Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay.

Protein therapeutics are an expanding area for research and drug development, and lipid nanoparticles (LNPs) are the most prominent nonviral vehicles for protein delivery. The most common methods for assessing protein delivery by LNPs include assays that measure the total amount of protein taken up by cells and assays that measure the phenotypic changes associated with protein delivery. However, assays for total cellular uptake include large amounts of protein that are trapped in endosomes or are otherwise nonfunctional. Assays for functional delivery are important, but the readouts are indirect and amplified, limiting the quantitative interpretation. Here, we apply an assay for cytosolic delivery, the chloroalkane penetration assay (CAPA), to measure the cytosolic delivery of a (-30) green fluorescent protein (GFP) fused to Cre recombinase (Cre(-30)GFP) fusion protein by LNPs. We compare these data to the data from total cellular uptake and functional delivery assays to provide a richer analysis of uptake and endosomal escape for LNP-mediated protein delivery. We also use CAPA for a screen of a small library of lipidoids, identifying those with a promising ability to deliver Cre(-30)GFP to the cytosol of mammalian cells. With careful controls and optimized conditions, we expect that CAPA will be a useful tool for investigating the rate, efficiency, and mechanisms of LNP-mediated delivery of therapeutic proteins.

Investigation of Sequence-Penetration Relationships of Antisense Oligonucleotides.

A major limitation for the development of more effective oligonucleotide therapeutics has been a lack of understanding of their penetration into the cytosol. While prior work has shown how backbone modifications affect cytosolic penetration, it is unclear how cytosolic penetration is affected by other features including base composition, base sequence, length, and degree of secondary structure. We have applied the chloroalkane penetration assay, which exclusively reports on material that reaches the cytosol, to investigate the effects of these characteristics on the cytosolic uptake of druglike oligonucleotides. We found that base composition and base sequence had moderate effects, while length did not correlate directly with the degree of cytosolic penetration. Investigating further, we found that the degree of secondary structure had the largest and most predictable correlations with cytosolic penetration. These methods and observations add a layer of design for maximizing the efficacy of new oligonucleotide therapeutics.

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.

A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics.

RNA therapeutics are a promising strategy to treat genetic diseases caused by the overexpression or aberrant splicing of a specific protein. The field has seen major strides in the clinical efficacy of this class of molecules, largely due to chemical modifications and delivery strategies that improve nuclease resistance and enhance cell penetration. However, a major obstacle in the development of RNA therapeutics continues to be the imprecise, difficult, and often problematic nature of most methods used to measure cell penetration. Here, we review these methods and clearly distinguish between those that measure total cellular uptake of RNA therapeutics, which includes both productive and non-productive uptake, and those that measure cytosolic/nuclear penetration, which represents only productive uptake. We critically analyze the benefits and drawbacks of each method. Finally, we use key examples to illustrate how, despite rigorous experimentation and proper controls, our understanding of the mechanism of gymnotic uptake of RNA therapeutics remains limited by the methods commonly used to analyze RNA delivery.

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.

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.

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.

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.

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.

ß-Branched Amino Acids Stabilize Specific Conformations of Cyclic Hexapeptides.

Cyclic peptides (CPs) are a promising class of molecules for drug development, particularly as inhibitors of protein-protein interactions. Predicting low-energy structures and global structural ensembles of individual CPs is critical for the design of bioactive molecules, but these are challenging to predict and difficult to verify experimentally. In our previous work, we used explicit-solvent molecular dynamics simulations with enhanced sampling methods to predict the global structural ensembles of cyclic hexapeptides containing different permutations of glycine, alanine, and valine. One peptide, cyclo-(VVGGVG) or P7, was predicted to be unusually well structured. In this work, we synthesized P7, along with a less well-structured control peptide, cyclo-(VVGVGG) or P6, and characterized their global structural ensembles in water using NMR spectroscopy. The NMR data revealed a structural ensemble similar to the prediction for P7 and showed that P6 was indeed much less well-structured than P7. We then simulated and experimentally characterized the global structural ensembles of several P7 analogs and discovered that ß-branching at one critical position within P7 is important for overall structural stability. The simulations allowed deconvolution of thermodynamic factors that underlie this structural stabilization. Overall, the excellent correlation between simulation and experimental data indicates that our simulation platform will be a promising approach for designing well-structured CPs and also for understanding the complex interactions that control the conformations of constrained peptides and other macrocycles.

Cell Penetration Profiling Using the Chloroalkane Penetration Assay.

Biotherapeutics are a promising class of molecules in drug discovery, but they are often limited to extracellular targets due to their poor cell penetration. High-throughput cell penetration assays are required for the optimization of biotherapeutics for enhanced cell penetration. We developed a HaloTag-based assay called the chloroalkane penetration assay (CAPA), which is quantitative, high-throughput, and compartment-specific. We demonstrate the ability of CAPA to profile extent of cytosolic penetration with respect to concentration, presence of serum, temperature, and time. We also used CAPA to investigate structure-penetration relationships for bioactive stapled peptides and peptides fused to cell-penetrating sequences. CAPA is not only limited to measuring cytosolic penetration. Using a cell line where HaloTag is localized to the nucleus, we show quantitative measurement of nuclear penetration. Going forward, CAPA will be a valuable method for measuring and optimizing the cell penetration of biotherapeutics.

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.

Emerging Methods and Design Principles for Cell-Penetrant Peptides.

Biomolecules such as antibodies, proteins, and peptides are important tools for chemical biology and leads for drug development. They have been used to inhibit a variety of extracellular proteins, but accessing intracellular proteins has been much more challenging. In this review, we discuss diverse chemical approaches that have yielded cell-penetrant peptides and identify three distinct strategies: masking backbone amides, guanidinium group patterning, and amphipathic patterning. We summarize a growing number of large data sets, which are starting to reveal more specific design guidelines for each strategy. We also discuss advantages and disadvantages of current methods for quantifying cell penetration. Finally, we provide an overview of best-odds approaches for applying these new methods and design principles to optimize cytosolic penetration for a given bioactive peptide.

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.

Analysis of Loops that Mediate Protein-Protein Interactions and Translation into Submicromolar Inhibitors.

Effective strategies for mimicking α-helix and ß-strand epitopes have been developed, producing valuable inhibitors for some classes of protein-protein interactions (PPIs). However, there are no general strategies for translating loop epitopes into useful PPI inhibitors. In this work, we use the LoopFinder program to identify diverse sets of "hot loops," which are loop epitopes that mediate PPIs. These include loops that are well-suited to mimicry with macrocyclic compounds, and loops that are most similar to variable loops on antibodies and ankyrin repeat proteins. We present data-driven criteria for scoring loop-mediated PPIs, uncovering a trove of potentially druggable interactions. We also use unbiased clustering to identify common structures among the hot loops. To translate these insights into real-world inhibitors, we describe a robust, diversity-oriented strategy for the rapid production and evaluation of cyclized loops. This method is applied to a computationally identified loop in the PPI between stonin2 and Eps15, producing submicromolar inhibitors. The most potent inhibitor is well-structured in water and successfully mimics the native epitope. Overall, these computational and experimental strategies provide new opportunities to design inhibitors for an otherwise intractable set of PPIs.

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.

Structured cyclic peptides that bind the EH domain of EHD1.

EHD1 mediates long-loop recycling of many receptors by forming signaling complexes using its EH domain. We report the design and optimization of cyclic peptides as ligands for the EH domain of EHD1. We demonstrate that the improved affinity from cyclization allows fluorescence-based screening applications for EH domain inhibitors. The cyclic peptide is also unusually well-structured in aqueous solution, as demonstrated using nuclear magnetic resonance-based structural models. Because few EH domain inhibitors have been described, these more potent inhibitors will improve our understanding of the roles of EHD1 in the context of cancer invasion and metastasis.