A Genetically Encoded, Phage-Displayed Cyclic-Peptide Library

Article abstract of DOI:10.1002/anie.201908713

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded N^?-acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4- to 6-fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.

Full text of DOI:10.1002/anie.201908713

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
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Accepted Article
Title: A Genetically Encoded, Phage-Displayed Cyclic Peptide Library
Authors: Wenshe Liu, Xiaoshan Wang, Peng-Hsun Chen, J. Trae
Hampton, Jeffery Tharp, catrina Reed, Sukant Das, Duen-
Shiah Wang, Hamed Hayatshahi, Yang Shen, and Jin Liu
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201908713
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10.1002/anie.201908713
Angewandte Chemie International Edition
RESEARCH ARTICLE
A Genetically Encoded, Phage-Displayed Cyclic Peptide Library
Xiaoshan Shayna Wang,[a] Peng-Hsun Chase Chen,[a] J. Trae Hampton,[a] Jeffery M. Tharp,[a] Catrina
A. Reed,[a] Sukant K. Das,[a] Duen-Shian Wang,[c] Hamed S. Hayatshahi,[c] Yang Shen,[b] Jin Liu,[c] and
Wenshe Ray Liu*[a]
Abstract: Superior to linear peptides in biological activities, cyclic
peptides are considered to have great potentials used as therapeutic
agents. In order to identify cyclic peptide ligands for therapeutic
targets, selection from phage-displayed peptide libraries in which
cysteines are conjugated covalently through either the disulfide bond
or organic linkers has been widely adopted with great success. To
resolve some technical drawbacks related to cysteine conjugation, we
have invented a novel phage display technique in which its displayed
peptides are cyclized through a proximity-driven Michael addition
reaction between a cysteine and an amber codon-coded Ne-acryloyl-
lysine (AcrK). Using a randomized 6-mer library in which peptides
were cyclized at two ends through a cysteine-AcrK linker, we
demonstrated the successful selection of potent ligands for TEV
protease and HDAC8. All selected cyclic peptide ligands showed 4 to
6-fold stronger affinity to their protein targets than their linear
counterparts. As a new addition to the phage display technique, we
believe this novel approach will find broad applications in drug
discovery.
of manufacturing than biologics.[1] Peptides are also extremely
easy to screen. Using peptide display technologies,[2] such as
phage display, which link the displayed peptide phenotype to the
genotype, it is possible for a single researcher to screen a library
of greater than 1010 unique peptides in a matter of days. Despite
these advantages, peptide-based inhibitors have long been
avoided for two reasons. First, peptides are generally
unstructured in solution which leads to an entropic penalty upon
binding to a target. Second, peptides are highly susceptible to
proteolysis when applied in vivo.[3] It has long been known that
macrocyclization can help to overcome some of the
disadvantages of peptides.[4, 5] Macrocyclization imparts a degree
of conformational rigidity to an unstructured peptide, which often
increases the binding affinity of the peptide for its target.[6] Cyclic
peptides are also significantly more resistant to proteolysis.[7] In
several cases this has led to peptides so stable that they have
been successfully used for oral delivery.[8]
Introduction
Traditionally, therapeutic drugs have consisted of small
molecules that are exquisite at binding their receptors. However,
due to their small size, small molecules have achieved little
success at targeting proteins that involve large, relative flat
surfaces for interactions with other molecules. With the
development of the recombinant protein expression technology, a
new class of protein pharmaceuticals, dubbed as biologics, has
emerged. Because of their larger sizes, biologics display far
superior target affinity and selectivity compared to small
molecules. However, their increased size and protein-based
composition lead to poor tissue permeability and metabolic
stability. With their intermediate size between small molecules
and biologics, peptides offer a promising alternative to the two
established classes of pharmaceutics. Being larger than small
molecules, peptides offer increased potency and target selectivity
while maintaining a potential for cell permeability and a lower cost
Figure 1. Representative, existing and proposed cyclization strategies for
phage-displayed peptides. (A) Cyclization through the disulfide bond between
cysteines; (B) Cyclization through covalent conjugation of cysteines with
organic linkers; (C) Representative organic linkers used for cysteine conjugation
to generate mono- and bicyclic peptides; (D) A proposed proximity-driven
cyclization between a cysteine and an electrophilic noncanonical amino acid
(ncAA); (E) An amber suppression-based approach to link the phenotypic ncAA
with the genotypic TAG mutation. The production of a phage with a TAG
mutation at the coding region of its displayed peptide is produced in E. coli cells
that harbour an evolved aminoacyl-tRNA synthetase and amber suppressing
tRNA for the genetic incorporation of the designated ncAA.
Although peptide cyclization generally leads to better
pharmacological properties, cyclizing a linear peptide identified
through screening can have unknown consequences on the
ability of the peptide to bind to a target protein.[9] For this reason,
multiple binary coding systems have been developed for direct
selection of cyclic peptide libraries.[10] These include phage
[a]
X. S. Wang,[+] P.-H. C. Chen,[+] J. T. Hampton, J. M. Tharp, Dr. C. A.
Reed, Dr. S. K. Das and Prof. Dr. W. R. Liu
Department of Chemistry, Texas A&M University, College Station,
TX 77843-3255, USA
E-mail: wliu@chem.tamu.edu
Prof. Dr. Y. Shen
Department of Electrical and Computer Engineering, Texas A&M
University, College Station, TX 77843-3218, USA
D.-S. Wang, H. S. Hayatshahi and Prof. Dr. J. Liu
Department of Pharmaceutical Sciences, UNT Health Science
Center, Fort Worth, Texas 76107, USA
[b]
[c]
[+]
display, bacterial display, yeast display, traditional mRNA display,
11, 12]
RaPID, SICLOPPS, etc.[4,
Screening one-bead-one-
compound libraries is also an effective method for the
identification of potent cyclic peptide ligands.[13] Phages natively
display linear peptides. To cyclize phage-displayed linear
peptides, several chemical approaches have been developed.[14]
One involves the formation of a disulfide bond between two
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201908713
Angewandte Chemie International Edition
RESEARCH ARTICLE
cysteine residues (Figure 1A).[11, 15] There are many examples
about using this strategy to produce disulfide-cyclized peptides
conjugation when AcrK and cysteine are located in close proximity
in a peptide. By installing a cysteine and an AcrK at two ends of a
phage displayed peptide, an automatic cyclization of the peptide
is expected (Figure 2A). There is also an advantage to work with
AcrK. Its acrylamide moiety undergoes Huisgen 1,3-cycloaddition
reaction selectively with a non-fluorescent diphenylnitrilimine
moeity to form an intense blue fluorescent final product.[25] Using
HZC1 (Figure 2B) that undergoes rapid dehydrochloration in
water to release a diphenylnitrile,[26] protein or phage with intact
AcrK can be easily labeled and visualized. However, a proximity-
driven Michael addition reaction between AcrK and cysteine in a
peptide will annihilate the acrylamide moiety, leading to a cyclic
peptide that cannot be labeled by HZC1.
with higher affinity for
a target protein than their linear
counterparts.[16] While beneficial for some in vitro applications,
peptides cyclized in this way cannot be used in vivo as they
cannot withstand the reducing cellular environments. An
alternative strategy relies on the reactivity of nucleophilic thiols
towards small-molecule organic linkers to covalently connect two
cysteines (Figure 1B).[17] This strategy has been successfully
used for the formation of both mono- and bicyclic, phage-
displayed peptide libraries and used to select ligands with
inhibition constants as low as 2 nM (Figure 1C).[18] Although
effective at forming cyclized peptide libraries, this method
modifies native phage cysteines leading to low phage viability.
Attempts have been made to construct phage strains with no
surface cysteines.[19] However, these phages have low viability,
limiting the phage production. Due to the non-selective nature
while conjugating cysteines, most current organic linkers are
symmetric and achiral for avoiding heterogeneity in the phage-
displayed cyclic peptides that might pose significant challenges in
the following synthesis and characterization of selected cyclic
peptides. The requirement of using symmetric, achiral organic
linkers limits the structural diversity of cyclized peptides. To
resolve these limitations, we envisioned that an electrophilic
noncanonical amino acid (ncAA) and
a cysteine can be
genetically installed in phage-displayed peptides in close
proximity for peptide cyclization (Figure 1D). The incorporation of
the ncAA into phages can be achieved by suppressing an amber
mutation in the phage-displayed peptide coding region in E. coli
cells that harbor a ncAA-specific aminoacyl-tRNA synthetase-
amber suppressor tRNA pair and grow in the presence of the
ncAA (Figure 1E).[20] Using this new method for the construction
of a phage display library will afford a genetically encoded, phage-
displayed cyclic peptide library whose spontaneous peptide
cyclization requires neither the use of phage strains with no
surface cysteines nor an organic linker for cyclization.
Encouragingly, several cases featuring proximity-driven reactions
using a genetically encoded ncAA and a cysteine or another
amino acid has recently been demonstrated by Fasan, Suga,
Wang, and their coworkers.[21, 22]
Figure 2. Cyclization of phage-displayed peptides through Michael addition
between a cysteine and a genetically incorporated Ne-acryloyl-lysine (AcrK). (A)
A
diagram that illustrates proximity-driven peptide cyclization between a
cysteine and an electron deficient ncAA; (B) The structure of AcrK and HZC1
whose dissociation product in water is a nitrilimine that reacts selectively with
an acrylamide to show intense blue fluorescence; (C) Two superfolder green
fluorescent protein (sfGFP) derivatives, one with a N-terminal CA5X peptide and
the other with a N-terminal A6X peptide, and their fluorescent labeling with HZC1.
X denotes AcrK. Proteins were denatured first and then analyzed by SDS-PAGE.
In-gel fluorescence was recorded at the blue region with the excitation at 365
nm; (D) Two phage derivatives, one with a N-terminal CA5X peptide and the
other with a N-terminal A6X peptide, and their titer results after reaction with the
N-biotinyl-cysteamine probe and then capturing by streptavidin beads.
Uncaptured phages were analyzed in the titer assay.
To demonstrate the proximity-driven cyclization between a
genetically incorporated AcrK and an adjacent cysteine in a
protein, we expressed superfolder green fluorescent protein
(sfGFP) that had a N-terminal CA5X peptide (X denotes AcrK and
is coded by an amber codon). To express this protein, we
transformed E. coli BL21(DE3) cells with two plasmids. One was
a previous described pEVOL-PrKRS plasmid that contained both
PrKRS and tRNAꢃꢀꢄꢁꢅꢂ genes and the other was a pETduet plasmid
that contained a gene coding the CA5X-sfGFP protein. Growing
the transformed cells in the presence of AcrK afforded CA5X-
sfGFP. Labeling this protein with HZC1 led to no blue fluorescent
product. However, a control sfGFP protein with an N-terminal A6X
peptide, that we expressed similarly to CA5X-sfGFP and reacted
with HZC1, provided an intense blue fluorescent protein band in
a SDS-PAGE gel (Figure 2C). In parallel, we generated two
phages, one with a CA5X peptide and the other with an A6X
peptide at the N-terminus of the coating protein pIII. To construct
the CA5X phage, we inserted a CA5X-coding DNA fragment
between the PelB leader peptide-coding region and the phage
Results and Discussion
Pyrrolysine (Pyl) is a naturally occurring 22nd proteinaceous
amino acid that is genetically encoded by an amber codon.[23] Its
incorporation is mediated by pyrrolysyl-tRNA synthetase (PylRS)
and tRNA . In the past decade, a number of research groups
including ours have evolved PylRS for the genetic incorporation
of more than 100 ncAAs including both lysine and phenylalanine
derivatives into proteins in E. coli. One of these ncAAs is Ne-
acryloyl-lysine (AcrK), a Michael acceptor.[24] We previously
demonstrated that AcrK reacts slowly with a thiol (the second-
order rate constant is 0.004 M-1s-1) at physiological conditions but
can be stably incorporated into proteins in E. coli using an evolved
PylRS mutant (PrKRS) and tRNAꢃꢀꢄꢁꢅꢂ. The slow reaction between
AcrK and cysteine is desirable in that it avoids non-specific
reactions with regular protein cysteines but allow rapid
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10.1002/anie.201908713
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RESEARCH ARTICLE
pIII-coding (gIII) gene in the pADLg3 phagemid vector that we
purchased from Antibody Design Labs. We used the afforded
phagemid pADLg3-CA5X to transform E. coli Top10 cells that also
harbored a plasmid pEVOL-PrKRS-CloDF and a mutant helper
phage plasmid M13K07-g3TAA. pEVOL-PrKRS-CloDF was
derived from pEVOL-PrKRS by switching the replication origin
from p15a to CloDF for its compatible use with a helper phage
magnetic beads for undergoing selection. We carried out three
rounds of affinity-based selection. Eluted phages were clearly
enriched after each round (Supplementary Figure 8). After the
third round, we sequenced 25 phage clones that converged to
only three peptide sequences, CycTev1, CycTev2, and CycTev3
(Supplementary Figure 9). Using solid-phase peptide synthesis,
we synthesized 5-FAM-conjugated CycTev1, CycTev2, as well as
their linear counterparts and then measured their binding affinities
to TEV protease using fluorescence polarization assays. Our
results as shown in Figures 3B-E and Table 1 indicated that both
CycTev1 and CycTev2 bind to TEV protease with a single digit
µM dissociation constant and both cyclic peptides bind to TEV
protease significantly better than their linear counterpart (>6-fold).
These results established the feasibility of using our genetically
encoded phage-displayed cyclic peptide library to identify potent
ligands for protein targets and demonstrated that cyclization
contributes to the binding.
plasmid that typically has
a p15a replication origin. We
constructed M13K07-g3TAA by introducing a deleterious ochre
mutation at the Q350 coding site in the gIII gene of the M13K07
helper phage. Since M13K07-g3TAA had a non-functional gIII
gene, its use together with PADLg3-CA5X drove the synthesis of
a phage that contained pIII expressed only from the later plasmid.
Growing the transformed cells in the presence of AcrK afforded
the CA5X phage. We used the similar approach to produce the
control A6X phage. Following their separation, we incubated them
together with N-biotinyl-cysteamine, a probe that reacts with
acrylamide. We used 60°C to speed up the relative slow Michael
addition reaction. After reaction, we used streptavidin beads to
capture all biotin-conjugated phages and tittered uncaptured
phages. Our result (Figure 2D) showed that more than 90% A6X
phage was captured but about 65% CA5X phage remained in the
supernatant, supporting that the A6X phage had a free acrylamide
moiety but the CA5X phage underwent
a proximity-driven
cyclization to eliminate its acrylamide. The loss of phages during
substantial washing to remove residual N-biotinyl-cysteamine
might contribute to the observation that only about 65% of the
CA5X phage was recovered. To examine whether peptide chain
lengths may significantly impact the proximity driven cyclization
between a cysteine and a AcrK, we synthesized three Cys-
containing peptides (CA5AcrK, CA6AcrK, and CA7AcrK) and their
non-Cys control peptides (A6AcrK, CA7AcrK, and CA8AcrK).
Labeling all six peptides with HZC1 in PBS buffer showed strong
blue fluorescence for non-Cys peptides but low fluorescence for
Cys-containing peptides indicating that all Cys-containing
peptides cyclized (Supplementary Figure 4). Prolonging the
peptide chain length did not significantly deter the cyclization
process.
Figure 3. Selected TEV protease-binding cyclic peptides and their Kd
measurements. (A)
A diagram to show the phagemid structure for the
production of a phage-displayed 6-mer cyclic peptide library; (B) The structure
of 5FAM-CycTev1. CycTev1, highlighted in blue and red, was selected from
phage display; (C) Fluorescence polarization analysis of 5FAM-CycTev1
binding to TEV protease. Data for a linear counterpart of 5FAM-CycTev1 with
no linker is also included; (D) The structure of 5FAM-CycTev2; (E) Fluorescence
polarization analysis of 5FAM-CycTev2 binding to TEV protease. Data for a
linear counterpart of 5FAM-CycTev2 with no linker is also included.
Encouraged by the in vitro results, we advanced to construct
a phage-displayed 6-mer cyclic peptide library. To afford a
phagemid library for the production of phages with displayed
cyclic peptides, we inserted a 24 base-pair DNA fragment that
encoded six randomized amino acids flanked by an N-terminal
cysteine and a C-terminal AcrK between the PelB leader peptide-
coding region and the gIII gene of the pADLg3 phagemid (Figure
3A). 20 clones from this library were sequenced to confirm the
library diversity (Supplementary Figure 7). We used this
phagemid library to transform E. coli Top10 cells that also
contained pEVOL-PrKRS-CloDF and M13K07-g3TAA to afford
close to 109 transformants and then grew the transformed cells in
the presence of AcrK to produce phages. To demonstrate the
viability of using this library to select cyclic peptide ligands for a
protein target, we first tested it on a model protein, TEV protease
that we conjugated with biotin for its loading onto streptavidin
HDAC8 is a Zn2+-dependent histone deacetylase that has
been implicated as a therapeutic target in various diseases
including cancer, X-linked intellectual disability, and parasitic
infections.[27] Notable efforts have been made to identify potent
HDAC8 inhibitors.[28] In order to identify novel cyclic peptide
ligands for HDAC8, we carried out selection from our genetically
encoded, phage-displayed 6-mer cyclic peptide library similar to
that for TEV protease. Out of selected clones that we
subsequently sequenced, the majority converged to a single
sequence CycH8a (Table 1 and Supplementary Figure 10).
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RESEARCH ARTICLE
Table 1. Determined Kd and IC50 values of selected cyclic peptides and their
linear counterparts when binding to their protein targets.
Ligand
Sequence[a]
CWRDYLIX
CWRDYLIK
CQWFSHRX
CQWFSHRK
CQSLWMNX
CQSLWMNK
CQSLWMNNle
Protein target
TEV protease
TEV protease
TEV protease
TEV protease
HDAC8
Kd (µM)
8.2 ± 0.8
50 ± 5
IC50 (µM)
CycTev1
LinTev1
CycTev2
LinTev2
CycH8a
LinH8a
6.9 ± 0.9
39 ± 5
7.1 ± 0.7
> 50
9.7 ± 0.7
HDAC8
LinH8a’
HDAC8
31 ± 7
[a] X denotes AcrK.
Figure 5. The molecular docking results of CycH8a binding to the HDAC8 dimer.
The top panel show different CycH8a conformers binding at two grooves of the
HDAC8 dimer interface. The bottom panel presents the most favorable
conformer of CycH8a binding at each groove.
To determine the affinity of CycH8a to HDAC8, we
synthesized 5-FAM-conjugated CycH8a (Figure 4A) and then
characterized its binding to HDAC8 using the fluorescence
polarization analysis. The result indicated a 7.1 µM dissociation
constant (Figure 4B). We also synthesized 5-FAM-conjugated
LinH8a, a linear counterpart of CycH8a and tested its binding to
HDAC8. However, LinH8a bound very weakly to HDAC8. Due to
the fact that HDAC8 aggregated at a concentration higher than
100 µM, we were not able to collect enough data to accurately
determine Kd for LinH8a though it is estimated to be higher than
50 µM. LinH8a has a positively charged lysine residue; however,
CycH8a has this charge neutralized. To resolve a concern that
this charge difference might contribute to the binding disparity
between the two peptides, we synthesized 5-FAM-conjugated
LinH8a’ in which norleucine (Nle), a neutral amino acid spatially
similar to lysine was installed at the lysine position in LinH8a and
determined its dissociation constant toward HDAC8 as 31 µM
(Table 1 and Supplementary Figure 12). This Kd value is four-
fold higher than that of CycH8a, eliminating the possibility that the
neutral charge of CycH8a contributes significantly to its strong
binding to HDAC8. Therefore, the cyclization is critical to provide
high potency to CycH8a for its binding to HDAC8. For a protein
target, it doesn’t necessarily bind to the active site of the protein
for direct inhibition. To test whether CycH8a can directly inhibit
the deacetylation activity of HDAC8, we adopted a HDAC8 activity
assay[29] as shown in Figure 4C and synthesized the substrate
Boc-AcK-AMC.In this assay, HDAC8 catalyzed the deacetylation
of Boc-AcK-AMC to afford Boc-K-AMC that reacted with the
coupling enzyme trypsin to release the fluorescent AMC, a
compound that we could easily track in a fluorescent plate reader.
As shown in Figure 4D, providing CycH8a to the assay inhibited
the deacetylation of Boc-AcK-AMC by HDAC8. The determined
IC50 value in the conditions of 5 µM HDAC8 and 50 µM Boc-AcK-
AMC is 9.7 µM, close to the determined Kd value. Given that IC50
is not a direct binding affinity indicator and influenced by the
concentration of the substrate, its slightly higher value than Kd
was expected. Collectively, results in Figure 4 demonstrated a
successful application of using our genetically encoded, phage-
displayed cyclic peptide library in identifying a potent cyclic
peptide inhibitor for a therapeutic protein target.
To gain insight into how CycH8a might interact with HDAC8,
we virtually docked CysH8a on HDAC8. HDAC8 naturally occurs
in a dimeric form.[30] Therefore, we investigated both monomeric
and dimeric HDAC8 as the receptor for docking. The docking
results indicated that CycH8a binds weakly to monomeric HDAC8
but fits favorably in two grooves that are at the dimeric interface
of HDAC8 and close to the two active sites (Figure 5). Several
published crystal structures of HDAC8-substrate complexes have
shown that the two dimer interface grooves are also part of
channels for binding peptide substrates. The binding of CycH8a
at the two grooves will block the entry of a peptide substrate to
the two active sites, which provides an explanation for the
inhibition of HDAC8 by CycH8a. This is different from most
HDAC8 inhibitors that have been developed so far. Most HDAC8
inhibitors are hydroxamates or other metal binders that strongly
chelate the active site Zn2+. They bind strongly to the active site
tunnel of HDAC8 with IC50 values typically below 1 µM.[31] Our
modeling results showed that CycH8a doesn’t directly interact
Figure 4. A selected cyclic peptide ligand CycH8a and its binding and inhibition
of HDAC8. (A) The structure of 5FAM-CycH8a. The sequence of CycH8a,
highlighted in blue and red, was selected from phage display; (B) Fluorescence
polarization analysis of 5FAM-CycH8a binding to HDAC8. Data for a linear
counterpart of 5FAM-CycH8a is also included; (C) A diagram to show a
fluorogenic HDAC8 activity assay scheme; (D) The IC50 determination of 5FAM-
CycH8a inhibition of HDAC8 using the assay shown in C.
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10.1002/anie.201908713
Angewandte Chemie International Edition
RESEARCH ARTICLE
with Zn2+ and other residues in the active site, explaining its
relatively lower potency than hydroxamate inhibitors. However,
CycH8a binds close to the active site tunnel of HDAC8. One
possibility to develop more potent HDAC8 inhibitors is to
conjugate an active site-targeting hydroxamate inhibitor and
CycH8a to form a tight-binding, bidentate ligand. This is a
research direction that we are actively exploring right now.
of Texas (grant RP170797), and Welch Foundation (grant A-
1715). We thank Sunshine Z. Leeuwon who proofread the
manuscript.
Keywords: phage display • cyclic peptides • proximity-driven
cyclization • HDAC8 • Ne-acryloyl-lysine
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Conclusion
In summary, we have developed a novel phage display technique
that allows the construction of a genetically encoded, phage-
displayed cyclic peptide library. The cyclization of phage
displayed peptides are achieved by a proximity-driven Michael
addition reaction between a cysteine and an AcrK that flank a
randomized 6-mer peptide sequence. AcrK was encoded by an
amber codon and its incorporation into phages was mediated by
an evolved PylRS-tRNAꢃꢀꢄꢁꢅꢂ pair in E. coli. Applying the developed
library to selection against both TEV protease and HDAC8
afforded cyclic peptide ligands that bind to their protein targets
with single digit µM Kd values and significantly better than their
linear counterparts. As a proof of concept, the current study
involved relatively small size peptides that randomized only 6
residues. It is expected that a library with much bigger randomized
peptides will afford the selection of more potent ligands. Given
that many electrophilic ncAAs have been incorporated into
proteins using the amber suppression mutagenesis approach,[22,
[6] S. Chen, I. R. Rebollo, S. A. Buth, J. Morales-Sanfrutos, J. Touti, P. G. Leiman, C.
Heinis, J. Am. Chem. Soc. 2013, 135, 6562-6569.
[7] D. J. Craik, Science 2006, 311, 1563-1564.
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they can all potentially be used to construct genetically
encoded, phage-displayed cyclic peptide libraries. Since these
ncAAs are structurally diverse, their use will impart different
structural constraints to phage displayed cyclic peptides that will
provide diverse structural diversity beneficial for selection. Unlike
most other binary coding techniques for the construction of cyclic
peptide libraries, the reported method leads to direct, irreversible,
and simultaneous cyclization of displayed peptides right after their
translation. There is no additional chemical intervention
necessary. This feature, shared by the mRNA display-based
RaPID technique, significantly simplifies the construction of cyclic
peptide libraries. As a novel addition to the phage display
technique, we anticipate that this developed technique will find
broad applications in the identification of potent ligands for many
surface receptors and strong inhibitors for enzymes and protein-
protein/DNA/RNA binding interactions.
Experimental Section
Experimental details for the synthesis of all small molecules and peptides,
protein expression, construction of plasmids, phagemids, and the
phagemid library, selection, and characterization of selected peptides are
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RESEARCH ARTICLE
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Entry for the Table of Contents
Research Article
X. S. Wang, P.-H. C. Chen, J. T.
Hampton, J. M. Tharp, C. A. Reed, S. K.
Das, D.-S. Wang, H. S. Hayatshahi, Y.
Shen Y., J. Liu, and W. R. Liu*
Page No. – Page No.
A Genetically Encoded, Phage-
Displayed Cyclic Peptide Library
Using the amber suppression-based mutagenesis approach, Ne-acryloyl-lysine was
genetically encoded in a phage-displayed peptide library for cyclization with a
preinstalled cysteine. Selection from this novel phage display library afforded cyclic
peptide ligands that bind TEV protease and HDAC8 much stronger than their linear
counterparts.
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