Crystal Structures of Protein-Bound Cyclic Peptides - Chemical

Review pubs.acs.org/CR

Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

Crystal Structures of Protein-Bound Cyclic Peptides Alpeshkumar K. Malde,†,§ Timothy A. Hill,† Abishek Iyer,†,‡ and David P. Fairlie*,†,‡ †

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Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia ‡ Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia ABSTRACT: Cyclization is an important post-translational modification of peptides and proteins that confers key advantages such as protection from proteolytic degradation, altered solubility, membrane permeability, bioavailability, and especially restricted conformational freedom in water that allows the peptide backbone to adopt the major secondary structure elements found in proteins. Non-ribosomal synthesis in bacteria, fungi, and plants or synthetic chemistry can introduce unnatural amino acids and non-peptidic constraints that modify peptide backbones and side chains to fine-tune cyclic peptide structure. Structures can be potentially altered further upon binding to a protein in biological environments. Here we analyze three-dimensional crystal structures for 211 bioactive cyclic peptides bound to 65 different proteins. The protein-bound cyclic peptides were examined for similarities and differences in bonding modes, for main-chain and side-chain structure, and for the importance of polarity, hydrogen bonds, hydrophobic effects, and water molecules in interactions with proteins. Many protein-bound cyclic peptides show backbone structures like those (strands, sheets, turns, helices, loops, or distorted variations) found at protein−protein binding interfaces. However, the notion of macrocycles simply as privileged scaffolds that primarily project side-chain substituents for complementary interactions with proteins is dispelled here. Unlike small-molecule drugs, the cyclic peptides do not rely mainly upon hydrophobic and van der Waals interactions for protein binding; they also use their main chain and side chains to form polar contacts and hydrogen bonds with proteins. Compared to small-molecule ligands, cyclic peptides can bind across larger, polar, and water-exposed protein surface areas, making many more contacts that can increase affinity, selectivity, biological activity, and ligand−receptor residence time. Cyclic peptides have a greater capacity than small-molecule drugs to modulate protein−protein interfaces that involve large, shallow, dynamic, polar, and water-exposed protein surfaces.

CONTENTS 1. Introduction 1.1. Peptides and Post-translational Modifications 1.2. Key Advantages of Peptide Cyclization 1.3. Molecular Constraints and Bioactive Cyclic Peptides 1.4. Review Scope 2. Cyclic Peptide-Bound Proteins 2.1. Acetylcholine Binding Protein 2.2. Alkaline Proteinase Savinase 2.3. Antibody-Binding Fragments 2.4. Apelin Receptor 2.5. Apical Membrane Antigen 1 2.6. Asp/Asn β-Hydroxylase 2.7. Bacterial RNA Polymerase 2.8. Botulinum Neurotoxin 2.9. Casein Kinase 2 2.10. Caseinolytic Protein C1 (ClpC1) 2.11. Chemokine CXCR4 Receptor 2.12. Chitinase 2.13. Chymotrypsin 2.14. Complement C5a Receptor 2.15. Cyclin-Dependent Kinase-2 © XXXX American Chemical Society

2.16. 2.17. 2.18. 2.19. 2.20. 2.21. 2.22. 2.23. 2.24. 2.25. 2.26. 2.27. 2.28. 2.29. 2.30. 2.31. 2.32. 2.33. 2.34.

B B B D D D D E F F G G H H I I J K K K L

Cyclophilin D DnaN Elastase Endothelin Receptor Ephrin A4 Receptor Erythropoietin Receptor Estrogen Receptor α Growth Receptor Bound Protein 2 SH2 Growth-Receptor-Bound Protein 7 SH2 Hemagglutinin Histone Deacetylase 8 Histone Demethylase HIV-1 gp41 HIV-1 Integrase HIV-1 Protease Human Adaptor Protein 14-3-3 Integrin αVβ3 K-Ras Kallikrein-Related Peptidase 4

M M N N O O P P Q Q R R S S T T U U V

Special Issue: Macrocycles Received: December 31, 2018

A

DOI: 10.1021/acs.chemrev.8b00807 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews 2.35. 2.36. 2.37. 2.38. 2.39.

Lectin LecB Matriptase Menin Mouse Double Minute 2 Multidrug and Toxic Compound Extrusion Transporter 2.40. Neurophysin 2.41. P-Glycoprotein 2.42. Phosphoglycerate Mutase 2.43. Plexin 2.44. Polo-like Kinase-1 2.45. Programmed Cell Death 1 Ligand 1 2.46. 20S Proteasome 2.47. Protein Phosphatase 1 2.48. Protein Tyrosine Phosphatase 1B 2.49. Regulator Gene of Glucosyltransferase 2.50. β-Secretase 2.51. Signal Peptidase 2.52. Sirtuin 2 Deacetylase 2.53. Splicing Factor 45 2.54. SPRY Domain-Containing SOCS Box Protein 2 2.55. Streptavidin 2.56. Tankyrase 2.57. Thrombin 2.58. Thrombin Activatable Fibrinolysis Inhibitor 2.59. Toxin Protein−Immunity Protein Interaction Inhibitors 2.60. Transducin-like Enhancer Protein 2.61. Trypsin 2.62. Tumor Necrosis Factor 2.63. Urokinase-Type Plasminogen Activator 2.64. WD Repeat Domain 5 Protein 2.65. X-Chromosome Linked Inhibitor of Apoptosis Protein 3. Discussion 3.1. Proteins Bound to Cyclic Peptides 3.1.1. Enzymes 3.1.2. G-Protein-Coupled Receptors and Ion Channels 3.1.3. Other Proteins 3.2. Protein-Bound Cyclic Peptide Backbones 3.2.1. Ramachandran analysis 3.2.2. Multiple Cyclic Peptides Bound to One Protein 3.2.3. One Cyclic Peptide Bound to Multiple Proteins 3.2.4. Secondary Structures in Cyclic Peptide Backbones 3.3. Protein-Bound Cyclic Peptide Side Chains 3.4. Less Common Amino Acids 3.5. Protein-Bound vs Unbound Cyclic Peptide Structures 4. Conclusions and Future Prospects Author Information Corresponding Author ORCID Present Address Notes Biographies Acknowledgments Abbreviations References

Review

1. INTRODUCTION

V W X X

1.1. Peptides and Post-translational Modifications

Polypeptides are naturally occurring biopolymers composed of sequences of amino acids, connected together by condensation of the amine terminus of one amino acid with the carboxylic acid terminus of another to form −[R1NH-COR2]n−. Folded polypeptides over 50 amino acids in length are referred to as proteins, while smaller sequences are termed peptides. Polypeptides are synthesized mainly from 20 common amino acid building blocks on the ribosome through translation from RNA into diverse arrays of sequences and structures.1 All of the properties of polypeptides are dictated by their threedimensional structures, which are determined by folding of the polypeptide backbone as directed by the order of their amino acid sequences.2 The Protein Data Bank (PDB, www.rcsb.org/ pdb)3 contains around 150 000 deposited structures of proteins and polypeptides in 2019. Polypeptides are also synthesized non-ribosomally, especially in bacteria, fungi, and plants, by batteries of enzymes that often incorporate unnatural or modified amino acids and other components.4,5 Most proteins and peptides are now believed to be enzymatically engineered with post-translational covalent modifications to their side chains, main chains, or termini. There are over 200 different post-translational modifications known (http://www. uniprot.org/docs/ptmlist),6 including glycosylation, glycation, phosphorylation, lipidation, acylation, acetylation, amidation, palmitoylation, sulfation, methylation, hydroxylation, biotinylation, cysteinylation, myristoylation, farnesylation, ubiquitylation, sumoylation, and oxidation, to name just a few.4−6 Such modifications to the chemical composition of proteins and peptides confer a wide variety of chemical, physical, and biological properties. They affect protein/peptide folding, conformation, solubility, and stability; customize intra- and intercellular signaling functions; confer protection from or assistance with interaction with other proteins, enzymes, or macromolecules; and alter membrane permeability, intracellular transport, catalytic functions, sensor properties, tissue elasticity, metabolic and immunological properties, transcriptional regulation, etc. Many of these modifications are also reversible, providing another level of control over protein and peptide activity. One less studied but very important and common type of post-translational modification is cyclization, with many cyclic peptides produced by ribosomal or non-ribosomal syntheses in bacteria, fungi, plants, and animals.7−12 Peptides are cyclized through head-to-tail linkage of the N-terminal amine to the Cterminal carboxylic acid or through a myriad of different side chain-to-side chain, side chain-to-main chain, or main chain-toside chain linkages, to form a pseudocircular sequence of peptide bonds. Although many cyclic peptides are now known and their three-dimensional structures have been determined and analyzed, there are surprisingly few analyses to date of three-dimensional structures of cyclic peptides bound to proteins.13−16 This Review concerns three-dimensional protein-bound structures of cyclic peptides that have been reported in the PDB to date.

Y Y Z Z AA AA AB AB AC AC AD AD AE AE AF AF AG AG AH AI AI AJ AJ AK AK AL AL AM AM AM AM AM AM AM AN AO AO AO AP AP AP AR AR AR AR AR AR AR AS AS

1.2. Key Advantages of Peptide Cyclization

Peptides have a number of deficiencies for applications in science and medicine that can be overcome by cyclization.17,18 Peptides are highly f lexible and often soluble molecules in water, most small peptides of less than 20 amino acids adopting no well-defined specific structure. Their polar backbone amide B


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CO and NH components, and any polar side chains, are solvated by water molecules. Removing the positive charged Nterminus (−NH3+) and negative charged C-terminus (−CO2−) through end-to-end cyclization can alter the pH-dependent solubility of a peptide controlled by its zwitterionic properties. Cyclization also reduces the conformational freedom of peptides,19−21 making their structures less dynamic, less flexible, or more rigid and defined. This brings an advantage of pre-organizing polypeptides for receptor binding, the aqueous structure of a cyclic peptide having to change less than a random peptide structure in water to bind to a protein. Cyclization can lead to mimicry of secondary structure elements of proteins.19−21 Cyclization compresses a peptide structure, most often by encouraging intramolecular hydrogen bonds that can be transannular between main-chain amides (CO···NH) or side chain-to-main chain or exocyclic between side chains (Scheme 1). Hydrogen bonds between main-chain amides

Smaller polypeptides that do not fold into a well-defined conformation, or are dynamic and can elongate into extended conformations, are thus prone to proteolytic cleavage. Cyclization of short peptides, or incorporation of cyclization constraints in longer polypeptides, encourages formation of turns, helices, and sheets and thereby increases resistance to cleavage by proteolytic enzymes that can no longer fit the cleavable peptide sequence in its active site. Most peptides, including proteins, small peptides, and cyclic peptides, are not cell permeable.34−36 Polypeptides are synthesized inside cells, and their polar nature does not typically engender the properties needed to permeate the lipophilic cell membrane. This is a major impediment to using peptides as exogenous drugs or molecular probes of biological processes, since most polypeptides are not able to penetrate the cell membrane and translocate to where their biological target exists, usually inside cells, organisms, or animals. Indeed, proteins are a major constituent of the diet, and Nature has evolved mechanisms (proteolysis, oxidation, reduction) to degrade proteins into small peptides and amino acids that are absorbed through active transport across the intestinal and other membranes. We still know little about transport of peptides larger than 3−4 amino acids across a membrane, but there are examples known where larger peptides do permeate membranes via endocytosis-related mechanisms.37,38 Cyclization has been found to convey cell permeability to some peptides, especially those containing N-methyl amino acids, Damino acids, heterocycles, certain glycopeptides, lipidated peptides, amphipathic peptides, and others. In the case of cyclic peptides, there are examples of antibiotics, amphipathic molecules, immunosuppressants, and anti-inflammatory and anti-tumor drugs that have a degree of cell permeability.39 Cyclization has also been shown to improve cell uptake of some cationic cell-penetrating peptides40−43 and can lead to increased escape from endosomes.44,45 Most peptides, including most cyclic peptides, are not orally bioavailable.46 As mentioned above, proteins and polypeptides in our diet are deliberately degraded by proteolytic enzymes into amino acids and very small peptides for oral absorption from the gut. In medicine, most peptide and protein therapeutics to date have had to be administered as injectable medicines, often by health workers, and this has issues such as higher medical costs and poor patient compliance with dosing schedules, whereas self-administered oral medications are more easily administered, more effective, and compliance with dosing is much higher. No more than traces of polypeptides are normally absorbed after oral delivery, the remainder being excreted or digested. Oral bioavailability relies upon a number of different factors, such as solubility, proteolytic and metabolic stability in the gut, oral absorption, and membrane permeability, but also rate of clearance from plasma, tissue distribution, renal and hepatic clearance, and metabolism. Most peptides are cleared rapidly from the circulation, metabolized by P450 enzymes in the intestinal lining and liver, or cleared through proteolytic action in plasma and in the kidney. On the other hand, there is a growing number of cyclic peptides that are being identified as orally bioavailable.46 In those cases cyclization tends to cluster hydrophobic side chains, N-methyl substituents,47 branched hydrophobic side chains,48 aliphatic or aromatic linkers, or modifications of amino acids to create extended hydrophobic surfaces; to cluster hydrophilic and positive-charged amino acids to create amphipathic surfaces;49 or to favor structures that are not very

Scheme 1. Peptide Cyclizationa

a

(Left) End-to-end cyclization can bring peptide main-chain carbonyl oxygens and amide NHs into closer proximity, enabling folding into different turn conformations stabilized by intramolecular, transannular, main chain-to-main chain hydrogen bonds. (Right) Cyclization through side chain-to-side chain linkages (i to i+2, i to i +3, i to i+4, i to i+7) can respectively stabilize β-strands, turns (gamma, beta, alpha), or helices (alpha, 310, pi).

stabilize gamma, beta, or alpha turns, the latter typically (but not always) in multiples to form α-helices. Connecting side chains from alternating residues (e.g., i and i+2) leads to cyclic peptides that can mimic peptides in β-strand conformations,22,23 strands being most often found paired as β-sheets in proteins. Connecting amino acid residues in a peptide through a side chain-to-side chain link at positions (i, i+3) can stabilize a β-turn,19,21,24−27 while linking side chains at positions (i, i+4) can stabilize an α-helix.19−21,28−30 Thus, even small cyclic peptides can potentially mimic the major secondary structural motifs (strands/sheets, turns, helices) found in proteins.19−30 Peptides are unstable to proteolytic enzymes. Carboxypeptidases and aminopeptidases are exopeptidases or proteolytic enzymes (“proteases”) that respectively cut peptide bonds from the C- and N-termini of a polypeptide. The actions of these enzymes are blocked through end-to-end cyclization of peptides, which removes the C- and N-termini, thereby making peptides stable to the actions of exopeptidases. Other proteases (endopeptidases) degrade peptides by cutting peptide bonds within sequences, and there are over 550 human proteases (http://degradome.uniovi.es/numbers.html) that have evolved to recognize and cut particular ordered sequences of amino acids at specific sites in a sequence.31 Most proteases cut only the extended or stretched main-chain or backbone conformations of their polypeptide substrates,32,33 and so folded polypeptides need to unwind or unpair to adopt an extended conformation or random conformations capable of adopting an extended region. Thus, polypeptide folding into helices, sheets, turns, and combinations of these secondary structures usually protects those regions in proteins from proteolytic cleavage.32 C


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for which structural coordinates have been lodged in the Protein Data Bank (PDB) as of November 1, 2018, and each description that follows here links to a specific PDB code. For ease of description, the cyclic peptides are listed here according to which of the 65 different proteins they bind. The purposes of this Review are to: (i) assemble, document, and describe protein-bound cyclic peptide structures; (ii) identify any unusual components that confer structure or function to the cyclic peptides; (iii) compare and classify protein-binding modes and identify the extent to which the cyclic peptide backbone directly binds to the protein versus serving as a scaffold for directing side chains to bind to protein; and (iv) describe, where it is known, how the cyclic peptide structure influences function. Some surprises are reported about the nature of the interactions between cyclic peptides and proteins and what this means for design of new cyclic peptides as diagnostics, therapeutics, and biological probes. The amino acid interaction maps were generated using a distance cutoff of 5 Å between the cyclic peptide and surrounding protein. In particular, the final section of this Review analyzes this set of protein-bound cyclic peptide structures for similarities and differences in protein-binding modes and addresses some key questions. For example, how do enzymes (e.g., proteases, kinases, others), GPCRs, and other protein classes recognize cyclic peptides? Are there variations on a common theme? Do the cyclic peptides mimic helix, sheet, turn conformations of protein segments found at protein−protein binding interfaces? Are interactions between the macrocycles and proteins influenced more by side-chain or main-chain components of cyclic peptides? How do different types of cyclization influence binding to proteins? What are the key amino acid side chains that mediate macrocycle−protein binding? What roles do transannular endocyclic versus exocyclic hydrogen bonds play in influencing macrocycle−protein binding? Are hydrophobic or hydrophilic interactions more dominant or more important in binding? What do Ramachandran plots indicate about the protein-bound cyclic peptide main-chain backbones? Do multiple cyclic peptides bound to the same protein indicate any common pattern of binding? Does a particular cyclic peptide bind in different ways to different proteins? This Review takes an important step toward identifying patterns in how cyclic peptides form complementary interactions with proteins and opens up a new dialogue on the relative importance of hydrophobic versus hydrogen bonding and other interactions in rationally targeting the growing number of physiologically important protein−protein interactions.

stable for acyclic peptides in order to promote oral bioavailability. 1.3. Molecular Constraints and Bioactive Cyclic Peptides

There is a great deal of evidence to indicate that Nature uses cyclization in bacteria, fungi, plants, and animals to constrain peptides into conformations that can selectively bind proteins and exert functions.19−21,50−53 Numerous peptides are cyclized through one or more disulfide bonds, such as the many peptide hormones including GPCR agonists (endothelin, oxytocin, orexin-A, urotensin-II, vasopressin, somatostatin, melaninconcentrating hormone, relaxin 2, sarafotoxin, calcitonin, etc.), and there is evidence that they stabilize one or more turn conformations that mediate interaction with a GPCR.54,55 However, not only disulfide bonds have been used for cyclizationthere are numerous ways in which Nature has achieved cyclization in peptides, indeed virtually every part of an amino acid has been adapted and modified as a cyclization point to create mono- and multicyclic peptides.4−21,56−59 Both natural and synthetic cyclic peptides are potent modulators of numerous biological and physiological processes.19−21,60 Numerous conformational constraints, such as D-amino acids, N-methyl substituents, aromatic rings, heterocycles, to name just a few, have been found to fine-tune the structures of cyclic peptides to modulate biological activity.19−21 Such components remove NH donors and increase areas of hydrophobic surface patches that shelter cleavable peptide bonds from peptidases, leading to longer durations of action in biological environments and enhanced permeability of lipid membranes. Over the past 25 years in particular, the influences of different molecular constraints on the threedimensional structures of cyclic peptides have become better understood. This has enabled a better understanding of links between folding, structure, and reactivity, making it easier to rationally exploit the design of synthetic macrocycles for a specific protein target, for modulating its function, and for delivering it to the target inside cells and sometimes into a subcellular compartment. Some examples of around 50 current or previously marketed cyclic peptide drugs (and therapeutic category) are Atosiban (tocolytic), Cyclosporine (immunosuppressant), Caspofungan, Micafungin (f ungicidal), Dalfopristin, Quinupristine, Vancomycin, Tyrothricin, Daptomycin, Gramicidin S, Polymixin B, Colistin (antibiotic), Eptifibatide (anti-thrombotic), Octreotide, Lanreotide (acromegaly), Lepirudin (anti-coagulant), Ziconotide (analgesic, chronic pain), Eptifibatide (myocardial infarction), Oxytocin (uterine contraction), Vasopressin (antidiuretic), Rhomidepsin (cancer), with a number of others in late stage clinical trials.61,62 In most cases the target protein is now known but the three-dimensional structures of the protein-bound cyclic peptide has only been determined in a few cases. In most of those cases, the three-dimensional solidstate or solution structure of the unbound cyclic peptide is not reported. Rational design of therapeutically useful new cyclic peptides can benefit from analysis of their protein-bound structures and also from comparisons with unbound structures.

2. CYCLIC PEPTIDE-BOUND PROTEINS 2.1. Acetylcholine Binding Protein

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are therapeutic targets for a range of human diseases.63 Acetylcholine binding protein (AChBP) is the structural and functional homologue of the extracellular ligandbinding domain of nAChRs that has been used as a pharmacological tool to identify novel drug leads for AChRrelated disorders.64 A number of co-crystal structures of Aplysia californica AchBP in complex with different α-conotoxin cyclic peptides have been reported.65−68 A representative αconotoxin IMI is described here (PDB 2C9T, 2.25 Å). This cyclic peptide is comprised of H-[GCCSDPRCAWRC]-NH2, containing two disulfide bonds between Cys2-Cys8 and Cys3Cys12 residues, the largest ring size involving 10 amino acid

1.4. Review Scope

Most reviews of cyclic peptides concern their biological activities, some describe their structures, a few try to rationalize structure and function, and even fewer relate function to structure to the nature and effects of their natural and unnatural molecular components. This Review is based on the three-dimensional structures of protein-bound cyclic peptides, D


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residues and 22 heavy atoms in the ring. The α-conotoxin IMI exhibits high affinity toward human α7 nAChR (IC50 = 132 nM) and human α7β3 nAChR (IC50 = 41 nM) but much lower affinity for human α9 nAChR (IC50 = 1.8 μM). The interactions between α-conotoxin IMI and AchBP are shown in Figure 1.

protein in this section are predominantly through side chains Arg11, Trp10, and Ala9. Clearly, the disulfide bonds have compressed the cyclic peptide structure to fix these hydrogen bonds and fold the peptide into a high-affinity protein-binding conformation. Comparison with the unbound NMR-derived solution structure for α-conotoxin IMI (PDB 1CNL) indicates that mainly the side-chain orientations, but not the main chain, are altered by protein binding, and this is also supported by other NMR structures.69−72 2.2. Alkaline Proteinase Savinase

Savinase is a bactericidal enzyme secreted by the alkalophilic bacterium Bacillus lentus.73,74 It is a subtilisin-like serine endopeptidase, with very broad substrate specificity, that displays stable and maximal proteolytic activity within the pH 8−12 range and functions optimally between 20 and 60 °C.73 In industry, savinase preparations are commonly used in detergent formulations to remove various protein-based stains, including from grass, mucous, feces, and foods such as eggs, lentils, and gravy.74 A crystal structure (PDB 1TK2, 1.54 Å) for the antibiotic decapeptide Gramicidin S in complex with the alkaline proteinase savinase shows the interactions summarized in Figure 2. Gramicidin S is cyclo-[Val-Orn-Leu-Dpn-Pro-Val-

Figure 1. 2D representation of interactions of α-conotoxin IMI with AchBP (PDB 2C9T, 2.25 Å). Ligand is black, while protein residues and water molecules are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Protein residue of alternate chain in italics. 3D views of the bound cyclic peptide (PDB 2C9T, middle panel) and unbound NMR-derived solution structure (PDB 1CNL, bottom panel) are also given. Superimposition of bound and unbound cyclic peptide backbone structures indicated little difference (RMSD = 2.2 Å).

Figure 2. 2D representation of interactions of Gramicidin S with alkaline proteinase savinase (PDB 1TK2, 1.54 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. A 3D view of the bound cyclic peptide is shown below.

The bound structure of α-conotoxin IMI can be viewed in two distinct macrocyclic parts. On the left side of the compound (Figure 1), five intramolecular hydrogen bonds are formed. One of these (Cys2 CO···HN Asp5) is an i to i+3 transannular 10-membered hydrogen bond that defines a βturn. A 13-membered i to i+4 transannular hydrogen bond from the same aspartate (Asp5 NH···OC Gly1) kinks the conformation at the N-terminus. Two exocyclic hydrogen bonds occur between Asp5 and Arg7 side chains, along with a hydrogen bond from Asp5 O to the main chain NH of Arg7. These bonds may help position the Arg7 side chain to make hydrogen bonds with the protein through Tyr91 and Ile194. This side of the macrocycle seems only to interact with the protein through side-chain hydrogen bonds and one hydrogen bond to the Cys3 carbonyl oxygen. On the right side of the macrocycle, two C-terminal transannular 10-membered hydrogen bonds (Ala9 CO···HN Cys12, Trp10 CO···NH2 Cys12 amide terminus) and one between side-chain Trp NH to the carbonyl oxygen of Arg7 appear to stabilize a 310-helical backbone structure (gray ribbon below). Interactions with the

Orn-Leu-Dpn-Pro], where Dpn = D-phenylalanine and there is a repeat of the pentapeptide sequence VOLfP. The cyclic peptide presents an extended strand to the enzyme. This structure has two DPn-Pro turn-inducing motifs on opposite sides of the cycle, which induce two β-turns through forming i to i+3 transannular 10-membered hydrogen-bonded rings (Val1 NH···OC Leu8, Val6 NH···OC Leu3). Another two transannular hydrogen bonds (Leu3 NH···OC Val6, Leu8 NH···OC Val1) help to stabilize a β-sheet structure or extended conformation along the Val-Orn-Leu segments that connect the two β-turns. Even though two faces of the cycle contain the Val-Orn-Leu sequence, they interact with the target differently, with only the hydrophobic side chains contacting the protein. Interestingly, only the one β-turn contacts the target, and this is through hydrogen bonds to Ser128 from both Leu8 CO and Val1 NH. Hence, the constraining effect of the β-turn is important for both induction of the transannular hydrogen bonds with Ser128 E


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transannular hydrogen bonds between main-chain amides, four of which are key to stabilizing a β-hairpin structure while the other (Asn7 NH···OC Gly8) stabilizes a β-turn. Interestingly, the Thr6-OH makes two additional intramolecular hydrogen bonds, which may help to stabilize the structure but also position the Thr6-OH for interaction with Asp103. These additional bonds do not appear to distort the molecule from a classical β-hairpin structure. The majority of interactions with the protein are made by the turn region of tetrapeptide TNGS and the Trp10 on the C-terminal strand. Limited interactions are made with other residues involved in the strand region, with three out of four residues in the N-terminal strand making no interactions, indicating that this region is mainly being used to induce the β-hairpin structure. Mutation of the Asn-Thr-Asn section of the molecule to Ala-Ala-Ala did not affect binding affinity, while the Trp-His-Ile segment was identified as being crucial for antibody recognition.

and for optimizing interactions between hydrophobic side chains on the cyclic peptide with hydrophobic residues in the protein. The substrate-binding loops consist of residues at positions 99−104 and 125−131, with residues 101−103 and 126−128 in β-strand conformations.75 The crystal structure76 and NMR structure in water77 of free Gramacidin S reveal that the four intramolecular hydrogen bonds, two β-turns and antiparallel β-strands are present in the macrocycle and preserved upon binding to the protein alkaline proteinase savinase. 2.3. Antibody-Binding Fragments

Antibodies are glycoproteins called immunoglobulins, produced by immune cells in response to a foreign molecule.78 The primary function of antibodies is to protect the host from infection by binding specifically to an antigen and alerting the immune system.78 Antibody-binding fragment (Fab) is a region on an antibody that binds to specific antigens.78 Over the years, antibody drug designers have exploited the unique specificity of immunoglobulins and Fabs to engineer customized therapeutics with unique pharmacological properties optimized for specific applications.78 A number of crystal structures of cyclic peptide-bound antigen-binding fragments (Fab) of monoclonal antibodies have been reported for targets, such as hepatitis C virus (HCV) E2 epitope I,79 third variable region of HIV-1 gp120,80,81 N-terminal sequence of transforming growth factor alpha,82 Cetuximab,83 and CD20 cell surface marker.84 These structures have major implications for design of epitope-based vaccines. The structure of Fab in complex with a cyclic peptide derived from the epitope comprising residues 412−423 of viral E2 glycoprotein is described here (PDB 5EOC, 1.98 Å). The epitope adopts a βhairpin conformation when bound to neutralizing antibodies. The cyclic peptide mimic, cyclo-[CQLINTNGSWHIC], has a disulfide bond between terminal residues and interacts with Fab as shown in Figure 3. This macrocycle contains five

2.4. Apelin Receptor

Apelin receptor (AR) is a class A G-protein coupled receptor with a wide expression pattern throughout the human body.85,86 Apelin peptides are the endogenous ligands for human AR that plays a major role in regulating a variety of physiological functions, including vasoconstriction and dilation, angiogenesis, energy metabolism, and fluid homeostasis.85,86 More recently, dysregulated activation of AR has been implicated in a range of pathologies, including cardiovascular disease, diabetes, obesity, and cancer.85,86

Figure 3. 2D representation of interactions of the cyclic peptide with Fab (PDB 5EOC, 1.98 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Protein residues of the heavy chain of Fab are shown as normal text while the ones from the light chain of Fab are shown in italics. A 3D view of the bound cyclic peptide is shown below.

Figure 4. 2D representation of interactions of the cyclic peptide with apelin receptor (PDB 5VBL, 2.6 Å). Atoms missing in the crystal structure are shown in red. Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. A 3D view of the bound cyclic peptide is shown below. F


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C-terminal loop of RON2, derived from the crystal structure of RONsp1 bound to AMA1 (PDB 3ZWZ, 2.1 Å), was used as a starting point to investigate a series of cyclic peptides where the terminal Cys residues were linked to form a disulfide bridge.91 One of the cyclic peptides, where Phe2 was replaced by 6-chloro-tryptophan, had KD = 0.9 μM toward FVO ARA1, and the crystal structure of the protein−peptide complex (PDB 4Z0F, 2.3 Å) showed the interactions summarized in Figure 5. Formation of the disulfide bond to create the macrocycle results in a β-sheet structure, which is stabilized by five intramolecular hydrogen bonds, two of which are made by the side-chain OH of Thr10. The compound contains a Pro-Pro motif, which usually favors a turn conformation but not in this case. Instead the RMSPP region is only a loop region. Only one face of the β-sheet region of the molecule is predominantly involved in binding to the target. Within the loop region only the Arg and Met side-chain residues make interactions with the protein. Alanine scanning of the CFTTRMSPPQQIC sequence shows that Phe2, Arg5, and Pro9 are the key side chains. The crystal structure reveals the importance of the Arg side chain which makes six hydrogen bonds to the target. NMR studies from the same report showed that the 13-residue peptide displayed 30 amide resonances, indicating three predominant conformations of the peptide in solution. The slowly exchanging conformations were attributed to cis−trans isomerization of Pro residues in the sequence.

Structural studies can aid in the design of future therapeutics for this receptor. A co-crystal structure of the apelin receptor bound to a designed peptidomimetic agonist has been reported (PDB 5VBL, 2.6 Å).87 The peptidomimetic comprises 17 amino acid residues with four residues forming a cyclic peptide component, H-cyclo-(10,13)-Lys-Phe-Arg-Arg-Gln-Arg-ProhArg-Cha-[Glu-His-Lys-Lys]-Oic-Nle-Pro-(4-Cl)Phe-OH, (hArg = homoarginine, Cha = cyclohexylalanine, Oic = octahydroindole-2-carboxylic acid, Nle = norleucine), where side chains of Glu10 and Lys13 form a lactam bridge. Interactions between the cyclic peptide and the apelin receptor are summarized in Figure 4. The macrocyclic part of the molecule appears to have done little to stabilize structure, with the i to i+3 lactam bridge region of the molecule not forming any intramolecular hydrogen bonds. Interestingly, the Glu10 carbonyl oxygen of the lactam bridge makes a hydrogen bond with Thr176, and the Lys13 side chain makes hydrophobic interactions with the target. 2.5. Apical Membrane Antigen 1

Malaria is an infectious disease that infects millions of humans worldwide and is now responsible for about 500 000 deaths per annum.88 It is caused by mosquitoes that transmit protozoan parasites of the genus Plasmodium. Of five species of these single-celled parasites, Plasmodium falciparum accounts for the majority of malaria-related deaths.88 Apical membrane antigen (AMA1) is a protein antigen expressed on the surface of P. falciparum.89 Recent studies have highlighted that the interaction between AMA1 and rhoptry neck protein (RON) complexes plays a key role in the invasion of host red blood cells by Plasmodium parasites.89,90 Thus, disrupting host invasion by targeting the AMA1-RON protein−protein interaction is a promising new therapeutic avenue.89,90 A 13residue β-hairpin peptide (CFTTRMSPPQQIC) based on the

2.6. Asp/Asn β-Hydroxylase

Aspartyl (asparginyl) β-hydroxylase (AAH) enzyme specifically post-translationally hydroxylates one Asp or Asn residue in certain epidermal growth factor-like domains of a number of

Figure 6. 2D representation of interactions of the cyclic peptide with Asp/Asn β-hydroxylase (PDB 5JZZ, 2.292 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Atoms missing in the crystal structure are shown in red. A 3D view of the bound cyclic peptide is shown below.

Figure 5. 2D representation of interactions of the cyclic peptide with ARA1 (PDB 4Z0F, 2.3 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. A 3D view of the bound cyclic peptide is shown below. G


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bovine and human proteins. AAH notably hydroxylates Notch, stimulates cell motility, and may crosstalk with HIF-1α.92 A crystal structure of aspartyl/asparginyl β-hydroxylase and TPR domain in complex with a cyclic peptide (factor X substrate peptide fragment mimic) has been reported (PDB 5JZZ, 2.292 Å). Figure 6 summarizes the interactions between Asp/Asn βhydroxylase cyclic peptide, Ac-cyclo-(1,10)-[AKDGLGEYTC]-TSLEGFEGK-OH, where the N-terminal methyl group is linked to the side-chain of Cys to form a thioether link. The macrocyclic section of this molecule does not contain any intramolecular hydrogen bonds. One interesting feature is the hydrogen bonding to Glu617, which makes three hydrogen bonds to three consecutive amide NHs along the peptide backbone. Interestingly, this section of the peptide appears to be the most highly structured even though it adopts a loop like conformation. 2.7. Bacterial RNA Polymerase

Bacterial RNA polymerase, the central enzyme in bacterial gene expression, is a complex molecular machine that is a proven target for anti-microbials such as Rifampicin.93 GE23077 is a cyclic heptapeptide produced by the soil bacterium Actinomadura sp. DSMZ 13491. It has anti-bacterial activity against both Gram-positive and Gram-negative bacteria by selectively inhibiting RNA polymerase (Ki = 6 nM) from bacteria over human.94 It prevents the binding of initiating nucleotides and thereby prevents transcription initiation. The structures (PDB 4MQ9, 3.35 Å; PDB 4OIN, 2.8 Å) containing the promoter DNA95 of GE23077 complexed with RNA polymerase from Thermus thermophilus showed binding to the functionally critical residues of enzyme adjacent to the rifamycin binding site. GE23077 contains head-to-tail cyclized cyclo-[D-Apa-D-Ser-D-Val-(3R,4S)-L-dhGln-D-aThr-D-iSer-LAma]; where Apa = α,β-diaminopropanoic acid, dhGln = β,γdihydroxyglutamine, aThr = allothreonine, iSer = isoserine, Ama = α-aminomalonic acid. GE23077 is a mix of four major factors (A1, A2, B1, B2) which differ in the nature of the side chain of the α,β-diaminopropanoic acid residue, where the amino group forms an amide with 2-methyl-2-butenoic acid (factor A, MW = 803 Da) and 3-methylbutanoic acid (factor B, MW = 805 Da). Of 10 chiral centers, stereochemistry at five centers was assigned by degradation studies,96 while chirality at four centers was assigned on the basis of the co-crystal structure (PDB 4OIN), the remaining one (C3 of dhGln) was uncertain. The critical inter- and intramolecular interactions are depicted in Figure 7. The bound conformation of GE23077 displays four intramolecular hydrogen bonds of which three are made with the D-Thr-OH. This interesting motif appears to hold the cycle in a stabilized loop on the left side of the molecule (L-Ama-D-Apa-D-Ser). The remaining hydrogen bond (D-Ser2 CO···HN D-aThr5) stabilizes a β-turn on the right side of the molecule. Within this motif there are two further intramolecular hydrogen bonds formed from the L-dhGln side chain. This appears to help position this side chain for formation of a complex with Mg2+. Hence the binding mode appears to present two distinct structural motifs for binding to the target. This molecule binds to the target through a complex series of hydrogen bonds with all but one side chain (Ser) making interactions. Structure−activity relationships studies of derivatives of GE show that modification of the dhGln side chain eliminates activity. Removal of AMA reduces activity, and substitution of dmaDap had little effect on activity.

Figure 7. 2D representation of interactions of GE23077 with RNA polymerase (PDB 4OIN). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

2.8. Botulinum Neurotoxin

Botulinum neurotoxins (serotypes A, B, C1, C2, D, E, F, and G) are proteins produced by the bacterium Clostridium botulinum.97,98 These neurotoxins induce flaccid paralysis by inhibiting the neurotransmitter acetylcholine release, mainly at peripheral cholinergic nerve terminals of the skeletal and

Figure 8. 2D representation of interactions of the cyclic peptide with botulinum neurotoxin (PDB 4ZJX, 1.94 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. A 3D view of the bound cyclic peptide is shown below. H


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autonomous nervous systems.97,98 These neurotoxins derived from botulinum are routinely used in a wide variety of cosmetological (Botox) and sports medicine applications.97,98 A crystal structure of the catalytic domain of botulinum neurotoxin serotype A with a cyclic peptide inhibitor is reported (PDB 4ZJX, 1.94 Å). The protein prevents the release of the neurotransmitter acetylcholine from axon endings at the neuromuscular junctions and thus causes flaccid paralysis. The protein is commercially used under the name Botox. The cyclic peptide is H-cyclo-[Cys-(Dab)-Arg-Trp-Thr-Lys-Cys]-LeuNH2, where Dab = 2,4-diaminobutyric acid which condenses with the carboxylate of Cys1 through its 4-amino group, and both Cys residues form a disulfide bond. The interactions between the cyclic peptide and botulinum neurotoxin are shown in Figure 8. The two intramolecular hydrogen bonds in this structure stabilize an α-helical motif across residues ArgTrp-Thr-Lys-Cys-Leu. This α-helix presents side chains for interactions with the protein while the amide backbone is not involved. The terminal NH3+ forms a charged interaction with a glutamate residue in the protein, while the Dab-NH2 coordinates to Zn2+. 2.9. Casein Kinase 2

Casein kinase 2 (CK2) is a serine/threonine-selective kinase with many intracellular substrates, particularly acidic substrates like caseins, involved in cell cycle regulation, DNA repair, regulation of circadian rhythm, and others.99 In terms of cancer, elevated concentrations of CK2 have long been associated with increased cell proliferation in cancer cells.100 Furthermore, inhibition of CK2 activity induces apoptosis of cancer cells in both in vitro and in vivo xenograft cancer models, suggesting promising therapeutic potential.100 CK2 exists as a heterotetrameric holoenzyme with two catalytic chains (CK2α) attached to a dimer of non-catalytic subunits (CK2β).99 A 13-residue disulfide bridged peptide with 11 residues in a cycle, cyclo-G-[CRLYGFKIHGC]-G-OH, was derived from the C-terminus of the CK2β segment and is reported to be a CK2β-competitive inhibitor (Ki = 20 μM), with a binding constant KD = 0.56 μΜ compared to KD = 4.0 nM for the CK2α(1−335)/CK2β(1−193) interaction as measured by ITC experiments.101 The affinity is largely driven by the enthalpic contributions in both cases. A co-crystal structure of this cyclic peptide with CK2α is reported (PDB 4IB5, 2.2 Å), where some well-ordered water molecules, absent in the holoenzyme, are detected at the binding interface.102 The interactions between the cyclic peptide and CK2α are shown in Figure 9. The cyclic peptide adopts a type-I β-hairpin loop motif upon binding to CK2α. The crystal asymmetric unit contains three independent CK2α(1−335) chains (A, B, and C) and four cyclic peptides (chains D, E, F, and G), where each cyclic peptide interacts with a single CK2α subunit and one cyclic peptide (chain G) with no β-hairpin structure is found to be unspecifically intercalated between two CK2α subunits. The cyclic peptide in chain D had the best-defined electron density in the crystal structure, and so it is discussed here. The macrocycle presents two distinct structural motifs to the target: a β-hairpin in the region RLYGFK formed by two hydrogen bonds (Leu4 NH···OC Phe7, Phe7 NH···OC Leu) and a β-turn centered on IHGC stabilized by an Ile9 CO···NH Cys11 hydrogen bond. The β-hairpin is stabilized by two intramolecular hydrogen bonds with a β-turn (LYGF) capping two β-sheet structures. The side chains of each amino acid within this turn are

Figure 9. 2D representation of interactions of cyclic peptide and CK2α (PDB 4IB5, 2.2 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

involved in binding to the target. A second distinct β-turn based around His10 presents this amino acid to Ile57 and Gln40. The amide NH of the neighboring Ile is the only amide NH within the cycle that makes a direct hydrogen bond to the target (Leu41). Neither charged NH3+ groups of this molecule appear to make any interactions with the target. Alanine scanning of this peptide revealed Tyr5, Gly6, Phe7 are highly disrupted by alanine substitution, with Phe7 being a key driver of binding. This highlights the importance of the β-turn structure in this molecule. 2.10. Caseinolytic Protein C1 (ClpC1)

Caseinolytic protein C1 (ClpC1) is a molecular chaperone that belongs to the HSP100 group of AAA+ ATPases.103,104 Mycobacterium tuberculosis, the key pathogen that causes tuberculosis, has four Clp ATPases (ClpC1, ClpX, Clp2, and ClpB) together with two ClpP ATP-dependent proteases (ClpP1 and ClpP2) to regulate intracellular protein homeostasis via proteolysis.103,104 Inhibition of Clp mediated proteolysis could inhibit M. tuberculosis viability.103,104 A naturally occurring cyclic heptapeptide cyclomarin A (CymA) has been reported to have potent anti-tubercular activity (MIC50 = 1 μM) through binding to ClpC1 and suppressing M. tuberculosis viability.104 A more soluble derivative CymA1 (containing the open epoxide ring and an additional propylene diamine group) was used to measure binding affinity with various fragments of ClpC1.105 CymA binds to the N-terminal domain (residues 1−145) in the crystal structure. CymA1 was found to inhibit ClpC1 at nM concentrations, while CymA proved to be a validated lead compound against the new drug target ClpC1. A crystal structure for CymA-ClpC1 was solved at a high resolution (PDB 3WDC, 1.18 Å), and interactions are summarized in I


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derived factor (SDF)-1α/CXCL12.108,109 Dysregulation of CXCR4 in multiple human inflammatory diseases and cancers and the exploitation of CXCR4 by HIV-1 virus for T-cell entry make it an attractive therapeutic target.108,109 Crystal structures

Figure 10. The head-to-tail cyclized peptide contains unusual amino acids, such as 5-hydroxyleucine, β-methoxyphenyl-

Figure 10. 2D representation of interactions of cyclic peptide CymA with ClpC1 (PDB 5CS2, 1.65 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 11. 2D representation of interactions of cyclic peptide CVX15 with chemokine CXCR4 receptor (PDB 3OE0, 2.9 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

alanine, tert-prenylated β-hydroxytryptophan, and 2-amino3,5-dimethyl-4-hexenoic acid, with two N-methyl residues and three transannular hydrogen bonds that stabilize two β-turns on either side of the macrocycle. CymA has also been found to inhibit diadenosine triphosphate hydrolase from P. falciparum (PDB 5CS2, 1.65 Å) and was also proposed to be a potential anti-malarial lead.106 This heptapeptide macrocycle contains three intramolecular hydrogen bonds, which help to stabilize two distinct turn structures in the molecule. On the left side, a β-turn presents the Val and Leu side chains to make hydrophobic interactions with the target. On the right side, the two other hydrogen bonds stabilize an unusual turn, which has a different shape than the left side of the cycle but also presents the side chains for interactions with the target. 5Hydroxy-N-methylleucine and β-methoxyphenylalanine are both presented in an extended strand conformation in which 5-hydroxy-N-methylleucine interacts with Glu89 while the βmethoxyphenylalanine interacts with the protein through both hydrophobic contacts with its side chain and the adjacent alanine hydrogen bonding to Phe80 through its amide carbonyl group. The crystal structure of unbound cyclomarin A as the acetate salt also showed the unusual turn conformation on the right side of the molecule; however, this was stabilized by only one transannular hydrogen bond. The other transannular hydrogen bond on the left side of the molecule was also present.107

of CXCR4 receptors have been reported for small-molecule antagonists as well as with a competitive inhibitor, CVX15 (PDB 3OE0, 2.9 Å),110 a 16-residue cyclic peptide analogue of the horseshoe crab peptide polyphemusin found to be an HIVinhibiting and anti-metastatic agent.111−113 Cyclo-(4,13)RR(Nal)[CYQK(D-Pro)ProYR(Cit)C]RG(D-Pro) contains a Cys4−Cys13 disulfide bond, two D-amino acids, and two nonnatural amino acids. CVX15 potently inhibits the binding of CXCL12-Red to CXCR4 (Ki = 9 pM).114 The bound conformation of CVX15 reveals that the D-Pro8-L-Pro9 motif induced a β-turn structure and promoted an extended strandlike conformation along each side of the macrocycle. Within the macrocycle, four transannular hydrogen bonds were formed along with one additional exocyclic hydrogen bond. Two additional hydrogen bonds from the glutamine side chain to the amide carbonyls of L-Pro and Tyr distort the shape of the peptide away from a classical β-sheet conformation. In this case both β-sheets are involved in making interactions with the target, and the structure-inducing motif plays no role in forming interactions. Outside of the macrocycle, three arginine residues form interactions with Asp262, Asp171 and Asp 187. The bulky peptide fills most available space in its binding pocket. The C-terminal D-Pro is buried in a pocket close to the N-terminus of the peptide, enabling water-mediated hydrogen bonds with Asp288 (Figure 11).

2.11. Chemokine CXCR4 Receptor

The CXC chemokine receptor 4 (CXCR4) belongs to the G protein-coupled receptor (GPCR) superfamily of proteins and is activated by its natural chemokine ligand, stromal cellJ


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2.12. Chitinase

a compressed structure and helps position the histidine to make four hydrogen bonds and two hydrophobic interactions with the target.

Chitin is a linear polymer of β-1,4-N-acetylglucosamine (GlcNAC) found in the cytoskeleton of insects, fungi, yeast, algae, and other vertebrates.115 Chitinases are glycosyl hydrolase enzymes that degrade chitin. Apart from their ability to break down chitin, some evidence suggests that chitin and chitinases are used by various parasites to establish themselves in humans, causing diseases such as inflammatory lung diseases.115 Thus, developing potent chitinase inhibitors could possibly pave the way to novel insecticides, fungicides, and even human therapeutics.115 Argifin and argadin, natural cyclopentapeptides derived from Gliocladium and Clonostachys fungal cultures, respectively, have been characterized as chitinase inhibitors. A number of crystal structures of these two cyclic peptides with chitinase from different organisms have been reported. This includes Serratia marcescens chitinase B (PDB 1H0G, 2.0 Å, argadin; PDB 1H0I, 2.0 Å, argifin), Aspergillus f umigatus (PDB 1W9U, 1.85 Å, argadin; PDB 1W9 V, 2.0 Å, argifin), and human chitinase (PDB 1WAW, 1.75 Å, argadin; PDB 1WB0, 1.65 Å, argifin).116,117 Argadin is the most potent of these inhibitors of human chitinase (IC50 = 13 nM), and its interactions with human chitinase are shown in Figure 12. Argadin is a cyclic pentapeptide with 15 heavy

2.13. Chymotrypsin

Proteases are well known to bind to extended or β-strand backbone conformations of substrates and inhibitors.28,29,32,33,112,113,118,119 Based on extensive reports of cyclic peptides being able to mimic this backbone conformation and inhibit proteases,19−23,32,33,118,119 a cyclic peptide was designed, synthesized, and biologically tested against cysteine proteases (mouse calpain, Ki = 220 nM; human cathepsin L, Ki = 30 nM; human cathepsin S, Ki = 2.3 nM) and serine proteases (bovine α-chymotrypsin, Ki = 33 nM; human leukocyte elastase, Ki = 600 nM).120 Calpains are implicated in cataracts and traumatic brain injury, and cathepsins L and S play key roles in tumor progression and inflammatory and autoimmune disorders, while α-chymotrypsin and human leukocyte elastase are model serine proteases. The crystal structure for a cyclic peptide bound to α-chymotrypsin (PDB 4Q2K, 2.2 Å) showed interactions summarized in Figure 13.

Figure 13. 2D representation of interactions of macrocyclic peptidomimetic 5b with α-chymotrypsin (PDB 4Q2K, 2.2 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 12. 2D representation of interactions of argadin with human chitinase (PDB 1WAW, 1.75 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

The aldehyde covalently bound to Ser195, the Phe side chain buried in the S1 enzyme pocket, and the inhibitor adopted a βstrand conformation. The bound conformation showed βsheet-like hydrogen bonding of cyclic peptide residues with Gly216 and Ser 214. The hydrophobic nature of the linker also contributed to binding to Trp215 of the protein.

atoms in the macrocycle composed of L-histidine, L-2aminoadipic acid, Nω-acetyl-L-arginine, D-proline, and Laspartic β-semialdehyde, in which the aldehyde carbon is bonded to the histidyl α-amino residue. The bound structure of argadin shows that one (i, i+3) transannular 10-membered hydrogen-bonded ring (D-Pro NH···OC L-2-amino-adipic acid) that defines a β-turn is stabilized in the cyclic pentapeptide giving rise to a flattened cyclic structure. This compression allows the L-2-aminoadipic acid, Nω-acetyl-L-arginine to interact with each other giving rise to three intramolecular hydrogen bonds, which in turn may allow this motif to interact with Trp218 and Trp99. The unusual L-aspartic β-semialdehyde alongside the dPro allows the folding of the cycle into

2.14. Complement C5a Receptor

Complement protein C5a is a potent proinflammatory and chemotactic factor that primarily signals via a G-protein coupled receptor (GPCR), the C5a receptor C5aR.121,122 C5aR is expressed widely on immune cells, including neutrophils, monocytes, macrophages, eosinophils, and T cells, but also on other cells including of the liver, kidney, adipose, and central nervous system. C5aR activation is also now implicated in many functions besides immunity and inflammation, such as metabolic functions and dysfunction, K


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antagonist is that only the indole side chain of Trp (or an aromatic side chain replacement without the indole NH) is responsible for antagonist activity, with the other residues all conferring affinity rather than function.129 Thus, it would appear that the Trp···Arg175 π−cation interaction and/or occupancy of a hydrophobic pocket by the Trp side chain is crucial for preventing receptor conformational change that leads to C5aR1 activation, since phenylalanine or naphthylalanine at this position also confers antagonist potency while aliphatic residues like Leu or Cha confer agonist activity.129 Finally, another important property of this cyclic peptide is its long residence time on its receptor, a property directly related to the binding mode that leads to a long duration of antagonist action on cells and in rodents.130 Three NMR solution structures of PMX53 have been reported in DMSO-d6 and show the presence of intramolecular hydrogen bonding that stabilizes a turn structure. This is in contrast to no intramolecular hydrogen bonding in the receptor-bound crystal structure derived from aqueous media.129,131,132

crosstalk with TLR signaling, developmental biology, and cancer metastasis and progression.121,123 Thus, novel potent C5aR antagonists have predicted therapeutic activity in many human diseases. PMX53, originally named 3D53,122 is a cyclic peptide Ac-cyclo-(2,6)-Phe-[Orn-Pro-D-Cha-Trp-Arg] that we designed to mimic the C-terminal turn of C5a,124,125 acts as an insurmountable antagonist of C5aR1 but not C5aR2, and has been evaluated in Phase II clinical trials for arthritis and psoriasis.122,124,126 Notable features of the macrocycle are a pendant phenylalanine attached to the cyclic pentapeptide, adjacent hydrophobic side chains (Phe, Pro, D-Cha, Trp) presenting a connected hydrophobic face, and high affinity conferring D-Cha, Phe, and L-Arg residues. A crystal structure (PDB 6C1R, 2.2 Å) has been reported for human C5aR1 in complex with this cyclic peptide, as well as with a smallmolecule allosteric antagonist, avacopan.127 A separate structure of the protein with a different small-molecule allosteric antagonist has also been reported.128 The PMX53C5aR1 binding interactions are summarized in Figure 14.

2.15. Cyclin-Dependent Kinase-2

Cyclin-dependent kinase-2 (CDK2), or cell division protein kinase 2, plays a critical role in eukaryotic cell cycle regulation, and inhibitors of CDK-2 are being developed as potential anticancer agents.133 The cyclin groove recognition motif

Figure 14. 2D representation of interactions of cyclic peptide PMX53 with human C5a receptor (PDB 6C1R, 2.2 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 15. 2D representation of interactions of the cyclic peptide with CDK2 (PDB 1URC, 2.6 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Although the macrocycle does present a well-defined turn motif to the receptor, there is no intramolecular hydrogen bond present in the receptor-bound macrocycle. Numerous water-mediated interactions are observed between the hydrophilic side of PMX53 and C5aR. In addition to several hydrophobic interactions stabilizing Trp and D-Cha side chains, and several direct and water-mediated hydrogen bonds stabilizing backbone and polar side chains, the structure is unusual in having two π−cation and one cation−π ligand− protein interactions that stabilize the complex. PMX53 makes contacts with all seven transmembrane helices as well as extracellular loop 2 of the receptor. A key feature of this

(RNLFG) in the natural CDK-inhibitory tumor suppressor protein p27KIPI was used as the basis for design and characterization of a series of cyclic peptide inhibitors.134 The cyclic peptide Ac-cyclo-(2,5)-R-[KLFG], where the Lys side chain forms a lactam with the C-terminal carboxylic acid, has only 16 heavy atoms in the ring. It exhibited IC50 = 19.2 μM in a competitive cyclin A binding assay, and a co-crystal structure with the kinase was determined (PDB 1URC, 2.6 Å). The interactions between the cyclic peptide and CDK2 are shown in Figure 15. The bound cyclic peptide has one L


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intramolecular seven-membered (i to i+2) hydrogen-bonded ring indicative of a γ-turn. This turn brings the Phe4 side chain into the proximity of the neighboring Leu3 side chain and allows both groups to form hydrophobic interactions with Ile213 and Leu 214. The Lys-Asp side chain−side chain bridge of the macrocycle also contributes to binding and interacts with Thr285. Outside of the macrocycle the arginine side chain forms a hydrogen bond with Glu220.

potently inhibits a sub-genomic HCV replicon co-expressing a luciferase gene in Hyh7.5 cells (IC50 = 40 nM), and also inhibits OATP1B1 transporter activity in CHO cells (IC50 = 6 μM, 8-fluorescein-cAMP as substrate). Interactions between NIM258 and cyclophilin D are shown in Figure 16. No transannular hydrogen bonds are observed in this proteinbound macrocycle, in contrast to most structures reported for CsA alone in solution, where there are often three transannular and one exocyclic hydrogen bonds. Instead there is only one intramolecular hydrogen bond in the macrocycle, from an amide carbonyl oxygen to a side-chain OH, that appears to distort the open structure of one end of the cycle but is not enough to induce a well-defined structure. The top half of this molecule is solvent exposed and does not interact with the protein, whereas the side chains and backbone of the bottom half of the molecule all interact with the target.

2.16. Cyclophilin D

Cyclophilin D, a matrix peptidyl isomerase, is a mitochondrialspecific cyclophilin that is known to play an important role in the formation of mitochondrial permeability transition pore (mPTP) and is a drug target for various inflammatory and neurodegenerative diseases.135 Cyclosporin A (CsA) is an 11-

2.17. DnaN

DnaN is a DNA sliding clamp protein that interacts with polymerase III subunit (DnaE1) and plays a major role in bacterial DNA replication.139 Emerging studies have validated

Figure 16. 2D representation of interactions of NIM258 with cyclophilin D (PDB 4TOT, 2.39 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 17. 2D representation of interactions of the cyclic peptide with DnaN (PDB 5AGU, 2.173 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

residue cyclic peptide that binds strongly to cyclophilins and is an established immunosuppressant drug in the clinic.136 Toward non-immunosuppressive cyclophilin inhibitors, which are also potent inhibitors of hepatitis C (HCV) replication in vitro, a modified analogue called NIM258 was developed.137 NIM258 is a less potent inhibitor of drug transporters, retains anti-HCV activity, and has a reasonable pharmacokinetic profile in rats and dogs. A crystal structure of NIM258 bound to rat cyclophilin D (PDB 4TOT, 2.39 Å) has been reported. Cyclosporin A, c[Bmt-Abu-Sar-Mle-Val-Mle-Ala-D-Ala-MleMle-Mva], contains seven N-methylated amino acids [Bmt = (4R)-4-((E)-2-butenyl)-4,N-dimethylthreonine, Abu = α-aminobutyric acid, Sar = N-methylglycine, Mle = N-methylisoleucine, and Mva = N-methylvaline.138 In NIM258, Sar3 and Mle4 residues of cyclosporin A are replaced by N-methylD-alanine and (3R)-4-[4-(2-methoxyethyl)piperazine-1-yl]-Nmethyl-L-valine, respectively. The cyclic peptide binds tightly to cyclophilin A (KD = 1.2 nM, surface plasmon resonance),

DnaN as a feasible anti-bacterial target for small molecules and peptidic inhibitors.139 Derivatives of a Streptomyces-derived cyclic peptide, griselimycin, inhibited the DNA polymerase sliding clamp DnaN and showed anti-tubercular activity,139 with MIC values for one analogue of 0.06 μg/mL against M. tuberculosis strain H37Rv and 0.2 μg/mL in RAW264.7 mouse macrophages. The crystal structure of griselimycin bound to DnaN was determined (PDB 5AGU, 2.173 Å). Griselimycin is Ac-Mva-Mp8-cyclo-[Nzc-Leu-Mp8-Leu-Mva-Pro-Mlu-Gly], where Mva = N-methylvaline, Mp8 = (4R)-methylproline, Nzc = N-methylthreonine, and Mlu = N-methyl-D-leucine), where the side-chain hydroxyl group of Nzc forms a lactone with the C-terminal carboxylic acid. Interactions between the cyclic peptide and DnaN are shown in Figure 17. The binding conformation of griselimycin shows two transannular hydrogen M


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acid group of Val, and there is a cis-peptide bond between Ynm and Phe (Figure 18). The bound structure of lyngbyastatin reveals two atypical intramolecular hydrogen bonds that help to present two distinct structural motifs to the protein. On the left side the combination of a cis-amide bond between Ynm and Phe and the hydroxy homoproline allows the formation of the hydrogen bond between the OH and the amide NH. This motif presents one turn structure to the target. On the right side of the compound the side-chain OH to C-terminal ester allows an atypical transannular hydrogen bond presenting a second turn motif.

bonds within the macrocycle. One bond in the Mva-Pro-MluGly segment is in an i to i+4 position; however, the dihedral angles for N-methylleucine and N-methylvaline are distorted away from an α-turn-type structure. The other bond is in an i to i+2 position indicating an inverse γ-turn with φ = −98.4°, ψ = 59.2°. This tight turn is induced by the methylproline in this position and draws both neighboring leucine side chains into close proximity, allowing them to both interact with Leu264. The binding of this peptide to its target is dominated by hydrophobic interactions with no side chains form any hydrogen bonds. Four hydrogen bonds are made between the amide backbone and the protein. The NMR structure of the closely related methylgriselimycin in chloroform maintained the two transannular hydrogen bonds. However, the intramolecular hydrogen bond between the amide proton of Gly10 and the carbonyl group of Leu6 is shorter (2.1 Å) and stronger in the protein-bound crystal structure than in the NMR solution structure (2.9 Å).140

2.19. Endothelin Receptor

Endothelin receptors (ETA and ETB) are G-protein coupled receptors (GPCR) that are activated by endogenous endothelins, ET-1, ET-2, and ET-3.143 ET-1 and ET-2 show higher affinity for ETA than ET-3, while all three have similar affinity for ETB.143 Endothelin antagonists show utility in congestive heart failure, hypertension, atherosclerosis, stroke, kidney failure, asthma, pain, and cancer.143 ET-1 is a 21residue cyclic peptide H-cyclo-(1,15; 3,11)-[CS[CSSLMDKEC]VYFC]-HLDIIW-OH, with two disulfide bonds between Cys1-Cys15 and Cys3-Cys11, the largest ring being 47-membered. The crystal structure of ETB receptor in complex with endothelin-1 was determined to understand the structural basis for receptor activation (PDB 5GLH, 2.8 Å).144 The interactions between endothelin-1 and ETB receptor are shown in Figure 19. The central residues D8 to L17 (α-helical region) form approximately three α-helical turns, and the C1 to M7 residues (N-terminal region) are tightly anchored to the α-helical region by the disulfide pairs. Intramolecular hydrogen bonds are made along the α-helical region D8 to L17, while

2.18. Elastase

Various cyclic peptide metabolites of cyanobacteria of marine, terrestrial, or freshwater origin exhibit serine protease inhibitory activity against elastase, chymotrypsin, and trypsin. In a study involving isolation, structural characterization, and testing of anti-proteolytic activity of metabolites derived from cyanobacteria collected from Cetti Bay in Guam, a range of cyclic peptides including various symplostatins were identified as potent inhibitors of elastase with selectivity over chymotrypsin.141 A potent cyclic peptide, lyngbyastatin 7,142

Figure 18. 2D representation of interactions of lyngbyastatin 7 with porcine pancreatic elastase (PDB 4GVU, 1.55 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

was an inhibitor of porcine pancreatic elastase (IC50 = 30 nM) and human neutrophil elastase (IC50 = 23 nM). Lyngbyastatin 7 is cyclo-(1,6)-[Val-Ynm-Phe-Glj-Dbu-Thr]-Gln-Ha, where Ynm = N-methyl-L-tyrosine, Glj = 5,5-dihydoxy-L-norvaline, Dbu = (2Z)-2-aminobut-2-enoic acid, and Ha = hexanoic acid. A crystal structure of lyngbyastatin 7 bound to porcine pancreatic elastase (PDB 4GVU, 1.55 Å)141 showed that the side chain of Thr forms a backbone ester with the carboxylic

Figure 19. 2D representation of interactions of endothelin-1 with ETB receptor (PDB 5GLH, 2.8 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. N


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interactions of this region with the receptor are predominately with all the helical side chains. On the right side of the molecule a transannular hydrogen bond from Met-NH to the Ser carbonyl oxygen helps to stabilize a β-turn, which allows a hydrogen bond to form between the Ser OH and the Leu amide NH. A γ-turn is also present in the C-terminal section of the molecule outside of the macrocyclic constraints. Both the C-terminal tail and the bottom section of the molecule form hydrogen bonds with the receptor. Comparison with the crystal structure of unbound endothelin reveals that the disulfide-constrained region of residues 1−15 has the same backbone conformation as when endothelin is bound to its receptor; however, many side-chain rotamers are different. The C-terminal region spanning amino acid residues 16−21 is helical in the free ligand, but it is in a different conformation in the protein-bound form.145,146 NMR solution structures show that the unbound cyclic peptide had the same α-helical region, whereas the N- and C-terminal regions were unstructured.147 2.20. Ephrin A4 Receptor

Ephrin A4 (EphA4), a member of the Eph family of receptor tyrosine kinases, has been recently identified as a molecular target for Alzheimer’s disease.148 Inhibiting receptor activation by endogenous ephrins has been shown to alleviate disease in an Alzheimer’s disease transgenic mouse model.149 EphA4 antagonists could be potential new treatments for Alzheimer’s disease.148 EphA4 has a ligand-binding domain at the Nterminus of its extracellular region and a tyrosine kinase domain in its cytoplasmic region. The co-crystal structure (PDB 4W50, 2,42 Å) of a known disulfide-bridged cyclic peptide H-cyclo-(4,12)-APY[CVYRGSWSC]-NH2, with micromolar inhibitory potency as well as binding affinity with the ligand binding domain of EphA4 receptor, was used to design a more potent cyclic peptide inhibitor where Gly8 was replaced by a β-alanine.150 The resulting peptide exhibited IC50 = 31 nM and KD = 30 nM (ITC experiment). The cyclic peptide was H-cyclo-(4,12)-Ala-Pro-Tyr-[Cys-Val-Tyr-Arg-Bal-SerTrp-Ser-Cys]-NH2, where BAL = β-alanine and both Cys residues form a disulfide bridge (PDB 4W4Z, 2.41 Å). Both cyclic peptides bind to the ephrin-binding pocket of EphA4 receptor. Upon binding, the peptide locks the DE and JK loops of the protein in highly structured anti-parallel β-sheet conformations, where replacement of Gly8 by β-alanine allows for formation of a less strained β-turn. Interactions between the cyclic peptide and EphA4 are shown in Figure 20. The bound conformation of the cyclic peptide shows five intramolecular hydrogen bonds, three being transannular bonds within the macrocycle. The disulfide bridge also allowed formation of an exocyclic hydrogen bond between the C-terminal amide and the Tyr2 carboxygennyl. The three hydrogen bonds on either side of the disulfide bridge stabilize a β-sheet structure. The other transannular hydrogen bond is in an i to i+4 position, but due to the presence of β-alanine, this results in a 13-membered ring. One exocyclic hydrogen bond from Tyr5-OH to the βalanine carbonyl appears to have affected the position of the tyrosine instead of the turn/loop position. Amino acids in the β-sheet region make minimal interactions with the target. In this case the extended loop and the N-terminal amino acids outside the macrocycle make hydrogen bonds with the target. These hydrogen bonds are predominantly made through sidechain interactions, with only one direct hydrogen bond to the peptide backbone from Tyr5 to Gln71.

Figure 20. 2D representation of interactions of the cyclic peptide with EphA4 (PDB 4W4Z, 2.41 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

2.21. Erythropoietin Receptor

Erythropoietin (EPO) is a 166-residue glycoprotein cytokine secreted by the kidneys, and it is the primary regulator of erythropoiesis, promoting survival, proliferation, and differentiation of erythroid progenitor cells.151,152 EPO binds to erythropoietin receptor (EpoR), a member of the type I cytokine receptor family.151,152 Clinically, recombinant EPO is used to treat anemia in chronic kidney disease, myelodysplasia, and cancer chemotherapy.151,152 A 20-residue cyclic peptide unrelated in sequence to the natural ligand was discovered as a potent agonist (KD = 200 nM EPOr, EC50 = 400 nM EPO stimulated cell proliferation).153 A crystal structure of it with the ligand-binding domain of its class 1 cytokine receptor was determined (PDB 1EBP, 2.8 Å).154 The cyclic peptide is comprised of H-cyclo-(6,15)-GGTYS-[CHFGPLTWVC]KPQGG-OH, where side chains of both Cys residues form a disulfide bridge. The peptide dimer binds to the dimer of the receptor. Interactions between the cyclic peptide and the ligand-binding domain of the erythropoietin receptor are shown in Figure 21. The bound conformation of this peptide shows a β-hairpin conformation stabilized by five transannular hydrogen bonds, three of which are inside the macrocycle. This peptide interacts with the target as a dimer, with the top βstrand involved in the interactions between two peptides. The left side of the compound has an i to i+5 hydrogen bond indicating a loop instead of a turn in this part of the macrocycle. The loop contains one exocyclic hydrogen bond from Thr12-OH to its amide NH. Phe8 and Trp13 on opposite sides of the β-sheet are in close proximity, indicating potential for a hydrophobic interaction. Outside the macrocycle, two further exocyclic hydrogen bonds help extend the βsheet structure. There are interactions with the target protein from both the loop and sheet motifs of the cyclic peptide. Interestingly, hydrogen bonds to the target in the loop section are made through the amide carbonyl groups of each amino O


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Figure 22. 2D representation of interactions of cyclic peptide PERM1 with estrogen receptor α (PDB 1PCG, 2.7 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 21. 2D representation of interactions of the cyclic peptide with the ligand binding domain of erythropoietin receptor (PDB 1EBP, 2.8 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. Atoms missing in the crystal structure are in red. A 3D view of the bound cyclic peptide is shown below.

2.23. Growth Receptor Bound Protein 2 SH2

Growth-receptor-bound protein 2 (Grb2) is a 25 kDa adapter protein that consists of two Src homology 3 (SH3) domains flanking the SH2 domain.158 Grb2 plays a key role in the regulation of p21ras activation to growth factor receptors.152 Recent evidence suggests that Grb2 activation is involved in multiple tumor malignancies and developing selective inhib-

acid. Interactions of the β-sheet section with protein are mainly through amino acid side-chain residues of the protein. 2.22. Estrogen Receptor α

Estrogen receptor α belongs to the nuclear receptor (NR) superfamily of ligand-activated transcription factors that regulate a wide range of physiological and developmental processes.155 Many NR coactivator proteins contain a short pentapeptide motif LXXLL, known as NR box, which is involved in receptor recognition.156 A peptidomimetic was designed by retaining the α-helical structure of an NR box mimetic in a short peptide with NR specificity. PERM-1, cyclo(2,5)-K[(D-Cys)ILC]RLLQ-NH2, has a D-Cys2-Cys5 disulfide bond and just four amino acids in the cycle which has 14 heavy atoms in the ring. It makes several hydrogen bonds that nucleate helicity leading to inhibition of receptor/coactivator recognition of estrogens α and β (Ki = 25 and 390 nM, respectively).157 While PERM-1 was not α-helical in water, it was quasi-helical (Figure 22) in the crystal structure for PERM-1 bound to the estrogen receptor α (PDB 1PCG, 2.7 Å). The bound structure shows five intramolecular hydrogen bonds with the D-Cys-Ile-Leu-Cys segment providing carbonyl oxygens that all interact with amide NHs along the chain. However, these are both i+3 and i+4 in nature, which distorts the structure from a classical α-helix. One exocyclic hydrogen bond is also observed, but it is in an i to i+3 position consistent with a β-turn or 310-helical conformation. The peptide adopts an amphipathic structure with four aliphatic residues (I3, L4, L7, L8) on one hydrophobic face while polar residues (Lys, Arg, Gln) align to form a hydrophilic face. The (i, i+3) disulfide-bridged sequence was a better helix mimetic than the (i, i+4) bridged sequence.

Figure 23. 2D representation of interactions of the cyclic phosphopeptide with Grb2 SH2 (PDB 1BM2, 2.1 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. P


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itors of Grb2 could provide an anti-cancer therapy.159 Screening using different phosphopeptides led to identification of a cyclic phosphopeptide as a selective and potent inhibitor of Grb2 SH2. 160 The crystal structure of the cyclic phosphopeptide with Grb2 SH2 was solved (PDB 1BM2, 2.1 Å). The cyclic phosphopeptide is Ac-cyclo-[Slz-Ptr-Val-AsnVal-Pro] (Slz = L-thialysine, Ptr = O-phosphotyrosine), where the C-terminal carboxylic group of Pro6 forms a lactam with the side chain of L-thialysine (Slz1). The interactions between the cyclic phosphopeptide and Grb2 SH2 are shown in Figure 23. The bound peptide has two intramolecular hydrogen bonds, one transannular bond dissecting the cycle in the middle to give two unusual 14-membered hydrogen-bonded rings. The left side has four amino acids and a loop structure, while the right side has an open structure. The other intramolecular hydrogen bond is from the Asn side-chain carbonyl to the Asn amide NH. This restriction of the side chain could be due to the multiple interactions made by the Asn.

with G7-17NATE was solved to understand the basis of the specificity and inhibition (PDB 3PQZ, 2.413 Å).163,164 Interactions between G7-18NATE and Grb7 SH2 are shown in Figure 24. The crystal structure is composed of Grb7-SH2 dimers in which one monomer of each dimer is peptide bound (chains A and B) and the other is apo-Grb7-SH2 (chains C and D). Grb7-SH2 dimers are a biologically relevant form of the protein and can direct binding to receptor tyrosine kinases.165 The bound structure of this compound reveals four intramolecular hydrogen bonds, three transannular and one exocyclic. The bond in the i to i+3 position between FEGY indicates a β-turn conformation. The i to i+2 bond on the right Pro-Phe indicates a γ-turn. These turn motifs on opposite sides of the cycle promote formation of the third hydrogen bond. The Asn forms an exocyclic hydrogen bond from the sidechain carbonyl to amide NH backbone. This residue is also involved in forming three hydrogen bonds with the target. This macrocycle presents one binding face to the target, and interactions are made with both the side chains and the amide backbone along the bottom face of this cycle. NMR studies of G7-18NATE show that the unbound peptide exists in two conformations in solution and does not form a rigid structure.166

2.24. Growth-Receptor-Bound Protein 7 SH2

Similar to Grb2, growth-receptor-bound protein 7 (Grb7) is an SH2 domain containing an adapter protein that interacts with the cytoplasmic domain of numerous receptor kinases to modulate downstream signaling.161 Grb7 has been identified as

2.25. Hemagglutinin

There is a continuous need to develop new influenza therapeutics that can target newer proteins and have novel mechanisms of action to counter potential pandemics, emerging viruses, and strain mutations.167 Influenza hemagglutinin is a class I fusion glycoprotein found on the surface of influenza viruses that recognizes sialic acid and enables specificity of virus attachment to target cells.168 Based on protein−protein interactions of hemagglutinin with neutralizing antibodies and their complex structures, a series of cyclic peptides was investigated.167 They exhibited affinity at nM concentrations and neutralized influenza A group 1 virus, including the H1N1 strain and avian H5N1. A representative co-crystal structure with hemagglutinin and a 33-membered cyclic peptide (PDB 5W6T, 2.59 Å), with KD < 30 nM against group 1 hemagglutinins and a comparatively better pharmacokinetic profile, is shown in Figure 25. The cyclic peptide is Ac-Ph8-cyclo-[Orn-Mle-Glu-Tyr-Zcl-Glu-Trp-Leu-Ser-9wv], where Ph8 = 5-phenyl-L-norvaline, Orn = L-ornithine, Mle = N-methyl-L-leucine, Zcl = 3,4-dichloro-L-phenylalanine, 9wv = β-alanyl-3-amino-L-alanine), and the side-chain amine of Orn forms a lactam bridge with the C-terminal carboxylic acid group. The protein-binding conformation of this compound is stabilized by four transannular hydrogen bonds, which present four turn motifs to the target. On the left side of the molecule, two γ-turns are formed by seven-membered hydrogen-bonded rings, one enabling the valine side chain to make hydrophobic interactions with Ile45 and Ile48. The second γ-turn around Glu helps to position the neighboring Tyr side chain. On the right side of the molecule, two 10-membered hydrogenbonded rings define two β-turns. The uppermost of these is also influenced by an exocyclic hydrogen bond from Ser-OH to the amide backbone NH which helps position this side chain to form a hydrogen bond with Gln42. The second β-turn around Trp and Glu presents the Trp to form a hydrogen bond with Asp 19. Almost all interactions with the target protein are made from side chains on the macrocycle, and there is also one hydrogen bond formed with the protein by the carbonyl oxygen of 5-phenyl-L-norvaline outside of the macrocycle.

Figure 24. 2D representation of interactions of G7-18NATE with Grb7 SH2 (PDB 3PQZ, 2.413 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

a prognostic marker and therapeutic target in breast cancer. 1 6 1 , 1 6 2 The G7-18NATE peptide (H-WFEGYDNTFPC-OH cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. The G718NATE peptide is a specific inhibitor of Grb7 over Grb2, Grb10, and Grb14 proteins. Binding studies using SPR show affinity for Grb7-SH2 domain under physiological phosphate conditions (KD = 5.7 μM). The crystal structure of Grb7 SH2 Q


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Figure 26. 2D representation of interactions of cyclic peptide Trapoxin A with histone deacetylase 8 (PDB 5VI6, 1.237 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

edge-to-face geometry (within 3 Å). Isothermal titration calorimetry gave a dissociation constant, KD = 3 nM. The hydrogen bond interactions of this compound with the target are driven by three amide NHs within the macrocycle. All three available NHs form a hydrogen bond with Asp101. The hPro and two Phe side chains all form hydrophobic interactions with the target protein. Interactions between the two Phe side chains may help present the amide NHs and also form a connected hydrophobic patch on this part of the macrocycle.

Figure 25. 2D representation of interactions of the cyclic peptide with hemagglutinin (PDB 5W6T, 2.59 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. The residue names from the second monomer of protein are shown in italics. A 3D view of the bound cyclic peptide is shown below.

2.26. Histone Deacetylase 8

2.27. Histone Demethylase

Acetylation/deacetylation of lysine residues by histone acetylases/deacetylases (HATs/HDACs) constitutes a major post-translational modification akin in frequency to phosphorylation.169 There are 18 mammalian HDAC enzymes, subdivided into four classes on the basis of domain organization: class I (HDAC1, 2, 3, 8), class IIa (4, 5, 7, 9) and IIb (HDAC6, 10), class III (SIRT 1−7), and class IV (HDAC11), all except class III being Zn2+-containing enzymes.170 Dysregulation of HDACs has been associated with a variety of pathological conditions including many inflammatory diseases and cancers.171,172 Currently, individual isozyme-selective inhibitors are sought as anti-inflammatory and anti-cancer agents.169 Four HDAC inhibitors are currently approved for clinical use in cancer chemotherapy, including the cyclic depsipeptide Romidepsin.173 Trapoxin A, first isolated from the microbial parasite Helocoma ambiens, is a cyclic tetrapeptide [Phe-Phe-(D-hPro)-Aoe] with just 12 heavy atoms in the macrocycle, where hPro = homoproline and Aoe = (2S,9S)-2-amino-8-oxo-9,10-epoxydecanoic acid. It is an irreversible inhibitor of class I HDAC enzymes (KD = 3 nM). The crystal structure of trapoxin A bound to HDAC8 (PDB 5VI6, 1.237 Å)174 showed that the carbonyl group of the α,β-epoxyketone electrophile of Aoe had undergone nucleophilic attack by water upon binding to HDAC8, whereas the epoxide group remained intact (Figure 26). The Phe-hPro forms a cis-peptide bond. Although there are no intramolecular hydrogen bonds, both Phe side chains interact through an

Histone lysine (K) demethylases (KDMs) are enzymes that remove methyl groups from lysine residues of histone proteins.175 KDMs regulate many cellular processes, including altering chromatin structure and transcription.175 KDMs are important for normal embryonic development, and aberrant function has been linked to various diseases, including many cancers.175 Using the Rapid non-standard Peptides Integrated Discovery (RaPID) system, highly selective and potent cyclic inhibitors of KDMs have been identified. The crystal structure of the cyclic peptide CP2, a competitive inhibitor of histone substrate (IC50 = 42, 33, and 39 nM for KDM4A, -4B, and -4C isoforms, respectively) was determined bound to KDM4A (PDB 5LY1, 2.5 Å).176 Interactions between CP2 and KDM4A are shown in Figure 27. The cyclic peptide CP2 is Ac-cyclo[yVYNTRSGWRWYTC]-NH2, where the acetyl methyl at the N-terminus forms a thioether link with the side chain of Cys14. The 14 amino acid residues including a D-tyrosine in the macrocycle create a 45-membered ring. CP2 binds at the interface of the KDM4A homodimer and adopts a distorted βsheet with two turns in the bound state. The binding conformation of this macrocycle shows 10 intramolecular hydrogen bonds, seven being transannular and three exocyclic. On the left side of the macrocycle, an i to i+3 hydrogen bond forms around Arg and Ser, indicating a β-turn which presents the side chains and carbonyl oxygens from each amino acid to make hydrogen-bonds with the target. On the right side of the R


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Figure 27. 2D representation of interactions of CP2 with KDM4A (PDB 5LY1, 2.5 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. The residue names from the second monomer of KDM4A are shown in italics. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 28. 2D representation of interactions of PIE71 with IQN17 (PDB 3MGN, 1.4 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. Missing atoms of PIE71 in PDB 3MGN are shown in red. The residue names from alternate chain of the homotrimer protein are shown in italics and underlined. A 3D view of the bound cyclic peptide is shown below.

macrocycle, the carbonyl group of Tyr forms two transannular hydrogen bonds which, along with an exocyclic hydrogen bond from the Tyr OH group, help stabilize a non-classical turn on this part of the macrocycle. This turn does not make any direct hydrogen bonds with the target. The remaining four-transannular hydrogen bonds form a β-sheet structure. Two further exocyclic bonds are also observed within the β-sheet region of the macrocycle, one from Thr OH to amide carbonyl and the other from Tyr-OH to Arg side chain. This β-hairpin/β-sheet macrocycle forms interactions with the target along both faces of the β-sheets. Interactions with the target in this case are both driven by interactions by the amino acid side chains and by the amide backbone of the macrocycle.

bonds. Within the macrocyclic section of the compound, a helical structure has been stabilized by eight transannular hydrogen bonds between residues [D-Glu-D-Trp-D-Arg-D-TrpD-Leu-D-Cys]-D-Asp-D-Leu; however, this structure is distorted from a classical α-helix. A γ-turn is formed around D-Glu, distorting this end of the helix. 2.29. HIV-1 Integrase

HIV-1 integrase is a retroviral enzyme that enables viral genetic material to be integrated into the host DNA of the infected cell.180 HIV-1 integrase is a clinically validated target for antiHIV therapy.181 One of the host cofactors that regulates the function of HIV-1 integrase is the cell Lens EpitheliumDerived Growth Factor (LEDGF, also known as p75). Based on the linear sequence of p75, SLKIDNLD, a series of cyclic peptides were investigated as HIV integrase inhibitors by binding to the LEDGF site, and a number of co-crystal structures were solved.182 A representative crystal structure (PDB 3AVB, 1.94 Å) showed interactions between a cyclic octapeptide, cyclo-[SLKIDNLD], and HIV-1 integrase as summarized in Figure 29. The cyclic peptide is a 24-membered ring, which exhibited IC50 = 85 μM in an AlphaScreen assay and 70 μM in the assay for inhibition of LEDGF enhancement of HIV-1 integrase strand-transfer activity. The binding conformation of this macrocycle reveals six intramolecular hydrogen bonds, four being transannular and two exocyclic. At the bottom of the cycle, the first transannular hydrogen bond is (i, i+3) and forms a 10-membered hydrogen-bonded ring (Asn6 NH···OC Lys3), consistent with a β-turn that supports

2.28. HIV-1 gp41

The HIV-1 envelope protein mediates viral entry into cells and is composed of surface protein gp120 and transmembrane protein gp41 subunits.177 Molecules interacting with the gp41 pocket could be developed as HIV entry inhibitors.177,178 In an attempt to improve proteolytic stability of pocket-specific peptide binders, D-peptides were screened, and PIE71, a 15residue peptide containing a 29-membered cyclic nonapeptide, was identified as a potent inhibitory molecule with IC50 = 1 μM in the pseudovirion entry inhibition assay (JRFL strain).179 A related peptide PIE12 is Ac-cyclo (5,12)-D-Lys-Gly-D-Phe-DVal-[D-Cys-D-Pro-D-Pro-D-Glu-D-Trp-D-Arg-D-Trp-D-Leu-DCys]-D-Asp-D-Leu-NH2, where both D-Cys residues are linked to form a disulfide bond. The co-crystal structure of PIE12 bound to IQN17 protein, a gp41 pocket mimic, was solved (PDB 3MGN, 1.4 Å). Interactions between PIE71 (chain K from PDB 3MGN) and IQN17 are shown in Figure 28. The binding conformation involves 10 intramolecular hydrogen S


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1.6 Å) shows two monocyclic hydrolysis products (cyclic tripeptides) trapped in the active site (Figure 30).

Figure 30. 2D representation of interactions of a macrocyclic peptidomimetic with HIV-1 protease (PDB 3BXS, 1.6 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. The residue names from the second monomer of protein are shown in italics. A 3D view of the bound cyclic peptide is shown below.

Figure 29. 2D representation of interactions of the cyclic peptide with HIV integrase (PDB 3AVB, 1.94 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

important interactions between Ile and Asp side chains and the receptor. An exocyclic hydrogen bond from the Asp side-chain carbonyl to the Asp amide NH may help to position this side chain to make four hydrogen bonds with the target. The macrocycle backbone in this turn also makes hydrogen bonds with the target through the Ile amide NH and both the Ile and Asp carbonyl oxygens. A second transannular hydrogen bond (Lys NH···OC Asn) compresses the structure of the cycle through its midsection, allowing the Lys and Asn side chains to make hydrogen bonds with the target. At the top of the macrocycle as drawn, another β-turn is stabilized by a 10membered (i, i+3) hydrogen-bonded ring (Leu2 NH···OC Leu7). However, two additional hydrogen bonds from the Asn carbonyl oxygen cause this turn to kink. Interestingly, this intricate turn does not make any interactions with the target. A series of cyclic peptides were designed, investigated, and crystallized based on PDB 3AVB.

These monocycles had two amino acid components, but the aliphatic linker is equivalent to the side chain of a third residue, and the macrocycle has 16 heavy atoms in its backbone. Interestingly, the trapped monocyclic products were identical, indicating that two bicyclic substrates had been cleaved and two copies of one half had been captured in the enzyme. 2.31. Human Adaptor Protein 14-3-3

Human adaptor proteins 14-3-3 are a family of highly conserved ubiquitously expressed adaptor proteins that can interact with a wide variety of cellular proteins.189,190 Recent work has highlighted the involvement of these proteins in human disease including infectious, neurodegenerative diseases, and cancers.189,190 In an attempt to stabilize an irregular peptide structure using hydrophobic cross-links from alkane and alkyne ring-closing metathesis, a series of cyclic peptides that inhibit the interaction between human adaptor protein 143-3 and virulence factor exoenzyme S were investigated using X-ray crystallography and isothermal titration calorimetry.191,192 Exoenzyme S is a virulence factor of Pseudomonas aeruginosa, and inhibiting 14-3-3−exoenzyme S interactions is considered a promising therapeutic strategy. A linear sequence of 11-amino acids QGLLDALDLAS from exoenzyme S, mainly contributing to the binding, and its co-crystal structure with 14-3-3 (PDB 4N7G, 2.25 Å) was used to design a range of cross-linked cyclic peptides (PDB 4N7Y, 2.16 Å; PDB 4N84, 2.5 Å; PDB 5J31, 2.4 Å; and PDB 5JM4, 2.34 Å). A representative peptide, and the most potent in the series (KD = 0.1 μM binding affinity to 14-3-3 measured by fluorescence polarization), is the 10-residue cyclized peptide with the sequence H-Gly-Mkd-Leu-Asp-Mkd-Leu-Asp-Leu-Ala-Ser-OH (Mkd = 2(S)-2-amino-2-methyloctanoic acid), where Mkd2

2.30. HIV-1 Protease

HIV-1 protease is an aspartyl protease that is important for the replication of human immunodeficiency virus and a primary target for anti-AIDS drugs.183 A series of macrocyclic peptides and peptidomimetics were designed from the HIV-1 protease substrates H-LNFPIV-OH and H-LVFFIV-OH using computational modeling and investigated as HIV-1 protease inhibitors, with a number of co-crystal structures solved.184−188 A representative example is discussed where a hexapeptide substrate, containing two macrocyclic tripeptides constrained to mimic a β-strand conformation and linked by a scissile peptide bond, was crystallized with both inactive (D25N) and active HIV-1 protease. The structure of the substrate with inactive enzyme retains the intact bicyclic substrate structure, whereas the one with catalytically active enzyme (PDB 3BXS, T


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and Mkd5 are linked via aliphatic side chains to form a 12atom hydrophobic linker (PDB 4N84, 2.5 Å). Interactions between the cyclic peptide and 14-3-3 are shown in Figure 31.

Figure 32. 2D representation of interactions of the cyclic pentapeptide and αVβ3 integrin (PDB 1L5G, 3.2 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. Residue names from β-subunit are shown in italics. A 3D view of the bound cyclic peptide is shown below.

with the target are made through the Arg and Asp side chains, while one hydrogen bond is made to the amide NH of Gly. The arginine side chain interacts with two aspartic acid residues in the target. NMR-derived solution structures of the free cyclic peptide reveal conformations closely resembling the protein-bound X-ray crystal structure, whereas the structure of cilengitide recrystallized from methanol showed a different conformation controlled by the crystal packing lattice.196

Figure 31. 2D representation of interactions of the cyclic peptide and 14-3-3 (PDB 4N84, 2.5 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

The binding conformation of this peptide shows that the i to i +4 hydrocarbon staple linker has not stabilized a full α-helical structure. Only three intramolecular hydrogen bonds are formed within the macrocyclic part of the molecule helping to fold that section into a helix-like structure. The hydrocarbon linker itself is able to make hydrophobic interactions with multiple protein residues. Hydrogen bonds with the target are driven by both side-chain interactions and interactions with the amide backbone of the cycle.

2.33. K-Ras

The rat sarcoma (Ras) proteins are small guanine nucleotidebinding proteins that act as molecular switches in signaling pathways associated with cell growth, proliferation, and differentiation. K-Ras is a small G protein that functions as a signal transducer in many cellular pathways197 and is the most frequently mutated oncogene, and strategies that repress its functions are predicted to prevent a large number of human cancers although development of direct K-Ras inhibitors has thus far remained elusive.197,198 K-Ras protein is the most frequently mutated isoform, and its inhibitors are potential anti-cancer drugs. A 19-mer cyclic peptide, KRpep-2d, identified from a phage display library, was co-crystallized with K-Ras G12D mutant and its binding with wild type as well as mutants characterized (PDB 5XCO, 1.25 Å).199 The KRpep-2d is Ac-cyclo-(5,15)-RRRR[CPLYISYDPVC]-RRRRNH2, where both Cys residues form a disulfide bridge. The cyclic peptide binds to K-Ras(G12D) with KD = 8.9 nM and exhibits ∼6-fold binding selectivity over the wild-type K-Ras. This peptide exhibits potent non-covalent K-Ras(G12D) inhibitory activity, with selectivity against wild-type (WT) KRas and the K-Ras(G12C) mutant in both cell-free enzyme (IC50 = 1.6 nM) and cell-based assays.200,201 Interactions between KRpep-2d and K-Ras(G12D) are summarized in Figure 33. The peptide binds near Switch II of K-Ras and allosterically blocks protein−protein interactions with the guanine nucleotide exchange factor. The electron density of the four Arg residues at N- and C-termini was ambiguous, with all exposed to solvent. The bound conformation of this macrocycle reveals five intramolecular hydrogen bonds, but

2.32. Integrin αVβ3

Integrins are a class of heterodimeric cell adhesion receptors, connecting the cytoskeleton with the extracellular matrix or other cells to mediate vital bidirectional signals during morphogenesis, tissue remodeling, and repair.193 Integrin αVβ3 also plays an important role in tumor angiogenesis, and αVβ3 antagonists and integrin-targeted delivery systems are currently pursued as potential cancer therapeutics.193 A crystal structure was reported for extracellular αVβ3 integrin in complex with a cyclic pentapeptide cyclo-[Arg-Gly-Asp-(DPhe)-(N-methyl-Val)] (PDB 1L5G, 3.2 Å) derived from the RGD recognition motif.194,195 The cyclic peptide (IC50 = 0.58 nM) binds at the major interface between the αV and β3 subunits. Comparison of this structure to αVβ3 integrin bound to the metal ion Mn2+ reveals that the bound cyclic peptide induces tertiary and quaternary changes in the integrin structure, specifically, it changes the orientation of αV relative to β3. Interactions between the cyclic pentapeptide and αVβ3 integrin are shown in Figure 32. One transannular hydrogen bond is observed in the macrocycle in an i to i+2 position, stabilizing a seven-membered hydrogen-bonded ring indicative of a γ-turn around the Asp residue. Hydrogen bonds formed U


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Figure 34. 2D representation of interactions of SFTI-1 and KLK4 (PDB 4K8Y, 1.0 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. 3D views of the proteinbound cyclic peptide (middle panel, PDB 4K8Y, Pro13 and parts of Asp14 missing) and unbound NMR solution structure (bottom panel, PDB 1JBL) are also given. When bound and unbound cyclic peptides are superimposed using backbone atoms (RMSD = 1.3 Å), there is little conformational difference in the main chain.

Figure 33. 2D representation of interactions of KRpep-2d with KRas(G12D) (PDB 5XCO, 1.25 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Pro13 and side chain of Asp 14 were not resolved clearly in the electron density map. The bound conformation of this macrocycle shows eight intramolecular hydrogen bonds, which stabilize two distinct structural motifs. On the left side of the macrocycle, three transannular hydrogen bonds cooperate with the disulfide bridge to form a β-sheet structure. On the right side of the macrocycle, a network of four intramolecular hydrogen bonds centered around Thr OH and Ser OH groups creates a loop-like structure. Interestingly, the Pro-Pro motif in the peptide sequence does not induce any turn structure or promote any intramolecular hydrogen bonding. Interactions with the target are made along the bottom strand section of the macrocycle and involve hydrogen bonds with both the amino acid side chains and the amide backbone. Comparison with the unbound NMR-derived solution structure of SFTI-1 (PDB 1JBL)206 shows that the peptide backbone conformation is quite rigid and maintains its transannular hydrogen bonds, only the side-chain conformations changing to accommodate binding to the protein.

only one is transannular (i to i+3). This helps stabilize a β-turn around Tyr and Ser. Other hydrogen bonds are from side chain to backbone in the macrocycle and seem to have little effect on binding to target. Hydrogen bonding of this macrocycle to the target is predominantly through the amide backbone of the cycle, with only the Asp and Ser side chains forming direct hydrogen bonds. Hydrophobic side chains Ile, Leu, and Pro all make hydrophobic interactions. 2.34. Kallikrein-Related Peptidase 4

Kallikreins are a group of 15 serine proteases implicated in a variety of human inflammatory and neurodegenerative diseases, skin conditions, asthma and cancers.202 Selective inhibitors of different kallikrein isozymes are being developed as potential therapeutics.203 Crystal structures for KLK4 bound to sunflower trypsin inhibitor 1 (SFTI-1) and rationally designed derivatives have been determined in order to understand the basis of the inhibition.204 SFTI-1 and analogues are inhibitors of KLK4 (IC50 = 221 nM).205 SFTI1 is a 14 amino acid bicyclic peptide, cyclo-(3,11)-GR[CTKSIPPIC]-FPD with a disulfide linkage between two Cys residues. Interactions between SFTI-1 and KLK4 (PDB 4K8Y, 1.0 Å) are summarized in Figure 34, but the backbone of

2.35. Lectin LecB

Lectin LecB is a bacterial virulence factor involved in bacterium adherence to host cells, biofilm formation, and cytotoxicity.207,208 Inhibiting LecB could serve as a potential new anti-bacterial strategy.207,208 A series of amphiphilic V


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CO, and D-Trp9 CO). The transannular hydrogen bond on the right side (D-Lys5 CO···NH L-Lys8) stabilizes a β-turn across residues 5−8. At the top of the macrocycle a second β-turn is formed around residues 8−11. The two other hydrogen bonds from the C-terminal amide cause a kink in the bottom face of the cycle. This macrocycle forms very few direct hydrogen bonds to the target. The bound cyclic peptides are mainly stabilized through direct and water-mediated intramolecular hydrogen bonds and a few interactions with LecB protein. In addition, the fucose moiety interacts with the protein by coordinating to two Ca2+ ions.

membrane-disrupting cyclic anti-microbial peptides were investigated, where lysine and tryptophan were used as cationic and hydrophobic residue pairs in an alternating D,Lsequence, and terminal Cys residues were cyclized via a xylene double thioether bridge.209 RH11 with the sequence H-cyclo[CwWkKkKkWwC]-NH2, cyclized with p-xylene thioether linker, was found to be active against Gram-negative Pseudomonas aeruginosa and Gram-negative Bacillus subtilis BR151 bacterial strains. The enantiomer of RH11, containing an α-L-c-fucosylacetyl group at the N-terminus of the peptide and terminal Cys residues linked via a o-xylene thioether link, FdRH11o (H-cyclo-[cWwKkKkKwWc]-NH2, MIC = 8 μg/ mL PA01, MIC = 1 μg/mL BR 151), was used to obtain a cocrystal structure with fucose-specific P. aeruginosa lectin LecB protein (PDB 5NF0, 1.271 Å). Additional co-crystal structures include fucosylated analogue with o-xylene thioether link (FRH11o, PDB 5NEY, 1.54 Å) and fucosylated analogue with m-xylene thioether link (FdRH11m, PDB 5NES, 1.6 Å). The crystal symmetry resulted in four non-equivalent LecB monomers and the corresponding complexed fucosyl ligands in each LecB tetramer. Interactions between FdRH11o and lectin LecB (from PDB 5NF0, chain B of LecB and chain E of FdRH11o) are shown in Figure 35. These cyclic peptides adopt amphiphilic structures in both free and bound states. The binding conformation of this macrocycle shows five intramolecular hydrogen bonds, two being transannular (DTrp3 NH···OC D-Cys11, D-Lys5 CO···NH L-Lys8) and three others from the C-terminal D-Cys11-NH2 terminal amide to carbonyl oxygens in the amide backbone (D-Trp3 CO, D-Lys8

2.36. Matriptase

Matriptase is a type II transmembrane-bound serine protease that is broadly expressed by all human epithelial cells.210

Figure 36. 2D representation of interactions of SFTI-1 with matriptase (PDB 3P8F, 2.0 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. 3D views of the protein-bound cyclic peptide (middle panel, PDB 3P8F) and unbound solution structure (bottom panel, PDB 1JBL) are also given. When superimposed on each other using backbone atoms, there was little difference in the main-chain conformations (RMSD = 0.75 Å). Figure 35. 2D representation of interactions of FdRH11o and Lectin LecB (PDB 5NF0, 1.271 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. Two Ca2+ ions (not shown) coordinate to the fucose moiety of the ligand as well as to Glu195 and Asp101 of the protein.

Dysregulated expression and activation of matriptase have been associated with a variety of inflammatory conditions and cancers.211 Sunflower trypsin inhibitor-1 (SFTI-1), a 14residue cyclic peptide, cyclo-(3,11)-GR-[CTKSIPPIC]-FPD with a disulfide linkage between the two Cys residues and isolated from sunflower seeds, was found to be a potent inhibitor of matriptase (Ki = 0.9 nM), and a co-crystal W


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structure was determined (PDB 3P8F, 2.0 Å).212 Interactions between SFTI-1 and matriptase are shown in Figure 36. SFTI1 binds to matriptase similar to how it binds to trypsin, with a β-sheet and a loop stabilized by the OH side-chain groups of Thr and Ser. Four intramolecular transannular hydrogen bonds are made in the loop section and, along with three proline rings help to stabilize a β-hairpin or sheet conformation. Binding to the target is from the bottom face of the macrocycle, and both side chains and the amide backbone are involved in making hydrogen bonds to matriptase. Comparison with the unbound NMR solution structure of SFTI-1 (PDB 1JBL)200 shows that the peptide backbone maintains its rigid conformation, with the transannular hydrogen bonds preserved. Only the side chains move to accommodate binding to the protein.

of L-lysine with seven instead of four methylene groups). The resulting cyclic peptide (Ki = 24 nM) was 3 times more potent than the linear octapeptide and 91 times more potent than the corresponding acyclic control compound.215 The co-crystal structure of the macrocyclic peptide with menin (PDB 4I80, 3.1 Å) showed mainly hydrophobic interactions (Figure 37), with binding modes and interactions being similar for macrocyclic and linear octapeptides. The binding conformation of the macrocyclic part of this molecule shows one i to i+3 intramolecular hydrogen bond (Phe4 CO···HN Arg7), indicating the presence of a β-turn in the Phe4-Pro5-Ala6Arg7 segment. This turn helps present the carbonyl oxygen of Ala6 to make a hydrogen bond with Tyr323 in menin. The Arg side chain makes hydrogen bonds with two glutamate residues in menin.

2.37. Menin

2.38. Mouse Double Minute 2

Chromosomal translocation of mixed lineage leukemia gene (MLL) occurs in aggressive acute leukemias.213 MLL fusion

The tumor suppressor p53 is one of the most frequently mutated oncogenes and is implicated in a wide variety of human cancers.216 Mouse double minute 2 (MDM2) is an

Figure 38. 2D representation of interactions of the stapled peptide with MDM2 (PDB 5AFG, 1.9 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 37. 2D representation of the interaction of the cyclic octapeptide with menin (PDB 4I80, 3.1 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

important negative regulator of p53. MDM2 is over-expressed in numerous cancers amplifying p53-dependent functions, including apoptosis and cell-cycle arrest.216 Inhibitors of MDM2-p53 interactions are being developed as anti-cancer agents.216 MDM2-binding peptides have been stabilized in helical structures and characterized.217 One peptide Ac-cyclo(4,11)-LTF-[AEYWAQLA]-S-NH2, where Ala4 and Ala11 side chains were replaced with a dibenzo-ditriazolo-cyclooctatetraene linker to create a cyclic component, bound tightly to MDM2 (KD = 12 nM, ITC) and inhibited p53-MDM2 in cells (EC50 = 2.9 μM). A co-crystal structure was determined (PDB 5AFG, 1.9 Å), and interactions are summarized in Figure 38.

proteins require menin for leukogenic activity, and targeting menin−MLL interaction using inhibitors could provide an effective treatment for MLL leukemias.213,214 Starting from a linear MLL1 octapeptide, a class of macrocyclic peptidomimetic inhibitors was designed, synthesized, and biologically tested, and subsequently one of the inhibitors of menin-MLL1 protein−protein interactions was co-crystallized with menin. Based on the available crystal structure of a conserved region of the menin binding motif (a linear octapeptide, AcRWRFPARP-NH2), a macrocyclic peptide (Ac-cyclo-(3,8)RW-[XFPARP], with Arg1 replaced by X, which is an analogue X


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bonds made from the Ser-OH to the amide backbone (Ser6 OH···NH Leu6) and another Ser-OH (Ser6 OH···OH Ser8). This creates a kinked turn-based structure. Interestingly, this turn is not involved in forming interactions with the MATE protein, and hence its key role is in helping to stabilize the βsheet structure. The bottom face of the β-sheet (Y10T11W12F13L14F15) is the key protein-binding domain which uses both the side chains and the amide backbone of the macrocycle to interact with MATE. One hydrogen bond interaction is made from the top β-strand with the target through the Tyr5-OH.

The protein-bound conformation of this stapled peptide retains α-helical structure, with an extensive network of intramolecular hydrogen bonds representative of an α-helix. Interestingly, two additional intramolecular hydrogen bonds are made from the linker to the side-chain NH and the amide backbone NH of Trp7. This may contribute to helical stabilization but also distorts the linker position. Interactions with the target are made through the helical section of the peptide and involve protein interactions with both the side chains and the amide backbone of the peptide. 2.39. Multidrug and Toxic Compound Extrusion Transporter

2.40. Neurophysin

Multidrug and toxic compound extrusion (MATE) proteins are a family of transporter proteins that export xenobiotics and confer multidrug resistance to drugs used in the treatment of a variety of diseases.218 Co-crystal structures were determined for three thioether-macrocyclic peptides, identified from a random non-standard peptide integrated discovery (RaPID), bound to MATE from Pyrococcus furious.219 One of the cyclic peptides MaL6 has the sequence Ac-FTFRYSPSLYTWFLFPCG-NH2, where the N-terminal acetyl methyl is linked to the side chain of Cys to form a thioether linker (PDB 3WBN, 2.45 Å). There are 17 amino acid residues in the macrocycle creating a 54-membered ring size. Interactions between MaL6 and MATE are shown in Figure 39. This

Neurophysins are small (93−95 residues) acidic carrier proteins that transport the hormones oxytocin and vasopressin to the posterior pituitary.220 Crystal structures of neurophysin in complex with oxytocin221 and vasopressin222 are reported. The interactions between oxytocin hormone and neurophysin are shown in Figure 40 (PDB 1NPO, 3.0 Å). Oxytocin is a

Figure 39. 2D representation of interactions of MaL6 with MATE (PDB 3WBN, 2.45 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. Atoms missing in the crystal structure are shown in red. A 3D view of the bound cyclic peptide is shown below.

Figure 40. 2D representation of interactions of oxytocin with neurophysin (PDB 1NPO, 3.0 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. 3D views of the bound cyclic peptide (middle panel, PDB 1NPO) and unbound NMR solution structure (bottom panel, PDB 2MGO) are also given. When superimposed on each other using backbone atoms there were small changes in the main-chain structure (RMSD = 1.4 Å).

macrocyclic structure reveals 10 intramolecular hydrogen bonds, five being transannular hydrogen bonds that help stabilize a β-sheet structure within the macrocycle. On the right side of the macrocycle, a turn motif is centered at Ser6Pro7-Ser8-Leu9-Tyr10, stabilized by two transannular hydrogen bonds in i to i+3 (Ser6 CO···HN Leu9) and i and i+4 (Ser6 CO···HN Tyr10) positions, as well as two hydrogen

nine-residue peptide with a cyclic hexapeptide component, Hcyclo-(1,6)-[CYIQNC]-PLG-NH2, where the two Cys residues form a disulfide bond. In the isolated crystal structure of oxytocin, residues 2−5 form a type II β-turn and residues 6−9 form a type-III β-turn; neither of these are observed in the neurophysin−oxytocin complex. The bound conformation of the oxytocin shows four intramolecular hydrogen bonds within Y


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later (PDB 4M2S and 4M2T with QZ59-RRR and QZ59-SSS, respectively).230 The new structures improved from ∼57% of the residues in favorable Ramachandran space to ∼95%, and a shift of transmembrane helixes (rotations and translations) revealed previously unrecognized amino acids involved in drug binding. P-gp distinguished between the enantiomers, with different binding locations and stoichiometry. QZ59-RRR binds to one site, while QZ59-SSS binds to two sites on P-gp. The representative interactions of QZ59-RRR with P-gp (from PDB 4M2S) are shown in Figure 41, where the cyclic peptide binds within a deep hydrophobic cavity of P-gp. Among other protein−cyclic peptide structures used in our Ramachandran analysis in section 4, there is also a crystal structure (PDB 3WMG) for a 19-residue cyclic peptide (anti-CmABCB1) bound to P-glycoprotein.231

the macrocyclic part of the molecule. Three of these bonds are transannular, the carbonyl oxygen of Tyr2 forming two hydrogen bonds with amide NHs of Asn and Cys (Tyr2 CO···HN Asn5, Tyr2 CO···HN Cys6), while the neighboring Ile carbonyl also forms a hydrogen bond to Cys (Ile3 CO···HN Cys6). These three hydrogen bonds in the same part of the macrocycle help stabilize a pseudohelical turn. An exocyclic hydrogen bond within Gln4 helps position its side chain for interaction with Asp76. Hydrogen-bonding interactions with the target are made through both the amino acid side chains and the macrocycle backbone. The protein-bound conformation of oxytocin is in agreement with the X-ray crystal structure223 of the unbound oxytocin analogue, but it differs from the protein-bound NMR structure224 with regard to orientation of Tyr2 involved in neurophysin recognition. Comparison with the unbound NMR-derived structure in solution (PDB 2MGO)225 shows that the both peptide backbone as well as side-chain conformations change to accommodate binding to the protein.

2.42. Phosphoglycerate Mutase

Cofactor-independent nematode phosphoglycerate mutase (iPGM) is an important metabolic enzyme that catalyzes the reversible conversion of 3-phosphoglycerate and 2-phosphoglycerate during glycolysis.232 Interestingly, iPGM is structur-

2.41. P-Glycoprotein

P-Glycoprotein (P-gp), a member of the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, is a transporter protein that determines the cellular uptake and efflux of many drugs.226 P-gp detoxifies cells by exporting

Figure 41. 2D representation of interactions of QZ59-RRR with Pglycoprotein (PDB 4M2S, 4.4 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. Figure 42. 2D representation of interactions of Ce-2d with iPGM (PDB 5KGN, 1.95 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

hundreds of chemically unrelated toxins and xenobiotics but has also been implicated in inducing multidrug resistance (MDR) in the treatment of cancers.227,228 The crystal structures have been solved for two enantiomers of cyclic hexapeptide inhibitors, cyclic-tris-(R)-valineselenazole (QZ59RRR) and cyclic-tris-(S)-valineselenazole (QZ59-SSS), bound to P-gp.228 The cyclic peptides incorporate alternating selenazole rings in their backbones, producing three-fold symmetry and inhibiting calcein-AM export (IC50 low μM). QZ59-SSS competed with substrates, and QZ59-RRR noncompetitively inhibited daunorubicin transport (K(I,app) = 1.9 μM).229 Significant deviations between the P-gp protein crystal structures (PDB 3G60 and 3G61 with QZ59-RRR and QZ59SSS at 4.4 and 4.35 Å, respectively) led to refinement 4 years

ally distinct from the mammalian cofactor-dependent (dPGM) isozyme, making iPGM a potential anti-helmintic drug target.232,233 A series of cyclic peptides and analogues were investigated as potent and isozyme-selective inhibitors of iPGM orthologues (pIC50 = 9, C. elegans iPGM; 7.27, B. malayii PGM; 7.13, O. volvulus iPGM; 6.3, D. immitis PGE; 7.98, E. coli PGM).233 The cyclic peptide Ce-2d has the sequence Ac-yDYPGDYCYLY-NH2, where the N-terminal Z


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NH2, where the N-terminal acetyl methyl is linked to the side chain of Cys to form a thioether bond. The 15 amino acid residues in the macrocycle create a 47-atom ring size. Interactions between PB1m6 and plexin B1 are shown in Figure 43. The bound macrocycle has six intramolecular hydrogen bonds, three being transannular and helping to stabilize a β-sheet structure through the middle of the macrocycle. In contrast to other similar examples in this Review, the macrocycle does not have a defined turn structure on opposite sides; instead both sides are loop-like structures. On the right side, an exocyclic hydrogen bond from Thr-OH to a carbonyl oxygen in the macrocycle backbone gives this loop a kinked conformation which helps bring the L-Trp NH within hydrogen-bonding distance to Gly415; however, sidechain functional groups in this kink do not interact with plexin. The loop on the left side of the macrocycle is heavily involved in interactions with plexin, forming hydrogen bonds between both the Arg side chains and the amide NH. Another exocyclic hydrogen bond in this region does not cause a large kink in the loop. Interactions between the β-sheet region and the target are formed on both faces of the β-sheet and involve side-chain and macrocycle backbone interactions.

acetyl methyl group forms a thioether bond with the side chain of Cys8. The co-crystal structure of Ce-2d with iPGM from Caenorhabditis elegans (PDB 5KGN, 1.95 Å) shows interactions depicted in Figure 42. The macrocycle is cradled in a pocket shaped from the hinge peptides and adjacent phosphatase and transferase domain surfaces of iPGM. The bound conformation of Ce-2d displays nine intramolecular hydrogen bonds. Within the macrocyclic section of the molecule, one transannular hydrogen bond (Asp6 NH···OC Tyr3) stabilizes a β-turn across Tyr3-Pro4-Gly5-Asp6. The carboxylate side chain of Asp6 forms four hydrogen bonds to amide backbone NHs (D-Tyr1, Asp2, Tyr3) in the cycle. These bonds help position the Y7C8Y9L10Y11-NH2 section into a helix-like structure. All Tyr residues of the cyclic peptide were involved in protein bonding. Hydrogen bonding to the target is predominantly through interactions with the peptide amide backbone. Only two side chains make direct hydrogen-bonding interactions. 2.43. Plexin

Class A plexins are large type 1 transmembrane proteins that act as semaphorin receptors. They were initially identified to have important roles in developmental processes, such as axon guidance in the neural system.234,235 More recently, accumu-

2.44. Polo-like Kinase-1

Polo-like kinase 1 (PLK1), a serine/threonine protein kinase, is a well-established mitotic regulator that is essential for successful cell division.237 PLK1 has a range of biological

Figure 43. 2D representation of interactions of PB1m6 with plexin B1 (PDB 5B4W, 2.6 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. The residue names from the second monomer of protein are shown in italics. A 3D view of the bound cyclic peptide is shown below.

Figure 44. 2D representation of interactions of cyclic phosphopeptide with PLK-1 polo-box domain (PDB 4X9R, 1.398 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

lating evidence suggests that semaphorin−plexin-mediated signaling is also crucial for regulating physiological and pathological immune responses including autoimmune diseases, allergies, and congenital bone diseases.236 A high-affinity (KD = 3.5 nM) plexin B1-binding macrocyclic peptide, PB1m6, was found to be a non-competitive allosteric inhibitor of plexin B1, and its structure is reported (PDB 5B4W, 2.6 Å). The macrocyclic peptide is Ac-cyclo-[wRPRVARWTGQIIYC]-S-

functions in the cell cycle, and inhibitors of PLK1 are being developed as potential anti-cancer agents.238 In an attempt to design peptidomimetics, phospho-dependent His N(τ)-alkylation was used for macrocyclization, and a series of cyclic phosphopeptides was investigated. IC50 values for inhibition of PLK-1 polo-box domain were measured, and three co-crystal structures were obtained.239 Based on the C-proximal residues AA


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molecular hydrogen bonds and has very little defined structure. Only two hydrogen bonds are formed with the target, both with the amide backbone of the macrocycle. All other interactions are hydrophobic and involve the amino acid side chains. Thus, this cycle appears to be a relatively flexible scaffold that projects substituents for protein binding.

of the polo-box interacting protein 1 (PBIP1), a cyclic peptide derivative was devised by modifying the sequence Ac-Pro-LeuHis-Ser-pThr-NH2, in which the His side chain was tethered via an ether linkage to the hydroxyl group of 4-hydroxyproline and the cycle acts as a scaffold in projecting both phenyloctanyl and dipeptidyl substituents. A representative structure (PDB 4X9R, 1.398 Å) with IC50 = 14 nM is shown in Figure 44. No intramolecular hydrogen bonds are observed in this structure, and mainly hydrogen bond interactions are made with the protein.

2.46. 20S Proteasome

Programmed cell death 1 ligand 1 (PD-L1) is a 40-kDa type 1 transmembrane protein that binds to a cell surface programmed death protein 1 (PD-1), an inhibitory immune checkpoint receptor that is a key component of programmed death signaling.240,241 PD-L1 is unregulated in various types of solid tumors, and the PD-L1/PD-1 signaling axis plays a major role in suppressing the immune system that in turn inhibits host anti-tumor responses.240,241 Based on reported macrocyclic peptide inhibitors of PD-L1, a series of cyclic peptides were investigated biochemically and structurally.242 One cyclic peptide (IC50 = 9 nM) was crystallized with PD-L1 (PDB 5O45, 0.99 Å). The 14-residue cyclic peptide was Ac-cyclo(1,13)-[Phe-Mea-9kk-Sar-Asp-Val-Mea-Tyr-Sar-Trp-Tyr-LeuCys]-Gly-NH2, where Mea = N-methylphenylalanine, 9kk = Nmethylnorleucine, Sar = sarcosine, and the N-terminal methyl group is linked to the Cys13 side chain to form a thioether bond. Interactions between the cyclic peptide and PD-L1 are shown in Figure 45. This macrocycle displays no intra-

Proteasomes are highly sophisticated threonine protease complexes that enable cells to selectively regulate the concentration of particular intracellular proteins and also degrade misfolded or damaged proteins via proteolysis.243 Proteasomes control a wide variety of basic cellular functions including cell cycle progression, signal transduction, cell death, immune response, metabolism, and others by degrading shortlived regulatory or structurally aberrant proteins.244 The proteasome is made up of a catalytic core particle (20S proteasome) and one or two terminal 19S regulatory particles. Inhibition of proteasome activity is emerging as a potential anti-tumor therapeutic strategy, and inhibitors are currently used to treat multiple myeloma.245 The biochemical characterization and crystal structure of a natural product cyclic peptide TMC-95A, a selective inhibitor of 20S proteasome, were reported (PDB 1JD2, 3.0 Å).246 TMC95-A is derived from Apiospora montagnei and contains modified amino acids forming a biaryl ring system. It is composed of (1QQ)-TyrAsn-(R4K)-(AKK), where 1QQ = (3S)-3-methyl-2-oxopentanoic acid, R4K = (2S,3R)-2-azanyl-3-hydroxy-3-[(3S)-3hydroxy-2-oxo-1H-indol-3-yl]propanoic acid, AKK = (1Z)prop-1-en-1-amine, and aromatic rings of Tyr and modified Trp residues form a biphenyl cyclic link. Interactions between TMC-95A and 20s proteasome are shown in Figure 46. The bound conformation of TCA-95A shows that even with a highly constrained cyclic structure, the rotation available in the

Figure 45. 2D representation of interactions of the cyclic peptide with PD-L1 (PDB 5O45, 0.99 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 46. 2D representation of interactions of TMC-95A with 20s proteasome (PDB 1JD2, 3.0 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

2.45. Programmed Cell Death 1 Ligand 1

AB


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hydrogen bond interactions (Figure 47). Only one intramolecular hydrogen bond (β-MeAsp CO2−···HN D-Glu) is observed in the bound conformation of this peptide. This bond appears to pull both carboxylates of the compound to one face of the macrocycle, allowing both groups to make interactions with Arg96. Motuporin binds to PP-1c in a manner similar to other toxins in that its macrocyclic ring occupies most of the active site region and its hydrophobic tail occupies the hydrophobic groove of PP-1c that is adjacent to the active site. Binding affinity is driven by a significant number of hydrogenbonding contacts between MOT and PP-1c, including strong interactions with Arg96 (three potential bonds involving guanidinium nitrogen atoms), Y134, R221, and Y272. The NMR solution structure of motuporin has been reported and is quite different from the protein-bound crystal structure.249,250

biaryl ring system allows a strand-like extended conformation of the amide backbone. The hydroxyl groups on R4K appear to only be involved in interactions with water, while the carbonyl oxygen on the side chain makes interactions with three distinct amino acids within the target. Due to the synthetic complexity of TCA-95A, this crystal structure has been used to design and develop chemically simpler compounds. 2.47. Protein Phosphatase 1

Reversible protein phosphorylation of serine/threonine residues by kinases and phosphatases is a major post-translational modification that is a significant component of the cell signaling machinery.247,248 Protein phosphatase 1 (PP1) is one

2.48. Protein Tyrosine Phosphatase 1B

Protein tyrosine phosphatases (PTP) are signaling enzymes that control a diverse array of cellular processes and catalyze phosphorylation of specific Tyr residues that form a recognition motif for many protein−protein interactions. PTP1B is a negative regulator of leptin and insulin signaling pathways.251 Inhibiting PTP1B activity has been suggested as an attractive target for obesity and type 2 diabetes.251,252 In an attempt to design a non-hydrolyzable analogue of the phosphotyrosine residue from the peptide fragment, a pTyr (phosphor L-tyrosine) mimic called FOMT (fluoromalonylLtyrosine) was introduced into a cyclic heptapeptide sequence with Ki = 170 nM.253 The cyclic peptide was crystallized with PTP1B (PDB 1BZH, 2.1 Å) to understand the structural basis of the mechanism in which a malonyltyrosine group mimics PTP1B-phosphotyrosine interactions (Figure 48).254 Unlike complexes of PTP1B with phosphotyrosine-containing peptides, the binding of FOMT containing cyclic peptide is not accompanied by closure of the catalytic site WPD loop. The fluorine substituent was proposed to reduce the pKa of the carboxylic acid of the malonyl moiety and hence increase the

Figure 47. 2D representation of interactions of motuporin and protein phosphatase 1 (PDB 2BCD, 2.1 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

such key signaling enzyme that regulates diverse cellular processes, including cell cycle progression, glycogen metabolism, protein synthesis, gene transcription, muscle contraction, and neuronal signaling.247,248 Two cyclic peptide hepatotoxins, microcystin and nodularin, are cyanobacterial metabolites found worldwide both in freshwater and in marine environments and are potent inhibitors of protein phosphatase 1 and 2A catalytic domains. Microcystins are covalent inhibitors of protein phosphatase 1. Motuporin, also called nodularin-V due to the presence of Val in its variable position, is a cyclic pentapeptide and non-covalent inhibitor of protein phosphatase 1 (IC50 = 0.06 nM). Motuporin is a cyanobacteria metabolite composed of the unusual β-amino acid (2S,3S,8S,9S,4E,6E)-3-amino-9-methoxy-2,6,8-trimethyl-10phenyldeca-4,6-dienoic acid (Adda), an α,β-unsaturated amino acid N-methyldehydrobutyrine (NMeΔBut), an isolinked Dglutamate, and a β-methyl D-aspartate (β-MeAsp) residue. The crystal structure of motuporin in complex with protein Ser/ Thr phosphatase 1c (γ isoform) is reported (PDB 2BCD, 2.1 Å)249 and shows structure stabilization mainly by polar and

Figure 48. 2D representation of interactions of cyclic peptide X with PTP1B (PDB 1BZH, 2.1 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. AC


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bacterial pheromone receptors such as rgg proteins have potential for therapeutic applications.256 Cyclosporin A (CsA), a cyclic undecapeptide known to be an inhibitor of cyclophilins, is an antagonist (inhibitor of SHP2 binding to Rgg2Sp and RggSd in vitro, IC50 = 0.4 μM) of pheromone signaling, and its co-crystal structure with RGG from Streptococcus was solved (PDB 4YV9, 1.95 Å).256 Interactions between CsA and RGG protein are shown in Figure 49. Cyclosporin A is a 33-membered macrocycle comprising 11 amino acids, seven of which are N-methylated, cyclo-[DAlaNMeLeu-NMeLeu-NMeVal-BMT-Abu-Sar-NMeLeu-ValNMeLeu-Ala], where DAL = D-alanine, MLE = N-methylleucine, MVA = N-methylvaline, BMT = 4-methyl-4-[(E)-2butenyl]-4,N-methylthreonine, ABA = α-aminobutyric acid, SAR = sarcosine. Two alternate conformations of CsA are observed in different monomers of the asymmetric unit, where the binding site is identical; however, interactions with Arg153 of protein are observed in one conformation, while interactions with Tyr222 are observed for the other three monomers. Two intramolecular, transannular hydrogen bonds in CsA define one (i, i+3) β-turn (10-membered hydrogen-bonded ring) and one (i, i+2) γ-turn (seven-membered hydrogen-bonded ring) in the structure. Hydrogen bonding to the target is through interactions with the peptide amide backbone. Interactions of the protein with side chains of the macrocycle are all hydrophobic. NMR solution structures of CsA display different conformations in hydrophobic257,258 and polar257 solvents and are also altered by bonding to metal ions259−261 These conformations are different from the structure observed for the protein-bound peptide. The conformation of CSA bound to RGG protein differs significantly from the one bound to cyclophilin (see Figure 71 ahead) with an all-atom RMSD = 4.0 Å and backbone atoms RMSD = 2.5 Å. This shows that the both peptide backbone as well as side-chain conformations change to accommodate binding to the given protein. On the other hand, a water-soluble analogue [D-MeSer3-D-Ser8-(OGly)]-CsA has been found to adopt the same structure in water as when bound to cyclophilin.262

negative charge density, making it more similar to that of a phosphate group. The binding conformation reveals two intramolecular hydrogen bonds: An exocylic hydrogen bond (Asp CO2−···HN Ala) on the left side does not promote any interactions with the target and is solvent exposed. On the right side, an i to i+2, seven-membered hydrogen bond (Tyr CO···HN hCys) stabilizes a γ-turn around the Leu. Interestingly, this turn does not position the side chain to make interactions with the target but does allow the Leu NH to form a hydrogen bond with Asp48. Most interactions with the target come from the substituted tyrosine and two glutamic acids, all of which form extensive hydrogen bonding and salt bridges with Arg47. 2.49. Regulator Gene of Glucosyltransferase

Quorum sensing pheromones help bacteria coordinate their actions as a colony when under survival pressure.255 Cytoplasmic transcription factors known as regulator gene of glucosyltransferase (RGG) proteins belong to a family of receptors that bind directly to pheromones.256 Targeting

2.50. β-Secretase

β-Secretase (β-amyloid converting enzyme, or BACE-1) is a membrane-associated aspartyl protease that has been thought to be crucial in the etiology of Alzheimer’s disease.263,264 BACE-1 is required for the production of the neurotoxic βamyloid peptide, and Alzheimer’s disease is associated with increased accumulation of amyloid plaques.263,264 Inhibitors of BACE-1 have held promise as therapeutics for Alzheimer’s disease.263,264 Based on a small peptide-like lead structure, a series of macrocyclic peptidomimetics were investigated as BACE-1 inhibitors.265 One of these cyclic inhibitors (IC50 = 27 nM) was co-crystallized with β-secretase and a structure solved (PDB 3DV5, 2.1 Å). Interactions between the macrocycle and BACE-1 are summarized in Figure 50. Attached to the macrocycle are the classic hydroxyethylamine transition state isostere, that interacts via hydrogen bonds with the catalytic Asp32 and Asp228, and an aromatic substituent for occupying a hydrophobic S1′ pocket formed by Tyr198, Val69, Pro70, and Tyr71. The cyclic component of this compound occupies S1, S2, and S3 hydrophobic pockets or indentations in the protease. Cyclization has provided a structural constraint that maintains the bis(amide)-containing backbone in an extended strand conformation, unaffected by N-methylation of the alanine, and projects its aliphatic components into positions

Figure 49. 2D representation of interactions of cyclosporin A with RGG protein (PDB 4YV9, 1.95 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. 3D views of the cyclic peptide bound to RGG protein (middle panel, PDB 4YV9) and cyclophilin (bottom panel, PDB 2WFJ) are also given. When superimposed on each other using backbone atoms (RMSD = 2.5 Å), there were changes in both backbone and side-chain atoms. AD


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Bacterial SPases utilize a Ser/Lys catalytic dyad mechanism instead of the classical Ser/His/Asp catalytic triad mechanism.266,267 Bacterial SPases are currently being considered as potential novel antibiotic targets at the bacterial membrane surface.266,267 The inhibition of the catalytically active fragment of SPase (Δ2-75) from E. coli by the antibiotic Arylomycin A2 has been studied using ligand binding studies including fluorescence spectroscopy, stopped-flow kinetics, differential scanning calorimetry, isothermal titration calorimetry, and X-ray crystallography (PDB 1T7D, 2.47 Å).268 Arylomycin A2 is a cyclic lipohexapeptide with the sequence Ac-cyclo-(4,6)-D-MeSer-D -Ala-Gly-[L-MeHpg- L-Ala-L-Tyr]OH (MeHpg = N-methyl-4-hydroxyphenylglycine). It has just three amino acid residues in the cycle forming a ring with 14 heavy atoms. It also has a 12-carbon branched fatty acid attached via an amide bond to the N-terminus and orthocarbon atoms of MeHpg and Tyr forming a (3,3)-biaryl bridge. Interactions between Arylomycin A2 and SPase (Δ2-75) are shown in Figure 51, and it has an apparent KD = 0.94 μM. No electron density was observed for the fatty acid, which is likely near the proposed SPase membrane association surface. The bound conformation of the macrocyclic part of the molecule reveals that the bridge between the two tyrosine side chains has helped constrain the peptide backbone of the cycle into an extended strand conformation. Hydrogen bonds are formed with enzyme from amide NHs inside and outside the macrocycle as well as with the C-terminal carboxylate substituent, which makes hydrogen bonds to four amino acids in the protein.

Figure 50. 2D representation of interactions of the macrocyclic peptidomimetic with BACE-1 (PDB 3DV5, 2.1 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

that enable hydrophobic interactions with secretase residues Leu30, Ille118, and Trp115. The extended conformation permits one of the amides within the cycle to be a key contributor to binding, making three hydrogen bonds with the protein.

2.52. Sirtuin 2 Deacetylase

2.51. Signal Peptidase

Sirtuins are nicotinamide adenine dinucleotide (NAD+)dependent protein deacetylases and/or mono-ADP-ribosyltransferases that are highly conserved from bacteria to humans.172 Humans contain seven sirtuins (SIRT1−7) that have high sequence homology across their catalytic and NAD+ binding domains.172 SIRTs regulate a wide variety of biological processes including gene silencing, cell-cycle regulation, immunological, and metabolic pathways and has been suggested as a potential therapeutic target for aging, obesity, cancer, and metabolic and neurodegenerative diseases.269,270 Owing to the lack of specific and potent SIRT inhibitors, a library of ε-trifluoroacetylamide-lysine containing macrocyclic peptides were screened for binding to SIRT2, leading to the identification of S2iL5 peptide (KD = 1 nM, IC50 = 3 nM; surface plasmon resonance). S2iL5 consists of 14 amino acids, cyclo-[AcYHTYHV(Fak)RRTNYYC]; Fak = N-6-(trifluoroacetyl)-L-lysine cyclized via a thioether bond (Figure 52). A crystal structure was determined for S2iL5 bound to SIRT2 (PDB 4L3O, 2.518 Å).271 A number of mutants of both S2iL5 and SIRT2 were studied.271,272 The comparison of SIRT2S2iL5 and free SIRT2 crystal structures revealed that the binding of S2iL5 induces an open-to-closed domain movement and an unexpected helix-to-coil transition in a SIRT2-specific region. The binding of the macrocyclic peptide induces a significant structural change in the SIRT2 protein, which may not be achieved by small-molecule inhibitors. The bound conformation of S2iL5 shows five intramolecular hydrogen bonds, two being transannular (Ac CO···NH His2, Thr3 CO··· HN Val6) and three from amino acid side chains back to the peptide backbone. At the top of the molecule a gamma turn (Ac CO···NH His2) is stabilized around Tyr1, which allows a second bond to form from the amide NH to the His2 side

Type 1 bacterial signal peptidases (SPase) are membranebound serine proteases that catalyze the cleavage of the aminoterminal signal peptide from secretory and membrane proteins that are translocated across biological membranes.266,267

Figure 51. 2D representation of interactions of Arylomycin A2 with SPase (Δ2-75) (PDB 1T7D, 2.47 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. AE


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RKSRWDETP-OH resulted in a cyclic hexapeptide H-cyclo(1,6)-[KSRWDE]-OH, where the side chain of Lys1 forms a lactam bridge with the side chain of Glu6. This cyclic peptide bound to SPF45 (KD = 1.4 μM), and a crystal structure of the complex (PDB 5LSO, 2.22 Å)273 showed multiple hydrogenbonding interactions between the cyclic peptide and SPF45 UHM domain (Figure 53). The bound conformation of this

Figure 53. 2D representation of interactions of the cyclic peptide and UHM domain (PDB 5LSO, 2.22 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. Figure 52. 2D representation of interactions of S2iL5 with SIRT2 (PDB 4L3O, 2.518 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

macrocycle reveals three intramolecular hydrogen bonds. An i to i+3 transannular hydrogen bond (Arg3 CO···NH Glu6) stabilizes a 10-membered hydrogen-bonding ring indicating a β-turn motif. Trp4 and Asp5 residues within this turn form hydrogen bonds with the protein through their side chains. The amide NH of Asp5 also forms a hydrogen bond with Tyr376. Hydrogen bonds at i to i+2 from the N-terminus (amine NH···OC Ser2), and i+2 to i+4 (Ser2 HO···NH Trp4) cooperate to form a second turn-like structure in the macrocycle that helps position the arginine side chain to interact with Asp319 and Glu325. Hydrogen bonds with the protein are formed both by the amino acid side chains (Arg3, Trp4, and Asp5) and the backbone of the macrocycle (Arg3 NH, Arg3 CO, Asp5 NH, and Glu6 CO2−).

chain. This motif does not interact with the protein. The second transannular hydrogen bond helps stabilize a β-turn across Thr3-Tyr4-His5-Val6, the central Tyr and His residues making both hydrogen bonds and hydrophobic interactions with the protein. The side chain of the conserved Arg8 folds back toward the center of the ring and forms two intramolecular interactions and extensive water-mediated interactions, allowing the side-chain of Fak7 to be presented outside the cyclic scaffold to bind to SIRT2. Extensive hydrogenbonding interactions with the target through both side chains and backbone amides are a feature of the protein−macrocycle interface despite, or perhaps because of, the solvent-exposed nature of the ligand binding site in SIRT2.

2.54. SPRY Domain-Containing SOCS Box Protein 2

SPRY domain-containing SOCS box protein 2 belongs to a family of proteins (SPSB1−4) composed of a central SPRY protein interaction domain and a C-terminal SOCS box.275,276 Recently, SPSB2 has been identified as a novel negative regulator of cellular nitric oxide concentrations by targeting iNOS for proteasomal degradation.266,267 Disrupting SPSB2iNOS interactions has a potential to lead to novel anti-bacterial and anti-cancer drugs.275,276 A cyclic octapeptide cyclo[RGDINNNV], denoted cR8, was designed from a short sequence motif (DINNV) in the N-terminus of inducible nitric oxide synthetase (iNOS) that mediates its interaction with SPSB2.276 The RGD motif was incorporated to enhance

2.53. Splicing Factor 45

U2AF homology motifs (UHMs) are emerging protein− protein interaction modules that play a major role in the regulation of the alternative pre-mRNA splicing process.273,274 Splicing factor 45 kDa (SPF45) is an alternative splicing factor within the UHM domain and is implicated in breast and lung cancers.273,274 Optimization of the sequence length and cyclization of the native UHM ligand motif HAF


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binding to αvβ3 integrins expressed in cancer cells, since cyclic RGD containing peptides have relatively higher binding affinity to integrins than their linear counterparts. The Asp residue is shared between two motifs in this chimeric peptide. Isothermal titration calorimetry (ITC) and chemical shift perturbation data indicated a 1:1 stoichiometric interaction (KD = 671 nM, pH 7.5) equivalent to a binding energy of ∼35.2 kJ/mol. The X-ray crystal structure (PDB 5XN3, 1.34 Å) along with titration data from [1H, 15N]-HSQC NMR spectroscopy, using 15 N-labeled SPSB2 in the presence and absence of cR8 peptide, validated the binding of cR8 to the iNOS binding site on SPSB2 (Figure 54). The cyclic peptide contains only natural L-

detergents, high temperatures, and organic solvents and has been used extensively in various molecular biology and drug discovery applications.277 The observation that cyclic peptides containing His-Pro-Gln bind to streptavidin with much greater affinity (∼3 orders of magnitude) than their linear counterparts led to crystallographic studies.278,279 The binding affinity (KD) for a series of cyclic peptides varied over ∼3 log units depending upon the pH of the medium, and streptavidinmacrocycle crystal structures varied after crystallization under different pH conditions. This was attributed to the low pKa of His in the cyclic peptides in their bound states, where it was proposed that deprotonation of ligand His was required for high-affinity binding. The crystal structure of the 20-membered cyclic peptide Ac-cyclo-(1,6)-[CHPQFC]-NH2, where the Cys residues form a disulfide bond, was determined at pH 7.5 (PDB 1SLD, 2.5 Å) and the binding affinity measured (KD = 230 nM). The interactions between the cyclic peptide and streptavidin are summarized in Figure 55. The bound cyclic

Figure 54. 2D representation of interactions of cyclic peptide cR8 with SPRY domain-containing SOCS box protein 2 (PDB 5XN3, 1.34 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 55. 2D representation of interactions of the cyclic peptide with streptavidin (PDB 1SLD, 2.5 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

amino acids with end-to-end cyclization. One transannular hydrogen bond (Val8 CO···HN Asn5) was formed to divide the macrocycle into unusual 14- and 16-membered hydrogenbonded rings, along with two other intramolecular hydrogen bonds between main-chain amide NHs and the carboxylate side chain of Asp3 (Asn7 NH···O2C···Asp3, Asn5 NH···Asp3). Surprisingly few interactions were made with the protein, suggesting the importance of the hydrogen-bonding side chains of Asp3 and Asn5−7 for binding affinity. The binding of SPSB2 to cR8 was compared to that with a linear iNOS peptide (Ac-KDINNNVKK-NH2, unpublished crystal structure), revealing similar conformations upon binding to SPSB2.

peptide had three intramolecular hydrogen bonds. Two of these are endocyclic and involve the carbonyl oxygen of His2 forming 10- and 13-membered hydrogen-bonded rings via His2 CO···HN Phe5 and His2 CO···NH Cys6, stabilizing a βturn and an α-turn, respectively. This structure allows formation of a third, exocyclic, 10-membered hydrogen bond from the His2 side chain to the Gln4 main-chain amide NH. These bonds pull the His2, Gln4, and Phe5 side chains to one face of the cycle which makes van der Waals, hydrophobic, and hydrogen-bonding interactions with the protein. At the top of the molecule the proximity of two carbonyl oxygens allows interaction with Arg84.

2.55. Streptavidin

Streptavidin is a ∼53 kDa protein isolated from the bacterium Streptomyces avidinii.277 Binding of the water-soluble vitamin, biotin, to streptavidin is one of the strongest non-covalent interactions known in biology.277 The streptavidin−biotin complex is highly resistant to proteolytic enzymes, denaturants,

2.56. Tankyrase

Tankyrase 1 (TNKS) is an enzyme that belongs to the poly(ADP-ribose) polymerase (PARP) superfamily of proteins that catalyze the transfer of ADP-ribose moieties onto their AG


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protein substrates.280 Recent studies highlight a role for tankyrase 1 in the control of WNT signaling, cell proliferation, and differentiation and have encouraged the development of enzyme inhibitors to target this protein.280 Click chemistry has been used to engineer macrocyclic peptides that can target the substrate-binding domain of tankyrase.281 One such cyclic peptide is Ac-cyclo-(3,7)-RE-[AGDGA]-E-NH2, where the side chains of both Ala residues are modified and linked via a 4,4′-propane-1,3-diylbis(1-methyl-1H-1,2,3-triazole) connector. This macrocycle bound to tankyrase (KD = 0.6 μM) and disrupted the TNKS−Axin interaction in a dose-dependent manner (IC50 ≈ 20 μM). A co-crystal structure was determined (PDB 5BXO, 1.33 Å), and interactions between the cyclic peptide and TNKS are summarized in Figure 56. No

2.57. Thrombin

Thrombin is a trypsin-like serine protease that converts fibrinogen into fibrin in blood coagulation.282 Thrombin plays crucial roles in regulating thrombosis and hemostasis.282 Various direct thrombin inhibitors have been developed and investigated for venous thromboembolism, thrombocytopenia, acute coronary syndromes, and other conditions.283 Cyclotheonamide A is a 19-membered cyclic pentapeptide isolated from marine sponge Theonella sp., a slow-binding inhibitor of thrombin (IC50 = 100 nM) that dose-dependently inhibits aggregation of human platelets (IC50 = 1.5 μM).284 It contains a Pro-Arg motif that correlates with the P2−P1 positions of known tripeptide substrates and inhibitors, a D-Phe at P1′, and an α-keto amide that makes the compound a transition-state analogue inhibitor of serine proteases (Ki = 180 nM, human thrombin; 23 nM, trypsin; 35 nM, streptokinase). Its crystal structure bound to human α-thrombin (PDB 1TMB, 2.3 Å, Figure 57)285 showed both aromatic (D-Phe, Tyr) side chains

Figure 56. 2D representation of interactions of the cyclic peptide with tankyrase (PDB 5BXO, 1.33 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 57. 2D representation of interactions of cyclotheonamide A with human α-thrombin (PDB 1TMB, 2.3 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

intramolecular hydrogen bonds are observed in the bound macrocycle. Hydrogen bonding from the macrocyclic section to the protein is driven by interactions with the amide backbone through both carbonyl oxygens and amide NHs. The double triazole linker does not form any direct interactions with the target. The affinity derives mainly from formation of salt bridges and hydrogen bonds with the protein. Salt bridges are made from Arg1 to Glu598 and Asp589, and between Glu2 and Lys557. The imidazole side chains at positions 3 and 7 of the ligand do not interact with protein but rather project away from the protein surface and into solvent, allowing them to be linked together to form a macrocycle. The intervening Gly4, Asp5, and Gly6 of the peptide occupy a groove on the protein surface, enabling hydrogen bonds between Asp5 and Ser527, the phenol rings of Tyr569 and Tyr536 stacking parallel on either side of Gly6, while Glu8 interacts with Lys604 via a salt bridge.

interacting in an edge-to-face geometry (within 4 Å), while the potentially reactive vinyl group in the backbone did not form a covalent bond to enzyme. An intramolecular 10-membered hydrogen bond ring, facilitated by the proline turn-inducing constraint, defines a β-turn motif. Hydrogen bonding to the target is driven by interactions with both the side chains and the backbone of the macrocycle. The arginine side chain of the cyclic peptide forms a classic high-affinity (Arg···Asp) hydrogen bond with Asp189 of thrombin. The NMR solution structure of cyclotheonamide A in water shows that residues important for active site binding (D-Phe, Arg, Pro) are in a conformation nearly identical to that in the enzyme-bound crystal structure.286 AH


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2.58. Thrombin Activatable Fibrinolysis Inhibitor

and ligand polar side chains and main-chain atoms from the appendage to the macrocycle, with its HTyr, Val, and Lys side chains making some van der Waals contacts.

Thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots.287,288 TAFI circulates in plasma as an inactive precursor until thrombin or plasmin induces it to adopt its active form. TAFI is an important link between coagulation and fibrinolysis, and the development of TAFI inhibitors may help to prevent thrombosis.287,288 Anabaenopeptides, cyclic peptides derived from cyanobacteria, were identified as potent inhibitors of TAFIa. 289 The co-crystal structures of TAFIa bound individually to three naturally occurring cyclic peptides, Anabaenopeptins B, C, and F (IC50 = 1.5−2 nM), were determined. Anabaenopeptin B is a hexapeptide containing an 18-membered cyclic pentapeptide component, cyclo-[D-LysVal-HTyr-NMA-Phe] (HTyr = homotyrosine, NMA = Nmethylalanine), where the side chain of D-Lys forms a lactam with the C-terminal Phe carboxylic acid, fused to an Arg residue linked to the N-terminal amine group of the cyclic pentapeptide via a urea bridge. Interactions between Anabaenopeptin B and TAFIa (PDB 5LRG, 2.02 Å) are summarized in Figure 58. The bound conformation of this

2.59. Toxin Protein−Immunity Protein Interaction Inhibitors

Contact-dependent growth inhibition (CDI) is a strategy used by some Gram negative bacteria to inhibit the competing growth of neighboring bacteria.290,291 Gram-negative bacteria

Figure 59. 2D representation of interactions of the macrocyclic peptide MAC with CdiA-CT (PDB 4ZQW, 2.00 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

deploy a variety of CdiA-CT toxins into target cells which, in turn, can protect themselves by producing small Cdil immunity proteins that bind to CdiA-CT and block its toxin activity.290,291 Crystal structures of the CdiA-CT/Cdil complexes from Escherichia coli EC869 and Yersinia pseudotuberculosis YPIII reveal that the protein−protein interactions are governed by β-augmentation, in which the toxin domain extends a β-hairpin to complete a six-stranded anti-parallel βsheet within the immunity protein. A 13-residue macrocyclic peptide mimic of the β-hairpin (called MAC) from CdiA-CT from E. coli EC869 was designed, synthesized, and crystallized with Cdil immunity protein (PDB 4ZQW, 2.00 Å).292 The MAC peptide contains residues corresponding to Lys242− Ser253 of CdiA-CT from E. coli EC869 connected through a δlinked ornithine residue giving the sequence of cyclo[SOKEYALSGRELT]. Biolayer interferometry was used to measure the dissociation constant (KD = 18 nM) for the CdiACT/Cdil complex from E. coli EC869. Even though binding between MAC and Cdil protein could not be detected using this approach, the complex could be crystallized, indicating low binding affinity. The MAC peptide forms a two-stranded β-

Figure 58. 2D representation of interactions of Anabaenopeptin B and TAFIa (PDB 5LRG, 2.02 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

macrocycle shows one intramolecular i to i+3 10-membered hydrogen-bonded ring (Val2 CO···NH Phe5), which stabilizes a β-turn around Val-HTyr-NMA-Phe. This turn is solvent exposed and does not make hydrogen-bonding interactions with the target. The complex shows that the cyclic peptide presents in an extended strand-like backbone structure that mostly makes hydrogen-bonding interactions between protein AI


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βF[WRP]W-OH, where the side-chain nitrogen of Trp2 is linked to the α-carbon of Pro via a four-carbon linker. The interactions between the cyclic peptide and WDR domain of TLE1 are summarized in Figure 60. The bound conformation of this macrocycle does not show any intramolecular hydrogen bonds. The macrocycle appears to have helped stabilize an extended ligand backbone conformation in the FWR residues. This conformation allows the Arg and Trp residues to present on the same face, enabling the arginine side chain and carbonyl oxygen of Trp to interact with adjacent protein residues. All other interactions made with the target are hydrophobic.

sheet, as in the CdiA-CT/Cdil structure from E. coli EC869. The interactions between MAC and CdiA-CT are shown in Figure 59. The bound conformation of this macrocycle shows four intramolecular hydrogen bonds. These help to present three distinct structural motifs to the target. On the right side of the molecule an i+3 to i transannular hydrogen bond (Arg10 NH··· OC Leu7) helps stabilize a β-turn. Through the middle section of the compound three transannular hydrogen bonds (Arg10 CO···HN Leu7, Leu12 HN···OC Tyr5, Leu12 CO···HN Tyr5) stabilize a β-sheet. A water-mediated hydrogen bond connects Lys3 CO to Leu 12 CO to further stabilize this structure. The left side of the macrocycle presents a loop like structure. Binding to the target takes place on both sides of the β-sheet, mostly through a combination of hydrophobic interactions, hydrogen bonds, and salt bridges. Three hydrogen bonds are made between the protein and amide carbonyl oxygens of the macrocycle. There are a number of prominent water-mediated hydrogen bonds.

2.61. Trypsin

Trypsin is an important digestive serine protease that cleaves peptides at the C-terminal side of arginine or lysine.295 Microviridins, serine protease inhibitors derived from cyanobacteria metabolites, are 12- to 14-membered cyclic peptides containing non-canonical lactone and lactam rings. Microviridin J, a 13-residue containing tricyclic depsipeptide inhibits trypsin (IC50 = 90 nM, KD = 680 nM from ITC), and two co-crystal structures are reported (PDB 4KTU, 1.35 Å, pH 6.5; PDB 4KTS, 1.3 Å, pH 8.5).296 Microviridin J is reportedly composed of Ac-cyclo-IS[TRKYPSDWEEW]-OH, with Thr3 side-chain hydroxyl and Asp9 side-chain carboxylic acid connected via a lactone, Ser8 side-chain hydroxyl and Glu11 side-chain carboxylic acid connected via a lactone, and sidechain amine of Lys5 forming a lactam bond with side-chain carboxylic acid of Glu12. Interactions between microviridin J and trypsin (PDB 4KTU) are summarized in Figure 61. Despite the intricate structure, only two intramolecular

2.60. Transducin-like Enhancer Protein

Transducin-like enhancer protein 1 (TLE 1) is a developmental and oncogenic transcriptional co-repressor protein that directly binds to transcription factors to regulate cellular Notch and WNT networks.293,294 Targeting TLE1-tran-

Figure 60. 2D representation of interactions of the cyclic peptide with the WDR domain of TLE1 (PDB 5MWJ, 2.04 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

scription factor interaction represents a putative anti-cancer target.293,294 A constrained cyclic peptide, containing a linker of four methylene groups inserted into the peptide epitope as found in many TLE1 binding partners, was investigated and exhibited KD = 25 nM (∼5 fold higher than the unconstrained peptide).294 A co-crystal structure of the constrained cyclic peptide in complex with the WDR domain of TLE1 was solved (PDB 5MWJ, 2.04 Å). The cyclic peptide is H-cyclo-(2,4)-

Figure 61. 2D representation of interactions of Microviridin J with trypsin (PDB 4KTU, 1.35 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below. AJ


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NH···OC Cys5). These stabilize two consecutive β-turns in a 310 like helix structure. The smaller right side cycle, a 19 heavy atom macrocycle, remains more flexible despite the constraining Pro-Pro motif. Only one direct hydrogen bond is made between the bicyclic ligand and the target protein (Trp7 NH··· O Tyr119). No other intermolecular hydrogen bonds or salt bridges are observed, suggesting that the high-affinity interaction is largely driven by hydrophobic and van der Waals interactions and shape complementarity. Thus, the interaction of M21 with TNF more closely resembles protein− drug interactions than conventional antibody−antigen interactions.

hydrogen bonds are present in the bound macrocycle. One at the top of the figure (Glu11 NH···O Ser8) is an unusual amide to ester hydrogen bond. Another intramolecular amide to ester hydrogen bond (Lys5 NH···OC Asp9) at the bottom of the compound helps position Arg4 as part of an extended Ile-SerThe-Arg strand conformation. Interactions with the target are formed mainly along the bottom strand-like face of the macrocycle and are both side chain and amide backbone mediated. 2.62. Tumor Necrosis Factor

Tumor necrosis factor (TNF) is a cytokine that belongs to the superfamily of type II transmembrane proteins and is involved in regulating systemic inflammatory processes.297,298 TNF can bind to and activate two different receptors, TNFR1 and TNFR2, that can initiate both distinct and overlapping cell signaling pathways.297,298 Antibodies, soluble receptors that target TNF have shown remarkable efficacy in a variety of immune-mediated inflammatory diseases, especially rheumatoid arthritis.297,298 A series of bicyclic peptides were investigated as TNF inhibitors.299 A bicyclic peptide M21, cyclo-[ACPPCLWQVLCG] where all three Cys side chains are linked via 2,4,6-trimethylmesitylene to form a bicyclic structure, was found to bind to TNF (KD = 10 nM). It also inhibited TNF effector functions at μM concentrations in a L929 fibrosarcoma necroptosis assay.300 A co-crystal structure (PDB 4TWT, 2.85 Å) showed interactions between M21 and TNF as depicted in Figure 62. M21 has two loops covalently

2.63. Urokinase-Type Plasminogen Activator

Urokinase-type plasminogen activator (uPA), also known as urokinase, is a trypsin-like serine protease that is involved in tissue remodeling and cell migration.301,302 Increased ex-

Figure 63. 2D representation of interactions of UK18 with uPA (PDB 3QN7, 1.9 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

Figure 62. 2D representation of interactions of M21 with TNF (PDB 4TWT, 2.85 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. The residue names from the second monomer of protein are shown in italics. A 3D view of the bound cyclic peptide is shown below.

pression and activity of uPA have been linked to tumor progression, metastasis, and decreased survival in cancer patients.301,302 A series of cyclic and bicyclic peptides have been investigated as uPA inhibitors, and a number of co-crystal structures have been reported.303−306 The bicyclic peptides were designed to contain a central hydrophobic linker or dithiols to make two disulfides, with the aim of providing additional restraints on the peptide backbone flexibility. One

anchored to a 2,4,6-trimethylmesitylene core that bisects the bicyclic structure, which interferes with assembly of (or disassembles) TNF trimers by binding to the dimer/trimer interface. The bound conformation of M21 reveals that the left side cycle, which is a 28 heavy atom macrocycle has two i+3 to i intramolecular hydrogen bonds (Leu10 NH···OC Trp7, Gln8 AK


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1 nM).309 MM-589 is cell permeable and a potent and selective inhibitor of cell growth in human leukemia cell lines harboring MLL translocations. A crystal structure (PDB 5VFC, 1.64 Å) was reported for WDR5 bound to MM-589, isobutyryl-cyclo[αMeOrn-Arg-Abu-Phg], where αMeOrn = α-methylornithine, Abu = L-α-aminobutyric acid, and Phg = Lphenylglycine (Figure 64). The structure showed a ligand binding conformation with three intramolecular hydrogen bonds in the cycle (isobutyryl CO···NH αMeOrn1, isobutyryl CO···NH Arg2, and αMeOrn1 CO···NH Phg4). The latter transannular hydrogen bond forms a 10-membered hydrogenbonded ring that defines a β-turn in the macrocycle. The other two intramolecular hydrogen bonds bring the hydrophobic Nterminus into closer proximity with the aminobutyric acid side chain to present a hydrophobic patch to the target. The β-turn presents the arginine side chain which makes multiple interactions with the protein.

potent bicyclic inhibitor (Ki = 53 nM) is UK18, a 17-residue bicyclic peptide with the sequence ACSRYEVDCRGRGSACG, where all three Cys residue side chains are linked via a symmetric trimethylbenzene to form a bicyclic peptide. A cocrystal structure of UK18 with human uPA has been solved (PDB 3QN7, 1.9 Å), and their interactions are summarized in Figure 63. The bound conformation of UK18 contains four intramolecular hydrogen bonds; however, none are transannular main chain-to-main chain in this structure. Within the left side cycle, two hydrogen bonds are formed from the Ser14 OH side chain to the carbonyl oxygen groups of Gly11 and Cys9, and a third hydrogen bond connects Gly13 NH···OC Asp8; all three hydrogen bonds draw the left side of the molecule closer to the right cycle and compress the bicyclic structure. This face of the molecule does not make any direct interactions with the target. These three hydrogen bonds allow the carbonyl groups of Val7 and Arg12 to be in close proximity and form hydrogen bonds with Gln192. In the right cycle, only a Glu6···Arg4 side chain-to-side chain intramolecular hydrogen bond is observed. The Arg12 and Arg10 side chains at the bottom form multiple hydrogen bonds with protein. Binding to the target is mostly side-chain directed except toward Gln192.

2.65. X-Chromosome Linked Inhibitor of Apoptosis Protein

Activation of caspases is central to the execution of programmed cell death.310 X-chromosome-linked inhibitor of apoptosis protein (XIAP) binds and inhibits the activation of initiator and effector caspases common to both intrinsic and extrinsic pathways.311 XIAP antagonists targeting its baculovirus IAP repeat domains BIR2 and BIR3 are potential anticancer compounds.312 Using a DNA-programmed chemistry approach, a series of macrocyclic peptides were investigated as XIAP BIR2 and BIR3 antagonists and co-crystal structures were reported.313 A macrocyclic peptide with the sequence cyclo-(3,5)-MeAla-Val-[Pro-Phe-Hox]-OH (Hox = 4aminophenylalanine), where a triazolylpropionic acid is linked to the 4-amino substituent of Hox5 and a triazolyl nitrogen connects to C4 of Pro3, inhibited BIR2 (IC50 = 1.97 μM) and BIR3 (IC50 = 0.11 μM), and a co-crystal structure was solved (PDB 4WVU, 2.02 Å). Interactions between the macrocyclic peptide and XIAP BIR2 are shown in Figure 65. No

2.64. WD Repeat Domain 5 Protein

Mixed lineage leukemia protein (MLL1) is a histone H3 lysine 4 methyltransferase that is frequently dysregulated in aggressive acute leukemias.307,308 WD repeat domain 5 protein

Figure 64. 2D representation of interactions of MM-589 with WDR5 (PDB 5VFC, 1.64 Å). Ligand is black, while protein residues and solvent regions are blue. Hydrogen bonds are shown as black (intramolecular) and blue (intermolecular) dotted lines. Amino acid residues in the binding pocket are shown. A 3D view of the bound cyclic peptide is shown below.

(WDR5) is an adaptor protein that binds to MLL and dramatically increases its enzyme activity.307,308 Targeting the WDR5-MLL interaction using inhibitors could provide an effective treatment for MLL leukemias.307,308 From a 3-residue peptide Ac-ARA-NH2 that bound to WDR5 (Ki = 120 nM), a cyclic peptidomimetic MM-589 was derived as a potent inhibitor of MLL H3K4 methyltransferase (IC50 = 0.9 nM, Ki

Crystal Structures of Protein-Bound Cyclic Peptides - Chemical

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