RNA Display Methods for the Discovery of Bioactive Macrocycles

Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

pubs.acs.org/CR

RNA Display Methods for the Discovery of Bioactive Macrocycles Yichao Huang, Mareike Margarete Wiedmann, and Hiroaki Suga*

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Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ABSTRACT: The past two decades have witnessed the emergence of macrocycles, including macrocyclic peptides, as a promising yet underexploited class of de novo drug candidates. Both rational/computational design and in vitro display systems have contributed tremendously to the development of cyclic peptide binders of either traditional targets such as cell-surface receptors and enzymes or challenging targets such as protein−protein interaction surfaces. mRNA display, a key platform technology for the discovery of cyclic peptide ligands, has become one of the leading strategies that can generate natural-product-like macrocyclic peptide binders with antibody-like affinities. On the basis of the original cell-free transcription/translation system, mRNA display is highly evolvable to realize its full potential by applying genetic reprogramming and chemical/ enzymatic modifications. In addition, mRNA display also allows the follow-up hit-to-lead development using high-throughput focused affinity maturation. Finally, mRNA-displayed peptides can be readily engineered to create chemical conjugates based on known small molecules or biologics. This review covers the birth and growth of mRNA display and discusses the above features of mRNA display with success stories and future perspectives and is up to date as of August 2018. 4.1. N-Methyl Amino Acid-Rich Peptide Libraries 4.2. Peptides Targeting Membrane Proteins 4.3. Macrocyclic Peptides Featuring a Peptidic Tail 4.4. Membrane-Permeable Macrocyclic Peptides 4.5. Dual-Peptide Selection for Binding Affinity and Protease Resistance 4.6. PPI Inhibitor Peptides 4.7. Peptide Agonists of Membrane Receptor Proteins 5. Growing Potential of mRNA Display 5.1. Identifying Cell-Permeable Peptides 5.2. Peptide Ligands as Cocrystallization Chaperones 5.3. Custom-Made Focused Library for Unique Targets 5.4. High-Throughput Affinity Maturation 5.5. Peptide Dimerization 5.6. Grafting onto a Scaffold Protein 6. Future Perspectives Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Abbreviations References

CONTENTS 1. Introduction 1.1. Current Methods To Discover Bioactive Macrocycles 1.2. Display Systems 1.2.1. In Vivo Display Systems 1.2.2. In Vitro Display Systems 1.3. mRNA Display 1.4. Advantages of mRNA Display 1.5. Disadvantages of mRNA Display 1.6. Review Outline 2. Historical Perspective: Birth and Growth of mRNA Display 2.1. mRNA Display of Canonical Linear Peptides 2.2. Optimization of Puromycin Linker Ligation 2.3. Use of Covalent mRNA-cDNA-Peptide Fusions 2.4. Translation of Peptides Containing Noncanonical Amino Acids 2.5. Flexible in Vitro Translation (FIT) System 2.6. mRNA Display Based on the RaPID System 3. Toolbox of the FIT System 3.1. Genetic Code Reprogramming 3.2. Post-Translational Enzymatic Modification 3.2.1. N-Terminal Modification 3.2.2. Backbone Modification 3.3. Post-Translational Chemical and ChemoEnzymatic Modification 3.3.1. Thioether-Based Cyclization 3.3.2. Nonthioether-Based Cyclization 3.4. Amino Acids Compatible with mRNA Display 4. Case Studies © XXXX American Chemical Society

B B E E E F G G H H H I J J M N N N P P P P P Q

R R R T U V V V V W W W W X X Y Y Y Y Y Y Y Z

Special Issue: Macrocycles

R R

Received: July 8, 2018

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DOI: 10.1021/acs.chemrev.8b00430 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. (A) Scheme showing the size difference of different drug modalities, exemplified by aspirin, cyclosporin A, human insulin, and an IgG2a antibody. (B) Macrocyclic drugs have characteristics of both small-molecule and biological drug classes by interacting via more extended binding interactions but having more small-molecule-like drug properties. Macrocyclic drugs benefit from generally higher potency, target selectivity, binding interaction size, and low toxicity but are also more metabolically stable and have lower manufacturing costs.

1. INTRODUCTION

has been shown to enhance resistance to degradation by proteases since the macrocyclic peptides do not fit into the endopeptidase active site as well as linear peptides.7,8 A further benefit of macrocyclization is the preorganization of the peptides, which may reduce the amount of entropy lost upon adopting the target binding conformation. Interest in the development of macrocyclic peptide drugs stems from the discovery of the many biologically active natural product macrocycles such as vancomycin, romidepsin, sirolimus, and amphotericin B.9 Furthermore, in the case of cyclosporine it was shown that the macrocycle exposes its polar groups when in aqueous medium but efficiently folds itself with its polar groups being shielded inside and apolar groups facing out to allow membrane permeability.10 Due to their effective interactions at large-shallow surfaces, macrocycles have been shown to be able to interact more effectively with challenging target classes.11 Doak and co-workers found that disk- and sphere-like macrocycles tend to be better binders of flat binding sites, whereas rod-like macrocycles more frequently target groove-shaped binding sites.11 This demonstrates the versatility of macrocycles as potential drugs. However, macrocycles used to be considered less favorable by the pharmaceutical industry, since they do not adhere to “Lipinski’s rule of five”, which used to be the accepted gold standard to assess drug likeness.12 Interestingly, several orally available cyclic peptide drugs have been approved that violate this dogma with a typical molecular weight > 500 Da. This trend in pharmaceutical chemistry has established the concept of beyond rule of 5 (bRo5). Cyclosporin A, as shown in Figure 1, as well as other macrocyclic drugs including Desmopressin, Alisporivir, and Linaclotide reside in this field.11 Although general solutions to cell permeability and oral bioavailability of macrocyclic peptides remain to be explored

1.1. Current Methods To Discover Bioactive Macrocycles

When comparing target classes that drugs were being developed for over the past 20 years compared with now, it becomes evident that there has been a gradual shift away from traditional druggable targets, such as G-protein-coupled receptors, ion channels, and enzymes.1,2 Because of the availability of new biological entities, the pharmaceutical industry has been able to move toward different and less conventional target classes. Nonetheless, target classes such as transcription factors and intracellular protein−protein interactions (PPIs) still represent a challenge for small-molecule or large-molecule drug discovery approaches.3,4 Macrocyclic molecules are a very attractive type of chemical class to target challenging targets such as protein−protein interactions, protein−nucleic acid interactions, as well as transcription factors.5,6 Macrocycles have been defined to contain one or more rings of at least 12 atoms, which results in the preorganization and a semirigid character of this chemical class.6 They combine the therapeutic benefits of small molecules as well as larger biomolecules with their lack of immunogenicity as well as high potency, selectivity, and reasonably low manufacturing costs. Figure 1 highlights the difference in the size of small-molecule drugs, macrocycles, and biologicals and the resulting chemical and pharmacological properties. Macrocyclic peptides, in particular, can be synthesized readily from orthogonally protected amino acid building blocks via chemical synthesis. However, it is the biological synthesis of macrocyclic peptide libraries through the use of DNA- or RNA-encoded libraries that has allowed the screening of highly diverse macrocyclic peptide libraries for drug discovery. Importantly, the cyclic nature of macrocycles B


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Table 1. Current Companies Involved in the Development of Bioactive Macrocycle Drugs company

category

founding year

Aileron Therapeutics Bicycle Therapeutics

stapled peptides macrocyclic peptides

2005

Cyclenium Pharma Circle Pharma

macrocycles

2013

macrocyclic peptides macrocyclic peptides macrocycles

2014 2013

macrocycles

2004

macrocyclic peptides

2011

Fog Pharma

macrocycles

2014

Lanthio Pharma

stapled peptides macrocyclic peptides macrocyclic peptides macrocycles

2010

Cypralis Encycle Therapeutics Ensemble Therapeutics EvoRX

Pepticom Peptidream Polyphor

Protagonist stapled Therapeutics peptides RA macrocyclic Pharmaceuticals peptides

2009

2015

2011 2006 1996 2007 2008

representative original publication

leading drug candidate

web site

platform technology

https://www.aileronrx. com/ https://www. bicycletherapeutics. com/ http://www.cyclenium. com/ https://circlepharma. com/ http://www.cypralis. com/ http:// encycletherapeutics. com/ http://www. ensembletx.com/ http:// evorxtechnologies. com/ http://fogpharma. com/ http://www. lanthiopharma.com/ http://pepticom.com/

rational design of stapled α-helical peptides

Nature 200917

phage display and bicyclization

Nat. Chem. Biol. 200918

ALRN-6924 (Phase I/IIa) BT1718 (Preclinical)

rational design of macrocycles

N.A.

N.A.

rational design of macrocycles

J. Med. Chem. 201719 N.A.

N.A.

https://www. peptidream.com/ https://www.polyphor. com/ http://www. protagonist-inc.com/ http://rapharma.com/

RNA display and spontaneous cyclization; flexible incorporation of npAAs rational design of macrocycles (MacroFinder and PEMfinder) phage display and virtual screening of disulfide-rich peptides RNA display and cyclization

targeting peptidyl-prolyl isomerases and cyclophilin inhibitors computational design of macrocycles

N.A. Nature 201420

CC-1233 (Preclinical) N.A.

DNA-programmed macrocycle synthesis and selection RNA display and cyclization

Sci. Rep. 201421

IDO-1 (Preclinical) N.A.

cell-permeable mini proteins

N.A.

N.A.

rational design of stapled natural peptides

Nat. Commun. 201722 N.A.

MOR107 (Phase I) N.A.

J. Am. Chem. Soc. 201323 N.A.

BMS (Phase I)

computational design of macrocycles

PLOSOne 201524 J. Am. Chem. Soc. 201225

POL7080 (Phase III) PTG-100 (Phase II) RA101495 (Phase II)

(3) only big pharmaceutical companies have the necessary resources to conduct HTS projects successfully. FBDD has become part of mainstream drug discovery since its development over 20 years ago.28 Smaller chemical fragments, which are able to weakly bind the target of choice, are identified and combined covalently to give higher-affinity lead molecules, a process often guided by X-ray crystallography.29 Since FBDD library sizes are smaller and hence easier to maintain and screen, academic laboratories as well as smaller pharmaceutical companies are able to undertake lead discovery using FBDD. Furthermore, the smaller fragments which are used are often more soluble, and a combination of these may result in the production of drugs with better pharmaceutical properties.29 Today, dozens of drugs derived from FBDD have entered the clinic.28 The way drug discovery is conducted has changed drastically in the first decade of the 21st century due to the invention of innovative technologies.30 Innovations emerged both in the area of pure organic synthetic chemistry (DNA-encoded chemical libraries, DELs) as well as in chem-biohybrid strategies such as mRNA display, phage display, and split intein-mediated circular ligation of peptides (SICLOPPS).31−36 A comparison of DEL screening, mRNA display, and phage display is given in Figure 2. DEL screening approaches differ from mRNA and phage display technologies since DELs are constructed by split-pool synthesis, and only one round of library screening is conducted per target. The latter part of the described technologies is mainly characterized by macrocycles that are peptidic in nature. All of these librarybased display technologies benefit significantly from the

and solved, this chemical class has witnessed ever-increasing research interest from academia and industry. While ongoing discussions about this topic can be found in reviews elsewhere,5,13−16 in general, two directions to engineer the chemical structures of macrocycles exist. First, macrocyclic peptides can be engineered to be more small-molecule-like, e.g., by incorporating N-methylated residues or stapling into a scaffold, to become more hydrophobic and metabolically stable. Second, macrocycles can be engineered to be more biological like, for example, by creating high molecular weight peptide conjugates, to become more potent and target specific. In an effort to tackle the remaining challenges surrounding macrocyclic drugs and to promote their further growth, several companies were established in the first decade of this century. Table 1 summarizes leading biotechnology venture companies which are involved in the development of bioactive macrocycle drugs. In the past, the two main methods for the discovery of new drug entities, including macrocycles, were high-throughput screening (HTS) and fragment-based drug discovery (FBDD). Two decades ago HTS became the dominant drug discovery approach used by the pharmaceutical industry to identify new lead compounds.26 HTS has yielded many approved drugs, in particular, against the established target classes. When applying large HTS libraries to more difficult target classes, fewer hits are recovered on average and false-positive hits can be a problem.27 Other disadvantages of HTS include that (1) it is time and money intensive, (2) only library sizes of up to 106 compounds can be screened realistically and logistically, and C


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Figure 2. Comparison of DNA-encoded library (DEL) screening, mRNA display, and phage display selections. (A) DELs are constructed by splitand-pool synthesis. Initial small-molecule warhead with a DNA tag is enzymatically ligated to DNA coding for the subsequent chemical modifications. After chemical synthesis the DNA products are pooled, precipitated, and split for another round of split-and-pool synthesis. DEL screens are characterized by consisting of only one round of affinity selection. (B) Phage display selections begin with the transformation of a plasmid library into bacteria. Helper phages are used to infect the plasmid-containing bacteria. This results in the assembly of the phage library with the library peptides expressed on the relevant phage coat proteins. As with mRNA display selections, affinity selection is followed by several rounds of reamplification and reselection until sufficient enrichment is observed. C) For mRNA display selections an initial mRNA library is fused to a linker using various methods, which upon in vitro translation results in the covalent attachment of the peptides to their mRNA coding tag. Affinity selection is followed by iterative rounds of mRNA/cDNA tag recovery, library reamplification, and reselection until sufficient enrichment is observed.

binders.42−44 DNA-encoded small-molecule libraries are synthesized using a split-and-pool approach, and because each synthesized library molecule is linked to a DNA tag it is possible to trace back the synthetic history of the attached molecule. DNA-encoded libraries are screened against an immobilized target, and the identity of binders is confirmed by sequencing of the covalently attached DNA tag. New developments in the chemical toolbox, such as enzymatic synthesis,45 and the development of additional DNAcompatible reactions in water may help in constructing additional and more diverse DELs in the near future.46 Importantly, the development of DNA-encoded libraries has made it possible to investigate broader areas of chemical space since libraries of 107−1010 molecules can be screened with DELs.47 In another direction, progress in the biotechnology area has been highlighted by the successful establishment of various display techniques for the selection of novel peptide-based macrocyclic binders targeting previously undruggable targets. Among them, in vitro mRNA display has also contributed to the expansion of “druggable target space” when compared with what is accessible if only small molecules and biologicals are used (Figure 1). The use of DNA-encoded libraries has

development of next-generation sequencing (NGS) technologies.37 Sanger sequencing suffers from the disadvantage that only a very small fraction of the display library can be sequenced (approximately only 10 to a few hundred sequence clones).38 NGS technologies offer a much greater sequencing depth of more than 106 sequence clones per run.38 This makes it possible to obtain sequence information on the majority of clones during each round of display selection and hence allows an extensive analysis of the selection library. Molecular biologist Sydney Brenner and chemist Richard Lerner were the first, in 1992, to imagine the creation of chemical libraries with individual compounds connected to DNA fragments, which serve as amplifiable identification tags or barcodes (Figure 2A).39 Brenner and Lerner discussed the possibility of simultaneously synthesizing polypeptide and oligonucleotide sequences on beads.39 Two research groups demonstrated shortly afterward the feasibility of the proposed concepts. Janda and co-workers as well as Needels and coworkers successfully used bead-based libraries to identify known antibody epitopes.40,41 The initial idea was further improved upon in 2004, when several groups constructed DNA-encoded combinatorial libraries of organic molecules devoid of beads and discovered a selection of novel D


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allowed HTS to expand beyond the typical library size of ∼106 for small-molecule libraries and has become part of mainstream hit identification, while mRNA display is an excellent method for harnessing the power of the DNA-encoded library technology, which has further pushed the library size limit to ∼1013.48

Each library member is genetically encoded to be fused to the Aga2p protein, which is located on the outside of the yeast cell. Transformation and subsequent induction of expression with galactose results in the surface display of the library on the yeast cells. YSD is compatible with flow cytometry and fluorescence-activated cell sorting (FACS) for the determination of equilibrium constants. After each selection round, the yeast cells isolated by FACS are amplified in culture to give an enriched library and are then reselected. Since YSD is a eukaryotic expression system, expression of complex mammalian proteins with the correct post-translational modifications, such as disulfide bonds, is possible. Limitations due to the yeast transformation efficiency restrict this type of display system to 108−109 yeast cells, as opposed to 1012−1014 observed with phage, ribosome, and mRNA display.61 YSD has been particularly successful for the selection of higher affinity antibodies.62,63 The bacterial surface display system was introduced in 1986 by Freudl et al. and Charbit et al., 1 year after the discovery of phage display. The bacterial surface proteins OmpA and LamB were used to display the initial peptide libraries.64,65 Since then a variety of protein carriers have been developed.66 An E. colibased display system has the advantage that thousands of peptide copies can be displayed on a single E. coli bacterium. Disadvantages of bacterial cell surface display are that it is limited by the transformation efficiency of the library and that it suffers from issues when expressing proteins inside a prokaryotic host, which is poorly equipped to the posttranslational processing of mammalian proteins. Similar to YSD, bacterial surface display is often coupled with FACS and magnetic-activated cell sorting and can be used for the selection of peptide libraries and live vaccine development by exposing heterologous epitopes on bacterial cells to elicit an immune response.58,67 1.2.2. In Vitro Display Systems. Although in vivo display methods, especially phage display, have become widely popular to select peptides or protein fragments,68,69 it is mandatory to perform a transformation of the PCR products into host cells after each selection round. This transformation step limits the diversity of display libraries. In addition, incorrect in vivo folding and membrane insertion usually results in a biased sequence library. To overcome these limitations, in vitro or cell-independent display methods have been developed. Although in vitro display methods require an RNase- and protease-free environment for selections, which is technically more demanding than some in vivo methods, a huge advantage is that a 1012−1015 sequence diversity can be covered compared with around 107−1010 for in vivo display methods such as phage and yeast display. A larger library increases the chance of identifying target-binding peptides or nucleotide sequences with higher affinity and specificity, sometimes with diverse binding mechanisms. Ribosome display is one of the in vitro display methods that couples the genotype (mRNA) to the phenotype (nascent peptide) through a noncovalent peptide/protein-ribosomemRNA ternary complex.70,71 This complex can be stabilized through stop codon mutation, magnesium cation addition, and delicate temperature control. Transcription and translation can be coupled into one step or conducted separately. Peptide/ protein expression is carried out by an in vitro cell-free translation system using cell lysates or prepurified translation components. After biopanning-based selection steps, the sequence information is obtained through PCR and high-

1.2. Display Systems

1.2.1. In Vivo Display Systems. The most commonly used in vivo display systems are SICLOPPS, phage display, yeast display, and bacterial cell surface display. An example of a cell-based intracellular selection strategy is SICLOPPS, which refers to the expression of peptides via a “split intein-mediated circular ligation of peptides and proteins”. This method allows the formation of intracellular head-to-tail cyclized peptides in bacteria.49,50 SICLOPPS consists of a library of random DNA sequences, which have DNA sequences coding for selfexercising proteins known as inteins on either side of the library DNA sequence in the plasmid. During translation, the split-inteins come into close proximity, which promotes an intracellular chemical ligation reaction. The intein is released, and a cyclic peptide is generated.33 Using this methodology, libraries of 106−108 cyclic peptides can be generated.33 The SICPLOPPS system has been successfully coupled to the reverse two-hybrid system for the discovery of novel PPI inhibitors and was successful in the discovery of HIF1 dimerization inhibitors.51,52 The main advantage of the SICLOPPS system is that peptides are discovered for their inhibitory activity as opposed to their binding affinities, and hence, bioactive species can be isolated.53 Phage display is an in vivo selection technique in which the translated peptides are attached to a bacteriophage cell surface protein, such as the M13 phage protein III (pIII). This is achieved by inserting the cDNA for the library peptides into the phage coat protein gene. Peptides displayed at the Nterminus of pIII do not interfere with the folding of the three domains of pIII.54 Helper phages are used to infect the E. coli, which contain the library plasmids (Figure 2B). The transformation efficiency of this step currently limits the number of independent sequences to 109.55,56 During the infection the phage DNA is translated by the E. coli and the library peptide is attached to the pIII protein, which is displayed on the outside of the phage during phage assembly. This results in a selectable system with a genotype−phenotype link. These modified phage viruses can be selected against an immobilized target, and selected sequences can be subjected to further rounds of phage display to increase the recovery of tightly binding sequences. An advantage is that phage display can be coupled with directed evolution using the phageassisted continuous evolution (PACE) system.57 Issues that can be encountered, which decrease the number of sequences represented, include poor expression or incorrect folding of the gene product and failure of the modified pIII protein to migrate to the phage surface. Most of these limitations come from E. coli not being able to express some eukaryotic proteins. This is because E. coli lack certain foldases and chaperones required for efficient folding.58−60 In yeast surface display (YSD), a protein or peptide is expressed on the outside surface of yeast (Saccharomyces cerevisiae) as a fusion to an abundant cell surface anchor protein such as the α-agglutinin mating protein (Aga2p).61 Each yeast cell is transformed with a single vector that contains a gene coding for a particular peptide-based library member. E


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Figure 3. (A) Structure of tyrosyl tRNA. (B) Antibiotic puromycin is a structural analogue of tyrosyl tRNA. Structural differences are highlighted in pink. Key difference is the amide bond connecting the tyrosine amino acid to the ribose, which confers greater stability. (C) mRNA display requires the use of a covalent fusion of coding mRNA and translated peptide (i) During translation, charged tRNAs enter the ribosomal A site and the amino acids are covalently transferred onto the growing peptide chain. (ii) When the ribosome reaches the amber stop codon (UAG) translation stalls due to the absence of release factor 1 in the in vitro translation system. (iii) Puromycin enters the ribosomal A site and is covalently attached to the translated peptide via the alpha amino group on puromycin due to the peptidyl transferase activity of the ribosome. (iv) Translated peptidepuromycin-mRNA fusion is released from the ribosome and can be used for target selection in mRNA display.

called covalent antibody display (CAD) uses another cis-acting protein, bacteriophage endonuclease P2A, to covalently link its coding DNA to the protein.74

throughput sequencing. Unlike mRNA display, one limitation of ribosome display is that magnesium-rich and low-temperature conditions (4 °C) are necessary, which can result in loss of potentially good binders. Single-stranded mRNA, an RNase-labile structure, is used in both ribosome display (noncovalently attached) and mRNA display (covalently attached) to encode the polypeptide chain. Some DNA-based in vitro selection methods have also been developed. For example, CIS display takes advantage of the cis activity of a replication initiator protein RepA, which binds to its own encoding DNA. By ligating the random DNA sequences to that of RepA, random peptide sequences can be attached to RepA through in vitro transcription and translation. The resulting peptide/protein-DNA complexes can then undergo affinity selections.72,73 A similar display method

1.3. mRNA Display

mRNA display has become an increasingly popular technique for the screening of vast libraries of peptides or proteins, which are covalently attached to their coding mRNA tag (Figure 2C). First, a cDNA library is transcribed into mRNA, which is then enzymatically ligated to a synthetic spacer, a strand of DNA with a puromycin molecule at its 3′ end. The ribosome, which is contained in the in vitro translation mixture, reads mRNA in the 5′ to 3′ direction and has both a P (peptidyl) and an A (aminoacyl) site to which peptidyl-tRNA and aminoacyl-tRNA bind. Most commonly, the P site contains the tRNA with the growing peptide chain, whereas the A site houses the tRNA F


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with the new amino acid to be added to the peptide chain. When the ribosome reaches the stop codon at the end of the coding mRNA, translation stalls and the ligated puromycin enters the A site, since the relevant release factor has been excluded from the in vitro translation system. The puromycin molecule is then attached covalently onto the polypeptide chain in the P site. Most commonly, the polypeptide is linked to its coding mRNA tag via a DNA spacer and the puromycin molecule (Figure 3). The covalent linkage allows for a specific mRNA sequence to be enriched based on the properties of its encoded peptide. Usually the mRNA-peptide fusion molecule is reverse transcribed next to give a library of cDNA-mRNA duplexes attached to their peptides. This is followed by the selection and pulldown of binding peptides via an immobilized protein target. cDNA sequences are then recovered from the binders and are amplified by PCR, and the next round of mRNA display selection takes place until an enrichment of sequences is observed. For the directed evolution in combination with mRNA display, it is possible to include a mutational strategy of choice during the PCR amplification step to generate further variability. Variability in an mRNA library is achieved through the initially designed cDNA library. The cDNA library can either be fully or partially randomized with some amino acid positions remaining fixed. The ability to include non-natural amino acids adds to the diversity that can be achieved with mRNA display. These include, but are not limited to, Nmethylated amino acids to improve cell permeability, nonnatural amino acids containing a reactive “warhead”, or amino acids that allow macrocyclization of the translated peptides.75,76 mRNA also allows the translated peptides to be further modified with other bridging small molecules such as 1,3,5-tris(bromomethyl)benzene (TBMB), which can react with cysteine residues present in the translated peptide to yield mono- or bicyclic peptides.18,77,78 Further linkers that have been used are 1,3,5-triacryloyl-1,3,5-triazinane and N,N′,N″′(benzene-1,3,5-triyl)-tris(2-bromoacetamide).79,80 These posttranslation modifications introduce a different topology and conformational constraints before the library is screened.79,80 Either natural or combinatorial peptide libraries can be screened for peptides with desired characteristics.81−83 mRNA display has been used successfully, in both academia and industry, for the selection of tightly binding molecules from both synthetic as well as natural libraries for drug discovery, for investigations of molecular interactions, and to interfere with or understand biological processes. The possibility to incorporate large numbers of nonstandard amino acids by genetic code reprogramming has increased the usability of mRNA display.

The most important advantage of in vitro display technologies, such as mRNA display, is the ease at which PCR-based amplification and randomization techniques can be incorporated, since PCR is used for the direct amplification after each selection round. Random mutagenesis by PCRbased methods such as error-prone PCR, random insertion or deletion, DNA shuffling, and random insertion−deletion strand exchange can be combined with mRNA display. During this procedure mutations are accumulated gradually, while a selective pressure via affinity selections is applied. This is useful for the directed evolution to isolate sequences with desirable characteristics.85 With in vivo display methods, amplification requires transformation of the mutated PCR products into cells. Compared with other screening technologies, mRNA display results are more reliable in the way sequencing results correspond to the binding ligand, since the genotype and phenotype are covalently linked. Furthermore, compared with ribosome display, mRNA display selection conditions can be optimized more easily and more stringent selection conditions may be applied to increase the recovery of enriched binding sequences. Ribosome display can be more limiting in terms of the assay conditions that can be used in order to not interfere with the ternary, noncovalent linkage. In mRNA display, 1012−1014 unique library sequences have been tested so far, but in theory cost and sample handling are the only limiting factors, since the entire display system is scalable. mRNA display is hence ideally suited for the identification of rare sequences as well as for giving a greater diversity of isolated sequences as the selected output. A further advantage of mRNA display is that it is not limited to shorter peptide sequences but that also larger proteins can be identified from selections. For example, using mRNA display, the interaction partners of a target protein can be identified from an mRNA-displayed proteome library using random priming, as is the case for the Ca2+-binding protein (CaM).86,87 1.5. Disadvantages of mRNA Display

The main disadvantage of any display system is nonspecific binding between the display system and the target to give false positive hits. In the case of mRNA display, highly positively charged target proteins may interfere during mRNA-display selections due to their high affinity for the negatively charged mRNA tag.88 Positively charged target molecules represent an estimated 35% of all proteins in the human genome, and all of these will show a particularly high, nonspecific binding of the negatively charged surfaces of mRNA.88 Attempts to circumvent this problem have involved the development of chemical (poly lysine) and genetic wrappers by Lamboy and coworkers.88 A covalent linkage between phenotype and genotype also has drawbacks associated with it: The relatively large mRNA tag may interfere with the translated peptide sterically or functionally. The issue of the mRNA tag interfering with the selection is attenuated by reverse transcription prior to selection to give the mRNA/cDNA hybrid to prevent the formation of secondary structures, but this may still be an issue for proteins that are known to bind nucleic acids, such as transcription factors. Several factors may also bias the enrichment of sequences during mRNA display selections, such as the formation of

1.4. Advantages of mRNA Display

In vitro display systems are represented by ribosome display and mRNA display systems. Since in vitro systems do not require a transformation step, they are not limited by the library transformation efficiency. Hence, the peptide or protein library complexity is nearly identical to that of the initial mRNA library complexity. Large libraries with diversities of >1012 can be screened. In contrast, a phage library may contain more than 1012 individual phage particles, but since a typical E. coli transformation efficiency is only 109 per microgram of DNA, the typical diversity that can be achieved using a phage display library is only around 109.84 G


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Table 2. Recent Reviews and Protocols Covering mRNA Display Since 2010 topic general review of selections

historical accounts flexizymes

FIT system

RaPID system

peptide delivery protein cocrystallization

publication title

ref

Annu. Rev. Biochem. 201492 Exploring sequence space: harnessing chemical and biological diversity toward new peptide leads Curr. Opin. Chem. Biol. 201755 mRNA display: from basic principles to macrocycle drug discovery Drug Discov. Today 201493 Max−Bergmann award lecture: A RaPID way to discover bioactive nonstandard peptides assisted by the flexizyme J. Pept. Sci. 201894 and FIT systems Flexizymes: Their Evolutionary History and the Origin of Catalytic Function Acc. Chem. Res. 201195 Flexizymes for genetic code reprogramming Nat. Protocols 201196 Flexizymes as a tRNA Acylation Tool Facilitating Genetic Code Reprogramming Methods. Mol. Biol. 201297 Flexizymes: Their Evolutionary History and Diverse Utilities Top. Curr. Chem. 201498 tRNA engineering for manipulating genetic code RNA Biol. 201899 Ribosomal synthesis of backbone macrocyclic peptides Chem. Commun. 2011100 Reprogramming the genetic code in vitro Trends. Biochem. Sci. 2014101 Discovering functional, nonproteogenic amino acid containing, peptides using genetic code reprogramming Org. Biomol. Chem. 2015102 Recent Developments of Engineered Translational Machineries for the Incorporation of Non-Canonical Amino Int. J. Mol. Sci. 2015103 Acids into Polypeptides Ribosome-mediated synthesis of natural product-like peptides via cell-free translation Curr. Opin. Chem. Biol. 2016104 Charging of tRNAs Using Ribozymes and Selection of Cyclic Peptides Containing Thioethers Methods Mol. Biol. 2012105 Flexizyme-Mediated Genetic Reprogramming As a Tool for Noncanonical Peptide Synthesis and Drug Discovery Chem. Eur. J. 2013106 Technologies for the Synthesis of mRNA-Encoding Libraries and Discovery of Bioactive Natural Product-Inspired Molecules 2013107 Non-Traditional Macrocyclic Peptides A RaPID way to discover nonstandard macrocyclic peptide modulators of drug targets Chem. Commun. 2017108 others31,109−115 Strategies for transitioning macrocyclic peptides to cell-permeable drug leads Curr. Opin. Biotechnol. 201713 Protein cocrystallization molecules originating from in vitro selected macrocyclic peptides Curr. Opin. Struct. Biol. 2014116 Selection-Based Discovery of Druglike Macrocyclic Peptides

development of the FIT system (section 3) and several case studies on the selection of bioactive macrocycles (section 4), which are distinctive in structure and/or function. Other technical advances and remaining challenges are discussed in this review (sections 5 and 6).

mRNA-protein adducts, how well the peptides are expressed, biased PCR amplification, and the initial abundance of sequences in the first selection round mRNA library.89 Also, using mRNA display as an in vitro method, it may be difficult to screen proteins that require post-translational modifications.90 In vitro display methods furthermore require clean working environments and clean handling techniques to give an RNase-free environment to prevent degradation of the mRNA library during selections.91 Previous obstacles of in vitro display systems, for example, the screening of difficult targets such as membrane proteins due to their limited expression and misfolding when expressed in vitro, have now become possible through the use of membrane protein-embedded nanodiscs.90

2. HISTORICAL PERSPECTIVE: BIRTH AND GROWTH OF MRNA DISPLAY 2.1. mRNA Display of Canonical Linear Peptides

Ribosome display systems suffered from the instability of the ternary complex of ribosome, mRNA, and peptide. A breakthrough discovery in 1997 by two separate groups, Szostak and Roberts in addition to Yanagawa and co-workers, involves the use of puromycin as opposed to chloramphenicol to stall translation. This resulted in the discovery of mRNA display, in which the mRNA is covalently and directly linked to its translated peptide through a puromycin linker (Table 3 v1.0).81,82 The antibiotic puromycin is a mimetic of the aminoacyl end of a tRNA molecule, and it prevents peptide chain elongation.117 Puromycin also releases the peptide from the ribosome, and it is incorporated into the C terminus of the translated peptide.118 Despite the similarities between acylated tRNA and puromycin there are some key functional differences (Figure 3A and 3B). During peptide bond formation the amino acid molecule in AA-tRNA undergoes rapid acyl migration, whereas the tyrosyl group in puromycin is inert due to the amide bond at the 3′ position of the nucleoside (Figure

1.6. Review Outline

Dozens of papers around RNA display have been published, which cover topics ranging from methodology development to application examples. This review is up to date as of August 2018. Table 2 provides a list of selected key reviews on mRNA display since 2010. General reviews of mRNA display-based selection of peptides as well as the development of the flexible in vitro translation (FIT) system and the random nonstandard peptide integrated discovery (RaPID) system are included. Readers are encouraged to choose topics of interest for further reading. This review focuses on the basic principles of mRNA display and its derived RaPID system (section 2) with the H


3C).118 Yanagawa and co-workers refer to their technique as the “in vitro virus”.82 The Szostak and Yanagawa groups for the first time highlight the synthesis of a covalent fusion between a strand of mRNA and its linear canonical translated peptide via a puromycin linker, allowing subsequent mRNA display affinity selections. The Szostak group applied their methodology to the enrichment of myc epitope peptides from a library of random sequences by immunoprecipitation.81 Up to then, the main issue with the development of a robust system for affinity selections was the development of a method that would allow the recovery of the sequence information after the peptides or ligands have been translated and selected successfully. Such a method would require the translated peptide to be attached to its coding sequence. This was shown to be possible in phage display, which has the disadvantage of being limited to less than 109 library members due to transfection efficiencies.81 An alternative method could be ribosome display, in which translated peptide sequences are displayed on the surface of the ribosome. This method, however, suffers from the indispensable drawback that selection conditions have to be used that guarantee the integrity of the ribosome-mRNApeptide complex. Covalent linkage of the mRNA to its translated peptide is in theory possible by using an mRNA template with a charged tRNA molecule at its 3′-end, but it would consist of an unstable aminoacyl ester. The Szostak and Yanagawa groups recognized that this problem could be solved by the use of puromycin. When puromycin is linked to the 3′end of the mRNA template, it can enter the ribosomal A site and subsequently be transferred onto the C-terminal end of the translated peptide by the peptidyl transferase activity of the ribosome resulting in the formation of a stable amide bond.81 It was confirmed that the attachment of the mRNA-puromycin linker to the translated peptide was due to the peptidyl transferase activity of the ribosome by adding specific inhibitors of peptidyl transferase as well as translocation inhibitors.81 Further evidence comes from pull-down experiments of a translated myc peptide.81 Yanagawa and co-workers showed that peptides are especially well translated and linked to their mRNA strand via the puromycin linker in the absence of a stop codon but in the presence of a DNA spacer. They hypothesized that upon reaching the DNA spacer during translation the ribosome would pause for long enough, allowing the puromycin to enter the A site of the ribosome to be transferred onto the translated peptide.82 Furthermore, no cross-transfer of the translated peptides was observed during the cotranslation of different template sequences.81 This important discovery shows that it is possible to enrich for a certain peptide using a randomized pool of mRNA sequences in combination with antibody immunoprecipitation.81,82

first example of TRAP display (one-pot transcription/noncovalent puromycin linker conjugation/translation allows faster selections)

macrocyclic peptides with spontaneous thioether cyclization, contain d-amino acids and backbone n-methylation macrocyclic peptides containing a warhead residue macrocyclic peptides v3.0

2.2. Optimization of Puromycin Linker Ligation

Before mRNA display could become a more broadly used technique, the synthesis of the mRNA-puromycin conjugate had to be improved. Previously, the preparation required a roughly 200-fold molar excess of the costly puromycin-linker DNA molecule with respect to the library mRNA due to a very poor ligation efficiency.119 To overcome this issue, different methods such as splint ligation, photo-cross-linking ligation, and the Y-ligation method have been developed (Figure 4). Photo-cross-linking of the puromycin-DNA linker to the mRNA template is fast and efficient with yields consistently exceeding 80%, but it suffers from the drawback that mRNA

v3.2

linear peptides v2.2

v3.1

macrocyclic peptides with thioether cyclization v2.1

first example of tailor-made warhead library

Suga lab Angew. Chem. Int. Ed. 201276 Murakami lab J. Am. Chem. Soc. 201323

for TRAP selection with highly diverse libraries: see Murakami lab ACS Chem. Biol. 2013167

for FIT system (wPURE system + Flexizyme-mediated aminoacylation of tRNA), see: Suga lab Nat. Methods 2006;164 Suga lab ACS Chem. Biol. 2008;173 Suga lab Chem. Biol. 200875

linear peptides containing npAAs v2.0

first example of high-throughput binding kinetics measurement by MiSeq sequencing first example of the RaPID system (mRNA display + FIT system)

Szostak lab J. Am. Chem. Soc. 2012a;151 Szostak lab J. Am. Chem. Soc. 2012b25 Roberts lab Angew. Chem. Int. Ed. 2016155 Suga lab Chem. Biol. 2011172

disulfide-rich peptides v1.1

first example of cDNA display (covalent mRNA-cDNA-protein ternary complex), which is derived from traditional mRNA display (covalent mRNA-protein fusion) first example demonstrating the compatibility of mRNA display with the wPURE system (customizable PURE system allowing genetic code reprogramming for the translation of noncanonical amino acids) representative examples of the incorporation of post-translational enzymatic or chemical modifications

Szostak lab J. Am. Chem. Soc. 2005171

for examples of cDNA display, see: Nemoto lab ACS Comb. Sci. 2011;168 Sakai lab Proc. Natl. Acad. Sci. 2012;169 Nemoto lab ACS Omega 2016170 for PURE system (protein synthesis using recombinant elements system), see: Ueda lab Nat. Biotechnol. 2001148

related documents key references

Szostak lab Proc. Natl. Acad. Sci. 1997,81 Yanagawa lab FEBS Lett. 199782 Nemoto lab Nucleic Acid Res. 2009123

comments and definition

first two examples of mRNA display (in vitro reconstituted translation system to synthesize mRNA-fused peptide libraries for selection) linear peptides

displayed structures

Review

v1.0

version

Table 3. Major Technical Advancements of mRNA Display in Its 20 Year History

for phage display and ribosome display, see: Smith lab Science 1985,68 Pluckthun lab Proc. Natl. Acad. Sci. 199771

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Figure 4. Summary of techniques for the attachment of the puromycin linker for mRNA display. (A) Ligation by UV-cross-linking using a reactive group. (B) Ligation by splint ligation using a strand of DNA complementary to both mRNA and puromycin linker sequence. (C) Y-ligation: More commonly used technique nowadays. (D) TRAP system: Added puromycin linker anneals to the complementary portion of the mRNA. Upon reaching a stop codon during translation puromycin enters the ribosomal A site and is covalently attached to the translated peptide. TRAP method results in a noncovalent complex between the translated peptide and the mRNA tag.

strands, a primer region for reverse transcription to form the covalent cDNA/mRNA fusions, a biotin moiety for the attachment of the conjugates to streptavidin-coated magnetic beads, and a PvuII restriction site for final release of the molecules. The developed system results in the cDNA being covalently linked to the translated peptide, which also allows expression and refolding of disulfide-rich proteins under different conditions, while the molecules are immobilized via their biotin tag. The release of the final peptide-mRNA/cDNA construct is achieved by enzymatic cleavage.123 The mRNA tag can then be digested with RNase H to convert from an mRNA display to a cDNA display system.123 This method was applied successfully to the discovery of novel peptides containing multiple disulfide bonds targeting the Interleukin-6 receptor (IL-6R).123

degradation can occur due to the UV irradiation used for ligation (Figure 4A).120 A disadvantage of splint ligation is that it requires a denaturing gel purification step in order to separate the remaining splint-oligo DNA from the successfully ligated mRNA template (Figure 4B).121 The third method involves the use of T4 RNA ligase, which can join complementary RNA and DNA strands using ATP. This method is known as the Y-ligation method and is favored due to it being fast (∼1 h) and proceeding under mild reaction conditions at 25 °C (Figure 4C).122 A fourth method has also been developed, which is referred to as the TRAP system (Figure 4D). A key difference is that the TRAP system results in noncovalent base pairing of the mRNA to the puromycin linker.123,124 2.3. Use of Covalent mRNA-cDNA-Peptide Fusions

A further improvement of the mRNA display technology involves the stabilization of the mRNA coding tag by conversion into the respective mRNA/cDNA dimer by reverse transcription (Table 3 v1.1).123 Due to the labile nature of the mRNA tag, mRNA display up to that point had been considered challenging, in particular for applications involving cell-based systems, due to the ribonuclease activity of the cells.123 Furthermore, conversion of the mRNA tag into the mRNA/cDNA fusion minimizes unwanted interactions of the mRNA due to secondary structure formation with the selection target.123 The puromycin-linker DNA developed by Nemoto and co-workers consists of a ligation site for the mRNA

2.4. Translation of Peptides Containing Noncanonical Amino Acids

In 2005, Szostak and co-workers used a reconstituted E. coli translation system to synthesize a single peptide containing 10 different unnatural amino acids. Instead of only using the natural genetically encoded AA/tRNA pairs (Figure 5A), using genetic code reprogramming, 35 of the 61 sense codons could be reassigned to 12 unnatural amino acid analogues (Figure 5B).125 Importantly, the developed system is compatible with mRNA display, allowing the selection of vast libraries of noncanonical peptides. J


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Figure 5. Various methods have been developed for the synthesis of aminoacylated tRNAs. (A) Natural aaRS-catalyzed synthesis of natural aminoacylated tRNA. Only refers to proteogenic amino acids. (B) RNA ligase-catalyzed synthesis of unnatural aminoacylated tRNA. (C) Natural aaRS-catalyzed synthesis of unnatural aminoacylated tRNA. Only suitable for proteogenic amino acid mimetics. (D) Evolved aaRS-catalyzed synthesis of unnatural aminoacylated tRNA. Limited to Tyr and Pyl mimetics (pyrrolysine). (E) Flexizyme-catalyzed synthesis of unnatural aminoacylated tRNA. Amino acids and their respective leaving groups have to be paired with the appropriate flexizyme.

which is incapable of synthesizing a particular amino acid or that has been starved of the natural amino acid. The bacterial host has then been shown to incorporate a chemically similar amino acid provided in the culture media instead.140−143 In vitro, this has been achieved using chemically charged tRNAs, demonstrating that the ribosome can be used for the synthesis of noncanonical combinatorial libraries.144,145 A further option involves the chemical modification of canonical charged tRNAs allowing the incorporation of α-hydroxy acids and Nmethylated amino acids.146,147 The field of genetic code reprogramming was advanced significantly by the development of fully reconstituted translation systems using highly purified E. coli ribosomes and His-tagged translation factors. The system is referred to as the Protein synthesis Using Recombinant Elements (PURE) system. It contains 32 components, which are all purified individually. These include initiation factors (IF1, IF2, IF3), elongation factors (EF-G, EF-Tu, EF-Ts), release factors (RF1, RF3), ribosome recycling factor (RRF), all 20 aaRSs, methionyl-tRNA transformylase (MTF), T7 RNA polymerase, and purified E. coli ribosomes. Furthermore, the system contains 46 tRNAs, NTPs, creatine phosphate, 10-formyl5,6,7,8-tetrahydrofolic acid, all canonical 20 amino acids, creatine kinase, myokinase, nucleoside-diphosphate kinase, and pyrophosphate.148,149 The advantage of the system is that there is full control over all included components of the translation machinery, such as tRNAs, amino acids, aaRSs, and release factors. This allows the simultaneous reprogramming of several amino acids by the omission of certain amino acids and the corresponding aaRSs and instead adding chemically charged tRNAs. Forster et al. showed that using this system it is

Up to then, the most common approach for the incorporation of noncanonical amino acids was via the use of the amber stop codon (UAG) to include one additional unnatural amino acid in addition to the canonical 20 amino acids. In this approach, the noncanonical amino acid is chemically attached to an amber suppressor-tRNA molecule via an ester bond using T4 ligase (Figure 5C).126 Translation is achieved using an in vitro translation system obtained from cellular extracts.127,128 This approach demonstrated that the ribosome is capable of translating a wide variety of noncanonical amino acids, including amino acids with bulky side chains and N-methyl amino acids.129−131 Schultz and co-workers showed that it is even possible to use this technique for the in vivo incorporation of noncanonical amino acids when a stop codon is encountered. They used orthogonal pairs (absent in the host E. coli strain but function efficiently in translation) of suppressor-tRNAs and mutant aminoacyl-tRNA-synthetases (aaRSs) (Figure 5D).132,133 Unfortunately, both of these nonsense suppression approaches suffer from the significant drawback that only a maximum of two nonsense codons may be reassigned to two different noncanonical amino acids since one stop codon must remain intact for functional translation termination. Two alternative approaches have been developed to overcome this limitation. The first approach involves the expansion of the genetic code by using four- and five-residue codons or by using three-residue codons which contain unnatural base pairs.134−139 As opposed to the expansion of the genetic code, the second approach involves the reassignment of sense codons to noncanonical amino acids (ncAA). In vivo, this has been accomplished by using a bacterial host, K


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Table 4. Summary of Representative Amino Acids and Their Derivatives Which Have Been Incorporated via the FIT System index

representative amino acids

aminoacylation leaving group

features

ref

1 2 3 4

p-benzoylphenylalanine glycine ε-N-biotinyl-lysine glycolic acid

CME DBE/CME/CBT DBE DBE/CME

Phe derivatives proteinogenic 20 aa biotinylated amino acids α-hydroxy acids

5

D-alanine

DBE/CME/CBT

D-stereo

6

4-(2-chloroacetyl)aminobutyric acid L-azidohomoalanine propargylglycine N-alkyl glycines N-acetylated lysine N-chloroacetylated phenylalanine lysine (ε-N-methylation/acetylation) N-methylated alanine 2-amino-3-(octanamido)propanoic acid D-Phe-L-Phe dipeptide 2-mercaptoethanol-homocysteine 5-hydroxy-tryptophan 4-aminomethyl-phenylalanine L-vinylglycine

DBE

side-chain chloroacetyl amino acids

Murakami et al. Chem. Biol. 2003177 Murakami et al. Nat. Methods 2006164 Murakami et al. Nat. Methods 2006164 Murakami et al. Nat. Methods 2006;164 Ohta et al. Chem. Biol. 2008174 Murakami et al. Nat. Methods 2006;164 Goto et al. RNA 2008212 Sako et al. J. Am. Chem. Soc. 2008200

DBE DBE DBE DBE CME DBE

side-chain azide amino acids side-chain alkyne amino acids peptoid building block N-acylation amino acids N-acylation amino acids Lys with side chain modifications

Sako et al. J. Am. Chem. Soc. 2008200 Sako et al. J. Am. Chem. Soc. 2008200 Kawakami et al. J. Am. Chem. Soc. 2008213 Goto et al. ACS Chem. Biol. 2008173 Goto et al. ACS Chem. Biol. 2008173 Kang et al. Chem. Biol. 2008214

DBE/CME/CBT ABT

N-methylated amino acids very hydrophobic amino acids

Kawakami et al. Chem. Biol. 200875 Niwa et al. Bioorg. Med. Chem. Lett. 2009163

CME/CBT DBE CME CME DBE

Goto et al. J. Am. Chem. Soc. 2009215 Nakajima et al. ChemBioChem 2009205 Yamagishi et al. ChemBioChem 2009204 Yamagishi et al. ChemBioChem 2009204 Goto et al. Chem. Commun. 2009198

γ-amino acid-phenylalanine ε-N-trifluoroacetyl-L-lysine L-thiazolidine-4-carboxylic acid N-acetyl-S-12-(ClAc)farnesyl cysteine N-(2-azidoethyl)-glycine N-(2-carboxylethyl)-glycine N-(5-carboxyfluorescein)L-phenylalanine β-homoglycine 4-(chloromethyl)benzoic acid 2-aminoisobutyric acid 3,4-dihydroxy-L-phenylalanine

CME DBE DBE/CME CME

exotic peptides masked thio side chain amino acids precursor amino acids for oxidative coupling precursor amino acids for oxidative coupling precursor amino acids of dehydrobutyrine for Michael addition diverse N-terminal structures warhead amino acids Pro-like N-alkylated cyclic amino acids amino acid with fatty acid side chain

DBE DBE CME

masked positively charged amino acids masked negatively charged amino acids fluorescent amino acids

Murakami et al. Chem. Sci. 2014210 Murakami et al. Chem. Sci. 2014210 Terasaka et al. Nat. Chem. Biol. 2014218

DBE CME DBE CME

Murakami et al. J. Am. Chem. Soc. 2016219 Kawakami et al. ACS Chem. Biol. 2016220 Katoh et al. Nucleic Acid Res. 2017182 Jongkees et al. Cell Chem. Biol. 2017221

quinoline/pyridine-based glycyl phenylalanine

CME

β-amino acids chloromethylbenzene α,α-disubstituted α-amino acids amino acids containing an o-dihydroxyphenyl ring foldamer oligomers

7 8 9 10a 11a 12 13 14 15a 16 17 18 19 20a 21 22 23 24 25 26a 27 28a 29 30 31a

19 amino acids

Ohshiro et al. ChemBioChem 2011209 Morimoto et al. Angew. Chem., Int. Ed. 201276 Kawakami et al. J. Am. Chem. Soc. 2013216 Torikai et al. J. Am. Chem. Soc. 2014217

Rogers et al. Nat. Chem. 2018222

a

Incorporation at initiator position only.

v2.2).155 This method combines high-throughput sequencing with mRNA display to characterize kon and koff rates of thousands of peptides simultaneously. First, starting with the enriched pool of a PCR-amplified DNA library that encodes the active peptide binders, the authors performed the routine transcription, puromycin ligation, translation, and reversetranscription to obtain the cDNA-attached peptide library. Second, the resulting library was mixed with target proteinimmobilized magnetic beads. Next, at certain time points, bead aliquots were taken, washed, and PCR amplified for sequencing. The hypothesis was that sequences with high kon rates should result in a higher sequencing recovery compared with an equivalent sample taken before incubation with the immobilized protein. This method was referred to as the highthroughput sequencing kinetics (HTSK) method. This semiquantification strategy was confirmed to be valid by an experiment in which the authors compared koff rates deducted from HTSK versus koff rates directly calculated from translated radiolabeled peptides. Overall, the HTSK method provides a

possible to simultaneously reassign three sense codons to noncanonical amino acids.150 Szostak and co-workers showed in 2005 that their method of genetic code reprogramming and in vitro translation is compatible with mRNA display (Table 3 v2.0).125 The diversity of the library that can be screened using this technology is limited only by the feasibility of handling the associated translation volume due to the single turnover of the ribosome in the presence of the puromycin linker. This method was further developed to incorporate post-translational enzymatic and/or chemical modifications to obtain versatile peptide libraries with unique structural features (Table 3 v2.1).25,151 By combining the PURE system, mRNA display, and sense codon reassignment, 152−154 dozens of pAA analogues have been incorporated for peptide binder selections. Very recently, Roberts and co-workers developed a new high-throughput method to determine binding affinities of ligand peptides selected out from mRNA display (Table 3 L


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Figure 6. Library construction and selection of mRNA display. (A) Linear peptide library for mRNA display can be generated using cell lysates containing ribosomes and all necessary components for peptide translation. (B) Cyclic peptide libraries can be prepared using the FIT system. Flexizyme-catalyzed aminoacylation to give charged tRNAs in combination with recombinantly expressed and purified translation components allow genetic code reprogramming and tailored peptide translation to give cyclic peptide mRNA display libraries. (C) Classic mRNA display selection cycle. RaPID system allows the synthesis of cyclized peptide libraries. cDNA of recovered peptide binders is PCR amplified, and iterative rounds of RaPID selection result in the enrichment of peptide binders, which are then identified by sequencing.

further randomization evolution to obtain a new ribozyme with lower substrate selectivity but high aminoacylation activity.159−161 A flexizyme prototype, Fx3, was discovered subsequently, which accepted aromatic amino acid activated with a CME leaving group and could aminoacylate multiple tRNAs.162 Through applying new selection pressure by changing the amino acid substrate structures, Suga et al. later successfully identified several second-generation flexizyme families, i.e., dinitro-flexizyme (dFx) with 46 nucleotides, enhanced flexizyme (eFx) with 45 nucleotides, and aminoflexizyme (aFx) with 47 nucleotides.157,162,163 With these three flexizymes, all tRNAs with a 3′-terminal CCA motif are able to undergo aminoacylation if provided with a suitable amino acid substrate. Due to different evolution pressures applied during the selection experiments, eFx favors CME-activated amino acids containing an aromatic group or bulky hydrophobic residue close to the carbonyl site or 4-chlorobenzyl thioester (CBT)-activated general amino acids.153 dFx prefers 3,5dinitrobenzyl ester (DBE)-activated general amino acid substrates,148 and aFx prefers 2-(aminoethyl)amidocarboxybenzyl thioester (ABT)-activated hydrophobic amino acids.154 Due to the different substrate preferences of eFx, dFx, and aFx, it is important to match the appropriate flexizyme partner with the desired amino acid for tRNA charging and incorporation into translated peptides using the PURE system. Up to now, hundreds of amino acids including β-amino acids, D-amino acids, and N-alkylated amino acids have been charged onto tRNAs using flexizymes. Some nonamino acids such as α-hydroxy acids, benzyl acids, and oligopeptides and foldamer structures are also well tolerated by flexizymes. The powerful flexible nature of the flexizymes’ substrate selectivity is summarized in Table 4. Recent

practical approach for the quick identification of very-highaffinity sequences as well as of false-positive DNA clones instead of having to perform labor-intensive low-throughput measurements. 2.5. Flexible in Vitro Translation (FIT) System

Although the methods described above by Szostak et al. have significantly extended the versatility of peptide libraries used in mRNA display, the efficiency and fidelity of peptide synthesis in this system is highly dependent on the substrate promiscuity of recombinant aaRS toward ncAAs.125 Insufficient aminoacylation may limit the diversity of the unnatural residue pool (currently up to 90 unnatural building block), which can be used for library construction.156 Another method to prepare tRNAs aminoacylated with ncAAs was reported by Suga and co-workers in 2006.157 The authors discovered a ribozyme aaRS (45 nucleotides), which was referred to as a flexizyme (flexible ribozyme), to perform the aminoacylation (Figure 5E). The name is based on the flexible, broad substrate scope of amino acids as well as the variety of uncharged tRNAs that can be aminoacylated. An early report demonstrated that a robust ribozyme, identified through an in vitro RNA selection, could catalyze the aminoacylation of tRNA using an amino acid substrate, Nbiotinylated-Phe-cyanomethyl ester (CME).158 This ribozyme, called r24 (90 nucleotides), displayed cis activity since it could aminoacylate itself. Further structural characterization of r24 determined that a truncated r24 mini (57 nucleotides) did not lose its catalytic capacity and showed trans aminoacylation selectivity to charge L-Phe onto a tRNA. This r24 family ribozyme clearly showed that the 5′-leader sequence of the tRNA could aminoacylate the 3′-tail of a tRNA with a specific amino acid. On the basis of this finding, r24 mini was used for M


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Figure 7. (A) Genetic codon table of native peptide translation. Codon redundancy means that 64 distinct codons only encode 20 proteogenic amino acids and three stop codons. (B) Codon redundancy is reduced by only considering NNS codons (where N = A/U/G/C and S = G/C), which results in 32 codons encoding all 20 proteogenic amino acids and only one stop codon. There are several amino acid families (highlighted in light gray) that are decoded by different tRNAs either by Watson−Crick base-pairing or by wobble base pairings. (C) In this example, the Val, Ser, and Gly codon boxes are divided artificially by excluding tRNAGlyGCC, tRNAArgGCG, and tRNAValGAC from the translation reaction but adding the respective three nonproteogenic-tRNAAsnE2GNNs, prepared by flexizyme-catalyzed aminoacylation. This results in 29 codons encoding all 20 proteogenic amino acids in addition to the insertion of three nonproteogenic amino acids. (D) Iwane et al. showed that two different peptides could be translated based on the specific FIT system used. With the FIT-32t system, native translation gave an unmodified peptide. Using the FIT29t system, three nonproteogenic amino acids were incorporated correctly.175

technological advancements of the FIT system are illustrated in section 3.

To streamline mRNA display, Murakami et al. developed an updated version of the RaPID system, referred to as the transcription-translation coupled with association of puromycin linker (TRAP) system (Table 3 v3.2, Figure 4D).85 In this process, four key steps in each round of selection, i.e., transcription, puromycin-linker ligation, translation, and puromycin-peptide conjugation are highly integrated to proceed continuously in a one-pot fashion, a protocol that can accelerate a typical RaPID selection to less than 3 h per round. Key to this streamlined process is the replacement of the T4-mediated puromycin ligation step with the mRNA library with a noncovalent base-pairing step of the Pu-linker instead. The authors confirmed the stability of this complex and showed that it can survive the subsequent selection cycles. Nanomolar binding macrocyclic peptides were identified by using this TRAP display protocol.23,167

2.6. mRNA Display Based on the RaPID System

With the development of the PURE system148 and the FIT system164 in the 2000s, an updated mRNA display platform was established by Suga and co-workers, who called it the RaPID (random nonstandard peptide integrated discovery) system (Table 3 v3.0, Figure 6). The essential advancement of the RaPID system is that certain natural aminoacyl-tRNAs can be excluded from the in vitro translation mixture by removing their cognate natural amino acids and/or natural aminoacyl tRNA synthetases. The evacuated mRNA codon box can then be refilled with unnatural aminoacyl-tRNAs, which are prepared through flexizyme-catalyzed tRNA charging with various nonstandard amino acids. In this manner, PURE components can be combined in a tailored fashion to generate peptide libraries with distinctive structural features for mRNA display. These libraries can contain D-amino acids, Nmethylated amino acids, and also warhead-embedded (Table 3 v3.1) linear or cyclic peptide libraries may be constructed. Importantly, many of these chemical features are also found in natural product macrocyclic peptides such as cyclosporine A.165 In this context, the RaPID system is an appealing platform, which allows the diversity-oriented selection of bioactive natural-product-like peptides with high affinities. Importantly, RaPID selections have been successfully applied to the discovery of selective and potent inhibitors against several previously undruggable protein targets such as the AKT protein.166 In the case of the AKT protein other screening or selection platforms did not yield any high-affinity binders.166

3. TOOLBOX OF THE FIT SYSTEM 3.1. Genetic Code Reprogramming

Although organisms only use 22 naturally occurring amino acids (20 basic natural amino acids in addition to selenocysteine and pyrrolysine), usually termed canonical or proteogenic amino acids (pAAs) to construct peptides and proteins, extensive efforts have been made to extend the ribosome-based translation machinery to incorporate nonproteogenic residues. While genetic code expansion through stop codon UAG suppression or canonical amino acid analogue replacement has been widely used for in vivo engineering of translation, genetic code reprogramming in vitro through adjustment of translation components such as amino acids, aaRSs, RFs, and EFs allows precise control of the N


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Figure 8. Post-translational enzymatic and chemical modification. (A) Chemoenzymatic backbone amide cyclization through NCL mechanism in which a PDF/MAP enzyme pair is used to free an N-terminal cysteine and a CPE motif is used to form a C-terminal thioester.184 (B) Chemical side-chain-to-side-chain cyclization through CuAAC reaction between side-chain azide and alkyne groups.200 (C) Chemical tricyclization through triple thioether formation by using a TBMB scaffold.195 (D) Thioether formation through spontaneous cyclization.173 (E) Chemical/enzymatic decaging with TCEP and carboxylesterase to allow the incorporation of N-methylated charged amino acids.210 (F) Enzymatic cyclodehydration by using an enzyme cascade consisting of PatD, GodD, and GodE catalysis.190

stituted while tRNAArgCCG and tRNAGlyCCC remained. Therefore, the revised codon box table contains 29 tRNA transcripts and 3 Naa-charged tRNAs, which allows the incorporation of “20 + 3” amino acids (Figure 7C). Various model peptides were successfully translated using the new set of genetic codes, and no codon-crossover contamination was observed (Figure 7D). In principle, the FIT-32t system enables the relocation of 11 vacant codon boxes to reach the maximum sequence diversity of in vitro translation. Another issue of the incorporation of npAAs is how to achieve high translational efficiency and low interference. Three types of orthogonal tRNAs, tRNAAsnE2, tRNAGluE2, and tRNAProE2, have been used in the FIT system. To ensure high translational fidelity, the npAA-charged orthogonal tRNA should not be recognized by any endogenous aaRSs. In this regard, tRNAAsnE2 was first developed to be inert to E. coli aaRSs by introducing several mutations in the tRNA acceptor stem.176,177 In the Suga lab, this artificial tRNA was successfully used to incorporate non-natural L-amino acids, D-amino acids, N-alkyl amino acids, and β-amino acids. However, the translation yield of peptides containing npAAs such as D-amino acids and β-amino acids is sometimes too low to find practical applications in mRNA display. Certain bulky N-methyl amino acids and negatively charged amino acids are also poor substrates for the ribosome. One way to improve this is to modify the structure of the tRNA to have a higher affinity toward EF-Tu, which is responsible for accommodating the charged tRNA in the A site of the ribosome. Inspired by a previous report that L-Val-tRNAGlu has a higher affinity than LVal-tRNAAsn toward EF-Tu,178 Katoh et al. designed a secondgeneration artificial tRNAGluE2 which is superior to tRNAAsnE2 for the incorporation of D-amino acids.179 By optimizing the concentration of translation factors such as elongation factor

translation process. As described in the previous sections, the flexizyme-supported FIT system in combination with the reconstituted in vitro PURE translation system can harness the ribosome to produce npAA-incorporated peptides and even artificial polymers such as polyesters.174 Although this platform technology has demonstrated convincing efficiency in conjunction with mRNA display, a potential drawback is that some pAAs have to be sacrificed by replacing them with npAAs, which limits the chemical diversity of mRNA display libraries. Thus, an ideal codon box table is anticipated to hold multiple npAAs in addition to the 20 pAAs and allow their incorporation (partial or all) into a single peptide sequence. Due to the fact that 20 pAAs correspond to 61 sense codons, which are decoded by 45 native tRNAs in the E. coli translation machinery (Figure 7A), a tricky question is how to partially release the redundancy of tRNAs to permit addition of orthogonal flexizyme-charged aa-tRNAs and finally facilitate translation with high fidelity. To answer this, in 2016, Iwane and co-workers developed an elegant strategy called the “artificial division of codon boxes” as depicted in Figure 7.175 Instead of simple amino acid exchange, a redundant tRNA of a specific amino acid was replaced with a precharged tRNAAsnE2NNN. The specific proteogenic amino acid and other tRNAs corresponding to it were kept in the translation mixture. In this manner, a free codon box was specifically created for the npAA by reducing tRNA redundancy. In the simplified FIT-32t system, 32 tRNA transcripts cover NNS (S = G or C) codons, which can decode all of the 20 pAAs (each pAA → one to three codons) (Figure 7B). Next, three codon boxes were artificially divided. For the Val codon box, for example, tRNAValGAC was replaced with Naa-charged tRNAEnAsnGAC and tRNAValCAC was kept untouched. Similarly, tRNAArgGCG and tRNAGlyGCC were subO


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PatD could also be premixed with translation components to give similar results. To investigate the substrate tolerance of PatD mutation studies of the core motif and flanking sequences were conducted. This recently developed methodology may give rise to the preparation and selection of peptides with improved pharmacokinetic properties such as passive cell permeability. In a second work to investigate the biosynthetic machinery of ribosomally synthesized and post-translationally modified peptides (RiPPs),189 a new in vitro translation system, referred to as the FIT-GS system, was established to express the native sequence and derivatives of goadsporin (GS), a member of RiPPs containing multiple azoles and dehydroalanines in its backbone chain.190 In this system, enzymes originated from the biosynthetic cluster of GS were added to the translation mixture. Enzymes GodD and GodE produced unsaturated thiazole, oxazole, and methyloxazole from Cys, Ser, and Thr residues, respectively. Ser-to-Dha conversion was then observed through catalysis of GodF and LazF (a stable analogue of GodG). Finally, treatment with GluC and GodH resulted in the target compound, the native GS peptide. Similarly, over 50 analogues of GS could be expressed with the FIT-GS system to further investigate the mechanism of posttranslational modifications of GS.190

Tu (EF-Tu), EF-G, and IF2, a peptide with up to 10 consecutive D-Ser could be translated. It should be noted that similar strategies aiming for higher binding affinity between npAA-tRNA and EF-Tu were also developed by other groups.180,181 To further enhance the incorporation efficiency of D-amino acids, Katoh and co-workers devised tRNAPro1E2, in which the T-stem of tRNAPro1 was replaced with that of tRNAGluE2.182 In this chimera tRNAPro1E2, the grafted T-stem should promote accommodation by EF-Tu onto the P site of the ribosome while the original D-arm can facilitate peptidyl transfer from the A site to the P site. An up to 9.5-fold improvement of consecutive D-amino acids incorporation was observed by changing tRNAGluE2 to tRNAPro1E2, which verified this hypothesis. Thus, multiple incorporations of D-amino acids were found to be enhanced with the help of elongation factors EF-P and EF-Tu as well as the new tRNA constructs. Very recently, orthogonal tRNAs were engineered for enhanced binding affinity to EF-Tu (via T-stem) and EF-P (via D-arm). By integration into the FIT system with optimized translation factor concentrations, up to seven consecutive β-amino acids residues were successfully translated in vitro.183 This deeply optimized and tailored FIT system should enable RaPID selections of macrocyclic peptides containing certain D-amino acids and/or β-amino acids, which are quite well tolerated by the ribosome. 3.2. Post-Translational Enzymatic Modification

3.3. Post-Translational Chemical and Chemo-Enzymatic Modification

3.2.1. N-Terminal Modification. In an effort to synthesize backbone-cyclized peptides with ribosomes in vitro, the Suga lab developed a backbone-cyclic peptide synthesis using recombinant elements (bcPURE) system.184 A key element of this system is the generation of a free N-terminus for cyclization because bacterial ribosomal expression normally starts with a formylated methionine at the N-terminus. It has been reported that peptide deformylase (PDF) can remove the formyl group selectively and that methionine aminopeptidase (MAP) can cleave off Met to release the N-terminus of the second N-terminal residue (Figure 8). To test the compatibility of these two enzymes with the PURE system, the authors succeeded to use PDF or PDF/MAP to release 14 kinds of amino acids. For the PDF manipulation, the initiator tRNA is charged with Met intact or reprogrammed to be charged with another natural amino acid. After translation and spontaneous formylation through formyltransferase (MTF),173 the Nterminus can then be exposed by one-step deformylation using PDF. Seven amino acids (Phe, Leu, Tyr, Ile, Gln, Trp, and Met) can be accessed in this way.184 For the PDF/MAP manipulation, formylmethionine is removed by consequential deformylation and demethionylation to expose additional seven amino acids (Gly, Ala, Pro, Ser, Thr, Val, and Cys). 3.2.2. Backbone Modification. Heterocycles are distributed widely among natural product peptides. With the help of enzymes such as cyclodehydratases, Cys, Ser, and Thr residues can undergo dehydration to give thiazoline, oxazoline, and methyloxazoline structures.185,186 Further derivatives can be obtained from these azoline backbones through enzymatic or chemical processes (Figure 8).187 Goto et al. utilized a cyclodehydratase, PatD, and integrated it into the FIT system.188 This FIT-PatD system enabled a one-pot generation of azoline-peptide derivatives. The authors first demonstrated that a close-to-native substrate of PatD, termed PatE, could be transformed in the FIT system. By incubation with PatD at 25 °C overnight, cleanly converted product could be detected.

3.3.1. Thioether-Based Cyclization. To further increase the diversity of in vitro-translated peptides, versatile chemical modification methods have been developed.104 To date, headto-side chain thioether cyclization mediated by an N-terminal 2-chloroacetyl group and a Cys side chain thiol group has been the most successful method used in combination with the RaPID system (Figure 8).173 A critical advantage of this method is that intramolecular cyclization can happen spontaneously during translation and proceed with quantitative yield (essentially no intermolecular polymerization is observed), eliminating the need for further chemical additives.173 Kinetics of the intramolecular reaction between the chloroacetyl group and the thiol group proved to be highly compatible with the FIT system when using micromolar concentrations in aqueous buffer at 37 °C. Similar thioether-based peptide cyclization methods have also been tested by several other groups.191,192 Szostak et al. reported cyclization of mRNA-displayed peptide libraries using two separate predetermined Cys residues and treatment with dibromoxylene.25 The reaction was highly efficient and produced several low nanomolar binders of the model protein target thrombin. In the related field of phage display, Heinis et al. replaced dibromoxylene with another symmetrical alkylating compound, TBMB, which reacted with three intramolecular Cys residues.18 This method was later found to give even more diverse architectures by changing the chemical linker based on different activated bromoalkyl compounds.80,193,194 Inspired by the merits of using TBMB, Suga also reported the in vitro production of tricyclic peptides.195 This strategy took advantage of the interesting finding that the N-terminal chloroacetyl group on the reprogrammed initiator residue prefers to react with the first and closest out of three incorporated Cys residue to give the smaller macrocycle.196 The second Cys residue is entropically disfavored and also prohibited from reacting due to steric hindrance, while the first Cys downstream is translated by the ribosome machine earlier P


Chemical Reviews

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Figure 9. Chemical structures of amino acids and derivatives thereof that have been incorporated into the FIT system.

method to construct a cyclic peptide library.202 The fusion complex of peptide-mRNA was incubated with a bis-NHS reagent, disuccinimidyl glutarate, at pH 8. Using this method they reported a cyclic peptide, cycGiBP, that binds to Gαi1 with a KD of 2.1 nM.203 In the Suga lab, we have reported a mild oxidation mediated cyclization method.204 Benzylamine and 5-hydroxytryptophan were incorporated into the FIT system and treated with potassium ferricyanide, K3Fe(CN)6, to afford a fluorescent heterocyclic linkage. A head-to-tail amide cyclization strategy was also reported.184 The acyl donor is produced either through a C-terminal γ-thiolactone205 or through a CPE (cysteinyl prolyl ester) motif,206,207 which acts as an autoactivating unit through a tandem reaction involving an N-to-S acyl shift, diketopiperazine formation, and glycolic acid release. Therefore, during translation the resulting thioester can be generated in situ. The acyl acceptor can be a Cys residue or non-Cys residue at the N-terminus prepared by PDF/MAP treatment as mentioned above.184 For cysteine, the intramolecular cyclization is chemoselective and happens through a native chemical ligation (NCL) mechanism involving S-to-S and S-to-N acyl transfers.208 For non-Cys residues, direct rearrangement can also occur spontaneously if no other Lys or Cys residues are present in the whole sequence.205,209 With this strategy in hand, backbone macrocyclic peptides containing γ-amino acids were synthesized ribosomally. Very recently, Murakami et al. presented a new method of the incorporation of charged N-alkyl amino acids. 210 Previously these residues were difficult to introduce likely due to the incompatibility of EF-Tu and/or the ribosome itself with non-native amino or carboxylate groups. To solve this problem, the authors prepared azide and ester protecting group building blocks, respectively. These precursors were found to undergo smooth aminoacylation and ribosomal incorporation. After translation, tris(2-carboxyethyl)phosphine (TCEP) treat-

than the remaining Cys residues. The tricyclization process is carried out through translation of linear peptides with an Nterminal chloroacetyl amino acid and four Cys residues (one of them at the second position, which is unreactive to the Nterminus). Spontaneous monocyclization of the chloroacetyl group occurs with the closest Cys residue in the peptide sequence followed by the formation of the next two cycles after addition of TBMB and cyclization of the remaining three Cys residues (Figure 8). It is also possible to generate another type of thioether cyclic structure, known as the lanthionines, using in vitro-translated peptides.151 In nature, lanthionine is generated by the dehydratases and cyclases of the lantipeptide family. Starting from serine and threonine residues, dehydration gives an intermediate dehydroalanine (Dha) or dehydrobutyrine (Dhb) residue.197 Then a nearby Cys residue can attack the double bond to give a Michael addition product. On the basis of the knowledge of this mechanism, precursor amino acids Dha and Dhb were introduced into peptides and were converted into artificial lantipeptides in an enzyme-free manner using an intramolecular Cys residue.198,199 Seebeck et al. established a thioether-cross-linked lantipeptide library.151 Using this library, selection against Sortase A gave binders with moderate KDs with a high dependency on the (2S,6R)-lanthionine moiety. 3.3.2. Nonthioether-Based Cyclization. Other cyclization or stapling methods, such as the copper-catalyzed azide− alkyne cycloaddition (CuAAC) chemistry, also allow construction of bicyclic peptide libraries for mRNA display. In 2012, Sako et al. first used CuAAC and thioether chemistry together on in vitro translated peptides to form bicyclic structures (Figure 8).200 Later, the Hartman group introduced a similar bicyclic framework in mRNA display.201 They confirmed that Cu(I) was compatible with the selection conditions of mRNA display, and they successfully obtained submicromolar binders toward a model protein, streptavidin. Roberts and co-workers introduced a diamide cross-linking Q


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ment transformed the azide into an amine,205 while carboxylesterase incubation released the free carboxylic acid. Both reactions proceeded with nearly quantitative yield according to MALDI-TOF-MS analysis (Figure 8). Thus, Nalkyl residues containing free amine or carboxylate groups can be incorporated indirectly using the PURE system through post-translational modifications. The authors also demonstrated that this masking strategy was compatible with the TRAP display system.23 Disulfide bridging is another convenient way to diversify the topology of linear peptides. Under translation conditions, air oxidation generally facilitates disulfide bridge formation without any additives. This was demonstrated in the ribosomal synthesis of thioether/disulfide bicyclic peptides.196 Twodisulfide-containing peptides were also produced in a cDNA display platform by Nemoto and co-workers to provide good binders targeting IL6R with nanomolar to submicromolar affinities.123,211 A summary of post-translational enzymatic and chemical modifications is given in Figure 8.

potency (KD = 0.6 nM), while the linear form, LM11-1, and non-N-methylated form, CP11-1, had a weaker binding affinity (KD = 180 nM and KD > 1000 nM) according to surface plasmon resonance (SPR) assays. The stability of the CM11 family peptides in human plasma was also confirmed by comparison with the control sequences. CM11-1 peptides demonstrated excellent in vitro binding affinity and plasma stability. In another ubiquitination assay, by detecting the formation of ubiquitin-thioesters, CM11-1 was found to inhibit p53 polyubiquitination in a dose-dependent manner in the micromolar range, while the linear control peptide LM11-1 did not. In summary, this work demonstrates that the RaPID system used in conjunction with N-methylated amino acids enables natural-product-like inhibitor discovery for previously thought nondruggable protein targets. 4.2. Peptides Targeting Membrane Proteins

In membrane protein crystallography, the limiting step often is the preparation of high-quality crystals for X-ray diffraction studies. To increase the chance of obtaining diffraction-quality crystals, versatile detergents are routinely used to help solubilize and stabilize the tertiary protein structure in a rigid conformation. For the same purpose, cocrystallization ligand proteins can also be prepared and incubated with membrane proteins to decrease their local heterogeneity. While many display techniques such as phage display and ribosome display have been used to produce antibody Fab fragments and designed ankyrin repeat proteins (DARPins) to target membrane proteins,240,241 mRNA display provides an alternative method. During the collaboration of the Hiroaki Suga and the Osamu Nureki lab, four thioether-macrocyclic peptides were identified using the RaPID system (N-(2-chloroacetyl)-Lphenylalanine and N-(2-chloroacetyl)-D-phenylalanine initiated (NNK)7−15 libraries), and all of them facilitated the cocrystallization of the Pyrococcus f uriosus multidrug and toxic compound extrusion (PfMATE) transporter in a straight conformation.223,224 The strongest binder, MaD5, has a small cyclic head and a linear tail. The crystal structure shows that the minicycle protrudes into a deep cleft and the tail portion extends along the rest of cavity. Note that these four macrocycles bound to different regions of PfMATE to lock it in its outward-facing conformation. This may be attributed to the high diversity of peptides that can be screened using the RaPID system. In another example, a similar strategy was employed to target the ABC-drug transporter CmABCB1. This also yielded a tightly binding peptide inhibitor (IC50 = 65 nM), which improved the crystallization properties and resulted in a crystal structure with a 2.4 Å resolution.225 By comparison with the apo-crystal structure of CmABCB1, this study revealed that the transport mechanism of CmABCB1 proceeds through an outward-opening motion. In summary, the robustness and versatility of the RaPID system provides a promising solution for proteins that are difficult to crystallize, and the system is possibly applicable to all other proteins.242

3.4. Amino Acids Compatible with mRNA Display

Table 4 and Figure 9 list residues that have been successfully incorporated into translated peptides using the FIT system as reported in the literature since 2006. These residues range from α-amino acids (D-stereochemistry, N-acyl, N-alkyl), βamino acids, γ-amino acids, α-hydroxy acids, α-mercapto acids, thioacids, and other acids.

4. CASE STUDIES Applications of mRNA display toward a wide selection of protein targets are listed in Table 5. Seven unique examples of peptide discovered by mRNA display selections are introduced below (sections 4.1−4.7). In Table 5 seven unique examples of peptide discovered by mRNA display selections are introduced, and X-ray structures of example cocrystals of thioether-macrocyclic peptides and their target proteins are shown in Figure 10. 4.1. N-Methyl Amino Acid-Rich Peptide Libraries

In 2011, Yamagishi et al. described the first proof-oftechnology example of the selection of macrocyclic peptides using the RaPID system.172 The key feature of this work is the thioether macrocycle and the multiple N-methylations contained in the backbone structure of the peptides. To achieve that the authors used a finely tuned PURE system by removing certain amino acids and their corresponding aaRSs from the translation mixture. This allowed the reassignment of the AUG, UUU, CUU, AUU, and GCU codons to N-(2chloroacetyl)-D-tryptophan, N-methylphenylalanine, N-methylserine, N-methylglycine, and N-methylalanine, respectively. Cyclization between the N-terminal chloroacetyl group and the side chain of a cysteine residue proceeds near quantitatively. The customized (NNU)8−15 library was then used to select against the E6AP HECT domain, an E3 ligase drug target involved in the p53-dependent apoptosis pathway. E6AP is thought to promote the degradation of tumor suppressor proteins, and to date there has been no report of the successful HTS-based discovery of any selective inhibitors.239 Using the RaPID system, after six rounds of positive selections with E6AP HECT GB1-tag-immobilized streptavidin beads and four rounds of counter selections with GB1-tag-immobilized beads, eight sequence families were identified following highthroughput sequencing. The top-performing peptide, CM11-1, contained four N-methylated residues and showed high

4.3. Macrocyclic Peptides Featuring a Peptidic Tail

Novel macrocyclic peptide ligands, referred to as iperglycermides, were discovered by the screening of mRNA display selections containing >1012 unique sequences. The inhibitors target the glycolytic phosphoglycerate mutase, or short iPGM, which is an attractive target for various diseases ranging from African trypanosomiasis, lymphatic filariasis to the S. aureus toxic shock syndrome.243,244 iPGM catalyzes the interconversion of phosphoglycerate isomers (2- to 3-phosphoglycerate) R


S

E6AP

SrtA

thrombin

Akt2

SIRT2

VEGF2

PfMATE

amino groups on solid phase CmABCB1

HGF receptor Met EpCAM Plexin B1

Gαi1

Her2

Ebola viral protein 24 (VP24) human pancreatic α-amylase (HPA) iPGM KDM4A, KDM4B, and KDM4C KDM4A and KDM4C TET1CCD

CM11-1

Sequence 2(2S,6R) N1

Pakti-L1

S2iD7

BD1

MaL6

CP1

aCAP

aMD4 Epi-1 PB1m6

GαSUPR

SUPR4

eVpeD2

PHD2

Plexin B1

NTCP

PHD2_4C

PB 1d6-28A

WD1

TiP2

CP 2f-7

Ce-1 CP2

piHA-D1

IL-6R

S1-Cys2-6

target

Gαi1

cycGiBP

peptide name

biotinylated Avitag/streptavidin beads biotin tag/streptavidin beads biotin tag/streptavidin beads His6-tag/NTA beads

His6-tag/NTA beads

Biotin tag/streptavidin beads His10-tag/NTA beads His6-tag/NTA beads

His10-tag/NTA beads

Fc tag/protein G beads Fc tag/protein G beads biotin tag/streptavidin beads biotin tag/NeutrAvidin beads Fc tag/protein G beads

His10-tag/NTA beads

N.A.

His6-tag/NTA beads

Fc tag/protein G beads

His6-tag/NTA beads

biotin tag/NeutrAvidin beads biotin tag/streptavidin beads biotin tag/streptavidin beads biotin tag/streptavidin beads biotin tag/streptavidin beads His6-tag/NTA beads

selection tag-bead pair

dimerization after selection thioether cyclization + backbone N-methylation

KD = 30 pM KD = 15 nM, IC50 = 0.85 μM

pIC50app = 8.04 nM IC50 = 42, 33, and 39 nM

thioether cyclization

thioether cyclization thioether cyclization

Ki = 1.0 nM

KD = 0.17 nM


KD = 76 nM (toward Her2) and 13 nM (toward Her2-overexpressing cells) KD = 3.2 nM


amide−amide bridging + N-methyl alanine amide−amide bridging + N-methyl alanine thioether cyclization

KD = 60 nM

KD = 48.6 nM, IC50 = 1.1 μM

dimerization after selection thioether cyclization thioether cyclization

KD = 2.4 nM KD = 1.7 nM KD = 3.5 nM



Ki = 65 nM (with 0 μM R6G)

IC50 = 6.0 and 2.2 nM

two disulfides

N.A.

N.A.

KD = 2 nM

KD = 3.7 nM, IC50 = 3.7 nM

IC50 = 110 nM

KD = 1.5 nM, Kiapp = 6.3 nM

thioether cyclization + Lys(Tfa) warhead thioether cyclization + backbone N-methylation thioether cyclization

thioether cyclization + backbone N-methylation lanthionine-type thioether cyclization thioether−thioether bridging thioether cyclization

KD = 0.6 nM KD = 3 μM

one disulfide

KD = 4 nM

structural features amide−amide bridging

KD = 2.1 nM

potency

Table 5. Publication Summary of Macrocyclic Peptides Discovered Using mRNA Display selection system

ref

RaPID system

RaPID system

RaPID system

RaPID system

RaPID system

RaPID system RaPID system

RaPID system

Bashirudin et al. Bioconjugate Chem. 2018236 Passioura et al. Cell Chem. Biol. 2018237

McAllister et al. Chem. Sci. 2018235

Jongkees et al. Cell Chem. Biol. 2017221 Yu et al. Nat. Commun. 2017231 Kawamura et al. Nat. Commun. 2017232 Passioura et al. Bioorg. Med. Chem. 2018233 Nishio et al. ChemBioChem 2018234

mRNA display + chemical modification Hofmann et al. J. Am. Chem. Soc. 2012151 mRNA display + npAA mimics Guillen Schlippe et al. J. Am. Chem. reassignment + chemical modification Soc. 201225 RaPID system Hayashi et al. ACS Chem. Biol. 2012166 RaPID system Morimoto et al. Angew. Chem., Int. Ed. 201276 RaPID system Murakami et al. ACS Chem. Biol. 2013167 RaPID system Tanaka et al. Nature 2013223 and Hipolito et al. Molecules 2013224 cDNA display Mochizuki et al. Chem. Commun. 2014211 RaPID system Kodan et al. Proc. Natl. Acad. Sci. 2014225 RaPID system Ito et al. Nat. Commun. 2015226 RaPID system Iwasaki et al. J. Mol. Evol. 2015227 RaPID system Matsunaga et al. Cell Chem. Biol. 2016228 mRNA display + npAA mimics Fiacco et al. ChemBioChem 2016229 reassignment + chemical modification mRNA display + npAA mimics Fiacco et al. ChemBioChem 2016229 reassignment + chemical modification RaPID system Song et al. Org. Biomol. Chem. 2017230

mRNA display + chemical modification Millward et al. ACS Chem. Biol. 2007203 cDNA display Yamaguchi et al. Nucleic Acid Res. 2009123 RaPID system Yamagishi et al. Chem. Biol. 2011172

Chemical Reviews Review


Passioura et al. J. Am. Chem. Soc. 2018238

in the metabolic pathway of many pathogenic microorganisms but is absent in the human host. Two thioether-cyclized peptide libraries initiated by either LTyr or D-Tyr were screened. The most promising macrocycles were obtained by using C. elegans iPGM as the target,using DTyr as the initiator for peptide synthesis and by conducting seven rounds of RaPID selection. This resulted in the discovery of two peptides, iperglycermide A and B, which have pIC50s of 8.40 and 8.65 nM, respectively. The crystal structure revealed an extensive hydrophilic binding surface, which highlights the importance of using macrocyclic peptides to identify novel binders of challenging targets lacking deep hydrophobic binding pockets. Recent HTS small-molecule drug discovery endeavors have only produced two low-potency compounds, most likely ion chelators.245 The successful discovery of a tightly binding small-molecule inhibitor of iPGM through HTS is considered unlikely due to the absence of a hydrophobic druggable pocket. Hao and co-workers discovered an 8-membered ring macrocyclic peptide inhibitor, which has a linear tail of 7 amino acids, which is crucial for its inhibitory activity.231 Hao and co-workers showed that the macrocyclic core is crucial for the binding affinity to its target protein iPGM in the nematode organism C. elegans. The macrocycle effectively holds the protein in a locked-open conformation, preventing the dynamic movement required during phosphatase and phosphotransferase catalysis. However, the inhibitory effect only exists if the linear tail attached to the macrocycle is present. Crystallography data suggests that this may be due to a Cys residue in the tail region of the inhibitor perturbing the coordination of the transition metal ion cluster inside the iPGM protein. The crystal structure also shows that the inhibitor binds to iPGM allosterically, sitting at the junction between both functional domains and trapping iPGM in a locked-open conformation.

RaPID system

selection system structural features

Review

thioether cyclization + hydrophobic library

ref

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potency

4.4. Membrane-Permeable Macrocyclic Peptides

selection tag-bead pair

IL-6R peptide name

αIL6R-1

Table 5. continued

target

EDC/NHS coupling/carboxylic acid beads

KD = 44 nM

Kawamura and co-workers used mRNA display and a ribosomally synthesized library of cyclic peptides to discover a highly selective inhibitor, CP2, of the JmjC histone demethylases (KDMs), which are linked to tumor cell proliferation. 232 Methyltransferases and demethylases (KDMs) regulate histone lysine methylation. cDNA sequences resulting from the fifth and sixth rounds of the mRNA-display selection were cloned into a pGEM-T Easy Vector by Kawamura and co-workers, and the resulting individual clones were sequenced to determine the identity of the binding macrocyclic peptides. A major challenge in the field has been the discovery of inhibitors, which are both selective and potent for specific JmjC-KDMs.246 The discovered macrocyclic peptides are inhibitors of KDM4A-C and are selective even over other closely related KDM isoforms. CP2 has a KD value of 29.8 nM (KDM4A binding) and is much more potent than previously discovered inhibitors, which have IC50 values in the micromolar range.247 The inhibitor competes with substrate binding in the active site but does not bind in the same way as the histone substrate. X-ray crystallography revealed that inhibitor CP2 binds to the histone-binding groove of KDM4A but not to the 2OGbinding pocket, which is targeted by the most recently developed set of inhibitors.247 Interestingly, the inhibitor CP2 adopts a distorted β-sheet with two turns. CP2 contains an important residue, Arg6, which is essential for potent T


Chemical Reviews

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Figure 10. Cocrystal X-ray structures of proteins and thioether-macrocyclic peptides discovered by mRNA display. (A) Transporter protein PfMATE in complex with peptide MaD3S (PDB 3VVS). Macrocyclic peptide binds inside a deep cleft of the transporter protein, locking PfMATE in its outward-facing conformation. (B) Ce iPGM in complex with its discovered truncated macrocyclic peptide inhibitor (PDB 5KGN) featuring a linear tail. Peptide binds allosterically, trapping iPGM in a locked-open conformation, preventing phosphatase and phosphotransferase activity. (C) KDM4A in complex with a highly selective inhibitor, CP2 (PDB 5LY1). CP2 inhibits the JmjC histone demethylases (KDMs) and has a KD of 29 nM. Inhibitor adopts a distorted β-sheet with two turns upon binding the histone-binding groove of KDM4A. (D) PlxnB1 in complex with macrocyclic peptide PB1m6 (PDB 5B4W). Interesting is the tight binding affinity (KD = 3.5 nM) and that the peptide inhibits the PlxnB1-Sema4D PPI allosterically.

KDM4A inhibition. This suggests that arginine residues are able to compete with the substrate, methylated lysine, binding to KDM4A. Modification of the inhibitor CP2 using structure- and mass spectrometry-guided modifications, such as backbone amide N-methylation, resulted in greater proteolytic stability. Residues not involved in critical H bonding with the target were modified. Mass spectrometry analysis of CP2 degradation patterns was used to confirm the sites, which are prone to proteolysis. Further modifications, which were explored, include the substitution of D-Ala for Gly as well as the incorporation of 4-fluorophenylalanine to improve cellular uptake by increasing hydrophobicity. CP2 was also turned into a cellular probe by attaching a fluorescein dye via a strainpromoted azide−alkyne cycloaddition. Upon treatment of HeLa cells with this fluorescent probe, a time-dependent increase in intramolecular fluorescence was observed, indicat-

ing cellular uptake. It was shown that CP2 is active in cells and is hence cell permeable. This research demonstrates the importance of mRNA display for the discovery of potent binders against challenging targets. 4.5. Dual-Peptide Selection for Binding Affinity and Protease Resistance

To discover peptides with high potency and stability to proteases, Roberts et al. developed a modified mRNA display which involves treating peptide libraries with a protease cocktail before routine target binding selection.248 In a proofof-principle example, they first created a focused library in which multiple codons were mutated to “UAG” to insert an Nmethyl residue, N-methylalanine, by genetic code reprogramming. Then the peptide library was expressed, cyclized, and subjected to an immobilized protease mixture. After collection of peptides that bound to the target protein, Gα1-GDP on solid phase, mRNA display was carried out again in the next U


Chemical Reviews

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mRNA display to the development of various artificial agonists specific for their corresponding membrane receptors.

round. Peptides selected by using this protocol were termed “SUPR (scanning unnatural protease resistant) peptides”. Next, the authors performed both in vitro and in vivo protease digestion assays. As anticipated, both cyclic structure and Nmethylalanine substitution were crucial for improving half-life values with chymotrypsin, proteinase K, human serum, human microsomes, and mouse serum. In another SUPR peptide selection, SUPR4 with 13 nM binding affinity was identified through selection against on-cell Her2. Two N-methylnorvaline residues were incorporated in SUPR4, and a peptide analogue of SUPR4 showed the ex vivo serum half-life of up to 5600 min. Hence, mRNA display can be used for the dual selection for binding potency and protease resistance to yield bioactive and stable macrocycles.248

5. GROWING POTENTIAL OF MRNA DISPLAY 5.1. Identifying Cell-Permeable Peptides

The majority of peptides targeting intracellular targets are cellpermeable natural product or natural product-derived peptides.251,252 Cyclosporine A, an immunosuppressant, is the most well-known orally available cyclic peptide.165 Most polypeptides are too large and polar to be able to passively diffuse through lipid membranes, and it has been shown that most active transport mechanisms facilitate the uptake of only very small peptides.253 “Lipinski’s rule of five”, which has been used with great success to predict the passive permeability of small molecules, is not a good indicator for the cell permeability of peptides.12 Due to the amide backbone of peptides and the resulting ability to form many H bonds, an entropic and enthalpic penalty needs to be taken upon the peptide passing through the hydrophobic lipid bilayer during passive diffusion.55 On the basis of a comparison of 125 orally absorbed cyclic peptides, it was shown that a lower number of hydrogen-bond donors and reduced flexibility generally increases oral bioavailability.253 Furthermore, macrocyclization allows some peptides to adapt to their local environment and to expose their polar functional groups when in aqueous medium but adopt a folded conformation to bury their polar groups through intramolecular hydrogen bonding on the inside of the macrocycle when in a hydrophobic environment. This has been referred to as “chameleonic” behavior, and it enables cyclosporine to diffuse through the lipid membrane.254 The incorporation of certain amino acid classes, such as D-amino acids, N-alkyl amino acids, or β-hydroxy-χ-amino acids, has also been identified to contribute to improved cell permeability.14,251,255 Yamagishi and co-workers successfully combined mRNA display with genetic code reprogramming for the discovery of bioactive macrocyclic peptides containing four Nmethyl backbone nitrogens.172 Other chemical motifs, such as azoles, have also been identified in natural and cell-permeable peptides.256 Recent publications on the integration of enzymatic peptide backbone modifications using cyclodehydratase PatD to produce heterocycles such as thiazoline, oxazoline, and methyloxazoline with the RaPID system hold great promise for the generation and selection of novel and more natural product-like peptide scaffolds with improved pharmacokinetic properties. This technique may be expanded in the future to be used in conjunction with mRNA display and with a wide variety of post-translational enzymes for increased scaffold and topology diversity. A further strategy is the replacement of amino acids with hydroxy acids to potentially reduce the external hydrogen-bonding network and hydrophilicity of the peptides to improve their membrane permeability.174 For peptide drugs to become a success in the clinic it is critical to develop an understanding of the criteria that favor the discovery of cell-permeable peptides. Despite progress in the area, there is a need for reliable methods and further guidelines beyond “Lipinski’s rule of five” to predict the cell permeability of macrocycles in addition to the development of high-throughput assays screening for cell permeability. This may also require the development of new chemistries and synthesis of novel scaffolds with improved cell permeabilities.

4.6. PPI Inhibitor Peptides

The mRNA display methodology can be applied to the discovery of peptide inhibitors targeting PPIs. PPIs are of importance in biological functions such as metabolism and signal transductions, but many are also implicated in diseases and are hence promising drug targets. PPIs normally occur at large and flat interfaces with few hydrophobic binding pockets, making it difficult for small molecules to bind.249 For example, the PPI between semaphorins and plexins is an important target for various diseases since it is involved in not only axon guidance but also cancer cell proliferation. Matsunaga et al. discovered a macrocyclic peptide (PB1m6) that binds plexin B1 (PlxnB1) with high affinity (KD = 3.5 nM) and that inhibits the interaction between PlxnB1 and Semaphorin 4D (Sema4D).228 Interestingly, a crystal structure analysis showed that the binding interface of PB1m6 on PlxnB1 was far from that of Sema4D, indicating that PB1m6 allosterically inhibits the PlxnB1−Sema4D interaction. This study highlights the potential of mRNA for the discovery of PPI inhibitors that bind to previously unknown allosteric regions on the target proteins. 4.7. Peptide Agonists of Membrane Receptor Proteins

mRNA display is not limited to the discovery of only inhibitors but can also be used for the discovery of an activator for a target protein. Ito et al. reported the development of artificial Met-activating dimeric macrocycles.226 Met is a hepatocyte growth factor (HGF) receptor which belongs to the receptor tyrosine kinase (RTK) family. Met is dimerized through its interaction with HGF, which subsequently activates Met signaling cascades, which are involved in embryonic development and wound healing. As such, a molecule that activates Met signaling pathways could be a potent drug in regenerative medicine. Although recombinant HGF and HGF gene therapies have been used, there is the risk of an unexpected immunological response and potentially irreversible damage to the gene pool.250 Therefore, alternative approaches that can activate Met signaling pathway are required. By means of the RaPID system, Ito and co-workers first identified monomeric macrocyclic peptides (named as aML5, aMD4, and aMD5) that bind to Met with high affinities (KD = 2−19 nM), and then they rationally designed macrocycle dimers (aML5-PEG3, aMD4-PEG11, and aMD5-PEG11) to dimerize Met artificially. These dimerized macrocycles function as Met agonists; they specifically activate Met signaling pathways through phosphorylation of Met, without affecting phosphorylation of other RTKs, and induce cellular responses such as cell migration and branching morphogenesis as strongly as HGF. This study emphasizes the application of V


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5.2. Peptide Ligands as Cocrystallization Chaperones

identification and quantification of all sequences bound to the beads at specific time points. By fitting the data to a simple kinetic on-rate equation each ligand’s kinetic on rate could be calculated. Ligands that had a high fractional composition at early time points had faster on rates for target binding. A similar methodology was used to also obtain the kinetic off rates of the ligands. This methodology was applied successfully to two enriched pools of peptides targeting the B-cell lymphoma extra-large protein (Bcl-xL).155 The KD values obtained by this method were highly reproducible and compared well with KDs from previously published data. This method allows the testing of thousands of sequences simultaneously without the need to individually synthesize and purify each library member on a larger scale. The further development of mRNA display as an in vitro selection technique in combination with high-throughput sequencing for the affinity maturation of peptide ligands holds great promise for the future.

mRNA display can also be used for the selection of peptides to be used for cocrystallization with difficult targets. As already described in detail in section 4.2, using the RaPID system, a cyclic peptide MaL6 was discovered, which upon cocrystallization with the MATE transporter improved the quality of the obtained crystals.223 This allowed the structure determination of two different conformations, depending on the pH, with the TM1 helix in a straight or bent conformation, at 2.4 and 2.5 Å resolution, respectively. The discovered peptides may also be used as a starting point for the development of potent antagonists. 5.3. Custom-Made Focused Library for Unique Targets

mRNA display can also be used to develop mechanism-based warhead inhibitors against difficult targets such as posttranslational-modification enzymes by the design of special libraries. This approach has been employed successfully by Morimoto et al. to discover isoform-selective inhibitors targeting the human deacetylase SIRT2.76 Sirtuins (SIRT1− 3) act to deacetylate the ε-N-acetyl group on LysAc using NAD + as a cosubstrate both in prokaryotes and in eukaryotes.257,258 The SIRT2 isoform specifically deacetylates both LysAc in α-tubulin and the lysine histone residue H4K16.257,259 An mRNA display library was constructed by Morimoto and co-workers that contains an ε-N-trifluoroacetyl lysine residue (KTfa) as a mechanism-based warhead in the middle of the mRNA library, as opposed to the natural substrate residue, εN-acetyl lysine (KAc). KTfa was used instead of the natural KAc residue in the mRNA library since it is unreactive toward deacetylation but serves as an isostere. Using genetic code reprogramming and an in vitro translation system, potent isoform-selective inhibitors with low nanomolar KDs and IC50s (approximately 3−4 nM) were discovered.76 The mRNA library ((AUG-(NNC)m-AUG-(NNC)n-UGC)) contains the initiator AUG codon, which was reprogrammed to D- or LClAcTyr residues, the elongator AUG codon, which was reprogrammed to the ε-N-trifluoroacetyl lysine (KTfa) residue, and the terminal UGC codon encoded a nucleophilic Cys residue, which spontaneously cyclized with the chloro-acetyl group on the translation initiator amino acid to form a macrocyclic peptide via a thioether linkage. The use of NNC codons for the random mRNA library region eliminated the presence of five amino acids (M, Q, E, K, and W), but more importantly, it suppresses the existence of any additional AUG codons in the random region. This ensured that KTfa only occurred at a single specified position. In this example, the “warhead” of the natural substrate of lysine deacetylase SIRT2 is a KAc residue, and this is converted into an unreactive equivalent, KTfa, in the mRNA screening library design.76 This general design may be applied to inhibitor discovery of various other post-translational-modification enzymes.

5.5. Peptide Dimerization

Dimerization is a common method to rationally improve the apparent binding affinity of monomeric peptides selected out from display technologies. Ito’s work,226 as introduced in the previous section, highlights the usefulness of this method to direct a dimer of ligand peptides to induce dimerization of cellsurface receptors, which can further induce intracellular responses. Not surprisingly, only appropriate linkers gave rise to higher activity, in this case, the Met phosphorylation level. Dimeric macrocycle aMD5-PEG11, which contains a longer 11-mer PEG linker, showed the highest activity, almost on the same level as that of the native receptor agonist hHGF. On the other hand, an aML5 dimer gave the highest activity only when dimerized by a shorter PEG3 linker. These results strongly indicated that the linker length has to be decided in a case-bycase manner. The identification of optimal linkers may be less time consuming if the mRNA display technology was expanded to allow the direct selection and maturation of dimeric macrocyclic peptides. Recently, Bashiruddin et al. reported a branched solid-phase synthesis protocol of PEG-linked dimeric macrocyclic peptides.236 Fmoc-protected bifunctional 2,3-diaminopropionic acid (Dap), Fmoc-Dap(Fmoc)-OH, was used as a relatively symmetric node residue to allow the simultaneous extension of linear sequences on solid phase. It was assumed that after cleavage the PEG linkers (30−200 Å) would form a highly diluted local environment to ensure correct selfmacrocyclization. Indeed, at concentrations lower than 10 mM, cyclization occurred to give a unanimous dimerization product of smaller rings without scrambling intramolecular or intermolecular thioether formations. With the dimer peptides in hand, the authors found that dimerization of PB1m6 (a previously reported low nanomolar binder of PlxnB1)228 generated PB1d6-28A, a 30 pM tighter binder. The 300-fold affinity enhancement highlighted the contribution of dimerization to give slower dissociation rates, a mechanism which is similar to the binding mode of bivalent antibodies. In another direction, heterodimerization of peptides to achieve multispecific binding effects can potentially be useful to function in a similar way to antibody-drug conjugates (ADC).

5.4. High-Throughput Affinity Maturation

mRNA display can be used in combination with highthroughput sequencing for the affinity maturation of peptide ligands. Recently, Jalali-Yazdi and co-workers applied this methodology to determine the kinetic on and off rates of over 20 000 ligands.155 For the determination of binding kinetics of the ligand libraries, the mRNA-peptide fusions were applied to target-immobilized beads. At various time points after the mixing a proportion of the beads was removed, washed, amplified by PCR, and then sequenced. This allowed the W


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Figure 11. Four main areas of development envisioned to extend the applicability of mRNA display for the discovery of more advanced, higher affinity, and more cell-permeable macrocyclic peptides. These include affinity maturation, N-methylation, peptide grafting, and macrocycle dimerization.

of antibodies has been reported in patents,263 which are called FcLoop peptibodies. Compared with normal peptibodies, in which peptide epitopes are directly fused to the C-terminus of each Fc chain, FcLoop peptibodies have improved pharmacokinetic properties mainly because the loop-embedded epitope becomes more rigid and more immune to proteolysis.264,265

5.6. Grafting onto a Scaffold Protein

In contrast to conjugation, the concept of grafting involves the insertion of a bioactive linear peptide, referred to as the epitope, into one of the loops of a structurally stabilized scaffold peptide or protein, connected through amide bonds. Such constructs share advantages with macrocyclic peptides as the conformation of the linear epitope can be locked, and the overall stability usually depends on the character of the protein framework. Several phage display-derived linear peptides have already been grafted onto different scaffold proteins, which display some “pseudo-macrocyclic effects”.260 On the basis of this feasible approach, one can envision that macrocyclic peptides selected through mRNA display can be incorporated into a protein scaffold with improved pharmacokinetic profiles. Haberkorn and co-workers demonstrated an example of grafting a phage-display-selected epitope onto the scaffold of the SFTI-I peptide.261 For phage display, they used Min-23, a well-established structural template with two disulfide bridges for stabilization, to build a peptide library, in which 10 random amino acids were inserted into its loop. This Min-23 phagedisplay library was applied to select against Dll4, a membrane protein which is highly expressed on the cell surface of tumor cells. They first identified an eight-residue binding motif, LFHLFIYI. Then the sequence was grafted onto SFTI-I to obtain a chimera construct GRCT-LFHLFIYI-CFPD crosslinked with an intramolecular disulfide bond. Compared with the parent peptide derived from Min-23, the new SFTI-I complex folds well and shows good Dll4-binding and tumortargeting activity in vitro and in vivo. In another example, Camarero and co-workers engineered MCoTI-I, a threedisulfide containing backbone-cyclized cyclotide family member, by inserting a p53-derived helical peptide onto its sixth loop.262 The resulting construct MCo-PMI was able to bind HDM2 and HDMX with nanomolar affinity. Moreover, MCoPMI showed good serum stability, membrane permeability, and strong cytotoxic potency against p53 cancer cell lines by targeting the intracellular p53 tumor suppressor pathway. Besides small-protein scaffolds, grafting peptide onto Fc loops

6. FUTURE PERSPECTIVES This review provides a summary of the historical development of mRNA display and how it enables the efficient discovery of novel bioactive macrocyclic peptides. Recent examples were discussed, highlighting the success and broad range target applicability of mRNA display. The power of mRNA display is supplemented by a variety of techniques that allow the further development and modification of the discovered peptides. As summarized in section 5 and Figure 11, we envision four main directions for the future discovery of bioactive macrocyclic peptides. Peptide binding affinities may be further optimized using techniques such as affinity maturation, in which sequence diversity is reintroduced into a preselected pool of sequences. Selection pressure can be increased to identify the highestaffinity peptides in the library. Pharmacokinetic properties in addition to cell permeability can be modulated by selective Nmethylation of peptide residues. A more recent development is the grafting of peptides discovered by phage display onto a scaffold protein. A similar approach can be envisioned to further advance peptides discovered by mRNA display. Once the peptide epitope has been determined it can be inserted into a peptide loop region of the scaffold protein and inherit its favorable pharmacokinetic properties. A further exciting prospect is the development of dimeric activator macrocyclic peptides using mRNA display. A monomeric macrocyclic peptide is discovered first by mRNA display and is then dimerized synthetically to increase the binding affinity of the dimeric target protein. Current drug discovery is about increasing the chemical space that can be screened to increase the success rate of identifying binders for challenging targets. In this review, we X


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AUTHOR INFORMATION

have shown that through mRNA display it is possible to screen vast libraries of ribosomally synthesized peptides with affinities often in the nanomolar range. A further area of future development is that of the automation of drug discovery. Most big pharmaceutical companies are already running highcapacity facilities, which are handled by robots. These robot systems are able to complete both high- and mediumthroughput screening of up to 10 000 samples per day.266 This is possible through the use of multiple arms and flexible workstations. One may envision the impact that the automation of mRNA display selections would have on the rate at which novel bioactive macrocyclic peptides could be discovered. Hence, an area of further development involves the automation of mRNA display, in which the repetitive rounds of selections are automated and conducted by a robot. Screening libraries of even higher diversities would be a simple matter of reaction scale-up. Fully automated systems could be coupled with an NGS platform and a peptide synthesizer to deliver the top hit macrocyclic peptides ready for biological screening. Such automation would hold great promise for the future of mRNA display. As an alternative to conventional antibodies to target extracellular proteins, macrocyclic peptides are anticipated to find wide application such as in molecular imaging and diagnostics in addition to therapeutic use. A recent example is by the Suga lab in 2015.227 Using the RaPID selection system, a 14-mer macrocyclic peptide binder Epi-1 targeting the extracellular domain of EpCAM (KD = 1.7 nM) was identified. Interestingly, the performance of fluorescent Epi-1 probe staining of EpCAM-expressing MCF7 cells under high cell density conditions is superior to conventional antibody staining. In a following work, Epi-1 was incorporated in a liposomal siRNA delivery system by decoration of lipid nanoparticles with this peptide. The resulting delivery system was found to target EpCAM-expressed cells with ∼100 fold improvement to transport therapeutic cargos such as siRNA and doxorubicin.267 Compared with traditional antibodies, the mRNA display technology yields nonstandard macrocyclic peptide binders that (1) show comparable or sometimes higher affinity, specificity, and stability than antibodies, (2) are faster to discover, (3) are easily accessible through solid-phase peptide synthesis (SPPS) for structural optimization, and 4) can be discovered without animal use. Therefore, with robust peptide binders and their derivatives in hand, it is possible to carry out common antibody-involved biochemical assays such as immunoblotting, immunoprecipitation, immunoaffinity chromatography, and immunofluorescence staining and drug delivery probes. In summary, over the past two decades, mRNA display has substantially grown into a competitive platform technology in the field of in vitro evolution-based drug discovery, accompanying phage display and DNA-encoded library technologies. In contrast to rational/virtual screening-based drug design, mRNA display provides a rapid and robust process to identify early hit compounds that can be further optimized using focused-selection and/or deep-screening approaches to obtain better pharmacokinetics parameters. With increasing enthusiasm from academia and industry about drugging previously nondruggable targets, we anticipate that the promising growth of mRNA display together with other fast-booming technologies only demonstrates the dawn of the golden era of macrocyclic drug entities toward this goal.

Corresponding Author

*E-mail: [email protected]. ORCID

Hiroaki Suga: 0000-0002-5298-9186 Notes

The authors declare no competing financial interest. Biographies Mareike Wiedmann obtained her Master’s degree in Chemistry under the supervision of Dr. Jonathan Burton at the University of Oxford. She received her Ph.D. degree in Chemistry and Chemical Biology from the University of Cambridge under the supervision of Prof. David Spring (Department of Chemistry) and Dr. James Brenton (CRUK CI) in 2016 working on the synthesis and development of constrained peptides. She then joined the research group of Prof. Hiroaki Suga at the University of Tokyo as a JSPS Postdoctoral Researcher. Her expertise is in chemical biology, organic synthesis, and peptide chemistry. Her current research interests are in the areas of further development of mRNA display of macrocyclic peptides, DNA-encoded library screening, and targeting infectious diseases. Yichao Huang was born in Shanghai, China. He obtained his B.S. degree in Chemical Biology (2011) and Ph.D. degree in Bioorganic Chemistry (2016) from Tsinghua University under the supervision of Prof. Lei Liu. During this time his research focus was on the preparation of functional protein derivatives by total chemical synthesis, semisynthesis, or chemoenzymatic synthesis. Currently, he is working as a postdoctoral researcher hosted by Prof. Hiroaki Suga at the University of Tokyo, where he is interested in advancing the RaPID selection technology for use against versatile targets. Hiroaki Suga received his Ph.D. degree in Chemistry (1994) from the Massachusetts Institute of Technology. He was Assistant Professor at the Department of Chemistry in the State University of New York at Buffalo (1997) and was promoted to Associate Professor (2002). He became Professor of the Research Center for Advanced Science and Technology at the University of Tokyo (2003) and later (2010) of the Department of Chemistry, Graduate School of Science. He is the recipient of the Akabori Memorial Award 2014, the Japanese Peptide Society and Max-Bergmann Gold Medal 2016, and the Nagoya Medal 2017 Silver. He is also a founder of PeptiDream Inc., Tokyo, a publicly traded company, which has many partnerships with pharmaceutical companies worldwide.

ACKNOWLEDGMENTS H.S. thanks AMED BINDS (18am0101XXXj0002) and the Basic Science and Platform Program for Innovative Biological Medicine (18am0301001h0004), the Program on the Innovative Development and the Application of New Drugs for Hepatitis B (18fk0310103j0001), and JST CREST (JPMJCR12L2). M.M.W. is an International Research Fellow of the Japanese Society for the Promotion of Science (P16810). The authors thank Hisaaki Hirose and Toby Passioura for helpful discussions. ABBREVIATIONS AA/aa amino acid ncAA noncanonical amino acid npAA nonproteinogenic amino acid pAA proteinogenic amino acid Ala/A alanine Y


Chemical Reviews Arg/R Asn/N Asp/D Cys/C Dha Dhb Gln/Q Glu/E Gly/G His/H Ile/I Leu/L Lys/K Met/M Phe/F Pro/P Pyl Ser/S Thr/T Trp/W Val/V aaRS/AARS/ARS ABT Ac ADC aFx ATP Bcl-xL bcPURE Bn CAD CaM CBT ClAc CME CPE CRISPR CuAAC DARPin DBE DEL dFx EF eFx EpCAM FACS FBDD Fc FIT Fx GS HECT HGF HIF1 HTS HTSK IF IL-6R iPGM

Review

KDM MATE MALDI MAP Me MTF NCL NGS NHS NNC

arginine asparagine aspartic acid cysteine dehydroalanine dehydrobutyine glutamine glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline pyrrolysine serine threonine tryptophan valine aminoacyl-tRNA synthetase 2-(aminoethyl)amidocarboxybenzyl thioester acetyl antibody-drug conjugate amino flexizyme adenosine triphosphate B-cell lymphoma extra-large protein backbone-cyclic PURE benzyl covalent antibody display calmodulin (calcium-modulated protein) 4-chlorobenzyl thioester chloroacetyl cyanomethyl ester cysteinyl prolyl ester clustered regularly interspaced short palindromic repeats copper-catalyzed azide−alkyne cycloaddition designed ankyrin repeat protein 3,5-dinitrobenzyl ester DNA-encoded chemical library dinitrobenzyl flexizyme elongation factor enhanced flexizyme epithelial cell adhesion molecule fluorescence-activated cell sorting fragment-based drug discovery fragment crystallizable flexible in vitro translation flexizyme (flexible ribozyme) goadsporin homologous to E6AP C-terminus hepatocyte growth factor Hypoxia-inducible factor 1 high-throughput screening high-throughput sequencing kinetics initiation factor interleucin-6 receptor cofactor-independent phosphoglycerate mutase

NNK NNU NTCP NTP PACE PDF PEG Pf pIII PlxnB1 PPI PSA PTM PURE RaPID RepA RF RiPP RRF RTK Sema4D SICLOPPS SIRT2 SPPS SPR SUPR TBMB TCEP TET1CCD Tfa TOF TRAP TSS wPURE YSD

lysine demethylase multidrug and toxic compound extrusion matrix-assisted laser desorption/ionization methionine aminopeptidase methyl methionyl-tRNA transformylase native chemical ligation next-generation sequencing N-hydroxysuccinimide codon = any base A/C/G/U + any base A/ C/G/U + base C codon = any base A/C/G/U + any base A/ C/G/U + keto base G/U codon = any base A/C/G/U + any base A/ C/G/U + base U sodium taurocholate cotransporting polypeptide nucleoside triphosphate phage-assisted continuous evolution peptide deformylase polyethlyene glycol Pyrococcus f uriosus M13 phage protein III plexin B1 protein−protein interaction polar surface area post-translational modification protein synthesis using recombinant elements random nonstandard peptide integrated discovery replication initiation protein release factor ribosomally synthesized and post-translationally modified peptide ribosome recycling factor receptor tyrosine kinase semaphorin 4D split intein−mediated circular ligation of peptides and proteins NAD-dependent deacetylase sirtuin 2 solid-phase peptide synthesis surface plasmon resonance scanning unnatural protease resistant 1,3,5-tris(bromomethyl)benzene tris(2-carboxyethyl)phosphine ten-eleven translocation protein 1 compact catalytic domain trifluoroacetyl time-of-flight transcription−translation coupled with association of puromycin linker toxic shock syndrome withdrawn PURE yeast surface display

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