Design of target specific peptide inhibitors using generative deep learning and molecular dynamics simulations.

We introduce a computational approach for the design of target-specific peptides. Our method integrates a Gated Recurrent Unit-based Variational Autoencoder with Rosetta FlexPepDock for peptide sequence generation and binding affinity assessment. Subsequently, molecular dynamics simulations are employed to narrow down the selection of peptides for experimental assays. We apply this computational strategy to design peptide inhibitors that specifically target ß-catenin and NF-κB essential modulator. Among the twelve ß-catenin inhibitors, six exhibit improved binding affinity compared to the parent peptide. Notably, the best C-terminal peptide binds ß-catenin with an IC50 of 0.010 ± 0.06 µM, which is 15-fold better than the parent peptide. For NF-κB essential modulator, two of the four tested peptides display substantially enhanced binding compared to the parent peptide. Collectively, this study underscores the successful integration of deep learning and structure-based modeling and simulation for target specific peptide design.

Design, synthesis, and analysis of macrobicyclic peptides for targeting the Gαi protein.

Bicyclic peptides are important chemical tools that can function, for example, as bioactive ligands switching on/off signaling pathways mediated by guanine nucleotide-binding proteins as bicycles are more broadly applicable. Despite their relevance in medicinal chemistry, the synthesis of such peptides is challenging, and the final yield is highly dependent on the chemical composition and physicochemical properties of the scaffold. We recently discovered novel, state-specific peptide modulators targeting the Gαi protein, namely, GPM-2/GPM-3, by screening a one-bead-two-compound combinatorial library. A more detailed analysis, including sequence alignments and computer-assisted conformational studies based on the hit compounds, revealed the new peptide 10 as a potential macrobicyclic Gαi ligand sharing high sequence similarity to the known Gαi modulators. The Gαs protein was included in this study for comparison and to unravel the criteria for the specificity of modulator binding to Gαi versus Gαs. This work provides in-depth computer-assisted experimental studies for the analysis of novel macrobicyclic, library-derived Gαi protein ligands. The sequence and structural comparison of 10 with the lead compounds GPM-2 and GPM-3 reveals the importance of the size and amino acid composition of one ring of the bicyclic system and suggests features enhancing the binding affinity of the peptides to the Gαi protein.

Bicyclic Peptide Library Screening for the Identification of Gαi Protein Modulators.

Noncanonical G protein activation and inactivation, particularly for the Gαi/s protein subfamilies, have long been a focus of chemical research. Combinatorial libraries were already effectively applied to identify modulators of the guanine-nucleotide exchange, as can be exemplified with peptides such as KB-752 and GPM-1c/d, the so-called guanine-nucleotide exchange modulators. In this study, we identified novel bicyclic peptides from a combinatorial library screening that show prominent properties as molecular switch-on/off modulators of Gαi signaling. Among the series of hits, the exceptional paradigm of GPM-3, a protein and state-specific bicyclic peptide, is the first chemically identified GAP (GTPase-activating protein) modulator with a high binding affinity for Gαi protein. Computational analyses identified and assessed the structure of the bicyclic peptides, novel ligand-protein interaction sites, and their subsequent impact on the nucleotide binding site. This approach can therefore lead the way for the development of efficient chemical biological probes targeting Gαi protein modulation within a cellular context.

Inhibition of Ehrlichia chaffeensis infection by cell-permeable macrocyclic peptides that bind type IV secretion effector Etf-1.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects monocytes and macrophages, and causes human monocytic ehrlichiosis, an emerging life-threatening infectious disease. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system effector, is essential for Ehrlichia infection of host cells. Etf-1 translocates to mitochondria to block host apoptosis; furthermore, it can bind Beclin 1 (ATG6) to induce cellular autophagy and localize to E. chaffeensis-inclusion membrane to obtain host-cell cytoplasmic nutrients. In this study, we screened a synthetic library of over 320,000 cell-permeable macrocyclic peptides, which consist of an ensemble of random peptide sequences in the first ring and a small family of cell-penetrating peptides in the second ring, for Etf-1 binding. Library screening followed by hit optimization identified multiple Etf-1-binding peptides (with K D values of 1-10 µM) that efficiently enter the cytosol of mammalian cells. Peptides B7, C8, B7-131-5, B7-133-3, and B7-133-8 significantly inhibited Ehrlichia infection of THP-1 cells. Mechanistic studies revealed that peptide B7 and its derivatives inhibited the binding of Etf-1 to Beclin 1, and Etf-1 localization to E. chaffeensis-inclusion membranes, but not Etf-1 localization to the mitochondria. Our results not only affirm the critical role of Etf-1 functions in E. chaffeensis infection, but also demonstrate the feasibility of developing macrocyclic peptides as powerful chemical probes and potential treatment of diseases caused by Ehrlichia and other intracellular pathogens.

Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases.

Efficient, site-specific, and bio-orthogonal conjugation of chemical functionalities to proteins is of great utility in fundamental research as well as industrial processes (e.g., the production of antibody-drug conjugates and immobilization of enzymes for biocatalysis). A popular approach involves reacting a free N-terminal cysteine with a variety of electrophilic reagents. However, current methods for generating proteins with N-terminal cysteines have significant limitations. Herein we report a novel, efficient, and convenient method for producing recombinant proteins with free N-terminal cysteines by genetically fusing a Met-Pro-Cys sequence to the N-terminus of a protein of interest and subjecting the recombinant protein to the sequential action of methionine and proline aminopeptidases. The resulting protein was site-specifically labeled at the N-terminus with fluorescein and a cyclic cell-penetrating peptide through native chemical ligation and a 2-cyanobenzothiazole moiety, respectively. In addition, the optimal recognition sequence of Aeromonas sobria proline aminopeptidase was determined by screening a combinatorial peptide library and incorporated into the N-terminus of a protein of interest for most efficient N-terminal processing.

Membrane translocation of folded proteins.

An ever-increasing number of proteins have been shown to translocate across various membranes of bacterial as well as eukaryotic cells in their folded states as a part of physiological and/or pathophysiological processes. Herein, we provide an overview of the systems/processes that are established or likely to involve the membrane translocation of folded proteins, such as protein export by the twin-arginine translocation system in bacteria and chloroplasts, unconventional protein secretion and protein import into the peroxisome in eukaryotes, and the cytosolic entry of proteins (e.g., bacterial toxins) and viruses into eukaryotes. We also discuss the various mechanistic models that have previously been proposed for the membrane translocation of folded proteins including pore/channel formation, local membrane disruption, membrane thinning, and transport by membrane vesicles. Finally, we introduce a newly discovered vesicular transport mechanism, vesicle budding and collapse, and present evidence that vesicle budding and collapse may represent a unifying mechanism that drives some (and potentially all) of folded protein translocation processes.

Discovery of a Cyclic Cell-Penetrating Peptide with Improved Endosomal Escape and Cytosolic Delivery Efficiency.

Cyclic cell-penetrating peptide 12 (CPP12) is highly efficient for the cytosolic delivery of a variety of cargo molecules into mammalian cells in vitro and in vivo. However, its cytosolic entry efficiency is substantially reduced at lower concentrations or in the presence of serum proteins. In this study, CPP12 analogs were prepared by replacing its hydrophobic residues with amino acids of varying hydrophobicity and evaluated for cellular entry. Substitution of l-3-benzothienylalanine (Bta) for l-2-naphthylalanine (Nal) resulted in CPP12-2, which exhibits up to 3.8-fold higher cytosolic entry efficiency than CPP12, especially at low CPP concentrations; thanks to improved endosomal escape efficiency. CPP12-2 is well suited for the cytosolic delivery of highly potent cargos to achieve biological activity at low concentrations.

Targeting Gαi/s Proteins with Peptidyl Nucleotide Exchange Modulators.

Chemical probes that specifically modulate the activity of heterotrimeric G proteins provide excellent tools for investigating G protein-mediated cell signaling. Herein, we report a family of selective peptidyl Gαi/s modulators derived from peptide library screening and optimization. Conjugation to a cell-penetrating peptide rendered the peptides cell-permeable and biologically active in cell-based assays. The peptides exhibit potent guanine-nucleotide exchange modulator-like activity toward Gαi and Gαs. Molecular docking and dynamic simulations revealed the molecular basis of the protein-ligand interactions and their effects on GDP binding. This study demonstrates the feasibility of developing direct Gαi/s modulators and provides a novel chemical probe for investigating cell signaling through GPCRs/G proteins.

How Do Biomolecules Cross the Cell Membrane?

Biomolecules such as peptides, proteins, and nucleic acids generally cannot cross a cell membrane by passive diffusion. Nevertheless, cell-penetrating peptides (CPPs), bacterial protein toxins, certain eukaryotic proteins, viruses, and many synthetic drug delivery vehicles have been shown to enter the cytosol of eukaryotic cells with varying efficiencies. They generally enter the cell by one or more of the endocytic mechanisms and are initially localized inside the endosomes. But how they cross the endosomal membrane to reach the cytosol (i.e., endosomal escape) has been a mystery for decades, and this knowledge gap has been a major bottleneck for the development of efficient drug delivery systems. In addition, many bacterial and eukaryotic proteins are transported across the plasma membrane in their native states into the periplasmic/extracellular space through the twin-arginine translocation (TAT) and unconventional protein secretion (UPS) systems, respectively. Again, the mechanisms underpinning these protein export systems remain unclear.In this Account, I introduce a previously unrecognized, fundamental membrane translocation mechanism which we have termed the vesicle budding-and-collapse (VBC) mechanism. Through VBC, biomolecules of diverse sizes and physicochemical properties autonomously translocate across cell membranes topologically (i.e., from one side to the other side of the membrane) but not physically (i.e., without going through the membrane). We have demonstrated that CPPs and bacterial protein toxins escape the endosome by the VBC mechanism in giant unilamellar vesicles as well as live mammalian cells. This advance resulted from studies in which we labeled the biomolecules with a pH-sensitive, red-colored dye (pHAb) and phosphatidylserine with a pH-insensitive green dye (TopFluor) and monitored the intracellular trafficking of the biomolecules in real time by confocal microscopy. In addition, by enlarging the endosomes with a kinase inhibitor, we were able to visualize the structural changes of the endosomes (i.e., endosomal escape intermediates) as they went through the VBC process. I postulate that bacterial/viral/eukaryotic proteins, nonenveloped viruses, and synthetic drug delivery vehicles (e.g., polyplexes, lipoplexes, and lipid nanoparticles) may also escape the endosome by inducing VBC. Furthermore, I propose that VBC may be the mechanism that drives the bacterial TAT and eukaryotic UPS systems. Our findings fill a long-standing gap in cell biology and provide guiding principles for designing more efficient drug delivery vehicles.

Designing Cell-Permeable Peptide Therapeutics That Enter the Cell by Endocytosis.

Intracellular protein-protein interactions (PPIs) represent a large class of exciting as well as challenging drug targets for traditional drug modalities (i.e., small molecules and biologics). Peptides (especially cyclic peptides) have proven highly effective as PPI inhibitors in vitro but are generally impermeable to the cell membrane. The recent discovery of a family of highly active cyclic cell-penetrating peptides (CPPs) has enabled the delivery of peptides into the cytosol of mammalian cells at therapeutically relevant levels. This chapter describes the various strategies that have been developed to conjugate or integrate different types of peptidyl cargoes (e.g., linear, cyclic, and stapled peptides) with cyclic CPPs to generate cell-permeable, metabolically stable, and biologically active macrocyclic peptides against intracellular targets including PPIs.

Assessing the Cellular Uptake, Endosomal Escape, and Cytosolic Entry Efficiencies of Cyclic Peptides.

Intracellular biologics such as cyclic peptides are an emerging class of macromolecular drugs that are either intrinsically cell permeable or can be effectively delivered into the cell interior to modulate the activity of previously intractable drug targets. They generally enter the mammalian cell by endocytosis mechanisms and are initially localized inside the endosomes. They subsequently escape from the endosomes (and/or lysosomes) into the cytosol with varying efficiencies. In this chapter, we provide the detailed protocol for a flow cytometry-based assay method to quantitate the overall cellular uptake, endosomal escape, and cytosolic entry efficiencies of biomolecules (e.g., linear and cyclic peptides, proteins, and nucleic acids), by using cell-penetrating peptides as an example. The scope of applicability, strengths, and weaknesses of this assay are also discussed.

Targeting intracellular protein-protein interactions with macrocyclic peptides.

Intracellular protein-protein interactions (PPIs) are challenging targets for traditional drug modalities. Macrocyclic peptides (MPs) prove highly effective PPI inhibitors in vitro and can be rapidly discovered against PPI targets by rational design or screening combinatorial libraries but are generally impermeable to the cell membrane. Recent advances in MP science and technology are allowing for the development of 'drug-like' MPs that potently and specifically modulate intracellular PPI targets in cell culture and animal models. In this review, we highlight recent progress in generating cell-permeable MPs that enter the mammalian cell by passive diffusion, endocytosis followed by endosomal escape, or as-yet unknown mechanisms.

Bacterial Toxins Escape the Endosome by Inducing Vesicle Budding and Collapse.

Bacterial protein toxins autonomously enter the cytosol of the target cell where they modify the activities of host components to exert their toxic effects. Many of the toxins enter the host cell by endocytosis followed by endosomal escape. However, their mechanism of endosomal escape remains unresolved. We show herein that diphtheria toxin (DT) and NleC of enteropathogenic Escherichia coli exit the endosome by inducing budding and collapse of small toxin-enriched vesicles from the endosomal membrane.

Discovery of a Bicyclic Peptidyl Pan-Ras Inhibitor.

The Ras subfamily of small GTPases is mutated in ∼30% of human cancers and represents compelling yet challenging anticancer drug targets owing to their flat protein surface. We previously reported a bicyclic peptidyl inhibitor, cyclorasin B3, which binds selectively to Ras-GTP with modest affinity and blocks its interaction with downstream effector proteins in vitro but lacks cell permeability or biological activity. In this study, optimization of B3 yielded a potent pan-Ras inhibitor, cyclorasin B4-27, which binds selectively to the GTP-bound forms of wild-type and mutant Ras isoforms (KD = 21 nM for KRasG12V-GppNHp) and is highly cell-permeable and metabolically stable (serum t1/2 > 24 h). B4-27 inhibits Ras signaling in vitro and in vivo by blocking Ras from interacting with downstream effector proteins and induces apoptosis of Ras-mutant cancer cells. When administered systemically (i.v.), B4-27 suppressed tumor growth in two different mouse xenograft models at 1-5 mg/kg of daily doses.

An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.

Infection with obligatory intracellular bacteria is difficult to treat, as intracellular targets and delivery methods of therapeutics are not well known. Ehrlichia translocated factor-1 (Etf-1), a type IV secretion system (T4SS) effector, is a primary virulence factor for an obligatory intracellular bacterium, Ehrlichia chaffeensis In this study, we developed Etf-1-specific nanobodies (Nbs) by immunizing a llama to determine if intracellular Nbs block Etf-1 functions and Ehrlichia infection. Of 24 distinct anti-Etf-1 Nbs, NbD7 blocked mitochondrial localization of Etf-1-GFP in cotransfected cells. NbD7 and control Nb (NbD3) bound to different regions of Etf-1. Size-exclusion chromatography showed that the NbD7 and Etf-1 complex was more stable than the NbD3 and Etf-1 complex. Intracellular expression of NbD7 inhibited three activities of Etf-1 and E. chaffeensis: up-regulation of mitochondrial manganese superoxide dismutase, reduction of intracellular reactive oxygen species, and inhibition of cellular apoptosis. Consequently, intracellular NbD7 inhibited Ehrlichia infection, whereas NbD3 did not. To safely and effectively deliver Nbs into the host cell cytoplasm, NbD7 was conjugated to cyclized cell-permeable peptide 12 (CPP12-NbD7). CPP12-NbD7 effectively entered mammalian cells and abrogated the blockade of cellular apoptosis caused by E. chaffeensis and inhibited infection by E. chaffeensis in cell culture and in a severe combined-immunodeficiency mouse model. Our results demonstrate the development of an Nb that interferes with T4SS effector functions and intracellular pathogen infection, along with an intracellular delivery method for this Nb. This strategy should overcome current barriers to advance mechanistic research and develop therapies complementary or alternative to the current broad-spectrum antibiotic.

Cyclic Peptidyl Inhibitors against CAL/CFTR Interaction for Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.

A Peptidyl Inhibitor that Blocks Calcineurin-NFAT Interaction and Prevents Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.

Rational design of cell-permeable cyclic peptides containing a d-Pro-l-Pro motif.

Cyclic peptides are capable of binding to challenging targets (e.g., proteins involved in protein-protein interactions) with high affinity and specificity, but generally cannot gain access to intracellular targets because of poor membrane permeability. In this work, we discovered a conformationally constrained cyclic cell-penetrating peptide (CPP) containing a d-Pro-l-Pro motif, cyclo(AFΦrpPRRFQ) (where Φ is l-naphthylalanine, r is d-arginine, and p is d-proline). The structural constraints provided by cyclization and the d-Pro-l-Pro motif permitted the rational design of cell-permeable cyclic peptides of large ring sizes (up to 16 amino acids). This strategy was applied to design a potent, cell-permeable, and biologically active cyclic peptidyl inhibitor, cyclo(YpVNFΦrpPRR) (where Yp is l-phosphotyrosine), against the Grb2 SH2 domain. Multidimensional NMR spectroscopic and circular dichroism analyses revealed that the cyclic CPP as well as the Grb2 SH2 inhibitor assume a predominantly random coil structure but have significant ß-hairpin character surrounding the d-Pro-l-Pro motif. These results demonstrate cyclo(AFΦrpPRRFQ) as an effective CPP for endocyclic (insertion of cargo into the CPP ring) or exocyclic delivery of biological cargos (attachment of cargo to the Gln side chain).

Cell-Penetrating Peptides Escape the Endosome by Inducing Vesicle Budding and Collapse.

Cell-penetrating peptides (CPPs) are capable of delivering membrane-impermeable cargoes (including small molecules, peptides, proteins, nucleic acids, and nanoparticles) into the cytosol of mammalian cells and have the potential to revolutionize biomedical research and drug discovery. However, the mechanism of action of CPPs has remained poorly understood, especially how they escape from the endosome into the cytosol following endocytic uptake. We show herein that CPPs exit the endosome by inducing budding and collapse of CPP-enriched vesicles from the endosomal membrane. This mechanism provides a theoretical basis for designing CPPs and other delivery vehicles of improved efficiencies.