct from that of Tat. The solution structure of BC1 was investigated by NMR and MD simulations, which revealed discrete water-binding sites on BC1 that reduce exposure of backbone amides to bulk water. This represents an original and potentially general strategy for promoting cell uptake.

Solution structure of a designed cyclic peptide ligand for nickel and copper ions.

Nuclear magnetic resonance (NMR) spectroscopy was used to study a cyclic peptide derived from the amino-terminal copper-and-nickel-binding (ATCUN) motif. The three-dimensional structure of the unliganded peptide in aqueous solution was solved by simulated annealing using distance constraints derived from Nuclear Overhauser Effects. A structural model for the Ni(II)-bound complex was also produced based on NMR evidence and prior spectroscopic data, which are consistent with crystal structures of linear ATCUN complexes. Structural interpolation, or "morphing," was used to understand the transition of this highly structured cyclic peptide from its unliganded structure to its metal-ion-bound structure.

Comprehensive analysis of loops at protein-protein interfaces for macrocycle design.

Inhibiting protein-protein interactions (PPIs) with synthetic molecules remains a frontier of chemical biology. Many PPIs have been successfully targeted by mimicking α-helices at interfaces, but most PPIs are mediated by nonhelical, nonstrand peptide loops. We sought to comprehensively identify and analyze these loop-mediated PPIs by writing and implementing LoopFinder, a customizable program that can identify loop-mediated PPIs within all of the protein-protein complexes in the Protein Data Bank. Comprehensive analysis of the entire set of 25,005 interface loops revealed common structural motifs and unique features that distinguish loop-mediated PPIs from other PPIs. 'Hot loops', named in analogy to protein hot spots, were identified as loops with favorable properties for mimicry using synthetic molecules. The hot loops and their binding partners represent new and promising PPIs for the development of macrocycle and constrained peptide inhibitors.