Cyclic and Macrocyclic Peptides as Chemical Tools To Recognise Protein Surfaces and Probe Protein–Protein Interactions

Abstract Targeting protein surfaces and protein–protein interactions (PPIs) with small molecules is a frontier goal of chemical biology and provides attractive therapeutic opportunities in drug discovery. The molecular properties of protein surfaces, including their shallow features and lack of deep binding pockets, pose significant challenges, and as a result have proved difficult to target. Peptides are ideal candidates for this mission due to their ability to closely mimic many structural features of protein interfaces. However, their inherently low intracellular stability and permeability and high in vivo clearance have thus far limited their biological applications. One way to improve these properties is to constrain the secondary structure of linear peptides by cyclisation. Herein we review various classes of cyclic and macrocyclic peptides as chemical probes of protein surfaces and modulators of PPIs. The growing interest in this area and recent advances provide evidence of the potential of developing peptide‐like molecules that specifically target these interactions.


Introduction
Protein-protein interactions (PPIs) control and regulate cellular processesi ncluding enzyme catalysis, cell signalling and development, and protein homeostasis. PPIs strongly influencet he abundance and cellular localisation of the proteins involved, and often determine the specificity and fidelityo ft heir function. The importance of PPIs is further underscored by the highly organised and responsive protein networks that regulate most biological processes,w hich offer many opportunities for therapeutic intervention. In theory,e ach PPI in such networks may play ar ole in physiological and pathophysiological states, and could thus representaprospective drug target with potential therapeutic relevance. [1] In this context,t he development of chemical tools or probest hat can help us understand andd issect the mechanismsa nd biological roles of specific PPIs is of extreme relevance.
In spite of the vast opportunities provided by targeting PPIs, the physicochemical nature of protein-protein interfaces makes it as erious challenge to develop small molecules that bind to these sites and potentially disrupt thesec ontacts. PPIs usually involvef lat surfaces of extended area, in contrastt o the more traditional bindings ites defined at the actives ites of receptors or enzymes, whicht end to be more buried from sol-vent and contain well-defined and deep pockets.T herefore, the reliable identification and development of binding ligands to protein surfaces, whether they be direct or allostericm odulators of PPIs, remaind ifficult and unsolved problems. However,m uch progress has been made in recent years in this direction. In particular,i ti sb ecomingi ncreasingly clear that the development of drug-like PPI inhibitors, and small-molecule ligands to protein surfaces in general, greatlyb enefits from the availability of ap eptidic ligand to that binding site, either from the natural interacting partner or from synthetic sources. Some notable successful examples of using peptides from the natural protein partner as as tartingp oint for drug design include such well-characterised systemsa sM DM2-p53 [2] and VHL-HIF1a. [3] Such peptidic ligandsc an provide information about the nature and detailso ft he key interactions required to achieve affinity at the targeted binding site, and can furnish crucial displacementt ools to ensure the specificity of interactions of the chemical series developed in the drug development process. Peptides also offer an interesting alternative in their own right as PPI modulators, with an umber of advantages over nonpeptidic small molecules:b iocompatibility,p resenting low toxicity to the organism;c hemical flexibility,w hich enhances the capacity to adapt to large ando ften flexible surfaces; and modularity,w hich expands structural diversity,t hereby facilitating selectivity and high potency. [4] Additionally,p eptides are able to more closely mimic the features of ap rotein interface, and as such they constitute suitable candidates as chemical tools to target PPIs. On the other hand, the use of peptides as drugs themselves has major drawbacks, especially in comparisonw ith proteins, antibodies, or drug-like compounds; these drawbacks include low plasma stability,c leavageb yc ellular proteolytic enzymes, andi nv ivo clearance by hepatic and renal activities. [5] Attempts to use short peptides to mimic pro-Ta rgeting protein surfaces and protein-protein interactions (PPIs) with small molecules is af rontier goal of chemical biology and provides attractive therapeutic opportunities in drug discovery. The molecular properties of protein surfaces, including their shallow features and lack of deep binding pockets, pose significant challenges, and as ar esult have provedd ifficult to target. Peptidesa re ideal candidates for this mission due to their ability to closely mimic many structural features of protein interfaces. However,t heir inherently low intracellular stabilitya nd permeability and high in vivo clearance have thus far limited their biological applications.O ne way to improve these properties is to constrain the secondary structure of linear peptides by cyclisation. Herein we review various classes of cyclic and macrocyclic peptides as chemical probeso fp rotein surfacesa nd modulators of PPIs. The growingi nterest in this area and recent advances provide evidenceo ft he potential of developing peptide-like molecules that specifically target these interactions. tein a helicesi mportant in ag iven PPI have had some success, but face limitations mainly due to the sequence and context dependence of peptideh elicity,o ften resulting in peptides that lack the desired helical structure in solution,w hich is required for productivei nteraction. This problem has been partially addressed by the peptide-stapling approach, which was extensively reviewed recently [6] and is not covered here. In ac omplementary strategy,n onpeptide compounds have been successfully developedt om imic the key interactions formed by the i, i + 4, and i + 7s ide-chains of a helices presenta t PPIs. [7] However, these approaches have tended to result in poorly soluble compounds that exhibit limited target selectivity and low cellularp otency, [8] andh ave not been applied across aw ide range of biological sequences andP PI targets.
To circumvent these issues and to enable the use of peptides as chemical tools and therapeutic leads, scientists have been creatively modifying biologically active peptides into molecules with more adequatestructural features and physicochemicalp roperties. [9] This reviewf ocuses on cyclic and macrocyclic peptidel igands as chemical tools to recognise protein surfaces and for use as chemical probes of PPIs.

Head-to-tail cyclic peptides
Naturally occurring peptides often presentc yclic conformations ( Figure 1). In the majority of cases,r ing structures are formed by disulfide bridges between cysteine residues. However,t his is not the only possibility and cyclisation can also be achieved through the formation of amide bonds or aryl-aryl linkages. Examples of such cyclic structures are found from animals to lower plants. [10] Head-to-tailc yclic peptides belong to this latter group, being the most common type, and typically form ar ing via amide bond formation between the N-terminal amine and the C-terminal carboxylic acid. In discussing headto-tail cyclic peptides, ar emarks hould be made about the Arg-Gly-Asp (RGD)-bearing peptides [11] as one of the first examples demonstrating the benefits of cyclisation in terms of increasingt he stability and affinity of cyclic peptides relative to their linear versions. [12] Through this sectionw ep resent and discusss ome of the approaches taken to obtain head-to-tail cyclic peptides and their applications in modulating PPIs.
The head-to-tail cyclisation can be reproduced synthetically by using liquid-phasep eptides ynthesis, solid-phase peptide synthesis (SPPS), or DNA-programmed chemistry, [13] and also biosynthetically,w itht ypical examples including phage display approaches [14] and split-intein circular ligation of peptides and proteins (SICLOPPS). [15] Several examples of head-to-tail cyclised peptides that effectively target PPIs have been described by the Ta vassoli research group. One of the examples is the identification of cyclic peptides that interferew ith the HIV Gag protein-TSG101 hostp rotein interaction, an importantc ontact involved in HIV virus outflow. [16] In this case the authors combined the use of SICLOPPS libraries with ab acterialr everse two-hybrid system (RTHS) [17] to identifyc yclic peptided isruptors of this PPI. Using this approach against ad ifferent target, the same group reported the identification of cyclic hexapeptides that inhibit hypoxiai nducible factor (HIF) heterodimerisation with high intracellular activity.O ft he four cyclic peptides retrieved from ap lasmid-encoded library of 3.2 10 6 cyclic peptides, cyclo-CLLFVY (or P1) revealed the capacity to disrupt HIF-1 heterodimerisation by binding the PAS-B domain of HIF-1a,w ithout affecting HIF-2a. [18] Using the same approach as in the previous examples, cyclic peptides were used to inhibit the dimerisation of the C-terminal binding protein (CtBP) transcriptional repressor. [19] The cyclic peptideC P61 was found to disrupt CtBP homo-and heterodimerisation at 20 mm in vitro and to inhibitt he cellular functions of CtBPs at 50 mm.H uman breastc ancer cells treated with the compound showed decreased mitotic fidelity,p roliferation, and colony-forming potential. The authorss uggest that ad i-or tripeptide motif is crucial for the inhibitory activity of the cyclic peptide, with the rest of the peptide acting as ab ackbonet hat presents the active motif to its target. [19] Another recent successful example of head-to-tail cyclic peptides as PPI inhibitors was reportedb yW ue tal.,t argeting the Ras-effector interaction. [20] The peptide identified as the most potent binder in vitro (IC 50 = 0.83 mm)i ncluded unnatural amino acids that were introduced to allow structural diversity www.chemmedchem.org and resistance to proteolytic cleavage, but showedp oor membrane permeability.T his peptide was subsequently optimised into ahigher potencyRas inhibitor (IC 50 = 0.12 mm)that exhibited high cell permeability and induced apoptosis in cancer cells, making it au seful lead for furtherd evelopment into therapeutic agents. [21] This work validates the strategy of integrating target-bindinga nd cell-penetrating motifs into as ingle cyclic peptidet od evelop biologically active inhibitors against other PPIs.

Side-chain-to-side-chain cyclic peptides
Side-chain-to-side-chainc yclisation is another approach that has been extensively exploredt oo btain cyclic peptides via linkage between amino acid side-chain groups. In particular, lactam-bridged peptides have been used to introducer igidity and defined a-helical secondary structure in short peptides.
Some effective examples of this approacha re featured. Using macrolactam constraints betweena mino acid side-chains, Mills et al. [22] reported the first a-helical peptidomimeticst argeting viral RNA.T heir synthetic approachi nvolved SPPS with orthogonal Pd 0 -labile protecting groups on the relevant lactam precursors,f ollowed by on-resin lactam formation. The tightest binder presented a K D value of 40 nm and, interestingly, showed2 5-fold improvement in its binding specificity toward the target, the HIV Rev-responsive element( RRE) RNA, relative to the corresponding linear precursor.T hese very encouraging results demonstrate the feasibility of developing peptidicl igands with binding affinities in the nanomolar range.U sing as imilara pproach, i.e.,l actam bridges between amino acid side-chains, the Fairlie research group developed as trategy for inducing peptide helicity by rationally linking togetherc yclic helical modulesa ss hort as five amino acids. [8] Thishelix pre-organisation resulted in significant enhancements in affinity and specificity over unconstrained peptides, and the functional responses were similart oo rg reater than those of the native proteins from which they wered erived. This approach holds potentialf or rational structure-based design using native protein structures and combinatorial helix libraries. Furthermore, it successfully demonstrates the ability to downsizep roteins from differents ources, including bacterial, viral, and human, to short synthetic peptides with strategically enforced waterstable a-helicalstructures. [8] Computational approaches can aid the rational design of cyclic peptides with biological activities.A ne xample of this is as tudy targetingt he post-synaptic density protein 95 (PSD-95), ap rotein that plays an important role in synaptic plasticity. Molecular modelling of ap rotein-peptide complex of the third PDZ domain (PDZ3) of PSD-95 supportedt he design of inhibitors of this PPI. Based on in silico studies, a-, b-, and g-amino acids were chosen as bridging elements to tether amino-a nd carboxy-functionalised residue side-chains. [23] The resulting cyclic peptides employed ab is-carboxylica cid as bridgingc onstraint between two amino-containing side-chains from the peptideb ackbone, and exhibited binding affinitiesi nt he single-to double-digit micromolar range. Different side-chain-to-side-chain cyclic peptides are those bridged by cysteine disulfide bonds, as illustrated by the followingt wo examples. The first reports cyclic peptides with greatera ffinity for the CREB binding protein (CBP) bromodomain than their biological ligands, including lysine-acetylated histones and tumours uppressor p53. [24] The authors used at arget-structure-guided and computer-aidedr ational design approacht od evelop as eries of disulfide-linked cyclic peptides for testing in af unctional assay.C oncerned with the stability of the disulfide bridges inside cells, the authors then modified the most promisingp eptides, replacing the disulfide by at hioether-like linker.Thiswork resulted in aseries of cyclic peptides in which the bestb inder exhibited a K D value of 8.0 mm,r epresenting a2 4-fold improvement in affinity in comparison with the linear lysine382-acetylated p53 peptide. [24] The second example concerns the development of ac yclic peptide with potentialu se as am odel for the inhibition of the recognition mechanism of HECT-E3 ligase. [25] The HECT-containing E3 ligase Itch mediates the degradation of severals ubstrate proteins, including p63, by recognizing as pecific region of p63 that contains aP PxY motif. In this work the authors developedastrategy for the stabilisation of the conformation of an 18-mer peptide derived from the recognition epitope on p63, ands howed that the cyclisation of this peptide enhanced its biological stability and binding affinity.T his is ag ood starting point for the development of drugs that inhibit the Itch E3 ligase complex in vivo.

Macrocyclics and macrocycle organopeptide hybrids
Merging the biologicala nd synthetic approaches for attaining macrocyclic ligands provides opportunities for ligand diversification in drug discovery.A na pproach developedb yF asan and co-workersu ses macrocyclic organopeptide hybrids (MOrPHs) incorporating non-proteogenic synthetic moieties into genetically encoded peptidic frameworks. [26] MOrPHs are based on the reactivity of intein proteins [27] and take advantage of the opportunity to introduce bioorthogonalf unctionalities into proteins by amber stop codon suppression. [28] These modular assemblies can be easily diversifiedb ya ltering the natureo f the synthetic or biosynthetic precursors. Ac hemoselective tandemr eaction can be performed in the presence of two functional groups with orthogonal reactivity to promote the formation of the organopeptide macrocycle.U sing this strategy,s everal MOrPHs have been prepared that exhibit various ring sizes, structures, and composition and molecular weight, ranging from 700 to 1800 Da. [26] Interested in probing the potential of MOrPHs for a-helix stabilisation and mimicry, the same researchg roup designed MOrPH-based peptides that target HDM2/X based on a1 2-mer linear peptide isolated by phage display:P MI (Figure 2). [29] The availablec rystal structure of PMI bound to HDM2 highlighted the presence of two solvent-exposedr esidues as viable sidechain attachment points for the MOrPH. The resulting molecules are the first examples of side-chain-to-tail peptide cyclisation to aid a-helix stabilisation and led to the discovery of sub-ChemMedChem 2016, 11,787 -794 www.chemmedchem.org micromolar inhibitors of the p53-HDM2/X interaction. [30] The work also describes the influence of the nonpeptidic moietyi n the modulation of the functional, conformational, and stability properties of a-helical MOrPHs, demonstrating the potential of coupling this approachw ith ad isplay methoda satool to identify MOrPH compounds with tuned protein binding properties.
In ad istinct strategy, using the DNA-programmed chemistry (DPC) method [31] for directly translating DNA sequences into small molecules, Seigal et al. developed extensive libraries of DNA-encoded macrocycles. [32] The authors describe the construction of macrocycle libraries using 20 different aminoa cid buildingb locks for each of the derivatisable positions and five azido-substituted amino acids in ap recise position to allow covalent attachment to the DNA-encoding template. The screening of these libraries against the BIR2 and BIR3 domains of the X-linked inhibitor of apoptosis protein (XIAP) led to the identificationo fn ovel macrocycles with high affinity.T his example shows that producing libraries by DPC is an efficient way to screen for novel macrocyclic inhibitors and to generate meaningful structure-activity relationships(SAR).
Finally,w er eport thioether macrocyclic N-methyl-peptide inhibitorso ft he E6AP E3 ubiquitinl igase identified from ar ibosome-expressed de novo library. [33] Using translation machinery under reprogrammed genetic code coupled with an in vitro display technique called RaPID (random nonstandard peptides integrated discovery), the macrocyclic N-methyl-peptides were screened, resulting in as election of stronga nti-E6AP binders (K D values ranging from sub-nanomolart os ingle-digit nanomolar), one of which with the ability to inhibit poly-ubiquitination of substrate proteins.

Bicyclic peptides
Bicyclic peptides can be simply defined as cyclic peptides containing two loops. Among possible ways to form such bicyclic structures are side-chain-to-side-chain linkages on monocyclic peptides as well as covalent linking units attached to three different points on al inear peptide. Bicyclic peptides tend to have am ore constrained structure than their monocyclic counterparts. One of the most attractive features of bicyclic peptides is the display of relativelyf lexible loops constrained to am ore rigid central scaffold, which allows closer mimicryo f antibodies in terms of their molecular recognition, binding af-finity,a nd specificity properties. In addition, theses tructural features have been shown to introduce improvements in binding affinities as well as resistance to intracellulard egradation and metabolic activities. Several examples have been reported for different and successful constructions of bicyclic peptides, some of which are discussed here.
The introduction of side-chain-to-side-chain staples in peptide macrocycles can be used as am eans of stabilising them. Quartaro and colleagues [34] reported as trategy for the generation of bicyclic peptides using this approach. Thes tarting point was an 11-mer disulfide-bridgedm acrocycle named G1 that binds the Src-homology 2( SH2) domain of growth-factorboundp rotein 2( Grb2).G 1h ad been subjected to several rounds of optimisation, allowing the authors to takeadvantage of previouslyk nown SAR [35] to help design bicyclic peptidea nalogues.T he approachr esulted in ab icyclic macrocycle with a6 0-foldi mprovement in inhibitory potencya nd 200-fold increased selectivity relative to the original peptide after only two rounds of iterative design. [34] To impart generality and application of this strategy to any given cyclic peptidew ill, however,r equire systematic exploration of cross-link positions to optimise functional properties and to identify ideal staples for each cyclic peptide.
Substantial research has been directed in the Heinis laboratory towardt he development and optimisation of bicyclic peptides to target differentp roteins.I n2 009 Heinis et al. [36] presented an ew strategy for selecting ligandsb ased on bicyclic peptides attached covalently to an organic core scaffold. The peptides contain three reactive cysteine residues, which allow conjugation with 1,3,5-tris-(bromomethyl)benzene (TBMB), thereby giving rise to two peptidic loops linked by amesitylene core. The cysteiner esiduesw ere spaced apart by av ariable number of random amino acids, and the peptides were fused to the phage gene-3-protein, permitting the screeningo fv ast libraries of bicyclic peptides with the convenience of phage display.O ne of the first successful examples described the screening of al ibrary of 4.0 10 9 different chemically constrained bicyclic peptides to identify as electivea nd potent (K i = 53 nm)i nhibitor (UK18)o fh uman urokinase-type plasminogen activator( uPA). [37] The crystal structure of the peptide-protein complex revealed that this peptide( < 2kDa) resembles features typical of PPIs, such as al arge interface of interaction with the targeta nd multiple hydrogen bonds and electrostatic interactions from both peptidic loops, contributingt oi ts binding affinity and specificity (Figure 3). [37] The same group tested the effect of differentc hemical cores in the variation of the backbonec onformation adopted by the peptidel oops. Studies with three different scaffolds led to the conclusion that different alkylating reagents imposed ifferent constraints on the backbonec onformation of bicyclic peptides. [38] This strong structurale ffect was not entirely expected due to the relative flexibility of the cysteine side-chains that reactw ith the chemical core. The authorsp roposed that this dominant effect is related to the chemical moiety being the central location and forming the branching pointo ft he peptide. Other protein surfaces successfully targeted since then, using the same bicyclic peptidea pproach, include the human plasma kallikrein (hPK), [39] the coagulation factor XII, [40] the epidermal growth factor receptor Her2, [41] and more recently the Notch1 receptor. [42] The characterisation of these phage-selected bicyclic peptides revealed high proteolytic stability relative to linear or monocyclic analogues, and most of them showedg ood stability in blood plasma. [43] In addition, to prevent renal clearance, ab icyclic peptide inhibitor of the uPAw as successfully conjugated to albumin-binding peptides, which resulted in an extended half-life of~24 hi nm ice. [44] Lian ando thers developed an approach to obtain bicyclic peptidel ibraries including non-proteinogenic (and certain unnatural aminoa cids) andn onpeptidic building blocks, thereby expanding their structurald iversity. [45] The main difference regardingt he previouse xamples relies on the fact that these librariesa re entirely synthetic. The screening of al ibrary containing the trimesic acid as as caffold against tumour necrosis factor-a (TNFa)y ielded1 2b icyclic peptide hits, of which six showedd issociation constantsb etween 1a nd 8 mm.T he most active peptidee xhibited high selectivity toward the target and ab inding affinity of 0.45 mm,r epresenting the most potent non-protein TNFa inhibitor reported until then. [45] The same group reportedt he development of bicyclic peptides with cell permeability by attachingt hem to cell-penetratingp eptides (CPPs), [46] whichh ave been widely used to deliver drugs,D NA, RNA, proteins, and nanoparticlesi nto mammalian cells and tissues. Protein tyrosinep hosphatase 1B (PTP1B)w as selected as am odel system, and PTP1B inhibitors were identified in as creen of bifunctionalc yclic peptides featuring the CPP motif and ar andom pentapeptide sequence. [47] It is suggestedt hat this delivery methodc ombining cell-penetrating and targetbinding sequences into cell-permeable inhibitors could be generalisable and extended to other intracellular targets. Taken together,t hese results encourage future development and optimisation of bicyclic peptides into potential therapeutic drugs.

Grafted peptides
The use of small proteins thata re ablet op erform the same functions as large proteins has gained popularity in recent years, leading to the expression and even synthesis of modified miniature proteins, called mini-proteins. Disulfide-rich mini-proteins were found to occur naturally in plants (called cyclotides), and due to their size and stabilityr epresent an attractives tartingp oint for the development of novel PPI modulators.I nf act, grafted peptides are moleculest hat use cyclotides as scaffolds for the introductiono fe pitopes with biological activities. [4] Examples of effective application of grafted peptidesf or targeting PPIs have been described. Ji et al. engineeredacyclotide that is able to activate the p53 tumour-suppressor pathway in vivo. [48] The resulting molecule was found to be an anomolar-affinity binder with high stabilityi nh uman serum.I nad ifferent study,t he Brunsveld research group developed mini-proteins as androgen receptorc o-activator mimics through ac omputational design approach. [49] The authors subsequently performed SAR studies of mini-proteins in which synthetic point mutations were made in two of the most potent inhibitors from the previouss eries, in order to achieve higher potency. [50] These mutations were designedt o maintain the secondary structure of the backbonew hilei ncreasingt he binding affinity through additional favourable interactions at the receptor surface, and resulted in at enfold gain in potency.
Exciting work publishedi n2 012 by Zoller et al. [51] reports the combination of phage display and molecular graftingt og enerate tumour-targeting mini-proteins. Ad ouble-disulfide-stabilised mini-protein called Min-23 [52] was used as am olecular scaffold in ap hage displayc ombinatorial library,l eadingt ot he identification of new disruptors of the interaction between the angiogenesis marker d-like ligand 4( Dll4) and Notch1. To overcome synthetic issues of the peptideh its, the expected binding residues of the Min-23p hage-display-evolved peptide were transferred to the variable loop of STF-1 (sunflowert rypsin inhibitor), ac yclic 14-residue peptidew ith as ingle disulfide bridge. Several mini-proteins were synthesised and tested for binding,a nd only one showeds pecific binding interactions with a K D value of 22 nm.T his study serves as ap roof of concept of applying molecular grafting to the development of new molecular entities for therapeutic use. These mini-proteins were active and specific in vitro and in vivo and were shown to accumulate in tumourt issue, although furthero ptimisation is warranted.

Photoswitchable peptide PPI inhibitors
The ability to monitorand regulate the activity of PPI inhibitors with spatiotemporal control opens up the possibility to control their kinetics and site of action in cells. Photoswitchable inhibitors are molecules that only adopt their active/inhibitory conformationw hen exposed to light of ac ertain wavelength ( Figure 4). Therefore, they offer tunable perturbations of biomolecular interactions, allowing regulation of timing and period of activity of the modulators, using light as an external stimulus with high temporal and spatial precision. [53] This strategy has been appliedb ys everal groups,a nd af ew examples are discussed below.
To control clathrin-mediated endocytosis (CME) in living cells, Nevola et al. [54] developed photoswitchablepeptide inhibitors that target AP2, ac omplex responsible for internalising cargo in this pathway.D isruptor molecules were designed by startingf rom ac rystal structure of the C-terminal peptide of the native interaction partner, b-arrestin,b ound to AP2. The binding mode consists of an a-helical structure with four conserved residues facing the binding pocket. The stabilityo ft he secondary structure of peptides is strongly linked with the ability to interactw ith their target, and this observationl ed to the hypothesis that one could reversibly regulate the peptides' affinity by controlling their secondary structure. To achievet his objective, ap hotoisomerisable cross-linker,3 ,3'-bis(sulfonate)-4,4'-bis(choloroacetamido)azobenzene (BSBBA), was conjugated between pairs of cysteines introduced in the peptides e-quence, so that the stabilityo ft he helix could be reversibly altered by using 380 nm or 500 nm light. The developed cellpermeable peptides TL-1 and TL-2 were able to disrupt the target PPI in ap hotocontrolled manner. [54] Using the same azobenzene linker (BSBBA) acting as ac yclising unit for peptides, by attaching them to the chemical moiety from two different points, am ethod to screen cyclic peptidel ibraries by phage display was developed by Heinis and co-workers. [55] The authors described the cyclisation of al ibrary of the format ACX 5 CG (whereXare random amino acid residues) with BSBBA and screening against streptavidin. The peptides were exposed to UV light prior to affinity panning with the aim of isolating ligands that bind preferentiallyi nt he cis conformation. The best binder identified showed a K D value of 2.2 mm,b ut the changes in affinity by exposure to UV light did not enhance the affinity drastically.I nt he future this limitation could be overcome by applyingd ifferent peptidel ibrary formats or different photoswitchable moieties. [55] The last application described here reports photoactive phosphopeptide mimeticsaspotent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. [56] Ab enzoyl phosphonate containing amino acid, 4-phosphonocarbonyl phenylalanine, was used to replace the native phosphotyrosiner esidue. Irradiation of this benzoylphosphonate under the right conditions and subsequentr ecognition by ap hosphotyrosine binding pocket led to photocross-linking of the target protein.
The peptide mimetics synthesised were validated as inhibitors of PTP1B, and it was shown that irradiation with 365 nm light strongly enhanced the inhibitory effects. PTP1B deactivation was found to occur via ar adicalm echanisma nd could be reverted by the additiono fd ithiothreitol( DTT) as reducing agent.

Summary and Outlook
The relevance of developing peptide-like molecules that target specific protein-protein interactions has been underpinned by approaches to obtain cyclic peptides and organopeptide hybrids and their respective applications.T he achievements in this area,i ncluding an increasing number of chemical strategies for constraining peptides econdary structure, will no doubtr einforce the importance of peptideb inding epitopes as lead structures.
One of the biggest challenges faced in the field remains surpassingt he poor oral bioavailability and liabilities associated with poor pharmacokinetics (PK), pharmacodynamics (PD), and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties that any type of peptidic ligand inherently suffers when used in cellular models and in vivo. Advances have been made towardo ptimisation of the oral bioavailability and membrane permeability of peptidic ligandsi nrecent years [57] and corroborate the enhanced properties of constrained peptideso ver their linear vectors. Improved in vivo lifetime has also been achieved, for example by replacing a-aminoa cid residues with homologous b-residues [58] or by coupling them to small molecules that bind reversibly to serum proteins. [59]  The peptideislinked to an isomerisablechemical moiety,which,upon irradiation with light of ac ertain wavelength, changesconformation and forces the peptide to adopt as pecific structure.
ChemMedChem 2016, 11,787 -794 www.chemmedchem.org Successful targeting of PPIs often requires ab ilateralr elationship between the protein and the ligand involved;t herefore, efforts have also been pursued in understanding which characteristics of ap rotein target might make it more suitable for productive binding by macrocyclic ligands. [60] This knowledge would definitely provide guidelines for the development of macrocyclic ligands with enhanced structural and physicochemicalproperties and better bioavailability.
Although there is still al ong road ahead, the advances reported herein have increased our understanding of the requirements imposed on peptidic PPI modulators as potential therapeutics. These advancements have significantly boosted the field, as reflected by the increasing number of publications, buildingc onfidence that the approachi sf easible and likely to deliver major breakthroughs in terms of novel chemical tools and potentialnew drugs in the near future.