Recent Advances in Synthesis and Identification of Cyclic Peptides for

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REVIEW ARTICLE ISSN: 1568-0266 eISSN: 1873-5294

Recent Advances in Synthesis and Identification of Cyclic Peptides for Bioapplications

Impact Factor: 3.402

Current Topics in Medicinal Chemistry

BENTHAM SCIENCE

Yong Siang Ong1, #, Liqian Gao1, #, Karunakaran A. Kalesh2, Zhiqiang Yu3,*, Jigang Wang4, Chengcheng Liu5, Yiwen Li6, Hongyan Sun7,** and Su Seong Lee1,*** 1

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore; Department of Chemical Engineering, Imperial College London, South Kensington Campus, London - SW7 2AZ, United Kingdom; 3School of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University, Guangzhou, Guangdong 510515, China; 4College of Life Sciences, The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 163 Xianlin Avenue, Nanjing, 210023, China; 5 Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore; 6 College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, China; 7Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China 2

ARTICLE HISTORY Received: February 24, 2016 Revised: July 28, 2016 Accepted: July 31, 2016 DOI: 10.2174/1568026617666170224121658

Abstract: Cyclic peptides, owing to their good stability, high resistance to exo- and to some extent endo-peptidases, enhanced binding affinity and selectivity towards target biomolecules, are actively investigated as biochemical tools and therapeutic agents. In this review, we discuss various commonly utilized synthetic strategies for cyclic peptides and peptoids (peptidomimetics), their important screening methods to identify the bioactive cyclic peptides and peptoids such as combinatorial beadbased peptide library, phage display, mRNA display etc. and recent advances in their applications as bioactive compounds. Lastly, we also make a summary and provide an outlook of the research area.

Keywords: Cyclic peptide, Cyclization, Bead-based library, Phage display, mRNA display, Computational. 1. INTRODUCTION Identification of a new drug-lead with sufficient structural novelty to push through the intellectual property space is one of the most challenging early-stage hurdles in drug discovery programs,although there are different ways to improve the efficicacy for many drugs available [1-11]. So far, although small-molecules are still the mainstay of therapeutic market, many large-size biological entities such as proteins, nucleic acids, antibodies and peptides are increasingly gaining momentum as drug candidates recently. Peptides offer high structural diversity, large surface area for interaction with strong binding affinity, excellent selectivity towards their biological targets, relatively straight forward *

Address correspondence these authors at the School of Pharmaceutical Sciences, Guangdong Provincial Key Laboratory of New Drug Screening, Southern Medical University, Guangzhou, Guangdong 510515, China; Tel: +86-20-62789465; Fax: +86-20- 61648655; E-mail: [email protected]; ** Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China; Tel: +852-3442 9537; Fax: +852-3442 0522; E-mail: [email protected]; ***Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore; Tel: +65-6824-7132; Fax:+65-6478-9080; E-mail: [email protected] # Equal contribution 1873-4294/17 $58.00+.00

synthesis, better safety profiles and tolerability [12-15]. However, linear peptides suffer many limitations for development as therapeutic agents. These include poor oral bioavailability, poor membrane permeability, high susceptibility to proteolysis, susceptibility to oxidation, short half-life and fast elimination [16]. Cyclic peptides could potentially overcome many of these limitations. For instance, typically, cyclic peptides, compared to their linear counterparts, demonstrate improved in vivo stability as they are resistant to exopeptidases and to a lesser extent to endopeptidases as well [17-19]. Cyclization typically decorates these entities into bioactive conformation, ensuring good target selectivity, whereas the minimized entropy penalty during their target binding ensures stronger binding [20, 21]. Cyclization, in some cases, has been found to be beneficial towards cell penetration as well [22, 23]. Therefore, although still in its infancy, cyclic peptide scaffolds are gaining traction in the pharmaceutical industry [24]. Natural cyclic peptides, called cyclotides, derived from plants, contain three disulfide bonds connected in a knotted topology [25]. These cyclotides have been reported to possess potent pharmacological properties such as anti-HIV, © 2017 Bentham Science Publishers

Recent Advances in Synthesis and Identification

antimicrobial and cytotoxic activities [26]. Examples of therapeutic cyclic peptides include bacitracin, vancomycin and octreotide which are derived from nature or a synthetic analog of a natural substrate. Along with natural cyclic peptides, many efforts have been put into the discovery of cyclic peptides as bioactive compounds using highthroughput screening methods. There have been two main approaches to generate and screen bioactive cyclic peptides– chemical and biological. The chemical approach mainly uses combinatorial one-bead-one-compound (OBOC) libraries that form a direct platform for high-throughput screening. Cyclic peptides can be chemically synthesized by many different strategies including side chain-to-tail, head-to-tail, side chain-to-side chain and head to side chain linkages [27, 28]. Biological methods including phage display, split-intein circular ligation of peptides and proteins (SICLOPPS) are also well-established. In this review, we will discuss various synthetic cyclization methods, screening strategies for bioactivity and noteworthy biological applications of cyclic peptides and peptoids from the recent literature. Lastly, we provide a summary and outlook of the cyclic peptide research area in the realm of applied biological research. 2. COMBINATORIAL BEAD-BASED PEPTIDE LIBRARY Compared to other high-throughput screening technologies (such as microplates and microarray etc.) [29-37], the OBOC library developed by Lam, et al. [38] in 1991 is also a versatile and powerful tool for screening a large quantity of peptide ligands against target proteins. Cyclic peptide libraries are usually prepared by synthesizing the linear peptides on beads using Fmoc or Boc chemistry, followed by cyclization. The beads act as a solid support for synthesis and screening can be done with the beads or with the cleaved peptides. Positive hits yielded from the screening are identified by Edman degradation or mass spectrometry [39]. Key advantages of OBOC libraries are their customizability and diversity. For a typical hexamer library generated with full 20 natural amino acids at each position, the total number of diverse peptides is 206 (64 million). Peptide libraries with mixture of D and L-amino acids can also be constructed [40] or the library diversity can be exponentially increased using post-translationally modified amino acids [41]. Modification at the N-terminus also allows for tagging peptides with fluorescent dyes or other substrates. The disadvantage of using OBOC is the difficulty in structural determination of positive hits. While the Edman degradation process [42] can be automated, the method fails to identify peptides which have their N-terminus modified. MADLI-TOF/TOF mass spectrometry can also be used to identify the positive hit sequences. However, isobaric amino acids such as lysine/glutamine (K/Q) and leucine/isoleucine (L/I) cannot be distinguished by this method. One of the solutions is to dope the I and Q with 10% glycine (G) so that their MS/MS spectra show a small satellite signal corresponding to the glycine version peptide in addition to the correct molecular mass (e.g. parent molecular peak – FQPNV and satellite peak – FGPNV) [39]. High-throughput screening of OBOC libraries is often conducted with fluores-

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cently labeled targets. One problem associated with dye labeled target proteins for screening is the potential nonspecific interactions between the dye molecules and peptides. It was shown that, in some cases, these unwanted interactions could be removed by using zwitterionic dyes. [43] 2.1. One-Bead-one-Compound (OBOC) Peptide Library For cyclic peptide synthesis, linear peptide is usually synthesized using Fmoc or Boc chemistry and cyclization is done towards the end before the final deprotection step. One of the most common strategies for cyclization is the formation of lactam. The linear peptide is firstly synthesized on the bead (usually TentaGel bead) with the first amino acid bearing a protected carboxylic acid (Fig. 1A). The protecting group is finally deprotected to yield a free carboxyl group which can form a lactam with the N-terminus of the linear peptide. A cleavable linker is used to facilitate the release of the peptide from the solid support after screening for further peptide sequencing or validation. α-allylic protection of acidic amino acids is an orthogonal method for the synthesis of cyclic peptides via the lactam formation [44]. Once the linear peptide synthesis is completed, the α-allyl group is removed by tetrakis(triphenylphosphine)-palladium(0) and cyclization is completed by using a coupling reagent such as benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (Fig. 1A). Many strategies were developed for sequencing cyclic peptides on beads including a “core sequencing peptide” approach where the bead surface has lactam cyclic peptides whereas the inner core of TentaGel beads contains their corresponding linear encoding peptides. This approach has been used to find inhibitors for K-Ras protein [45]. Phenyl isopropyl protecting group is also compatible with Fmoc chemistry. Camperi and co-workers managed to synthesize cyclic peptides using this strategy [46, 47] Cyclization through disulfide bond formation is done using cysteine residues. Linear peptides starting and ending with cysteines are synthesized and then cyclized by disulfide bond between the two flanking cysteine residues (Fig. 1B). While head-to-tail cyclization is fairly common, side chainto-side chain cyclization is also possible [48]. These cyclic peptides were used to find α3β1 integrin binders through incubation with cancer cells [49]. The beads were incubated with the A549 lung cancer cells and positive beads were sieved out using a tetrazolium (MTT) dye which is metabolically converted to formazan by the living cells attached onto the beads surface. The cNGRFEQc beads did not induce growth of A549 cells when antibodies against α3 and β1 integrins were applied hence the authors concluded that the cyclic peptide is a ligand for integrin α3β1. Non-natural amino acids can also be included to expand the diversity of the peptide library. Peptide ligands incorporating non-natural amino acids were found for α3 integrin of CAOV-3 human ovarian adenocarcinoma cell line [50] and MDA-MB-231 breast cancer cell lines, and the later exhibited an IC50 of 57 nM [51]. Besides acting as a peptide ligand, disulfide ligated cyclic peptides were also found to inhibit the stimulated Gα0 GTPase activity of Regulation of G protein signaling 4 (RGP4) at 25-50 µM [48]. The thiol-ene click reaction is another noteworthy cyclization strategy (Fig. 1C). Anseth and co-workers used this

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SH

S Mmt

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SH

O

O

DMSO

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peptide

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peptide

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N

N

Fig. (1). Synthesis of cyclic peptides on beads A) via lactam formation; B) via disulfide bond formation; C) via thio-ene click reaction; D) via azide-alkyne Huisgen cycloaddition.

method to construct RGD peptide derivatives which exhibited IC50 values of 0.2-0.36 µM against inhibition of fibrinogen binding to GPIIb/IIIa [52]. The peptide is first constructed with a monomethoxytrityl (MMT) protected cysteine and an alkene-functionalized lysine. After deprotection of the MMT, a photoinitiator was added to the resin mixture for the click reaction under 365 nm light. Another method is to use the well-established copper (I) catalyzed alkyne-azide cycloaddition click reaction (CuAAC) (Fig. 1D) using alkyne- and azide-functionalized non-natural amino acids [53]. While all the above mentioned examples of cyclic peptides are constructed through chemical means, Pentelute and coworkers demonstrated the use of glutathione S-transferase (GST) to catalyze the macrocyclization process between cysteine and perfluoroaromatic moieties in aqueous buffer [54]. Using this method, the researchers managed to generate a 40mer with 70% yield.

synthesize an OBTC library. This method, pioneered by Liu et al. in 2002, involves controlled synthesis of a coding compound in the interior of the resin while the testing compound is fabricated on the outer layer of the bead [59]. The synthesis (Fig. 2) starts with the swelling of TentaGel NH2 beads in water for 24 h followed by filtering off the water and adding 9-Fluorenylmethyloxycarbonyl-Nhydroxysuccinimide (Fmoc-OSu) in DCM/diethyl ether. In this biphasic solvent environment, the amino groups in the interior of the beads is not affected by the Fmoc-OSu, leaving the surface of the beads exposed and thus derivatized. A coding scaffold moiety (Boc-AA3-(PG2)AA2-(PG1)AA1) is then fabricated onto interior while the exterior protected amino group is spared. AA3’, which is a bi-functional building block that is coded by amino acid AA3, facilitates the connection between the scaffold (S) and the bead at the outer layer. Next, the protecting groups (PG1 and PG2) are removed and the coupling moieties (R1 and R2) are added. Through sequencing, the (R1)AA1 and (R2)AA2 amino acids can be identified and the active compound can be deduced.

2.2. One-Bead-Two-Compound (OBTC) Peptide Library After screening of cyclic peptide libraries, peptides can be cleaved from the beads using a variety of methods such as methionine/CNBr [55], HMBA/NH4OH [56] and ANP/UV [57]. Although sequencing cyclic peptide is possible [58], sequencing linear peptide is still preferred. Conventional OBOC libraries with cyclic peptides fabricated on their surface are not the most ideal method for high-throughput screening. One of the ways to streamline this process is to (i)

NH2 H2N

NH2

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This method has been utilized for identification of bioactive cyclic peptides through the assembly of linear peptide in the interior of the bead (encoding peptide) whilst the surface of the beads displaying cyclic peptides (termed “core sequence peptide” approach). Wu and co-workers have synthesized 4mer, 5mer and 6mer cyclic peptides using this strat-

PG2 R1 Boc AA3 AA2 AA1 PG2

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(vi)

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, AA3

R1

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S R1

(iv)

, AA3

(vii)

R2

R1

H2N AA3 AA2 AA1 R2

S

, AA3

R1

Fig. (2). Synthesis of OBTC libraries with the encoding peptide and binding molecule. (i) Addition of Fmoc-OSu; (ii),(iii) and (iv) Standard solid-phase peptide synthesis; (v) Deprotection of PG1 followed by coupling of R1; (vi) Deprotection of PG2 followed by coupling of R2; (vii) Deprotection of Boc.



egy [45]. The random cyclic peptides were fused with DkbPip-D-homoPhe moiety (Dkb = 3.3-dimehtyl-2-ketobutyryl, Pip = L-pipecpolinate. D-homoPhe = (R)-3-amino-5phenylpentanoic acid), which was shown to bind FK506binding protein 12 (FKBP). The authors used a large-sized cyclic peptide-FKBP complex to create a steric block and inhibit the protein-protein interaction activity of G12V KRas. Confocal image of positive hit beads showed labelled target proteins forming a fluorescent “shell” around the beads while the macro-sized proteins are unable to penetrate into the interior and reach the encoding linear peptide. From a 3 million compounds library screening, 20 sequences were selected as hits and the sequencing results showed preference to larger macrocycles rich in aromatic and basic residues. The best peptide, cyclic D-Nle-Fpa-Arg-D-Nal-Arg-Arg (where D-Nle = D-Norleucine, Fpa = L-4fluorophenylalanine and D-Nal = D-β-napthylalanine) showed an IC50 of 0.70 µM and KD of 0.83 µM although it has poor cell penetration properties. To improve the cell permeability, an amphipathic motif, Arg-Arg-D-Nal -Arg-

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Fpa, was exploited for a second generation library screening with the structure X1-5-D-Nle-Fpa-Arg-D-Nal-Arg-Arg (where X1-5 means 1 to 5 amino acids with 28 diversity each) [60]. Subsequent screening yielded Arg-Arg-D-Nal -ArgFpa-D-Nle-D-Ser-Trp-Thr-D-Ala-Gln which had a slight improvement in IC50 (0.65 µM) and better cell permeability. The sequence, Trp-Thr-D-Ala-Arg-Arg-Arg-D-Nal-Arg-FpaD-Nle-Gln, obtained after Alanine scanning, resulted in further improvement in cell permeability in A549 lung cancer cells and was shown to inhibit the growth of the cancer cells by binding to Ras-GTP and eventual apoptosis. Liu and co-workers [40] have improved the “core sequencing peptide” approach by fabricating an alkyne handle on the linker of the cyclic peptide, improving the earlier works of Hintersteiner and co-workers [61]. The sorting schematic is shown in Fig. (3). After incubation with biotinylated calcineurin (Cn), the positive hits are fished out using streptavidin-coated magnetic beads. The bound protein was washed away and the beads were incubated again with : Calcineurin biotin

:

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N3

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N Linker

Linear peptide

Cleavage Pra Linker

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Linear peptide

Linker

,

R

,

CL

Fig. (3). Sorting schematics using magnetic-colorimetric screening. An OBTC library is first incubated with biotinylated calcineurin. Positive hits bind the magnetic beads which are coated with streptavidin. After magnetic sorting, the positive beads are washed and incubated with biotinylated calcineurin again. Streptavidin-alkaline phosphatase was added, followed by 5-bromo-4-chloro-2-indiyl phosphate (BCIP). Beads with turquoise coloration were chosen and conjugated with tetramethylrhodamine azide (TMR-N3). Because the linkers for cyclic portion (CL) and linear portion (CL’) are orthogonal, they can be cleaved separately for its intended purpose. The linear portion is cleaved using cyanogen bromide (methionine linker) for sequencing and the dye-labelled cyclic peptide is cleaved using sodium hydroxide (base cleavable linker) for fluorescent anisotropy assay.

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biotinylated Cn, followed by addition of streptavidinalkaline phosphatase (SA-AP). Colorimetric assay using 5bromo-4-chloro-2-indoyl phosphate (BCIP) produced turquoise coloration on the bead surface. A third round of screening was done using Texas-red labeled Cn. Validation was done by attaching tetramethyl rhodamine (TMR) azide onto the peptide’s alkyne handle through the artificial amino acid propargylglycine (Pra) using ‘click’ chemistry and cleaving the cyclic peptide from the bead for measurement of its dissociation constant (KD) using fluorescence anisotropy. The linear peptide in the inner core is then cleaved for identification of its sequence. The cyclic peptides were found to bind to the substrate-docking site for nuclear factor of activated T cells (NFAT) with K D values ranging from 0.74-30 µM. The library exemplifies the versatility of OBOC peptide libraries by combining the ability to do screening, sequencing and validation without the hassle of re-synthesis.

naphthylalanine), which was found earlier [64], was incorporated together with a pentamer peptide to form a monocyclic macrocycle library for screening against one of the important signaling enzyme-protein tyrosine phosphatase 1B (PTP1B). While one of the identified hits was potent (IC50 = 31 nM), its cellular uptake was affected due to the increase in ring size. The monocyclic peptide was then converted to a bicyclic peptide which has a cell-penetrating peptide in one ring and a pentamer binding peptide on the other. The bicyclic variant performed much better than the monocyclic peptide in terms of cell permeability and stability in human serum. When the bicyclic inhibitor was applied to A549 cells, phosphotyrosine (pY) levels of a large number of proteins rose in a dose-dependent manner. Although it was demonstrated with only two peptides, this method is a potentially powerful way to fuse two unique properties of cyclic peptides into one molecule.

Lian and co-workers used a similar magneticcolorimetric screening method for the screening of bicyclic peptide library [62]. By using the same OBTC approach, a bicyclic peptide was screened against the proinflammatory protein-tumor necrosis factor-α (TNFα) and the corresponding linear peptide, as an encoding component, was used for sequencing. The synthesis of bicyclic peptide involves the addition of Dap which has a free amine group in the side chain for reaction with the trimesoyl group (Fig. 4). After four rounds of screening and fluorescence anisotropy validations, bicyclo (Phg-Tyr-D-Ala-Lys-Tyr-D-Phe-Gly-D-LysHis-Dap) (termed Anticachexin C1 by the authors) was obtained with a KD of 6.6 µM. Interestingly, the monocyclic variant did not show appreciable binding to TNFα. Anticachexin C1 was also able to inhibit the TNFα-TNFR1 interaction with an IC50 value of 3.1 µM.

2.3. Cyclic Peptoid Library on Beads Peptoids are attractive alternative to peptides as they are resistant to proteolysis [65] and are easy to synthesize. The first successful screening of a combinatorial peptoid library was reported by Zuckermann and co-workers in 1994 [38]. This work identified two novel ligands with Ki of 5 nM and 6 nM for α 1-adrenergic receptor and µ -opiate receptor, respectively. A recent comparison between macrocyclic and linear peptoid libraries also showed superiority of the former over the linear library [66]. In another noteworthy example, selective substitution of a N-methylated cyclic hexapeptide to a peptoid form was found to improve cell-permeability up to 3-fold [67]. Due to their relatively simple synthesis and similarities to peptides, cyclic peptoids have also found their applications in high affinity binding ligands.

Since the bicyclic peptide synthesis is customizable, it was employed as a synergistic enhancement approach for cell permeability by Lian and co-workers [63]. A cellpenetrating peptide, cyclo(FΦRRRRQ) (where Φ is L-

Cyclic peptoids are traditionally synthesized using the submonomer strategy (Fig. 5), developed by Zuckermann [68]). Firstly, a secondary amine is acylated using bromoacetic acid in the presence of N,N’-diisopropylcarbodiimide O

OH H N

HO O

peptide

O

H N

peptide

O

H N

O

pe

pt

NH

HN

NH2

NH2

O

pep

O

tide

H N

id

e

NH

H N

O

O

O

Fig. (4). Two portions of the bicyclic peptide are flanked by a non-natural amino acid, Dap, which provides a free amine group for lactamization with trimesoyl group at the N-terminal. Reaction is completed by using phenol: water: thioanisole: ethanedithiol: anisole: triisopropylsilane: trifluoroacetic acid = 7.5: 5: 5: 2.5: 1: 1: 78. P

O NH + Br

(i) OH

P (iii)

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Br

+ R

NH2

DMSO

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NH O

O

R

H

P (iv)

N Coupling

DIC DMF

P (ii)

N O

H

R n

N

N

Deprotection O

H

R n

Fig. (5). Submonomer strategy. (i): acylation using bromoacetic acid; (ii): nucleophilic displacement of halogen by a primary alkyl amine; (iii): repeat step (i) and (ii). (iv): deprotection of the protecting group (P).



(DIC). The side chain of the N-substituted glycine (NSG) is introduced through the addition of excess of primary amine in DMSO. The peptoid can then be extended to desired length by repeating step 1 and 2; the final deprotection step yields the desired peptoid. While cyclic peptoids were commonly synthesized on beads, most screening procedures are done with cleaved peptoides as the diversity is usually not large to pair them with high-throughput screening strategies like flow cytometry.

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toid analogues with high net positive charge showed better antifungal activity [71]. Similar to the OBTC method of cyclic peptide synthesis, Kwon and Kodadek synthesized a cyclic peptoid with functionalized cysteine handle for immobilization to maleimide-activated slides together with its corresponding linear peptoid for sequencing. As a proof of concept, biotinylated peptides with the same cysteine handle were synthesized, cleaved off from the solid support and serially diluted. The diluted biontinylated peptides were spotted onto the PEGlyated, maleimide-activated glass microscope slides and tested with Cy3-labeled streptavidin. The fluorescence detected was proportional to the amount of peptoid spotted, thus confirming the viability of this highthroughput screening application.

Similar to the case of cyclic peptides, lactamization is one of the most popular cyclization methods used in the synthesis of cyclic peptoids. Peptoids as large as 55 atomic members have been synthesized through side chain-to-tail lactamization [69]. A neutral N-benzyloxyethyl cyclohomohexamer peptoid which possesses high affinity for the first group alkali metal ion [70], was regarded as a viable antimicrobial agent due to its similarity to valinomycin, an excellent K+-carrier which is also a potent antibiotic. Cationic analogues of both linear and cyclic peptoids were reported by Comegna and co-workers and they found that cyclic pep-

Carbon-carbon bond formation using olefin metathesis is well-studied and has been applied in many chemical syntheses. Khan and co-workers analyzed the use of ring-closing metathesis (RCM) to form cyclic peptoids (Fig. 6A) using commercially available catalysts Grubbs’ first and second generation catalysts (G1 and G2 respectively) and Hoveyda-

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Fig. (6). Common cyclization methods for cyclic peptoids. A) Ring-closing metathesis; B) Boronic ester formation; C) Nucleophilic substitution of halogenated triazine; D) Nucleophilic substitution of halogenated triazine yielding bicyclic peptoids; E) Nucleophilic substitution of halogenated alkyl group.


Ong et al.

Grubbs second generation catalyst (HG2) [72]. Using 2% HG2 in methylene chloride at 40 °C for 2 h, cyclic peptoids were formed in good yields with varying ring sizes (4 to 7). Unlike the OBTC strategies used for sequencing, the cyclic peptoids generated by RCM can be oxidatively cleaved using ozone to yield a linear molecule (Fig. 7A) which can be sequenced using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

duction of many bulky cyclization groups into the cyclic peptoid, which may compromise the use of these molecules in biological applications. Cyclization of peptides on solid-phase through nucleophilic substitution using halogenated triazine was described by Scharn et al. in 2001 [75]. The same chemistry can be employed to cyclize peptoids on solid-phase; for instance, via the reaction between cysteine and activated cyanuric chloride at the N-terminus (Fig. 6C). Lee and co-workers used this method to synthesize a cyclic pentamer peptoid library [76]. Cyclic peptoids can be linearized using mchloroperoxybenzoic acid (mCPBA) in the presence of 1N NaOH (Fig. 7B); and cleaved using UV irradiation of a photosensitive ANP linker between the bead and peptoid. Nucleophilic substitution of halogenated triazine has also been used for the construction of triazine-bridged, conformationally constrained and bicyclic peptidomimetic peptoids (Fig. 6D) [77]. These bicyclic peptoids can be converted to their linear forms using the mCPBA oxidation reaction (Fig. 7C) as described before. Similar cyclization technique was also utilized by Oh and co-workers [78] for generating a cyclic pentameric peptoid inhibitor library of Skp2/p300 interaction. One of the identified cyclic peptoids had a KD value of

Synthesis of cyclic peptoids can also be accomplished through intramolecular condensation reaction between phenylboronic acid and a diol in the vicinity [73]. Firstly, the Nterminus is functionalized with a propargyl handle which is used to incorporate the phenylboronic acid using 1,3-dipolar cycloaddition reaction. The peptoids then cyclize spontaneously via the formation of boronic ester between the phenylboronic acid at the N-terminus and the cis-diol of the galactose in aqueous solutions with an excellent yield (80-90%) (Fig. 6B). Using Alizirin Red S assay [74] to assess the free boronic acid concentrations, the authors concluded that the reaction occurs primarily in an intramolecular fashion and that the sugar unit should be in the same molecule as the boronic acid for effective esterification (hence ruling out dimer formation). A downside of this reaction is the intro-

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,,

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Fig. (7). Ring opening methods for sequencing. A) Ozonolysis; B) and C) Oxidation cleavage using mCPBA; D) Through CNBr cleavage of the thioether bond of homocysteine.



3.85 µM when tested with Skp1-Skp2 complex. Using dihydroxyphenylalanine (DOPA)-mediated chemical crosslinking technology [79], the authors showed that the cyclic peptiod interacts exclusively with Skp2 and not Skp1. Further validations revealed that the cyclic pentameric peptoid is able to inhibit Skp2/p300 interaction and thereby promotes p300-mediated p53 acetylation and induces p53-mediated apoptosis in HeLa cells. However, a drawback for this method is the formation of cyclic sulfone peptoid as a minor product after the ring opening step. Moreover, mCPBA, being a strong oxidizing agent, could affect other functional groups besides the thioether. Lee and co-workers, on the other hand, replaced the cysteine residue to a homocysteine residue and functionalized the Nterminus of the peptoid using an alkyl chloride [80]. Cyclization occurs via a nucleophilic substitution reaction between the thiol of homocysteine and the alkyl chloride (Fig. 6E). Due to the similarity of homocysteine to methionine, cyanogen bromide (CNBr) ensures the cleavage of the thioether linkage and generates a linear peptide with a homoserine lactone at the C-terminus (Fig. 7D), which can then be easily and efficiently sequenced by tandem mass-spectrometry. The use of these specialized cyclization methods brings about exciting opportunities in peptoid library synthesis and subsequently in their applications in high-throughput screening for bioactive cyclic peptoids which are more advantageous than conventional cyclic peptides. 3. PHAGE DISPLAY The most widely used phage for peptide display is the filamentous bacteriophage M13. Depending on the ligation of the DNA encoding the peptide of interest onto either pIII or pVIII gene, the peptide can be encoded on the minor or major coat protein, respectively. While peptides fused to pIII have higher copies than pVIII, problems associated with multivalent binding make it difficult to differentiate mediocre binding peptides and peptides with high binding affinity. Usually, target molecules are immobilized onto a solid support and phages with peptides that bind to the target molecule remain on the solid support after washing (Fig. 8). The phages are then eluted and amplified for encoding, and the cycle repeats so as to enrich the phages with high binding affinity. Panning can also be done on cells [81] or in vivo [82]. The success of this method can be seen by the numerous amount of approved or late-stage clinical drugs it has generated [83]. 3.1. Monocyclic Peptides Unlike the OBOC libraries, most phage display cyclic peptides are cyclized by intramolecular disulfide bonds using flanking cysteine residues (CXn C format where X does not contain cysteine). Other cyclization methods includes a linear peptide preceding the cyclic peptide (XiCXnCXj format where X does not contain cysteine) or a random ring sized cyclic peptide (XiCXj where the X residues contain cysteine for cyclization). This method has been successful in sieving out several good binding cyclic peptides [84, 85]. A cyclic nonapeptide was found to bind to the extradomain-B fibronectin (EDB-

2309

Binding

Repeat

Amplifying

Washing

Eluting

Fig. (8). General process for the phage display method (panning).

FN) [86], a marker for angiogenesis and epithelial mesenchymal transition (EMT). It has been reported that EMT relates to the high-grade prostate tumors [87]. This cyclic peptide is able to differentiate human prostate specimens from benign prostatic hyperplasia. One way to streamline the screening process is to conjugate the target molecule to magnetic beads and phages that bind to them are easily selected using magnets [88]. Biotin and streptavidin conjugation are widely used for this purpose. Shomin and co-workers have made use of this method to conjugate biotinlylated Aurora A onto streptavidin modified beads [89]. However, they found that such a method produced cyclic peptides that contained the motif HPQ, which binds to streptavidin matrix more strongly than to Aurora A. To remove the non-specific binding, they tried increase the amount of BSA and Tween 20 for blocking and screened against Aurora A and streptavidin alone. This condition proved to be too harsh as the HPQ motif for streptavidin alone screening was absent and no clear trends emerged from the positive hits from the Aurora A screening. The phage library upon rescreening with more number of washings in buffer containing free streptavidin led to identification of two Aurora A inhibitors with IC50 values of 6 and 7 µM. While not as versatile as OBOC libraries, phage display is also able to find more stable D-peptides binders using a method first described by Schumacher and co-workers called “mirror-image phage display” [90]. The library construction remains unchanged but the screening is done with the mirror image of the original target (i.e. D-protein). The L-peptide is then translated to its D-form and its binding should be specific for the L-protein target. The authors identified a cyclic D-peptide binding with the c-Src homology 3 (SH3) domain of chicken-Src kinase with a K D of 63 µM. L- and Denantiomers of chicken c-Src domain were produced by bacterial expression and synthetically, respectively. The screening showed no obvious similarity between sequences from


Ong et al.

the two protein enantiomers. It is interesting to note that the D-SH3 domain screening produced ligands containing a pair of cysteine residues while not for the L-SH3 domain screening. The formation of disulfide bonds might have produced ligands with favorable binding thermodynamics through lowering of the entropy penalty. While this method is plausible, it is not feasible to chemically synthesize large Dprotein. Screening multi-domain proteins may be practical by finding peptides that bind to one or more of their constituent domains followed by stringing the corresponding peptides as a multi-ligand. Another disadvantage of this method is that it cannot be used to find a D/L mixture peptide. Others have also made use of this method to find Dpeptide ligands. Welch and co-workers have used this technique to find a cyclic octameric D-peptide, PIE7, that binds the gp41 N-trimer pocket and inhibits the HIV-1 entry (BaL strain) with IC50 of 650 pM [91]. Due to the trimeric nature of gp41, the authors tested the idea of multimeric ligand by crosslinking PIE7 using polyethylene glycol (PEG) to form dimer and trimers which have vastly improved IC50 values of 1.9 nM and 250 pM against HXB2, respectively.

in 2009 [94]. Using a disulfide-free gene-3-protein phage, a linear peptide with Cys-X6-Cys-X6-Cys was displayed and cyclized using tri-(bromomethyl)-benzene through nucleophilic substitution reaction (Fig. 9). As a proof of concept, a bicyclic peptide was identified through this method and the fusion protein showed an IC50 of 20 nM with plasma kallikrein while the linear peptide exhibited 250-fold decrease in potency. The authors again used this method to find a bicyclic peptide, UK18, for urokinasetype plasminogen activator (uPA) which inhibits with a Ki of 53 nM [95]. UK18 showed superior proteolytic stability compared to the monocyclic and linear counterparts. Although UK18 remained intact during the first hour of circulation in vivo, its relatively short half-life is still insufficient for it to be used as a therapeutic peptide. The authors tethered UK18 to SA21 (a cyclic peptide with KD of 266 nM for rat albumin [96] and this was found sufficient to increase the conjugated bicyclic peptide’s elimination half-life in mouse by 50-fold to 24 h. Through a replacement of glycine residue at position 13 to D-serine, a new peptide, UK202, showed an increase in potency by 1.75-fold and stability by 4-fold [97]. This shows that while phage display inherently has some limitations, some customization (replacement with D-amino acids, non-natural amino acids or modification, etc.) can still be done after screening to further improve on its function and stability.

Besides targeting proteins and cells, a new technique called ‘internalizing phage’ (iPhage) was introduced by Rangel and co-workers [92]. It integrates peneratin (pen: sequence RQIKIWFQNRRMKWKK) with the recombinant major coat protein (rpVIII) and displays random peptide libraries on the minor coat protein (pIII). Pen is a motif extracted from the third helix of the homeo-domain of Drosophila antennapedia protein and it facilities the transport of prokaryotic viral particles into mammalian cells. Using the parental phage (f88-4) and mutated iPhage that contains the loss-of-function form of pen as controls, the authors were able to show that the peptide-less iPhage enters cells of different species, irrespective of transformation status and tumor type and the mechanism of internalization was receptor independent. A 9mer peptide iPhage library was then panned against live KS1767 cells and a single peptide was isolated from the mitochondria/ER-enriched fraction. Affinity chromatography experiment with the peptide, YKWYYRGAA, showed that it binds to RPL29; and the YKWYYRGAA-pen treated KS1767 cells undergo cell death. As the peptide sequences fabricated on the phage are independent of their ability to internalize, cyclic peptide libraries can also be formed using this method [93].

The same bicyclization method was used to screen for competitive inhibitor of HECT domain’s binding to E2 enzyme [98]. The bicyclic library was incubated with HECT domain of Smurf2, Nedd4, Mule/Huwe1 and WWP1. Because the HECT domains are His-tagged, the proteins were immobilized on Ni-NTA beads and positive hits were eluted using imidazole. Since the goal was to find inhibitor of HECT and E2 enzyme interaction, E2 enzyme UbcH7 was used for elution to pick out the competitive inhibitors. A specific sequence converged for one of the bicyclic rings and was thus used as a fixed template for a second generation library to identity a peptide for the second ring. Further screening also showed convergence of the variable loop to similar motifs. The bicyclic peptides hits for all the five HECT domains (Smurf2, Nedd4, Mule/Huwe1 and WWP1) showed KD and IC50 in the low micromolar range. 4. mRNA DISPLAY Another powerful display system is mRNA display. One of its advantages is its ability to generate a huge diversity of ~1013 unique peptide sequences [99]. mRNA is mainly an in

3.2. Bicyclic Peptides Combinatorial bicyclic peptides are also made possible in phage display by the works pioneered by Heinis and Winter HS

S

SH

Br

HS

S

Br Br

Fig. (9). Bicyclization of a linear peptide displayed on phage.

S



vitro technique like the phage display and combinatorial bead-based peptide screening. Protein synthesis using recombinant elements (PURE) system was used for this purpose [100]. The system is entirely defined – one can customize the number of amino acids, translation factors and aminoacyl-tRNA synthetases to add or omit. By selectively customizing its component, the codons encoding the omitted amino acids can be replaced with non-proteinogenic amino acids with the help of so called flexible tRNA acylation ribozymes (flexizymes). Flexizymes facilitate the charging of non proteinogenic amino acids onto tRNA for transcription. Vacant codons generated through PURE system by omission of the proteinogenic amino acids can now be reassigned with non-proteinogenic charged tRNAs whose anticodons are complementary of the vacant codons. The cDNA template first undergoes transcription to form the mRNA library (Fig. 10). Puromycin’s ability to accept a nascent peptide as a result of the catalytic activity of the ribosome was exploited by Szostak [101] and Yanagawa [102] to develop the puromycin linker-based mRNA display technique independently. The puromycin moiety ligated at the 3’-terminus of the mRNA would enter the ribosome and form mRNA-peptide fusion after the ribosome reaches the end of the RNA open reading frame (ORF). The mRNApeptide fusion would be screened with the target protein carrying an affinity tag or immobilized on a solid support after reverse transcription. Positive hits are isolated, amplified and transcribed for the next round of screening. The combined system was referred to as the Random non-standard Peptides Integrated Discovery (RaPID) system by Suga and coworkers [103].

Because of the wide variety of artificial amino acids that is able to be fabricated, cyclic peptides aside from the classical disulfide linked ones are also possible. Several macrocyclization methods were recently reviewed [104-106]. Both cyclic peptides and bicyclic peptides are attainable with the vast variety of cyclization techniques available. This method was met with success in the identification of a cyclic 12mer peptide for thrombin [107]. A 12mer library consisted of peptides with 10 diversified positions with 2 flanking cysteine residues for cyclization using dibromoxylene. 12 out of the 20 natural amino acids were replaced with unnatural amino acids that display unique functional groups like alkyne, thiaozolidine, etc. Peptide-mRNA fusions that bind to biotinylated thrombin was selected using streptavidin beads and washed with a powerful inhibitor of thrombin called hirudin. In the last three rounds of the ten rounds screening, more stringent washing conditions were used. The mRNA-peptide fusion which remained bound to thrombin for 1 h in the presence of hirudin was amplified and used for the next round. The best cyclic peptide obtained from the 1013 unique combination library had a K D value of 4.5 nM. Both the replacement of the unnatural amino acids and the linear form showed loss of binding. This shows that large diversity libraries can be screened efficiently using the RaPID method. Despite the high-throughput screening capacity, the preparation steps are often long [108] due to construction of the library in a stepwise manner. Ishizawa and co-workers [109] have circumvented this situation by introducing the transcription-translation coupled with association of puromycin linker (TRAP) system. In this system, step from the

(A)

(G)

(F)

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: mRNA : cDNA

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: Ribosome : Polypeptide/ Cyclic peptide : Target protein : Puromycin : Affinity tag/solid support

(E)

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(D) Fig. (10). Schematics for mRNA display screening using RaPID (Random non-standard Peptides Integrated Discovery) system. (A) Transcription of cDNA templates to mRNA library; (B) Ligation of puromycin and ribosomal display of the polypeptide; (C) End of translation; (D) Nascent peptide transfers to puromycin through the A site of ribosome; (E) Reverse transcription; (F) Screening with the target protein which can be linked to an affinity tag or solid support for the downstream selection process; (G) Hit peptide’s cDNA are amplified and transcribed for next round of screening.


Ong et al.

transcription of DNA to mRNA to the formation of mRNApeptide fusion proceeds continuously akin to a one-pot synthesis concept in chemistry (Fig. 11).

5. SPLIT-INTEIN CIRCULAR LIGATION OF PEPTIDES AND PROTEINS (SICLOPPS) SICLOPPS makes use of the Synechocystis sp PCC6803 DnaE split intein to generate cyclic peptide libraries containing up to 108 cyclic peptides [111]. The active cis-intein (fusion protein in Fig. (12) produces cyclic peptides after splicing and subsequent rearrangements. The vector for SICOPPS is generated by inserting an encoding oligonucleotide between the IC and IN genes and an affinity tag gene is sometimes added for characterization and purification [112].

As a proof-of-concept, the TRAP system was used in the screening against human serum albumin (HSA). Using a 10–12mer cyclic peptide library, the authors managed to identify two hits with nanomolar-range binding affinity in 14 h. As a further study, the same group of authors successfully incorporated 16 N-alkylated amino acids into their TRAP system using flexizymes [110]. This study exemplifies the high-throughput screening ability of mRNA display method in sieving out high binding affinity ligands in a vast library.

(A)

SICLOPPS is often integrated with a bacterial reverse two-hybrid system (BRTHS) to identify inhibitors of protein-protein interactions. This system, developed by Horswill, Savinov and Benkovic, facilitates the selection of cy-

(B) : mRNA : cDNA : Ribosome : Puromycin

(C)

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Fig. (11). TRAP system (transcription-translation coupled with association of puromycin linker). (A) The system makes use of T7 RNA polymerase to transcribe the DNA to mRNA; (B) “Trapping” of puromycin-linker which is complementary for the 3’-untranslated region of the mRNA; (C) UAG blank codon placed (due to the omission of release factor 1) immediately before the puromycin linker/mRNA duplex region slows down the dissociation of the puromycin-linker from the mRNA caused by the ribosome helicase activity; (D) Nascent peptide transfer to puromycin-linker/mRNA complex.

peptide

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Fig. (12). Mechanism of SICLOPPS. The first amino acid must be a nucleophilic cysteine or serine for the intein chemistry to work. (i) Formation of intein; (ii) N-to-S acyl shift to produce thioester; (iii) Transesterification to produce lariat; (iv) Asparagine side chain cyclization, leaving the lactone product; (v) X-to-N acyl shift yields the thermodynamically favored lactam (cyclic peptide) product.



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Fig. (13). Schematic representation of RTHS. (Top) Protein fusions (A and B) containing DNA-binding domains (X and Y) are expressed after induction. These fusion proteins bind to their respective DNA-domains (X’ and Y’) and repress a promoter that directs expression of three reporter genes HIS3, KanR and lacZ, inhibiting growth on minimal media. (Bottom) When a cyclic peptide (P) inhibits the interaction between A and B, transcription and translation of the reporter genes rescues growth through induction of reporter genes.

clic peptides that can disrupt interaction between two proteins (Fig. 13) [113]. In the event when two interacting proteins (each fused to a DNA-binding domain) come to close proximity, the downstream transcription of the reporter genes is suppressed, stopping the growth. Conversely, introduction of cyclic peptide that restores growth suggests the disruption of the protein-protein interactions.

contained the cyclic peptides SGWXXXXX and SGWXXPXXPXX were screened. The second library was constructed based on PTAP binding motif discovered previously [118]. One of the hit peptide has inhibitory effect on virus-like particle (VLP) production with an IC50 of 7 µM. Besides using BRTHS, another method developed by Keiler and co-workers combines SICLOPPS and fluorescence activated cell sorting (FACS) for high-throughput screening of cyclic peptide inhibitors. This method was first used to find cyclic peptides that inhibit the proteolysis of tmRNA-tagged proteins in E. coli [119]. It was modified later to find inhibitors of the sRNA that affect the Hfq-RybB pathway (Fig. 14) [120, 121]. In order to find an inhibitor for Hfq-sRNA pathway, a ompC’-yfp fusion gene was constructed. The transcription of rybB allows Hfq-RybB to suppress yellow fluorescent protein (YFP) production through repression of ompC’-yfp mRNA and facilitates its degradation. Conversely, if a cyclic peptide were to inhibit the association of Hfq-RybB and ompC’-yfp mRNA, transcription of ompC’-yfp mRNA would proceed and cells would be bright due to the production of YFP. Positive peptides are then selected using the FACS by detecting the YFP fluorescence intensity. Cells are then sorted by FACS using a cell sorter tuned to detect the YFP fluorescence.

This method has been met with much success in finding cyclic peptides that inhibit protein-protein interactions [114116]. Miranda, Nordgren and co-workers used this system to identify cyclic peptides that inhibit hypoxia inducible factor1 (HIF-1) dimerization [116]. HIF-1 is a heterodimer comprised of an alpha and a beta subunit. The formation of HIF1 transcription factor complex invokes the hypoxia response network and inhibition of HIF-1 is a potential cancer therapy method [117]. A cyclic peptide, cyclo-CLLFVY, was found to have an IC50 of 19 µM in HIF-1-dependent luciferase reporter assay. Through a series of binding tests similar to ELISA, the binding domain was narrowed down to PAS-B domain of HIF-1α. An in vivo test also confirmed the binding to PAS-B domain of HIF-1α, without affecting the HIF2. Tavassoli and co-workers also used the SICLOPPS/RTHS approach to find peptides that can disrupt the interaction between HIV Gag protein and tumor susceptibility gene 101 (TSG101) to inhibit HIV egress [114]. Two libraries which rybB

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(ii)

(i)

No YFP production

rybB

ompC yfp RybB

Transcription

ompC-yfp mRNA Hfq (i)

(ii) YFP production

Cyclic peptide inhibitor

Fig. (14). Schematics of cyclic peptide inhibition screening using SICLOPPS/FACS. (Top) (i) Association of Hfq-RybB and omC’-yfp mRNA; (ii) Degradation of RybB; (iii) No YFP production. (Bottom) (i) Cyclic peptide inhibits the association of Hfq-RybB to omC’-yfp mRNA; (ii) YFP production.


This method found a cyclic peptide inhibitor, albeit a weak one, with an apparent Ki of 111 µM [120]. The mode of inhibition is through the disruption of Hfq and RybB binding. A cyclic peptide inhibitor, S124, for σE pathway was also found using similar method [121]. S124 was able to inhibit the binding of σE to RNAP with a KD of ~32 µM and inhibit transcription from σE-dependent promoters in vitro and in vivo. Similar to SICLOPPS/BRTHS, SICLOPPS/FAC relies on positive readout for detection, the survival of the cells in the case of BRTHS and the gain of fluorescence for FAC. Many recent advances in improving the diversity of SICLOPPS-based screening have been made, including the synthesis of organo-peptide hybrids [122, 123] and formation of bicyclic peptides [124]. These advances would certainly improve and fuel the development of SICLOPPS as a well-rounded high throughput screening platform. 6. COMPUTATIONAL SCREENING Computational modeling assists the drug discovery process by providing critical information about the structureactivity relationship (SAR) [125], cell permeability [126, 127] and solubility of drug candidates [128]. Due to the appreciable time and cost savings, computational screening and structure refinements are often paired with conventional high-throughput screening to hasten the discovery process. By understanding how the protein-protein interaction (PPI) works, one could use the knowledge to design specialized libraries targeting the specific interactions [48]. Computational modeling can also be used after screening to improve the binding of peptides with their target biomolecules. Hao, Serohijos and co-workers used phage display to identify a cyclic peptide binder of cysteine-rich intestinal protein 1 (CRIP1) [129], a marker for breast cancer [130]. One of the cyclic peptides, CLKDNHRSC, with relatively higher binding affinity to CRIP1 and occurrence in multiple phagotypes was chosen as a potential lead for computational refinement. Docking of the peptide on 48 different conformations of CRIP1 revealed the binding site of the peptide; mutations of the bound peptide were then carried out to search for the best peptide with lowest freeenergy change upon binding. The computationally redesigned peptide, CLDGGGKGC, was found to be the most energetically favorable peptide due to the prevalence of glycine residues in its sequence, which contributed to a more extensive van der Waals interaction between the peptide and CRIP1. In the saturation binding experiment, the new peptide performed slightly better than CLKDNHRSC (K D apparent = 2.5 µM and 34.4 µM, respectively). Also, it performed 27.5 times better CLKDNHRSC in the competitive binding assay using FITC-CLDGGGKGC as the substrate for CRIP1. Besides paring with high-throughput screening methods, Gerona-Navarro and coworkers have used computation studies to improve on an existing biological ligand, p53K382ac, for the acetyl-lysine binding bromodomain (BRD) of CREB binding protein (CBP) [131]. The β-turn like octapeptide portion (379-RHK-Kac-LMFK-386) of p53K382ac that is responsible for the binding recognition was explored using molecular dynamics (MD) simulations.

Ong et al.

Based on the results, R379, H380 and M384 were found to be non-critical residues for binding, so R379/H30 and M384 were replaced by cysteines to form a disulfide bond and stabilize the peptide. R379C, M384 mutant peptide performed the best in the competition fluorescent polarization (FP) assay and was chosen for the second round of computational refinement. In this MD simulation, the H380 residue was found to be interacting with different residues in the same acetyl-lysine-binding pocket at a different site. The replacement of H380 to tryptophan was predicted by simulation to improve interaction through the hydrophobic interactions between the H380W mutant peptide and Leu, Val and Ile in the CBP BRD ZA loop. FP assay showed that the second generation mutant peptide is almost 3-fold improvement in IC50 value compared to the first generation mutant peptide and a 24-fold improvement compared to the original linear octapeptide. With the setup of many databases, a plethora of PPI information has fuelled the advancement of in silico screening. Duffy and co-workers relied solely on computational screening to identify a weak binding candidate for thrombin (KD of 545 µM) [132]. A database consisting of 372 pharmacophores (short linear motifs, protein-binding peptides, turns at protein-protein interfaces) was first constructed. The pharamacophores were broken down to interactions like hydrogen bonding, hydrophobic and aromatic regions, and charged interactions. These critical interactions were then used to screen with virtual cyclic peptide libraries with CysX4-Cys and Cys-X3-Cys (X: 20 naturally occurring amino acids) structure. Hits were chosen based not just on similarity in pharamcophore with the reference database but also on their size. The normalized pharamacophore score predicted 958 hits that could block the exosite-1 of thrombin. It was then reduced to 17 potential candidates after second virtual screening using the USRCAT package [133]. One out of the 17 candidates, CEPKFC, showed anti-thrombin activity in a platelet aggregation assay. Despite the low binding affinity, this method has shown that it is possible to identify a cyclic peptide lead through virtual screening. SUMMARY AND CONCLUSION Cyclic peptides have recently gained significant attention in drug discovery and development programs by virtue of their unique structural features. In this review, we have discussed the key cyclization methods used in the synthesis of cyclic peptides/peptoids libraries and important screening methods used to characterize their bioactivity. Cyclic peptides usually show enhanced binding affinity with their biological targets, excellent target selectivity, improved stability and sometimes even better cell permeability compared with the linear peptides of the same or very similar sequences. Due to all the advantages and scopes mentioned above, cyclic peptides have been developed as receptor agonists/antagonists, RNA binding molecules, enzyme inhibitors and a multitude of therapeutic possibilities, including anticancer, immunosuppressive and antibacterial activities [134, 135]. Combinatorial bead based library screening is one of the most customizable screening methods available today. Although the theoretical peptide diversity on the beads is


high, it is usually restricted to the number of beads used during the screening. Another drawback of this technique is the difficulty in the peptide sequence identification. Encoding peptides have been used for this purpose but unwanted interactions can potentially affect the screening results. Fortunately, some newer generation macrocycles can now be converted into their linear sequences through several ring opening mechanisms to do away the need of encoding peptide. Biological methods could offer the advantage of having the ability to do in vitro and in vivo screening. While the diversity of phage display technique and SICCLOPS is very much dependent on their transformation efficiencies, the mRNA display approach does not suffer from this limitation as it does not rely on living organism for transformation to cyclic peptides. mRNA display is capable of generating and screening 1013 unique sequences in one cycle, making it one of the most diversified screening platforms. Due to its unique screening workflow, SICCLOPS-coupled methods are very useful in finding inhibitors for proteinprotein interactions. Today, cyclic peptides can be chemically synthesized by straightforward methods, including cyclizing the two ends of a linear peptide via lactam formation or other chemically stable bonds such as lactone, ether, thioether, disulfide etc.. However, the N-terminus-to-C-terminus (or head-to-tail) cyclization by amide bonds is more predominant in many biologically active cyclic peptides. Upon planning a synthesis, one needs to select a suitable cyclization method to meet the specific requirement of the designated application [136]. The ring size of the cyclic peptide has been identified as an important factor affecting the level of improvements possible in the bioactivity [19]. It is also important to keep in mind that although typically cyclization improves the bioactivity of a linear peptide, it does not always guarantee to provide all intended improvements, for example, cyclization may improve the binding affinity but decrease the cell permeability. In silico methods and experimental high-throughput screening methods, as in the case of small organic molecule-based drug lead developments, greatly facilitate refinement of lead cyclic peptide candidates to highly potent and target specific compounds. It appears that cyclic peptides also hold great promise for overcoming or minimizing the roadblock of oral bioavailability and membrane permeability issues associated with the development of peptide-based therapeutics. As solid phase methods still remain the main explored strategy for experimental-scale synthesis of cyclic peptides, scaling up for industrial-scale production could be a challenge that largely remain unaddressed. Cyclic peptides and peptoids are now considered as a promising class of compounds in drug discovery programs. They are also being actively investigated for tackling some of the difficult classes of therapeutic targets such as protein-protein interactions. Guided by all these important findings together with the advent of various high-throughput screening methods, it is envisioned that more and more cyclic peptides/peptoids could be developed as drug leads for different therapeutic uses or as assay kits for various diagnostic uses.


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CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise. ACKNOWLEDGEMENTS The authors acknowledge the funding support from the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore), and financial support from the Hong Kong Early Career Scheme Grant (No. 21300714), National Natural Science Foundation of China (No. 21572190), and also grants from Natural Science foundation of China (NSFC) Project (81541086), and the Natural Science Foundation of Guangdong Province (2014A030310310). REFERENCES [1]

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[4]

[5]

[6]

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[8] [9]

[10]

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