ß-Hairpin Alignment Alters Oligomer Formation in Aß-Derived Peptides.

Amyloid-ß (Aß) forms heterogeneous oligomers, which are implicated in the pathogenesis of Alzheimer's disease (AD). Many Aß oligomers consist of ß-hairpin building blocks─Aß peptides in ß-hairpin conformations. ß-Hairpins of Aß can adopt a variety of alignments, but the role that ß-hairpin alignment plays in the formation and heterogeneity of Aß oligomers is poorly understood. To explore the effect of ß-hairpin alignment on the oligomerization of Aß peptides, we designed and studied two model peptides with two different ß-hairpin alignments. Peptides Aßm17-36 and Aßm17-35 mimic two different ß-hairpins that Aß can form, the Aß17-36 and Aß17-35 ß-hairpins, respectively. These hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues that they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. In the crystal state, Aßm17-36 forms triangular trimers that further assemble to form hexamers, while Aßm17-35 forms tetrameric ß-barrels. In SDS-PAGE, Aßm17-36 assembles to form a ladder of oligomers, while Aßm17-35 either assembles to form a dimer or does not assemble at all. The differences in the behavior of Aßm17-36 and Aßm17-35 suggest ß-hairpin alignment as a source of the observed heterogeneity of Aß oligomers.

Palladium-Mediated Incorporation of Carboranes into Small Molecules, Peptides, and Proteins.

Carboranes represent a class of compounds with increasing therapeutic potential. However, few general approaches to readily embed carboranes into small molecules, peptides, and proteins are available. We report a strategy based on palladium-mediated C-X (X = C, S, and N) bond formation for the installation of carborane-containing moieties onto small molecules and peptides. We demonstrate the ability of Pd-based reagents with appropriate ligands to overcome the high hydrophobicity of the carborane group and enable chemoselective conjugation of cysteine residues at room temperature in aqueous buffer. Accordingly, carboranes can be efficiently installed on proteins by employing a combination of a bis-sulfonated biarylphosphine-ligated Pd reagent in an aqueous histidine buffer. This method is successfully employed on nanobodies, a fully synthetic affibody, and the antibody therapeutics trastuzumab and cetuximab. The conjugates of the affibody ZHER2 and the trastuzumab antibody retained binding to their target antigens. Conjugated proteins maintain their activity in cell-based functional assays in HER2-positive BT-474 cell lines. This approach enables the rapid incorporation of carborane moieties into small molecules, peptides, and proteins for further exploration in boron neutron capture therapy, which requires the targeted delivery of boron-dense groups.

Effects of N-Terminal Residues on the Assembly of Constrained ß-Hairpin Peptides Derived from Aß.

This paper describes the synthesis, solution-phase biophysical studies, and X-ray crystallographic structures of hexamers formed by macrocyclic ß-hairpin peptides derived from the central and C-terminal regions of Aß, which bear "tails" derived from the N-terminus of Aß. Soluble oligomers of the ß-amyloid peptide, Aß, are thought to be the synaptotoxic species responsible for neurodegeneration in Alzheimer's disease. Over the last 20 years, evidence has accumulated that implicates the N-terminus of Aß as a region that may initiate the formation of damaging oligomeric species. We previously studied, in our laboratory, macrocyclic ß-hairpin peptides derived from Aß16-22 and Aß30-36, capable of forming hexamers that can be observed by X-ray crystallography and SDS-PAGE. To better mimic oligomers of full length Aß, we use an orthogonal protecting group strategy during the synthesis to append residues from Aß1-14 to the parent macrocyclic ß-hairpin peptide 1, which comprises Aß16-22 and Aß30-36. The N-terminally extended peptides N+1, N+2, N+4, N+6, N+8, N+10, N+12, and N+14 assemble to form dimers, trimers, and hexamers in solution-phase studies. X-ray crystallography reveals that peptide N+1 assembles to form a hexamer that is composed of dimers and trimers. These observations are consistent with a model in which the assembly of Aß oligomers is driven by hydrogen bonding and hydrophobic packing of the residues from the central and C-terminal regions, with the N-terminus of Aß accommodated by the oligomers as an unstructured tail.

Anthrax Protective Antigen Retargeted with Single-Chain Variable Fragments Delivers Enzymes to Pancreatic Cancer Cells.

The nontoxic, anthrax protective antigen/lethal factor N-terminal domain (PA/LFN ) complex is an effective platform for translocating proteins into the cytosol of cells. Mutant PA (mPA) was recently fused to epidermal growth factor (EGF) to retarget delivery of LFN to cells bearing EGF receptors (EGFR), but the requirement for a known cognate ligand limits the applicability of this approach. Here, we render practical protective antigen retargeting to a variety of receptors with mPA single-chain variable fragment (scFv) fusion constructs. Our design enables the targeting of two pancreatic cancer-relevant receptors, EGFR and carcinoembryonic antigen. We demonstrate that fusion to scFvs does not disturb the basic functions of mPA. Moreover, mPA-scFv fusions enable cell-specific delivery of diphtheria toxin catalytic domain and Ras/Rap1-specific endopeptidase to pancreatic cancer cells. Importantly, mPA-scFv fusion-based treatments display potent cell-specific toxicity in vitro, opening fundamentally new routes toward engineered immunotoxins and providing a potential solution to the challenge of targeted protein delivery to the cytosol of cancer cells.

Effects of charge and hydrophobicity on the oligomerization of peptides derived from IAPP.

Changes in pH resulting in modifications of charge can dramatically alter the folding and interaction of proteins. This article probes the effects of charge and hydrophobicity on the oligomerization of macrocyclic ß-sheet peptides derived from residues 11-17 of IAPP (RLANFLV). Previous studies have shown that a macrocyclic ß-sheet peptide containing this IAPP sequence (peptide 1Arg) does not form oligomers in aqueous solution at low millimolar concentrations. Replacing arginine with the uncharged isostere citrulline generates a homologue (peptide 1Cit) that forms a tetramer consisting of a sandwich of hydrogen-bonded dimers. The current study probes the role of charge and hydrophobicity by changing residue 11 to glutamic acid (peptide 1Glu) and leucine (peptide 1Leu). Diffusion-ordered spectroscopy (DOSY) studies show that peptides 1Glu and 1Leu form tetramers in solution. NOESY studies confirm that both peptides form the same sandwich-like tetramer as peptide 1Cit. 1H NMR spectroscopy at various concentrations reveals that peptide 1Leu has the highest propensity to form tetramers. The effects of pH and charge on oligomerization are further probed by incorporating histidine at position 11 (peptide 1His). DOSY studies show that peptide 1His forms a tetramer at high pH. At low pH, peptide 1His forms a new species that has not been previously observed by our research group-a dimer. These studies demonstrate the importance of charge and hydrophobicity in the oligomerization of IAPP-derived peptides.

A Tetramer Derived from Islet Amyloid Polypeptide.

Aggregation of the islet amyloid polypeptide (IAPP) to form fibrils and oligomers is important in the progression of type 2 diabetes. This article describes X-ray crystallographic and solution-state NMR studies of peptides derived from residues 11-17 of IAPP that assemble to form tetramers. Incorporation of residues 11-17 of IAPP (RLANFLV) into a macrocyclic ß-sheet peptide results in a monomeric peptide that does not self-assemble to form oligomers. Mutation of Arg11 to the uncharged isostere citrulline gives peptide homologues that assemble to form tetramers in both the crystal state and in aqueous solution. The tetramers consist of hydrogen-bonded dimers that sandwich together through hydrophobic interactions. The tetramers share several features with structures reported for IAPP fibrils and demonstrate the importance of hydrogen bonding and hydrophobic interactions in the oligomerization of IAPP-derived peptides.

Coassembly of Peptides Derived from ß-Sheet Regions of ß-Amyloid.

In this paper, we investigate the coassembly of peptides derived from the central and C-terminal regions of the ß-amyloid peptide (Aß). In the preceding paper, J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b06000 , we established that peptides containing residues 17-23 (LVFFAED) from the central region of Aß and residues 30-36 (AIIGLMV) from the C-terminal region of Aß assemble to form homotetramers consisting of two hydrogen-bonded dimers. Here, we mix these tetramer-forming peptides and determine how they coassemble. Incorporation of a single 15N isotopic label into each peptide provides a spectroscopic probe with which to elucidate the coassembly of the peptides by 1H,15N HSQC. Job's method of continuous variation and nonlinear least-squares fitting reveal that the peptides form a mixture of heterotetramers in 3:1, 2:2, and 1:3 stoichiometries, in addition to the homotetramers. These studies also establish the relative stability of each tetramer and show that the 2:2 heterotetramer predominates. 15N-Edited NOESY shows the 2:2 heterotetramer comprises two different homodimers, rather than two heterodimers. The peptides within the heterotetramer segregate in forming the homodimer subunits, but the two homodimers coassemble in forming the heterotetramer. These studies show that the central and C-terminal regions of Aß can preferentially segregate within ß-sheets and that the resulting segregated ß-sheets can further coassemble.

Assembly of Peptides Derived from ß-Sheet Regions of ß-Amyloid.

In Alzheimer's disease, aggregation of the ß-amyloid peptide (Aß) results in the formation of oligomers and fibrils that are associated with neurodegeneration. Aggregation of Aß occurs through interactions between different regions of the peptide. This paper and the accompanying paper constitute a two-part investigation of two key regions of Aß: the central region and the C-terminal region. These two regions promote aggregation and adopt ß-sheet structure in the fibrils, and may also do so in the oligomers. In this paper, we study the assembly of macrocyclic ß-sheet peptides that contain residues 17-23 (LVFFAED) from the central region and residues 30-36 (AIIGLMV) from the C-terminal region. These peptides assemble to form tetramers. Each tetramer consists of two hydrogen-bonded dimers that pack through hydrophobic interactions in a sandwich-like fashion. Incorporation of a single 15N isotopic label into each peptide provides a spectroscopic probe with which to elucidate the ß-sheet assembly and interaction: 1H,15N HSQC studies facilitate the identification of the monomers and tetramers; 15N-edited NOESY studies corroborate the pairing of the dimers within the tetramers. In the following paper, J. Am. Chem. Soc. 2016, DOI: 10.1021/jacs.6b06001 , we will extend these studies to elucidate the coassembly of the peptides to form heterotetramers.

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Antigen processing is critical for therapeutic vaccines to generate epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often limits the quantity of epitopes released. We address this challenge using machine learning to ascribe a proteasomal degradation score to epitope sequences. Epitopes with varying scores were translocated into cells using nontoxic anthrax proteins. Epitopes with a low score show pronounced immunogenicity due to antigen processing, but epitopes with a high score show limited immunogenicity. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by these vaccines in clinical settings.

Design of Cytotoxic T Cell Epitopes by Machine Learning of Human Degrons.

Antigen processing is critical for producing epitope peptides that are presented by HLA molecules for T cell recognition. Therapeutic vaccines aim to harness these epitopes for priming cytotoxic T cell responses against cancer and pathogens, but insufficient processing often reduces vaccine efficacy through limiting the quantity of epitopes released. Here, we set out to improve antigen processing by harnessing protein degradation signals called degrons from the ubiquitin-proteasome system. We used machine learning to generate a computational model that ascribes a proteasomal degradation score between 0 and 100. Epitope peptides with varying degron activities were synthesized and translocated into cells using nontoxic anthrax proteins: protective antigen (PA) and the N-terminus of lethal factor (LFN). Immunogenicity studies revealed epitope sequences with a low score (<25) show pronounced T-cell activation but epitope sequences with a higher score (>75) provide limited activation. This work sheds light on the sequence-activity relationships between proteasomal degradation and epitope immunogenicity, through conserving the epitope region but varying the flanking sequence. We anticipate that future efforts to incorporate proteasomal degradation signals into vaccine designs will lead to enhanced cytotoxic T cell priming by vaccine therapeutics in clinical settings.

Enhanced Vaccine Immunogenicity Enabled by Targeted Cytosolic Delivery of Tumor Antigens into Dendritic Cells.

Molecular vaccines comprising antigen peptides and inflammatory cues make up a class of therapeutics that promote immunity against cancer and pathogenic diseases but often exhibit limited efficacy. Here, we engineered an antigen peptide delivery system to enhance vaccine efficacy by targeting dendritic cells and mediating cytosolic delivery. The delivery system consists of the nontoxic anthrax protein, protective antigen (PA), and a single-chain variable fragment (scFv) that recognizes the XCR1 receptor on dendritic cells (DCs). Combining these proteins enabled selective delivery of the N-terminus of lethal factor (LFN) into XCR1-positive cross-presenting DCs. Incorporating immunogenic epitope sequences into LFN showed selective protein translocation in vitro and enhanced the priming of antigen-specific T cells in vivo. Administering DC-targeted constructs with tumor antigens (Trp1/gp100) into mice bearing aggressive B16-F10 melanomas improved mouse outcomes when compared to free antigen, including suppressed tumor growth up to 58% at 16 days post tumor induction (P < 0.0001) and increased survival (P = 0.03). These studies demonstrate that harnessing DC-targeting anthrax proteins for cytosolic antigen delivery significantly enhances the immunogenicity and antitumor efficacy of cancer vaccines.

Palladium-peptide oxidative addition complexes for bioconjugation.

The synthesis of palladium oxidative addition complexes derived from unprotected peptides is described. Incorporation of 4-halophenylalanine into a peptide during solid phase peptide synthesis allows for subsequent oxidative addition at this position upon treatment with a palladium precursor and suitable ligand. The resulting palladium-peptide complexes are solid, storable, water-soluble, and easily purified via high-performance liquid chromatography. These complexes react with thiols in aqueous buffer, offering an efficient method for bioconjugation. Using this strategy, peptides can be functionalized with small molecules to prepare modified aryl thioether side-chains at low micromolar concentrations. Additionally, peptide-peptide and peptide-protein ligations are demonstrated under dilute aqueous conditions.

IgG-Engineered Protective Antigen for Cytosolic Delivery of Proteins into Cancer Cells.

Therapeutic immunotoxins composed of antibodies and bacterial toxins provide potent activity against malignant cells, but joining them with a defined covalent bond while maintaining the desired function is challenging. Here, we develop novel immunotoxins by dovetailing full-length immunoglobulin G (IgG) antibodies and nontoxic anthrax proteins, in which the C terminus of the IgG heavy chain is connected to the side chain of anthrax toxin protective antigen. This strategy enabled efficient conjugation of protective antigen variants to trastuzumab (Tmab) and cetuximab (Cmab) antibodies. The conjugates effectively perform intracellular delivery of edema factor and N terminus of lethal factor (LFN) fused with diphtheria toxin and Ras/Rap1-specific endopeptidase. Each conjugate shows high specificity for cells expressing human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR), respectively, and potent activity across six Tmab- and Cmab-resistant cell lines. The conjugates also exhibit increased pharmacokinetics and pronounced in vivo safety, which shows promise for further therapeutic development.

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids.

Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

Automated Flow Synthesis of Tumor Neoantigen Peptides for Personalized Immunotherapy.

High-throughput genome sequencing and computation have enabled rapid identification of targets for personalized medicine, including cancer vaccines. Synthetic peptides are an established mode of cancer vaccine delivery, but generating the peptides for each patient in a rapid and affordable fashion remains difficult. High-throughput peptide synthesis technology is therefore urgently needed for patient-specific cancer vaccines to succeed in the clinic. Previously, we developed automated flow peptide synthesis technology that greatly accelerates the production of synthetic peptides. Herein, we show that this technology permits the synthesis of high-quality peptides for personalized medicine. Automated flow synthesis produces 30-mer peptides in less than 35 minutes and 15- to 16-mer peptides in less than 20 minutes. The purity of these peptides is comparable with or higher than the purity of peptides produced by other methods. This work illustrates how automated flow synthesis technology can enable customized peptide therapies by accelerating synthesis and increasing purity. We envision that implementing this technology in clinical settings will greatly increase capacity to generate clinical-grade peptides on demand, which is a key step in reaching the full potential of personalized vaccines for the treatment of cancer and other diseases.

Square channels formed by a peptide derived from transthyretin.

High-resolution structures of peptide supramolecular assemblies are key to understanding amyloid diseases and designing peptide-based materials. This paper explores the supramolecular assembly of a macrocyclic ß-sheet peptide derived from transthyretin (TTR). The peptide mimics the ß-hairpin formed by the ß-strands G and H of TTR, which form the interface of the TTR tetramer. X-ray crystallography reveals that the peptide does not form a tetramer, but rather assembles to form square channels. The square channels are formed by extended networks of ß-sheets and pack in a "tilted windows" pattern. This unexpected structure represents an emergent property of the peptide and broadens the scope of known supramolecular assemblies of ß-sheets.