Natural Proline-Rich Cyclopolypeptides from Marine Organisms: Chemistry, Synthetic Methodologies and Biological Status

Peptides have gained increased interest as therapeutics during recent years. More than 60 peptide drugs have reached the market for the benefit of patients and several hundreds of novel therapeutic peptides are in preclinical and clinical development. The key contributor to this success is the potent and specific, yet safe, mode of action of peptides. Among the wide range of biologically-active peptides, naturally-occurring marine-derived cyclopolypeptides exhibit a broad range of unusual and potent pharmacological activities. Because of their size and complexity, proline-rich cyclic peptides (PRCPs) occupy a crucial chemical space in drug discovery that may provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites. Diverse pharmacological activities of natural cyclic peptides from marine sponges, tunicates and cyanobacteria have encouraged efforts to develop cyclic peptides with well-known synthetic methods, including solid-phase and solution-phase techniques of peptide synthesis. The present review highlights the natural resources, unique structural features and the most relevant biological properties of proline-rich peptides of marine-origin, focusing on the potential therapeutic role that the PRCPs may play as a promising source of new peptide-based novel drugs.


Introduction
An interesting class of marine cyclic peptides is represented by the proline-rich compounds usually containing more than six or seven amino acid residues. The role of proline in these molecules has been linked to the control of the conformation of the molecule in solution because of the restricted φ of proline. The proline-rich cyclic peptides (PRCPs) are formed by linking one end of the peptide and the other with an amide bond or other chemically-stable bonds. Some of them are used in the clinic, e.g., gramicidin S and tyrocidine with bactericidal activity, while others are in clinical trials, e.g., dehydrodidemnin B, and most of them originate from natural resources. Although the literature is enriched with reports concerned with marine-derived linear proline-rich bioactive peptides [1][2][3][4][5], e.g., dolastatin 15, kurahyne B, jahanyne, cemadotin, koshikamide A 1 , etc., PRCPs from marine

Stability and Comparison with Linear Peptides
Linear peptides that contain less than 10 amino acid residues are especially flexible in solution. Once the length of linear peptides extends to between 10 and 20 amino acid residues, random linear peptide sequences can begin to obtain secondary structures, including α-helices, turns and β-strands. These secondary structures impose constraints that reduce the free energy of linear peptides and limit their conformations to those that may be more biologically active. The constraints imposed by cyclization force cyclic peptides to adopt a limited number of molecular conformations in solution. Generally, if cyclization limits conformations to those required for optimum receptor binding, these cyclic peptides would be more useful compared with their linear counterparts that can adopt more conformations, which are not useful for receptor binding. Cyclization has been shown to increase the propensity for β-turn formation in peptides, which is of vital utility since β-turns are often found in native proteins. Although peptide cyclization generally induces structural constraints, the site of cyclization within the sequence can affect the binding affinity of cyclic peptides.
In the case of proline, which is a proteinogenic amino acid with a secondary amine that does not follow along with the typical Ramachandran plot, the ψ and φ angles about the peptide bond have fewer allowable degrees of rotation due to the ring formation connected to the beta carbon. As a result, it is often found in "turns" of peptides/proteins, as its free entropy (∆S) is not as comparatively large as other amino acids, and thus, in a folded form vs. unfolded form, the change in entropy is less. Furthermore, proline is rarely found in α and β structures, as it would reduce the stability of such structures, because its side chain α-N can only form one hydrogen bond.
Further, the hydroxylation of proline by prolyl hydroxylase and other additions of electron-withdrawing substituents, such as fluorine, increases the conformational stability of collagen significantly. Hence, the hydroxylation of proline is a critical biochemical process for maintaining the connective tissue of higher organisms. Polypeptide chains containing proline lack the flexibility of other peptides, because the proline ring has only one available angle for backbone rotation. Rotation occurs around the angles φ, ψ and ω [68,69].
The cyclization of linear peptide sequences can create constrained geometries that can alter the specificity of cyclic peptides to different isoforms or subtypes of targeted receptors. Peptides can be cyclized in order to reduce the overall numbers of interchanging conformers in the hope of limiting them to those selective for the desired receptors while avoiding degradation by not forming conformers susceptible to interacting with proteolytic enzymes [70].
In general, cyclization often increases the stability of peptides [71,72], which can prolong their biological activity. This prolonged activity may even be the result of additional resistance to enzymatic degradation by exoproteases that preferentially cleave near the Nor C-termini of peptide sequences. In particular, cyclization can create peptides with the ability to penetrate tumors in order to enhance the potency of anticancer drugs [73]. Cyclic peptides can potentially obtain desirable constrained geometries that are responsible for increasing their binding affinity, specificity or stability compared with their linear counterparts. Cyclic peptides are of considerable interest as potential protein ligands and might be more cell permeable than their linear counterparts due to their reduced conformational flexibility. However, it is important to note that cyclization does not necessarily lead to improvements in all of these properties, e.g., linear peptides can contain sequences that can support rigid structures without the need for cyclization [74].

Structural Features
The distinctive cyclic structure of proline's side chain gives proline an exceptional conformational rigidity compared to other amino acids, which affects the rate of peptide bond formation between proline and other amino acids. The exceptional conformational rigidity of proline affects the secondary structure of proteins near a proline residue and may account for proline's higher prevalence in the proteins of thermophilic organisms. Proline acts as a structural disruptor in the middle of regular secondary structure elements, such as alpha helices and beta sheets; however, proline is commonly found as the first residue of an alpha helix and also in the edge strands of beta sheets. Multiple prolines and hydroxyprolines in a row can create a polyproline helix, the predominant secondary structure in collagen [75].
Furthermore, unusual amino acid residues like pHSA and pyroGlu were found to be part of the structure of cyclothiopeptide gombamide A, which possess moderate inhibitory activity against Na + /K + -ATPase [22]. Further, thiazoline-based proline containing doubly-prenylated cyclopeptides like trunkamide A contain reverse prenylated ethers of serine and threonine together in their composition. Heterocyclic amino acids like histidine and tryptophan also form part of the structures of proline-rich cyclic peptides, such as wainunuamide, phakellistatin 15, 17 and stylissatin B [23,25,97]. Moreover, cytotoxic phakellistatin 3 and isophakellistatin 3 represent a new class of proline-rich cycloheptapeptides containing an unusual amino acid unit "Hpi" that apparently derived from a photooxidation product of tryptophan [100].
Moreover, five-residue cystine-linked cyclic peptides like eudistomides A, B are flanked by a C-terminal methyl ester and a 12-oxo-or 12-hydroxy-tetradecanoyl moiety [42]. The structure of proline containing cytotoxic peptide scleritodermin A incorporates a novel conjugated thiazole moiety 2-(1-amino-2-p-hydroxyphenylethane)-4-(4-carboxy-2,4-dimethyl-2Z,4E-propadiene)-thiazole (ACT) and unusual amino acids O-methyl-N-sulfo-D-serine, keto-allo-isoleucine [26]. The proline unit may be part of a cyclic peptide and/or may be part of a side chain, e.g., scleritodermin A, didemnin B, C and plitidepsin [26,43,45], or may be part of a linear peptide, e.g., dolastatin 15 and koshikamide A 1 [1,5]. The methyloxazoline ring is the part of the composition of cyclohexapeptides ceratospongamides [10]. In addition, trichormamide A contains β-amino acid residue viz. β-aminodecanoic acid, in addition to two D-amino acid residues (D-Tyr and D-Leu) [49]. The wewakpeptins, proline-rich cyclic depsipeptides contain unusual moieties, like "Dhoya", "Maba" and "Hiva" [46], and prenylagaramides B and C contain a rare "Ptyr" unit. Moreover, nostophycin bears a novel β-amino acid moiety "Ahoa" in its structure [50]. Macrocyclic depsipeptides, homodolastatin 16 and dolastatin 16 contain the new and unusual amino acid units "Pval" and "Admpa" [51,52]. Besides this, structural features for pahayokolides A and B include a pendant N-acetyl-N-methylleucine, both Eand Z-dehydrobutyrines, a homophenylalanine and an unusual polyhydroxy amino acid [48]. Oxazole and methyloxazole rings were found to be part of the structures of cyclopolypeptides myriastramides A-C and haliclonamide A [28,93], whereas N,O-dimethyl tyrosine and "Hiv" moieties were found in the structures of cytotoxic depsipeptides, tamandarins A and B [44]. The presence of two dimethylallyl threonines (or one threonine and one serine) side chains and one thiazoline ring in the backbone of the patellins is the most important feature of these compounds termed as "cyanobactins", which have sparked attention due to their interesting bioactivities and for their potential to be prospective candidates in the development of drugs [101,102].

Stereochemical Aspects
Structurally, proline is the only unusual amino acid with a secondary amino group based on a pyrrolidine, which forms a ring structure with rigid conformation and a secondary amine compared to the other twenty natural amino acids. This significantly reduces the structural flexibility of the polypeptide chain, and the nitrogen in the pyrrolidine ring cannot participate in hydrogen bonding with other residues [103]. Many biologically-important cyclic peptide sequences and natural products contain multiple proline residues. As seen previously for peptide bonds, the proline amide bond can also exist in trans or cis conformations (Figure 4). Peptide bonds to proline, and to other N-substituted amino acids, are able to populate both the cis and trans isomers. Most peptide bonds overwhelmingly adopt the trans isomer (typically 99.9% under unstrained conditions), because the amide hydrogen (trans isomer) offers less steric repulsion to the preceding C α atom than does the following C α atom (cis isomer). By contrast, the cis and trans isomers of the X-Pro peptide bond (where X represents any amino acid) both experience steric clashes with the neighboring substitution and are nearly equal energetically. Hence, the fraction of X-Pro peptide bonds in the cis isomer under unstrained conditions ranges from 10% to 40%; the fraction depends slightly on the preceding amino acid, with aromatic residues favoring the cis isomer slightly. Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding [104]. Furthermore, proline cis-trans isomerization controls autoinhibition of a signaling protein [105].
X represents any amino acid) both experience steric clashes with the neighboring substitution and are nearly equal energetically. Hence, the fraction of X-Pro peptide bonds in the cis isomer under unstrained conditions ranges from 10% to 40%; the fraction depends slightly on the preceding amino acid, with aromatic residues favoring the cis isomer slightly. Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding [104]. Furthermore, proline cis-trans isomerization controls autoinhibition of a signaling protein [105]. Although the trans amide bond is more common, the occurrence of cis geometry is more frequent for the proline peptide bond than for other amino acids. The frequency of the cis proline peptide bond is higher in cyclic peptides than in linear peptides. As per a statistical study performed on the Cambridge Structural Database, 57.4% of proline residues present in cyclic peptides were in the cis conformation as compared to only 5.6% in acyclic peptides [106]. The reason for this high proportion of cis proline in cyclopeptides is due to the conformational restrictions during the cyclisation step. The geometry of the proline amide can be determined on the basis of the difference in 13 C chemical shifts between Cβ and Cγ signals (Δδβγ = δβ − δγ). A small 13 C chemical shift difference indicates that the proline peptide bond is trans, while a large 13 C chemical shift difference indicates a cis proline residue. The change in conformation of a cyclopolypeptide from "trans" to "cis" can result in loss of activity [10], e.g., the trans, trans-isomer of cyclic heptapeptide ceratospongamide showed potent inhibition of sPLA2 expression in a cell-based model for anti-inflammation, whereas the cis, cis-isomer was inactive ( Figure 5). The distribution of the peptide bond angle omega for peptidyl-prolyl bonds in proteins shows significant peaks at 180° (trans peptide bond) and 0° (cis peptide bond). Investigations on "peptidyl-prolyl bonds and secondary structure" showed that trans petidyl-prolyl bonds are distributed in all types of secondary structure, whereas cis peptidyl is found primarily in bends and turns, suggesting a specific structural role for this type of bonding.  Although the trans amide bond is more common, the occurrence of cis geometry is more frequent for the proline peptide bond than for other amino acids. The frequency of the cis proline peptide bond is higher in cyclic peptides than in linear peptides. As per a statistical study performed on the Cambridge Structural Database, 57.4% of proline residues present in cyclic peptides were in the cis conformation as compared to only 5.6% in acyclic peptides [106]. The reason for this high proportion of cis proline in cyclopeptides is due to the conformational restrictions during the cyclisation step. The geometry of the proline amide can be determined on the basis of the difference in 13 C chemical shifts between Cβ and Cγ signals (∆δβγ = δβ − δγ). A small 13 C chemical shift difference indicates that the proline peptide bond is trans, while a large 13 C chemical shift difference indicates a cis proline residue. The change in conformation of a cyclopolypeptide from "trans" to "cis" can result in loss of activity [10], e.g., the trans, trans-isomer of cyclic heptapeptide ceratospongamide showed potent inhibition of sPLA 2 expression in a cell-based model for anti-inflammation, whereas the cis, cis-isomer was inactive ( Figure 5). The distribution of the peptide bond angle omega for peptidyl-prolyl bonds in proteins shows significant peaks at 180 • (trans peptide bond) and 0 • (cis peptide bond). Investigations on "peptidyl-prolyl bonds and secondary structure" showed that trans petidyl-prolyl bonds are distributed in all types of secondary structure, whereas cis peptidyl is found primarily in bends and turns, suggesting a specific structural role for this type of bonding.
following Cα atom (cis isomer). By contrast, the cis and trans isomers of the X-Pro peptide bond (where X represents any amino acid) both experience steric clashes with the neighboring substitution and are nearly equal energetically. Hence, the fraction of X-Pro peptide bonds in the cis isomer under unstrained conditions ranges from 10% to 40%; the fraction depends slightly on the preceding amino acid, with aromatic residues favoring the cis isomer slightly. Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding [104]. Furthermore, proline cis-trans isomerization controls autoinhibition of a signaling protein [105]. Although the trans amide bond is more common, the occurrence of cis geometry is more frequent for the proline peptide bond than for other amino acids. The frequency of the cis proline peptide bond is higher in cyclic peptides than in linear peptides. As per a statistical study performed on the Cambridge Structural Database, 57.4% of proline residues present in cyclic peptides were in the cis conformation as compared to only 5.6% in acyclic peptides [106]. The reason for this high proportion of cis proline in cyclopeptides is due to the conformational restrictions during the cyclisation step. The geometry of the proline amide can be determined on the basis of the difference in 13 C chemical shifts between Cβ and Cγ signals (Δδβγ = δβ − δγ). A small 13 C chemical shift difference indicates that the proline peptide bond is trans, while a large 13 C chemical shift difference indicates a cis proline residue. The change in conformation of a cyclopolypeptide from "trans" to "cis" can result in loss of activity [10], e.g., the trans, trans-isomer of cyclic heptapeptide ceratospongamide showed potent inhibition of sPLA2 expression in a cell-based model for anti-inflammation, whereas the cis, cis-isomer was inactive ( Figure 5). The distribution of the peptide bond angle omega for peptidyl-prolyl bonds in proteins shows significant peaks at 180° (trans peptide bond) and 0° (cis peptide bond). Investigations on "peptidyl-prolyl bonds and secondary structure" showed that trans petidyl-prolyl bonds are distributed in all types of secondary structure, whereas cis peptidyl is found primarily in bends and turns, suggesting a specific structural role for this type of bonding.  Most amino acids occur in two possible optical isomers, called D and L ( Figure 6). The L-amino acids represent the vast majority of amino acids found in proteins. L-proline is a natural non-essential amino acid, and D-proline is an unnatural amino acid, with one basic and one acidic center each. In proline, only the L-stereoisomer is involved in the synthesis of mammalian peptides/proteins. Most amino acids occur in two possible optical isomers, called D and L ( Figure 6). The L-amino acids represent the vast majority of amino acids found in proteins. L-proline is a natural non-essential amino acid, and D-proline is an unnatural amino acid, with one basic and one acidic center each. In proline, only the L-stereoisomer is involved in the synthesis of mammalian peptides/proteins. The racemization of L-proline to D-proline proceeds through a planar transition state, where the tetrahedral α-carbon becomes trigonal as a proton leaves the L-proline. The transition-state analog for this step is pyrrolidin-2-ide-2-carboxylate (2 − ). The absolute configuration of proline residue can be determined by Marfey's method using reagent 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide (FDAA) [107]. The absolute configuration of amino proline was determined by comparing the retention time with the standard FDAA-derivatized amino acids, e.g., the structure of cyclooctapeptide reniochalistatin E contains three L-proline units with trans conformation [20] whereas the structure of cycloheptapeptide euryjanicin E contains three L-proline units with cis conformation [88]. Further, a novel cyclic tetrapeptide isolated from a Pseudomonas sp. (strain IM-1) associated with the marine sponge Ircinia muscarum was found to contain two proline units, one with L-configuration and the other with D-configuration [77].

Steric and Lipophilicity Parameters
In order to describe the intermolecular forces of drug receptor interaction, as well as the transport and distribution of drugs in a quantitative manner, various steric and lipophilicity parameters, like molar refractivity (MR 20

Steric and Lipophilicity Parameters
In order to describe the intermolecular forces of drug receptor interaction, as well as the transport and distribution of drugs in a quantitative manner, various steric and lipophilicity parameters, like molar refractivity (MR 20 ), molar volume (MV 20 ), parachor (P r ), index of refraction (n 20 ), surface tension (γ 20 ), density (d 20 ), polarizability (α), etc., need to be calculated for natural cyclic peptides. Diverse parameters were calculated for proline-rich cyclopolypeptides of marine origin using ACD/ChemSketch software (Version 2.0, Toronto, ON, Canada) (Table S1, Supplementary Materials).

Synthetic Methodologies
Many proline-rich cyclic peptides were synthesized successfully by various research groups employing different techniques of peptide synthesis. The literature is enriched with reports explaining the synthesis of euryjanicin A [108], delavayin C [109], cherimolacyclopeptide G [110], psammosilenin A [111], hymenamide E [112], stylisin 1 [113], stylisin 2 [114], hymenistatin and yunnanin F [115], pseudostellarin B [116], segetalin E [117], rolloamide B [118] and pseudostellarin G [119] using the solution-phase method utilizing different carbodiimides as coupling agents, TEA/NMM as the base and the synthesis of euryjanicin B [120], mollamide [121], met-cherimolacyclopeptide B [122], axinellin A [123], phakellistatin 7 [124], phakellistatin 12 [125], petriellin A [126], hymenamide C [127], gombamide A [128] and scleritodermin A [129] by the solid-phase method of peptide synthesis. Solid-phase peptide synthesis (SPPS) results in high yields of pure products and works more quickly than classical synthesis, i.e., liquid-phase peptide synthesis (LPPS). Through the replacement of a complicated isolation procedure for each intermediate product with a simple washing procedure, much time is saved using SPPS. In addition, SPPS has proven possible to increase the yield in each individual step to 99.5% or better, which cannot be attained using conventional synthetic approaches. However, solution phase synthesis continues to be especially valuable for large-scale manufacturing and for specialized laboratory applications [130,131]. Moreover, in some cases, a mixed solid-phase/solution synthesis strategy is employed to accomplish total synthesis of the cyclopolypeptide [132], e.g., during the total synthesis of the naturally-occurring proline-rich cyclic octapeptide stylissamide X, the linear octapeptide was assembled first by standard Fmoc solid-phase peptide synthesis (SPPS), and cyclization was carried out subsequently by the solution method. Total synthesis can also be achieved via a convergent native chemical ligation-oxidation strategy [133], e.g., polydiscamides B-D, or utilizing diethyl phosphorocyanidate/BOP-Cl chemistry [134], e.g., axinastatins 2 and 3.

Biological Status
L-proline itself is an osmoprotectant and is used in many pharmaceutical and biotechnological applications, whereas the proline analogue cis-4-hydroxy-L-proline has been clinically evaluated as an anticancer drug. Although proline-rich cyclopolypeptides of marine origin are associated with a number of bioactivities, including anti-cancer, anti-tuberculosis, anti-inflammatory, anti-viral, immunosuppressive and anti-fungal activities, still the majority of them were found to exhibit cell growth inhibitory activity [135,136]. Various pharmacological activities associated marine-derived proline-rich cyclopeptides along with susceptible cell line/organism with minimum inhibitory concentration are compiled in Table 2.

Mechanism of Action
In drug development, a good antimicrobial candidate should exhibit highly specific biological activity followed by a good pharmacokinetic profile and low immunogenicity. Studies have demonstrated that the members of the proline-rich peptide group and their derivatives act with a completely divergent mechanism than the lytic amphiphilic antimicrobial peptides. Retaining highly potent antimicrobial activities, proline-rich antimicrobial peptides subsequently act in a divergent way, including stereospecific interaction with the membrane translocation system followed by intracellular targeting, compared with the more general membrane disruption mode of action of traditional antimicrobial peptides. It has been further suggested that proline-rich antimicrobial peptides stereo-specifically bind to intracellular targets, such as the bacterial heat shock DnaK protein, and this binding can be correlated with the observed antimicrobial activity. Moreover, proline-rich peptides are characterized by good water solubility, high potency against bacteria killing and low cytotoxic effects at high concentrations, making them attractive lead candidates for the development of novel antimicrobial therapeutic agents [103].
Further, proline-rich antimicrobial peptides are actively transported inside the bacterial cell where they bind and inactivate specific targets like the bacterial ribosome and, thereby, inhibit protein synthesis. This implies that they can be used as molecular hooks to identify the intracellular or membrane proteins that are involved in their mechanism of action and that may be subsequently used as targets for the design of novel antibiotics with mechanisms different from those now in use. Didemnin B is a heterodetic non-polar cyclic peptide associated with antiviral, antitumor, immunomodulating properties, potently inhibits protein and DNA synthesis by binding to the eukaryotic translation elongation factor EF-1α in a GTP-dependent manner, and the formation of the didemnin B-GTP-EF-1α complex may be responsible for the observed inhibition of protein synthesis [139]. Inhibition of protein synthesis by didemnin B occurs by stabilization of aminoacyl-tRNA to the ribosomal A-site, preventing the translocation of phenylalanyl-tRNA from the A-to the P-site, but not preventing peptide bond formation. Tamandarin A may act by the same mechanism as didemnin B. Aplidine's (dehydrodidemnin B) mechanism of action involves several pathways, including cell cycle arrest and inhibition of protein synthesis. Aplidine induces early oxidative stress and results in a rapid and persistent activation of JNK and p38 MAPK phosphorylation with activation of both kinases occurring very rapid, long before the execution of apoptosis [140]. Didemnin B induces the death of a variety of transformed cells with apoptotic morphology, DNA fragmentation within the cytosol and the generation of DNA ladders. Scleritodermin A acts by tubulin polymerization inhibition [26].
The immunosuppressive activity of cyclolinopeptide A results from the formation of the complex with cyclophilin and inhibition of the phosphatase activity of calcineurin, a phosphatase that plays an important role in T lymphocyte signaling [141]. Cemadotin (LU103793) is a water-soluble synthetic analogue of linear peptide dolastatin 15, which is believed to act on microtubules involving binding to tubulin and strong suppression of microtubule dynamics.

Peptide Market and PRCPs in Clinical Trials
Currently, there are more than 60 U.S. Food and Drug Administration (FDA)-approved peptide medicines on the market, and this is expected to grow significantly, with approximately 140 peptide drugs currently in clinical trials and more than 500 therapeutic peptides in preclinical development. In terms of value, the global peptide drug market has been predicted to increase from US$14.1 billion in 2011 to an estimated US$25.4 billion in 2018, with an underlying increase in novel innovative peptide drugs from US$8.6 billion in 2011 (60%) to US$17.0 billion (66%) in 2018 [74]. Currently, most peptide drugs are administered by the parental route, and approximately 75% are given as injectables. However, alternative administration forms are gaining increasing traction, including oral, intranasal and transdermal delivery routes, according to the respective technology developments. The use of alternative administration forms could also enable greater usage of peptide therapeutics in other disease areas, such as inflammation, where topical administration of peptides could be the basis for highly efficacious novel treatments.
The cyclic depsipeptide didemnin B was the first marine-derived cyclopolypeptide to undergo clinical trials targeted at oncological patients. However, high toxicity, poor solubility and short life span led to the discontinuation of clinical trials of didemnin B and rendered it unsuitable for further drug development [142]. The linear depsipeptide kahalalide F is known for its antifungal and antitumor activities, and its phase II clinical trials are underway. Another cyclic depsipeptide plitidepsin (dehydrodidemnin B or aplidine) is in clinical development. In 2003, plitidepsin was granted orphan drug status by the European Medicines Agency for treating acute lymphoblastic leukemia. In 2007, it was undergoing multicenter phase II clinical trials, and in 2016, early results in a small phase I trial for multiple myeloma were announced. The two most promising peptides of antimitotic dolastatins group, dolastatin 10 and 15, were selected for development and are currently undergoing phase II clinical trials. Cemadotin, the synthetic analogue of dolastatin 15, is also in phase II clinical trials as a promising cancer chemotherapeutic agent [143,144].

Conclusions and Future Prospects
There is increased evidence of the emergence of resistance to conventional drugs illustrating the importance of research on natural peptide-based drug development. PRCPs have several structural features making them good drug leads, and there are several naturally-occurring cyclic peptides in clinical use and in clinical trials. In addition, biologically-active proline-rich cyclic peptides have been developed with synthetic approaches, and they are useful as therapeutics and biochemical tools. With the introduction of new high throughput screening methods, there will be more availability of marine-based PRCPs with interesting biological properties. PRCPs can work on their targets very selectively, as the interaction with the targets is very specific compared to small molecules. In addition to the merits of peptides, especially "proline-rich cyclic structures" as drug molecules, cyclopolypeptides could make even better peptide drugs for future use. Moreover, the future development of peptide drugs will continue to build upon the strengths of naturally-occurring proline-rich peptides, with the application of traditional rational design to improve their weaknesses, such as their chemical and physical properties. Further, emerging peptide technologies will help broaden the applicability of PRCPs as therapeutics. While still in the early stages of development, PRCPs drug leads have started gaining the attention of the pharmaceutical industry; however, their true potential is still very much unknown.