Design, synthesis, and diversification of ribosomally derived peptide macrocycles

Available online at www.sciencedirect.com

Design, synthesis, and diversification of ribosomally derived peptide macrocycles John R Frost, Jessica M Smith and Rudi Fasan Ring topologies are widespread structural motifs among biologically active peptides found in nature. The recurrence of this motif is linked to the inherent advantages resulting from backbone cyclization, which include increased resistance against proteolytic degradation, improved cell permeability, and tighter and more specific interaction with the respective biomolecular target. Inspired by these natural product topologies, a number of groups have recently focused on developing methodologies that hinge upon the chemical elaboration of ribosomally derived polypeptides toward the synthesis and diversification of macrocyclic peptide structures. In this review, we highlight recent advances in this emerging new area and discuss the opportunities created by these methods toward the discovery of new functional entities. Addresses Department of Chemistry, University of Rochester, Rochester, NY 14627, USA Corresponding author: Fasan, Rudi ([email protected])

proteolytic degradation as well as enabling them to adopt a certain degree of structural pre-organization. The latter is often associated with the ability of these compounds to interact with their respective biomolecular targets with high affinity and specificity, as a result of pre-organization of the scaffold in a bioactive conformation and reduced entropic costs upon binding. In addition, as exemplified by the complex of cyclosporin A (Figure 1) with its target proteins, cyclophilin and FK506-binding protein [5], the structural and functional complexity of these molecules makes them well suited for interacting with extended biomolecular interfaces that characterize challenging targets such as protein–protein or protein–nucleic acid interactions. Owing to the biomedical potential of this structural class, there has been a growing interest in developing strategies to generate collections of novel macrocyclic peptides which are either derived from or inspired by natural cyclopeptide architectures as a source of relevant molecular diversity.

Current Opinion in Structural Biology 2013, 23:571–580 This review comes from a themed issue on Engineering and design Edited by Florian Hollfelder and Stefan Lutz For a complete overview see the Issue and the Editorial Available online 12th July 2013 0959-440X/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.sbi.2013.06.013

Introduction Macrocyclic peptides have emerged as a relevant structural class for the search and development of novel therapeutic agents [1–3]. Since the discovery of the first bioactive cyclopeptide, Gramicidin S, in 1944 [4], a large number of macrocyclic peptides have been isolated from organisms spanning all kingdoms of life that possess a broad spectrum of interesting biological properties, ranging from hormonal to antibacterial, antifungal, immunosuppressive, and anticancer activity [1–3]. A common structural motif among natural bioactive peptides is a ring topology, which is achieved through various modalities, namely via backbone (N-terminus to C-terminus) cyclization (e.g. cyclosporine) and/or linkage of a side-chain functionality to the N-/C-terminus (e.g. daptomycin, polymixin) or to another side-chain (e.g. vancomycin). The recurrence of this structural feature is linked to the inherent advantages it can confer to these molecules, which include providing increased resistance against www.sciencedirect.com

One interesting approach in this area has involved the manipulation of biosynthetic machineries and pathways implicated in the biogenesis of naturally occurring macrocyclic peptides, such as those produced by the action of non-ribosomal peptide synthetases (NRPSs) [6,7] or those belonging to the expanding class of ribosomally synthesized and post-translationally modified peptides (RiPPs) [8]. Non-ribosomal peptide synthetases operate as a multi-enzyme assembly line catalyzing the sequential activation, condensation, and ‘tailoring’ of amino acid building blocks to give rise to a diverse array of natural products [6,7]. The modular structure of NRPSs makes them potentially useful for the combinatorial biosynthesis of new natural product analogs, in particular via swapping of the adenylation domains within NRPS modules which are primarily involved in recognition of the amino acid building blocks [9,10]. Although this process has proven to be much more challenging than originally envisioned, recent reports have described the successful application of directed evolution to re-engineer NRPS systems for generating tailored-made compounds [11–13]. RiPPs constitute another important class of natural cyclopeptides, which comprise more than 20 distinct subclasses of compounds as emerged from a recent systematic classification [8]. These natural products are produced via the post-translational processing of a precursor polypeptide via a battery of enzymes, which include dehydratases, peptidases, and methyl- or farnesylCurrent Opinion in Structural Biology 2013, 23:571–580

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Figure 1

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Chemical structures of peptide natural products nisin A, cyclosporin A, and trunkamide and of representative peptide macrocycles obtained via the cyclization of ribosomally derived polypeptides according to the methodologies highlighted in the text. In the latter compounds, the structural elements involved in the cyclization are highlighted in red.

transferases. The genetically encoded nature of RiPPs and relatively simpler organization of post-ribosomal peptide synthesis (PRPS) pathways (compared to NRPSs) have facilitated the manipulation of these systems for generating new natural product analogs, as demonstrated by recent studies in the context of lanthipeptides [14–19], cyanobactins [20,21], thiopeptides [22,23], lasso peptides [24], and cyclotides [25,26]. Inspired by the ring topologies observed in the natural world, other groups have recently begun to explore alternative strategies for generating artificial, ‘natural product-like’ and ‘natural product-inspired’ macrocyclic peptides (Figure 1). These approaches rely on the design and implementation of non-natural cyclative processes to convert ribosomally derived polypeptides into conformationally constrained peptide macrocycles. Peculiar advantages inherent to these approaches include the possibility to define and modulate the ring topology of the final products (e.g. beyond the boundaries imposed by the NRPS or PRPS machineries) combined with the opportunity to exploit the combinatorial potential of ribosomal synthesis toward the generation of large and diverse chemical libraries. In this review, we highlight recent developments in this emerging area with representative examples of the opportunities created by these strategies toward the discovery of biologically active molecules. Current Opinion in Structural Biology 2013, 23:571–580

Cysteine-mediated macrocyclization Owing to its peculiar chemical reactivity, cysteine has been widely exploited in nature for constraining the conformational flexibility of ribosomal peptide sequences. For example, its ability to create disulfide bridges across distant positions in a polypeptide is at the basis of the structural stabilization of many naturally occurring cyclopeptides (e.g. defensins [27]) and mini-proteins (e.g. cyclotides [28]). Cysteine residues also play a key role in the structural organization of lanthipeptides (e.g. nisin A, Figure 1) via the formation of inter-side-chain (methyl)lanthionine bridges, which result from the enzymatically assisted Michael addition of cysteine sulfhydryl groups onto dehydroalanine (Dha) or dehydrobutyrine (Dhb) residues generated from the dehydration of serine or threonine, respectively. Finally, cysteines are involved in the formation of thiazoli(di)ne rings in a variety of other RiPPs, such as cyanobactins (e.g. trunkamide, Figure 1) and thiopeptides [8]. Similarly, exploitation of the nucleophilic reactivity of cysteines has become a hallmark of many recent strategies directed at generating macrocyclic peptides from ribosomal precursors via unnatural routes, as outlined in the sections below. Lanthipeptide-like macrocyclic peptides

In recent reports by the Suga and Szostak groups, two alternative methods for generating ‘lanthipeptide-like’ macrocyclic peptides (structure ‘a’, Figure 1) from in www.sciencedirect.com

Ribosomally derived peptide macrocycles Frost, Smith and Fasan 573

vitro translated polypeptide precursors have been described. In one case, the unnatural amino acid vinylglycine was incorporated into a short peptide sequence utilizing codon reprogramming [29] in a reconstituted in vitro translation system [30]. Vinylglycine was isomerized to (Z)-dehydrobutyrine (Dhb) by heating at 958C, thereby enabling the conjugate addition of a downstream cysteine to the Dhb residue to form an intramolecular lanthionine bridge [31]. As a proof-of-principle experiment, this method was applied to reproduce the B and C rings of the lantibiotic nisin (as a diastereomeric mixture). An alternative strategy was introduced by Szostak and coworkers [32], in which a selenium-containing lysine analog, 4-seleno-Lys, was incorporated into a ribosomal peptide via in vitro translation and subsequently oxidized by H2O2 to generate Dha via b-elimination. Upon deprotection of a neighboring cysteine, formation of the desired lanthionine bridge could be achieved. This methodology was later integrated with mRNA display to isolate a 3 mM binder for Sortase A upon screening of a 1011-member library of lanthionine-constrained peptides [33]. Binding studies with the isolated peptide, LWY-Lan-LS-LanWGRI, revealed the critical importance of the stereochemical configuration of the lanthionine bridge for interaction with the enzyme. Thioether-linked cyclopeptides via in vitro translation

Taking inspiration from cyclization strategies implemented in the context of synthetic peptides [34,35], other viable approaches for the cyclization of ribosomal peptides have been recently developed that rely on the reaction of a cysteine with an electrophilic functionality installed in the sequence by means of unnatural amino acids. In a first example, Suga and coworkers reported the successful formation of head-to-side-chain cyclopeptides (structure ‘b’, Figure 1) via the ribosomal incorporation of Na-(2-chloroacetyl)-Trp at the N-terminal end of a cysteine-containing peptide through reassignment of the initial AUG codon via genetic code reprogramming in the presence of a methionine-free reconstituted in vitro translation system [36]. Nucleophilic substitution of the a-halo-amide moiety by the cysteine sulfhydryl group resulted in the desired intramolecular thioether linkage (Figure 2). Notably, this methodology could be later coupled to mRNA display to create a large (1012) library of natural product-like peptide macrocycles (10mer to 15mer) containing a N-terminal D-Trp residue and various N-methylated amino acids [37]. Library screening against ubiquitin ligase E6AP enabled the isolation of a potent inhibitor for this enzyme (Kd = 0.6 nM). More recently, similar studies have been conducted in the context of other medically relevant target enzymes, resulting in the successful identification of potent inhibitors for both Akt2 kinase (IC50: 110 nM) [38] and human deacetylase SIRT2 (IC50: 3.2 nM) [39]. Exploiting a similar cysteine-alkylation strategy, the same group was able to generate cyclopeptides constrained by an www.sciencedirect.com

inter-side-chain thioether linkage through the incorporation of Ng-(2-chloroacetyl)-a,g-diaminobutyric acid (Cab) into in vitro translated peptides containing a C-terminal cysteine [40]. Interestingly, this approach could be applied to prepare a more stable analog of the human hormone urotensin II, in which the disulfide bridge was substituted by a redox stable thioether bond. The aforementioned Cab-based and ClAc-AA-based cyclization strategies were recently used in conjunction with CuI-catalyzed azide/alkyne cycloaddition [41] and oxidative disulfide formation [42], respectively, to yield constrained peptides featuring a bicyclic topology. In the latter report, the regioselectivity of thioether/disulfide bond formation in peptide sequences containing multiple cysteine residues was systematically investigated [42]. This work showed that the N-terminal ClAcPhe residue reacted preferentially with the most proximal Cys residue in the downstream sequence. On the basis of the observed reactivity trend, ‘‘overlapping’’ and dumbbell-type bicyclic peptides could be obtained with the expected intramolecular connectivities. Using a variation of Suga’s ClAc-AA-mediated cyclization strategy, Murakami and coworkers have recently described the creation of a library of in vitro translated thioether-constrained cyclopeptides (Figure 2) [43]. In this case, a N-benzoylated Phe derivative, (Na-[(3-(2chloroacetamido)benzoyl]-L-Phe or ClAB-L-Phe), was installed at the N-terminus of a cysteine-containing peptide by genetic reprogramming of the AUG codon in a Met-depleted system. This approach could be integrated with a modified (and improved) version of mRNA display as a screening platform, called TRAP display [43]. Using human serum albumin (HSA) as model target protein, the authors demonstrated the efficiency of the new in vitro selection method to enable the isolation of nanomolar HSA binders at a much greater speed than required when using the conventional mRNA display protocol. In another notable contribution, Murakami and coworkers produced a panel of peptide macrocycle libraries via ClAc-(L/D)-Phe-mediated and ClAB-(L/D)-Phe-mediated cyclization and replacing four of the 20 natural amino acid building blocks with backbone-modified derivatives (i.e. N-methylated Phe and His, cycloleucine, D-Tyr). Using the TRAP system, these libraries were screened against the therapeutically relevant vascular endothelial growth factor receptor 2 (VEGFR2), resulting in the identification of several nanomolar binders for the target protein, some of which were found capable of inhibiting VEGFinduced VEGFR2 autophosphorylation and angiogenesis of vascular endothelial cells [44]. Thioether-linked cyclopeptides via cysteine crosslinking agents

Another relevant strategy for the cyclization of ribosomal peptides has relied on the alkylation of multiple Current Opinion in Structural Biology 2013, 23:571–580

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Figure 2

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Generation of cyclopeptides via in vitro translation and genetic code reprogramming with flexible tRNA acylation ribozyme-based system. After ribosomal translation, peptide-mRNA conjugation/complexes are generated via a puromycin (Pu) linker, enabling the screening of cyclopeptide libraries via mRNA or TRAP display.

cysteine residues installed at pre-defined positions of the peptide sequence using bifunctional or trifunctional cross-linking reagents (structure ‘c’, Figure 1) [45]. In a first example, Szostak and coworkers demonstrated the viability of this method to cyclize in vitro translated peptides via cross-linking two cysteines with 1,3-dibromo-xylene [46]. More recently, the same group demonstrated the possibility to combine this method with mRNA display to create and screen a large library of cyclopeptides containing various amino acid analogs and identify potent inhibitors for thrombin (Ki = 20 nM) [47]. Current Opinion in Structural Biology 2013, 23:571–580

Using an analogous chemical cross-linking approach, Heinis and Winter demonstrated the successful display on phage of a library of bicyclic peptides obtained via alkylation of the linear peptide sequence, Cys-(Xxx)6Cys-(Xxx)6-Cys, with tris-1,3,5-(bromomethyl)benzene (TBMB) (Figure 3) [48]. This platform allowed for the discovery of a bicyclic nanomolar inhibitor of human plasma kallikrein [48], including binders with increased protease stability [49]. More recently, the same library of bicyclic 12mer peptides proved useful toward the discovery of a potent inhibitor of human urokinase-type plasminogen activator (uPA) (UK18, Kd: 53 nM) [50]. www.sciencedirect.com

Ribosomally derived peptide macrocycles Frost, Smith and Fasan 575

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(a) Chemically cross-linked bicyclic peptides on phage particles are generated via cysteine-alkylation with a tris-(bromomethyl)benzene reagent. (b) Crystal structure of human urokinase-type plasminogen activator (uPA, blue) in complex with a bicyclic peptide inhibitor (UK18, green/orange) isolated after screening of the phage display library. The amino acid residues in UK18 involved in the interaction with the target enzyme are highlighted.

Solution of the crystal structure of the complex revealed that the inhibitor covers an extended area of the enzyme surface (700 A˚2), forming multiple hydrogen bonds (14) with the protein (Figure 3). The larger size and more extensive interactions established by the bicyclic peptide explained the higher inhibitory potency of this compound compared to a phage-display disulfide-constrained peptide previously isolated against the same target enzyme [51]. More recently, Heinis and coworkers expanded the range of cross-linking reagents available for the generation of bicyclic peptides via the aforementioned strategy [52]. Specifically, three reagents alternative to TBMB, namely 1,3,5-triacryloyl-1,3,5-triazinane N,N0 ,N00 -(benzene-1,3,5-triyl)tris(2-bromo(TATA), acetamide) (TBAB), and N,N0 ,N00 -benzene-1,3,5-triyltrisprop-2-enamide (TAAB), were tested for efficiency in cyclization using the kallikrein inhibitor peptide, PK15. Interestingly, TATA and TBAB were able to promote quantitative cyclization of the linear peptide (similarly to TBMB), whereas poor yields (10%) were achieved with TAAB. NOESY spectra and a reduction in the kallikrein inhibitory potency for the PK15-TATA and PK15-TBAB conjugates supported the ability of these alternative scaffolds to affect the conformational properties of the bicylic peptide [52].

Split intein-mediated peptide cyclization The use of split intein-mediated peptide cyclization (called SICLOPPS) has represented another powerful www.sciencedirect.com

approach for producing head-to-tail cyclic peptides (structure ‘d’, Figure 1) [53]. This methodology and its application toward the discovery of cyclopeptide-based inhibitors for various protein and enzyme targets has been extensively reviewed elsewhere [54–56], so only most recent contributions in this area will be mentioned here. Briefly, this strategy involves the genetic encoding of a target peptide sequence inserted in between the C-terminal (InC) and N-terminal (InN) domain of a split intein (e.g. Synechocystis sp. DnaE). Upon expression, the InC and InN domains undergo a trans-splicing reaction resulting in the release of a cyclopeptide encompassing the target sequence [53]. A key feature of this method is the possibility to produce the target cyclopeptide within the host cell, be it Escherichia coli [53], yeast [57], or human cells [58], thus providing the opportunity to couple library generation with a genetic selection [59,60,61] or intracellular reporter systems [58,62]. Within this approach, sequence-dependent factors have been found to affect both the efficiency of peptide cyclization and the rate of this process in vivo [57,63,64]. For example, systematic analyses of libraries with randomized target sequences showed that about 50–70% of the sequences undergo cyclization [63,64]. These limitations notwithstanding, cyclopeptide libraries prepared by SICLOPPS remain sufficiently large to enable the isolation of effective inhibitors for the target protein(s) [54–56]. Recent contributions in this area include the combination of SICLOPPS with unnatural amino acid mutagenesis via the amber stop codon suppression method developed in Current Opinion in Structural Biology 2013, 23:571–580

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the Schultz’s group [65] to isolate cyclopeptide inhibitors of the HIV protease [61]. Interestingly, the genetically encoded unnatural amino acid ( p-benzoylphenylalanine) was found to play a key role in the mechanism of inhibition. Another notable example is the recent application of SICLOPPS in a yeast-based model of Parkinson’s disease [57]. In this case, cyclopeptides capable of reducing a-synuclein-dependent toxicity could be identified by selecting yeast colonies that survived after induction of a-syn expression. Finally, Soumillion and coworkers recently reported a strategy to direct the formation of SICLOPPS cyclopeptide in the periplasmic space of E. coli [64]. Although not yet applied for this purpose, this approach could prove valuable to select bioactive cyclopeptides that can act on extracellular targets on the host (or a co-cultured organism) or that exhibit increased cell permeability.

Enzyme-mediated peptide cyclization Other groups have investigated the possibility to exploit enzyme-catalyzed reactions to obtain cyclic peptides from linear polypeptide precursors. In a recent report, Taki and co-workers described a method that takes advantage of leucyl/phenylalanyl tRNA-protein transferase (L/F transferase) from E. coli, an enzyme able to catalyze the transfer of a hydrophobic amino acid from tRNA to the N-terminal end of a peptide/protein chain with a N-terminal lysine or arginine [66]. Using this system in combination with aminoacylated-tRNAPhe reagents (called NEXT-A), the group was able to extend the N-terminus of a model peptide with various unnatural amino acids bearing an electrophilic sidechain group to mediate peptide cyclization via the reaction with a downstream cysteine [67]. Most efficient cyclization was observed in the presence of p-(chloroacetylamino)-Lphenylalanine as the reactive unnatural amino acid (structure ‘e’, Figure 1). Importantly, this cyclization strategy was found to work with reasonable efficiency (75%) also when the target sequence was fused to a C-terminal protein (GFP), suggesting its compatibility with phage display. Another contribution in this area has involved the use of transglutaminases (TGases) for peptide cyclization [68]. This class of enzyme catalyzes transamination reactions between the side-chain amide group of a glutamine residue with amines [69]. Using a microbial TGase from Streptomyces mobaraensis [70], Heinis and coworkers demonstrated the possibility to generate cyclic peptides via TGasecatalyzed coupling of Gln and Lys side chains contained in the peptide sequence. Although these studies mainly focused on polyglycine target sequences, fairly good cyclization efficiencies (50%) were achieved also in the context of more complex sequences, suggesting that the substrate tolerance of the enzyme is rather broad.

Macrocyclic organo-peptide hybrids While the aforementioned strategies have enabled the cyclization of ribosomal peptides through a variety of Current Opinion in Structural Biology 2013, 23:571–580

covalent linkages, none of them allows for the incorporation of arbitrary non-proteogenic building blocks into the final peptide macrocycles as a means to alter and diversify the topology of these molecules. In an effort to address this gap, the Fasan group has developed strategies for the creation of Macrocyclic Organo Peptide Hybrids (MOrPHs) via the cyclization of ribosomal precursor polypeptides by means of synthetic linker units (structures ‘f–g’, Figure 1) [71,72]. MOrPHs are generated via a dual, bio-orthogonal ligation between bifunctional synthetic precursors (SPs) and recombinant protein precursors (BPs), in which a variable peptide target sequence (TS) is framed between an unnatural amino acid (UAA) installed by amber stop codon suppression [65], and an engineered intein (Mycobacterium xenopi GyrA(N198A)) lacking C-terminal splicing ability. The UAA carries a bioorthogonal functional group to provide a first ligation site to the synthetic molecules, whereas a second ligation point is created by interception of the intein-catalyzed thioester bond at the C-terminus of the target sequence by an appropriate nucleophilic functionality on the SP. This general concept was first implemented by utilizing azide/hydrazide-based SPs in combination with protein precursors bearing an alkyne-containing tyrosine derivative (OpgY). Hybrid macrocycles were generated via a side-chain CuI-catalyzed azide/alkyne cycloaddition (CuAAC) followed by ring closure via SP-hydrazide attack on the intein-thioester (Figure 4) [71]. In this work, structurally different aryl-based SPs were made to react with precursor polypeptides containing target sequences of varying length (4mer to 12mer), to rapidly afford the desired MOrPHs as the major product (80– 100%). In the presence of the 4mer target sequence and/ or SPs with short azido/hydrazide distances (<6 A˚), more modest yields (50–60%) were observed possibly due to an increase in conformational strain during cyclization. Variation of the N-terminal tail preceding the UAA in the protein precursor enabled the production of cyclic, lariatshaped, and protein-fused MOrPHs [71]. A complementary method to access MOrPHs was later developed [72], which exploits a catalyst-free dual oxime/intein-mediated ligation strategy involving an oxyamino/1,3-amino-thiol-based SP and a protein precursor bearing a side-chain keto group provided by pacetyl-Phe (pAcF) (Figure 4). Hybrid macrocycles encompassing 4–12 amino acid residues were obtained in good yields (50–80%), with no side-products, and within relatively short times (<5 hours) [72]. Expanding on the latter approach, a panel of four structurally diverse and more conformationally rigid SPs containing the reactive 1,3-amino-thiol-aryl (AMA) moiety were investigated and found to induce MOrPH formation across 4– 15 amino acid long target sequences with even higher efficiency (80–95% yield, 5 hours) [73]. Systematic mutagenesis of the terminal amino acid residue www.sciencedirect.com

Ribosomally derived peptide macrocycles Frost, Smith and Fasan 577

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Chemobiosynthetic methods for the synthesis of macrocyclic organo-peptide hybrids (MOrPHs). Macrocycle formation is achieved upon reaction of a recombinant intein-fusion polypeptide (equipped with a side-chain alkyne or keto group) with a bifunctional (azide/hydrazide or oxyamine/amino-thiol) synthetic precursor.

preceding the intein revealed that 12 out of the 20 possible substitutions at this site are compatible with MOrPH formation, the highest yields (70–90%) being achieved with Phe, Tyr, Ala, and Thr at this site. Additionally, in contrast to the former CuAAC/hydrazide-mediated www.sciencedirect.com

strategy, which was found to proceed exclusively via side-chain ! C-end ligation [71], the oxyamine/aminothiol-mediated cyclization involves primarily a C-end ! side-chain tandem ligation. Overall, these complementary methods provide a unique means to modulate the Current Opinion in Structural Biology 2013, 23:571–580

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topology, ring size, and functionalization pattern of hybrid organo-peptide compounds and are expected to facilitate future applications toward the identification of bioactive macrocycles via the combinatorial assembly and screening of diverse MOrPH libraries.

5.

Jin L, Harrison SC: Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophilin. Proc Nat Acad Sci U S A 2002, 99:13522-13526.

6.

Finking R, Marahiel MA: Biosynthesis of nonribosomal peptides. Annu Rev Microbiol 2004, 58:453-488.

7.

Fischbach MA, Walsh CT: Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem Rev 2006, 106:3468-3496.

Conclusion The contributions highlighted above illustrate the recent progress toward making available versatile methodologies to generate macrocyclic peptides, and diversified libraries thereof, by exploiting the combinatorial potential inherent to the ribosomal polypeptide synthesis in conjunction with creative chemical cyclization strategies. From a structural standpoint, a particularly attractive feature of these approaches is the potential to give access to molecular scaffolds that encompass regions of the chemical space which have so far remained unexplored in chemical biology and drug discovery, overlapping with that of peptide natural products (Figure 1) and lying in between that covered by the conventional classes of probes and therapeutics, namely small molecules and biologics (e.g. antibodies) [1,2]. As illustrated by some of the reviewed studies, the ability to integrate these methodologies with molecular display/functional selection platforms has provided valuable tools toward the isolation of bioactive molecules against a variety of therapeutically important targets, including kinases, proteases, and membrane receptors. Further expansion and application of the methodological repertoire available to create and functionally interrogate macrocyclic peptide libraries is expected to disclose new exciting opportunities toward the discovery of functional entities, in particular against biomolecular targets which have remained difficult to target via conventional classes of therapeutic agents.

Acknowledgements This work was supported by the U.S. National Science Foundation Grant CHE-1112342. J.M.S. also acknowledges the U.S. NSF Graduate Research Fellowship program for financial support.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

2.

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580 Engineering and design

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transferase-mediated chemoenzymatic coupling of N-terminal Arg/Lys units in post-translationally processed proteins with nonnatural amino acids. Chembiochem 2006, 7:1676-1679. 67. Hamamoto T, Sisido M, Ohtsuki T, Taki M: Synthesis of a cyclic  peptide/protein using the NEXT-A reaction followed by cyclization. Chem Commun (Camb) 2011, 47: 9116-9118. New chemoenzymatic approach to generate cyclic peptides that relies on the N-terminal modification of ribosomal peptides with Leu/Phe-tRNAprotein transferase followed by cysteine-mediated alkylation. 68. Touati J, Angelini A, Hinner MJ, Heinis C: Enzymatic cyclisation of peptides with a transglutaminase. Chembiochem 2011, 12:38-42. 69. Beninati S, Piacentini M: The transglutaminase family: an overview: minireview article. Amino Acids 2004, 26:367-372. 70. Yokoyama K, Nio N, Kikuchi Y: Properties and applications of microbial transglutaminase. Appl Microb Biotechnol 2004, 64:447-454. 71. Smith JM, Vitali F, Archer SA, Fasan R: Modular assembly of  macrocyclic organo-peptide hybrids using synthetic and genetically encoded precursors. Angew Chem Int Ed 2011, 50:5075-5080. Novel strategy for the synthesis of organo-peptide macrocycles via a tandem CuAAC/hydrazide-mediated ligation between a recombinant protein and a bifunctional synthetic reagent. The topology of these macrocycles could be readily diversified by varying the nature of the genetically encoded and synthetic precursors. 72. Satyanarayana M, Vitali F, Frost JR, Fasan R: Diverse organopeptide macrocycles via a fast and catalyst-free oxime/inteinmediated dual ligation. Chem Commun (Camb) 2012, 48: 1461-1463. 73. Frost JR, Vitali F, Jacob NT, Brown MD, Fasan R: Macrocyclization of organo-peptide hybrids through a dual  bio-orthogonal ligation: insights from structure–reactivity studies. Chembiochem 2013, 14:147-160. Systematic analysis of the scope and mechanism of a catalyst-free, oxime-/intein-mediated ligation method for MOrPH synthesis. This methodology was found to constitute a robust and versatile platform for the construction of structurally diverse, natural product-inspired macrocycles.

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