Backbone-cyclized Peptides

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Send Orders for Reprints to [email protected] Current Topics in Medicinal Chemistry, 2018, 18, 526-555

REVIEW ARTICLE ISSN: 1568-0266 eISSN: 1873-4294

Backbone-cyclized Peptides: A Critical Review

Impact Factor: 3.374

The international journal for in-depth reviews on Current Topics in Medicinal Chemistry

Samuel J.S. Rubina,§ and Nir Qvitb,§,* a

Stanford Immunology Program, School of Medicine, Stanford University, 269 Campus drive, Stanford CA 94305-5174, USA; bThe Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, Henrietta Szold St. 8, POB 1589, Safed, Israel

ARTICLE HISTORY Received: July 24, 2017 Revised: January 24, 2018 Accepted: January 24, 2018

Abstract: Backbone-cyclized peptides and peptidomimetics integrate the biological activity and pharmacological features necessary for successful research tools and therapeutics. In general, these structures demonstrate improved maintenance of bioactive conformation, stability and cell permeability compared to their linear counterparts, while maintaining support for a variety of side chain chemistries. We explain how backbone cyclization and cycloscan techniques allow scientists to cyclize linear peptides with retained or enhanced biological activity and improved drug-like features. We discuss head-to-tail (Cterminus to N-terminus), building unit-to-tail, building unit-to-side chain, building unit-to-building unit, and building unit-to-head backbone cyclization, with examples of building blocks, such as Nα-(ωthioalkylene), N α-(ω-aminoalkylene) and Nα-(ω-carboxyalkylene) units. We also present several methods for recombinant expression of backbone-cyclized peptides, including backbone cyclic peptide synthesis using recombinant elements (bcPURE), phage display and induced peptidyl-tRNA drop-off. Moreover, natural backbone-cyclized peptides are also produced by cyanobacteria, plants and other organisms; several of these compounds have been developed and commercialized for therapeutic applications, which we review. Backbone-cyclized peptides and peptidomimetics comprise a growing share of the pharmaceutical industry and will be applied to additional problems in the near future.

Pe N rs ot on fo al rD U is se tri O bu n tio ly n

Current Topics in Medicinal Chemistry

BENTHAM SCIENCE

DOI: 10.2174/1568026618666180518092333

Keywords: Backbone cyclization, Cyclization, Cycloscan, Peptides, Peptidomimetics, Protein-protein interactions, Therapeutic. 1. INTRODUCTION

Protein interactions are the broad basis of signaling pathways and other cellular processes, which maintain physiological or mediate pathological conditions. Crosstalk between receptors and ligands is involved in the control of cellular homeostasis, from proliferation, differentiation, migration, and cell death, to large-scale angiogenesis and the immune responses. Deregulation of Protein-Protein Interactions (PPIs) causes many forms of human disease, such as cancer, cardiovascular disease, infection, and neurodegeneration. This type of signaling requires tight spatial and temporal regulation of protein complex formation, which is mediated by specific protein interaction domains. Complexes are usually reversible and transient, and their stability and duration of association may be critical for the biological outcome. Therefore, modulating PPIs is an attractive option for basic science and therapeutic applications [1].

Protein complexes are held together by their contact interface, which is usually flat and large, ranging from approximately 300 to 4,800 Å2 [2, 3], and typically lacking small deep pockets that would bind small molecules (~300*Address correspondence to this author at the Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, P.O. Box: 1311502, Henrietta Szold St. 8, Safed, Israel; Tel: +972-72-264-4849; E-mails: [email protected]; [email protected] § Equal contribution 1873-4294/18 $58.00+.00

500 Å2) [4-7]. Peptides offer attractive features for targeting protein-protein interfaces; these molecules can mimic the properties of one interface as a competitive inhibitor, therefore interrupting the binding of partner proteins [5, 8-10]. Peptides derived from such intermolecular protein-protein interfaces frequently serve as antagonists; however, if a peptide binds to the site of an intramolecular protein-protein interaction, the molecule could potentially serve as an agonist as well [11, 12]. Recent studies suggest that 15-40% of known protein-protein interactions are modulated by endogenous linear peptides [13, 14]. Moreover, peptides demonstrate several advantages for targeting protein complexes compared to small molecules, such as conformational flexibility (induced fit) [15] and increased selectivity [16-18]. Nevertheless, linear peptides are limited by poor stability, reduced selectivity and poor cell membrane permeability. Various modifications can be employed to overcome these limitations, such as N-terminal and C-terminal modifications [19], peptide bond modifications [20], incorporation of nonproteinogenic amino acids [21], and cyclization [22-24]. These alterations are used to create peptidomimetics (modified peptides), a term henceforth used interchangeably with peptides for the purposes of this work. Purification and treatment of a diabetic child with bovine insulin almost 100 years ago demonstrated that peptides could be used to treat human disease [25]. The first cyclic peptide antibiotic, gramicidin S, was identified several dec-

© 2018 Bentham Science Publishers

Backbone-Cyclized Peptides

Backbone cyclization is a method to develop focused, novel peptide libraries. Using backbone cyclization, a small library of peptides can be developed to mimic, for example, a target protein epitope. A small library of peptides is typically screened for bioactivity and the most active peptides are selected as lead compounds. For each lead compound, a focused library of cyclic peptides may be further synthesized. In general, each cyclic peptide in the focused library is derived from the same natural/primary sequence, while factors such as size, location and chemistry of the cyclization diversify the library. In this review, we discuss various approaches for the synthesis and development of backbonecyclized peptides using alpha-amino acid derivative building units, as well as the applications of backbone-cyclized peptides in basic research and drug discovery. 2. BACKBONE CYCLIZATION AND CYCLOSCAN Several main formats have been proposed for cyclization of linear peptides, such as head-to-tail (C-terminus to Nterminus), side chain-to-tail, side chain-to-side chain, and side chain-to-head (Fig. 1) [17]. Various synthetic ap-

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proaches are used to covalently link linear peptides in a process termed as macrocyclization, although the most common methods for the final ring-closing reaction, especially in naturally occurring cyclic peptides, are cyclization via lactam bridges [42, 43], lactone bridges [27, 44] and disulfide bridges [45]. Cyclic peptides usually demonstrate high biological activities, as well as improved potency and increased selectivity compared to their linear analogs, making them ideal candidates for biological research as well as therapeutic lead compounds [46, 47]. R

O H N

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ades later [26], and since then cyclic peptides with increased selectivity and bioactivity compared to their linear analogs have shown promise in basic and translational research settings [27-29]. Three main approaches have been described to identify bioactive cyclic peptides: isolation of naturally occurring cyclic peptides, screening of cyclic peptide libraries, and rational design. Naturally occurring cyclic peptides have been isolated from tissues and organisms ranging from prokaryotes to humans. These natural cyclic peptides, such as antimicrobial peptides [30], cyclotides [31], conotoxins [32], knottins [33], and some human hormones [17], play critical roles in cell signaling pathways and demonstrate potent bioactivity and efficacy as pharmacological compounds. The use of cyclic peptide libraries represents another important approach to identifying bioactive peptides, and these resources can be divided into three main classes: biological, semisynthetic, and fully synthetic. Biological libraries can be formed by phage display [34], the yeast two-hybrid assay [35], or Split Intein Cyclization of Peptides and Proteins (SICLOPPS) [36]. Various approaches have been developed for semisynthetic and fully synthetic libraries, which can incorporate random synthesis by SPOT-synthesis or oligonucleotide-encoded expression, as well as microbial display systems [37, 38]. Importantly, rational design is highly attractive for developing novel peptide compounds. Several strategies have been used to rationally design cyclic peptides; many employ bioinformatic methods to integrate data derived from the sequence and/or structure of target proteins to design cyclic peptides with enhanced bioactivity and stability [39, 40]. For instance, bioactivity can be prioritized by using algorithms to design mimics of the small exposed surfaces on proteins that mediate PPIs; thus, sequence and structural information about target epitopes greatly enhances design opportunities [41]. Peptide cyclization is another feature that can enhance rationally designed compounds for therapeutic applications. In this article, we discuss three main approaches to identify cyclic peptides: Isolation of naturally occurring cyclic peptides, screening of cyclic peptide libraries and rational design. Our primary focus is on the rational design of backbone-cyclized peptides.

Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7

O

R

O H N

N H O

R H N

N H R

n

O

O R

c. Side-chain to head/tail

Fig. (1). “Classical” modes of peptide cyclization.

However, cyclization can interfere with the bioactive epitopes of a linear compound if the process compromises their chemistries. One common reason for failed development of highly active cyclic peptides is the use of important bioactive residues in cyclization, which blocks critical interaction sites. As mentioned above, cyclization can be done from head-to-tail of the peptide; although this format is common in naturally occurring cyclic peptides, it offers only one option for cyclization from the C-terminus to the N-terminus, and that results in a single cyclic peptide. Using other modes of cyclization, such as side chain-to-tail, side chain-to-side chain and side chain-to-head, one can create a variety of molecules. However, it is important to keep in mind that many members of such a library are frequently and inevitably inactive due to obstruction of side chains necessary for peptide bioactivity, which is particularly important for shorter peptides. Another common failure in the development of bioactive cyclic peptides is a result of limited bioactivity imposed by conformational constraint. Several models were suggested previously to describe the binding interaction between two proteins or between a peptide and a protein. The “Lock and Key” model was introduced by Emil Fisher in 1894, where the lock represents an enzyme and the key represents a substrate. In this model, it is assumed that both proteins have a specific, fixed geometric shape and orientation that is complementary to the other protein. Their respective shapes allow two proteins to fit perfectly into each other as a lock and a key [48]. However, the Lock and Key model was later replaced by the “Induced fit” model, presented by Daniel E. Koshland Jr., which takes into account the flexibility of proteins and the influence of a substrate on the shape of an enzyme [15]. Since cyclic peptides are restricted in their conformation, their induced fit upon binding to a target is limited - either preventing a cyclic peptide from achieving the biologically active conformation, or in an ideal scenario,

528 Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7

maintaining a bioactive conformation. Consequently, to identify an active cyclic peptide(s) with desirable biological activity, a library of cyclic peptides should be screened [49].

different building blocks and bridge elements can be used to efficiently screen the conformational space of the parent peptide. Importantly, most of the compounds in such a library are inactive since they do not exhibit a bioactive conformation. The peptide(s) that do maintain a biologically active conformation are highly potent and possess the pharmacological advantages of cyclic peptides. Although we do not discuss this in detail, substitution of the natural amino acids can be done in some cases to further diversify the library [23]. Moreover, there are eight modes of cyclization amenable to the cycloscan technique, including four natural modes: head-to-tail (C-terminus to N-terminus), side chain-to-tail, side chain-to-side chain, and side chain-to-head; and four modes of backbone cyclization: backbone-to-backbone, backbone-to-N terminus, backbone-to-C terminus, and backbone-to-side chain (Fig. 2). R

O H N

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To overcome limitations associated with traditional peptide cyclization, Chaim Gilon presented two important methodologies: backbone cyclization [50] and cycloscan [51]. Backbone cyclization is a technique that enables the development of cyclic peptides without utilizing the residues that are part of the natural linear peptide, which may be essential for the biological activity of the peptide, particularly if short. By definition, backbone cyclization is a method in which functionalized alkyl spacers anchored to the peptide backbone are connected to another functionalized spacer, to a functional amino acid side chain on the peptide, or to the Nor C-terminus. As mentioned earlier, avoiding the use of functional groups in the natural residue side chains for cyclization may be particularly valuable in short peptides and/or peptides in which all the amino acids are important for the biological activity [52]. In backbone cyclization, a building unit composed of a functional atom, such as sulfur, and nitrogen or carbon with an orthogonal protecting group, covalently bonded via spacer to the backbone, is used to form a cyclic peptide from a linear parent molecule. The main advantage of this method is that the cyclization linkage is formed between backbone atoms and not the atoms of the side chain functional groups, which are typically critical for binding and biological function. In summary, backbone cyclization uses unnatural building blocks with an extra linker of customizable length covalently bonded to a backbone functional group for the peptide cyclization. This arrangement maintains the natural amino acid functional groups to support biological activity.

Rubin and Qvit

Several other methods, such as click chemistry [53, 54], Ring Closing Metathesis (RCM) [55], stapled peptides [56], and native chemical ligation [57], are also used to create cyclic peptides. The use of click chemistry and RCM was reviewed recently [58]. Stapled peptides are predominantly used to mimic and stabilize known secondary structural elements, such as alpha helices, in peptides through alkenebased ‘staples’ [39, 59]. Native chemical ligation is mainly used for linking peptide fragments [60], although this technique has also been extended to the realm of solid phase macrocyclization chemistry [61]. In this review, we focus on the development of backbone-cyclized peptides, based on the knowledge of native sequence and use of alpha-amino acidderived Nα-(ω-Y-alkyl) building units. Using functional knowledge of linear peptides (parent compounds) and/or target proteins, backbone-cyclized peptides can be developed to address challenges in basic and translational sciences.

The variety of building blocks used for backbone cyclization offers opportunities to explore the conformational space of cyclic peptides using the cycloscan methodology. Cycloscan is a method in which a library of cyclic peptides is prepared using an identical primary sequence. The cyclic peptides in the library differ from each other in their conformation, as a result of the cyclization approach and bridge features. Cycloscan is particularly advantageous when the native sequence of a natural epitope or the pharmacophore is known. For a library in which all the backbone-cyclized peptides have the same primary sequence (the natural peptide),

O

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O

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O N O

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R

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a. N-backbone to side chain

R

R

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R

N

N H O

R H N

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n

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c. N-backbone to head/tail

Fig. (2). Modes of backbone cyclization demonstrated by examples of N-backbone cyclization.

In addition to the cyclization approach (e.g. location of cyclization anchor and linkage of building unit to Nterminus, C-terminus, side chain, or another building unit), other parameters related to the cyclization bridge can be used to diversify a backbone-cyclized peptide library. These features include the bridge position, size and chemistry, which are critical for binding and biological activity of backbonecyclized peptides. The position of the building block unit in the primary sequence that defines the bridge also determines the specific atoms which are part of the cyclic motif of the peptide. For example, Hurevich et al. tested the effect of bridge position on bioactivity, and developed a library of backbone-cyclized peptides to mimic the helical conformation region of the chemokine (C-C motif) receptor 2 (CCR2) dimerization site, which is important for immune responses and has been implicated in the pathogenesis of immunemediated diseases such as multiple sclerosis. In a bridge position scan, systematic replacement of the building units that form the bridge and adjustment of their position revealed that one backbone-cyclized peptide, M3D-1, specifically inhibited CCR2-mediated chemotactic migration in cells [62].

Altstein et al. optimized the cyclization bridge size while maintaining building unit location. The authors evaluated different bridge sizes derived from the length of functionalized spacers, another important factor in determining bioactivity. They developed libraries derived from pheromone biosynthesis activating neuropeptide (PBAN), which regulates sex pheromone biosynthesis in Heliothis peltigera female moths. Using various linkers, the authors demonstrated


that the bridge determined biological activity. Backbonecyclized peptides in which the bridge amide bond was after two or four carbons (peptides 21 and 25, respectively) were highly active compared to an inactive backbone-cyclized peptide with the same sequence and ring size but an amide bond bridge after three carbons (peptide 23) [63]. Another study of bridge size was conducted by Qvit et al., who developed a small library of backbone-cyclized peptides derived from the Leishmania sp. parasite scaffold protein LACK (Leishmania's receptor for activated Ckinase). One of these peptides, p4d (with 10 atoms in the bridge), exhibited leishmanicidal activity in culture and was also effective at reducing parasitemia and increasing survival in infected mice, while other peptides with a longer bridge (11, 12 or 14 atoms) were much less active [64].

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dure in a reactant solution (Fig. 3). A major advantage of this method is that an excess of reagents can be used to drive reactions to completion, and later these reagents can be removed easily by washing. Another key advantage of the solid phase synthesis is the extended set of molecules, such as D-amino acids, N-methyl amino acids, and nonproteinogenic amino acids (hundreds of such building blocks are commercially available), which can be incorporated in the chemical synthesis. The possibility to add nonproteinogenic amino acids in the solid phase approach is a significant benefit compared to the recombinant expression of peptides and proteins, which is limited in compatibility with non-proteinogenic amino acids [96, 97]. The use of diverse building blocks often improves peptide stability, since these units are generally more resistant to proteolysis. Moreover, non-proteinogenic amino acids can maintain desirable features of natural alpha-amino acids, but often enhance bioactivity of the natural compounds. As a result, solid phase synthesis is widely used for the synthesis of peptides, short proteins, nucleic acids, and oligosaccharides.


Bridge chemistry is also critical for the bioactivity of the cyclic peptides. This feature is defined by the type(s) of functional groups used to form the bridge. Several different approaches have been introduced for cyclization, including backbone disulfide bridges [23, 65], backbone amide bridges [63], backbone metal bridges [66, 67], backbone urea bridges [68], and backbone azo bridges [69]. For instance, replacement of lactam bridges with backbone disulfide bridges was used to support on-resin backbone cyclization and enhance peptide selectivity [65]. Furthermore, backbone metal cyclization via two hemi-chelating groups on the peptide was used to implement simultaneous cyclization and metal labeling for targeted imaging [66, 67]. This approach could also be utilized for further development of peptide receptor radionuclide therapy [70]. All these formats have significant effects on the biological activity and functional capacity of peptide libraries.


Since the introduction of backbone cyclization and cycloscan, these approaches have been used to modify a myriad of bioactive natural peptides and peptides derived from target proteins, such as substance P [71, 72], somatostatin [65, 67, 73, 74], Pheromone Biosynthesis Activating Neuropeptide (PBAN) [75], Nuclear Factor-Kappa B (NF-κB) [76, 77], Insulin-Like Growth Factor-1 Receptor (IGF-1R) [23, 78], Human Immunodeficiency Virus (HIV)-1 integrase [79], Bovine Pancreatic Trypsin Inhibitor (BPTI) [80], protein kinase B [81], protein kinase C and its substrates [12, 82-87], as well as LACK (Leishmania’s receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase) proteins [64, 88-90]. In many cases, backbone-cyclized peptides have been shown to improve the pharmacological selectivity of a given peptide, as demonstrated, for example, with substance P [72] and somatostatin analogs [91]. In addition to the selectivity of the backbonecyclized peptides, their stability [64, 92] and paracellular permeability [93, 94] are frequently improved compared to linear analogs. 3. BUILDING BLOCKS FOR BACKBONE CYCLIZATION

The chemical synthesis of peptides and peptidomimetics is most frequently performed using the Solid Phase Synthesis (SPS) approach pioneered by Merrifield [95]. In SPS, amino acids and/or other building blocks are bound to an insoluble material (e.g. a bead) and synthesized via a stepwise proce-

Currently, there are numerous building blocks with a variety of orthogonal protecting groups that are available commercially for solid phase synthesis. The most common groups are derived from alpha-amino acids, for example, beta- and gamma-amino acids that have one or two additional methylene groups, respectively, between the carbonyl and the nitrogen of the amino acid [98-100]. Additional common alpha-amino acid derivatives are N-substitutions (N-Me and N-alkyl) [101] and alpha-carbon modifications, including replacement of the alpha-carbon atom (mostly by nitrogen), replacement of the alpha-hydrogen atom (often by an alkyl group) and inversion of the alpha-carbon configuration [102, 103]. In addition to alpha-amino acid derivatives, a plethora of non-proteinogenic building blocks are currently available for chemical peptide synthesis, such as aldol building units [104], beta-lactam building blocks [105, 106] and sugar-conjugated amino acids [107, 108]. In this review, we focus on building units derived from alpha-amino acids, which are Nα-(ω-Y-alkyl) building units, where Y is an orthogonally protected thiol, amino, or carbonyl group, which in many cases are analogs of the original amino acids at the sites chosen for cyclization (Fig. 4). The diversity of building blocks allows for the development of a variety of backbone-cyclized peptides. There are four main components in each building block that determine its chemical properties. The first element is the amino acid from which the building block is derived (the pharmacophore of the unit), the second element is the functional group that is used to form the bridge, the third element is the protecting group of the functional group, and the final element is the length of the alkyl chain. Nα-(ω-functionalized alkylated) building unit derivatives of natural amino acids are used as building blocks for the synthesis of peptidomimetics, such as backbone-cyclized peptides [50], peptoids [94], pseudo peptides [109], and Peptide Nucleic Acids (PNAs) [110] (Fig. 4). 3.1. Nα-(ω-thioalkylene) Building Units Nα-(ω-thioalkylene) building units can be incorporated into peptides for targeting parent protein sequences. Several methods for the synthesis of Nα-(ω-thioalkylene) building


Rubin and Qvit Amino acid

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5. Cyclization, cleavage and deprotection of the semipermanent protecting group

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NH

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= Polymer bead TPG = Temporary protecting group SPG = Semipermanent protecting group X = NH or O

Fig. (3). The chemical synthesis of peptides and peptidomimetics using solid phase synthesis.


Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7 SPG

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Y (CH2)n R

TPG

N

COOH

R = Pharmacophore of the unit Y = Functional group that is used to form the bridge SPG = Protecting group of the functional group (Semipermanent protecting group) n = Length of the alkyl chain TPG = Temporary protecting group

Fig. (4). General structure of Nα-(ω-Y-alkyl) building units. O

O N

(CH2)n

Br

Bzl-S Na+

N

(CH2)n

S

Bzl

N2H4, EtOH

(CH2)n

H 2N

Bzl Bzl COOCH3

DCM

S

Bzl

+ F3C

S

O

R

O

1N NaOH

H

S (CH2)n R

S Fmoc-OSu

(CH2)n R

Fmoc


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(CH2)n

Bzl

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O

H 2N

S

1N HCl

NMP

N

COOH H

COOH

HN

H

(Boc)2O

Bzl

Boc

S (CH2)n R N

COOH H

Fig. (5). Synthesis of Nα-(ω-thioalkylene) building units by nucleophilic substitution [111].

units have been demonstrated previously for generating backbone-cyclized peptides. Bitan and Gilon synthesized Nα(ω-thioalkylene) building units by alkylation of ω thioalkylamines with trifluoromethanesulfonate (triflate) of alpha-hydroxy acids. The thiol building units were prepared from ω -bromoalkylphtalimides by nucleophilic substitution with benzyl mercaptan in the presence of sodium hydride to form ω-benzylthioalkylamines, which were then reacted with D-alpha-hydroxy acid triflate to form the Nα-(ωthioalkylene) building units. The benzyl protecting group was used as an orthogonal S-protecting group to facilitate the synthesis and incorporation of the building unit into peptides using the solid phase synthesis methodology (Fig. 5) [111].

Since complicated syntheses involve many steps, costly D-amino acids and double alkylation side products often limit the use of the aforementioned technique. Another approach by the same group took advantage of the reductive alkylation method developed earlier by Fehrentz and Castro [112]. (ω-Benzylthio) aldehydes were prepared from ω halogeno acetals in which the halogen was substituted with toluene-alpha-thiol, and then either (ω-benzylthio) acetals were hydrolysed to give the desired aldehydes, or ( ωbenzylthio) carboxylic acids were converted to N,O-dimethyl hydroxamates and then reduced to aldehydes using lithium aluminum hydride (LiAlH4). Finally, the Nα-(ω-thioalkylene) building units were obtained from alkylation of amino acids with aldehydes (Fig. 6) [113]. Gazal et al. developed general approaches for the synthesis of Nα-(ω-thioalkylene) building units using reductive alkylation. Glycine S-functionalized N-alkylated building units were prepared based on two techniques. In the first method, an Acm (acetamidomethyl) protecting group was introduced by reacting cysteamine hydrochloride with

acetamido methanol, which was next reacted with glyoxylic acid, and an Fmoc (Fluorenylmethyloxycarbonyl) protecting group was introduced using Fmoc-OSu (9-Fluorenylmethyl N-succinimidyl carbonate) (Fig. 7a) [65, 114]. In some cases, an alternative approach was used that requires an additional step, as some of the thioalkylamines are not available commercially. In these cases, nucleophilic substitution of bromo alkylamine hydrobromide with trityl mercaptan generated the ω -protected thiamine. The remainder of the synthesis was as described above (Fig. 7b) [114].

Other sulfur-building units have been prepared using reductive alkylation of the amino acid with ω -protected thio aldehydes. Thiol building units were prepared by nucleophilic substitution of the halogen by trityl mercaptan, which was next condensed with N,O-dimethylhydroxylamine hydrochloride using PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) and then reduced by LiAlH4. Reductive alkylation of the appropriate amino acids, followed by Fmoc introduction, yielded the desired building units (Fig. 8) [114]. 3.1.1. Nα-(ω-Thioalkylene) Building Unit Applications 3.1.1.1. Substance P Substance P is a member of the neurokinin family, a subfamily of the tachykinin peptides that also include neurokinin A and neurokinin B (Fig. 9). Substance P is an elvenamino acid peptide with an amidation at the C-terminus. This neuropeptide is produced from a polyprotein precursor after splicing of the preprotachykinin A gene. It is ubiquitous in the human body, throughout the central and peripheral nervous systems, as well as in the liver, lung, and placenta. Substance P has been associated with transmission of pain stimuli, related to inflammation, hepatitis and cancer, as well as

532 Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7 OEt X

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Rubin and Qvit

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S

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S (CH2)n R COOH

N

Fmoc

H X = Br, Cl

α

Fig. (6). Synthesis of N -(ω-thioalkylene) building units by reductive alkylation [113]. a.


TFA

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NH2

Br

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Acm

CH3CONHCH2OH

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NH2

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C H2

COOH

NH2

S

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H2 C

COOH

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N

Fmoc-OSu, Et3N

Fig. (7). Synthesis of Nα-(ω-thioalkylene) building units by reductive alkylation [65, 114]. Trt

Br

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DMF

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Trt-SH, NaH

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MeO

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Trt

R

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+

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Me

Trt

S

MeOH NaBH3CN

O

Et2O

LiAlH4

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COOH

Trt

S

(CH2)3

HN

DCM

R

(R)

S

(CH2)3

R

Fmoc-Cl, DIEA

N

Fmoc

COOH

(R)

COOH

H

Fig. (8). Synthesis of Nα-(ω-thioalkylene) building units by reductive alkylation [114]. Substance P

H

Neurokinin A

H

Neurokinin B

H

Arg

His

Asp

Pro

Lys

Pro

Lys Thr

Asp

Met

Asp

His

Gln

Gln

Ser

Phe Val

Phe Phe

Phe

Val

Phe Gly

Leu

Met

Met

NH2

Gly

Leu

Gly

Leu Met

NH2

NH2

Fig. (9). Linear structures of Substance P and two other tachykinin peptides.

bacterial and viral infection. Each neurokinin receptor maintains specificity via selective peptides (neurokinin 1 by Substance P, neurokinin 2 by neurokinin A and neurokinin 3 by neurokinin B), although the selectivity of each receptor for the corresponding peptide is not high [115, 116]. Despite studies of substance P dating to the 1930s, only small molecule NK1 antagonists (e.g. aprepitant) with limited efficacy have been approved for treatment of nausea [117]. There have been many efforts to identify alternative modes of drugging substance P and/or its receptor, NK1, which have yet to reconcile in vitro activity with clinical efficacy [118].

Bitan et al. developed a biased library containing backbone bicyclic peptide analogs of Substance P. Several backbone-cyclized analogs with the Nα-(ω-thioalkylene) building unit demonstrated relevant bioactivity (EC50 20 nM towards neurokinin 1) and specificity (they were not active for neurokinin 2 and neurokinin 3 receptors) (Fig. 10) [119]. In a follow-up study, Bitan et al. prepared a biased library of bicyclic peptides in which the backbone peptides were cyclized on resin using Nα-(ω-thioalkylene) building units to produce lactam and disulfide rings. The activities of the peptides were about three orders of magnitude lower than the



native agonist (EC50 in the low micromolar range), probably due to over-rigidification from bicyclization (Fig. 11) [120]. O HN

CH2

Arg

Phe

Phe

N

Leu

Gly

N

X

Hcy

(CH2)p

NH2

S

HN (CH2)n

(CH2)m Arg

S

Trp

S

Lys Ser

Thr

Phe

Thr

Gazal et al. synthesized a library of somatostatin analogs that all share a common core sequence. Diversity in the library was derived from the mode of cyclization, either from the N- or C- terminus to an Nα-(ω-thioalkylene) building unit or from one building unit to another building unit. Some of the analogs had high binding affinity to hsst2 and hsst5 in the low nanomolar range and demonstrated significant metabolic stability compared to somatostatin (Fig. 14) [65, 114]. 3.1.1.3. Insulin-like Growth Factor I Receptor

Phe

Phe

N

CH2

Leu

Xaa

O

Tos

Phe Phe

Insulin-like growth factor-I (IGF-I) receptor (IGF-IR) is a cell surface receptor tyrosine kinase activated by insulin-like growth factor-I and insulin-like growth factor-II endocrine hormones. IGF-IR is key for regulation of cell growth and metabolism, and its activation is critical in various cancerrelated signaling pathways. Moreover, overexpression of IGF-IR is related to the development of resistance to anticancer therapeutics, and in some cases, it can be used as a biomarker for cancer. IGF-I, also somatomedin C, is a 70amino acid single chain polypeptide with three disulfide bridges, which is produced mainly by the liver and also in some other target tissues. IGF-I plays a significant role in human physiology and development [124]. Mecasermin (Increlex) is a synthetic analog of insulin-like growth factor-I, which is used for the treatment of growth failure in children with IGF-I deficiency [125].


HN

Asn

Fig. (12). Structure of somatostatin.

S

O

(CH2)i

O

Cys Lys

Cys (CH2)2

Fig. (10). Structure of the backbone cyclic analog I of Substance P [119]. O

Gly

S

(CH2)3 Ac

Ala

533

X = CH or N i = 3-4 m = 3-4 n = 1-2 p = 1-3 Xaa = Cys, HCys, Gly(S2)

Fig. (11). Structure of the backbone bicyclic analog of Substance P [120].

3.1.1.2. Somatostatin

Somatostatin, also Growth Hormone-Inhibiting Hormone (GHIH) or Somatotropin Release-Inhibiting Factor (SRIF), is an endogenous cyclic tetradecapeptide hormone (Fig. 12), which also has an alternative active form of 28 amino acids (the short form with another 14 amino acids at one end). The molecule is produced by many tissues, and it regulates a wide variety of physiological processes by inhibiting the secretion of other hormones, such as growth hormone, thyroid stimulating hormone, cholecystokinin, and insulin. Somatostatin regulates the endocrine system via interactions with five different ubiquitously distributed G-proteincoupled seven transmembrane receptors for somatostatin (sst1-sst5). Although somatostatin exerts multiple biological activities and has broad inhibitory effects (anti-secretory, anti-proliferative and anti-angiogenic), its clinical use is limited by a broad spectrum of off-target biological responses and a short half-life (1-3 min). In the 1980s, octreotide (a cyclic octapeptide somatostatin analog, brand name Sandostatin) was introduced to the clinic. Several additional cyclic peptides soon followed, including pasireotide (Signifor), lanreotide (Lanreotide) and vapreotide (Sanvar), all of which demonstrated longer half-lives and increased pharmacologic efficacy (Fig. 13). The majority of these analogs bind strongly to two out of the five human somatostatin receptors (hsst2 and hsst5), and have a moderate or low affinity for the other receptors (hsst3 and hsst1, respectively). The affinity for hsst4 varies between the different analogs, and the compounds are used to treat patients with neuroendocrine tumors and pituitary adenomas [74, 121-123].

Qvit et al. developed a 34-member library of macrocyclic peptides derived from the sequence of the IGF-IR activation loop. Synthesis was performed either using Nα-(ωthioalkylene) building units and commercial amino acids or using functionalized aldehydes. The library was based on tyrosine residues of the IGF-IR activation loop. The best compound inhibited the activity of IGF-IR (IC50 6 μM), while no inhibitory effect was detected on the closely related tyrosine kinase epidermal growth factor receptor (EGFR) (Fig. 15). These macrocyclic molecules have properties of peptides, such as modularity, flexibility, and stability, and also possess drug-like properties of small molecules that comply with Veber [126] and Lipinski [127] rules. The novel approach presented in this study for the conversion of active protein sequences into small macrocyclic peptides was another important step towards incorporation of peptides and peptidomimetics into the drug discovery toolkit [23, 78]. 3.2. Nα-(ω-Aminoalkylene) Building Units Nα-(ω-aminoalkylene) building units are the most common building units for backbone cyclization and have been used in many applications, including substance P, somatostatin and protein kinases. An early synthesis of Nα-(ωaminoalkylene) building units was demonstrated by Byk and Gilon, for alkylation of alkylenediaminies with alpha-


Rubin and Qvit

Octerotide

Pasireotide

O

H 2N OH HO

OH

OH

N S

S

N

OH

N

H

OH

N

OH HN

OH

N

OH HO

H

H 2N

N

N

N

N

OH

H

O

N

OH HO OH

N H

H N

N

N

O

H

NH2

OH

H

OH HN


NH2

Vapreotide

Lanreotide

NH2

NH

H

OH

H

N

HO

HO

N

N

N

HO

NH2

OH H

HO

HO

H

HO

HO

H

N

S

N

N

S

H

H

HO OH

N

S

NH

HO

N

N

OH

HO

OH

HO

N

N

NH

N

H

NH2

OH

OH

NH

NH2

Fig. (13). Structures of octreotide, pasireotide, vapreotide, and lanreotide. S

S

(H2C)2 H

AA

N

(CH2)2 Phe

CH2

Trp

Lys

(D)Trp

Thr Phe N

O AA = 0, (D)Phe, (D)Nal

Fig. (14). Structures of the backbone cyclic analogs of somatostatin [65, 114]. S (CH2)5 O

N

S (CH2)4 O N NH2

O O

OH

Fig. (15). Structure of the backbone cyclic analog of IGF-IR [23, 78].

O CH2

C

OH

N

S

NH2

NH



halogeno acids. Nucleophilic substitution of alkylene diamines was accomplished by reacting the appropriate alkyelene diamine with unsubstituted alpha-halogeno carboxylic acids, producing Nα-(ω-aminoalkylene) building units. Protection of the amine groups by the orthogonal protecting groups Fmoc or Boc (tert-butyloxycarbonyl) was performed with an intermediate step including a third orthogonal protecting group (benzyloxycarbonyl, or carboxybenzyl/Cbz/Z) (Fig. 16) [128].

units; after removal of the benzyl protecting group, the alpha-amine was protected by Fmoc (Fig. 17) [130]. Bitan et al. developed another approach for the synthesis of Nα-(ω-aminoalkylene) building units. Instead of using nucleophilic substitution of ω -substituted amines on halogeno acids or their esters, the authors used reductive alkylation with aldehydes in the presence of reducing agent, which was simpler and economical. The reaction of amino acids with the appropriate aldehydes was performed based on the procedure of Ohfune et al. [131] with some minor modifications. Different amino acids were N-alkylated with ω-Boc-NH-aldehydes to produce Nα-(ω-aminoalkylene) building units with a variety of functional groups and alkyl chains. Since the introduction of the Fmoc protecting group for protection of the secondary alpha-amino group was challenging due to solubility issues, an alternative approach, in which the trimethylsilyl group served as a temporary protecting group using N,O-bis(trimethylsilyl)acetamide (BTSA) as the silylating agent, was used to introduce the Fmoc protecting group (Fig. 18) [113].


Muller et al. improved the synthesis of N α-(ωaminoalkylene) building units presented by Byk et al. [128] by replacing the poor chloride and bromide leaving groups. One option to overcome this limitation is to replace the halogen with a triflate leaving group, which results in a reduction of synthesis time as well as increase in yield and purity [129]. The first part of the synthesis included protection of the nucleophilic amine with a Boc protecting group on one amine and a temporary protecting group (benzyl) on the other amine, which made purification of the product more feasible. A benzyl ester of the alpha-hydroxy carboxylic acid ester triflate was used for alkylation of the protected ω alkylamines to produce Nα-(ω-(Boc-amino)alkyl) building

Gellerman et al. developed a general approach for the synthesis of a variety of Nα-(ω-aminoalkylene) building NH2

R

H2N

NH2 +

(CH2)n

(CH2)n

COOH

X

Z

(CH2)n HN

R

HN

X = Br, Cl R = H, Me, i-Bu n = 2, 3, 6

NH2

Z-O(p-NO2)Ph

R

COOH

COOH

NH

(CH2)n

HN

R

COOH

R = H, i-Bu n = 2, 3, 6

Z

NH (CH2)n

Fmoc-OSu

Fmoc

Z (Boc)2O

Boc H2/Pd/C

R COOH

Fmoc

NH (CH2)n

Fmoc H2/Pd/C

R

α

N

COOH

Boc

Fig. (16). Synthesis of N -(ω-aminoalkylene) building units by nucleophilic substitution [128].

R COOH

N

NH (CH2)n

Fmoc-OSu Boc

NH (CH2)n

(Boc)2O

N

535

N

R COOH

536 Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7 (CH2)n

NH2

(Boc)2O

NH2

Boc

Rubin and Qvit

NH (CH2)n

NH2

Ph-CHO NaBH4

Boc

NH (CH2)n

N H

Bzl

R Boc

NH (CH2)n

Boc

N H

Bzl

X

+

COOBzl

Boc

NH

NH

H2/Pd/C (CH2)n Bzl

N

(CH2)n

Fmoc-OSu

R

Fmoc

COOBzl

R

N

COOH


X = Br, OTf R = H, Me, Bzl n = 2-6

Fig. (17). Synthesis of Nα-(ω-aminoalkylene) building units [130]. Boc

(CH2)n-1 O H

Boc

R

NH

+

H2N

NH

Boc

NH

BTSA

COOH

NaBH3CN

n(H2C)

n(H2C)

Fmoc-Cl

R

R

MeOH

HN

COOH

Fmoc

N

COOH

BTSA = N,O-bis(trimethylsilyl)acetamide

Fig. (18). Synthesis of Nα-(ω-aminoalkylene) building units by reductive alkylation [113].

units. Alloc (allyloxycarbonyl) bromo alkyl amines were obtained by reacting bromo alkyl amines with N-(4bromobutyl)-phthalimide (for n=4) or with Alloc chloride (for n=2 or 3). Next, N-alkylation of tert-butyl amino acid esters with the suitable bromo alkyl group produced Nalkylated amino acid tert-butyl esters, which were converted to the Fmoc derivative using Fmoc-Cl, and after tert-butyl removal, yielded the final building block (Fig. 19) [132].

Barda et al. developed a novel procedure for the synthesis of Nα-(ω-aminoalkylene) glycine building units using the two aforementioned methods. However, in this case they used an Alloc protecting group instead of Acm [114]. This novel procedure is an improvement on previous methods [132], allowing the use of building units with a variety of alkyl chain lengths (Fig. 20) [66]. Hurevich et al. presented an improved technique for the large-scale synthesis of Nα-(ω-aminoalkylene) glycine building units. A gram-scale synthesis was based on mono alkylation of diamines of various lengths, followed by reductive alkylation in the presence of glyoxylic acid and lastly protection with the orthogonal Fmoc protecting group (Fig. 21) [68]. 3.2.1. Nα-(ω-Aminoalkylene) Building Unit Applications 3.2.1.1. Substance P

As described above, Substance P is a neuropeptide with pleiotropic functions and has been targeted using approaches

to incorporate several different building blocks. Bitan et al. developed a library of backbone-cyclized peptides and performed Structure-Activity Relationship (SAR) studies. In early systematic studies where each peptide bond at the Cterminus was replaced by an N-methylated peptide bond, the authors identified methylations important for activity and selectivity for a single receptor [133-135]. Bitan et al. developed metabolically stable neurokinin-1-specific activators (e.g. SP6, EC50 20 nM), which completely lacked activity towards off-target receptors (neurokinin-2 and neurokinin-3) (Fig. 22) [71]. In order to gain insight into the relationship between conformation and bioactivity the authors solved the Nuclear Magnetic Resonance (NMR) structures of two backbone-cyclized analogs (in collaboration with Horst Kessler), which were similar to other bioactive substance P analogs [136]. Based on the same scaffold, Byk et al. developed and characterized a backbone hexapeptide library derived from the C-terminal hexapeptide of substance P. The library was designed using NMR data and molecular modeling of a short, selective peptide analog of neurokinin-1, WS-septide, which was identified previously (Fig. 23) [134, 135]. The authors determined the bioactivity and specificity of the backbone-cyclized peptides for neurokinin-1 receptor, and also identified a correlation between bioactivity and peptide ring size. An analog with a ring size of 20 atoms was the most active and selective backbone-cyclized peptide. Importantly, this analog was also more metabolically stable compared to the linear peptide (Fig. 24) [72].


Br

(CH2)n


n = 2,3

Br

Alloc-Cl, TEA

NH3Br-

Br

DCM

(CH2)n

NH

Alloc

O

(CH2)4

HBr Alloc-Cl

N

Br

(CH2)4

NH

Alloc

O

Boc

R Br

(CH2)n

NH

Alloc

+

CH3CN, Na2CO3 CH2Cl2

COOBut

H2N

NH (CH2)n

R COOBut

HN

Boc

NH

NH


Boc Fmoc-Cl, NaHCO3 CH2Cl2

(CH2)n

Fmoc

TFA, CH2Cl2

R

(CH2)n

Fmoc

COOBut

N

R

N

COOH

n = 2-4

Fig. (19). Synthesis of Nα-(ω-aminoalkylene) building units [132].

Alloc

NH2

H 2N

(CH2)6

NH2

(Boc)2O

Boc

Alloc

TFA

NH

(CH2)6

Allylchloroformate

(CH2)6

Alloc

Glyoxylic acid

Fmoc-OSu Et3N

NaCNBH3

HN

NH

Alloc

NH

(CH2)6

NH2

(CH2)6

Boc

NH

NH

CH2

Fmoc

COOH

NH (CH2)6 N

CH2

COOH


Alloc

Allylchloroformate

H 2N

(CH2)n

NH2

NH

(CH2)n NH2

Alloc Glyoxylic acid

Alloc

NH (CH2)n

Fmoc-OSu

HN

CH2

n = 2-4, 6


(CH2)n

Et3N

NaCNBH3 COOH

NH

Fmoc

N

CH2

COOH

537


Rubin and Qvit

NH

O (CH2)3 O

(CH2)4 Arg

Phe

Phe

N

O

CH2

Leu

Met

NH2

Fig. (22). Structure of the backbone cyclic analog SP6 of Substance P [71].

Ac

Arg

Phe

Phe

Pro

Met

NH2

Fig. (23). Linear structure of the WS-septide[134, 135]. NH

O (CH2)m O

(CH2)n Arg

Phe

Phe

N

CH2

O Leu

Met

NH2

n = 2, 3, 6 m = 2-4

In addition to facilitating cellular import through the PTD, the TAT protein contains a Nuclear Localization Signal (NLS) with an Arginine-Rich Motif (ARM) that mediates nuclear localization within the cell. Peptide mimicry of this sequence can either facilitate or inhibit nuclear import depending on the context, and several groups have applied backbone cyclization to develop enhanced analogs. Friedler et al. developed a backbone-cyclized peptide library using the Nα-(ω-aminoalkylene) building unit, which mimics the Arginine-Rich Motif (ARM) of the HIV-1 TAT protein, based on the NMR structure of TAT; the molecules were screened for their ability to mediate nuclear import of corresponding bovine serum albumin conjugates in cells. The authors identified one backbone-cyclized peptide, TAT11, with active nuclear localization signal properties. They also studied the mechanism of nuclear import, concluding that the peptide used the same pathway as the native TAT NLS. The backbone-cyclized peptide inhibited nuclear import mediated by the TAT NLS in a selective manner, and also inhibited the binding of the HIV-1 Rev arginine-rich motif to its RNA binding element (Rev response element) (Ki = 5 nM) (Fig. 27) [148].


Fig. (24). Structure of the backbone cyclic analog of Substance P [72].

linear sequence has been used as a CPP to deliver small molecules, peptides and proteins into cells. For example, the fusion of the TAT PTD to the 120 kDa beta-galactosidase protein resulted in delivery of the functional protein to all tissues in mice, including the brain [142]. Although it is clear that TAT allows these molecules to cross the cell membrane, there is significant debate in the field over the mechanism [143]. Nevertheless, CPPs have demonstrated efficacy and safety in clinical trials for diagnostic (e.g. contrast agent delivery) and therapeutic (e.g. drug delivery) indications [144]. These molecules are effective vehicles for the transport of diverse cargos into cells, yet their use is limited by the inherent lack of target cell specificity. Thus, it is important to consider the potential toxicity of any CPPconjugated payload; several enhanced CPP systems are being developed to address this issue [145], and there are also other novel approaches for delivery cargo into cells [146, 147].

3.2.1.2. Bovine Pancreatic Trypsin Inhibitor (BPTI)

Bovine pancreatic trypsin inhibitor (BPTI) is a small protein (58 residues) that was approved as an antifibrinolytic drug under the trade name Trasylol. However, the drug was withdrawn from the market in 2007 due to risk of complications or death [137]; BPTI has since become available for limited use. As a result, there was an opportunity to develop smaller peptide compounds with drug activity and increased safety. The BPTI protein inhibits serine proteases, such as trypsin, kallikrein, chymotrypsin, and plasmin, and is often sourced from bovines [138]. A short domain creates a looplike structure stabilized by a disulfide bond, which is critical for binding of bovine pancreatic trypsin inhibitor to trypsin [139]. Equipped with this information, Kasher et al. developed a backbone bicyclic nonapeptide antagonist. The authors implemented a new thioproline building unit in addition to the Nα-(ω-aminoalkylene) building unit. The resultant peptide contains cis-thioproline and acts as an inhibitor of trypsin (Ki ~ 76 µM) (Fig. 25). Compared to the natural protein, bovine pancreatic trypsin inhibitor (Ki ~ 10−13 M) [140], inhibition by this peptidomimetic is modest. Differences in the biological activity may be due to the replacement and omission of several residues, as well as conformational restriction [80]. In a follow-up study, Kasher et al. prepared a backbone-cyclized peptide library based on the same short domain that creates a loop-like structure. Although the bioactivity of the backbone-cyclized peptides in this library did not improve significantly, the authors suggested using the structure of the peptides that they solved to further develop more bioactive compounds (Fig. 26) [141]. 3.2.1.3. Human Immunodeficiency Virus (HIV)

In another study based on the NMR structure of HIV-1 Matrix Protein (MA) (Fig. 28), Friedler et al. developed a library of backbone-cyclized peptides using the Nα-(ωaminoalkylene) building unit for cyclization. The authors screened for bioactivity by inhibition of nuclear import by an NLS on bovine serum albumin in cells. In the initial screen, they identified a lead compound (IC50 = 3 µM). Based on the lead compound, they developed a second library that led to the discovery of a very potent backbone-cyclized peptide, BCvir (IC50 = 35 nM, compared to the linear parent HIV-1 MA NLS peptide IC50 = 12 µM). In addition, the novel peptide reduced HIV-1 production by 75% in cultured human cells and was highly stable (Fig. 29) [149].

Human immunodeficiency virus (HIV) has been the target of numerous therapeutic programs making use of backbone-cyclized peptides, and its study also led to the identification of Cell-Penetrating Peptides (CPPs). The HIV transActivating Transcriptional Activator (TAT) can be taken up from media into cells by its Protein Transduction Domain (PTD), which led to development of many CPPs [136]. The

Hariton-Gazal et al. constructed a library of backbonecyclized peptides with Nα-(ω-aminoalkylene) building units, based on the previously identified peptide BCvir, derived from the NLS of HIV-1 MA [149]. In this library, the scaffold was constant, but the amino acid residue at the Cterminus was different in each backbone-cyclized peptide. The authors found that while the C-terminus was occupied



539

H O

N

O H NH

S

(CH2)4 O

(CH2)2

S CH2

Pro

Cys

O Lys

Ala

Arg

N

CH2

Ile

Gly

NH2

Fig. (25). Structure of the backbone cyclic analog of BPTI [80]. HN

Cys

O


CH2 (CH2)m

S

(CH2)n

S

O

CH2

Pro

Cys

O

Lys

Ala

Arg

N

CH2

Ile

Gly

NH2

n = 2-5 m = 1-5

Fig. (26). Structure of the backbone cyclic analog of BPTI [141].

NH

O

(CH2)4

O

(CH2)4

Arg

Lys

Lys

Arg

Arg

N

O

CH2

Arg

Arg

Arg

Cys

NH2

Fig. (27). Structure of the backbone cyclic analog of the ARM in the HIV-1 TAT [148].

Val

O

NH

(CH2)4

O

(CH2)6

Lys

Lys

Lys

N

CH2

O Lys Leu

NH2

Fig. (29). Structure of the backbone cyclic analog of the HIV-1 MA, BCvir [149].

with bulky hydrophobic amino acids, such as Phe and Nal (naphthyl Ala), the backbone-cyclized peptides were highly bioactive, inhibiting nuclear import in vitro (inhibition of 70% and 90%, respectively); polar amino acid residues, such as Asn and Cys, did not exhibit an inhibitory effect (Fig. 30) [150].

Fig. (28). NMR structure of the HIV-1 matrix protein (MA). The protein backbone is shown in black (PDB: 1TAM) [212]. Peptides were derived from the domain shown in red, amino acid sequence: H-Gly-Lys-Lys-Lys-Tyr-Lys Leu-Lys-His-Ile-NH2. [213, 149]. (The color version of the figure is available in the electronic copy of the article).

Rev is an attractive drug target because it provides an essential signal for HIV-1 mRNAs to be exported from the nucleus for translation. Chaloin et al. developed a library of backbone-cyclized peptides using Nα-(ω-aminoalkylene) building units and simultaneous multi-peptide solid phase synthesis. The authors evaluated in vitro inhibition of HIV-1 replication in chronically infected T lymphocytes. The backbone-cyclized peptide Rev-BCP 14 bound Rev responsive

540 Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7 Val

O

NH

(CH2)4 O

(CH2)6 Lys

Lys

Rubin and Qvit

Lys

N

O

CH2

Lys

AA

Cys

NH2

AA= Tyr, Ser, Gly, Ala, (D)Leu, Phe, Val, Lys, Asn, Nal (naphthyl alanine), MeLeu, No AA, Lys5Ψ(CH2-NH)Leu6 (a reduced bond between Lys5 and Leu6)

Fig. (30). Structure of additional backbone cyclic analogs of the HIV-1 MA [150].

element and distorted the major groove of viral RNA in molecular dynamics simulations (IC50 = 6 µM). Further, the compound interfered with the synthesis of gag and env proteins, likely by preventing intracellular accumulation of unspliced viral RNA (Fig. 31) [151].

3.2.1.5. Alpha-melanocyte-stimulating Hormone (α-MSH)


HIV-1 Integrase (IN) is another drug target because it mediates integration of retroviral DNA into the host genome. Hayouka et al. developed a backbone-cyclized library using Nα-(ω-aminoalkylene) building units that mimic the HIV-1 IN binding loop which interacts with its cellular cofactor, Lens Epithelium-Derived Growth Factor (LEDGF/p75). Biological screening identified one backbone-cyclized peptide, c(MZ 4-1), as a potent and stable inhibitor of HIV-1 IN activity. c(MZ 4-1) binds (low micromolar range) and inhibits integrase catalytic activity by 90% (at 100 nM) in vitro, and also inhibits HIV-1 replication in cells (Fig. 32) [79]. In a follow-up study, Hayouka and colleagues evaluated the effect of backbone cyclization combining N-alkylation with side chain cyclization, for which the building units are commercially available and therefore easier to implement in synthesis. Using a similar biological assay, they identified inhibitors of integrase and found that backbone-cyclized peptides were generally more stable due to the effects of Nalkylation. However, the most potent side chain cyclic peptide was almost as potent as some backbone-cyclized peptides and could serve as an alternative to backbone cyclization when synthesis is prohibitive (Fig. 33) [152].

based on the sequence of the lead Arg-Tyr-Phe-(D)Phe-ProArg-Leu-NH2 antagonist identified previously [154]. Out of the two libraries, four backbone-cyclized peptides (all from the second library with (D)Phe) exhibited antagonist activity and completely inhibited the sex pheromone biosynthesis at 1 nmol. Interestingly, substitution of (D)Phe with Ser in these analogs completely abolished their bioactivity (Fig. 34) [63, 75].

3.2.1.4. Pheromone Biosynthesis Activating Neuropeptide (PBAN)

Pheromone Biosynthesis Activating Neuropeptide (PBAN) is a 33-amino acid neuropeptide member of the pyrokinin/pheromone biosynthesis activating neuropeptide family, which activates the biosynthesis of various pheromones in moths. Members of this family play a key role in a variety of physiological and behavioral functions, including contraction of the locust hindgut, egg diapause in the larvae and stimulation of sex pheromone biosynthesis in female moths. Female moths release PBAN into their hemolymph during the scotophase to stimulate the biosynthesis of neuropeptides for attracting conspecific males [153].

Structure activity relationship studies revealed that the Cterminal of PBAN is conserved in numerous members of the pyrokinin/pheromone biosynthesis activating neuropeptide family and may be important for its bioactivity. Two backbone-cyclized peptide libraries using Nα-(ω-aminoalkylene) building units were developed to identify antagonists capable of inhibiting sex pheromone biosynthesis in female Heliothis peltigera moths. The first library with Ser amino acids was designed based on the C-terminal hexapeptide sequence TyrPhe-Ser-Pro-Arg-Leu-NH2, while the second library was

Alpha-melanocyte-stimulating hormone (α-MSH, or melanotropin) is an endogenous tridecapeptide hormone and member of the melanocortin family (Fig. 35). α-MSH is involved in feeding behavior and melanogenesis, which is responsible for pigmentation of the hair and skin through a process mediated by melanocortin receptor MC1. The synthetic linear α-MSH analog afamelanotide was recently approved in Europe and is currently under review by the FDA for use in preventing skin damage from sun exposure [155]. Hess et al. developed a library of backbone-cyclized peptides using Nα-(ω-aminoalkylene) building units, derived from the tetrapeptide sequence His-Phe-Arg-Trp shown to be critical for α -MSH bioactivity. Peptide 1 was selective for the melanocortin-4 receptor, highly stable and demonstrated transcellular penetration in enterocytes. The peptide was found in the brain following oral administration to rats, and reduced food consumption by approximately 50% when administered to mice (0.5 mg/kg) (Fig. 36) [156]. Linde et al. used a similar approach to mimic alphamelanocyte-stimulating hormone, in which they developed a backbone-cyclized peptide library and N-methylated backbone cyclic libraries using Nα-(ω-aminoalkylene) building units derived from the pentapeptide sequence Phe-(D)PheArg-Trp-Gly. However, all backbone-cyclized peptides had a similar or lower bioactivity compared to the lead compound, which was identified in the previous study by Hess et al. [156]. Moreover, all the N-methylated backbone-cyclized peptides in this study had significantly reduced biological activity compared to the most active backbone-cyclized peptide. The authors suggest that the reduced bioactivity was due to departure from the active conformation and an increase in rigidity of the backbone-cyclized peptides (Fig. 37) [92]. A follow-up study by Ovadia et al. illustrated an interesting approach with hybrid compounds composed of peptides and peptoids, the latter of which are constructed from amino acids where the side chain of the alpha-carbon is moved to the nitrogen. Based on the same Phe-(D)Phe-Arg-Trp-Gly pentapeptide sequence derived from α -MSH, the authors developed a backbone-cyclized peptide-peptoid library using



NH

O (CH2)m O

(CH2)n Arg

Gln

Ala

Arg

Arg

N

O

CH2

Arg

Arg

Arg

Cys

NH2

n = 2-4, 6 m = 2-5 Fig. (31). Structure of the backbone cyclic analog of Rev, Rev-BCP 14 [151]. NH

O Fluorescein

NH CH2

(CH2)4

O

NH

Trp

Asn

Ser

Leu

Lys

Ile

Asp

Asn

Leu

Asp

Val

N

O

CH2

NH2


Fig. (32). Structure of the backbone cyclic analog of the HIV-1 IN, c(MZ 4-1) [79]. c(MZ 4K-1)

NH

O

H2N

CH2

O

NH

Trp

Asn

Ser

Leu

Lys

Ile

Asp

Asn

Leu

Asp

Val

NH

(CH2)4

O

CH

NH2

c(MZ 4-1)

NH

O H2N

CH2

O

(CH2)4

Trp

NH

Asn

Ser

Leu

Lys

Ile

Asp

Asn

Leu

Asp

Val

N

O NH2

CH2

Fig. (33). Structure of additional backbone cyclic analogs of the HIV-1 IN [152].

NH

O

(CH2)m

O

(CH2)n

Arg

Tyr

Phe

(D)Phe

N

O

CH2

Arg

Leu

NH2

n = 2-4, 6 m = 2-3 Fig. (34). Structure of the backbone cyclic analog of PBAN [63, 75]. Ac

Ser

Tyr

Ser

Met

Glu

His

Phe

Arg

Trp

Gly

Lys

Pro

Fig. (35). Linear sequence of α-MSH. NH

O (CH2)2 O

(CH2)2 Phe

(D)Phe

Fig. (36). Structure of the backbone cyclic analog of α-MSH [156].

Arg

Trp

N

CH2

O NH2

Val

NH2

541


Rubin and Qvit

O

achieving approximately 90% inhibition of IκB ubiquitylation at a concentration of 3 µM (Fig. 39) [76, 77]. (CH2)m

O

(CH2)n (D)Phe

Phe

Arg

Trp

N

O

CH2

NH

O

NH2

(CH2)2 O

n = 2-4 m = 2-5

Fig. (37). Structure of another backbone cyclic analog of α-MSH [92].

NH

O (CH2)m O

O

Phe

N CH2

n=2 m = 2-4

Asp

Ser

Gly

Phe

CH2

(CH2)n

Arg

Trp

N

CH2

O

NH2

N

CH2

NH2

Protein kinase B (PKB, or Akt) is a serine/threonine kinase member of the AGC subfamily that has three mammalian isoforms, PKBα, PKBβ and PKBγ, which all have a conserved structure. PKB regulates cellular metabolism, survival, motility, transcription, and cell cycle progression by binding and phosphorylating various substrates. Moreover, PKB signaling is dysregulated in several human diseases, such as cancer, diabetes and schizophrenia [158]. Phospholipid and small molecule inhibitors of PKB have been developed for treatment of cancer but demonstrated no success in clinical trials thus far; only small molecule inhibitors of the broader phosphoinositide 3-kinase (PI3K)/Akt pathway have been approved [158].

Hurevich et al. synthesized a library of urea backbonecyclized peptides using Nα-(ω-aminoalkylene) building units, creating analogs of glycogen synthase kinase 3 to target PKB as lead compounds for treating cancer. Several peptides inhibited PKB activity, one of which was particularly active (c(YM3-6) IC50 = 0.16 µM) (Fig. 40) [68]. O

HN

(CH2)m

3.2.1.6. Nuclear Factor-kappa B (NF-κB)

Qvit et al. developed an approach for the synthesis of backbone-cyclized phosphorylated peptides derived from the conserved phosphorylated sequence of inhibitor kappa B, DS(PO3)GXXS(PO3) where X is any amino acid, which binds β -TrCP. These novel backbone-cyclized peptides prevented the release of NF-κB and its translocation to the nucleus, which could be used to reduce its negative effects in disease. Several backbone-cyclized phosphopeptides with Nα-(ω-aminoalkylene) building units demonstrated high bioactivity in vitro, with the best compound, IκB 31-37 (pBC-2,3),

pSer

3.2.1.7. Protein kinase B (PKB, or Akt)

Fig. (38). Structure of an additional backbone cyclic analog of α MSH [94].

Nuclear factor-kappa B (NF-κB) is a protein complex transcription factor that controls cytokine production and cell survival through regulation of DNA transcription, and is present in almost all cell types. Over-activation of NF-κB is related to inflammatory diseases, infection and cancer. The protein is involved in many signal transduction cascades and plays a pivotal role in the ubiquitin-proteasome system. NFκB forms a complex with Inhibitor kappa B (IκB), which binds the beta-transduction repeat-containing protein (βTrCP), enabling NF-κB to enter the cell nucleus [157]. Several approved small molecule and monoclonal antibody therapeutics have been shown to modulate the NF-κB signaling cascade, but it is believed that none of these molecules directly interact with NF-κB [157].

Ile

O

Fig. (39). Structure of the backbone cyclic analog of NF-κB, IκB3137(pBC-2,3) [76].


Nα-(ω-aminoalkylene) building units to target the Melanocortin-4 Receptor (MC4R). Two backbone-cyclized peptidepeptoid analogs demonstrated high stability and increased intestinal permeability compared to their linear counterparts, and one backbone cyclic peptide-peptoid analog was highly bioactive (nanomolar range) for MC4R (Fig. 38) [94].

(CH2)3

H

Arg

N

NH

O

CH2

Arg

Nva

(CH2)3 Tyr

Dap

Hol

N

CH2

O NH2

m = 2-4, 6 Nva - Norvaline Dap - 2,3-Diaminopropionic Acid Hol - Homoleucine

Fig. (40). Structure of the backbone cyclic analog of PKB, c(YM36) [68].

Tal-Gan et al. synthesized two backbone-cyclized peptide libraries using Nα-(ω-aminoalkylene) building units to study the effect of cyclization approach, bridge chemistry, and ring size on the bioactivity of backbone-cyclized peptides. Interestingly, backbone-cyclized peptides in which the cyclization was done by backbone-to-backbone urea bridge were more potent than N-terminus-to-backbone amide peptides. Two analogs, compound 43 and compound 46, were ten-fold more active than the parent linear peptide (IC50 = 0.16 and 0.17 µM, respectively) (Fig. 41) [81]. 3.2.1.8. Human Leukocyte Antigen Class II Histocompatibility, D Related Beta Chain (HLA-DRB1) Human leukocyte antigen class II Histocompatibility, D Related Beta Chain (HLA-DRB1) is expressed on AntigenPresenting Cells (APCs), and plays a critical role in the immune system by presenting antigenic peptides processed from extracellular proteins to T and B cells. HLA-DRB1



shared epitope alleles have been associated with an increased incidence of Rheumatoid Arthritis (RA), which is an autoimmune inflammatory disease that primarily affects joints, causing pain, swelling, stiffness, and loss of function. The cause of RA is not known, but likely involves a combination of genetic and environmental factors. Treatment generally includes lifestyle changes, pharmacological immunosuppression, and/or surgery to slow joint damage and reduce pain, but there is no curative regimen [159]. O HN

NH

(CH2)m H

Arg

N

O

(CH2)n

CH2

Arg

Nva

Tyr

Dap

Hol

N

O

CH2

NH2

Fig. (41). Structure of an additional backbone cyclic analog of PKB [81].

Many rheumatoid arthritis patients have a five-amino acid shared epitope motif in the DRB1-chain of the HLA protein, which may activate nitric oxide production. Naveh et al. developed a library of backbone-cyclized peptides to stabilize the alpha-helix structure of the DRB1 domain. They synthesized a small library of backbone-cyclized peptides to mimic the shared epitope motif. Although the study identified a stable backbone-cyclized peptide, c(HS4-4), which activated nitric oxide (NO) production in the low nanomolar range, the authors suggest that this lead compound could be useful for development of potent inhibitory antagonists as well (Fig. 42) [41, 160]. O

NH

HN (CH2)n H

Trp

n=2, 4, 6

N

CH2

Hurevich et al. synthesized peptidomimetics using Nα(ω-aminoalkylene) building units and microwave assisted urea backbone cyclization to construct a “Helix-walk” library based on the CCR2 dimerization region sequence. The authors identified potent dimerization sites from the CCR2 G-protein-coupled receptor (GPCR), which they used to synthesize a short linear peptide. Since linear peptides do not form helical structures in solution, they performed backbone cyclization to assemble a cyclic helix mimetic from the short linear peptide. The “Helix-walk” refers to construction of the cyclic library, whereby the dimerization site mimetics were cyclized between ring positions i and i+4, i and i+7, or i and i+3, which allowed for frame shift backbone cyclization scanning. Hurevich et al. used this cycloscan approach to identify the optimal characteristics for the backbone-cyclized peptidomimetic to inhibit binding of CCR2 to its ligand. The stable helix structure of backbone-cyclized peptidomimetics produced library hits such as peptides hCCR2(61-67), which specifically inhibited CCR2-mediated chemotactic migration in cultured monocytes (Fig. 43) [62].


Nva - Norvaline Dap - 2,3-Diaminopropionic Acid Hol - Homoleucine Compound 43 c(YM3-6) m=6, n=3 Compound 46 c(YM4-4) m=4, n=4

O

(CH2)4

Gln

Lys

Arg

N

O

CH2

Ala

NH2

Fig. (42). Structure of the backbone cyclic analog of the HLADRB1-chain, c(HS4-4) [160].

3.2.1.9. Chemokine (C-C motif) Receptor 2 (CCR2) Chemokine (C-C motif) receptor 2 (CCR2) mediates monocyte chemotaxis, in cases of both beneficial (antitumor or antimicrobial) and pathological (autoimmune) inflammatory infiltration. Moreover, dysregulation of CCR2 has been linked to multiple sclerosis and rheumatoid arthritis, amongst other diseases. CCR2 binds via a helical dimerization site to its ligand (CCL2) by helix-helix interactions. An approach to inhibit these potentially pathogenic interactions involved the development of backbone-cyclized peptides, which are an optimal scaffold for helical peptidomimetics [161]. Despite limited historical efficacy of other CCR2 antagonists [162], several small molecules targeting CCR2, such as PF-6309 and CCX872 for pancreatic cancer, have shown promise in recent clinical trials [163].

543

O

HN

(CH2)2

H

Met

Leu

Val

N

CH2

NH O

(CH2)4 Leu

Ile

N

CH2

O NH2

Fig. (43). Structure of a backbone cyclic analog based on the CCR2 dimerization region sequence [62].

3.2.1.10. Bradykinin

Bradykinin is a nonapeptide thought to rely on a turn structure for its biological activity. The peptide induces vasodilation, mediating inflammation through vasculature and likely pain as well. Bradykinin binds B1 receptors (constitutively expressed) and B2 receptors (expressed as a result of tissue injury). Icatibant is a cyclic peptidomimetic bradykinin B2 receptor antagonist, which was approved in 2008 by the European Commission for treatment of Hereditary Angioedema (HAE) in adults with C1-esterase-inhibitor deficiency [164].

Schumann et al. synthesized backbone-cyclized peptide analogs with N-carboxy alkylated, N-amino alkylated amino acids and dipeptide building blocks synthesized in solution beforehand. The authors used these compounds to study the proposed bioactive turn conformation. Indeed, only compound 3, which was backbone-cyclized between positions 2 and 3, exhibited significant agonist activity in a rat uterus assay. This validated the hypothesis that the bradykinin turn structure is important for its biological activity (Fig. 44) [165]. 3.2.1.11. Src Homology Region 2 Domain-containing Phosphatase-1 (SHP-1) SHP-1 is a protein tyrosine phosphatase expressed by hematopoietic cells and is involved in signaling pathways that regulate cellular activation, growth, proliferation, differentiation, cell cycle, and oncogenesis. Intracellular adaptor proteins help recruit SHP-1, and its tandem Src homology (SH2) domains mediate binding to phospho-tyrosines on target proteins; SHP-1 then dephosphorylates transmem-


Rubin and Qvit

brane receptor cytoplasmic domains and removes phosphates from other intracellular kinases. Several small molecule SHP-1 antagonists have been suggested for development as anticancer agents [166, 167]. NH

O (CH2)2

(CH2)3

(N-OC)ΨArg

Phe

Pro

GlyΨ(CO-N)

Phe

Ser

Pro

Phe

Arg

Fig. (44). Structure of the backbone cyclic analog of bradykinin, compound 3 [165].

HN

Gly

(CH2)3

H

Glu

Gly

Nle - norleucine

Gazal et al. prepared modified Gly amino acids using reductive alkylation. Allyl esters of several amino acids were reacted with the glyoxylic acid in the presence of sodium cyanoborohydride (NaBH3CN) and protected with Fmoc via an in situ reaction with Fmoc-Cl, producing modified Gly building units of several residues (Fig. 49) [170]. 3.3.1. Nα-(ω-Carboxyalkylene) Building Unit Applications 3.3.1.1. Somatostatin Somatostatin has been the subject of numerous peptide design studies (see above). Gilon et al. synthesized a backbone-cyclized analog of somatostatin, PTR 3046, using Nα(ω-carboxyalkylene) building units. The compound binds selectively to the seven-transmembrane domain somatostatin receptor 5 (sst5) with an IC50 of 67 nM for sst5 and an IC50 greater than 1000 nM for sst1-4, and is highly resistant to enzymatic degradation. PTR 3046 inhibited bombesin- and caerulein-induced amylase and lipase release from the pancreas, but not growth hormone or glucagon release, in animal studies (Fig. 50) [73].


Zoda et al. synthesized backbone-to-side chain cyclized octapeptides based on the ligand consensus sequence, which binds the N-terminal SH2 domain of SHP-1 and activates the protein. The authors used dipeptide building blocks and Nfunctionalized tyrosine derivatives to overcome challenges associated with condensation of N-protected amino acids. Using these building blocks, they performed structure activity relationship studies, which incorporated ring size, flexibility and hydrophobicity of the bridge. The authors found that backbone-cyclized peptide activity increased (up to 14fold greater than basal activity) with bridge chain length due to greater flexibility and presumably more favorable binding conformations. (Fig. 45) [168].

carboxylates. Next, N-alkylation of tert-butyl amino acid esters was performed by nucleophilic substitution with the appropriate bromo alkyl compounds, and the secondary amine was protected by Fmoc using Fmoc-Cl, followed by removal of tert-butyl (Fig. 48) [132].

Leu

Abu

pTyr

Nle

Asp

Leu

NH2

Fig. (45). Structure of the backbone cyclic analog of SHP-1 [168].

3.3. Nα-(ω-Carboxyalkylene) Building Units

Nα-(ω-carboxyalkylene) units are an additional set of building blocks for backbone cyclization. Muller et al. synthesized Nα-(ω-carboxyalkylene) building units using a similar approach to their synthesis of N α-(ω-aminoalkylene) building units. With an orthogonal tert-butyl protecting group and a second nucleophilic amine protected by benzaldehyde via reductive alkylation, the authors built on previous work of Bergeron et al [169]. Next, alpha-hydroxy carboxylic ester triflates were used for alkylation of the Nα-(ω-(tertbutyl-carboxy)alkyl) amino acids. The benzyl protecting group was removed from the alpha-amine, and the alphaamine was protected by Fmoc (Fig. 46) [130].

Another approach for the synthesis of Nα-(ωcarboxyalkylene) building units was performed by initially preparing ω -(tert-butoxycarbonyl) aldehydes. Cyclic anhydride was reacted with tert-butyl alcohol to produce monoprotected diacid, which was converted to N,O-dimethyl hydroxamate and later reduced to an aldehyde. Next, Nα-(ωaminoalkylene) building units (such as alanine, isoleucine and methionine) were N-alkylated with aldehydes to produce Nα-(ω-carboxyalkylene) building units. The last step was introduction of an Fmoc protecting group (Fig. 47) [113]. Gellerman et al. used a different approach taking advantage of the reaction between bromo alkyl carboxyl chlorides or bromides and allyl alcohol, which produced allyl bromo

Afargan et al. also developed a stable backbone-cyclized analog of somatostatin, PTR 3173, using Nα-(ωcarboxyalkylene) building units. This molecule bound with high affinity to three somatostatin seven-transmembrane domain receptors (3 nM for hsst2, 7 nM for hsst4 and 6 nM for hsst5) and inhibited accumulation of forskolin-stimulated cAMP (cyclic adenosine monophosphate). In animal studies, PTR 3173 selectively and potently inhibited growth hormone release, by 1000-fold and over 10,000-fold more than glucagon and insulin, respectively (Fig. 51) [91]. Falb et al. developed a bicyclic backbone peptide analog of somatostatin, PTR 3205, with N α-(ω-carboxyalkylene) building units. PTR 3205 was specific and selective for hsst2 (IC50 3.7 nM) and at least two orders of magnitude lower for other somatostatin receptors. The 3D structure of PTR 3205 revealed a similar region to other known somatostatin analogs, such as compound L-363,301 (Merck) and Sandostatin, located in the pharmacophoric region containing the Phe(D)Trp-Lys-Thr motif (Fig. 52) [171].

Backbone metal cyclization is a unique approach for synthesizing backbone-cyclized peptides, which can simultaneously support radiolabeling of peptides for imaging and/or therapeutic purposes [172, 173]. Backbone metal cyclic peptide combinatorial libraries were developed using two hemichelating arms and Nα-(ω-carboxyalkylene) building units to bind the metal and to perform the backbone peptide cyclization. Fridkin et al. synthesized a library of 48 rhenium cyclic somatostatin analogs, in which five demonstrated high bioactivity via in vitro binding to hsst2 with IC50 values between 1 and 3 nM (Fig. 53) [67]. 3.3.1.2. Gonadotropin-releasing Hormone (GnRH) Gonadotropin-Releasing Hormone (GnRH) is a 10-amino acid peptide hormone produced in the hypothalamus, which


Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7 COOBut

COOBut HOOC

(CH2)n

NH

t-Bu-OH, POCl3

Z

Z

COOBut

H2, Pd/C

(CH2)n

Pyridine

(CH2)n NH2

NH

R

COOBut

COOBut H2/Pd/C

Ph-CHO NaBH4

(CH2)n Bzl

+ X

COOBzl

(CH2)n

NH

Bzl

R

(CH2)n

Fmoc-OSu

N

Fmoc

COOBzl

R

N

COOH

R = H; X = Br R = Me, Bzl (benzyl); X = OTf (trifluoromethanesulfonate) n = 1-5

(CH2)n

NH

t-Bu-OH, POCl3

Z

But OOC

Pyridine

(CH2)n

NH

Z


HOOC

H2, Pd/C

But OOC

(CH2)n

Ph-CHO NaBH4

NH2

But OOC

(CH2)n

NH

Bzl

COO But

R

(CH2)n

+

But OOC

(CH2)n

NH

Bzl

COOBzl

X

Bzl

R COOBzl

N

COO But

H2, Pd/C

(CH2)n

Fmoc-OSu

Fmoc

R

N

COOH

R=H; X=Br R= Me, Bzl; X= OTf n=1, 2, 4, 5

Fig. (46). Synthesis of Nα-(ω-carboxyalkylene) building units [130]. O

O

COO But

COO But

ButOH, ZnCl2

(CH2)3

(CH2)3

MeONHMe Cl BOP, TEA

N

OH

O

LiAlH4

O

O

COO But

(CH2)3

Et2O

OCH3

O H

CH3

COO But (CH2)3

COO But R

+

NaBH3CN MeOH

O

H2N

COOH

(CH2)3 HN

H

BTSA = N,O-bis(trimethylsilyl)acetamide

Fig. (47). Synthesis of Nα-(ω-carboxyalkylene) building units [113].

COO But BTSA Fmoc-Cl

R COOH

(CH2)3 Fmoc

N

R COO H

545


Rubin and Qvit O

O Allyl-OH Br

(CH2)n

O

Br

X

(CH2)n

O

Allyl O

X = Br, Cl n = 1, 3-5

Allyl

O

CH3CN, Na2CO3

Br

(CH2)n

O

(CH2)n

R

+

O

H2N

Allyl

HN

COOBu'

O Allyl

Allyl (CH2)n

CH2Cl2 Fmoc

COOBu'

O

O

Fmoc-Cl, NaHCO3

R

O

TFA, CH2Cl2

R

(CH2)n

N

COOBu'

Fmoc

R

N

COOH

α


Fig. (48). Synthesis of N -(ω-carboxyalkylene) building units [132].

O

HO

NaBH3CN

R

O

+

H

CH2

Fmoc-Cl

COOH H2N

COOAl

Fmoc

R

O

N

O

Al

Al = allyl

Fig. (49). Synthesis of Nα-(ω-carboxyalkylene) building units by reductive alkylation [170].

HN

O

(CH2)2

O

HN

(CH2)3

Tyr CH2

(D)Trp

Lys

Val

O

N

Thr

NH2

which was then cyclized by backbone metal cyclization. The compound bound with high affinity to rat pituitary membranes, which express gonadotropin-releasing hormone receptors, with an IC50 in the nanomolar range (similar to native GnRH) (Fig. 54) [66].

CH2

O

GABA

Phe

(CH2)3

H 2N

Fig. (50). Structure of the backbone cyclic analog of somatostatin, PTR 3046 [73].

stimulates the release of Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) from the anterior pituitary. Receptors for GnRH are overexpressed in breast, ovarian and prostate cancers, which suggests its utility as a therapeutic target. Originally, natural GnRH was used for therapeutic purposes, but analogs have since been developed [174]; leuprolide is used to treat cancer, endometriosis and precocious puberty, but often requires continuous infusion due to its short half-life [175]. Barda et al. designed a backbone-metal cyclized 99mTcradiolabeled analog of gonadotropin-releasing hormone to target receptors on malignant cells. They synthesized a precyclic GnRH analog with two hemi-chelator groups,

Gly

Trp (D)Trp

Phe

Thr

Lys

GABA = Gamma-Aminobutyric acid Fig. (51). Structure of another backbone cyclic analog of somatostatin, PTR 3173 [91]. NH

O (CH2)3 HN

O

S Cys

S Phe

(D)Trp

Lys

Thr

Cys

(CH2)3 N

O NH2

Fig. (52). Structure of another backbone cyclic analog of somatostatin, PTR 3205 [171].

Backbone-Cyclized Peptides S

Current Topics in Medicinal Chemistry, 2018, Vol. 18, No. 7 O

S

Re

O

O N H

N H (CH2)m O

(CH2)n Phe

Trp

(D)Trp

Lys

Thr

Phe

N

O

CH2

NH2

m = 0-3 n = 2,3 or 6

Fig. (53). Structure of an additional backbone cyclic analog of somatostatin [67].

4. RECOMBINANT EXPRESSION OF BACKBONECYCLIZED PEPTIDES

dipeptide is expressed with a cysteine-proline sequence followed by glycolic acid, which rearranges to form a Cterminal diketopiperadine-thioester, resulting in the desired cyclization without an enzymatic requirement. Kawakami and colleagues demonstrated backbone cyclic peptide synthesis using recombinant elements by producing the natural backbone-cyclized peptides rhesus-theta defensin-1 (an antimicrobial) and sunflower trypsin inhibitor-1 (a small, potent 14-amino acid bicyclic peptide inhibitor of trypsin found in sunflower seeds). In this proof of concept study, they also developed a synthetic library of backbone-cyclized peptides, which they used to identify the pharmacophoric regions within each molecule [180]. 4.2. Peptide Display Phage display of cyclic peptides has been developed for M13 phage N-terminal pIII protein fusion, but is primarily limited to non-eukaryotic or extracellular applications [181]. Thus, Kritzer et al. addressed a gap by introducing a system for display of backbone-cyclized peptide libraries within eukaryotic cells for use in chemical genetics. The technique employs a Split Intein Cyclization of Peptides and Proteins (SICLOPPS) construct for expression of the backbonecyclized peptide in eukaryotic cells, which can support human disease models for rapid phenotypic selection of library hits. The authors used alpha-synuclein toxicity in S. cerevisiae as a model for Parkinson's Disease (PD) in a study of five million library transformants. This allowed them to identify two related backbone-cyclized peptides, which reduced the toxicity of human alpha-synuclein and also prevented dopaminergic neuron loss in a C. elegans model of Parkinson's disease. This established an efficient method for backbone-cyclized peptide library expression in cellular models, which can support high-throughput screens for biological function [182].


A number of recent advances have allowed the development of experimentally feasible recombinant expression systems for backbone-cyclized peptides, which employ protein ligation, intein-mediated protein trans-splicing, genetic reprogramming, or protease-catalyzed trans-peptidation [176]. These methods involve the ribosomal production of a precursor peptide, which is then processed to produce the target molecule. For example, Expressed Protein Ligation (EPL) is amenable to a variety of sequences, including disulfide-containing peptides in both prokaryotic and eukaryotic systems, but requires the use of modified inteins and thioester-mediated ligation [176-178]. Protease-mediated trans-peptidation requires both specific sortases and proteases and is somewhat limited in sequence adaptability [176]. Genetic reprogramming also relies on thioester-mediation ligation, is best for in vitro applications and can accommodate unnatural amino acids, but can be difficult for screening libraries or producing large yields [176]. Protein transsplicing involves split-inteins for self-processing, and although this technique can be used for complex peptides in both prokaryotic and eukaryotic systems, the intein-extein junction requirements constrain sequence parameters [176]. While limitations to individual techniques exist, progress in library screening and selection has improved access to backbone cyclization for both basic research and pharmaceutical applications [179]. 4.1. Backbone Cyclic Peptide Synthesis Using Recombinant Elements (bcPURE)

Backbone cyclic peptide synthesis using recombinant elements (bcPURE) allows for incorporation of nonproteinogenic amino acids through genetic reprogramming and ribosomal synthesis of backbone-cyclized peptides, pioneered by Kent and colleagues (The University of Chicago) as well as Suga and colleagues (The University of Tokyo). A linear O

S

4.3. Induced Peptidyl-tRNA Drop-off Kang and colleagues developed a creative one-pot production system for backbone-cyclized peptide synthesis. The technique involves the use of programmed peptidyl-tRNA drop-off, with recycling of tRNA but without incorporation of nonproteinogenic amino acids. The authors used as an example a vacant His codon (CAC) for the peptidyl-tRNA drop-off site, after which nonenzymatic intramolecular rearrangements released the peptidyl-tRNA and allowed assembly of the backbone-cyclized peptide by enzymes in the recombinant system. As a proof of concept, they produced the sunflower trypsin inhibitor-1 backbone-cyclized peptide (see above) [183].

S

Tc99m

O

O NH

N H

NH

NH

CH2 O

(CH2)6 His

Trp

Ser

Tyr

(D)Ala

Fig. (54). Structure of the backbone cyclic analog of GnRH [66].

547

Leu

Arg

Pro

N

CH2

O NH2


5. BACKBONE-CYCLIZED NATURAL PRODUCTS

PEPTIDES

Rubin and Qvit

FROM

6. CYCLIC PEPTIDES FOR THERAPEUTIC APPLICATIONS Despite significant improvements in science and technology over the last 65 years, the number of new drugs approved by global regulators such as the US Food and Drug Administration (FDA), the European Medicines Agency (EMA) and Japan's Pharmaceutical and Medical Devices Agency (PMDA), has decreased per billion US dollars spent on Research and Development (R&D) [191]. The number of new FDA-approved drugs per billion US dollars of R&D spending in the drug industry has halved approximately every 9 years since 1950, raising the estimated average cost per newly approved drug to $1,395 million [192]. Scannell et al. coined this phenomenon as Eroom’s Law (the reverse spelling of Moore - as in Moore’s Law, which describes the doubling every two years from the 1970s to 2010 of the number of transistors which can be placed at a reasonable cost onto an integrated circuit), and discussed several factors that decrease R&D productivity in the pharmaceutical industry [193, 194]. As a result, the pharmaceutical industry is exploring various approaches to decrease R&D expenses and increase productivity.


A variety of backbone-cyclized peptides are found in nature - from flora, to microorganisms, to mammals. We discussed several examples above (e.g. somatostatin), related to studies aimed at enhancing or otherwise modifying aspects of these natural compounds. Antimicrobial cyclic peptides can also be found in microorganisms as well as invertebrates [184]. For instance, scorpions produce a 34-amino acid peptide called maurotoxin, which contains four disulfide bridges between Cys3 and Cys24, Cys9 and Cys29, Cys13 and Cys19, and Cys31 and Cys34 (Fig. 55) [185]. Cyanobacteria produce cyclic peptides with additional activities, such as cryptophycin-1, which inhibits microtubule assembly (antitumor), and majusculamide C, which depolymerizes microfilaments (antifungal) [186]. Some plants (Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, and Solanaceae) produce cyclotides, or backbone-cyclized peptides, with three conserved disulfide bonds that form a cyclic cysteine motif.

molluscicidal potency and decreased toxicity to off-target species for cyclotide extracts compared to pesticides targeting the Southeast Asian rice pest Pomacea canaliculata [189]. Cyclotides can also be classified into bracelet and Möbius groups based on biological activities, the former of which is thought to exhibit anti-HIV activity. However, the Möbius cyclotide Kalata B1 also inhibits HIV (Fig. 56), via a mechanism likely related to membrane disruption based on its cyclization format rather than specific residues; study of its acyclic analogs revealed that the compound's anti-HIV activity is dependent on cyclization. In cultured T lymphocytes, the EC50 was approximately 140 nM, while the cytotoxic IC50 was over 3500 nM, revealing a wider potential therapeutic window than typical bracelet cyclotides [190].

Fig. (55). Structure of maurotoxin. The protein backbone is shown in red and the disulfide bridges in yellow (PDB: 1TXM) [210], using Pymol [211]. (The color version of the figure is available in the electronic copy of the article).

As a mechanism of host defense, plants express between 10 and 100 cyclotides of about 30 amino acids each, some of which have uterotonic, anti-HIV, antimicrobial, and insecticidal activities [187, 188]. Plan et al. demonstrated increased

One approach to improving pharmaceutical R&D is to revive interest in peptides as potential drug candidates. Peptides are expected to be cell permeable similarly to small molecules, but also to possess the high selectivity and potency characteristic of antibodies. Furthermore, peptides can take on different modulatory roles as agonists, antagonists and allosteric modulators. As a result, there is growing interest in peptides and peptidomimetics in the pharmaceutical industry. Novel synthetic strategies and alternative routes of administration have emerged [195-197], resulting in an increase in the average number of peptides entering clinical studies, from 1.2 per year (1970s) to over 16.8 per year (2000s) [198, 199]. Based on a recent publication by Bhat et al., peptides have higher success rates in transitioning from

Gly

Leu

Pro

Val

Cys20

Gly

Glu

Thr

Cys40

Val

Gly

Asn

Arg

Thr

Cys100

Val

Pro

Trp

Ser

Cys80

Thr

Cys78

Fig. (56). Structure of the Kalata B1 cyclotide.

Gly

Pro

Thr

Asn

Cys60

Thr

Gly



phase 1 to phase 2 trials (83%) compared to small molecules and antibodies (63% and 77%, respectively), and in transitioning from phase 3 to regulatory review (68%, compared to 61% of small molecules and 63% of antibodies) [200]. Taken together, it is not surprising that the number of available therapeutic peptides and peptidomimetics is increasing, with over 60 approved compounds on the market to date, and approximately 140 peptide drugs currently in clinical trials [47]. We focus on a limited selection of approved cyclic peptides in this review, as several excellent reviews recently discussed the current status of peptide drug discovery and development [47, 199, 201-203].

549

was subsequently shown to induce severe and irreversible side effects, such as cardiac arrhythmias, angina and abdominal cramps, which led to discontinuation of use in the USA and limitation of use worldwide (Fig. 59) [207]. HO

O

O N H

NH

S S

O

6.1. Vasopressin

NH

H 2N

H N O

O

O H2N

O

O

NH2 O

NH HN O

O

N

H2N

NH2

Fig. (58). Structure of desmopressin. O

H 2N

O

O

H 2N

O

O

N H

H 2N

NH

O H 2N O

S

HN

N

O

O

H N

O N

O

N H

O

O

NH2

N H

NH

O

S S

NH

O

H N

O

NH

S

O

NH2 H N

N H

O

NH O

N

O

HO

H 2N

HN


Vasopressin, or antidiuretic hormone (commercialized as arginine vasopressin, or Argipressin), is a natural cyclic peptide consisting of nine amino acids (Cys-Tyr-Phe-Gln-AsnCys-Pro-Arg-Gly-NH2) with a disulfide bond between the cysteine residues at positions 1 and 6. Vasopressin is a vital neurohypophysial hormone secreted from the posterior pituitary that regulates water, glucose, and salts in the blood. The antidiuretic activity of vasopressin is ascribed to increases in water reabsorption by the renal tubules, which also increases peripheral vascular resistance and arterial blood pressure. Vasopressin is used to treat diabetes insipidus and to improve vasomotor tone and blood pressure (Fig. 57) [204].

O H N

N H

NH2

O

NH2

O

O

NH

HN

HO

Fig. (59). Structure of terlipressin.

O

N

H 2N

NH2

Fig. (57). Structure of vasopressin.

Desmopressin (3-Mercaptopropionyl(Mpr)-Tyr-Phe-GlnAsn-Cys-Pro-(D)Arg-Gly-NH2 with a disulfide bridge between Mpr1 and Cys6) is a synthetic analog of vasopressin, with prolonged antidiuretic but little pressor activity [205]. Due to its decreased metabolism (half-life 1.5-2.5 hours compared to 10-20 minutes for vasopressin), the drug can be administered nasally, intravenously, or as an oral sublingual tablet (Fig. 58) [206]. Terlipressin (Gly-Gly-Gly-Cys-TyrPhe-Gln-Asn-Cys-Pro-Lys-Gly-NH2 with a disulfide bridge between Cys4 and Cys9) is another synthetic analog of vasopressin, which is used as a vasoactive drug in the management of hypotension. This compound has a prolonged half-life (~6 hours), which initially made it more popular, but

6.2. Somatostatin

Somatostatin (see above) has been commercialized due to its clinical importance as a regulatory hormone. Several synthetic analogs, including pasireotide, lanreotide and octreotide, are on the market for clinical use (Fig. 13). Pasireotide (cyclo(-Hyp(2-aminoethyl-carbamoyl)-Phg-(D)Trp-Lys-Tyr (Bzl)-Phe) is a six-membered cyclic peptide analog, which was approved as an orphan drug in the USA and Europe for treatment of Cushing’s disease in patients who fail or are ineligible for surgical intervention. The drug has a 40-fold increased affinity for somatostatin receptor 5 (sst5) compared to other somatostatin analogs [208]. Lanreotide ((D)β-Nal-Cys-Tyr-(D)Trp-Lys-Val-Cys-ThrNH2 with a disulfide bridge between Cys2 and Cys7) is a long-acting analog of somatostatin (half-life 2 hours, compared to 2-3 minutes for somatostatin) that acts primarily by binding to somatostatin receptor 2 (sst2) (Fig. 13). The drug is used for management of acromegaly and symptoms caused


Rubin and Qvit

O

O OH

O

HO

OH

NH2

HO

NH2 O

O H2N

O OH

OH

OH

NH2

NH2

Fig. (60). Structure of glatiramer acetate. H 2N N

HN N O H N

O

O

HO

H N

N H

NH2

O H N

N H

N H

O

O N

N H

OH

O

O

O

HN


O

O H N

NH

OH

Fig. (61). Structure of leuprolide acetate.

H2N

HN

N

O

H N

O

O

H N

N H

HO

O

O

H N

N H

O

O

N

O

H N

N H

NH2

O

N H

O

NH

OH

N

HN

O

NH

H2N

O

Fig. (62). Structure of goserelin.

by neuroendocrine tumors [209-211]. Octreotide acetate (Sandostatin, (D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-ol with a disulfide bridge between Cys2 and Cys7) is a synthetic octapeptide analog of somatostatin. This compound is also long-acting, with an increased plasma half-life (1.7 hours) and pharmacologic activity similar to natural somatostatin, mediated via binding to somatostatin receptor 2 (sst2) (Fig. 13). Octreotide acetate is one of four peptides that reached global sales of over US $1 billion in 2008; glatiramer acetate (Fig. 60; $3.18 billion), leuprolide acetate (Fig. 61; $2.12 billion) and goserelin acetate (Fig. 62; $1.14 billion) also reached global sales in excess of US $1 billion in the same year [198].

antibiotics, will be revived in part by the adoption of peptide or peptidomimetic formats - particularly through the integration of backbone-cyclized peptides, which possess important therapeutic qualities. Further applications of rationally designed or otherwise synthetic peptides may be demonstrated in related fields such as agricultural sciences and environmental microbiology, where numerous examples of natural backbone-cyclized peptides can already be seen. We anticipate that the diverse chemistries, which can be integrated into backbone-cyclized peptides, will present opportunities for incorporation of more complex features, such as photo- or enzyme-cleavable units or small molecule drug conjugates.

CONCLUSION

CONSENT FOR PUBLICATION

Over the past century, scientists have used peptides and peptidomimetics to address a variety of problems in biology, ranging in application from research tools to therapeutic leads, and from broad conceptual to focused mechanistic studies. We predict that fields hindered by obstacles to pharmaceutical innovation, such as the development of new

Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.



ACKNOWLEDGEMENTS

[25]

The authors thank Dr. Moshe Dessau for his help with Figure 55.

[26]

REFERENCES

[27]

[1]

[28]

[3] [4] [5]

[6] [7]

[8] [9]

[10] [11] [12]

[13] [14] [15] [16]

[17] [18]

[19]

[20]

[21] [22] [23]

[24]

[29] [30]

[31] [32] [33]

Banting, F.G.; Best, C.H.; Collip, J.B.; Campbell, W.R.; Fletcher, A.A. Pancreatic extracts in the treatment of diabetes mellitus. Can. Med. Assoc. J., 1922, 12(3), 141-146. Consden, R.; Gordon, A.H.; Martin, A.J.P. Gramicidin S; the sequence of the amino-acid residues. Biochem. J., 1947, 41, (4), 596602. White, C.J.; Yudin, A.K. Contemporary strategies for peptide macrocyclization. Nat. Chem., 2011, 3(7), 509-524. Roxin, A.; Zheng, G. Flexible or fixed: a comparative review of linear and cyclic cancer-targeting peptides. Future Med. Chem., 2012, 4(12), 1601-1618. Russo, A.; Aiello, C.; Grieco, P.; Marasco, D. Targeting "undruggable" proteins: Design of synthetic cyclopeptides. Curr. Med. Chem., 2016, 23(8), 748-762. Fjell, C.D.; Hiss, J.A.; Hancock, R.E.W.; Schneider, G. Designing antimicrobial peptides: form follows function. Nat. Rev. Drug Discov., 2012, 11(1), 37-51. Jagadish, K.; Camarero, J.A. Cyclotides, a promising molecular scaffold for peptide-based therapeutics. Biopolymers, 2010, 94(5), 611-616. Essack, M.; Bajic, V.B.; Archer, J.A.C. Conotoxins that confer therapeutic possibilities. Mar. Drugs, 2012, 10(6), 1244. Moore, S.J.; Leung, C.L.; Cochran, J.R. Knottins: disulfide-bonded therapeutic and diagnostic peptides. Drug Discov. Today Technol., 2012, 9(1), e1-e70. Wu, C.H.; Liu, I.J.; Lu, R.M.; Wu, H.C. Advancement and applications of peptide phage display technology in biomedical science. J. Biomed. Sci., 2016, 23, 8. Barreto, K.; Geyer, C.R. Screening combinatorial libraries of cyclic peptides using the yeast two-hybrid assay. Methods Mol. Biol., 2014, 1163, 273-309. Tavassoli, A.; Benkovic, S.J. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nat. Protoc., 2007, 2(5), 1126-1133. Needels, M.C.; Jones, D.G.; Tate, E.H.; Heinkel, G.L.; Kochersperger, L.M.; Dower, W.J.; Barrett, R.W.; Gallop, M.A. Generation and screening of an oligonucleotide-encoded synthetic peptide library. Proc. Natl. Acad. Sci. U. S. A., 1993, 90(22), 10700-10704. Frank, R. The SPOT-synthesis technique. J. Immunol. Methods, 2002, 267(1), 13-26. Tan, Y.S.; Lane, D.P.; Verma, C.S. Stapled peptide design: principles and roles of computation. Drug Discov. Today, 2016, 21(10), 1642-1653. Bhardwaj, G.; Mulligan, V.K.; Bahl, C.D.; Gilmore, J.M.; Harvey, P.J.; Cheneval, O.; Buchko, G.W.; Pulavarti, S.V.; Kaas, Q.; Eletsky, A.; Huang, P.S.; Johnsen, W.A.; Greisen, P.J.; Rocklin, G.J.; Song, Y.; Linsky, T.W.; Watkins, A.; Rettie, S.A.; Xu, X.; Carter, L.P.; Bonneau, R.; Olson, J.M.; Coutsias, E.; Correnti, C.E.; Szyperski, T.; Craik, D.J.; Baker, D. Accurate de novo design of hyperstable constrained peptides. Nature, 2016, 538(7625), 329335. Fu, J.; Ling, S.; Liu, Y.; Yang, J.; Naveh, S.; Hannah, M.; Gilon, C.; Zhang, Y.; Holoshitz, J. A small shared epitope-mimetic compound potently accelerates osteoclast-mediated bone damage in autoimmune arthritis. J. Immunol., 2013, 191(5), 2096-2103. Montalbetti, C.A.; Falque, V. Amide bond formation and peptide coupling. Tetrahedron, 2005, 61(46), 10827-10852. Pattabiraman, V.R.; Bode, J.W. Rethinking amide bond synthesis. Nature, 2011, 480(7378), 471-479. Parenty, A.; Moreau, X.; Campagne, J.M. Macrolactonizations in the total synthesis of natural products. Chem. Rev., 2006, 106(3), 911-939. Gongora-Benitez, M.; Tulla-Puche, J.; Albericio, F. Multifaceted roles of disulfide bonds. Peptides as therapeutics. Chem. Rev., 2014, 114(2), 901-926. Tsomaia, N. Peptide therapeutics: targeting the undruggable space. Eur. J. Med. Chem., 2015, 94, 459-470. Fosgerau, K.; Hoffmann, T. Peptide therapeutics: Current status and future directions. Drug Discov. Today, 2015, 20(1), 122-128. Fischer, E. Einfluss der configuration auf die wirkung der enzyme. Ber. Dtsch. Chem. Ges., 1894, 27, 2985-2993 Powell, G.B.; Brown, K.C. Combinatorial peptide libraries: Mining for cell-binding peptides. Chem. Rev., 2014, 114(2), 1020-1081. Gilon, C.; Halle, D.; Chorev, M.; Selinger, Z.; Byk, G. Backbone cyclization - a new method for conferring conformational constraint on peptides. Biopolymers, 1991, 31(6), 745-750.


[2]

Pawson, T. Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell, 2004, 116(2), 191-203. Pal, A.; Chakrabarti, P.; Bahadur, R.; Rodier, F.; Janin, J. Peptide segments in protein-protein interfaces. J. Biosci., 2007, 32(1), 101111. Hwang, H.; Vreven, T.; Janin, J.; Weng, Z. Protein-protein docking benchmark version 4.0. Proteins, 2010, 78(15), 3111-3114. Arkin, M.R.; Wells, J.A. Small-molecule inhibitors of proteinprotein interactions: progressing towards the dream. Nat. Rev. Drug Discov., 2004, 3(4), 301-317. Fuller, J.C.; Burgoyne, N.J.; Jackson, R.M. Predicting druggable binding sites at the protein-protein interface. Drug Discov. Today, 2009, 14(3-4), 155-161. Arkin, M.R.; Tang, Y.; Wells, J.A. Small-molecule inhibitors of protein-protein interactions: progressing toward the reality. Chem. Biol., 2014, 21(9), 1102-1114. Cukuroglu, E.; Engin, H.B.; Gursoy, A.; Keskin, O. Hot spots in protein-protein interfaces: towards drug discovery. Prog. Biophys. Mol. Biol., 2014, 116(2-3), 165-173. Jones, D.S.; Silverman, A.P.; Cochran, J.R. Developing therapeutic proteins by engineering ligand-receptor interactions. Trends Biotechnol., 2008, 26(9), 498-505. Scott, D.E.; Bayly, A.R.; Abell, C.; Skidmore, J. Small molecules, big targets: drug discovery faces the protein-protein interaction challenge. Nat. Rev. Drug Discov., 2016, 15(8), 533-550. Wojcik, P.; Berlicki, L. Peptide-based inhibitors of protein-protein interactions. Bioorg. Med. Chem. Lett., 2016, 26(3), 707-713. Churchill, E.N.; Qvit, N.; Mochly-Rosen, D. Rationally designed peptide regulators of protein kinase C. Trends Endocrinol. Metab., 2009, 20(1), 25-33. Cunningham, A.D.; Qvit, N. Specific kinase inhibition using peptides to target kinase-substrate docking. Chimica Oggi/Chemistry Today, 2016, 34(5), 22-25. Petsalaki, E.; Russell, R.B. Peptide-mediated interactions in biological systems: new discoveries and applications. Curr. Opin. Biotechnol., 2008, 19(4), 344-350. London, N.; Raveh, B.; Schueler-Furman, O. Modeling peptideprotein interactions. Methods Mol. Biol., 2012, 857, 375-398. Koshland, D.E. The key-lock theory and the induced fit theory. Angew. Chem. Int. Ed., 1995, 33(23-24), 2375-2378. Bruno, B.J.; Miller, G.D.; Lim, C.S. Basics and recent advances in peptide and protein drug delivery. Ther. Deliv., 2013, 4(11), 14431467. Rubin, S.; Qvit, N. Cyclic peptides for protein-protein interaction targets: Applications to human disease. Crit. Rev. Eukaryot. Gene Expr., 2016, 26(3), 199-221. Cunningham, A.D.; Qvit, N.; Mochly-Rosen, D. Peptides and peptidomimetics as regulators of protein–protein interactions. Curr. Opin. Struct. Biol., 2017, 44, 59-66. Brinckerhoff, L.H.; Kalashnikov, V.V.; Thompson, L.W.; Yamshchikov, G.V.; Pierce, R.A.; Galavotti, H.S.; Engelhard, V.H.; Slingluff, C.L., Jr. Terminal modifications inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide: implications for peptide vaccines. Int. J. Cancer, 1999, 83(3), 326-334. Ahn, J.M.; Boyle, N.A.; MacDonald, M.T.; Janda, K.D. Peptidomimetics and peptide backbone modifications. Mini Rev. Med. Chem., 2002, 2(5), 463-473. Yin, H. Constrained peptides as miniature protein structures. ISRN Biochem., 2012, 2012, 1-15. Sewald, N.; Jakubke, H. In Peptides: chemistry and biology; VCH, 2002, 311-334. Qvit, N.; Reuveni, H.; Gazal, S.; Zundelevich, A.; Blum, G.; Niv, M.Y.; Feldstein, A.; Meushar, S.; Shalev, D.E.; Friedler, A.; Gilon, C. Synthesis of a novel macrocyclic library: discovery of an IGF1R inhibitor. J. Comb. Chem., 2008, 10(2), 256-266. Qvit, N. Microwave-assisted synthesis of cyclic phosphopeptide on solid support. Chem. Biol. Drug Des., 2014, 85(3), 300-305.

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42] [43] [44] [45]

[46] [47] [48] [49] [50]

551


[52]

[53] [54]

[55]

[56]

[57]

[58] [59]

[60] [61] [62]

[63]

[64]

[65]

[66]

[67]

[68]

[69] [70]

Gilon, C.; Muller, D.; Bitan, G.; Salitra, Y.; Goldwasser, I.; Hornik, V. Cycloscan: Backbone cyclic conformational libraries of peptides. In 24th Conference of the European, Peptide Society, Edinbrough, Scotland, 1998. Demmer, O.; Frank, A.O.; Kessler, H. In Peptide and protein design for biopharmaceutical applications; John Wiley & Sons, Ltd, 2009, 133-176. Kolb, H.C.; Finn, M.G.; Sharpless, K.B. Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed., 2001, 40(11), 2004-2021. Tornoe, C.W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1, 2, 3]-triazoles by regiospecific copper (i)-catalyzed 1, 3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem., 2002, 67(9), 3057-3064. Kazmaier, U.; Hebach, C.; Watzke, A.; Maier, S.; Mues, H.; Huch, V. A straightforward approach towards cyclic peptides via ringclosing metathesis-scope and limitations. Org. Biomol. Chem., 2005, 3(1), 136-145. Bird, G.H.; Crannell, W.C.; Walensky, L.D. Chemical synthesis of hydrocarbon-stapled peptides for protein interaction research and therapeutic targeting. Curr. Protoc. Chem. Biol., 2011, 3(3), 99117. Clark, R.J.; Craik, D.J. Native chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins. Biopolymers, 2010, 94(4), 414-422. Hill, T.A.; Shepherd, N.E.; Diness, F.; Fairlie, D.P. Constraining cyclic peptides to mimic protein structure motifs. Angew. Chem. Int. Ed. Engl., 2014, 53(48), 13020-13041. Blackwell, H.E.; Grubbs, R.H. Highly efficient synthesis of covalently cross-linked peptide helices by ring-closing metathesis. Angew. Chem. Int. Ed., 1998, 37(23), 3281-3284. Dawson, P.E.; Muir, T.W.; Clark-Lewis, I.; Kent, S.B. Synthesis of proteins by native chemical ligation. Science, 1994, 266(5186), 776-779. Tulla-Puche, J.; Barany, G. On-resin native chemical ligation for cyclic peptide synthesis. J. Org. Chem., 2004, 69(12), 4101-4107. Hurevich, M.; Ratner-Hurevich, M.; Tal-Gan, Y.; Shalev, D.E.; Ben-Sasson, S.Z.; Gilon, C. Backbone cyclic helix mimetic of chemokine (C-C motif) receptor 2: A rational approach for inhibiting dimerization of G protein-coupled receptors. Bioorg. Med. Chem. 2013, 3958-3966. Altstein, M.; Ben-Aziz, O.; Daniel, S.; Schefler, I.; Zeltser, I.; Gilon, C. Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths. J. Biol. Chem., 1999, 274(25), 17573-17579. Qvit, N.; Schechtman, D.; Peña, D.A.; Berti, D.A.; Soares, C.O.; Miao, Q.; Liang, L.; Baron, L.A.; Teh-Poot, C.; Martínez-Vega, P.; Ramirez-Sierra, M.J.; Churchill, E.; Cunningham, A.D.; Malkovskiy, A.V.; Federspiel, N.A.; Gozzo, F.C.; Torrecilhas, A.C.; Manso Alves, M.J.; Jardim, A.; Momar, N.; Dumonteil, E.; Mochly-Rosen, D. Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors. Int. J. Parasitol. Drugs Drug Resist., 2016, 6, 74-84. Gazal, S.; Gelerman, G.; Ziv, O.; Karpov, O.; Litman, P.; Bracha, M.; Afargan, M.; Gilon, C. Human somatostatin receptor specificity of backbone-cyclic analogues containing novel sulfur building units. J. Med. Chem., 2002, 45(8), 1665-1671. Barda, Y.; Cohen, N.; Lev, V.; Ben-Aroya, N.; Koch, Y.; Mishani, E.; Fridkin, M.; Gilon, C. Backbone metal cyclization: novel 99mTc labeled GnRH analog as potential SPECT molecular imaging agent in cancer. Nucl. Med. Biol., 2004, 31(7), 921-933. Fridkin, G.; Bonasera, T.A.; Litman, P.; Gilon, C. Backbone metalcyclization: a novel approach for simultaneous peptide cyclization and radiolabeling. Application to the combinatorial synthesis of rhenium-cyclic somatostatin analogs. Nucl. Med. Biol., 2005, 32(1), 39-50. Hurevich, M.; Tal-Gan, Y.; Klein, S.; Barda, Y.; Levitzki, A.; Gilon, C. Novel method for the synthesis of urea backbone cyclic peptides using new Alloc-protected glycine building units. J. Pept. Sci., 2010, 16(4), 178-185. Fridkin, G.; Gilon, C. Azo cyclization: peptide cyclization via azo bridge formation. J. Pept. Res., 2002, 60(2), 104-111. Dash, A.; Chakraborty, S.; Pillai, M.R.; Knapp, F.F., Jr. Peptide receptor radionuclide therapy: an overview. Cancer Biother. Radiopharm., 2015, 30(2), 47-71.

[71]

[72]

[73]

[74]

[75]

[76]

Bitan, G.; Behrens, S.; Matha, B.; Mashriki, Y.; Hanani, M.; Kessler, H.; Gilon, C. New backbone cyclic substance P analogs. Lett. Pept. Sci., 1995, 2(3-4), 121-124. Byk, G.; Halle, D.; Zeltser, I.; Bitan, G.; Selinger, Z.; Gilon, C. Synthesis and biological activity of NK-1 selective, N-backbone cyclic analogs of the C-terminal hexapeptide of substance P. J. Med. Chem., 1996, 39(16), 3174-3178. Gilon, C.; Huenges, M.; Matha, B.; Gellerman, G.; Hornik, V.; Afargan, M.; Amitay, O.; Ziv, O.; Feller, E.; Gamliel, A.; Shohat, D.; Wanger, M.; Arad, O.; Kessler, H. A backbone-cyclic, receptor 5-selective somatostatin analogue: Synthesis, bioactivity, and nuclear magnetic resonance conformational analysis. J. Med. Chem., 1998, 41(6), 919-929. Ovadia, O.; Greenberg, S.; Laufer, B.; Gilon, C.; Hoffman, A.; Kessler, H. Improvement of drug-like properties of peptides: the somatostatin paradigm. Expert Opin. Drug Discov., 2010, 5(7), 655-671. Zeltser, I.; Ben-Aziz, O.; Schefler, I.; Bhargava, K.; Altstein, M.; Gilon, C. Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists. J. Pept. Res., 2001, 58(4), 275-284. Qvit, N.; Hatzubai, A.; Shalev, D.E.; Friedler, A.; Ben‐Neriah, Y.; Gilon, C. Design and synthesis of backbone cyclic phosphorylated peptides: The IκB model. Biopolymers, 2009, 91(2), 157-168. Qvit, N.; Hatzubai, A.; Shalev, D.; Ben-Neriah, Y.; Gilon, C. Design and synthesis of backbone cyclic phosphopeptides: The IκB model. In 20th American Peptide Symposium; Advances in Experimental Medicine and Biology: Montreal, Canada., 2009, 611, 139140. Qvit, N.; Gazal, S.; Feldstein, A.; Meoshar, S.; Peretzman-Shemer, A.; Blum, G.; Niv, M.Y.; Reuveni, H.; Gilon, C. Novel macrocyclic libraries with spatial diversity (Sibs): Application for the discovery of IGF-1R inhibitor. In 28th European Peptide Symposium & 3rd International; Journal of Peptide Science: Prague, Czech Republic, 2004, 10, 95-95. Hayouka, Z.; Hurevich, M.; Levin, A.; Benyamini, H.; Iosub, A.; Maes, M.; Shalev, D.E.; Loyter, A.; Gilon, C.; Friedler, A. Cyclic peptide inhibitors of HIV-1 integrase derived from the LEDGF/p75 protein. Bioorg. Med. Chem., 2010, 18(23), 8388-8395. Kasher, R.; Bitan, G.; Halloun, C.; Gilon, C. Synthesis of a bicyclic BPTI mimetic containing 4-thioproline replacing Cys(38). Lett. Pept. Sci., 1998, 5(2-3), 101-103. Tal-Gan, Y.; Gilon, C.; Hurevich, M.; Klein, S.; Ben-Shimon, A.; Rosenthal, D.; Hazan, C.; Shalev, D.E.; Niv, M.Y.; Levitzki, A. Backbone cyclic peptide inhibitors of protein kinase B (PKB/Akt). J. Med. Chem., 2011, 54(14), 5154-5164. Qvit, N.; Disatnik, M.; Mochly-Rosen, D. Peptide regulators of protein-protein interactions with selectivity for specific sub-cellular compartments. In: 21th American Peptide Symposium; Biopolymers: Bloomington, IN, USA, 2009; Vol. 92, pp. 322-322. Qvit, N.; Disatnik, M.H.; Mochly-Rosen, D. A second generation peptide activator derived from the C2 domain of delta protein kinase C and regulates its function in the mitochondria. In: 31th European Peptide Symposium; Journal of Peptide Science: Copenhagen, Denmark, 2010; Vol. 16, pp. 115-116. Qvit, N.; Disatnik, M.H.; Mochly-Rosen, D. Novel selective peptide regulator of protein kinase C based on rational approach design. Biosens. Bioelectron., 2012, 2012, 142. Qvit, N.; Disatnik, M.H.; Mochly-Rosen, D. Engineering a delta protein kinase C inhibitor that selectively inhibits interaction and phosphorylation of one substrate of the multi-substrate kinase. In: American Society for Biochemistry and Molecular Biology Annual Meeting: San Diego, 2014; Vol. 28, pp. 1. Qvit, N.; Joshi, A.U.; Cunningham, A.D.; Ferreira, J.C.; MochlyRosen, D. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) protein-protein interaction inhibitor reveals a non-catalytic role for GAPDH oligomerization in cell death. J. Biol. Chem., 2016, 291(26), 13608-13621. Qvit, N.; Disatnik, M.H.; Sho, J.; Mochly-Rosen, D. Selective phosphorylation inhibitor of delta protein kinase C-pyruvate dehydrogenase kinase protein-protein interactions: Application for myocardial injury in vivo. J. Am. Chem. Soc., 2016, 138(24), 76267635. Qvit, N.; Miguel, D.C.; Bonano, V.I.; Schechtman, D.; Uliana, S.R.; Mochly-Rosen, D. Targeting neglected diseases based on rational approach design; Proof of concept: Novel peptides for inhib-


[51]

Rubin and Qvit

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84] [85]

[86]

[87]

[88]


[90] [91]

[92]

[93]

[94]

[95] [96] [97] [98] [99]

[100] [101]

[102] [103]

[104] [105]

[106]

[107] [108]

[109]

[110]

[111]

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

[122]

[123]

[124] [125]

[126]

[127]

[128]

[129]

553

Bitan, G.; Gilon, C. Building units for N-backbone cyclic-peptides .2. Synthesis of protected N-(omega-thioalkylene) amino-acids and their incorporation into dipeptide units. Tetrahedron, 1995, 51(38), 10513-10522. Fehrentz, J.A.; Castro, B. An efficient synthesis of optically active alpha-(t-butoxycarbonylamino)-aldehydes from alpha amino acids. Synthesis, 1983, (8), 676-678. Bitan, G.; Muller, D.; Kasher, R.; Gluhov, E.V.; Gilon, C. Building units for N-backbone cyclic peptides .4. Synthesis of protected Nalpha-functionalized alkyl amino acids by reductive alkylation of natural amino acids. J. Chem. Soc., Perkin Trans. 1, 1997, (10)1501-1510. Gazal, S.; Gellerman, G.; Glukhov, E.; Gilon, C. Synthesis of novel protected N-alpha(omega-thioalkyl) amino acid building units and their incorporation in backbone cyclic disulfide and thioetheric bridged peptides. J. Pept. Res., 2001, 58(6), 527-539. Munoz, M.; Covenas, R. Involvement of substance P and the NK-1 receptor in human pathology. Amino Acids, 2014, 46(7), 17271750. Mander, K.; Harford-Wright, E.; Lewis, K.M.; Vink, R. Advancing drug therapy for brain tumours: a current review of the proinflammatory peptide Substance P and its antagonists as anti-cancer agents. Recent. Pat. CNS Drug Discov., 2014, 9(2), 110-121. Aziz, F. Neurokinin-1 receptor antagonists for chemotherapyinduced nausea and vomiting. Ann. Palliat. Med., 2012, 1(2), 130136. Keller, M.; Montgomery, S.; Ball, W.; Morrison, M.; Snavely, D.; Liu, G.; Hargreaves, R.; Hietala, J.; Lines, C.; Beebe, K.; Reines, S. Lack of efficacy of the substance P (neurokinin1 receptor) antagonist aprepitant in the treatment of major depressive disorder. Biol. Psychiatry, 2006, 59(3), 216-223. Bitan, G.; Zeltser, I.; Byk, G.; Halle, D.; Mashriki, Y.; Gluhov, E.V.; Sukhotinsky, I.; Hanani, M.; Selinger, Z.; Gilon, C. Backbone cyclization of the C-terminal part of substance P. Part 1: The important role of the sulphur in position 11. J. Pept. Sci., 1996, 2(4), 261-269. Bitan, G.; Sukhotinsky, I.; Mashriki, Y.; Hanani, M.; Selinger, Z.; Gilon, C. Synthesis and biological activity of novel backbonebicyclic substance-P analogs containing lactam and disulfide bridges. J. Pept. Res., 1997, 49(5), 421-426. Theodoropoulou, M.; Stalla, G.K. Somatostatin receptors: From signaling to clinical practice. Front. Neuroendocrinol, 2013, 34(3), 228-252. Rai, U.; Thrimawithana, T.R.; Valery, C.; Young, S.A. Therapeutic uses of somatostatin and its analogues: Current view and potential applications. Pharmacol. Ther., 2015, 152, 98-110. Narayanan, S.; Kunz, P.L. Role of somatostatin analogues in the treatment of neuroendocrine tumors. Hematol. Oncol. Clin. North Am., 2016, 30(1), 163-177. Farabaugh, S.M.; Boone, D.N.; Lee, A.V. Role of IGF1R in breast cancer subtypes, stemness, and lineage differentiation. Front. Endocrinol., 2015, 6, 59. Khwaja, O.S.; Ho, E.; Barnes, K.V.; O'Leary, H.M.; Pereira, L.M.; Finkelstein, Y.; Nelson, C.A., 3rd; Vogel-Farley, V.; DeGregorio, G.; Holm, I.A.; Khatwa, U.; Kapur, K.; Alexander, M.E.; Finnegan, D.M.; Cantwell, N.G.; Walco, A.C.; Rappaport, L.; Gregas, M.; Fichorova, R.N.; Shannon, M.W.; Sur, M.; Kaufmann, W.E. Safety, pharmacokinetics, and preliminary assessment of efficacy of mecasermin (recombinant human IGF-1) for the treatment of Rett syndrome. Proc. Natl. Acad. Sci. U. S. A., 2014, 111(12), 4596-4601. Veber, D.F.; Johnson, S.R.; Cheng, H.Y.; Smith, B.R.; Ward, K.W.; Kopple, K.D. Molecular properties that influence the oral bioavailability of drug candidates. J. Med. Chem., 2002, 45(12), 2615-2623. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Del. Rev., 1997, 23(1-3), 3-25. Byk, G.; Gilon, C. Building units for N-backbone cyclic-peptides 1. Synthesis of protected N-(omega-aminoalkylene)amino acids and their incorporation into dipeptide units. J. Org. Chem., 1992, 57(21), 5687-5692. Effenberger, F.; Burkard, U.; Willfahrt, J. Trifluoromethanesulfonates of α -hydroxycarboxylates-educts for the racemization-free


[89]

iting leishmaniasis. In: 61st Annual Meeting of the American Society of Tropical Medicine and Hygiene: Atlanta, GA, USA, 2012. Qvit, N.; Crapster, J.A. Peptides that target protein-protein interactions as an anti-parasite strategy. Chim. Oggi Chem. Today, 2014, 32, (6), 62-66. Qvit, N.; Kornfeld, O.S. Development of a backbone cyclic peptide library as potential antiparasitic therapeutics using microwave irradiation. J. Vis. Exp., 2016, 107, e53589. Afargan, M.; Janson, E.T.; Gelerman, G.; Rosenfeld, R.; Ziv, O.; Karpov, O.; Wolf, A.; Bracha, M.; Shohat, D.; Liapakis, G.; Gilon, C.; Hoffman, A.; Stephensky, D.; Oberg, K. Novel long-acting somatostatin analog with endocrine selectivity: potent suppression of growth hormone but not of insulin. Endocrinology, 2001, 142(1), 477-486. Linde, Y.; Ovadia, O.; Safrai, E.; Xiang, Z.; Portillo, F.P.; Shalev, D.E.; Haskell-Luevano, C.; Hoffman, A.; Gilon, C. Structureactivity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides. Biopolymers, 2008, 90(5), 671-682. Hess, S.; Ovadia, O.; Shalev, D.E.; Senderovich, H.; Qadri, B.; Yehezkel, T.; Salitra, Y.; Sheynis, T.; Jelinek, R.; Gilon, C.; Hoffman, A. Effect of structural and conformation modifications, including backbone cyclization, of hydrophilic hexapeptides on their intestinal permeability and enzymatic stability. J. Med. Chem., 2007, 50(24), 6201-6211. Ovadia, O.; Linde, Y.; Haskell-Luevano, C.; Dirain, M.L.; Sheynis, T.; Jelinek, R.; Gilon, C.; Hoffman, A. The effect of backbone cyclization on PK/PD properties of bioactive peptide-peptoid hybrids: the melanocortin agonist paradigm. Bioorg. Med. Chem., 2010, 18(2), 580-589. Merrifield, R.B. Solid phase peptide synthesis I. The synthesis of a tetrapeptide. J. Am. Chem. Soc., 1963, 85, 2149-2154. Wang, Q.; Parrish, A.R.; Wang, L. Expanding the genetic code for biological studies. Chem. Biol., 2009, 16(3), 323-336. GroB, A.; Hashimoto, C.; Sticht, H.; Eichler, J. Synthetic peptides as protein mimics. Front. Bioeng. Biotechnol., 2016, 3, 211. Cheng, R.P.; Gellman, S.H.; DeGrado, W.F. Beta-Peptides: From structure to function. Chem. Rev., 2001, 101(10), 3219-3232. Seebach, D.; Beck, A.K.; Bierbaum, D.J. The world of beta- and gamma-peptides comprised of homologated proteinogenic amino acids and other components. Chem. Biodivers., 2004, 1(8), 11111239. Seebach, D.; Gardiner, J. Beta-peptidic peptidomimetics. Acc. Chem. Res., 2008, 41(10), 1366-1375. Fernandez-Llamazares, A.I.; Spengler, J.; Albericio, F. Review backbone N-modified peptides: How to meet the challenge of secondary amine acylation. Biopolymers, 2015, 104(5), 435-452. Avan, I.; Hall, C.D.; Katritzky, A.R. Peptidomimetics via modifications of amino acids and peptide bonds. Chem. Soc. Rev., 2014, 43(10), 3575-3594. Wu, H. Gamma-AApeptides as a new class of peptidomimetics: Synthesis, structures, and functions. Chemistry (Weinheim an der Bergstrasse, Germany), 2015, 21(6), 2501-2507. Sasmal, S.; Geyer, A.; Maier, M.E. Synthesis of cyclic peptidomimetics from aldol building blocks. J. Org. Chem., 2002, 67(17), 6260-6263. Palomo, C.; Aizpurua, J.M.; Ganboa, I.; Oiarbide, M. Asymmetric synthesis of beta-lactams through the Staudinger reaction and their use as building blocks of natural and nonnatural products. Curr. Med. Chem., 2004, 11(14), 1837-1872. Alcaide, B.; Almendros, P.; Aragoncillo, C. Beta-lactams: versatile building blocks for the stereoselective synthesis of non-beta-lactam products. Chem. Rev., 2007, 107(11), 4437-4492. Gruner, S.A.W.; Locardi, E.; Lohof, E.; Kessler, H. Carbohydratebased mimetics in drug design: Sugar amino acids and carbohydrate scaffolds. Chem. Rev., 2002, 102(2), 491-514. Paolini, A.; Nuti, F.; Pozo-Carrero, M.D.; Barbetti, F.; Kolesinska, B.; Kaminski, Z.J.; Chelli, M.; Papini, A.M. A convenient microwave-assisted synthesis of N-glycosyl amino acids. Tetrahedron Lett., 2007, 48(16), 2901-2904. Ngyen, H.; Orlamuender, M.; Pretzel, D.; Agricola, I.; Sternberg, U.; Reissmann, S. Transition metal complexes of a cyclic pseudo hexapeptide: synthesis, complex formation and catalytic activities. J. Pept. Sci., 2008, 14(9), 1010-1021. Gambari, R. Peptide nucleic acids: a review on recent patents and technology transfer. Expert Opin. Ther. Pat., 2014, 24(3), 267-294.



[131]

[132]

[133] [134]

[135] [136]

[137]

[138]

[139]

[140]

[141] [142]

[143] [144] [145]

[146] [147]

[148]

[149]

[150]

[151]

[152]

[153]

[154]

[155]

[156]

Hariton-Gazal, E.; Friedler, D.; Friedler, A.; Zakai, N.; Gilon, C.; Loyter, A. Inhibition of nuclear import by backbone cyclic peptidomimetics derived from the HIV-1 MA NLS sequence. Biochim. Biophys. Acta., 2002, 1594(2), 234-242. Chaloin, L.; Smagulova, F.; Hariton-Gazal, E.; Briant, L.; Loyter, A.; Devaux, C. Potent inhibition of HIV-1 replication by backbone cyclic peptides bearing the Rev arginine rich motif. J. Biomed. Sci., 2007, 14(5), 565-584. Hayouka, Z.; Levin, A.; Hurevich, M.; Shalev, D.E.; Loyter, A.; Gilon, C.; Friedler, A. A comparative study of backbone versus side chain peptide cyclization: Application for HIV-1 integrase inhibitors. Bioorg. Med. Chem., 2012, 20(10), 3317-3322. Rafaeli, A. Pheromone biosynthesis activating neuropeptide (PBAN): regulatory role and mode of action. Gen. Comp. Endocrinol., 2009, 162(1), 69-78. Gilon, C.; Zeltser, I.; Daniel, S.; Aziz, O.B.; Schefler, I.; Alstein, M. Rationally designed neuropeptide antagonists: anovel approch for generation of environmentally friendly insecticides. Invert. Neurosci., 1997, 3, 245-250. Minder, E.I.; Barman-Aksoezen, J.; Schneider-Yin, X. Pharmacokinetics and pharmacodynamics of afamelanotide and its clinical use in treating dermatologic disorders. Clin. Pharmacokinet., 2017, 56(8), 815-823. Hess, S.; Linde, Y.; Ovadia, O.; Safrai, E.; Shalev, D.E.; Swed, A.; Halbfinger, E.; Lapidot, T.; Winkler, I.; Gabinet, Y.; Faier, A.; Yarden, D.; Xiang, Z.; Portillo, F.P.; Haskell-Luevano, C.; Gilon, C.; Hoffman, A. Backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administrated drug lead for treating obesity. J. Med. Chem., 2008, 51(4), 1026-1034. Zhang, Q.; Lenardo, M.J.; Baltimore, D. 30 years of NF-kappaB: A blossoming of relevance to human pathobiology. Cell, 2017, 168(12), 37-57. Schwartz, P.A.; Murray, B.W. Protein kinase biochemistry and drug discovery. Bioorg. Chem., 2011, 39(5-6), 192-210. Sardar, S.; Andersson, A. Old and new therapeutics for Rheumatoid Arthritis: in vivo models and drug development. Immunopharmacol. Immunotoxicol., 2016, 38(1), 2-13. Naveh, S.; Tal-Gan, Y.; Ling, S.; Hoffman, A.; Holoshitz, J.; Gilon, C. Developing potent backbone cyclic peptides bearing the shared epitope sequence as rheumatoid arthritis drug-leads. Bioorg. Med. Chem. Lett., 2012, 22(1), 493-496. Hurevich, M.; Talhami, A.; Shalev, D.E.; Gilon, C. Allosteric inhibition of G-protein coupled receptor oligomerization: strategies and challenges for drug development. Curr. Top. Med. Chem., 2014, 14(15), 1842-1863. Horuk, R. Chemokine receptor antagonists: overcoming developmental hurdles. Nat. Rev. Drug Discov., 2009, 8(1), 23-33. Nywening, T.M.; Wang-Gillam, A.; Sanford, D.E.; Belt, B.A.; Panni, R.Z.; Cusworth, B.M.; Toriola, A.T.; Nieman, R.K.; Worley, L.A.; Yano, M.; Fowler, K.J.; Lockhart, A.C.; Suresh, R.; Tan, B.R.; Lim, K.H.; Fields, R.C.; Strasberg, S.M.; Hawkins, W.G.; DeNardo, D.G.; Goedegebuure, S.P.; Linehan, D.C. Targeting tumour-associated macrophages with CCR2 inhibition in combination with FOLFIRINOX in patients with borderline resectable and locally advanced pancreatic cancer: a single-centre, open-label, dose-finding, non-randomised, phase 1b trial. Lancet Oncol., 2016, 17(5), 651-662. Bork, K.; Yasothan, U.; Kirkpatrick, P. Icatibant. Nat. Rev. Drug Discov., 2008, 7(10), 801-802. Schumann, C.; Seyfarth, L.; Greiner, G.; Paegelow, I.; Reissmann, S. Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues. J. Pept. Res., 2002, 60(2), 128140. Kundu, S.; Fan, K.; Cao, M.; Lindner, D.J.; Zhao, Z.J.; Borden, E.; Yi, T. Novel SHP-1 inhibitors tyrosine phosphatase inhibitor-1 and analogs with preclinical anti-tumor activities as tolerated oral agents. J. Immunol., 2010, 184(11), 6529-6536. Liu, C.Y.; Tseng, L.M.; Su, J.C.; Chang, K.C.; Chu, P.Y.; Tai, W.T.; Shiau, C.W.; Chen, K.F. Novel sorafenib analogues induce apoptosis through SHP-1 dependent STAT3 inactivation in human breast cancer cells. Breast Cancer Res., 2013, 15(4), R63. Zoda, M.S.; Zacharias, M.; Reissmann, S. Syntheses and activities of backbone-side chain cyclic octapeptide ligands with Nfunctionalized phosphotyrosine for the N-terminal SH2-domain of the protein tyrosine phosphatase SHP-1. J. Pept. Sci., 2010, 16(8), 403-413.


[130]

synthesis of N-substituted α -amino acids. Angew. Chem. Int. Ed., 1983, 22(1), 65-66. Muller, D.; Zeltser, I.; Bitan, G.; Gilon, C. Building units for Nbackbone cyclic peptides. 3. Synthesis of protected N(alpha)(omega-aminoalkyl) amino acids and N(alpha)-(omegacarboxyalkyl) amino acids. J. Org. Chem., 1997, 62(2), 411-416. Ohfune, Y.; Kurokawa, N.; Higuchi, N.; Saito, M.; Hashimoto, M.; Tanaka, T. An efficient one-step reductive N-monoalkylation of alpha-amino-acids. Chem. Lett., 1984, 3, 441-444. Gellerman, G.; Elgavi, A.; Salitra, Y.; Kramer, M. Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N-backbone cyclic peptide chemistry. J. Pept. Res., 2001, 57(4), 277-291. Wormser, U.; Laufer, R.; Hart, Y.; Chorev, M.; Gilon, C.; Selinger, Z. Highly selective agonists for substance P receptor subtypes. EMBO J., 1986, 5(11), 2805-2808. Laufer, R.; Gilon, C.; Chorev, M.; Selinger, Z. Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand. J. Biol. Chem., 1986, 261(22), 10257-10263. Laufer, R.; Gilon, C.; Chorev, M.; Selinger, Z., Desensitization with a selective agonist discriminates between multiple tachykinin receptors. J. Pharmacol. Exp. Ther., 1988, 245, (2), 639-643. Behrens, S.; Matha, B.; Bitan, G.; Gilon, C.; Kessler, H. Structureactivity relationship of the ring portion in backbone-cyclic Cterminal hexapeptide analogs of substance P. NMR and molecular dynamics. Int. J. Pept. Protein Res., 1996, 48(6), 569-579. Mangus, R.S.; Kinsella, S.B.; Fridell, J.A.; Kubal, C.A.; Lahsaei, P.; Mark, L.O.; Tector, A.J. Aminocaproic acid (Amicar) as an alternative to Aprotinin (Trasylol) in liver transplantation. Transplant. Proc., 2014, 46(5), 1393-1399. Chang, J.Y. Distinct folding pathways of two homologous disulfide proteins: bovine pancreatic trypsin inhibitor and tick anticoagulant peptide. Antioxid. Redox Signal., 2011, 14(1), 127-135. Huber, R.; Kukla, D.; Bode, W.; Schwager, P.; Bartels, K.; Deisenhofer, J.; Steigemann, W. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. J. Mol. Biol., 1974, 89(1), 73-101. Vincent, J.P.; Lazdunski, M. Trypsin-pancreatic trypsin inhibitor association. Dynamics of the interaction and role of disulfide bridges. Biochemistry, 1972, 11(16), 2967-2977. Kasher, R.; Oren, D.A.; Barda, Y.; Gilon, C. Miniaturized proteins: the backbone cyclic proteinomimetic approach. J. Mol. Biol., 1999, 292(2), 421-429. Schwarze, S.R.; Ho, A.; Vocero-Akbani, A.; Dowdy, S.F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science, 1999, 285(5433), 1569-1572. Bechara, C.; Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett., 2013, 587(12), 1693-1702. Guidotti, G.; Brambilla, L.; Rossi, D. Cell-penetrating peptides: From basic research to clinics. Trends Pharmacol. Sci., 2017, 38(4), 406-424. Huang, Y.; Jiang, Y.; Wang, H.; Wang, J.; Shin, M.C.; Byun, Y.; He, H.; Liang, Y.; Yang, V.C. Curb challenges of the "Trojan Horse" approach: smart strategies in achieving effective yet safe cell-penetrating peptide-based drug delivery. Adv. Drug Deliv. Rev., 2013, 65(10), 1299-1315. Blum, A.P.; Kammeyer, J.K.; Gianneschi, N.C. Activating peptides for cellular uptake via polymerization into high density brushes. Chem. Sci., 2016, 7(2), 989-994. Sharei, A.; Zoldan, J.; Adamo, A.; Sim, W.Y.; Cho, N.; Jackson, E.; Mao, S.; Schneider, S.; Han, M.J.; Lytton-Jean, A.; Basto, P.A.; Jhunjhunwala, S.; Lee, J.; Heller, D.A.; Kang, J.W.; Hartoularos, G.C.; Kim, K. S.; Anderson, D.G.; Langer, R.; Jensen, K.F. A vector-free microfluidic platform for intracellular delivery. Proc. Natl. Acad. Sci., 2013, 110(6), 2082-2087. Friedler, A.; Friedler, D.; Luedtke, N.W.; Tor, Y.; Loyter, A.; Gilon, C. Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif. J. Biol. Chem., 2000, 275(31), 23783-23789. Friedler, A.; Zakai, N.; Karni, O.; Broder, Y.C.; Baraz, L.; Kotler, M.; Loyter, A.; Gilon, C. Backbone cyclic peptide, which mimics the nuclear localization signal of human immunodeficiency virus type 1 matrix protein, inhibits nuclear import and virus production in nondividing cells. Biochemistry, 1998, 37(16), 5616-5622.

Rubin and Qvit

[157] [158] [159]

[160]

[161]

[162]

[163]

[164] [165]

[166]

[167]

[168]


[170] [171]

[172]

[173]

[174]

[175]

[176] [177] [178] [179]

[180] [181]

[182]

[183]

[184] [185]

[186] [187] [188] [189]

Bergeron, R.J.; Garlich, J.R.; Stolowich, N.J. Reagents for the stepwise functionalization of spermidine, homospermidine, and bis(3-aminopropyl)amine. J. Org. Chem., 1984, 49(16), 2997-3001. Gazal, S.; Gelerman, G.; Gilon, C. Novel Gly building units for backbone cyclization: synthesis and incorporation into model peptides. Peptides, 2003, 24(12), 1847-1852. Falb, E.; Salitra, Y.; Yechezkel, T.; Bracha, M.; Litman, P.; Olender, R.; Rosenfeld, R.; Senderowitz, H.; Jiang, S.; Goodman, M. A bicyclic and hsst2 selective somatostatin analogue: design, synthesis, conformational analysis and binding. Bioorg. Med. Chem., 2001, 9(12), 3255-3264. Giblin, M.F.; Wang, N.; Hoffman, T.J.; Jurisson, S.S.; Quinn, T.P. Design and characterization of alpha-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination. Proc. Natl. Acad. Sci. U.S.A., 1998, 95(22), 12814-12818. Melendez-Alafort, L.; Ferro-Flores, G.; Arteaga-Murphy, C.; Pedraza-Lopez, M.; Gonzalez-Zavala, M.A.; Tendilla, J.I.; GarciaSalinas, L. Labeling peptides with rhenium-188. Int. J. Pharm., 1999, 182(2), 165-172. Fujino, M.; Fukuda, T.; Shinagawa, S.; Kobayashi, S.; Yamazaki, I. Synthetic analogs of luteinizing hormone releasing hormone (LHRH) substituted in position 6 and 10. Biochem. Biophys. Res. Commun., 1974, 60(1), 406-413. Crowley, W.F. Jr.; Beitins, I.Z.; Vale, W.; Kliman, B.; Rivier, J.; Rivier, C.; McArthur, J.W. The biologic activity of a potent analogue of gonadotropin-releasing hormone in normal and hypogonadotropic men. N. Engl. J. Med., 1980, 302(19), 1052-1057. Borra, R.; Camarero, J.A. Recombinant expression of backbonecyclized polypeptides. Biopolymers, 2013, 100(5), 502-509. Vila-Perello, M.; Liu, Z.; Shah, N.H.; Willis, J.A.; Idoyaga, J.; Muir, T.W. Streamlined expressed protein ligation using split inteins. J. Am. Chem. Soc., 2013, 135(1), 286-292. David, R.; Richter, M.P.; Beck Sickinger, A.G. Expressed protein ligation. Eur. J. Biochem., 2004, 271(4), 663-677. Morioka, T.; Loik, N.D.; Hipolito, C.J.; Goto, Y.; Suga, H. Selection-based discovery of macrocyclic peptides for the next generation therapeutics. Curr. Opin. Chem. Biol., 2015, 26, 34-41. Kawakami, T.; Ohta, A.; Ohuchi, M.; Ashigai, H.; Murakami, H.; Suga, H. Diverse backbone-cyclized peptides via codon reprogramming. Nat. Chem. Biol., 2009, 5(12), 888-890. Giebel, L.B.; Cass, R.T.; Milligan, D.L.; Young, D.C.; Arze, R.; Johnson, C.R. Screening of cyclic peptide phage libraries identifies ligands that bind streptavidin with high affinities. Biochemistry, 1995, 34(47), 15430-15435. Kritzer, J.A.; Hamamichi, S.; McCaffery, J.M.; Santagata, S.; Naumann, T.A.; Caldwell, K.A.; Caldwell, G.A.; Lindquist, S. Rapid selection of cyclic peptides that reduce alpha-synuclein toxicity in yeast and animal models. Nat. Chem. Biol., 2009, 5(9), 655663. Kang, T.J.; Hayashi, Y.; Suga, H. Synthesis of the backbone cyclic peptide sunflower trypsin inhibitor-1 promoted by the induced peptidyl-tRNA drop-off. Angew. Chem. Int. Ed. Engl., 2011, 50(9), 2159-2161. Bulet, P.; Stocklin, R.; Menin, L. Anti-microbial peptides: from invertebrates to vertebrates. Immunol. Rev., 2004, 198, 169-184. Kharrat, R.; Mabrouk, K.; Crest, M.; Darbon, H.; Oughideni, R.; Martin-Eauclaire, M.F.; Jacquet, G.; el Ayeb, M.; Van Rietschoten, J.; Rochat, H.; Sabatier, J.M. Chemical synthesis and characterization of maurotoxin, a short scorpion toxin with four disulfide bridges that acts on K+ channels. Eur. J. Biochem., 1996, 242(3), 491-498. Moore, R.E. Cyclic peptides and depsipeptides from cyanobacteria: A review. J. Ind. Microbiol., 1996, 16(2), 134-143. Craik, D.J. Discovery and applications of the plant cyclotides. Toxicon, 2010, 56(7), 1092-1102. Weidmann, J.; Craik, D.J. Discovery, structure, function, and applications of cyclotides: Circular proteins from plants. J. Exp. Bot., 2016, 67(16), 4801-4812. Plan, M.R.; Saska, I.; Cagauan, A.G.; Craik, D.J. Backbone cyclised peptides from plants show molluscicidal activity against the rice pest Pomacea canaliculata (golden apple snail). J. Agric. Food Chem., 2008, 56(13), 5237-5241.

[190]

[191] [192]

[193] [194]

[195] [196]

[197] [198]

[199]

[200] [201]

[202]

[203]

[204]

[205]

[206]

[207] [208]

[209]

[210]

[211] [212] [213]

555

Daly, N.L.; Gustafson, K.R.; Craik, D.J. The role of the cyclic peptide backbone in the anti-HIV activity of the cyclotide kalata B1. FEBS Lett., 2004, 574(1-3), 69-72. DiMasi, J.A.; Feldman, L.; Seckler, A.; Wilson, A. Trends in risks associated with new drug development: Success rates for investigational drugs. Clin. Pharmacol. Ther., 2010, 87(3), 272-277. DiMasi, J.A.; Grabowski, H.G.; Hansen, R.W. Innovation in the pharmaceutical industry: New estimates of R&D costs. J. Health Econ., 2016, 47, 20-33. Scannell, J.W.; Blanckley, A.; Boldon, H.; Warrington, B. Diagnosing the decline in pharmaceutical R&D efficiency. Nat. Rev. Drug Discov., 2012, 11(3), 191-200. Scannell, J.W.; Bosley, J. When quality beats quantity: Decision theory, drug discovery, and the reproducibility crisis. PLoS One, 2016, 11(2), e0147215. Marzinzik, A.L.; Vorherr, T.E. Towards intracellular delivery of peptides. CHIM. Int. J. Chem., 2013, 67(12), 899-904. Uhlig, T.; Kyprianou, T.; Martinelli, F.G.; Oppici, C.A.; Heiligers, D.; Hills, D.; Calvo, X.R.; Verhaert, P. The emergence of peptides in the pharmaceutical business: From exploration to exploitation. EuPA Open Proteom., 2014, 4, 58-69. Vorherr, T. Modifying peptides to enhance permeability. Future Med. Chem., 2015, 7(8), 1009-1021. Reichert, J. Development trends for peptide therapeutics. In Peptide therapeutics symposium: San Diego, CA, 2010. Kaspar, A.A.; Reichert, J.M. Future directions for peptide therapeutics development. Drug Discov. Today, 2013, 18(17-18), 807-817. Bhat, A.; Roberts, L.R.; Dwyer, J.J. Lead discovery and optimization strategies for peptide macrocycles. Eur. J. Med. Chem., 2015, 94, 471-479. Joo, S.H. Cyclic peptides as therapeutic agents and biochemical tools. Biomol. Ther. (Seoul), 2012, 20(1), 19-26. Tapeinou, A.; Matsoukas, M.T.; Simal, C.; Tselios, T. Review cyclic peptides on a merry-go-round; towards drug design. Biopolymers, 2015, 104(5), 453-461. Qvit, N.; Rubin, S.J.S.; Urban, T.J.; Mochly-Rosen, D.; Gross, E.R. Peptidomimetic therapeutics: Scientific approaches and opportunities. Drug Discov. Today, 2017, 22(2), 454-462. Stoop, R.; Hegoburu, C.; van den Burg, E. New opportunities in vasopressin and oxytocin research: A perspective from the amygdala. Annu. Rev. Neurosci., 2015, 38, 369-388. Vande Walle, J.; Stockner, M.; Raes, A.; Norgaard, J.P. Desmopressin 30 years in clinical use: A safety review. Curr. Drug Saf., 2007, 2(3), 232-238. Ozgonenel, B.; Rajpurkar, M.; Lusher, J.M. How do you treat bleeding disorders with desmopressin? Postgrad. Med. J., 2007, 83(977), 159-163. Kam, P.C.; Williams, S.; Yoong, F.F. Vasopressin and terlipressin: Pharmacology and its clinical relevance. Anaesthesia, 2004, 59(10), 993-1001. Cuevas-Ramos, D.; Fleseriu, M. Pasireotide: A novel treatment for patients with acromegaly. Drug Des. Devel. Ther., 2016, 10, 227239. Lesche, S.; Lehmann, D.; Nagel, F.; Schmid, H.A.; Schulz, S. Differential effects of octreotide and pasireotide on somatostatin receptor internalization and trafficking in vitro. J. Clin. Endocrinol. Metab., 2009, 94(2), 654-661. Blanc, E.; Sabatier, J.M.; Kharrat, R.; Meunier, S.; el Ayeb, M.; Van Rietschoten, J.; Darbon, H. Solution structure of maurotoxin, a scorpion toxin from Scorpio maurus, with high affinity for voltagegated potassium channels. Proteins, 1997, 29(3), 321-333. DeLano, W.L. The PyMOL Molecular Graphics System, DeLano Scientific: San Carlos, 2002. Matthews, S.; Barlow, P.; Clark, N.; Kingsman, S.; Kingsman, A.; Campbell, I. Refined solution structure of p17, the HIV matrix protein. Biochem. Soc. Trans., 1995, 23(4), 725-729. von Schwedler, U.; Kornbluth, R.S.; Trono, D. The nuclear localization signal of the matrix protein of human immunodeficiency virus type 1 allows the establishment of infection in macrophages and quiescent T lymphocytes. Proc. Natl. Acad. Sci. USA., 1994, 91(15), 6992-6996.


[169]
