Selection-based discovery of druglike macrocyclic peptides.

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ANNUAL REVIEWS

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Further

Annu. Rev. Biochem. 2014.83:727-752. Downloaded from www.annualreviews.org by University of Leeds on 08/18/14. For personal use only.

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Selection-Based Discovery of Druglike Macrocyclic Peptides Toby Passioura,1 Takayuki Katoh,1 Yuki Goto,1 and Hiroaki Suga1,2 1 Department of Chemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan; email: [email protected], [email protected], [email protected], [email protected] 2 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan

Annu. Rev. Biochem. 2014. 83:727–52

Keywords

First published online as a Review in Advance on February 21, 2014

phage display, mRNA display, genetic code expansion, genetic code reprogramming, flexizyme

The Annual Review of Biochemistry is online at biochem.annualreviews.org This article’s doi: 10.1146/annurev-biochem-060713-035456 c 2014 by Annual Reviews. Copyright All rights reserved

Abstract Macrocyclic peptides are an emerging class of therapeutics that can modulate protein–protein interactions. In contrast to the heavily automated high-throughput screening systems traditionally used for the identification of chemically synthesized small-molecule drugs, peptidebased macrocycles can be synthesized by ribosomal translation and identified using in vitro selection techniques, allowing for extremely rapid (hours to days) screening of compound libraries comprising more than 1013 different species. Furthermore, chemical modification of translated peptides and engineering of the genetic code have greatly expanded the structural diversity of the available peptide libraries. In this review, we discuss the use of these technologies for the identification of bioactive macrocyclic peptides, emphasizing recent developments.

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Contents


INTRODUCTION . . . . . . . . . . . . . . . . . . CANONICAL GENETIC CODE–BASED SELECTIONS . . . Intracellular Selections . . . . . . . . . . . . . Phage Display–Mediated Selections . . . . . . . . . . . . . . . . . . . . . . mRNA Display–Mediated Selections . . . . . . . . . . . . . . . . . . . . . . GENETIC CODE MANIPULATION . . . . . . . . . . . . . . . . Genetic Code Expansion . . . . . . . . . . . Genetic Code Reprogramming . . . . . Aminoacyl–Transfer RNA Synthetase–Mediated Genetic Code Reprogramming . . . . . . . . . . Flexizyme-Mediated Genetic Code Reprogramming . . . . . . . . . . . . . . . . GENETICALLY ENGINEERED SELECTIONS OF TARGET-BINDING MACROCYCLIC PEPTIDES . . . . . Selections Involving Genetic Code Expansion . . . . . . . . . . . . . . . . . . . . . . Selections Involving Aminoacyl–Transfer RNA Synthetase–Mediated Genetic Code Reprogramming . . . . . . . . . . Selections Involving Flexible In Vitro Translation System–Mediated Genetic Code Reprogramming . . . . . . . . . . . . . . . . Selections Involving Mixed-Method Genetic Reprogramming . . . . . . . . SUMMARY AND OUTLOOK . . . . . . .

728 729 729 730 736 737 739 740

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743 743

744

744 745 746

INTRODUCTION Peptide-based molecules are appealing scaffolds for the development of novel drugs. Their high structural diversity, often combined with a large surface area for interaction with the target, enables very high affinity to the target with exceptional specificity, as evidenced by the success of peptide therapeutics derived from naturally occurring peptide hormones (1–4). 728

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Moreover, even relatively small peptides can bind to target sites that are not the active sites of enzymes and can modulate protein–protein interactions—characteristics that are not typical of traditional small-molecule drugs. However, despite these characteristics, some aspects of peptides are undesirable in potential therapeutics: Their susceptibility to proteases makes them unstable in vivo; they are not generally orally available; and they cannot cross cell membranes, making them unsuited for use against intracellular targets. Interestingly, several relatively small peptides (generally of the order of 15 residues or fewer) derived from natural sources circumvent these apparent limitations (Figure 1). The best example may be the immunosuppressant cyclosporine, a highly N-methylated, macrocyclic, 11-residue peptide that is orally available, exhibits acceptable pharmacokinetics and acts therapeutically by binding to intracellular target proteins named cyclophilins (5–9). Other clinically useful peptide-based drugs derived from natural sources include the echinocandins (a class of antifungals), actinomycin D (an antibiotic, but used clinically as a chemotherapeutic), and daptomycin and vancomycin (antibiotics) (10, 11). Beyond the clinic, several strongly bioactive, naturally occurring peptides are also known. The highly toxic amanitins, for example, are readily absorbed from the gastrointestinal tract following ingestion and are consequently responsible for approximately 90% of mushroom poisoning fatalities globally (12). Taken together, these examples demonstrate that these relatively small, naturally occurring peptides can exhibit druglike properties by combining the pharmacokinetic qualities of small molecules with the target-binding characteristics of larger peptides. Notably, almost all naturally occurring peptides with potent bioactivities and favorable pharmacokinetic properties possess noncanonical structural characteristics. The most frequently observed of these is some form of macrocyclic motif, which confers structural rigidity (and, thus, increased target-binding


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affinity) and resistance to proteases and plays an important role in biological membrane permeability (10, 13–15). N-Alkylation (commonly N-methylation) of the peptide backbone is also frequently observed and is important for membrane permeability (presumably by reducing the hydrophilicity of the amide backbone) and protease resistance (16–19). Additionally, D-stereochemistry, noncanonical side-chain structures, backbone heterocycles, and other nonstandard moieties are frequently observed in cyclic peptides derived from biological sources; they appear, similar to macrocyclization and N-alkylation, to imbue peptides with improved pharmacokinetic profiles relative to canonical peptides. The appeal of macrocyclic peptides as drug candidates has led to significant research efforts to develop screening methods that can isolate peptides with high target-binding affinities from complex libraries. Although combinatorial chemistry approaches have shown some promise, several recently developed techniques have employed biosynthesis of macrocyclic peptides from libraries of combinatorial genetic templates. Such strategies allow for the synthesis of very diverse libraries and the use of iterative selection/amplification-based screening techniques so that libraries of macrocyclic peptides containing greater than 1013 species can be rapidly screened for binding to a target of interest or activity in cell-based phenotypic assays. Furthermore, the application of genetic code manipulation techniques to these biosynthetic systems and/or posttranslational chemical reactions have greatly expanded the range of chemical moieties that can be synthesized, enabling the screening of libraries of noncanonical peptides that mimic the structural characteristics of naturally occurring bioactive peptides. In this review, we provide an overview of the use of selection methods for the isolation of small macrocyclic peptides with high targetbinding affinities and the development of in vitro genetic code engineering techniques, emphasizing recent developments in these areas.

CANONICAL GENETIC CODE–BASED SELECTIONS Selection-based methodologies are powerful tools for the identification and/or directed evolution of proteins and peptides with novel characteristics. Although a wide range of such techniques [including cell-based, in vitro compartmentalization–based, phage display, and messenger RNA (mRNA) display techniques] have been described in the literature, all are based on a common principle, namely the formation of a link (colocalization in the case of cell- or in vitro compartmentalization–based techniques and physical connection in the case of display techniques) between the genetic material (DNA or RNA) and the protein that it encodes. This link between genotype and phenotype allows for the selection of polypeptides that exhibit a specific property. With respect to studies aiming to identify potential therapeutic molecules, the specific property selected for is usually binding to a specific disease-related target. Such approaches have been particularly useful in the development of therapeutic antibodies (20, 21). However, for selections of small macrocyclic peptides from randomized libraries, cell- or display-based techniques have been the most widely used (Table 1); they form the basis of the material discussed herein.

Macrocyclic peptide: a peptide in which the amide backbone has been cross-linked to itself to generate a cyclic structure Split intein–mediated circular ligation of peptides and proteins (SICLOPPS): a genetic engineering approach for the intracellular synthesis of macrocyclic peptides

Intracellular Selections Some of the pioneering studies in the selection of bioactive macrocyclic peptides were performed using cell-based selection strategies. In these studies, the cyclic peptides were expressed using a so-called split intein–mediated circular ligation of peptides and proteins (SICLOPPS) strategy to form intracellular, head-to-tail, amide-cyclized peptides in situ in bacteria (22, 23). By coupling this expression system with a bacterial reverse two-hybrid selection system (in which disruption of a defined protein–protein interaction in a bacterial clone generates an antibiotic-resistant phenotype), the investigators selected cyclic peptide inhibitors of specific protein–protein interactions (Figure 2) (24, 25). www.annualreviews.org • Druglike Macrocyclic Peptides

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In the first such study, a library of cyclic hexamers consisting of five randomized amino acid residues and an invariant cysteine was screened for heterodimerization inhibitors of the murine ribonucleotide reductase protein, yielding molecules with detectable but modest dimerization inhibitory activities in the micromolar range (26). Subsequent studies using similar approaches identified inhibitors of the cancer-related purine biosynthetic enzyme ATIC and the human immunodeficiency virus (HIV) GAG protein; these findings demonstrated the general applicability of this technique (27, 28). Notably, the inhibitors derived from the latter two studies were also not very potent; the observed half-maximal inhibitory concentrations (IC50 values) were, again, in the micromolar range. In the case of one of the ATIC inhibitors, however, a small-molecule analog with nanomolar inhibitory activity was subsequently derived from the active motif (29). By using a SICLOPPS strategy in combination with slightly different bacterial selection methods from the two-hybrid schemes described above, investigators have also reported cyclic peptide inhibitors of the bacterial ClpXP protease and Dam methyltransferase (both with IC50 values in the micromolar range) (30, 31). Furthermore, the SICLOPPS technology has been extended to selection in eukaryotic cells, allowing for selections based on more complex phenotypes. Using this technique, investigators have found cyclic peptide inhibitors of interleukin-4 signaling in B cells and α-synuclein toxicity in yeast (a model of Parkinson’s disease pathology) (32, 33). A significant advantage of the SICLOPPS technology versus the display-based methodologies described below is that bioactive species can be isolated on the basis of not only affinity screening but also in-cell functional screening. Such screens select for peptides with inhibitory activity rather than peptides that simply exhibit target affinity (the latter may bind to the target without inhibiting its activity). Moreover, they enable screening against relatively complex phenotypes (e.g., the α-synuclein toxicity


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described above) and, as such, can be applied to disease models in which the underlying molecular pathology has not been completely elucidated. However, the necessity of a bacterial or eukaryotic host for SICLOPPS significantly limits the diversity of the library that can be generated; this technology usually permits generation and screening of libraries of fewer than 108 different backbone-cyclized peptides. Note that cyclizations of intein-fused peptides via chemoselective posttranslational chemical reactions, as reported in recent studies (34–38), enable the synthesis of macrocyclic organo–peptide hybrids encoded by DNA. Although such methodologies have not yet been used to select bioactive molecules, they demonstrate the potential of variant SICLOPPS techniques for the synthesis of more structurally diverse peptides.

Phage Display–Mediated Selections In phage display, expression of a randomized protein or peptide on the surface of a bacteriophage virion that contains its cognate genetic material (39) provides the link between phenotype and genotype that is required for selection, enabling the use of relatively large libraries of up to 1010 species. By panning a library of such phages against an immobilized target of interest, one can isolate those with target affinity, and the phages can then be passaged through a bacterial host to generate an enriched library (Figure 3a). Iterative rounds of this process yield phages with high affinity to the target of interest that can be subjected to DNA sequencing to identify the sequence of the expressed peptide. Phage display is a well-established technique that is particularly useful for the isolation or directed evolution of antibody fragments (20, 21, 40). However, phage display selection for smaller peptides had been challenging, given that linear, unconstrained peptides generally do not bind to protein targets with high binding affinities (41). Constraining a peptide library through the inclusion of cysteine–cysteine disulfide linkages can be an effective way to

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N O

O

O

HN

O

O O H N

O

O

N O

N

NH O NH2

O

O

O

HO H

N

N

OO HN

Figure 1

OH

O

N

HN

N HO

O

H N

N H

O O O S O NH

N O

H2N

O HN

N H

OH H HN O

O N H

O

α-Amanitin

Actinomycin D

NH2

O


HN OH O

N N

O

O

O O

NH

NH

O

O

N O

N N

O

O N H

N H

O O

O

HN O

NH2 O

O

N H

HO

Cyclosporin A

O O HN

O NH

NH

O

H N

N NH H

O

O

N

O

HN NH2 HO

O

HN O

N

O

HN

OH O NH

Representative bioactive macrocyclic peptides: the chemotherapeutic actinomycin D, the mycotoxin α-amanitin, the immunosuppressant cyclosporin A, the antibiotics daptomycin and vancomycin, the antifungal agent echinocandin D, and the cyanobacterial secondary metabolite patellamide A.

O O OH

O NH OH

O

Daptomycin

OH HO HO

O

N

N H HN O O H O O N N

HN

O

NH

S

OH O O

OH

OH

O

O N HN

NH N

O

N HN

NH

O

N O

Echinocandin D

Patellamide A HO HO

O HN

H N

O N H

O HO

HO

O

O

O Cl

O O

HO OH H2N

Vancomycin

OH

OH NH2 O HN H H N O N N H O O OH O

Cl

S

O OH

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Head-to-tail backbone macrocyclic Thioetherbridged bicyclic Thioetherbridged bicyclic Thioetherbridged bicyclic

CP1

PK15

UK18

FXII402

Coagulation factor XII

Human urokinase–type plasminogen activator

Human plasma kallikrein

α-Synuclein toxicity

Interleukin-4mediated signaling

Head-to-tail backbone macrocyclic

ClpXP protease

c(SARFV), etc.


Cyclic XB

HIV budding

Dam methyltransferase


c11 (fused with Tat peptide)

Homodimerization of ATIC

Heterodimerization of ribonucleotide reductase subunits

Target/bioactivity

GFP reporter selection in E. coli Methyltransferase activity–based selection in E. coli

Ki = 8 μM

IC50 = 50 μM

Viability selection in a yeast model of synucleinopathy Phage display

Phage display

Phage display

No quantitative study IC50 = 1.7 nM

Ki = 53 nM

Ki = 1.2 μM

Functional retroviral–based selection in A5T4 cells

Reverse two-hybrid selection in E. coli

IC50 = 7 μM

No quantitative study

Reverse two-hybrid selection in E. coli

Reverse two-hybrid selection in Escherichia coli

Selection method

Ki = 17 μM

Kd = 53 μM

Potency

Modification of cysteine by tris(bromomethyl)benzene



SICLOPPS cyclization







Posttranslational chemical reaction

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

Genetic code manipulation Reference

53

52

51

33

32

31

30

28

27

26

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c(SGWYVRNM) Head-to-tail backbone macrocyclic


c-1a


Structural features

ARI

c-RR130

Name

Table 1 Noncanonical peptides derived from selection against a protein target


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Macrocyclic closed via a chemical linker

scFv protein with pboronophenylalanine

scFv protein with sulfotyrosine

Linear peptide with azobenzoyllysine

Linear peptide with benzoylphenylalanine

Head-to-tail backbone macrocyclic with benzoylphenylalanine

Thioether-bridged macrocyclic with multiple noncanonical amino acids

Peptide with a lanthionine-closed cyclic structure

cycGiBP

172–6 (scFv protein)

66CC8-SY (scFv protein)

LA81

APCSSA (bzoPhe) DDV

G12

N1

2(2S,6R)

Sortase A

Thrombin

HIV protease

Streptavidin

Streptavidin

Cellular viability selection in E. coli

mRNA display

mRNA display

IC50 = 960 nM

Kd = 1.5 nM, Ki = 6.3 nM

Kd = 3.0 μM

mRNA display

Ribosome display

Kd = 6.3 μM


Phage display


Phage display

mRNA display

Oxidative elimination of selenalysine to Dha followed by intramolecular 1,4-addition of cysteine onto Dha to produce lanthionine

Modification of cysteine by dibromoxylene


N/A

N/A

N/A

N/A

Bridging two amino groups by bis-NHS cross-linker

ARS-mediated genetic code reprogramming with selenalysine

ARS-mediated genetic code reprogramming with 12 noncanonical amino acids

Amber suppression with benzoylphenylalanine

Four-base codon and amber suppression with multiple noncanonical amino acids

Amber suppression with azobenzoyllysine

Amber suppression with sulfotyrosine

Amber suppression with pboronophenylalanine

Amber suppression with Me Phe (note that Me Phe was not included in the selected peptide)


(Continued )

136

96

134

133

132

131

130

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gp120


Kd = 2.1 μM

ARI

NMethylglucamine

Gαi1


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733

734

Thioether-bridged macrocyclic with a warhead residue (trifluoroacetyllysine) and D-tyrosine

Thioether-bridged macrocyclic with a D-tryptophan and multiple N-methylated amino acids

Thioether-bridged macrocyclic with D-tyrosines

S2iD7

Passioura et al.

CM11 -1

nBL1

VEGFR2

E6AP

Sirtuin2

pfMATE

Akt2

mRNA display

mRNA display

mRNA display

TRAP display

Kd = 3.7 nM, IC50 = 3.7 nM

Kd = 600 pM

Kd = 33 nM

mRNA display


IC50 = 110 nM

Spontaneous cyclization between N-terminal chloroacetyl and cysteine





FIT system–mediated genetic code reprogramming with a chloroacetylated amino acid and ARS-mediated genetic code reprogramming with three noncanonical amino acids

FIT system–mediated genetic code reprogramming with chloroacetyl-Dtryptophan and four N-methylated amino acids

FIT system–mediated genetic code reprogramming with chloroacetyl-Dtyrosine and trifluoroacetyl-lysine

FIT system–mediated genetic code reprogramming with chloroacetyl-Dphenylalanine

FIT system–mediated genetic code reprogramming with chloroacetyltyrosine

manipulation

Genetic code Reference

138

110

127

137

126

30 April 2014

Abbreviations: ARS, aminoacyl–transfer RNA synthetase; Dha, dehydroalanine; FIT, flexible in vitro translation; GFP, green fluorescent protein; HIV, human immunodeficiency virus; Me Phe, N-methylphenylalanine; mRNA, messenger RNA; N/A, not applicable; NHS, N-hydroxysuccinimide; scFv, single-chain variable fragment; SICLOPPS, split intein–mediated circular ligation of peptides and proteins; TRAP, transcription-translation coupled with association of puromycin linker; VEGFR2, vascular endothelial growth factor receptor 2.

Thioether-bridged macrocyclic with D-phenylalanine

Thioether-bridged macrocyclic

Pakti-L1

Posttranslational Selection method

chemical reaction

Potency

Target/ bioactivity

ARI

MaD5

Structural features

Name

Table 1 (Continued)


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IC H2N

H O N

O

XH

N H O

N H O

HS

SH

i. Transformation

HN

O

O HX

N IC O H NH OHX

H2N

O

O

Cyclic peptides expressed in host cell

NH

O IN

O

IC

X

H2N

H2N

O


O

iv. Sequence analysis

O

O

O

Plasmid vector library

O

IN

IN

SH NH2 O

IN IC H2N

NH O X

O

HN

S

NH2 HX NH O N H

O

O

iii. Plasmid preparation ii. Binding selection

Target protein Repressor Operator

Reporter gene

O X H2N O Transcription Operator

Reporter gene

Reverse two-hybrid selection system

Figure 2 Intracellular selection of bioactive macrocyclic peptides using split intein–mediated circular ligation of peptides and proteins (SICLOPPS). A plasmid-based DNA library is used to transform bacteria (i ), in which subsequent expression of each peptide gene causes the formation of a specific macrocyclic peptide through intein-mediated splicing (IN and IC indicate N- and C-terminal inteins, respectively), an N-to-S acyl shift, nucleophilic transesterification (X = S or O), and subsequent removal of the fused intein to generate the lactam via the corresponding lactone. Selection for peptides that disrupt a protein–protein interaction of interest is then performed using a reverse two-hybrid reporter gene system (ii ). Bacteria expressing the reporter gene can be recovered and the peptide expressing plasmids isolated to generate an enriched library (iii ). Iterative rounds of this selection process isolate the bacteria expressing peptides with inhibitory activity against the targeted protein–protein interaction, the amino acid sequence of which can be inferred by sequencing individual clones (iv).

isolate peptides with high target-binding affinities (Figure 3b,c) (42–49); however, such molecules are usually unstable in biological settings due to reduction of the disulfide bonds (50). More recent approaches employing posttranslational chemical cross-linking to produce bicyclic peptides that can be screened by phage

display are useful techniques for the identification of constrained peptides with high binding affinities for protein targets (Figure 3d ). The first report of such an approach (using phage display; chemical cyclization of peptides for mRNA display had been reported previously and is discussed in the section titled mRNA Display–Mediated Selections, below) was from www.annualreviews.org • Druglike Macrocyclic Peptides

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a

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Phage library

Peptide i. Binding selection

iv. Amplification

Immobilized target protein


v. Sequence analysis

iii. Infection into host bacteria

ii. Washing and elution

b Cys

c S S Cys

d Cys S S

Cys

Cys S S

Cys

S

S

Cys

Cys

S Cys

with IC50 values in the high nanomolar–to–low micromolar range; further affinity maturation of the kallikrein inhibitors ultimately yielded inhibitors with IC50 values ranging from 20 to 50 nM. The same approach was subsequently used to identify similar inhibitors of two other human proteases, urokinase-type plasminogen activator and coagulation factor XII (52, 53). This thioether-closed bicyclic scaffold derived from phage displayed linear peptides containing three cysteine residues is an ingenious way to overcome the underlying issue of the reducible disulfide bond, and clearly, bicyclic peptides generated by this method have significantly improved target affinity and protease resistance relative to their linear counterparts. However, all the current reports using this method have employed extracellular proteases as the therapeutic targets (although these targets were carefully chosen to maximize the therapeutic potential of the bicyclic peptides); therefore, whether a more extensive range of proteins may be targeted in this manner remains unclear.

Cys S S Cys

mRNA Display–Mediated Selections Figure 3 Selection of peptide aptamers by phage display. During phage display, a library of peptides is fused to a bacteriophage coat protein, such that each peptide is displayed on the surface of the phage virion that contains its corresponding genetic material (a, top). Panning of the phage library against an immobilized target protein (i ), followed by washing and elution (ii ), is used to isolate phage virions that bind to the target of interest, which are then amplified by passage through a bacterial host (iii, iv). Iterative rounds of this selection process lead to the isolation of phage-expressing peptides with high affinity for the target protein, the amino acid sequence of which can be inferred by sequencing individual clones (iv). Exemplary cyclic peptide structures that have been screened using page display: peptides cyclized through intramolecular formation of (b) a single or (c) multiple disulfide bonds, along with a bicyclic peptide synthesized through the intermolecular reaction of tris-(bromomethyl)benzene with cysteine residues (d ).

Heinis et al. (51), who used a reaction with tris(bromomethyl)benzene to covalently link three cysteine residues separated by randomized regions to synthesize a library of bicyclic peptides expressed on phage virions. Panning of this library against the human plasma proteases kallikrein and cathepsin G yielded inhibitors 736

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The SICLOPPS and phage display studies described above demonstrate the utility of these techniques for the isolation of cyclic or bicyclic peptides with inhibitory activities against target proteins. However, these techniques have some limitations with respect to screening for peptidic drugs. In particular, in each case the requirement that the library pass through host cells for amplification substantially limits both the diversity of the library and the range of chemistries that can be employed. In consequence, alternative in vitro display techniques (such as mRNA display, ribosome display, and complementary DNA display), which do not require a transformation step, allow for both greater library diversity and, when combined with genetic code engineering, a broader range of potential chemistries (54–56). In mRNA display, the link between phenotype and genotype is provided by direct covalent linkage of the mRNA to the peptide


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that it encodes (Figure 4a) (57–59). This link is achieved through the use of a puromycin moiety that is chemically ligated to the 3 end of the mRNA prior to translation. Puromycin is an analog of the acylated 3 end of an aminoacylated transfer RNA (tRNA) in which the ester bond between the amino acid and the tRNA is substituted with an amide bond. Following translation, the puromycin moiety enters the A site of the ribosome and is catalytically ligated to the C terminus of the peptide chain, thereby forming the covalent link between the newly translated peptide and the mRNA that encoded it. As with phage display, peptide–RNA hybrids that bind to the target of interest can be isolated by panning against immobilized target, and the genetic material can be recovered and amplified by reverse transcription and polymerase chain reaction (PCR). The resulting DNA library can be transcribed into an enriched RNA library for further iteration of the screening procedure, or it can be sequenced to identify the peptides selected. A key attribute of mRNA display is that the translation of each mRNA into its cognate peptide can be performed in a cell-free translation system, obviating the need to introduce the genetic material into a host organism. Consequently, the diversity of the library is limited only by the mRNA concentration and the scale of the translation reaction that are technically feasible. In practice, the host organism’s independence allows for the screening of libraries of greater than 1013 species—a few orders of magnitude more diverse than can be achieved using phage display and far more diverse than can be achieved using SICLOPP-based techniques. This procedure allows one to select peptide aptamers to a specific target with very high binding affinities (typically, the low nanomolar range, although picomolar binding affinities have been observed). Indeed, the library diversity achievable using mRNA display is so great that even unconstrained linear peptides with high target-binding affinities can be isolated in some cases (60–66). However, as noted above, the structural rigidity conferred on peptides by cyclization

generally improves affinity for a target protein. Indeed, Millward et al. (67) performed chemical cyclization by using disuccinimidyl glutarate to cross-link the peptide N terminus to a downstream lysine, forming two amide bonds (Figure 4b). By means of mRNA display using such a library (estimated to contain more than 1012 different species), these authors isolated potent inhibitors against the Gαi1 protein, a signal transducer involved in G protein– coupled receptor signaling (68). Importantly, one of these inhibitors exhibited nanomolar dissociation constant (Kd ) and IC50 values without further affinity maturation, demonstrating the power of using the large libraries afforded by mRNA display in conjunction with a cyclization strategy for the isolation of peptides with extremely high target-binding affinities.

Noncanonical amino acid: any amino acid that differs structurally (including stereochemically) from the 20 canonical amino acids

GENETIC CODE MANIPULATION The selection schemes described above are highly effective techniques for the identification of linear or cyclic peptides composed of canonical amino acid residues with high affinities for specific protein targets. However, as mentioned in the Introduction, naturally occurring bioactive peptides nearly always include unusual structural features such as D-amino acids, noncanonical amino acid side chains, and N-alkylated amide linkages, and these unusual structures are critical for properties such as oral availability, protease resistance, and membrane permeability (10, 13, 14, 16, 17, 19). Thus, selection systems that are restricted to the incorporation of canonical amino acids may have limited applicability to the discovery of natural product–like peptides with favorable pharmacokinetic properties. In nature, some peptidic secondary metabolites (for example, the amanitins and patellamides) are synthesized via ribosomal translation followed by substantial posttranslational modification (69, 70). However, most are synthesized through the activity of nonribosomal protein synthetases (NRPSs), which are large, modular, multiprotein complexes that synthesize peptides in a stepwise, www.annualreviews.org • Druglike Macrocyclic Peptides

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mRNA-independent fashion (71). The peptide product of a given NRPS can be structurally altered through genetic engineering approaches (72, 73), and NRPS gene clusters are amenable to screening by phage display (74). However, the generation of NRPS genetic libraries that

a

DNA library

produce structurally diverse peptide products remains challenging, and to the best of our knowledge, screening of a randomized, NRPSsynthesized cyclic peptide library by phage display to identify novel bioactive peptides has not been reported.

i. Transcription

mRNA library

ii. Conjugation of puromycin linker


vi. Elution and PCR

P

Puromycin P

vii. Sequence analysis

Immobilized target protein

P P

P P

iii. Translation and puromycin reaction v. Binding selection P

Peptide P P

P

P

b

iv. Reverse transcription

P

c H N

H N

S Lys

Met O

Cys

O

e

d

Cys

H N

S

Trp S Cys

O

S

Cys

MeSer

Cys

MeGly MePhe MeAla

738

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Thus, neither canonical ribosomal translation (restricted to the 20 canonical amino acids) nor NRPS-mediated synthesis (not capable of being engineered for sufficient diversity) appears to be well suited to the synthesis of noncanonical peptide libraries for selection-based screening. However, this problem has largely been solved through genetic code manipulation approaches, which enable the introduction of diverse, noncanonical residues during ribosomal translation and can thus be used to synthesize macrocyclic peptides containing diverse structural features in a defined manner from a genetic template.

Genetic Code Expansion During ribosomal translation, the sequence of the synthesized polypeptide is dictated by the sequence of the substrate mRNA, mediated by specific hybridization between the codons of the mRNA and the anticodons of each successive tRNA. However, in addition to the codon–anticodon interactions, the fidelity of translation requires each tRNA to be specifically conjugated to its cognate amino acid in the form of an aminoacyl–transfer RNA (aatRNA). Consequently, by utilizing tRNAs conjugated to noncognate amino acids, one can alter the structure of the synthesized peptide without changing the nucleotide sequence of the substrate mRNA. To date, several different techniques have been developed for synthesis of such noncanonical peptides. Essentially, each technique consists of (a) a method for synthesiz-

ing the noncanonical aa-tRNA and (b) a translation technique to incorporate this species into the peptide. Such a technique was first reported more than 20 years ago; it involved the use of amino acids (both canonical and noncanonical) charged onto tRNAs by use of a chemoenzymatic approach, in which each amino acid was chemically conjugated to a dinucleotide and subsequently ligated onto a tRNA body via an RNA ligase (75, 76). The investigators then incorporated each amino acid into polypeptides through translation of an mRNA template in an Escherichia coli cell lysate system. They used a tRNA with an anticodon complementary to a stop codon, such as UAG (nonsense codon suppression), so the resulting polypeptides contained up to 21 different amino acid residues (20 canonical and 1 additional). Therefore, this technique was named genetic code expansion. This approach has been broadly applied to the expression of proteins with noncanonical functional groups and to diverse biochemical studies (77–80). Alternatively, four-base codons derived from so-called rare codons (the corresponding tRNAs are less abundant than other tRNAs in the species of the translation system) can be used for the assignment of additional amino acids (81–85). A technical limitation of such nonsense and four-base codon suppression methods arises from competing termination by release factors (RFs) and background elongation by the cognate aa-tRNAs, respectively. For instance, when certain N-alkyl-amino acids (76, 86, 87)

Aminoacyl–transfer RNA (aa-tRNA): an amino acid–tRNA hybrid molecule linked through an ester bond; the substrates for ribosomal translation Genetic code expansion: translational synthesis of peptides composed of all 20 canonical amino acids and 1 or more noncanonical amino acids

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure 4 Selection of peptide aptamers by messenger RNA (mRNA) display. (a) In mRNA display, a randomized mRNA library is synthesized from a DNA template through an in vitro transcription reaction (i ) and then ligated to a puromycin moiety (ii ). Translation of this library in an in vitro reaction leads to the synthesis of peptides covalently linked to their cognate mRNAs through the reaction of the puromycin with the C terminus of nascent peptide (iii ). Reverse transcription of the mRNA (iv) into complementary DNA (cDNA), followed by panning against immobilized target (v), isolates peptide–RNA/DNA molecules that bind to the target, which can be recovered and amplified by polymerase chain reaction (PCR) (vi ), followed by transcription and translation. Iterative rounds of this selection process isolate peptides with high affinity for the target protein, the amino acid sequence of which can be inferred by sequencing individual DNA clones (vii ). (b–e) Representative bioactive macrocyclic peptides identified by mRNA display: peptides posttranslationally cyclized by intermolecular reaction with disuccinimidyl glutarate (b) or dibromoxylene (c), and a lantipeptide (d ) and a thioether-cyclized N-methylated peptide (e), both derived from genetically reprogrammed translation reactions. www.annualreviews.org • Druglike Macrocyclic Peptides

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Protein synthesis using recombinant elements (PURE) system: an in vitro translation system composed entirely of a defined mixture of purified recombinant components aa-tRNA synthetase (ARS): one of several naturally occurring protein enzymes that conjugates amino acids onto tRNAs prior to ribosomal translation Genetic code reprogramming: translational synthesis of peptides under conditions in which codons have been completely reassigned to canonical or noncanonical amino acids

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and D-amino acids (75, 76, 86) were charged onto such tRNAs, translational incorporation into the peptide chain was nearly undetectable. Furthermore, the number of noncanonical amino acids that can be simultaneously used in such translation systems is restricted not only because of the limited number of nonsense or rare codons available for suppression but also because of the unavoidable background competition discussed above.

Genetic Code Reprogramming The obstacles to genetic code expansion techniques can be circumvented, however, through the use of a cell-free translation system composed entirely of purified components, such as the so-called protein synthesis using recombinant elements (PURE) system (88). In such a system, specific RFs, aminoacyl–transfer RNA synthetases (ARSs), and/or their cognate amino acids can be omitted from the reaction, which “vacates” the corresponding codon(s) and thereby greatly diminishes any potential competition from endogenous cellular components. In the first report of such an approach, Forster et al. (89) demonstrated that by introducing noncanonical aa-tRNAs (synthesized by the chemoenzymatic method described above) into a fully reconstituted translation reaction, three codons can be reassigned to encode amino acids with noncanonical side chains, creating a de novo genetic code (Figure 5a). Together, these studies demonstrated the expression of

pentamers containing two canonical and three noncanonical amino acids that were precharged onto the designated tRNAs enzymatically and chemoenzymatically, respectively, under single-turnover conditions (i.e., no ARSs or RFs were added to the translation system). Subsequent studies have shown that such reconstituted translation machinery containing the ribosome, elongation factors, ARS(s), and RF(s) has remarkable tolerance for noncanonical amino acids; this approach can be used to synthesize natural product–like peptides with a diverse range of residue structures, including N-methylated and side chain–substituted amino acids (Figure 5b–g) (18, 90–92). Such studies demonstrate the principle of genetic code reprogramming and its use for the synthesis of peptides with diverse chemical structures. In practice, however, the chemoenzymatic method used for the synthesis of aa-tRNAs in these early experiments is relatively complex and laborious, and alternative techniques based on the use of ARSs, or ribozymes with an analogous activity (referred to as flexizymes), have been developed. We discuss each of these methodologies in turn in the following sections.

Aminoacyl–Transfer RNA Synthetase–Mediated Genetic Code Reprogramming One alternative to chemoenzymatic synthesis of noncanonical aa-tRNAs relies on the moderate promiscuity of ARSs for their amino acid

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure 5 Genetic code reprogramming. (a) (Left) The standard codon usage table for translation using the 20 canonical amino acids. Translation of a messenger RNA (mRNA) under such conditions causes the synthesis of a polypeptide composed of canonical amino acid residues (area below the table). (Right) Removal of amino acids and/or their cognate amino acid–transfer RNA (tRNA) synthetases (ARSs) “vacates” the corresponding codons from the table, which can then be reprogrammed by inclusion of tRNAs preacetylated with noncanonical amino acids. This example (89) shows reprogramming of asparagine to 2-amino-4-pentynoic acid (yU), threonine to O-methylserine (mSer), and valine to 2-amino-4-pentenoic acid (eU). (b–g) Examples of noncanonical amino acids (or amino acid–like molecules) that can be incorporated into peptides through genetic reprogramming: (b) an α-hydroxy acid, (c) an alkyl-glycine, (d ) an N-methylated amino acid, (e) a D-amino acid, ( f ) a disubstituted amino acid, and ( g) an amino acid with a noncanonical side chain. (h–k) Four examples of intramolecular macrocyclization reactions that can be achieved through incorporation of noncanonical residues in peptides: (h) reaction of a chloroacetylated N terminus with a downstream cysteine residue to form a thioether bond; (i ) seamless amide cyclization through enzymatic liberation of the amino moiety at the N terminus (PDF refers to protein deformylase; MAP refers to methionine amino peptidase), followed by spontaneous rearrangement of ( j ) a cysteine–proline–glycolic acid sequence, hydroxyindole- and benzylamine-mediated oxidative coupling; and (k) Huisgen 1,3-cycloaddition click chemistry. 740

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Genetic code reprogramming

Canonical genetic code 1st U

2nd U Phe Leu

C Ser

A Tyr Stop

C

Leu

Pro

His Gln

A

Ile Met

Thr

Asn Lys

Val

Ala

G

Asp Glu

3rd G Cys U C Trp A stop G U Arg C A G Ser U C Arg A G U Gly C A G


H N

N H

N H NH2 O

O

C

A

3rd

G

U C A G U C A G U C A G U C A G

U

Withdrawal of ARSs and amino acids

C

Addition of aminoacyl tRNAs fMet–tRNAini yU–tRNAAsnB GUU mSer–tRNAAsnB GGU eU–tRNAAsnB GAC Glu–tRNAGlu

mSer

A G

OH

O

H

U

yU

Met eU Glu

c

d

R OH

HO O

e

O

H2N

g R1

R1 R2

OH

H2N O

O

H N

O N H

OH

N H

O

O

O

O

H N

N H

O

H N

O

O

OH O

O mRNA : AUG AAC ACC GUU GAA Peptide : Met Val Thr Asn Glu

h Trp

mRNA : AUG AAC ACC GUU GAA Peptide : Met yU mSer eU Glu

i

H N

Cl

SH

O

O Cys

O

Cys

PDF/MAP fMet

Pro

HS N

N H

O

O

O N

S HN

HOGly

NH2

H N

O

O

j

H N

S

Trp O

Cys

bzaPhe

HOTrp

k NH2

NH

Aha

Pgl

HO N3 K3Fe(CN)6

Cu(l)

HOTrp

bzaPhe

Aha N NH O

H N

HO O

O

Pgl N

N N


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OH

H2 N

O N H

R2

OH

OH

O

S H

OH

HN

O

f R

R

OH

HN R

Translation

S

O

2nd

1st

Translation

HO O

b

Reprogrammed genetic code

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Flexizymes: a family of in vitro evolved RNA enzymes (ribozymes) that function as versatile ARSs

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substrates. Although this promiscuity is not marked, ARSs act upon analogs of their cognate amino acids (e.g., monofluorinated analogues of phenylalanine or tyrosine) (93). Thus, if specific canonical amino acids are replaced with appropriate analogs in a fully reconstituted translation reaction, it is possible to introduce those analogs into the nascent peptide chain. To date, approximately 100 such amino acid analogs have been mischarged onto tRNAs by native ARSs (94). Moreover, it appears that, once charged onto tRNAs, more than half of these noncanonical amino acids are sufficiently compatible with ribosomal translation to allow their incorporation into peptides (95). Using such an approach, one can reassign numerous codons to noncanonical amino acids and thereby synthesize peptides containing 10 or more such nonstandard residues (93, 95–98). The range of chemistries accessible through this method includes N-methylated and α,α-disubstituted amino acids, as well as amino acids with a range of unnatural sidechain structures. Note, however, that amino acids with unnatural side chains appear to be better tolerated by the translational machinery than do backbone-modified amino acids; only two N-methylated (aspartate and histidine) and two α,α-disubstituted amino acids are efficient substrates for both aminoacylation and translation (95). To circumvent this limitation, investigators have developed a hybrid approach involving aminoacylation of tRNAs with canonical amino acids using ARSs followed by chemical N-methylation; this technique enables the synthesis of peptides containing at least N-methylated leucine, threonine, and valine (99–101). Similarly, dehydroamino acid– containing peptides, which cannot be directly synthesized by ribosomal translation, can be generated through posttranslational oxidative elimination of selenolysine (a lysine analog) residues incorporated via ARS-mediated genetic code reprogramming (Figure 4c) (97, 98). Compared with the chemoenzymatic synthesis of aa-tRNAs (described above), the use of ARSs to charge noncanonical amino acid analogs onto tRNAs has substantial advantages Passioura et al.

with respect to facility of use, given that many such analogs can be obtained from commercial suppliers and used in a reconstituted translation reaction without chemical alteration. However, this approach concedes some degree of flexibility because the moderate promiscuity of ARSs allows only for the use of specific analogs. Nevertheless, this technique is a powerful tool for the synthesis of diverse natural product– like libraries and is applicable to selectionbased screening for bioactive molecules, as discussed in the section titled Selections Involving Aminoacyl–Transfer RNA Synthetase– Mediated Genetic Code Reprogramming, below.

Flexizyme-Mediated Genetic Code Reprogramming The second alternative to classical chemoenzymatic synthesis of aa-tRNAs utilizes artificial ribozymes with highly promiscuous aminoacyltransferase activity known as flexizymes. The discovery of these ribozymes through in vitro selection experiments, and details of their properties and use, has been recently reviewed elsewhere (102–105). Flexizymes are small (∼46-nt) artificial RNAs that catalyze the aminoacylation of essentially any tRNA by using an amino acid substrate that has been chemically activated by a leaving group attached through an ester linkage (106, 107). Three such flexizymes are currently in use: eFx (which catalyzes reactions involving amino acids with aromatic side chains activated by a cyanomethyl ester or amino acids with diverse structures activated by a chlorobenzylthioester), dFx (which catalyzes reactions involving amino acids with diverse structures activated by a dinitrobenzylester) and aFx [which catalyzes reactions involving amino acids with diverse structures activated by an (aminoethyl)amidocarboxybenzyl thioester]. Between them, these three flexizymes can charge virtually any amino acid (or any other small molecule with a carboxy moiety that can be activated) onto diverse tRNAs bearing any anticodon desired.


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Importantly, the codon reassignments with noncanonical amino acids using flexizymes are executed by precharged, noncanonical aatRNAs. Therefore, one can create the vacant codons for reassignment by omitting not only the relevant amino acids but also their cognate ARSs. Because trace amounts of canonical amino acids are often carried into the reconstituted translation system with the purified ribosomes, translation factors, and/or tRNAs, the double exclusion of amino acids and cognate ARSs ensures complete knockdown of any potential competition for elongation. Moreover, aa-tRNA synthesis using flexizymes is far more simple to perform than the chemoenzymatic method, and flexizymes accept a much wider range of amino acid substrates than ARSs, including N-alkyl-amino acids (108–112), D-amino acids (112–114), N-acylated amino acids (112), and even exotic peptides containing β- and γ-amino acids (112); the latter two cases are particularly useful for reprogramming the initiation codon. Introduction of the resulting noncanonical aa-tRNAs into the custom-made translation system in which specific codons have been vacated thus enables the incorporation of any translatable amino acid(s) into a polypeptide at any desired position(s). Such a translation system integrated with flexizyme chemistry is referred to as a flexible in vitro translation (FIT) reaction. To date, more than 300 such noncanonical amino acids (and even hydroxy acids) have been introduced into polypeptides through FIT reactions (108–110, 112, 113, 115–127). Consequently, extremely diverse chemical structures based loosely on a peptide backbone have been generated. These structures have included peptoids (N-alkylated polyglycines), (poly)esters, and peptides with diverse patterns of N-methylation and noncanonical side chains. Furthermore, the diverse chemistries accessible through the FIT system permit the synthesis of peptides containing reactive moieties that can mediate intramolecular cyclization. Such incorporation of reactive moieties allows for the synthesis of macrocyclic peptides without the need for intermolecular chemical

cross-linking. As a specific example, the FIT system can be used to synthesize a chloroacetylated N terminus that will spontaneously react with a downstream cysteine residue to form a macrocycle through a nonreducible thioether bond (Figure 5h) (117, 119). Other examples of macrocyclization through introduction of reactive moieties by FIT synthesis include oxidative coupling (124), Huisgen 1,3-dipolar cycloaddition (click chemistry) (120), Michael addition (121), and native chemical ligation strategies (122); several of these approaches have been combined to produce bicyclic peptides with defined structures (Figure 5i–k) (120).

Flexible in vitro translation (FIT) reaction: a PURE reaction that has been genetically reprogrammed using flexizymes to generate the noncanonical aa-tRNAs

GENETICALLY ENGINEERED SELECTIONS OF TARGET-BINDING MACROCYCLIC PEPTIDES Genetic code reprogramming enables mRNA template–directed synthesis of noncanonical macrocyclic peptides with structures analogous to those of naturally occurring druglike molecules. The combination of such techniques with selection methodologies allows for the selection of druglike macrocyclic peptides with high binding affinities for specific targets.

Selections Involving Genetic Code Expansion Early reports of the combined use of selection techniques with noncanonical amino acid incorporation utilized stop codon suppression– mediated genetic code expansion rather than genetic reprogramming per se. For example, proof-of-concept experiments used mRNA display to select for linear peptides incorporating a biotin moiety (128, 129). Subsequently, investigators reported several techniques using different display methodologies to select for linear peptides or proteins containing noncanonical amino acids (130–133). However, to the best of our knowledge, only one study has used codon suppression–mediated genetic code expansion for the selection of macrocyclic peptides containing noncanonical structures (134). In that www.annualreviews.org • Druglike Macrocyclic Peptides

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Random nonstandard peptides integrated discovery (RaPID): a selection system in which nonstandard peptides are generated by FIT synthesis and target binders are isolated by mRNA display

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case, a SICLOPPS-based selection including a single unnatural amino acid was used to identify moderately potent inhibitors (IC50 ≈ 1 μM) of the HIV protease. A possible reason that few studies employ genetic code expansion to identify bioactive macrocyclic peptides with noncanonical structures is that such techniques do not readily allow for the synthesis of molecules with the chemical diversity of naturally occurring cyclic peptides (which often contain multiple noncanonical residues or backbone modifications). For the mRNA template–directed synthesis of such molecules, more radical genetic reprogramming techniques are generally required. Although selection of linear noncanonical peptides using chemoenzymatic synthesis of noncanonical aa-tRNAs has been reported (135), selections of macrocyclic noncanonical peptides have generally been performed with ARSmediated or flexizyme-mediated genetic code reprogramming. These selections are discussed individually in the following section.

Selections Involving Aminoacyl–Transfer RNA Synthetase–Mediated Genetic Code Reprogramming Using an ARS-mediated genetic code reprogramming technique in conjunction with mRNA display, investigators have demonstrated that macrocyclic, noncanonical peptides with strong binding affinity to a desired protein target can be identified. In one such example, Schlippe et al. (96) identified potent inhibitors of the human plasma protease thrombin. In this case, the peptide library consisted of 10 randomized amino acids flanked by cysteine residues and was translated in a genetically reprogrammed PURE translation system such that 8 canonical and 12 noncanonical amino acids were incorporated into the randomized region. Thereafter, posttranslational macrocyclization with dibromoxylene (Figure 4d ) was used to link the cysteine residues. Two of the peptides obtained were characterized in detail; 744

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they had Kd and inhibition constant (Ki ) values in the low nanomolar range. Using a similar technique, Hofmann et al. (136) isolated bioactive artificial lantipeptides that bind to the bacterial target Sortase A. For these experiments, dehydroalanine was incorporated into peptides via selenolysine, as described above, and a subsequent intramolecular Michael-type reaction with cysteine enabled synthesis of a lanthionine moiety, forming a macrocycle. In this case, the macrocyclic peptides bound the Sortase A target with only moderate affinity (Kd values above 1 μM), and inhibition was not observed. Nevertheless, this study demonstrates the feasibility of isolating functional lantipeptides from a genetically reprogrammed library.

Selections Involving Flexible In Vitro Translation System–Mediated Genetic Code Reprogramming The combination of the FIT system with an mRNA display–based screening method, known as random nonstandard peptides integrated discovery (RaPID), has been used to identify numerous bioactive macrocyclic peptides with high binding affinities to therapeutic targets of interest. Similar to the ARS-mediated genetically reprogrammed mRNA display selections described in the previous section, RaPID screening involves translation of a semirandomized puromycin-linked mRNA library in a genetically reprogrammed, fully reconstituted FIT system, such that the resulting nonstandard peptides are covalently linked to the mRNA that encodes them. Iterative rounds of selection and amplification are then performed against the desired target immobilized on magnetic beads, and the enriched mRNA libraries are translated in the same FIT system at each step. In their simplest incarnation, such selections can be performed using a FIT reaction that incorporates primarily canonical amino acids, initiated by a noncanonical D- or Lamino acid bearing a chloroacetyl moiety at


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the N terminus and a downstream cysteine to form a macrocycle through a thioether linkage. Using such an approach, researchers have created potent (IC50 ≈ 100 nM) and isoform-selective cyclic peptide inhibitors of the human AKT2 serine/threonine kinase (126). A similar approach was used to identify macrocyclic peptide inhibitors (again, active at nanomolar concentrations) of the bacterial drug transporter pfMATE. This finding is especially significant because proteins of this family are responsible for drug resistance in both bacterial pathogens and malignant cells (137). Note that this experiment involving three-dimensional X-ray structures of the macrocyclic peptide/pfMATE complexes provided the first example of how the series of macrocyclic peptides that are obtained by RaPID selection interact with a target protein. Selections using libraries including more diverse noncanonical amino acid residues have also been reported. For example, Morimoto et al. (127) identified cyclic peptide inhibitors of the human Sirtuin2 lysine deacetylase from a library of macrocyclic peptides engineered to include a trifluoroacetyl-lysine “warhead” that mimics the acetyl-lysine substrate of the enzyme. As for the AKT2 inhibitors described above, the derived Sirtuin2 inhibitors exhibited high potency (Kd and IC50 values in the singledigit nanomolar range) and significant isoform selectivity. RaPID selection using still more complex noncanonical libraries is also possible. Taking advantage of the FIT system’s ability to translate N-methylated amino acids, Yamagishi et al. (110) screened a library that was engineered to express N-methylated phenylalanine, serine, glycine, and alanine, as well as D-tryptophan (and macrocyclized through an intramolecular reaction between an N-terminal chloroacetyl group and a downstream cysteine, as described in the section titled Flexizyme-Mediated Genetic Code Reprogramming, above) for inhibitors of the human oncoprotein E6AP. Each of the macrocyclic E6AP inhibitors obtained included multiple N-methylated residues and was arguably more similar to

naturally occurring nonstandard macrocyclic peptides compared with any other nonnatural macrocyclic peptide inhibitor discovered to date (Figure 4e). Moreover, they exhibited extremely high affinity for E6AP, with Kd values ranging from subnanomolar to single-digit nanomolar values, and appeared able to disrupt the protein–protein interaction between E6AP and the p53 tumor-suppressor protein.

Selections Involving Mixed-Method Genetic Reprogramming Notably, the ARS-mediated and flexizymemediated genetic code reprogramming techniques described above are not mutually incompatible. That is, it is possible to construct an in vitro translation reaction so that some noncanonical amino acids are incorporated through ARS-mediated reprogramming and some through flexizyme-mediated reprogramming. However, to the best of our knowledge, to date only a single study using such a translation system has been reported (138). This experiment involved the use of several extensively reprogrammed libraries in combination with a modified mRNA display technique known as transcription-translation coupled with association of puromycin linker (TRAP) to identify macrocyclic inhibitors of human vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). In this case, the higher throughput of TRAP compared with conventional mRNA display (hours versus days) allowed for the simultaneous screening of eight different libraries, each of which contained different combinations of noncanonical amino acids (D-tyrosine, N-methylphenylalanine, N-methylhistidine, and cycloleucine, incorporated through ARS-mediated reprogramming), which were cyclized using one of four different N-terminal chloroacetylated noncanonical amino acids (incorporated through flexizyme-mediated reprogramming). As are nearly all of the above-discussed bioactive macrocyclic peptides discovered through the combined use of genetic reprogramming and selection, the identified molecules are www.annualreviews.org • Druglike Macrocyclic Peptides

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highly potent inhibitors of their target protein, with observed IC50 values as low as 20 nM. Moreover, like the E6AP inhibitors discussed above, this inhibition appeared to stem from alteration of the protein–protein interaction between VEGFR2 and the VEGF ligand.

SUMMARY AND OUTLOOK


The combination of high target affinity and specificity, the ability to alter protein–protein interactions, and the potential for small molecule–like pharmacokinetics makes relatively small macrocyclic peptides attractive drug candidates. As discussed above, selectionbased techniques allow for the screening of extremely large libraries of such peptides and, as such, are particularly powerful tools for their isolation. Moreover, it is now possible to screen libraries of natural product–like peptides containing extensive noncanonical structural features through the use of genetic code reprogramming in conjunction with such selection techniques. As described in this review, the state of the art in this field already permits relatively simple identification of macrocyclic peptide ligands that (a) have high affinities and specificities for their targets, (b) exhibit enzyme inhibition with high isoform selectivity, and (c) can disrupt protein–protein interactions. For this reason, the technologies described above are already being used to identify molecules for pharma-

ceutical purposes. However, we believe that further development will be required for the complete realization of these techniques’ potential. In particular, a greater understanding of the general medicinal chemistry of macrocyclic peptides will be required to generate molecules with truly druglike characteristics such as serum stability, cell membrane permeability, and oral bioavailability. Ultimately, we anticipate that such an understanding may lead to the derivation of general rules for the design of peptides with the desired pharmacological properties, which in turn will enable the construction of libraries biased toward such molecules. Undoubtedly, the development of such bioactive macrocyclic peptides will, in most cases, require noncanonical chemistries. As such, we believe that selection strategies that incorporate genetic code reprogramming will be particularly advantageous compared with those that utilize canonical peptide synthesis. However, it is likely that engineering peptides for optimal bioactivity will require the use of chemical structures that are not currently accessible using existing genetic code reprogramming techniques; therefore, we anticipate that improvements in the genetic code reprogramming methodologies will go hand in hand with advances in our understanding of peptide medicinal chemistry. Ultimately, these developments will give rise to a new class of therapeutic molecules identified through selection from highly engineered noncanonical peptide libraries.

SUMMARY POINTS 1. Macrocyclic peptides are an emerging class of therapeutics that can modulate protein– protein interactions. 2. Translational synthesis enables rapid screening of macrocyclic peptide libraries comprising more than 1013 species through the use of iterative selection–based approaches. 3. The use of genetic code manipulation techniques greatly increases the chemical diversity that can be achieved through translational synthesis, thereby allowing for the screening of natural product–like macrocyclic peptide libraries.

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FUTURE ISSUES 1. Investigators need a better understanding of macrocyclic peptide medicinal chemistry to design peptides with more favorable pharmacokinetics. 2. Further development of genetic manipulation and posttranslational modification systems will be required to fully exploit genetic code manipulation and iterative selection of macrocyclic peptides.


DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS Our work was supported by a Japan Society for the Promotion of Science Grant-in-Aid for Specially Promoted Research (21000005) to H.S. and partly supported by the Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Establishment of Molecular Technology towards the Creation of New Functions. LITERATURE CITED 1. Barnett AH, Owens DR. 1997. Insulin analogues. Lancet 349:47–51 2. Koretz RL, Pleguezuelo M, Arvaniti V, Barrera Baena P, Ciria R, et al. 2013. Interferon for interferon nonresponding and relapsing patients with chronic hepatitis C. Cochrane Database Syst. Rev. 1:CD003617 3. Takeda A, Cooper K, Bird A, Baxter L, Frampton GK, et al. 2010. Recombinant human growth hormone for the treatment of growth disorders in children: a systematic review and economic evaluation. Health Technol. Assess. 14:1–209 4. Drucker DJ, Nauck MA. 2006. The incretin system: glucagon-like peptide 1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 368:1696–705 5. Altschuh D, Vix O, Rees B, Thierry JC. 1992. A conformation of cyclosporin A in aqueous environment revealed by the X-ray structure of a cyclosporin–Fab complex. Science 256:92–94 6. Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, Schmid FX. 1989. Cyclophilin and peptidylprolyl cis-trans isomerase are probably identical proteins. Nature 337:476–78 7. Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW. 1984. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226:544–47 8. Holt DW, Mueller EA, Kovarik JM, van Bree JB, Kutz K. 1994. The pharmacokinetics of Sandimmun Neoral: a new oral formulation of cyclosporine. Transplant. Proc. 26:2935–39 9. Takahashi N, Hayano T, Suzuki M. 1989. Peptidyl-prolyl cis-trans isomerase is the cyclosporin A–binding protein cyclophilin. Nature 337:473–75 10. Driggers EM, Hale SP, Lee J, Terrett NK. 2008. The exploration of macrocycles for drug discovery—an underexploited structural class. Nat. Rev. Drug Discov. 7:608–24 11. Perlin DS. 2011. Current perspectives on echinocandin class drugs. Future Microbiol. 6:441–57 12. Berger KJ, Guss DA. 2005. Mycotoxins revisited: part I. J. Emerg. Med. 28:53–62 13. White TR, Renzelman CM, Rand AC, Rezai T, McEwen CM, et al. 2011. On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds. Nat. Chem. Biol. 7:810–17 14. Rezai T, Yu B, Millhauser GL, Jacobson MP, Lokey RS. 2006. Testing the conformational hypothesis of passive membrane permeability using synthetic cyclic peptide diastereomers. J. Am. Chem. Soc. 128:2510– 11 www.annualreviews.org • Druglike Macrocyclic Peptides

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Contents

Annual Review of Biochemistry Volume 83, 2014


Journeys in Science: Glycobiology and Other Paths Raymond A. Dwek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 Lipids and Extracellular Materials William Dowhan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p45 Topological Regulation of Lipid Balance in Cells Guillaume Drin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p51 Lipidomics: Analysis of the Lipid Composition of Cells and Subcellular Organelles by Electrospray Ionization Mass Spectrometry Britta Brugger ¨ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p79 Biosynthesis and Export of Bacterial Lipopolysaccharides Chris Whitfield and M. Stephen Trent p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p99 Demystifying Heparan Sulfate–Protein Interactions Ding Xu and Jeffrey D. Esko p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 129 Dynamics and Timekeeping in Biological Systems Christopher M. Dobson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 159 Metabolic and Nontranscriptional Circadian Clocks: Eukaryotes Akhilesh B. Reddy and Guillaume Rey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 165 Interactive Features of Proteins Composing Eukaryotic Circadian Clocks Brian R. Crane and Michael W. Young p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 191 Metabolic Compensation and Circadian Resilience in Prokaryotic Cyanobacteria Carl Hirschie Johnson and Martin Egli p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 221 Activity-Based Profiling of Proteases Laura E. Sanman and Matthew Bogyo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 249 Asymmetry of Single Cells and Where That Leads Mark S. Bretscher p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 275 Bringing Dynamic Molecular Machines into Focus by Methyl-TROSY NMR Rina Rosenzweig and Lewis E. Kay p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 291

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Chlorophyll Modifications and Their Spectral Extension in Oxygenic Photosynthesis Min Chen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 317 Enzyme Inhibitor Discovery by Activity-Based Protein Profiling Micah J. Niphakis and Benjamin F. Cravatt p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 341 Expanding and Reprogramming the Genetic Code of Cells and Animals Jason W. Chin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379 Genome Engineering with Targetable Nucleases Dana Carroll p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 409 Annu. Rev. Biochem. 2014.83:727-752. Downloaded from www.annualreviews.org by University of Leeds on 08/18/14. For personal use only.

Hierarchy of RNA Functional Dynamics Anthony M. Mustoe, Charles L. Brooks, and Hashim M. Al-Hashimi p p p p p p p p p p p p p p p p p p 441 High-Resolution Structure of the Eukaryotic 80S Ribosome Gulnara Yusupova and Marat Yusupov p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 467 Histone Chaperones: Assisting Histone Traffic and Nucleosome Dynamics Zachary A. Gurard-Levin, Jean-Pierre Quivy, and Geneviève Almouzni p p p p p p p p p p p p p p 487 Human RecQ Helicases in DNA Repair, Recombination, and Replication Deborah L. Croteau, Venkateswarlu Popuri, Patricia L. Opresko, and Vilhelm A. Bohr p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 519 Intrinsically Disordered Proteins and Intrinsically Disordered Protein Regions Christopher J. Oldfield and A. Keith Dunker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 553 Mechanism and Function of Oxidative Reversal of DNA and RNA Methylation Li Shen, Chun-Xiao Song, Chuan He, and Yi Zhang p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 585 Progress Toward Synthetic Cells J. Craig Blain and Jack W. Szostak p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 615 PTEN Carolyn A. Worby and Jack E. Dixon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 641 Regulating the Chromatin Landscape: Structural and Mechanistic Perspectives Blaine Bartholomew p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 671 RNA Helicase Proteins as Chaperones and Remodelers Inga Jarmoskaite and Rick Russell p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 697

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Contents

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ARI

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Selection-Based Discovery of Druglike Macrocyclic Peptides Toby Passioura, Takayuki Katoh, Yuki Goto, and Hiroaki Suga p p p p p p p p p p p p p p p p p p p p p p p p p 727 Small Proteins Can No Longer Be Ignored Gisela Storz, Yuri I. Wolf, and Kumaran S. Ramamurthi p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 753 The Scanning Mechanism of Eukaryotic Translation Initiation Alan G. Hinnebusch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 779 Understanding Nucleic Acid–Ion Interactions Jan Lipfert, Sebastian Doniach, Rhiju Das, and Daniel Herschlag p p p p p p p p p p p p p p p p p p p p p p 813


Indexes Cumulative Index of Contributing Authors, Volumes 79–83 p p p p p p p p p p p p p p p p p p p p p p p p p p p 843 Cumulative Index of Article Titles, Volumes 79–83 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 847 Errata An online log of corrections to Annual Review of Biochemistry articles may be found at http://www.annualreviews.org/errata/biochem

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Annual Reviews It’s about time. Your time. It’s time well spent.

New From Annual Reviews:

Annual Review of Statistics and Its Application Volume 1 • Online January 2014 • http://statistics.annualreviews.org

Editor: Stephen E. Fienberg, Carnegie Mellon University


Associate Editors: Nancy Reid, University of Toronto Stephen M. Stigler, University of Chicago The Annual Review of Statistics and Its Application aims to inform statisticians and quantitative methodologists, as well as all scientists and users of statistics about major methodological advances and the computational tools that allow for their implementation. It will include developments in the field of statistics, including theoretical statistical underpinnings of new methodology, as well as developments in specific application domains such as biostatistics and bioinformatics, economics, machine learning, psychology, sociology, and aspects of the physical sciences.

Complimentary online access to the first volume will be available until January 2015. table of contents:

• What Is Statistics? Stephen E. Fienberg • A Systematic Statistical Approach to Evaluating Evidence from Observational Studies, David Madigan, Paul E. Stang, Jesse A. Berlin, Martijn Schuemie, J. Marc Overhage, Marc A. Suchard, Bill Dumouchel, Abraham G. Hartzema, Patrick B. Ryan

• High-Dimensional Statistics with a View Toward Applications in Biology, Peter Bühlmann, Markus Kalisch, Lukas Meier • Next-Generation Statistical Genetics: Modeling, Penalization, and Optimization in High-Dimensional Data, Kenneth Lange, Jeanette C. Papp, Janet S. Sinsheimer, Eric M. Sobel

• The Role of Statistics in the Discovery of a Higgs Boson, David A. van Dyk

• Breaking Bad: Two Decades of Life-Course Data Analysis in Criminology, Developmental Psychology, and Beyond, Elena A. Erosheva, Ross L. Matsueda, Donatello Telesca

• Brain Imaging Analysis, F. DuBois Bowman

• Event History Analysis, Niels Keiding

• Statistics and Climate, Peter Guttorp

• Statistical Evaluation of Forensic DNA Profile Evidence, Christopher D. Steele, David J. Balding

• Climate Simulators and Climate Projections, Jonathan Rougier, Michael Goldstein • Probabilistic Forecasting, Tilmann Gneiting, Matthias Katzfuss • Bayesian Computational Tools, Christian P. Robert • Bayesian Computation Via Markov Chain Monte Carlo, Radu V. Craiu, Jeffrey S. Rosenthal • Build, Compute, Critique, Repeat: Data Analysis with Latent Variable Models, David M. Blei • Structured Regularizers for High-Dimensional Problems: Statistical and Computational Issues, Martin J. Wainwright

• Using League Table Rankings in Public Policy Formation: Statistical Issues, Harvey Goldstein • Statistical Ecology, Ruth King • Estimating the Number of Species in Microbial Diversity Studies, John Bunge, Amy Willis, Fiona Walsh • Dynamic Treatment Regimes, Bibhas Chakraborty, Susan A. Murphy • Statistics and Related Topics in Single-Molecule Biophysics, Hong Qian, S.C. Kou • Statistics and Quantitative Risk Management for Banking and Insurance, Paul Embrechts, Marius Hofert

Access this and all other Annual Reviews journals via your institution at www.annualreviews.org.

Annual Reviews | Connect With Our Experts Tel: 800.523.8635 (us/can) | Tel: 650.493.4400 | Fax: 650.424.0910 | Email: [email protected]

Selection-based discovery of macrocyclic peptides for the next generation therapeutics.

Macrocyclic Antimicrobial Peptides Engineered from ω-Conotoxin.

Macrocyclic cell penetrating peptides: a study of structure-penetration properties.

Disulfide-rich macrocyclic peptides as templates in drug design.

Protein cocrystallization molecules originating from in vitro selected macrocyclic peptides.

2 Kinase Inhibitors.

Tetrahydroquinoline-derived macrocyclic toolbox: the discovery of antiangiogenesis agents in zebrafish assay.

Synthesis of a large library of macrocyclic peptides containing multiple and diverse N-alkylated residues.

Highly selective inhibition of histone demethylases by de novo macrocyclic peptides.

4a Protease Inhibitor.

Bioinspired strategy for the ribosomal synthesis of thioether-bridged macrocyclic peptides in bacteria.

The renaissance era of peptides in drug discovery at the 14th Naples workshop on bioactive peptides.

Peptide array based discovery of synthetic antimicrobial peptides.

Macrocyclic peptides self-assemble into robust vesicles with molecular recognition capabilities.

Discovery of 12-mer peptides that bind to wood lignin.

Discovery of Short Peptides Exhibiting High Potency against Cryptococcus neoformans.

Construction and screening of vast libraries of natural product-like macrocyclic peptides using in vitro display technologies.

4A Protease with Improved Preclinical Plasma Exposure.

Discovery, Structure Elucidation, and Biological Characterization of Nannocystin A, a Macrocyclic Myxobacterial Metabolite with Potent Antiproliferative Properties.

A novel oxy-oxonia(azonia)-cope reaction: serendipitous discovery and its application to the synthesis of macrocyclic musks.

The effect of a macrocyclic constraint on electron transfer in helical peptides: a step towards tunable molecular wires.

Discovery of a neuroprotective chemical, (S)-N-(3-(3,6-dibromo-9H-carbazol-9-yl)-2-fluoropropyl)-6-methoxypyridin-2-amine [(-)-P7C3-S243], with improved druglike properties.

Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids.

Discovery of a Direct Ras Inhibitor by Screening a Combinatorial Library of Cell-Permeable Bicyclic Peptides.