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Catalyst transfer polymerization (CTP) is widely applied to the synthesis of well-defined π-conjugated polymers. Unlike other polymerization reactions that can be performed in water (e.g., controlled radical polymerizations and ring-opening polymerizations), CTP has yet to be adapted for the modification of biopolymers. Here, we report the use of protein-palladium oxidative addition complexes (OACs) that enable catalyst transfer polymerization to furnish protein-polyarene conjugates. These polymerizations occur with electron-deficient monomers in aqueous buffers open to air at mild (≤37 °C) temperatures with full conversion of the protein OAC and an average polymer length of nine repeating units. Proteins with polyarene chains terminated with palladium OACs can be readily isolated. Direct evidence of protein-polyarene OAC formation was obtained using mass spectrometry, and all protein-polyarene chain ends were uniformly functionalized via C-S arylation to terminate the polymerization with a small molecule thiol or a cysteine-containing protein.

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 Z(HER2) 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.

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

Here, we combined unsupervised machine learning (ML), non-natural amino acids, and affinity-selection mass-spectrometry (AS-MS) for the discovery of ultra-high affinity peptidomimetics that bind to a protein target. Peptides and peptidomimetics were discovered using AS-MS, encoded using diverse representations, and decomposed into two-dimensional “maps” of the chemical space by dimensionality reduction. These maps showed well-defined clusters of target-specific binders distinct from the remaining chemical space that included nonspecific and nonbinding peptides. Experimental testing of abiotic peptidomimetics confirmed the discovery of low nanomolar to picomolar binders and the accurate mapping of high-affinity binders across the co-learned sequence space. With ML and AS-MS, we anticipate this cartographic approach will accelerate the definition of chemical design spaces for the prediction and generation of functional peptidomimetics.

MicroRNAs (miRNAs) are implicated in the onset and progression of a variety of diseases. Modulating the expression of specific miRNAs is a possible option for therapeutic intervention. A promising strategy is the use of antisense oligonucleotides (ASOs) to inhibit miRNAs. Targeting ASOs to specific tissues can potentially lower the dosage and improve clinical outcomes by alleviating systemic toxicity. We leverage here automated peptide nucleic acid (PNA) synthesis technology to manufacture an anti-miRNA oligonucleotide (antagomir) covalently attached to a 12-mer peptide that binds to transferrin receptor 1. Our PNA-peptide conjugate is active in cells and animals, effectively inhibiting the expression of miRNA-21 both in cultured mouse cardiomyocytes and different mouse organs (heart, liver, kidney, lung, and spleen), while remaining well-tolerated in animals up to the highest tested dose of 30 mg/kg. Conjugating the targeting ligand to the PNA antagomir significantly improved inhibition of miRNA-21 in the heart by over 50% relative to the PNA alone. Given the modulation of biodistribution observed with our PNA-peptide conjugate, we anticipate this antagomir platform to serve as a starting point for pre-clinical development studies.Table of Contents EntrySynopsisConjugating T12, a peptide targeting transferrin receptor 1 (TfR1), to a peptide nucleic acid (PNA) oligonucleotide targeting microRNA-21 increases delivery of the PNA-T12 conjugate to cardiac tissue relative to PNA alone.

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Captions for Supplementary Figures and Supplementary Table

Antitumor T-cell responses have the potential to be curative in cancer patients, but the induction of potent T-cell immunity through vaccination remains a largely unmet goal of immunotherapy. We previously reported that the immunogenicity of peptide vaccines could be increased by maximizing delivery to lymph nodes (LNs), where T-cell responses are generated. This was achieved by conjugating the peptide to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG (DSPE-PEG) to promote albumin binding, which resulted in enhanced lymphatic drainage and improved T-cell responses. Here, we expanded upon these findings and mechanistically dissected the properties that contribute to the potency of this amphiphile-vaccine (amph-vaccine). We found that multiple linkage chemistries could be used to link peptides with DSPE-PEG, and further, that multiple albumin-binding moieties conjugated to peptide antigens enhanced LN accumulation and subsequent T-cell priming. In addition to enhancing lymphatic trafficking, DSPE-PEG conjugation increased the stability of peptides in serum. DSPE-PEG peptides trafficked beyond immediate draining LNs to reach distal nodes, with antigen presented for at least a week in vivo, whereas soluble peptide presentation quickly decayed. Responses to amph-vaccines were not altered in mice deficient in the albumin-binding neonatal Fc receptor (FcRn), but required Batf3-dependent dendritic cells (DCs). Amph-peptides were processed by human DCs equivalently to unmodified peptides. These data define design criteria for enhancing the immunogenicity of molecular vaccines to guide the design of next-generation peptide vaccines. Cancer Immunol Res; 6(9); 1025–38. ©2018 AACR.

Supplementary Table S1

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

Mirror-image biological systems have the potential for broad-reaching impact in health and diagnostics, but their study has been greatly limited by the lack of routine access to synthetic D-proteins. We demonstrate that automated fast flow peptide synthesis (AFPS) can reliably produce novel mirror-image protein targets without prior sequence engineering. We synthesized 12 D-proteins, along with their L-counterparts. All 24 synthetic proteins were folded into active structures in vitro, and characterized using biochemical and biophysical techniques. From these chiral protein pairs, we chose MDM2 and CHIP to carry forward into mirror-image phage display screens, and identified macrocyclic D-peptides that bind the recombinant targets. We report 6 mirror-image peptide ligands with unique binding modes: three to MDM2, and three to CHIP, each confirmed with X-ray co-crystal structures. Reliable production of mirror-image proteins with AFPS stands to enable not only the discovery of D-peptide drug leads, but to the study of mirror-image biological systems more broadly.

α-Klotho is a protein associated with aging that is expressed in the kidney, parathyroid gland, and choroid plexus. As a transmembrane protein, it acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. α-Klotho has an extracellular domain that can be cleaved, released and circulated in the blood stream as a soluble form. Decreased levels of α-Klotho are an indication of chronic kidney disease and other age-associated diseases. Detecting or labeling transmembrane and soluble α-Klotho is a longstanding challenge that has impeded the in-depth understanding of its role. Here we describe branched multimeric peptides that recognize α-Klotho with high affinity and selectivity in the biological milieu. The branched peptides are prepared in a single-shot synthesis by parallel automated fast-flow synthesis in under one hour. The branched α-Klotho-binding peptides show improvement in affinity relative to the monomeric versions and can be used to label Klotho for live imaging in kidney cells. Our results demonstrate the potential of automated flow technology to deliver peptide-based reagents with complex architecture and improved affinity for the selective binding of target proteins in physiological settings.

The increase of emerging drug resistant Gram-negative bacterial infections is of global concern. In addition, there is growing recognition that compromising the microbiota, through the use of broad spectrum antibiotics, may affect patient health in the long term. Therefore, there is the need to develop new -cidal strategies to combat Gram-negative infections that would consider these specific issues. In this study, we report and characterize one such approach, the antibody-drug conjugates (ADCs) that combine (i) targeting a specific pathogenic organism through a monoclonal antibody with (ii) the high killing activity of antimicrobial peptides. We focused on a major pathogenic Gram-negative bacterium associated with antibacterial resistance:Pseudomonas aeruginosaand designed an ADC by fusing an antimicrobial peptide at the C-terminal end of the VHand/or VL-chain of a monoclonal antibody, VSX, that targets the core ofP. aeruginosalipopolysaccharide (LPS). This ADC demonstrated appropriately minimal levels of toxicity to mammalian cells and rapidly killsP. aeruginosastrains through several mechanisms while protecting mice fromP. aeruginosalung infection when administered therapeutically. Furthermore, we found that the ADC was synergistic with several classes of antibiotics. This approach described in this study may result in a widely useful strategy to target specific pathogenic microorganisms without augmenting further antibiotic resistance.Author SummaryThe increasing of emerging drug resistant bacterial infections is a worldwide issue and infections caused by antibiotic resistant Gram-negative pathogens are particularly concerning. In addition, there is now growing recognition that disruption of the microbiota, through the use of broad spectrum antibiotics, may affect patient health in the long term. Therefore, there is the need to develop new -cidal strategies to combat Gram-negative infections while preserving the microbiota and also avoid enhancement of antibiotic resistance. We report and characterize here one such approach by using a specific monoclonal antibody associated with the potent killing activity of antimicrobial peptides in the form of an antibody-drug conjugate (ADC). The selected pathogenic bacterium wasPseudomonas aeruginosa,that presents numerous markers for both innate and acquired antibiotic resistance. The ADC lacked significant cytotoxicity against mammalian cells and was shown to be effective bothin vitroandin vivoagainstP. aeruginosa.

Therapeutic macromolecules such as proteins and oligonucleotides can be highly efficacious but are often limited to extracellular targets due to the cell's impermeable membrane. Cell-penetrating peptides (CPPs) are able to deliver such macromolecules into cells, but limited structure-activity relationships and inconsistent literature reports make it difficult to design effective CPPs for a given cargo. For example, polyarginine motifs are common in CPPs, promoting cell uptake at the expense of systemic toxicity. Machine learning may be able to address this challenge by bridging gaps between experimental data in order to discern sequence-activity relationships that evade our intuition. Our earlier data set and deep learning model led to the design of miniproteins (>40 amino acids) for antisense delivery. Here, we leveraged and expanded our model with data augmentation in the short CPP sequence space of the data set to extrapolate and discover short, low-arginine-content CPPs that would be easier to synthesize and amenable to rapid conjugation to desired cargo, and with minimal in vivo toxicity. The lead predicted peptide, termed P6, is as active as a polyarginine CPP for the delivery of an antisense oligomer, while having only one arginine side chain and 18 total residues. We determined the pentalysine motif and the C-terminal cysteine of P6 to be the main drivers of activity. The antisense conjugate was able to enhance corrective splicing in an animal model to produce functional eGFP in heart tissue in vivo while remaining nontoxic up to a dose of 60 mg/kg. In addition, P6 was able to deliver an enzyme to the cytosol of cells. Our findings suggest that, given a data set of long CPPs, we can discover by extrapolation short, active sequences that deliver antisense oligomers.

Information storage in DNA forms the fundamental basis of cellular life, but inherent limitations of information stability and density hamper innovative applications such as data storage or drug discovery. Here, we establish abiotic peptides for next-generation information storage and apply them for the encoding of diverse small molecule synthesis. The chemical stability of the peptide-based tag allows the use of palladium-mediated reactions to efficiently synthesize peptide-encoded libraries (PELs) with large chemical diversity and excellent purity. We demonstrate the successful application of PELs in drug discovery by affinity selection yielding small molecules with nanomolar affinity toward their target protein. Collectively, this work establishes abiotic peptides as carriers of information, leveraged herein for the encoding of small molecule synthesis.

There are more amino acid permutations within a 40-residue sequence than atoms on Earth. This vast chemical search space hinders the use of human learning to design functional polymers. Here we show how machine learning enables the de novo design of abiotic nuclear-targeting miniproteins to traffic antisense oligomers to the nucleus of cells. We combined high-throughput experimentation with a directed evolution-inspired deep-learning approach in which the molecular structures of natural and unnatural residues are represented as topological fingerprints. The model is able to predict activities beyond the training dataset, and simultaneously deciphers and visualizes sequence–activity predictions. The predicted miniproteins, termed ‘Mach’, reach an average mass of 10 kDa, are more effective than any previously known variant in cells and can also deliver proteins into the cytosol. The Mach miniproteins are non-toxic and efficiently deliver antisense cargo in mice. These results demonstrate that deep learning can decipher design principles to generate highly active biomolecules that are unlikely to be discovered by empirical approaches. Machine learning has now been shown to enable the de novo design of abiotic nuclear-targeting miniproteins. To achieve this, high-throughput experimentation was combined with a directed evolution-inspired deep-learning approach in which the molecular structures of natural and unnatural residues are represented as topological fingerprints. The designed miniproteins, called Mach proteins, are non-toxic and can efficiently deliver antisense cargo in mice.

Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases., PMOs (phosphorodiamidate morpholino oligomers) have huge potential for antisense therapy but complex and slow synthesis limits application. Here, the authors report the development of automated flow synthesis methods which reduce nucleobase coupling times from hours to minutes removing human errors and allow for high-throughput production.

The selective N-arylation of p-aminophenylalanine in polypeptides with pre-formed palladium oxidative addition complexes is described. The depressed pKa of the aniline NH2 group enables chemoselective C-N bond formation on peptides containing multiple other aliphatic amino groups at lysines or the N-terminus via Curtin-Hammett control under mild conditions. Using palladium complexes derived from electron-poor aryl halides, p-aminophenylalanine is fully arylated in aqueous buffer in as little as one hour at micromolar concentrations. A complementary protocol using the non-nucleophilic, organic base 1,5-diazabicyclo(4.3.0)non-5-ene (DBN), expands the substrate scope to tolerate electron-rich functional groups provides up to 97 % conversion. These procedures enable the chemoselective conjugation of functionally diverse small molecule pharmaceuticals to p-aminophenylalanine containing derivatives of cell-penetrating peptides.

Peptide-based cancer vaccines are widely investigated in the clinic but exhibit modest immunogenicity. One approach that has been explored to enhance peptide vaccine potency is covalent conjugation of antigens with cell-penetrating peptides (CPPs), linear cationic and amphiphilic peptide sequences designed to promote intracellular delivery of associated cargos. Antigen-CPPs have been reported to exhibit enhanced immunogenicity compared to free peptides, but their mechanisms of action in vivo are poorly understood. We tested eight previously described CPPs conjugated to antigens from multiple syngeneic murine tumor models and found that linkage to CPPs enhanced peptide vaccine potency in vivo by as much as 25-fold. Linkage of antigens to CPPs did not impact dendritic cell activation but did promote uptake of linked antigens by dendritic cells both in vitro and in vivo. However, T cell priming in vivo requiredBatf3-dependent dendritic cells, suggesting that antigens delivered by CPP peptides were predominantly presented via the process of cross-presentation and not through CPP-mediated cytosolic delivery of peptide to the classical MHC class I antigen processing pathway. Unexpectedly, we observed that many CPPs significantly enhanced antigen accumulation in draining lymph nodes. This effect was associated with the ability of CPPs to bind to lymph-trafficking lipoproteins and protection of CPP-antigens from proteolytic degradation in serum. These two effects resulted in prolonged presentation of CPP-peptides in draining lymph nodes, leading to robust T cell priming and expansion. Thus, CPPs can act through multiple unappreciated mechanisms to enhance T cell priming that can be exploited for cancer vaccines with enhanced potency.

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., Using tools from protein engineering and chemical biology, we combine antibodies and nontoxic anthrax proteins to develop new immunotoxins that target cancer cells and deliver therapeutic proteins.

We report aqueous, site-selective modification of proteins using a reactive peptide interface comprising a nine-residue sequence. This interface is the fastest (second-order rate constant of 152 M(−1) s(−1)) catalyst-free, cysteine-based method for modifying proteins available to date, and enables near-quantitative labeling of antibodies in cell lysate.

In-solution affinity selection (AS) of large synthetic peptide libraries affords identification of binders to protein targets through access to an expanded chemical space. Standard affinity selection methods, however, can be time-consuming, low-throughput, or provide hits that display low selectivity to the target. Here we report an automated bio-layer interferometry (BLI)-assisted affinity selection platform. When coupled with tandem mass spectrometry (MS), this method enables both rapid de novo discovery and affinity maturation of known peptide binders with high selectivity. The BLI-assisted AS-MS technology also features real-time monitoring of the peptide binding during the library selection process, a feature unattainable by current selection approaches. We show the utility of the BLI AS-MS platform toward rapid identification of novel nanomolar (dissociation constant, KD < 50 nM) non-canonical binders to the leukemia-associated oncogenic protein menin. To our knowledge, this is the first application of BLI to the affinity selection of synthetic peptide libraries. We believe our approach can significantly accelerate the use of synthetic peptidomimetic libraries in drug discovery., This work reports an automated affinity selection-mass spectrometry (AS-MS) approach amenable to both de novo peptide binder discovery and affinity maturation of known binders in a high-throughput and selective manner.

The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor (GPCR) and diabetes drug target expressed mainly in pancreatic β-cells that, when activated by its agonist glucagon-like peptide 1 (GLP-1) after a meal, stimulates insulin secretion and β-cell survival and proliferation. The N-terminal region of GLP-1 interacts with membrane-proximal residues of GLP-1R, stabilizing its active conformation to trigger intracellular signaling. The best-studied agonist peptides, GLP-1 and exendin-4, share sequence homology at their N-terminal region; however, modifications that can be tolerated here are not fully understood. In this work a functional screen of GLP-1 variants with randomized N-terminal domains reveals new GLP-1R agonists and uncovers a pattern whereby a negative charge is preferred at the third position in various sequence contexts. We further tested this sequence-structure-activity principle by synthesizing peptide analogues where this position was mutated to both canonical and non-canonical amino acids. We discovered a highly active GLP-1 analogue in which the native glutamate residue three positions from the N-terminus was replaced with the sulfo-containing amino acid cysteic acid (GLP-1-CYA). The receptor binding and downstream signaling properties elicited by GLP-1-CYA were similar to the wild type GLP-1 peptide. Computational modeling identified a likely mode of interaction of the negatively charged side chain in GLP-1-CYA with an arginine on GLP-1R. This work highlights a strategy of combinatorial peptide screening coupled with chemical exploration that could be used to generate novel agonists for other receptors with peptide ligands.

Nature has three biopolymers: oligonucleotides, polypeptides, and oligosaccharides. Each biopolymer has independent functions, but when needed, they form mixed assemblies for higher-order purposes, as in the case of ribosomal protein synthesis. Rather than forming large complexes to coordinate the role of different biopolymers, we dovetail protein amino acids and nucleobases into a single low molecular weight precision polyamide polymer. We established efficient chemical synthesis and de novo sequencing procedures and prepared combinatorial libraries with up to 100 million biohybrid molecules. This biohybrid material has a higher bulk affinity to oligonucleotides than peptides composed exclusively of canonical amino acids. Using affinity selection mass spectrometry, we discovered variants with a high affinity for pre-microRNA hairpins. Our platform points toward the development of high throughput discovery of sequence defined polymers with designer properties, such as oligonucleotide binding.

Capture and release of peptides is often a critical operation in the pathway to discovering materials with novel functions. However, the best methods for efficient capture impede facile release. To overcome this challenge, we report linkers based on secondary amino alcohols for the release of peptides after capture. These amino alcohols are based on serine (seramox) or isoserine (isoseramox) and can be incorporated into peptides during solid-phase peptide synthesis through reductive amination. Both linkers are quantitatively cleaved within minutes under NaIO4 treatment. Cleavage of isoseramox produced a native peptide N-terminus. This linker also showed broad substrate compatibility; incorporation into a synthetic peptide library resulted in the identification of all sequences by nanoLC-MS/MS. The linkers are cell compatible; a cell-penetrating peptide that contained this linker was efficiently captured and identified after uptake into cells. These findings suggest that such secondary amino alcohol based linkers might be suitable tools for peptide-discovery platforms., National Institutes of Health (Grants R01 GM110535, F32 GM133073), National Science Foundation (Grants 4000057398, 4000057441)

Peptide-mediated delivery of macromolecules in cells has significant potential therapeutic benefits, but no therapy employing cell-penetrating peptides (CPPs) has reached the market after 30 years of investigation due to challenges in the discovery of new, more efficient sequences. We developed a method for in-cell penetration selection-mass spectrometry (in-cell PS-MS) to discover peptides from a synthetic library capable of delivering macromolecule cargo to the cytosol. This method was inspired by recent in vivo selection approaches for cell-surface screening, with an added spatial dimension resulting from subcellular fractionation. A representative peptide discovered in the cytosolic extract, Pep1a, is nearly 100-fold more active toward antisense phosphorodiamidate morpholino oligomer (PMO) delivery compared to a sequence identified from a whole cell extract, which includes endosomes. Pep1a is composed of D-amino acids and two non-α-amino acids. Pulse-chase and microscopy experiments revealed that while the PMO-Pep1a conjugate is likely taken up by endosomes, it can escape to localize to the nucleus. In-cell PS-MS introduces a means to empirically discover unnatural synthetic peptides for subcellular delivery of therapeutically relevant cargo.