In Vitro Genetic Code Reprogramming for the Expansion of Usable Noncanonical Amino Acids.

Genetic code reprogramming has enabled us to ribosomally incorporate various nonproteinogenic amino acids (npAAs) into peptides in vitro. The repertoire of usable npAAs has been expanded to include not only l-α-amino acids with noncanonical sidechains but also those with noncanonical backbones. Despite successful single incorporation of npAAs, multiple and consecutive incorporations often suffer from low efficiency or are even unsuccessful. To overcome this stumbling block, engineering approaches have been used to modify ribosomes, EF-Tu, and tRNAs. Here, we provide an overview of these in vitro methods that are aimed at optimal expansion of the npAA repertoire and their applications for the development of de novo bioactive peptides containing various npAAs.

Selection-based discovery of druglike macrocyclic peptides.

Macrocyclic peptides are an emerging class of therapeutics that can modulate protein-protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptide-based macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 10(13) different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.

In Vitro Biosynthesis of Peptides Containing Exotic Azoline Analogues.

In nature, azolines produced by YcaO cyclodehydratases during the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs) are generally limited to thiazoline, oxazoline, and methyloxazoline, which are derived from the proteinogenic Cys, Ser, and Thr residues, respectively. To investigate whether YcaO cyclodehydratases precisely recognize the common structural characteristics and chirality of the modifiable residues, the "reprogrammed FIT-PatD system" has been established by combining a YcaO cyclodehydratase (PatD) with genetic code reprogramming powered by the flexible in vitro translation (FIT) system, in which precursor peptides bearing non-proteinogenic Cys/Ser/Thr analogues could be expressed through a reprogrammed genetic code and subsequently cyclodehydrated by PatD. The study has revealed remarkable stereo-, chemo-, and regioversatility for modifiable residues in PatD-catalyzed cyclodehydration, expanding the repertoire of backbone heterocycles in RiPPs to exotic azoline analogues.

Suppression of Formylation Provides an Alternative Approach to Vacant Codon Creation in Bacterial In Vitro Translation.

Genetic code reprogramming is a powerful approach to controlled protein modification. A remaining challenge, however, is the generation of vacant codons. We targeted the initiation machinery of E. coli, showing that restriction of the formyl donor or inhibition of the formyl transferase during in vitro translation is sufficient to prevent formylation of the acylated initiating tRNA and thereby create a vacant initiation codon that can be reprogrammed by exogenously charged tRNA. Our approach conveniently generates peptides and proteins tagged N-terminally with non-canonical functional groups at up to 99 % reprogramming efficiency, in combination with decoding the AUG elongation codons either with native methionine or with further reprogramming with azide- and alkyne-containing cognates. We further show macrocyclization and intermolecular modifications with these click handles, thus emphasizing the applicability of our method to current challenges in peptide and protein chemistry.

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation.

Bicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully-unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.

Reprogramming the genetic code in vitro.

The site-specific introduction of non-canonical amino acids into polypeptides through genetic code reprogramming has become a powerful tool for biochemical studies and bioorganic synthesis. Although a variety of such techniques have been developed, all are based on the 'mis-acylation' of tRNA molecules with non-canonical amino acids. Multiple strategies have been devised to synthesize such non-canonical aminoacyl-tRNAs; for example, those based on protein or ribozyme aminoacyl-tRNA synthetase enzymes are particularly useful. Such techniques have enabled the incorporation of hundreds of different non-canonical amino acids into polypeptides in vitro. This review discusses the development and application of in vitro genetic code reprogramming techniques, especially enzymatic mis-acylation, and examines recent efforts to engineer the translational machinery to increase the range of translatable non-canonical amino acids.

tRNA engineering for manipulating genetic code.

In ribosomal translation, only 20 kinds of proteinogenic amino acids (pAAs), namely 19 l-amino acids and glycine, are exclusively incorporated into polypeptide chain. To overcome this limitation, various methods to introduce non-proteinogenic amino acids (npAAs) other than the 20 pAAs have been developed to date. However, the repertoire of amino acids that can be simultaneously introduced is still limited. Moreover, the efficiency of npAA incorporation is not always sufficient depending on their structures. Fidelity of translation is sometimes low due to misincorporation of competing pAAs and/or undesired translation termination. Here, we provide an overview of efforts to solve these issues, focusing on the engineering of tRNAs.

Ribosome Subunit Stapling for Orthogonal Translation in E.†coli.

The creation of orthogonal large and small ribosomal subunits, which interact with each other but not with endogenous ribosomal subunits, would extend our capacity to create new functions in the ribosome by making the large subunit evolvable. To this end, we rationally designed a ribosomal RNA that covalently links the ribosome subunits via an RNA staple. The stapled ribosome is directed to an orthogonal mRNA, allowing the introduction of mutations into the large subunit that reduce orthogonal translation, but have minimal effects on cell growth. Our approach provides a promising route towards orthogonal subunit association, which may enable the evolution of key functional centers in the large subunit, including the peptidyl-transferase center, for unnatural polymer synthesis in cells.

Ribosomal Synthesis of Peptides Bearing Noncanonical Backbone Structures via Chemical Posttranslational Modifications.

Noncanonical peptide backbone structures, such as heterocycles and non-α-amino acids, are characteristic building blocks present in peptidic natural products. To achieve ribosomal synthesis of designer peptides bearing such noncanonical backbone structures, we have devised translation-compatible precursor residues and their chemical posttranslational modification processes. In this chapter, we describe the detailed procedures for the in vitro translation of peptides containing the precursor residues by means of genetic code reprogramming technology and posttranslational generation of objective noncanonical backbone structures.

Local Conformational Constraint of Firefly Luciferase Can Affect the Energy of Bioluminescence and Enzyme Stability.

Conformational dynamics contribute importantly to enzyme catalysis, such that targeted conformational constraint may affect catalysis. Firefly luciferases undergo extensive structural change during catalysis; key residues form a hydrophobic pocket, excluding water and enabling maximally energetic light production. Point mutants almost always luminesce at longer wavelengths (lower energy) than the wild type. Conformational constraint, using dipeptide analogue 3 at a position critical for optimized excited state structure, produced luciferase emission at a shorter wavelength by ~10 nm. In comparison, introduction of conformationally constrained analogues 4, 5, or 7 afforded luciferases emitting at longer wavelengths, while a related unconstrained luciferase (analogue 6) exhibited wild-type emission. The constrained luciferases tested were more stable than the wild type. Protein modeling demonstrated that the "inside" or "outside" orientation of the conformationally constrained dipeptide led to the shorter or longer emission wavelength, respectively. More broadly, these results suggest that local conformational constraint can control specific elements of enzyme behavior, both in vitro and in vivo. This represents the first example of studying enzyme function by introducing conformationally constrained dipeptides at a specific protein position. The principles discovered here in luciferase modification will enable studies to control the wavelength emission and photophysical properties of modified luciferases.

A Macrocyclic Peptide Library with a Structurally Constrained Cyclopropane-containing Building Block Leads to Thiol-independent Inhibitors of Phosphoglycerate Mutase.

Here we report the construction of an mRNA-encoded library of thioether-closed macrocyclic peptides by using an N-chloroacetyl-cyclopropane-containing exotic initiator whose structure is more constrained than the ordinary N-chloroacetyl-α-amino acid initiators. The use of such an initiator has led to a macrocycle library with significantly suppressed population of lariat-shaped species compared with the conventional libraries. We previously used a conventional library and identified a small lariat thioether-macrocycle with a tail peptide with a C-terminal free Cys whose sidechain plays an essential role in potent inhibitory activity against a parasitic model enzyme, phosphoglycerate mutase. On the other hand, the cyclopropane-containing macrocycle library has yielded a larger thioether-macrocycle lacking a free Cys residue, which exhibits potent inhibitory activity to the same enzyme with a different mode of action. This result indicates that such a cyclopropane-containing macrocycle library would allow us to access mechanistically distinct macrocycles.

Peptide Folding and Binding Probed by Systematic Non-canonical Mutagenesis.

Many proteins and peptides fold upon binding another protein. Mutagenesis has proved an essential tool in the study of these multi-step molecular recognition processes. By comparing the biophysical behavior of carefully selected mutants, the concert of interactions and conformational changes that occur during folding and binding can be separated and assessed. Recently, this mutagenesis approach has been radically expanded by deep mutational scanning methods, which allow for many thousands of mutations to be examined in parallel. Furthermore, these high-throughput mutagenesis methods have been expanded to include mutations to non-canonical amino acids, returning peptide structure-activity relationships with unprecedented depth and detail. These developments are timely, as the insights they provide can guide the optimization of de novo cyclic peptides, a promising new modality for chemical probes and therapeutic agents.

Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides.

Synthetic biology holds promise to revolutionize the life sciences and biomedicine via expansion of macromolecular diversity outside the natural chemical space. Use of non-canonical amino acids (ncAAs) via codon reassignment has found diverse applications in protein structure and interaction analysis, introduction of post-translational modifications, production of constrained peptides, antibody-drug conjugates, and novel enzymes. However, simultaneously encoding multiple ncAAs in vivo requires complex engineering and is sometimes restricted by the cell's poor uptake of ncAAs. In contrast the open nature of cell-free protein synthesis systems offers much greater freedom for manipulation and repurposing of the biosynthetic machinery by controlling the level and identity of translational components and reagents, and allows simultaneous incorporation of multiple ncAAs with non-canonical side chains and even backbones (N-methyl, D-, ß-amino acids, α-hydroxy acids etc.). This review focuses on the two most used Escherichia coli-based cell-free protein synthesis systems; cell extract- and PURE-based systems. The former is a biological mixture with >500 proteins, while the latter consists of 38 individually purified biomolecules. We delineate compositions of these two systems and discuss their respective advantages and applications. Also, we dissect the translational components required for ncAA incorporation and compile lists of ncAAs that can be incorporated into polypeptides via different acylation approaches. We highlight the recent progress in using unnatural nucleobase pairs to increase the repertoire of orthogonal codons, as well as using tRNA-specific ribozymes for in situ acylation. We summarize advances in engineering of translational machinery such as tRNAs, aminoacyl-tRNA synthetases, elongation factors, and ribosomes to achieve efficient incorporation of structurally challenging ncAAs. We note that, many engineered components of biosynthetic machinery are developed for the use in vivo but are equally applicable to the in vitro systems. These are included in the review to provide a comprehensive overview for ncAA incorporation and offer new insights for the future development in cell-free systems. Finally, we highlight the exciting progress in the genomic engineering, resulting in E. coli strains free of amber and some redundant sense codons. These strains can be used for preparation of cell extracts offering multiple reassignment options.

Discovery of Functional Macrocyclic Peptides by Means of the RaPID System.

Flexizymes, highly flexible tRNA aminoacylation ribozymes, have enabled charging of virtually any amino acid (including non-proteogenic ones) onto tRNA molecules. Coupling to a custom-made in vitro translation system, namely the flexible in vitro translation (FIT) system, has unveiled the remarkable tolerance of the ribosome toward molecules, remote from what nature has selected to carry out its elaborate functions. Among the very diverse molecules and chemistries that have been ribosomally incorporated, a plethora of entities capable of mediating intramolecular cyclization have revolutionized the design and discovery of macrocyclic peptides. These macrocyclization reactions (which can be spontaneous, chemical, or enzymatic) have all served as tools for the discovery of peptides with natural-like structures and properties. Coupling of the FIT system and mRNA display techniques, known as the random non-standard peptide integrated discovery (RaPID) system, has in turn allowed for the simultaneous screening of trillions of macrocyclic peptides against challenging biological targets. The macrocyclization methodologies are chosen depending on the structural and functional characteristics of the desired molecule. Thus, they can emanate from the peptide's N-terminus or its side chains, attributing flexibility or rigidity, or even result in the installation of fluorescent probes.

Consecutive Elongation of D-Amino Acids in Translation.

Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and D-amino acids. Despite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two D-amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous D-amino acid elongation by the use of engineered tRNAs and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive D-Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive D-amino acids consisting of D-Phe, D-Ser, D-Ala, or D-Cys closed by either a disulfide bond or a thioether bond.

Rapid Discovery of Potent and Selective Glycosidase-Inhibiting De Novo Peptides.

Human pancreatic α-amylase (HPA) is responsible for degrading starch to malto-oligosaccharides, thence to glucose, and is therefore an attractive therapeutic target for the treatment of diabetes and obesity. Here we report the discovery of a unique lariat nonapeptide, by means of the RaPID (Random non-standard Peptides Integrated Discovery) system, composed of five amino acids in a head-to-side-chain thioether macrocycle and a further four amino acids in a 310 helical C terminus. This is a potent inhibitor of HPA (Ki = 7 nM) yet exhibits selectivity for the target over other glycosidases tested. Structural studies show that this nonapeptide forms a compact tertiary structure, and illustrate that a general inhibitory motif involving two phenolic groups is often accessed for tight binding of inhibitors to HPA. Furthermore, the work reported here demonstrates the potential of this methodology for the discovery of de novo peptide inhibitors against other glycosidases.