Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para-aminophenylalanine.

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para-aminophenylalanine (pAF) in Escherichia coli. First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host's native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (∼20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

Protein nanowires with tunable functionality and programmable self-assembly using sequence-controlled synthesis.

Advances in synthetic biology permit the genetic encoding of synthetic chemistries at monomeric precision, enabling the synthesis of programmable proteins with tunable properties. Bacterial pili serve as an attractive biomaterial for the development of engineered protein materials due to their ability to self-assemble into mechanically robust filaments. However, most biomaterials lack electronic functionality and atomic structures of putative conductive proteins are not known. Here, we engineer high electronic conductivity in pili produced by a genomically-recoded E. coli strain. Incorporation of tryptophan into pili increased conductivity of individual filaments >80-fold. Computationally-guided ordering of the pili into nanostructures increased conductivity 5-fold compared to unordered pili networks. Site-specific conjugation of pili with gold nanoparticles, facilitated by incorporating the nonstandard amino acid propargyloxy-phenylalanine, increased filament conductivity ~170-fold. This work demonstrates the sequence-defined production of highly-conductive protein nanowires and hybrid organic-inorganic biomaterials with genetically-programmable electronic functionalities not accessible in nature or through chemical-based synthesis.

Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

Chemoselective restoration of para-azido-phenylalanine at multiple sites in proteins.

The site-specific incorporation of nonstandard amino acids (nsAAs) during translation has expanded the chemistry and function of proteins. The nsAA para-azido-phenylalanine (pAzF) encodes a biorthogonal chemical moiety that facilitates "click" reactions to attach diverse chemical groups for protein functionalization. However, the azide moiety is unstable in physiological conditions and is reduced to para-amino-phenylalanine (pAF). Azide reduction decreases the yield of pAzF residues in proteins to 50%-60% per azide and limits protein functionalization by click reactions. Here, we describe the use of a pH-tunable diazotransfer reaction that converts pAF to pAzF at >95% efficiency in proteins. The method selectively restores pAzF at multiple sites per protein without introducing off-target modifications. This work addresses a key limitation in the production of pAzF-containing proteins by restoring azides for multi-site functionalization with diverse chemical moieties, setting the stage for the production of genetically encoded biomaterials with broad applications in biotherapeutics, materials science, and biotechnology.

Peptide Shuttle-Mediated Delivery for Brain Gene Therapies.

The manipulation of an individual's genetic information to treat a disease has revolutionized the biomedicine field. Despite the promise of gene therapy, this treatment can have long-term sideeffects. Efforts in the field and recent discoveries have already led to several improvements, including efficient gene delivery and transfer, as well as inpatient safety. Several studies to treat a wide range of pathologies-such as cancer or monogenic diseases- are currently being conducted. Here we provide a broad overview of methodologies available for gene therapy, placing a strong emphasis on treatments for central nervous system diseases. Finally, we give a perspective on current delivery strategies to treat such diseases, with a special focus on systems that use peptides as delivery vectors.

The Role of Orthogonality in Genetic Code Expansion.

The genetic code defines how information in the genome is translated into protein. Aside from a handful of isolated exceptions, this code is universal. Researchers have developed techniques to artificially expand the genetic code, repurposing codons and translational machinery to incorporate nonstandard amino acids (nsAAs) into proteins. A key challenge for robust genetic code expansion is orthogonality; the engineered machinery used to introduce nsAAs into proteins must co-exist with native translation and gene expression without cross-reactivity or pleiotropy. The issue of orthogonality manifests at several levels, including those of codons, ribosomes, aminoacyl-tRNA synthetases, tRNAs, and elongation factors. In this concept paper, we describe advances in genome recoding, translational engineering and associated challenges rooted in establishing orthogonality needed to expand the genetic code.

A MALDI-TOF-based Method for Studying the Transport of BBB Shuttles-Enhancing Sensitivity and Versatility of Cell-Based In Vitro Transport Models.

In recent decades, peptide blood-brain barrier shuttles have emerged as a promising solution for brain drugs that are not able to enter this organ. The research and development of these compounds involve the use of in vitro cell-based models of the BBB. Nevertheless, peptide transport quantification implies the use of large amounts of peptide (upper micromolar range for RP-HPLC-PDA) or of derivatives (e.g. fluorophore or quantum-dot attachment, radiolabeling) in the donor compartment in order to enhance the detection of these molecules in the acceptor well, although their structure is highly modified. Therefore, these methodologies either hamper the use of low peptide concentrations, thus hindering mechanistic studies, or do not allow the use of the unmodified peptide. Here we successfully applied a MALDI-TOF MS methodology for transport quantification in an in vitro BBB cell-based model. A light version of the acetylated peptide was evaluated, and the transport was subsequently quantified using a heavy internal standard (isotopically acetylated). We propose that this MALDI-TOF MS approach could also be applied to study the transport across other biological barriers using the appropriate in vitro transport models (e.g. Caco-2, PAMPA).

HAI Peptide and Backbone Analogs-Validation and Enhancement of Biostability and Bioactivity of BBB Shuttles.

Low effectiveness and resistance to treatments are commonplace in disorders of the central nervous system (CNS). These issues concern mainly the blood-brain barrier (BBB), which preserves homeostasis in the brain and protects this organ from toxic molecules and biohazards by regulating transport through it. BBB shuttles-short peptides able to cross the BBB-are being developed to help therapeutics to cross this barrier. BBB shuttles can be discovered by massive exploration of chemical diversity (e.g. computational means, phage display) or rational design (e.g. derivatives from a known peptide/protein able to cross). Here we present the selection of a peptide shuttle (HAI) from several candidates and the subsequent in-depth in vitro and in vivo study of this molecule. In order to explore the chemical diversity of HAI and enhance its biostability, and thereby its bioactivity, we explored two new protease-resistant versions of HAI (i.e. the retro-D-version, and a version that was N-methylated at the most sensitive sites to enzymatic cleavage). Our results show that, while both versions of HAI are resistant to proteases, the retro-D-approach preserved better transport properties.

Next-generation genetic code expansion.

Engineering of the translation apparatus has permitted the site-specific incorporation of nonstandard amino acids (nsAAs) into proteins, thereby expanding the genetic code of organisms. Conventional approaches have focused on porting tRNAs and aminoacyl-tRNA synthetases (aaRS) from archaea into bacterial and eukaryotic systems where they have been engineered to site-specifically encode nsAAs. More recent work in genome engineering has opened up the possibilities of whole genome recoding, in which organisms with alternative genetic codes have been constructed whereby codons removed from the genetic code can be repurposed as new sense codons dedicated for incorporation of nsAAs. These advances, together with the advent of engineered ribosomes and new molecular evolution methods, enable multisite incorporation of nsAAs and nonstandard monomers (nsM) paving the way for the template-directed production of functionalized proteins, new classes of polymers, and genetically encoded materials.

Immunosilencing peptides by stereochemical inversion and sequence reversal: retro-D-peptides.

Peptides are experiencing a new era in medical research, finding applications ranging from therapeutics to vaccines. In spite of the promising properties of peptide pharmaceuticals, their development continues to be hindered by three weaknesses intrinsic to their structure, namely protease sensitivity, clearance through the kidneys, and immune system activation. Here we report on two retro-D-peptides (H2N-hrpyiah-CONH2 and H2N-pwvpswmpprht-CONH2), which are protease-resistant and retain the original BBB shuttle activity of the parent peptide but are much less immunogenic than the parent peptide. Hence, we envisage that retro-D-peptides, which display a similar topological arrangement as their parent peptides, will expand drug design and help to overcome factors that lead to the failure of peptide pharmaceuticals in pre- and clinical trials. Furthermore, we reveal requirements to avoid or elicit specific humoral responses to therapeutic peptides, which might have a strong impact in both vaccine design and peptide therapeutic agents.

Bifunctional Peptide-Based Opioid Agonist-Nociceptin Antagonist Ligands for Dual Treatment of Acute and Neuropathic Pain.

Herein, the opioid pharmacophore H-Dmt-d-Arg-Aba-ß-Ala-NH2 (7) was linked to peptide ligands for the nociceptin receptor. Combination of 7 and NOP ligands (e.g., H-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) led to binding affinities in the low nanomolar domain. In vitro, the hybrids behaved as agonists at the opioid receptors and antagonists at the nociceptin receptor. Intravenous administration of hybrid 13a (H-Dmt-d-Arg-Aba-ß-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2) to mice resulted in potent and long lasting antinociception in the tail-flick test, indicating that 13a was able to permeate the BBB. This was further supported by a cell-based BBB model. All hybrids alleviated allodynia and hyperalgesia in neuropathic pain models. Especially with respect to hyperalgesia, they showed to be more effective than the parent compounds. Hybrid 13a did not result in significant respiratory depression, in contrast to an equipotent analgesic dose of morphine. These hybrids hence represent a promising avenue toward analgesics for the dual treatment of acute and neuropathic pain.

MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery.

Drug delivery across the blood-brain barrier (BBB) is a formidable challenge for therapies targeting the central nervous system. Although BBB shuttle peptides enhance transport into the brain non-invasively, their application is partly limited by lability to proteases. The present study proposes the use of cyclic peptides derived from venoms as an affordable way to circumvent this drawback. Apamin, a neurotoxin from bee venom, was minimized by reducing its complexity, toxicity, and immunogenicity, while preserving brain targeting, active transport, and protease resistance. Among the analogues designed, the monocyclic lactam-bridged peptidomimetic MiniAp-4 was the most permeable. This molecule is capable of translocating proteins and nanoparticles in a human-cell-based BBB model. Furthermore, MiniAp-4 can efficiently deliver a cargo across the BBB into the brain parenchyma of mice.

Lipid bilayer crossing--the gate of symmetry. Water-soluble phenylproline-based blood-brain barrier shuttles.

Drug delivery to the brain can be achieved by various means, including blood-brain barrier (BBB) disruption, neurosurgical-based approaches, and molecular design. Recently, passive diffusion BBB shuttles have been developed to transport low-molecular-weight drug candidates to the brain which would not be able to cross unaided. The low water solubility of these BBB shuttles has, however, prevented them from becoming a mainstream tool to deliver cargos across membranes. Here, we describe the design, synthesis, physicochemical characterization, and BBB-transport properties of phenylproline tetrapeptides, (PhPro)4, an improved class of BBB shuttles that operates via passive diffusion. These PhPro-based BBB shuttles showed 3 orders of magnitude improvement in water solubility compared to the gold-standard (N-MePhe)4, while retaining very high transport values. Transport capacity was confirmed when two therapeutically relevant cargos, nipecotic acid and l-3,4-dihydroxyphenylalanine (i.e., l-DOPA), were attached to the shuttle. Additionally, we used the unique chiral and conformationally restricted character of the (PhPro)4 shuttle to probe its chiral interactions with the lipid bilayer of the BBB. We studied the transport properties of 16 (PhPro)4 stereoisomers using the parallel artificial membrane permeability assay and looked at differences in secondary structure. Most stereoisomers displayed excellent transport values, yet this study also revealed pairs of enantiomers with high enantiomeric discrimination and different secondary structure, where one enantiomer maintained its high transport values while the other had significantly lower values, thereby confirming that stereochemistry plays a significant role in passive diffusion. This could open the door to the design of chiral and membrane-specific shuttles with potential applications in cell labeling and oncology.

'À la carte' peptide shuttles: tools to increase their passage across the blood-brain barrier.

Noninvasive methods for efficient drug delivery to the brain is an unmet need. Molecular access to the brain is regulated by the blood-brain barrier (BBB) established by the endothelial cells of brain vessels. Passive diffusion is one of the main mechanisms that organic compounds use to travel through these endothelial cells. This passage across the BBB is determined mainly by certain physicochemical properties of the molecule such as lipophilicity, size, and the presence of hydrogen bond donors and acceptors. One emerging strategy to facilitate the passage of organic compounds across the BBB is the use of peptide shuttles.1 In using this approach the permeability in front the BBB is, clearly, determined by the combined physicochemical properties of both the cargo and the shuttle. Herein we report the synthesis of a series of variations of one of the more efficient peptide shuttles, (N-MePhe)n . These include diverse structural features such as various backbone stereochemistries or the presence of non-natural amino acids, including halogenated residues. In several cases, we assessed the BBB permeability of both the shuttles alone and linked to a few cargos. Our results show how factors such as stereochemistry or halogen content influences the passage across the BBB and, more importantly, opens the way to a strategy of peptide shuttles 'à la carte', in which a particular fine-tuned shuttle is used for each specific cargo.