Key words
Euphorbiaceae -
Croton
-
Jatropha
- orbitides - cyclic peptides
Abbreviations
AMPs:
antimicrobial peptides
CCK:
cyclic cystine knot
CIP:
Cahn-Ingold-Prelog
CPPs:
cyclic peptides precursors
IUPAC:
International Union of Pure and Applied Chemistry
OLP:
oligopeptidase
PCY:
peptide cyclase
PPIase:
peptidyl-prolyl cis-trans isomerase
RiPPs:
ribosomally synthesized and post-translationally modified peptides
SPPS:
solid-phase peptide synthesis
UniProt:
Universal Protein Resource
Amino Acids
Ala:
alanine
Arg:
arginine
Asn:
asparagine
Asp:
aspartic acid
Cys:
cysteine
Glu:
glutamic acid
Gln:
glutamine
Gly:
glycine
His:
histidine
Ile:
isoleucine
Leu:
leucine
Lys:
lysine
Met:
methionine
Phe:
phenylalanine
Pro:
proline
Ser:
serine
Thr:
threonine
Trp:
tryptophan
Tyr:
tyrosine
Val:
valine
Introduction
Nature continues to guide researchers in the discovery and development of novel drug
leads [1]. Cyclic peptides are structurally diverse and are found in all kingdoms of life.
Traditionally, they have been found in animals [2], fungi [3], bacteria [4], and marine [5] sources, although higher plants have recently been shown to be a rich source of
cyclic peptides with peculiar composition, stability, and structural and functional
diversity [6], [7]. As a source of bioactive compounds, plants still play an important role in drug
design and form the basis of many traditional medicines [1], [8]. Based on their structure, plant-derived cyclic peptides are classified into cyclotides
[9], [10], [11], [12], orbitides [7], [13], and sunflower-derived peptides [14]. Their pharmacological activities, applications, and structures have been extensively
investigated [7], [15], [16], [17], [18], [19]. However, in addition to the obtained information, it is estimated that less than
1% of naturally occurring cyclic peptides have been discovered [20]. Orbitides constitute a class of natural cyclic peptides presenting a low molecular
weight, ranging from 5 to 12 amino acids, ribosomally synthesized, with head-tail
cyclization and a lack of disulfide bonds [17], [18], [21]. Cys is reported to be the least abundant amino acid observed in orbitides [17].
Angiosperm families are characterized by the biosynthesis of a diversity of classes
of secondary metabolites. According to a literature survey, the orbitides are described
only in the families Annonaceae, Caryophyllaceae, Euphorbiaceae, Lamiaceae, Linaceae,
Phytolaccaceae, Rutaceae, Schisandraceae, and Verbenaceae [17].
The Euphorbiaceae family, comprising approximately 300 genera, is recognized due to
itʼs biosynthesizing of several classes of secondary metabolites, such as terpenoids,
flavonoids, tannins, alkaloids, and steroids [22], [23], [24]. However, the members of this family are also described as a source of bioactive
orbitides [13]. Even though Euphorbiaceae comprises most of the orbitides described in the literature,
they were isolated from the genera Jatropha
[25], [26], [27], [28], [29], [30], [31], [32], [33], [34] and Croton
[35], [36], [37]. The 1990s were considered the gold period of investigations of orbitides, with
the discovery of several new molecules, thus demonstrating that the search for their
mechanisms of action, structures, and biosynthesis needs to be expanded [7]. It should be noted that the majority of orbitides from Euphorbiaceae were isolated
from the plant latex. Only a few compounds have been described in the leaves, stem
bark, or aerial parts of the plant [25], [35].
In recent years, the discovery of bioactive cyclic peptides from different sources
has grown exponentially, making them outstanding as the most promising molecules in
the search for new drug leads [15], [16], [19], [38], [39]. The market for protein- and peptide-based drugs is over $40 billion per year, and
most of the available products are small peptides, ranging from 8 to 10 amino acid
residues. These data reflect the number of papers found in the literature that primarily
focus on peptide isolation, characterization, synthesis, pharmacological activities,
and mechanism of action. It should be highlighted that over the years, more than 100
peptide drugs have reached the market, demonstrating the pharmaceutical potential
of these compounds [19].
In this review, we discuss the biological activities, chemical synthesis, elucidation
techniques, classification, and biosynthesis of bioactive orbitides isolated from
the Euphorbiaceae family. All information shown was gathered via searching English
papers from scientific databases including PubMed, Elsevier, Google Scholar, Scopus,
Web of Science, Cybase, and SciFinder by using the key words Euphorbiaceae, cyclic
peptides, and orbitides.
Euphorbiaceae Family
The Spurge family (Euphorbiaceae) is one of the most complex, largest and most diverse
families of angiosperms and occurs in arid and semiarid environments [40], [41]. It comprises approximately 300 genera and 8000 species, and it is established as
an important source of medicines and toxins [42], [43], [44]. This family occurs mainly in the tropics, with the majority of the species in tropical
and subtropical areas of Africa and the Americas. As might be expected from such a
large and diverse family, there is a vast amount of information on its ethnomedical
use, phytochemical investigations, and bioactive constituents. Williamson and Hooper
[45] reported the use of Euphorbia polycarpa, Euphorbia hirta, and Acalypha indica L. for the treatment of different diseases in Ayurvedic medicine. In Chinese medicine,
Lai and co-authors [46] reported 33 other species belonging to 17 genera of Euphorbiaceae used in herbal
medicine. Among them, 17 species were used to treat snakebites, following the principle
of “combat toxins with toxins”. Several species are grown as ornamental plants (Euphorbia milli, Euphorbia tirucalli, Euphorbia obesa, and Euphorbia pulcherrima), and many others are of considerable economic importance, such as cassava (Manihot esculenta), the castor oil plant (Ricinus communis L.), the rubber tree (Hevea brasiliensis Willd. Ex. A. Juss), and a biodiesel source (Jatropha curcas L.) [42]. As several species within the Euphorbiaceae family contain toxic compounds, some
plants are usually described in the literature as poisonous, especially those from
the Jatropha
[43] and Euphorbia genera, which might induce dermatitis. Phorbol esters and curcins are described in
the literature as the most toxic components found in J. curcas L. and exhibit toxicity to many species, including microorganisms, animals, and humans
[47]. Many plants have oleaginous seeds, may contain latex, and provide a source of rubber
[24], [40]. Latex is described as a complex mixture of peptides, enzymes, triterpenoids, diterpenoids,
amino acids, and other compounds. Among the Euphorbiaceae family, latex is found primarily
in members of the subfamilies Crotonoideae and Euphorbioideae, but is generally lacking
in the Phyllanthoideae, Oldfieldioideae, and Acalyphoide subfamilies [24].
The Euphorbiaceae family presents a large variety of secondary metabolites, and the
major types include alkaloids [23], triterpenoids [48], [49], flavonoids (both C- and O-glycosides) [49], [50], diterpenoids [23], tannins, and peptides (cyclic and linear) [13], [24], [48]. According to the literature, all orbitides isolated from Euphorbiaceae to date
consist exclusively of L-amino acids. The D-isomers are not commonly available from
natural sources and are usually obtained by synthetic approaches [51].
Classification
The IUPAC provides rigorous recommendations for the nomenclature of linear peptides,
although many attempts have been made recently by several researchers to organize
and clarify the orbitides nomenclature [20]. In 2006, Tan and Zhou [7] reviewed and classified a range of plant-derived cyclic peptides into two classes,
five subclasses, and eight types based on their chemical structure and family origin.
Initially, the classification of these small natural cyclic peptides as Caryophyllaceae-type
cyclopeptides (type VI) was based on phytochemical studies of Caryophyllaceae species.
It was believed that small cyclopeptides were characteristic components of Caryophyllaceae
plants [7]. In general, this nomenclature was based on the botanical families, leading to an
unclear generalization. As demonstrated, naming a group as “Caryophyllaceae-type”
or “Rubiaceae-type” leads to the conclusion that all the peptides described in this
botanical family occur exclusively in this taxon, which is not true [18]. Thus, in 2013, Arnison and co-authors [17] published a review on RiPPs and proposed the name “orbitide” to replace the older
designation “Caryophyllaceae-type cyclopeptides”. In fact, RiPPs comprise a large
group of natural peptides that are classified into subfamilies according to their
biosynthetic pathway and the diversity of structural scaffolds. Caryophyllaceae-type
homomonocyclopeptides, orbitides, and cyclotides are defined as RiPPs. In a general
way, they can occur in many taxa and are genetically encoded and synthesized as precursor
proteins [17], [52]. Additionally, according to Arnison and co-authors [17], it is too early to classify the various types of orbitides when only a few examples
are reported in the literature. Recently, in 2015, Shim and co-authors [21], considering the disarray in the nomenclature of type VI cyclic peptides, proposed
a new systematic naming system that reflects the taxonomic name of the species and
a numbering system that enables systematic representation of the amino acid residues
and modifications. According to the authors, this proposed nomenclature was based
on IUPAC, UniProt, and the review published by Arnison and co-authors [17]. Following the instructions, the orbitide name should start with [1-#-NαC], which refers to the linkage between amino acid 1 and “#” through the α-amino group that is an N-C cyclization of the core peptide. Then add [#-Xaa, #-Xaa],
which refers to the modifications and specifies the position and type of modified
amino acid(s). A comma must be used to separate multiple post-translational modifications.
In the sequence, add the taxonomic name abbreviation to identify the origin of the
orbitide, which is based on the first three letters of the genus and the first two
letters of the species name. Then add the common suffix (-orb) for orbitide. Finally,
add the amino acid residue numbering (#) that begins at the N-terminal-most amino
acid residue, based on the DNA sequence. As demonstrated in this review, papers published
after this date (2015) describing the isolation of new orbitides started to adopt
this nomenclature suggested by Shim and co-authors [21]. The use of this taxonomic nomenclature system allows the differentiation of orbitides
with sequence diversity from closely related species.
Orbitides are described as small N-to-C terminal intramolecularly cyclized peptides
from plants that do not have disulfide bonds, ranging from 5 to 12 amino acid residues.
In contrast, cyclotides (type VIII) are natural peptides comprising 28 – 37 amino
acids, which are cyclized by both the fusion of the terminal amino and cysteine double
bonds, thus containing three conserved disulfide bonds, known as a CCK [7], [9], [10], [11]. It was noticed that many amino acid residues might be underrepresented in orbitides.
Cys is the least abundant amino acid observed, and some other residues such as Asp
and Glu, their amides Asn and Gln, basic residues Lys and Arg, and His are also rare.
Orbitides are found in higher plants but not in every taxon. So far, Annonaceae, Caryophyllaceae,
Euphorbiaceae, Lamiaceae, Linaceae, Phytolaccaceae, Rutaceae, Schisandraceae, and
Verbenaceae are the plant families described in the literature as containing orbitides
[17].
Based on the discussion of cyclic peptide nomenclature and classification, a task
group was recently designed by IUPAC to propose guidelines for homodetic cyclic peptides
and specify amino acid priority for consistent numbering. IUPAC defines homodetic
peptides as “cyclic peptides in which the rings consist only of amino acid residues
in eupeptide linkage”. The amino acid numbering of peptides can be determined by various
methods such as mass spectrum sequencing, alphabet, gene sequencing, and CIP rules.
According to Craik and co-authors [20], the CIP system is unequivocal and can be generally applied to proteinogenic, nonproteinogenic,
and unnatural amino acids. In the CIP rules, the priority of the amino acids is assigned
by comparing the atomʼs atomic number. A table containing the priority ranking of
the amino acids can be found in the mentioned paper [20], and according this rule, proline takes the highest position, as it is the only
amino acid that contains a secondary amine. The list of the 21 proteinogenic amino
acids in the CIP priority is described as: Pro, Sec, Cys, Thr, Ser, Ile, Val, Met,
Asp, Asn, His, Trp, Tyr, Phe, Leu, Glu, Gln, Arg, Lys, Ala, and Gly. Following this
procedure, first identify the highest-ranking amino acid. Then, in case of duplicate
amino acids, the next one on the C-terminal is compared. Finally, number the remaining
amino acids from the N-terminal to C-terminal. Also, according to this rule, an R amino acid presents higher priority over its S isomer. As this system is not a formal IUPAC recommendation, the task group encourages
further discussion and feedback from the scientific community [20]. In this review, compounds were numbered according to the CIP rules.
Biosynthesis
Understanding the biosynthetic mechanism is a challenge for all classes of peptides
[53]. Not all of them are completely understood; sometimes the exact mechanism still
remains unclear and needs to be further investigated. Regarding the biosynthesis of
cyclic peptides from plants, the cyclotides present the best-understood pathway so
far.
Based on their origins and biosynthetic pathways, in general, peptides can be organized
into three groups: ribosomal, nonribosomal, and protein derivatives. Ribosomally synthesized
peptides (RiPPs) are products of gene expression in which the amino acids are attached
to one another during mRNA translation. The main classes of ribosomally synthesized
cyclic plant peptides are the cyclotides, the SFTI-like peptides, also recognized
as PawS-derived peptides (PDPs), and the orbitides [17].
Both orbitides and cyclotides differ greatly in arrangement and sequence [17], [54], [55]. Unlike cyclotides, orbitides have no disulfide bond, have a high proportion of
hydrophobic residues, and are processed in two steps involving two separate enzymes:
cleavage by an oligopeptidase (OLP) and cyclization by a PCY defined as a serine protease-like
enzyme [16], [54], [56]. According to some assays with seed extracts using synthetic peptide substrates,
orbitides from Saponaria vaccaria are first ribosomally synthesized as linear precursors (32-amino acid linear precursor),
termed presegetalin A1 [54], [57]. Further, biochemical assays were carried out in S. vaccaria seed extracts to determine the enzymes involved in cyclic peptide formation in Caryophyllaceae.
The first enzyme from S. vaccaria is OLP1, a serine protease that recognizes and cleaves the sequence corresponding
to the junction between the N-terminal region and the incipient cyclic peptide sequence.
The OLP1 cleaves the first 15 amino acids to yield a product containing an N-terminal
glycine (presegetalin A1 [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]) [56]. A second enzyme (PCY1) acts on this product to excise the linear C-terminalʼs 13
residues (presegetalin A1 [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]), with the concomitant macrocyclization of the remaining N-terminalʼs six residues
to yield the c(Gly-Val-Pro-Val-Trp-Ala) segetalin A product. The biosynthesis of orbitides, as well
as other RiPPs such as cyclotides, commonly uses convergent strategies based on Ser
proteases that can catalyze the formation of a cyclic product via transamidation rather
than a linear product via hydrolysis [56]. Biosynthetic studies were also conducted on the Citrus genus (Rutaceae), and the obtained data were consistent with the ribosome-dependent
biosynthesis of orbitides via CPPs, which confirmed the role of the corresponding
genes and provided evidence for the genes encoding CPPs [57].
Orbitides are generally derived from proteinogenic amino acids. Prior to 2011, very
little was known about the biosynthesis of the orbitides and whether ribosomes were
involved or not [57]. Recently, they started to be better understood once many aspects of RiPP biosynthesis
and new insights were investigated [52]. The obtained data already described for systems such as Caryophyllaceae (S. vaccaria, Dianthus caryophyllus) and Rutaceae (Citrus spp.) are consistent and likely to provide further general insights into the mechanisms
of enzyme activation and orbitide biosynthesis [57]. In 2011, the whole genome of J. curcas L. was sequenced, thus generating information for future research for this species
and related plants. It was obtained using a strategy that combines the Sanger method
and the new-generation multiplex sequencing methods. Also, when compared with genes
from other plant species, it was indicated that 4% of genes are conserved specifically
in the Euphorbiaceae family [58]. Finally, future efforts to obtain a better understanding of the biosynthetic pathway,
genomic sequences, and the enzymes involved in RiPPs will contribute to the development
and discovery of novel peptides with potential pharmaceutical and biotechnological
applications [52], [54], [55], [56].
Peptides from the Croton Genus
Peptides from the Croton Genus
Croton is a large genus from the Euphorbiaceae family comprising approximately 1300 species
of trees, herbs, and shrubs commonly distributed in tropical and subtropical regions
[23]. The seeds, roots, volatile oil, leaves, and stem bark are reported to contain several
secondary metabolites [23] such as diterpenoids [59], [60], [61], [62], triterpenoids, steroids, alkaloids, lignoids, proanthocyanidins, and flavonoids
[37]. Some Croton species may contain latex, which is usually viscous and red colored [23]. Traditionally, the red sap, popularly known as dragonʼs blood or “sangre de gadro”,
is collected by cuts on the bark surface of several Croton species (Croton lechleri, Croton palanostigma, Croton draconoides, and Croton urucurana Baill.) and used by indigenous Amazon cultures and many others as a traditional medicine
to promote wound healing [63], [64]. The Croton species are frequently associated with popular medicines for gastrointestinal ulcers
and cancer, cicatrizants, and anti-inflammatory therapies [65], [66], and are reported to have a wide range of pharmacological properties such as mutagenic,
apoptosis, antioxidant, antitumor, cytotoxic, and antiproliferative activity [23], [65], [66], [67], [68], [69]. Cyclic peptides (orbitides) [35], [36], [37] (Table S1, Supporting Information) and some linear peptides [22] isolated from the Croton species have been described in the literature.
Studies on the latex of Croton gossypiifolius Vahl. led to the isolation of a cyclic nonapeptide named crotogossamide (1). This species was collected in Trinidad and Tobago but also occurs in Colombia,
Venezuela, Mexico, and Martinique. Crotogossamide (1) was obtained as white crystals, and the amino acid sequence was determined to be
cyclo(-Ser
1
-Gly
2
-Leu
3
-Asn
4
-Gly
5
-Ile
6
-Phe
7
-Gly
8
-Ala
9
-) [37]. C. urucurana Baill. is one of the dragonʼs blood species mentioned above [36], [63], [64] and is commonly found in midwestern Brazil (Cerrado and Pantanal biomes). Examining
the red latex of C. urucurana Baill. led to the isolation of an orbitide named [1–9-NαC]-crourorb A1 (2), a cyclic nonapeptide with an amino acid sequence defined as cyclo(-Ser
1
-Leu
2
-Gly
3
-Ala
4
-Phe
5
-Gly
6
-Asn
7
-Leu
8
-Gly
9
-) [36]. The proposed orbitide name for this compound followed the systematic nomenclature
recently suggested by Shim and co-authors [21]. From whole plant material of Croton sparsiflorus Morong, a shrub found in Asia and South America, a cyclic nonapeptide named crotosparsamide
(3) was isolated ([Fig. 1]). Crotosparsamide was obtained as white crystals, and the amino acid sequence was
determined to be cyclo(-Val
1
-Asn
2
-Gly
3
-Ala
4
-Phe
5
-Gly
6
-Leu
7
-Leu
8
-Gly
9
-) [35]. According to the literature data, all orbitides isolated from Croton species so far were defined as nonapeptides, and the absolute configurations of the
amino acids were shown to be all L, thus highlighting the biosynthetic pathway.
Fig. 1 The structures of orbitides (1–3) from Croton species.
In addition to the cyclic peptides already described, two linear peptides were isolated
from the aerial parts of Croton hieronymi Griseb., a plant native to northwestern Argentina [22]. Compound 4 was described as the peptide derivative aurentiamide acetate, and according to Catalan
and co-workers [22], it was previously found in Aspergillus species, two algae, and some higher plants. The other compound was a new linear peptide
derivative named N-benzoylphenylalaninyl-N-benzoylphenyl-alaninate (5), which differed from compound 4 in that the left half was esterified by N-benzoylphenylalaninol and a second benzamide
functionality was present. It should be highlighted that compounds of this type are
rare and have not previously been found in Croton species [22] ([Fig. 2]).
Fig. 2 The structures of linear peptides (4, 5) from C. hieronymi Griseb.
Peptides from the Jatropha Genus
Peptides from the Jatropha Genus
The genus Jatropha comprises 170 species of woody trees, shrubs, and subshrubs distributed in tropical
and subtropical countries. The name Jatropha derives from the Greek word jatrós, which means doctor, and the word trophé, which means food, thus implying the medicinal uses of these plants since ancient
times [13], [70]. Currently, Jatropha species are widely used as medicinal plants in several countries from Asia, Latin
America, and Africa, even considering the toxicity of different parts of the plant
[13], [43], [71], [72]. They are commonly known for their popular uses for purgative, anti-allergic, wound
healing, and anti-inflammatory effects [13]. Experimental studies described in the literature reveal antimicrobial, antineoplastic,
wound healing, and hypotensive activities [13], [71]. In the past two decades, studies on Jatropha seed oil, especially J. curcas L. and Jatropha gossypiifolia L., for biofuel have drawn attention, thus resulting in industrial-scale cultivation
and making Jatropha species prospective high-quality biodiesel yielding crops [43], [71], [73].
Phytochemical studies on Jatropha plants led to the isolation of several chemical constituents [71], [72] such as alkaloids, triterpenoids, diterpenoids, sesquiterpenoids, flavonoids, coumarins,
lignans, phorbol esters, phytosterols, and cyclic peptides. The Jatropha genus is characterized by the abundant production of latex, which is watery to white
and contains saponin, resin, peptides, tannin, wax, and enzymes [70], [71]. A protease named curcain was obtained from the latex of J. curcas L. and reported to have wound healing activity in mice [70], [74]. The majority of cyclic peptides from Jatropha species were isolated from the latex, except for Jatropha ribifolia (Pohl) Baill. [25].
Investigations on the latex of J. curcas L., which is commonly known as a medicinal plant [70], [75], led to the isolation of three orbitides named curcacycline A (6) [29], curcacycline B (7) [30], and jatrophidin I [28] (8). Curcacycline A (6) is an octapeptide that was first isolated from an Indonesian plant, and its amino
acid sequence was determined to be cyclo(-Thr
1
-Val
2
-Leu
3
-Leu
4
-Gly
5
-Leu
6
-Leu
7
-Gly
8
-) [29]. The structure of curcacycline B (7), a nonapeptide, was determined to be cyclo(-Pro
1
-Ile
2
-Leu
3
-Leu
4
-Gly
5
-Ile
6
-Leu
7
-Gly
8
-Ser
9
-), and the plant was obtained from Senegal [30]. Later, in 2012, Insanu and co-workers [76] applied a synthetic approach to confirm the structures of curcacycline A (6) and B (7), and also applied new pharmacological insights into these orbitides. Jatrophidin
I (8) was isolated from a Brazilian plant, and the amino acid sequence was determined
to be cyclo(-Pro
1
-Gly
2
-Leu
3
-Leu
4
-Asn
5
-Leu
6
-Trp
7
-Gly
8
-). This compound contains an asparagine residue, which is not very common in cyclic
peptides from plants. It was tested for some biological activities but was demonstrated
to be inactive in antimalarial, antifungal, and antioxidant assays [28]. The cyclic octapeptide named ribifolin (9), an amorphous white solid, was isolated from the aerial parts of J. ribifolia (Pohl) Baill. This plant was collected in the semiarid northeastern region of Brazil,
and the sequence of the orbitide was determined to be cyclo(-Ser
1
-Gly
2
-Leu
3
-Ile
4
-Gly
5
-Leu
6
-Ile
7
-Ile
8
-). Pinto and co-workers [25] also described the synthesis of the linear ribifolin analogue by an SPPS, which
was used in further studies to determine the role of cyclization in some bioactivity
assays. Studies on the latex of J. gossypiifolia L. led to the isolation of two orbitides, one heptapeptide and one octapeptide, named
cyclogossine A (10) [77] and cyclogossine B (11) [31], respectively. The structure of cyclogossine A (10) was determined to be cyclo(-Thr
1
-Ala
2
-Leu
3
-Val
4
-Gly
5
-Leu
6
-Trp
7
-), and it was isolated from a plant collected in Indonesia [77]. Cyclogossine B (11) was isolated from a plant from Senegal, and the amino acid sequence was determined
to be cyclo(-Ile
1
-Leu
2
-Gly
3
-Gly
4
-Trp
5
-Leu
6
-Ala
7
-Ala
8
-) [31] ([Fig. 3]).
Fig. 3 The structures of orbitides (6–11) from Jatropha species.
A study on the latex of Jatropha pohliana ssp. molissima led to the isolation of two cyclic heptapeptides, pohlianins A (12) and B (13), and one cyclic octapeptide, pohlianin C (14) [26]. This plant was collected in the semiarid northeastern region of Brazil. The amino
acid sequence of pohlianin A (12) was determined to be cyclo(-Pro
1
-Leu
2
-Gly
3
-Val
4
-Leu
5
-Leu
6
-Try
7
-), the sequence of pohlianin B (13) was determined to be cyclo(-Pro
1
-Leu
2
-Gly
3
-Leu
4
-Leu
5
-Leu
6
-Try
7
-), and the sequence of pohlianin C (14) was determined to be cyclo(-Thr
1
-Ile2-Ile
3
-Phe
4
-Gly
5
-Phe
6
-Gly
7
-Gly
8
-) [26]. From the latex of Jatropha chevalieri Beille, a small shrub collected in Senegal, researchers obtained three orbitides
named chevalierins A (15) and B (16), two cyclic octapeptides, and chevalierin C (17), a cyclic nonapeptide [34]. Locally, in folk medicine, the latex of this plant is used to treat infected wounds.
The structure of chevalierin A (15) was determined to be cyclo(-Pro
1
-Ile
2
-Leu
3
-Ala
4
-Ile
5
-Met
6
-Gly
7
-Ile
8
-), and the sequence of chevalierin C (17) was determined to be cyclo(-Thr
1
-Ile
2
-Phe
3
-Asp
4
-Ile
5
-Phe
6
-Gly
7
-Ala
8
-Tyr
9
-). Chevalierin B (16) was found to be very similar and to have the same amino acid sequence as chevalierin
A (15), and the only difference is the additional oxygen atom, suggesting the presence
of methionine sulfoxide (Mso) instead of methionine (Met). Thus, the structure of
chevalierin B (16) was described as cyclo(-Pro
1
-Ile
2
-Leu
3
-Ala
4
-Ile
5
-Mso
6
-Gly
7
-Ile
8
-) ([Fig. 4]). They also described the synthesis of the chevalierinsʼ linear precursors using
a solid-phase technique [34].
Fig. 4 The structures of orbitides (12–18) from Jatropha species.
From the latex of Jatropha multifida L. collected in Indonesia from leaf stalks, two orbitides, named labaditin (18) [32], a cyclic decapeptide, and biobollein (19) [78], a cyclic nonapeptide, have been reported. Its latex is commonly used in folk medicine
for the treatment of infected wounds and skin infections. The structure of labaditin
(18) was determined to be cyclo(-Thr
1
-Ile
2
-Ala
3
-Gly
4
-Val
5
-Trp
6
-Thr
7
-Val
8
-Trp
9
-Gly
10
-) and according to Kosasi and co-authors [32], it was the first time that a cyclic decapeptide was documented in higher plants.
The sequence of biobollein (19) was determined to be cyclo(-Ser
1
-Ile
2
-Leu
3
-Gly
4
-Leu
5
-Gly
6
-Trp
7
-Ala
8
-Ala
9
-) [78]. Examination of Jatropha mahafalensis Jum. & H. Perrier, a tree endemic to Madagascar and also used to treat infected wounds
in traditional medicine, led to the isolation of two cyclic heptapeptides named mahafacyclin
A (20) [79] and mahafacyclin B (21) [33]. The amino acid sequence of mahafacyclin A (20) was determined to be cyclo(-Thr
1
-Ile
2
-Leu
3
-Gly
4
-Val
5
-Phe
6
-Gly
7
-), and according to the authors it was the first natural cyclic heptapeptide without
proline to present the β-bulge characteristics [79]. It should be noted that the β-motif observed in cyclic heptapeptides usually contains at least one proline residue
[26], [33], [79]. Mahafacyclin B (21) was identified as a minor component of J. mahafalensis Jum. & H. Perrier latex, and the structure was determined to be cyclo(-Thr
1
-Phe
2
-Phe
3
-Gly
4
-Phe
5
-Phe
6
-Gly
7
-), highlighted by the presence of four phenylalanine residues. The linear precursor
of mahafacyclin B (21) was synthesized by a solution phase method [33]. Research on the latex of Jatropha podagrica Hook, a plant collected in Kenya, led to the isolation of two orbitides, podacycline
A (22), a cyclic nonapeptide, and podacycline B (23), a cyclic heptapeptide. The structure of podacycline A (22) was determined to be cyclo(-Val
1
-Trp
2
-Ala
3
-Gly
4
-Gly
5
-Leu
6
-Leu
7
-Gly
8
-Ala
9
-), and the structure of podacycline B (23) was determined to be cyclo(-Thr
1
-Ile
2
-Phe
3
-Gly
4
-Phe
5
-Ala
6
-Gly
7
-) [27]. Jatropha integerrima Jacq. was collected in Thailand, and two cyclic heptapeptides, integerrimides A (24) and B (25), were isolated from the latex. The orbitide integerrimide A (24) was obtained as a colorless solid and identified as cyclo(-Pro
1
-Trp
2
-Gly
3
-Leu
4
-Leu
5
-Leu
6
-Thr
7
-). Integerrimide B (25) was obtained as colorless needles and identified as cyclo(-Pro
1
-Trp
2
-Ala
3
-Leu
4
-Leu
5
-Val
6
-Ser
7
-) [80] ([Fig. 5]).
Fig. 5 The structures of orbitides (19–25) from Jatropha species.
In addition to the presence of cyclic peptides in the latex of J. curcas L., an investigation of the seeds led to the isolation of a new linear peptide named
PII-C, consisting of 14 amino acid residues and having a molecular mass of 1341.58 Da
[M + H]+. The amino acid sequence was found to be Ala-Pro-Thr-Leu-Ser-Gly-Gly-Ser-Val-Pro-Arg-Asp-Ala-Asp
[81].
It should be mentioned that all orbitides already described in the literature from
Jatropha species have an L-configuration, a low amino acid complexity, and a high proportion
of hydrophobic amino acids, which is commonly noticed in cyclic peptides isolated
from the Caryophyllaceae group (Table S1, Supporting Information) [17], [18]. Concerning cyclic peptides, we can assume that the Jatropha genus has been investigated in greater detail than the other representative genera
of the Euphorbiaceae family.
Peptides from Other Genera (Euphorbiaceae Family)
Peptides from Other Genera (Euphorbiaceae Family)
In addition to the orbitides predominantly described in Croton and Jatropha species, some other genera from the Euphorbiaceae family have also been identified
as sources of peptides and derivatives. Those peptides are usually differentiated
from orbitides by the presence of disulfide bonds, a lack of head-to-tail cyclization,
and a linear amino acid sequence.
Ricinus communis L. is a plant from the Euphorbiaceae family known for the presence of the protein
ricin, an extremely toxic natural poison found in high amounts in the plant. Ricin
can be toxic by ingestion, inhalation, or injection [82], [83]. Recently, three new peptides, designated R. communis L. biomarkers (RCB) 1 – 3, were identified in the plant seeds. Those peptides comprise
homologous sequences of 19 (RCB-1) or 18 (RCB-2 and RCB-3) amino acid residues, including
four cysteine moieties and disulfide bonds [82]. Thus, RCB-1 consists of 19 amino acids and has two disulfide bonds, and the structure
was determined to be (-Ala-Arg-Cys-Cys-Leu-Val-Met-Pro-Val-Pro-Pro-Phe-Ala-Cys-Val-Lys-Phe-Cys-Ser-)
[83]. It was synthesized by SPPS with the assistance of microwave heating during the
deprotection and coupling reaction steps, and then subjected to oxidative folding.
Additionally, the three-dimensional structure of RCB-1 was determined [82]. RCB-2 contains 18 amino acids and two disulfide bonds, and the sequence was determined
to be (-Ala-Arg-Cys-Cys-Leu-Val-Met-Pro-Val-Pro-Pro-Phe-Ala-Cys-Val-Lys-Phe-Cys-).
The peptide RCB-3 also has two disulfide bonds and has the sequence (-Ala-Arg-Cys-Cys-Leu-Val-Leu-Pro-Val-Pro-Pro-Phe-Ala-Cys-Val-Lys-Phe-Cys-)
[83]. Due to their similarity to the Ib-AMP peptide family, it was suggested that the
function of these RCB peptides might be related to plant defence, playing a role in
seed protection, although further investigations are still suggested [82], [83]. The genus Euphorbia also contains peptides. A molecule identified as a peptide derivative, aurentiamide
acetate, compound 4, was isolated from the roots of Euphorbia fischeriana Steudel [84]. As mentioned previously, this compound was also found in the aerial parts of C. hieronymi Griseb [22].
Biological Activities
Cytotoxicity
Cytotoxicity is a well-documented activity for orbitides. As found in the literature,
some orbitides are cytotoxic, and others are demonstrated to be nontoxic to the cells.
This different behavior may be related to their conformation, their sequence, the
presence of charges, the number of amino acid residues, and the type of tumor cells
involved. Thus, the mechanism of action needs to be further investigated. The orbitide
[1–9-NaC]-crourorb A1 (2) isolated from the latex of C. urucurana Baill. was evaluated against six human cancer cell lines, namely, 786 – 0 (human
kidney carcinoma), HT-29 (human colon carcinoma), MCF-7 (breast adenocarcinoma), NCI-ADR/RES
(ovary adenocarcinoma, multidrug-resistance phenotype), PC-03 (human prostate carcinoma),
and Hep-G2 (hepatocellular carcinoma). Compound 2 demonstrated selective inhibition against NCI-ADR/RES cells (GI50 = 4.8 µM) although according to the authors, further studies are necessary to determine
the effects on nontumour cells [36]. The orbitide ribifolin (9), isolated from the aerial parts of J. ribifolia (Pohl) Baill., and its synthetic linear analogue were assessed for their cytotoxicity
towards human embryonic kidney cells (HEK293T). No cytotoxicity was observed; normal
growth occurred in the tested concentration range (0.001 – 100 µM) [25]. Cytotoxicity testing of curcacyclines, isolated from J. curcas L., demonstrated that curcacycline A (6) presented low toxicity to OVCAR 3 (ovarium cancer cells) with an IC50 of 945 µM, while no effect was found on Colo205 (human colon cancer cells). Curcacycline
B (7) did not demonstrate any effect on either cell line. Additionally, they were evaluated
in a brine shrimp cytotoxicity assay, and curcacycline A (6) and B (7) showed LD50 values of 65.07 µM and 18.35 µM, respectively [76].
Antimalarial
The orbitide ribifolin (9) and its linear synthetic analogue were evaluated for antimalarial activity. Ribifolin
(9) demonstrated moderate activity with an IC50 of 42 µM against the Plasmodium falciparum 3D7 strain, while the linear analogue was inactive (IC50 = 519 µM), providing evidence that cyclization is important for the growth inhibitory
activity against P. falciparum
[25]. The pohlianins, isolated from J. pohliana ssp. molissima, were also evaluated for antimalarial activity, and moderate inhibition was obtained
for these compounds. Pohlianin A (12) and B (13) had IC50 values of 57 µM and 25 µM, respectively. Pohlianin C (14) showed an IC50 of 16 µM and was the most potent among the pohlianins [26]. The three chevalierins A (15), B (16), and C (17), obtained from J. chevalieri, were examined for antiproliferative activity against P. falciparum, but only chevalierin A (15) showed antimalarial activity, with an IC50 of 8.9 µM [34]. The mahafacyclins, obtained from J. mahafalensis Jum. & H. Perrier [33], [79], were evaluated for antimalarial activity. Mahafacyclin A (20) presented a moderate antimalarial activity with an IC50 value of 16 µM [79]. Mahafacyclin B (21) was the most potent and possessed an IC50 of 2.2 µM P. falciparum
[33]. Integerrimides A (24) and B (25), from the latex of J. integerrima Jacq., were evaluated in antimalarial assays but demonstrated to be inactive at test
concentrations of 12.8 µM and 13.0 µM, respectively [80]. Jatrophidin I (8) was also inactive in antimalarial assays [28].
Immunosuppressive
Autoimmune diseases can be classified as organ-specific or as systemic. On a molecular
level, autoreactive T cells play an important role in disease progression [85]. The orbitide curcacycline A (6), isolated from J. curcas L., showed a moderate dose-dependent inhibition of human T cell proliferation, although
direct cytotoxic effects could not be observed [29]. Also, an immunologically active peptide, labaditin, was isolated from the latex
of J. multifida L. [32]. Usually, the immunosuppressive activity of orbitides is characterized by weak affinity
to their molecular target, calcinerium. Other members of the orbitide famil,y such
as cyclolinopeptides, cycloleonurinins, and hymenistatins, also exhibited potential
immunosuppressive activity [85].
Enzymatic Evaluation
Jatrophidin I (8), isolated from J. curcas L., showed inhibitory activity in a fluorimetric protease inhibition assay using
pepsin as a molecular model for aspartic protease inhibition. Its IC50 value was 0.88 µM, although it did not inhibit the serine protease subtilisin, thus
suggesting a selective inhibition for aspartic proteases [28]. According to the authors, the structure of curcacycline B (7) was suggested to be a PPIase substrate. PPIase assays using human cyclophilin B,
based on an α-chymotrypsin-rotamase coupled enzymatic experiment, were carried out to determine
if curcacycline B (7) could bind human cyclophilin B. Curcacycline B (7) was shown to enhance the rotamase activity of human cyclophilin B. PPIase assays
using human cyclophilin B were carried out to determine if curcacycline B (7) could bind the human cyclophilin B. In the absence of cyclophilin B, curcacycline
B (7) had no effect on α-chymotrypsin activity, although when the experiment was carried out in the presence
of cyclophilin B, it enhanced the PPIase activity of cyclophilin B by 60% at 30 µM.
Antibacterial and antifungal
The search for new classes of antibiotics, such as AMPs, has become even more relevant
as the resistance of bacteria has reached higher levels worldwide. Investigation of
the antibacterial activity of labaditin (18), isolated from J. multifida L., demonstrated a reduction of viability in gram-positive bacteria (Streptococcus mutans), while its linear analogue is not active, thus demonstrating that cyclization might
contribute to the antibacterial activity. No effects were observed in gram-negative
bacteria (Aggregatibacter actinomycetemcomitans) for concentrations ranging up to 100 mM [86]. Labaditin (18) and its linear analogue were also evaluated against Staphylococcus aureus. It was shown that the cyclic compound (18) was highly effective against S. aureus, with no bacterial growth at a concentration as low as 0.23 mM. On the other hand,
the linear analogue required concentrations above 28.6 mM to be active. According
to Barbosa and co-authors [87], the antibacterial activity of labaditin (18) was related to its inducing leakage in the cell membrane. Leakage occurs with the
self-assembly of labatidin (18) into a nanotube owing to its preserved secondary structure in the membrane. Additionally,
this mode of action may represent a promising step towards the design of a new class
of antibiotics. The screening of curcacycline A (6) for antibiotic activity was done against five strains, Bacillus subtilis, S. aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans, using the paper disk (6 mm of diameter) diffusion method at a concentration of 1.3 mM.
Curcacycline A (6) inhibited the growth of B. subtilis and P. aeruginosa with inhibition zones of (10.3 ± 0.4 mm) and (8.3 ± 0.8 mm), respectively. Although
the mechanistic action of curcacyline A (6) still remains unclear, the antibacterial activity of cyclic peptides might be related
to their ability to cross membranes and their higher affinity to bind with some proteins
[76]. The orbitides integerrimides A (24) and B (25), obtained from the latex of J. integerrima Jacq., were evaluated for antifungal assays, although they were demonstrated to be
inactive at concentrations of 64.1 µM and 65.3 µM, respectively [80]. In addition, jatrophidin I (8) was inactive in antifungal assays [28]. The antibacterial and antifungal activity of peptide RCB-1, isolated from R. communis L., was tested against human and plant pathogens. Different concentrations of RCB-1,
ranging from 160 to 0.625 µM, were used. The compound RCB-1 was found to be active
against only P. aeruginosa, although it was tested against a set of human pathogens (E. coli, S. aureus, and C. albicans) and plant pathogens (Erwinia carotovora, Erwinia amylovora, and Aspergillus fumigatus) [82].
Other Families as Orbitide Sources
Other Families as Orbitide Sources
Plants in different angiosperm families are notorious for accumulating peptides, primarily
cyclic peptides [7]. According to the literature, in addition to being found in the Euphorbiaceae family,
orbitides can also be isolated from at least eight other plant families, including
Annonaceae, Caryophyllaceae, Lamiaceae, Linaceae, Phytolaccaceae, Rutaceae, Schisandraceae,
and Verbenaceae [17], [18].
In fact, the orbitides can be isolated not only from plant latex but also from seeds,
leaves, roots, twigs, and stem bark. The Rutaceae family is commonly known as the
citrus family, which is also the most economically important genus. Studies on Citrus species [88], [89], [90], [91] led to the isolation of orbitides, and they also might be found in the Zanthoxylum
[92], [93], Evodia
[94], and Clausena genera [95], [96]. Recently, in 2016, the orbitide named [1 – 8-NαC]-zanriorb A1 was isolated from the leaves of Zanthoxylum riedelianum Engl. (Rutaceae) [93]. The proposed orbitide name of this compound followed the systematic nomenclature
recently suggested by Shim and co-authors [21]. This compound induced cell death by apoptosis in Jukart leukemia T cells with an
IC50 of 218 nM [93].
The Linaceae family is also a good source of orbitides. In 1959, the first orbitide,
a cyclic nonapeptide from the flaxseed oil of Linum usitatissimum L. (Linaceae), was discovered by Kaufman and Tobschirble and named cyclolinopeptide
A, cyclo(-Ile-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile-) [97]. This cyclic nonapeptide exhibits several biological activities, and later on many
additional cyclic peptides were isolated from linseed (cyclolinopeptide B – E and
cyclolinopeptide F – I) [98], [99]. Actually, the cyclolinopeptides are assigned in the literature as significant compounds
found in flaxseed, but like many other orbitides, the role of these molecules in the
plants remains unknown [100]. The Annonaceae family comprises approximately 130 genera, but most of the orbitides
documented in the literature are isolated from the genus Annona (Annona squamosa, Annona cherimola, Annona glauca) [101], [102], [103]. Caryophyllaceae is a family of flowering plants with 81 genera, commonly called
the pink family. Some plants belonging to the Caryophyllaceae family contain cyclic
peptides, and they are most commonly found in the genera Drymaria
[104], Dianthus
[105], Pseudostellaria
[106], [107], [108], Stellaria
[109], [110], [111], and Vaccaria
[112].
In summary, more than 100 orbitides with various bioactivities have been discovered,
and this number continues to grow as natural products remain an important source of
bioactive compounds over the years [53]. Additionally, other classes of peptides might emerge with new bioactivities and
novel applications, thus attracting the attention of many researchers worldwide.
Elucidation Techniques and Chemical Synthesis
Elucidation Techniques and Chemical Synthesis
Orbitide structures are mainly identified by two techniques, NMR and MS, which are
very well established in this research field. In NMR spectroscopy, each resonance
must be associated with a specific nucleus in the investigated molecule, a process
called assignment. Thus, the data are assigned by one-dimensional (1D) 1H, 13C, and 15N and two-dimensional (2D) techniques such as COSY, TOCSY, NOESY, and ROESY (rotating
frame Overhauser effect spectroscopy) NMR. Several overlaps may occur in 1H and 13C NMR spectra due to the similarity of the chemical shifts from different amino acid
residues, which is the reason 2D NMR techniques became very suitable for structure
determination [51], [113], [114]. In general, assignment can be done using homonuclear spectra in two steps: a) the
sequential assignment of the amino acids in the peptide sequence and b) the assignment
of the amino acid side chains [115].
Using TOCSY and COSY, it is possible to establish the identification of certain amino
acid spin systems, with a characteristic pattern of cross signals. For example, Gly
is the only residue in which two protons interact with the amide proton. Val, Leu,
and Ile residues can be recognized by their two methyl groups, which give a characteristic
row of double signals. In contrast, His, Trp, Tyr, and Phe residues are difficult
to distinguish because Hα interacts with two methylene protons that do not show any J coupling to any of the ring protons (more than three bonds removed). Therefore, these
residues are also difficult to distinguish from Asp, Asn, Cys, and Ser residues [115], [116]. The next step in the assignment procedure concerns the identification of the sequence-specific
position of each amino acid by a NOESY experiment. Briefly, the spectra obtained through
this experiment are used to sequentially connect the spin systems. The connection
of an amino acid in the sequence (i) to its following one (i + 1) can be observed
in the NOESY because the distance of the amide proton of (i + 1) to the Hα, Hβ, or Hγ protons of (i) is frequently smaller than 5 Å. An exception is proline residues,
which have no amide proton and for which no HN(i)-Hα(i-1) cross signal can be observed. However, if the proline (i) is in its trans conformation, sequential HN(i-1)-Hδ(i) and Hα(i-1)-Hδ(i) cross signals can be observed [116].
MS measures the mass-to-charge ratio (m/z) of gas-phase ions and is important for the accurate mass determination and characterization
of peptides, only requiring small amounts of the compounds [117]. As it should be performed in the positive mode, it shows the protonated molecule
MH+ and frequently some adduct ions [M + Na]+ or [M + K]+ in the MS spectrum. The most used method for the ionization of orbitides is electrospray
ionization (ESI). ESI is a solution technique used in conjunction with mass analyzers
such as tandem mass spectrometry. MS is very sensitive and does not require peptides
to be purified. Finally, the MS data must be consistent with the amino acid sequence
derived from the NMR experiments. It was noticed that peptide amino acid sequences
are typically reported in the literature in the order in which they occur in MS/MS
fragmentation [21]. In addition, using NMR studies and X-ray crystallography, it is possible to assign
the 3D structures of the cyclic peptides, thus providing more detailed information
on the peptide structure [51], [113]. NMR has the advantage that it can be done in solution and has proven to be a robust
and widely applied technique [113]. In addition, crystallography gives the static structure of the molecule but requires
the production of crystals, which are not often easily obtained in orbitides. Finally,
the structures of orbitides have been proposed based on extensive 2D NMR investigation
and HRMS [51].
The accessibility of orbitides by chemical synthesis has facilitated studies of their
mechanism and activities, usually once just a few milligrams are obtained from the
natural sources. The synthesis of cyclic peptides can occur in two different ways,
in solution (classical synthesis) or in the presence of a polymeric support. The latter
method is known as SPPS. SPPS is the most commonly used strategy due to its practicality,
its higher speed, and the possibility of automation [114]. It is well established and applicable to any peptide synthesis. Thus, SPPS is considered
a useful tool that can be applied to obtain cyclic peptide libraries and assist in
biological assays, especially in vivo assays, which usually require higher amounts of compounds. Cyclic peptides differ
from linear peptides in that their backbones have one additional peptide bond that
joins the N- and C-terminal to make a circular backbone [6]. The cyclization of linear peptides can be achieved in different ways, including
head-to-tail, side chain-to-side chain, or terminus-to-side chain linkages, and, as
was mentioned before, the orbitides are obtained through head-to-tail cyclization
[118]. The synthesis of peptides containing a cyclic backbone is achieved by SPPS based
on the 9-fluorenylmethoxycarbonyl (Fmoc) amino protection strategy of an acyclic (linear)
precursor followed by an intramolecular reaction linking the C- and N-termini. Thus,
the linear chain is cyclized via amide bond formation [119]. SPPS was first developed by Merrifield [120], [121] and still remains an efficient strategy to obtain peptide libraries with chemodiversity.
Furthermore, this technique provided great improvement in the drug discovery of peptide
mimetics, especially ones involving changes in the backbones of novel molecules containing
unnatural amino acids, mutations, and chemical modifications, which might help to
drive their application as drug scaffolds.
Conclusions
In the current review, we have demonstrated that natural products play an important
role in pharmaceutical and biological research, in which peptide-based drugs have
a prominent place due to their chemical versatility, stability, and specificity. Although
the description of plant-derived cyclic peptides has increased in recent years, the
mechanisms of action of only a limited number of compounds have been reported. Additionally,
despite the increasing number of compounds over the years, it has become clear that
orbitides are still underexplored. Many cyclic peptides found in Euphorbiaceae and
other plant families await discovery and further investigation. In addition, according
to the literature, only the Jatropha and Croton genera have been documented as sources of orbitides in the Euphorbiaceae family.
As demonstrated, there are still many species that have received little to no attention.
Lastly, to address the disarray of orbitides nomenclature, which has been classified
three times in the past few years, much effort was devoted by researchers and improvement
was obtained in the classification and numbering of orbitides, highlighting the importance
of this class of compounds in higher plants.
