Scorpion venom peptides with no disulfide bridges: A review

Peptides 51 (2014) 35–45

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Review

Scorpion venom peptides with no disulfide bridges: A review Ammar Almaaytah a,∗ , Qosay Albalas b a b

Department of Pharmaceutical Technology, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan

a r t i c l e

i n f o

Article history: Received 5 September 2013 Received in revised form 15 October 2013 Accepted 15 October 2013 Available online 31 October 2013 Keywords: Scorpion Venom Non-disulfide-bridged peptides NDBPs Antimicrobial Anticancer Structural properties Classification

a b s t r a c t Scorpion venoms are rich sources of biologically active peptides that are classified into disulfide-bridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs). DBPs are the main scorpion venom components responsible for the neurotoxic effects observed during scorpion envenomation as they usually target membrane bound ion channels of excitable and non-excitable cells. Several hundred DBPs have been identified and functionally characterized in the past two decades. The NDBPs represent a novel group of molecules that have gained great interest only recently due to their high diversity both in their primary structures and bioactivities. This review provides an overview of scorpion NDBPs focusing on their therapeutic applications, modes of discovery, mechanisms of NDBPs genetic diversity and structural properties. It also provides a simple classification for NDBPs that could be adopted and applied to other NDBPs identified in future studies. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4.

5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General methods of scorpion NDBPs discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structural properties of scorpion NDBPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic applications of NDBPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Bradykinin potentiating activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Antibacterial, antifungal and cytolytic activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. NDBPs with antimicrobial activity against antibiotic resistant strains of microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Antiviral activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Antimalarial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6. Anticancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. Immune-modulatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular mechanisms for NDBPs diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Classification of NDBPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Scorpions are considered to be one of the oldest animals living on the planet and their existence dates back to more than 400 million years ago [32]. Scorpions are represented by around 1500 species and they are distributed geographically all over the world [46]. Scorpions have acquired the ability to defend

∗ Corresponding author. Tel.: +962 795550234; fax: +962 2 7201075. E-mail address: [email protected] (A. Almaaytah). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.10.021

35 36 36 36 37 37 40 40 40 41 42 42 42 43

themselves against predators and capture prey through the production of toxin loaded venoms that are secreted through specialized venom glands found at the end of the scorpion telson [57]. During their long evolutionary existence on this planet accompanied by the selective pressure applied on these organisms, scorpions managed to develop series of venom peptides that display diverse biological activities and pharmacological functions [52]. The scorpion venom peptides are generally classified into two main groups: the disulfide-bridged peptides (DBPs) which usually target membrane bound ion channels [9,10,54] and the non-disulfide-bridged peptides (NDBPs), a smaller group within the scorpion venom peptide

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arsenal that display multifunctional activities [68,69]. Most scorpion DBPs contain three to four disulfide bridges and are further sub-classified into four different families according the type of membrane channels they interact with. The membrane bound ion channels targeted by the DBPs family of scorpion peptides include the Na+ , K+ , Ca2+ and Cl− channels [46]. These channels play major roles in regulating the normal cellular physiology within many mammalian organisms and disrupting the function of these channels by interacting with scorpion venom peptides can result in significant alterations in their normal function leading to the several symptoms developed in mammals during scorpion envenomation [61]. The NDBPs represent a different group within the scorpion venom assembly of peptides that have gained interest only recently, several peptides belonging to this group have been identified and functionally characterized only in the last decade. The great interest in NDBPs was generated due to the fact that these peptides display diverse biological functions and exhibit multifunctional activities. Some of the biological functions NDBPs display includes antimicrobial, anticancer, hemolytic, anti-inflammatory, immune-modulatory and bradykinin potentiating activities. Unlike the DBPs that display conserved structure-function relationships, the scorpions NDBPs are structurally diverse and display their activity against numerous biological targets when compared to DBPs.

2. General methods of scorpion NDBPs discovery The scorpion group of NDBPs represents a significant component of the total scorpion peptide venom assembly [62,76]. Mass fingerprinting studies have revealed that NDBPs account for more than one third of all peptides present within scorpion venoms [42,52]. Despite representing a significant proportion of the total venom peptides, this group has the lowest number of functionally characterized peptides when compared with the membrane ion targeting DBPs that constitute the majority of functionally characterized scorpion venom peptides reported in the literature. This is attributed not least in part to the interest generated in this group as a result of their activity on membrane-associated ion channels and their often lethal effects on humans [76]. The importance of studying NDBPs lies in their biological and structural diversity. Several methods have been employed for the identification and discovery of novel NDBPs within scorpion venoms. Early methods for the identification of NDBPs depended on chromatographic separation techniques combined with mass spectrometry for the purification and identification of venom peptide components. These techniques were later followed by biological assays for the determination of the activities of the identified peptides [20]. These early peptide separation and proteomic techniques were later followed by peptide sequence determination employing Edman degradation, a technique that proved to be crucial in determining the full amino acid sequence of the peptides studied but posed technical limitations in identifying long chain peptides in addition to the technical challenges associated with the N-terminal modifications reported in some peptides [39,60]. One of the major breakthroughs in the study of scorpion venom peptidomics was the integration of molecular cloning techniques combined with HPLC fractionation and mass spectrometry for the characterization of venom peptide components. In this novel approach, even limited N-terminal peptide sequence information obtained through Edman degradation permits the design of degenerate primers suitable for the cloning of full-length cDNA sequences of the toxin precursor peptides [13]. Additionally, construction of scorpion mRNA derived cDNA libraries allowed the screening of several random cDNA generated clones that proved to be a successful strategy for the identification of several putative NDBPs reported in several studies [72]. The study of the transcriptomic profile of NDBPs

managed to provide additional information about the posttranslational processing and the evolutionary diversity of such peptides, factors which are proving to be important tools in the field of taxonomy as well [52]. 3. Structural properties of scorpion NDBPs The scorpion NDBPs are composed of 13–56 amino acid residues and exhibit marked diversity in their sequences. To date more than 40 peptides have been identified and functionally characterized from scorpion venoms so far. Table 1 lists all of these peptides with their corresponding number of amino acids, isoelectric point, charge, biological function and scorpion species. Regarding the secondary structure of NDBPs, the majority of NDBPs with the exception of Peptide T and peptide K-12 display a cationic amphipathic ␣-helical structure [20,37]. The information regarding the conformation of NDBPs was either generated from Circular dichroism studies or from the use of bioinformatics for the prediction of the secondary structure of peptides. The majority of NDBPs display an ␣-helical structure and fall in to three different classes regarding the organization of their ␣-helical regions within the major body of the mature peptide. The first family consists of a single ␣-helix domain and two random coiled regions at both C and T termini. This organization has been observed with Pandinin 2, BmKb1, BmKn2, IsCT, IsCT2, Meucin-24, Im-1, AamAP1, AamAP2, HsAP and Mauriporin [1,2,11,12,14,21,38,43,72]. The second class of helical organization for NDBPs suggests the presence of two alpha-helical regions within the major peptide body separated in the middle by a random coiled region and this orientation has been observed in Hadrurin, Pandinin 1, Opistoporin 1, Parabutoporin and BmKbpp [11,39,60,71]. The last class described is for peptides that display 100% helicity and this has been observed in a fewer number of NDBPs such as Imcroporin and StCT2 [8,74]. One of the characteristic features of NDBPs regarding their conformation is that all these peptides are present in an unordered random coil conformation when present in benign conditions such as aqueous solutions and only when these peptides are shifted to membrane mimicking solutions such as 50–60% aqueous trifluoroethanol (TFE) or in the presence of dodecylphosphocholine (DPC) micelles that the peptides dramatically change their conformation adopting an ␣-helical structure. This behavior and conformational transition which is observed with all studied NDBPs when exposed to membrane mimicking solvents reflects the potential ability and structural flexibility of this group of peptides to interact with anionic membranes of target cells as these solvents mimic the membrane environment of the cells and are responsible for stabilizing the hydrogen bonds within the peptide and its surrounding solutes which ultimately leads to the induction of an ␣-helical structure only in peptides that have the propensity to adopt such a conformation. Additionally, the majority of NDBPs carry a net positive charge in the range of (1–7), a feature that allows theses peptides to be attracted toward the negatively charged phospholipid head groups of the lipid membranes of target cells, a force that is mainly driven by electrostatic interactions. 4. Therapeutic applications of NDBPs The NDBPs group of scorpion peptides displays their activity against a wide range of biological targets leading to significant variation in their biological activity. Unlike the scorpion DBPs that target membrane bound ion channels with their function being predicted through sequence analysis of their mature peptides, the NDBPs do not seem to display conserved sequence–function relationships and some of these peptides even exhibit multifunctional activities without regard to their biological target. To date,

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Table 1 A list of all the functionally characterized NDBPs with their corresponding number of amino acids, isoelectric point, charge, biological function and scorpion species. NDBPs

Number of amino acids

Isoelectric point

Charge

Biological activities

Scorpion species

Peptide T K 12 Hadrurin Pandinin 1 Pandinin 2 Opistoporin 1 BmKb1 BmKn2 IsCT IsCT2 Parabutoporin Mucroporin Meucin-13 Meucin-18 Imcroporin StCT1 Meucin-24 Meucin-25 Im-1 HP 1090 Vejovine Ctriporin BmKbpp VmCT1 VmCT2 AamAP1 AamAP2 StCT2 HsAP Css54 UyCT1 UyCT2 UyCT3 UyCT5 Pantinin-1 Pantini-2 Pantinin-3 Mauriporin TsAP-1 TsAP-2

13 21 41 44 24 44 18 13 13 13 45 17 13 18 17 14 24 25 56 13 47 19 47 13 13 18 18 14 29 25 14 13 13 13 14 13 13 48 17 17

6.65 6.52 11.09 10.49 10.80 10.58 9.67 9.69 9.69 9.69 10.75 9.69 9.69 10.80 10.1 6.34 10.96 11.13 10.82 10.80 10.97 10.80 10.82 10.1 10.1 9.72 9.72 9.88 10.75 11.25 9.53 9.55 9.69 9.69 9.55 9.69 9.69 10.82 9.69 9.69

0 0 +5 +4 +3 +4 +1 +1 +1 +1 +7 +1 +1 +3 +1 0 +4 +4 +7 +2 +4 +2 +7 +1 +1 +2 +2 +2 +3 +4 +2 +2 +2 +2 +1 +1 +1 +6 +1 +1

Bradykinin-potentiating Bradykinin-potentiating Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, immune-modulatory, hemolytic Antimicrobial Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, immune-modulatory, hemolytic Antimicrobial Antimicrobial, hemolytic, cytolytic Antimicrobial, hemolytic, cytolytic Antimicrobial, hemolytic, cytolytic Antimicrobial, hemolytic Antimalarial Antimalarial Antimicrobial, hemolytic, cytolytic Antiviral Antimicrobial, hemolytic Antimicrobial Antimicrobial, hemolytic, bradykinin-potentiating Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Antimicrobial, hemolytic Anticancer Antimicrobial, anticancer Antimicrobial, hemolytic, anticancer

Tityus serrulatus Buthus occitanus Hadrurus aztecus Pandinus imperator Pandinus imperator Opistophtalmus carinatus Mesobuthus martensii Mesobuthus martensii Opisthacanthus madagascariensis Opisthacanthus madagascariensis Parabuthus schlechteri Lychas mucronatus Mesobuthus eupeus Mesobuthus eupeus Isometrus maculates Scorpiops tibetanus Mesobuthus eupeus Mesobuthus eupeus Isometrus maculatus Heterometrus petersii Vaejovis mexicanus Chaerilus tricostatus Mesobuthus martensii Vaejovis mexicanus Vaejovis mexicanus Androctonus amoreuxi Androctonus amoreuxi Scorpiops tibetanus Heterometrus spinifer Centruroides suffusus suffusus Urodacus yaschenkoi Urodacus yaschenkoi Urodacus yaschenkoi Urodacus yaschenkoi Pandinus imperator Pandinus imperator Pandinus imperator Androctonus mauritanicus Tityus serrulatus Tityus serrulatus

several biological functions have been reported to NDBPs including Bradykinin potentiating, antimicrobial, antimalarial, antiviral, hemolytic, insecticidal, Immune-modulatory and anticancer activities (Table 1). 4.1. Bradykinin potentiating activities Bradykinin is a vasoactive peptide that plays an important role in blood pressure regulation. Bradykinin is responsible for increasing vasodilation and capillary permeability and consequently lowering blood pressure [24]. The angiotensin converting enzyme (ACE) proteolytically inactivates bradykinin and the discovery of peptide inhibitors for this enzyme named the bradykinin potentiating peptides (Bpps) lead to the development of captopril, the first active site directed inhibitor of ACE currently used for the treatment human hypertension [7]. The first scorpion peptide discovered to display bradykinin potentiating activities was Peptide T which was isolated from the venom of the scorpion Tityus Serrulatus [20]. Peptide T is composed of 13 amino acid residues and was the first reported NDBP identified from scorpion venoms. Peptide T potentiated the contractile activity of bradykinin on the isolated ileum of guinea pigs and inhibited the proteolytic activity of ACE against Bradykinin [20]. Another Bpp, named peptide K12 was also isolated from the venom of the scorpion Buthus occitanus [37]. Peptide K12 was found to be composed of 21 amino acid residues and managed to potently inhibit ACE activity and behaved in a similar manner to the previously identified peptide T. Peptide K-12 did not share any sequence homology

with peptide T and any other proteins or peptides found in protein data banks [37]. BmKbpp is a 47 amino acid peptide identified from the venom of the Chinese scorpion Mesobuthus martensii Karsch that also displays bradykinin potentiating activities as the previous two peptides. Sequence homology analysis of BmKbpp revealed that the C-terminal region of BmKbpp shares a significant degree of homology (57%) with the Bpp K-12. BmKbpp was also found to display antimicrobial and immune-modulatory activities in addition to the activities mentioned earlier which highlights the multifunctional nature of this peptide [71].

4.2. Antibacterial, antifungal and cytolytic activities Antimicrobial peptides (AMPs) are considered to be a significant component of the innate defence system of a variety of eukaryotic organisms including humans, plants and insects [41]. This group of peptides are considered to be primitive in their nature and mechanism of action as they are one of the first immune defence mechanisms to evolve in nature lacking specific targets with their synthesis and secretion being induced by the entry of foreign invading agents or expressed constitutively within the organism as a result of environmental pressures [49]. Antimicrobial peptides display a broad spectrum of activity against a wide range of microorganisms including bacteria, protozoa, yeast, fungi and viruses [27]. The majority of scorpion NDBPs can be considered as members of the group of antimicrobial peptides as 34 peptides out of

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Table 2 The antibacterial, antifungal and hemolytic activities of scorpion NDBPs. NDBP

Hadrurin Pandinin 1 Pandinin 2 Opistoporin 1 BmKb1 BmKn2 IsCT IsCT2 Parabutoporin Mucroporin Meucin-13 Meucin-18 Imcroporin StCT1 Im-1 Vejovine Ctriporin BmKbpp VmCT1 VmCT2 AamAP1 AamAP2 StCT2 HsAP Css54 UyCT1 UyCT2 UyCT3 UyCT5 Pantinin-1 Pantini-2 Pantinin-3 TsAP-1 TsAP-2

MIC (␮M) Gram positive

Gram negative

10–50 1.3–5.2 2.4–4.8 12.5–50 16–81.5 0.6–8 0.7–16.6 0.7–17.1 6.3–50 25–50 0.25–2.9 0.25–0.6 20–50 12.5–100 0.8–25 – 5–10 5.7–70 10–20 10–20 20 48 6.5–25 11.8–46.5 12.5 15 ND 10 1 8–32 18–48 4–16 120 5

10–50 20.8–20.8 19.1–38.2 1.6–50 18.1–90.8 1.5–21.3 3.3–150 3.4–150 1.6–50 >100 6.2–50 1.5–10.9 >100 >100 0.4–0.8 4.4 >100 2.3–68.2 5–25 10–20 150 120 25–50 23.8–51.2 12.5 10 25–40 6–15 2–15 62–87 48–87 36–87 160 >320

Activity on resistant strains of bacteria

Antifungal activity (␮M)

Hemolytic activity

ND ND ND ND ND ND ND ND ND MRCNS ND ND MRSA, MRCNS, PRSE MRSA, MRCNS, PREF, PRSA ND E. coli, E. cloacae, A. baumannii, K. pneumonia, P. aeruginosa MRSA, MRCNS, PRSE ND ND ND ND ND PRSA, MRSA ND ND ND ND ND ND MRSA, VRE MRSA, VRE MRSA, VRE ND ND

ND ND ND 0.8–3.1 ND ND ND ND ND ND 14.1–50 1.9–8.3 ND ND ND ND 20 0.2–3.1 ND ND 64 64 ND 48.6 ND ND ND ND ND 16 16 17 160 10

+ + + ND ND ND + + + ND + + + + ND + ND + + + + + + + + + + + + + + + + +

MIC, minimal inhibitory concentration; ND, not determined; + positive activity; −, negative activity; MRCNS, methicillin-resistant coagulase-negative Staphylococci; MRSA, methicillin-resistant Staphylococcus aureus; PRSE, penicillin-resistant Pneumococci; PREF, penicillin-resistant Enterococcus faecalis; PRSA, penicillin-resistant Staphylococcus aureus.

the 40 that have been functionally characterized till now display antimicrobial activities (Table 2). Hadrurin, a 41 amino acid peptide isolated from the venom of the scorpion Hadrurus aztecus was the first scorpion NDBP to demonstrate antimicrobial activities as it was able to inhibit the growth of Gram-positive and Gram-negative bacteria at low micromolar concentrations (MIC 10–50 ␮M) [60]. Hadrurin was also found to display potent cytolytic and hemolytic activities at the same concentration range needed to inhibit the growth of bacteria. To assess the probable molecular mechanism of action of Hadrurin and if the peptide was targeting a specific cellular receptor, a Cterminal amidated replicate and an all d-enantiomer of Hadrurin was synthesized and tested against the same strains of microorganisms that were used previously. The results of these studies clearly indicate that the synthetic replicates of Hadrurin behaved in a similar manner to the native peptide with no differences in the mode of action detected and consequently excluding the possibility of receptor targeting and indicating that the most probable mechanism of action for Hadrurin is through membrane destabilization as reported with several cationic antimicrobial peptides discovered from other animal species [60]. Pandinin 1 and 2 are two antimicrobial peptides isolated from the venom of the scorpion Pandinus imperator. Both peptides displayed potent antimicrobial activities against Gram-positive bacteria with a MIC range of 2.4–5.2 ␮M and their activity was less potent against Gram-negative bacteria with a MIC range of 2.4–38.2 ␮M. Additionally, Pandinin2 managed to inhibit the growth of the yeast Candida albicans. Pandinin 1 also did not display hemolytic activity against sheep erythrocytes, in contrast with Pandinin 2 which exhibited strong hemolytic activity

[11]. Opistoporin 1 is 44 amino acid antimicrobial peptide that was isolated from the venom of the South African scorpion Opistophtalmus carinatus. Opsitoporin 1 was found to be most active in inhibiting the growth of Gram-negative bacteria with a MIC range of 1.6–50 ␮M and with less potent activity against Gram-positive bacteria and moderate hemolytic activity. Additionally Opistoporin 1 displayed potent antifungal activities, inhibiting 50% growth of both phytopathogenic fungi Botrytis cinerea and Fusarium culmorum at a concentration of 5 ␮M [39]. BmKb1 and BmKn2 are two antimicrobial peptides identified through molecular cloning from the cDNA library of the venom of the scorpion M. martensii. BmKb1 is composed of 18 amino acid residues while BmKn2 is composed of 13 residues. When the two peptides were assessed for their antimicrobial activity, BmKn2 displayed potent inhibitory activities against both Gram-positive and Gram-negative bacteria while Bmkb1 was weaker than Bmkn2 in inhibiting bacterial growth. These differences in the antimicrobial behavior between the two peptides could be explained by the number of positively charged residues found with BmKb1 which are significantly less in number when compared to BmKn2 [72]. IsCT is a short linear antimicrobial peptide isolated from the venom of the scorpion Opisthacanthus madagascariensis. IsCT demonstrated potent antimicrobial activities against both Gram-positive and Gram-negative bacteria with strong hemolytic activity against sheep erythrocytes. Additionally, IsCT is considered to be one of the shortest cytotoxic peptides ever to be identified [14]. Parabutoporin is a potent scorpion antimicrobial peptide that was isolated from the venom of the scorpion Parabuthus schlechteri. Parabutoporin was found to inhibit bacterial growth of Gram-negative bacteria with a MIC concentration

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as low as 1.6 ␮M. Additionally Parabutoporin was found to be hemolytic against human erythrocytes with an LD50 of 37 ␮M and it also was found to display potent cytotoxic activities against normal mammalian cells such as human neutrophils displaying an LD50 of 1.3 ␮M and thus limiting its potential for clinical application [63]. Mucroporin is a 17 amino acid antimicrobial peptide that was cloned and functionally characterized from the venom of the scorpion Lychas mucronatus. Mucroporin was effective in inhibiting the growth of Gram-positive bacteria with a MIC value of 25 ␮g/ml against Staphylococcus aureus while its activity against Gram-negative bacteria was minimal and concentrations above 100 ␮g/ml were needed to inhibit the growth of Escherichia coli. A synthetic analog of Mucroporin named Mucroporin-M1 was designed for the purpose of enhancing the net positive charge on the parent peptide and to assess the influence of increasing the cationic nature of Mucroporin on its antimicrobial behavior. The change in net positive charge which was designed for Mucroporin-M1 caused a significant improvement in the antimicrobial activity of the peptide against both Gram-positive and Gram-negative bacteria when compared with the parent peptide. Additionally Mucroporin-M1 demonstrated potent antibacterial activity against clinically isolated antibiotic-resistant pathogens such as methicillin-resistant S. aureus (MRSA) and methicillinresistant coagulase-negative Staphylococci (MRCNS). These results indicate that Mucroporin can be used successfully as an ideal template for ant-infective drug design [13]. Meucin-13 and Meucin-18 are two scorpion antimicrobial peptides that were identified from a cDNA library prepared from the venom gland of the scorpion mesobuthus eupeus and are composed of 13 and 18 amino acids each, respectively. Both peptides displayed potent cytolytic activities against both prokaryotic and eukaryotic cells including Gram-positive and Gram-negative bacteria, fungi, yeasts, rabbit erythrocytes and root ganglion cells at micromolar concentrations. Meucin-18 was found to display 2–14 fold higher potency than meucin-13 against all cells tested, a behavior that is attributed to differences in the cationic nature and hydrophilic/hydrophobic distribution of both peptides [21]. Im-1 is a 56 amino acid antimicrobial peptide isolated from the venom of the Japanese scorpion Isometrus maculates. Till date Im-1 is reported to contain the longest peptide chain within the group of scorpion NDBPs and the peptide proved to display potent bacterial inhibitory activities at submicromolar concentrations against the Gram-negative bacteria E. coli with modest activity against the Gram-positive strains such as Bacillus subtilis and S. aureus. Moreover Im-1 exhibited significant insecticidal activity causing rapid and reversible paralysis of crickets after injection making it the only scorpion NDBP reported to display insecticidal activity [38]. BmKbpp is a multifunctional peptide isolated from the venom of the scorpion Mesobuthus martenssii Karsch, Bmkbpp was found to exhibit potent antimicrobial, Bradykinin potentiating and immune-modulatory activities. BmKbpp possesses strong antimicrobial activity against both Gram-positive and Gram-negative bacteria with MIC values in the range of 2.3–68.2 ␮M. The highest antimicrobial activity for BmKbpp was against Gram-negative bacteria and specifically against the E. coli DH 5-␣ strain with a MIC of 2.3 ␮M. BmKbpp also displayed potent antifungal activity with IC50 values as low as 0.2 ␮M against the plant pathogen F. culmorum. Additionally, BmKbpp was found to display moderate hemolytic activity and the percentage hemolysis induced by BmKbpp never exceeded 39.5% at the highest concentration employed in the antimicrobial studies [71]. VmCT1 and VmCT2 are two short antimicrobial peptides identified from the cDNA library of the Mexican scorpion Vaejovis mexicanus smithi. Both peptides are composed of 13 amino acid residues and display broad spectrum activity against Gram-positive and Gram-negative bacteria with similar MIC values in the range of 5–25 ␮M. Although their antimicrobial activity was comparable,

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their hemolytic activity differed significantly as VmCT1 cause mild hemolysis with a maximum of 12% at 50 ␮M, while VmCT1 caused 84% hemolysis at the same concentration employed [48]. AamAP1 and AamAP2 are two C-terminally amidated antimicrobial peptides that were identified from the venom derived cDNA library of the North African scorpion Androctonus amoreuxi. Both peptides are composed of 17 amino acids and differ in primary structure at just two positions, both peptides displayed moderate broad spectrum antimicrobial activity in the range of 20–150 ␮M against representative strains of Gram-positive, Gram-negative bacteria and yeast. In regards to their hemolytic activity against sheep erythrocytes, both peptides displayed significant hemolytic activity at high concentrations [2]. HsAP is a scorpion antimicrobial peptide that was identified from the cDNA library of the Southeast Asian scorpion Heterometrus spinifer. HsAP is a C-terminally amidated peptide composed of 29 amino acid residues and displays moderate antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi with MIC values in the range of 11.8–51.2 ␮M. Moreover, HsAP also exhibited potent hemolytic activity against human erythrocytes at low concentrations and induced 100% hemolysis at a concentration of 6.4 ␮M suggesting that HsAP lacks any apparent target cell selectivity and displays high toxicity against mammalian cells limiting its therapeutic applications [43]. Recently another scorpion antimicrobial peptide named Css54 was isolated from the venom of the Mexican scorpion Centruroides suffusus suffusus. The primary structure of Css54 was determined using N-terminal sequencing combined with mass spectrometry and revealed that Css54 is composed of 25 amino acid residues. The antimicrobial activity of Css54 was determined against both representative strains of Gram-positive (S. aureus) and Gram-negative bacteria (E. Coli). The antimicrobial studies revealed that the peptide was displaying equal potency against both strains of bacteria with a MIC value of 12 ␮M, the hemolytic activity of Css54 was also evaluated against human erythrocytes and the peptide was found to display moderate hemolytic activity of approximately 30% at a concentration of 10 ␮M and caused 83% hemolysis at 25 ␮M [23]. Three scorpion antimicrobial peptides were identified recently from the venom of the Australian scorpion Urodacus yasochenokiby. The peptides were identified using a combination of both proteomic and transcriptome analytical techniques for the evaluation of scorpion venom components. The three peptides named UyCT1, UyCT3 and UyCT5 exhibit a significant degree of sequence similarity and their antimicrobial activity was evaluated against three microbial strains (S. aureus, E. Coli and P. aeruginosa). The antimicrobial studies revealed that all three peptides display potent activities at concentrations lower than 40 ␮M with UyCT5 being the most potent of all three inhibiting the growth of S. aureus with a MIC value of 1 ␮M. Additionally, the cytotoxicity of all three peptides was evaluated against human erythrocytes and revealed that the peptides exhibited moderate cytotoxicity as peptides UyCT1 and UyCT3 displayed 17–25%, and 5–35% hemolysis, respectively at their corresponding MICs while peptide UyCT5 does not show marked hemolysis at lower concentrations (1–2) ␮M and only at a MIC value of 15 ␮M that the peptide achieved 37% hemolysis indicating that the peptide is mildly hemolytic [35]. Another three scorpion antimicrobial peptides were also identified recently through a cDNA library screening strategy from the venom glands of the scorpion Panidnus imperator. The three peptides named Pantinin-1, Pantinin-2 and Pantinin-3 are composed of 13 or 14 residues respectively and all peptides display weak antimicrobial activity against Gram-negative bacteria but display potent activities against Gram-positive bacteria with Pantinin-1 being the most potent of all three peptides against non-resistant strains of S. aureus with a MIC value of 8 ␮M while Pantinin-3 being the most

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potent against antibiotic resistant strains such as MRSA and VRE with MIC values of 4 ␮M and 12 ␮M, respectively [73].

4.3. NDBPs with antimicrobial activity against antibiotic resistant strains of microorganisms During the last decade, there has been a progressive shrinkage in the range of antimicrobial drugs effective against common human pathogens due to the increased frequency of bacterial antibiotic resistance, especially the evolution of multidrug-resistant pathogens [53,65]. The widespread overuse and systemic misuse of those potent and relatively safe antibiotics contributed heavily to the issue of global microbial resistance [65]. Thus, new agents to treat bacterial infection are badly needed. The broad-spectrum activity combined with the low potential to induce resistance make AMPs an attractive group of molecules with the potential to be developed as therapeutic agents for the purpose of combating antibiotic resistant microorganisms [31]. The majority of NDBPs belong to the group of AMPs and several members were found to display potent activities against resistant strains of microorganisms. Imcroporin is a 17 amino acid peptide that was identified from the cDNA library of the scorpion Isometrus maculates. Imcroproin was the first peptide identified from scorpion venoms to display potent antimicrobial activity against antibiotic-resistant microorganisms such as MRSA, MRCNS and penicillin-resistant Pneumococci (PRSE). The MIC of Imcroporin against MRSA was 50 ␮g/ml, 40 fold lower than that of penicillin and 8 fold lower than Cefotaxime. Additionally, the cytotoxicity of Imcroporin against human erythrocytes was lower than 50% at a concentration of 50 ␮g/ml. Imcorporin antibacterial activity was also tested in the mouse model, where Imcroporin managed to rapidly decrease the bacterial counts within the infected mice and achieved a 100% cure rate after 7 days of treatment without affecting the overall health status of treated mice. All this data suggest that Imcroporin could be considered as a novel lead compound and a potential anti-infective agent that could be exploited for clinical use for the treatment of infections caused by antibiotic-resistant microorganisms [74]. Another scorpion NDBP with inhibitory activity against antibiotic-resistant pathogens is StCT1, a C-terminally amidated 14 amino acid residue peptide identified from the venom generated cDNA library of the Chinese scorpion Scorpiops tibetanus. The MICs of StCT1 against the antibiotic resistant pathogens such as MRSA, MRCNS, penicillin-resistant Enterococcus faecalis (PREF) and penicillin-resistant S. aureus (PRSA) were in the range of 50–250 ␮g/ml. However, no studies were performed on StCT1 to identify its cytolytic and hemolytic activity against mammalian and red blood cells, thus limiting the data regarding the toxicity of the peptide in order to determine the potential of exploiting StCT1 for clinical use against antibiotic-resistant infections [67]. Another scorpion NDBP reported to display potent antimicrobial activity against multi-drug resistant microorganisms is Ctriporin that was identified from the venom gland constructed cDNA library of the scorpion Chaerilustri costatus. Ctriporin is composed of 19 amino acids with an amidated C terminus and proved to exert potent antimicrobial activity against standard Gram-positive strains with MIC values in the range of 5–20 ␮g/ml. Moreover, Ctriporin managed to inhibit the growth of antibiotic-resistant pathogens such MRSA, MRCNS and PRSE strains with MIC values as low as 10 ␮g/ml. The potential of Ctriporin to be used as a topical antibiotic was also assessed using a S. aureus infected mouse skin model and the peptide managed to significantly reduce the number of bacterial counts and cure the infection in mice completely. These results indicate that Ctriporin could have the potential to be developed into a new topical antibiotic to treat MRSA skin infections [19].

StCT2 is a scorpion antimicrobial peptide with exclusive activity against Gram-positive bacteria. StCT2 was identified from the venom derived cDNA library of the Chinese scorpion Scorpiops tibetanus. StCT2 is C-terminally amidated and is composed of 14 amino acids. The peptide displayed potent antimicrobial activity against Gram-positive bacteria, but showed no activity against representative strains of Gram-negative bacteria. StCT2 inhibited the growth of different strains of S. aureus with MIC values in the range of 6.25–25 ␮g/ml including antibiotic resistant strains such as MRSA. Additionally, the in vivo bioactivity of StCT2 was assessed with a S. aureus mouse infected model and the peptide managed to achieve a 100% cure rate in the infected mice [8]. Vejovine is a 47 amino acid peptide isolated from the venom of the Mexican scorpion Vaejovis mexicanus. This peptide displayed significant potent antimicrobial activity against clinical isolates of Gram-negative multidrug resistant strains of Pseudomonas aeruginosa and Acinetobacter baumanii with MIC values as low as 4.4 ␮M. Additionally Vejovine displayed cytolytic and hemolytic activity against human erythrocytes reaching 50% hemolysis at 100 ␮M which clearly highlights the low hemolytic activity of Vejovine and demonstrates that this molecule is a good candidate for development as a new potent anti-infective agent against multidrug resistant strains of Gram-negative bacteria [29]. 4.4. Antiviral activity Viral diseases represent a major cause of morbidity and mortality worldwide and are considered a significant health burden facing the global human population [17]. Vaccination managed to eradicate and permanently disable several viral diseases successfully but a large number of viral pathogens responsible for current human viral infections remain with no vaccines available [44]. Additionally, the development of novel antiviral agents remains hampered by several obstacles such as the significant viral genetic adaptability, high mutation rate and the great cost of research associated with antiviral drug development [18]. All these factors highlight the importance of developing novel therapeutic strategies for combating viruses and viral infections. Cationic alpha-helical peptides displaying antiviral activities could prove to be good candidates for novel antiviral drug development due to their significant antiviral potency, low cost as low molecular weight molecules and low levels of side effects [59]. Hp1090 was the first and only scorpion NDBP to display potent antiviral activities in vitro [66]. Hp1090, a 13 amino acid peptide identified from the venom gland constructed cDNA library of the scorpion Heterometrus petersii managed to inhibit Hepatitis C virus (HCV) infection with an IC50 of 5 ␮M and also inhibited HCV RNA amplification in Huh7.5.1 cells with higher potency than IFN-␣. Additionally Hp1090 seemed to kill HCV directly by binding to the viral particle and disrupting the structural integrity of the virus [66]. 4.5. Antimalarial activity Malaria constitutes a major public health problem in the majority of tropical countries [58]. Malaria is caused by the female Anopheles mosquito-borne unicellular parasite Plasmodium falciparum and is responsible for 1.2 million of human deaths annually of which 200,000 are reported in infants [28]. Current effective drugs used for the treatment of malarial infections are dwindling rapidly due to the resistance acquired by the P. falcipaum parasite against these drugs and the resistance acquired by the female Anopheles mosquito to the pesticides currently in use for the eradication and control of the mosquitoes in tropical areas [16,47]. Several cationic alpha helical peptides identified from different animal species have been reported to display antimalarial activities against the Plasmodium genus of parasites [4]. Most of

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these peptides display potent antibacterial and hemolytic activities in addition to their antimalarial activities which could indicate that several antimicrobial cationic alpha helical peptides could have the potential to kill the plasmodium parasite if assessed against it. Meucin-24 and Meucin-25 were the first and only scorpion NDBPs reported to display selective antimalarial activity against the human parasite P. falciparum. Both peptides composed of 24 and 25 amino acids, respectively, were identified from the cDNA library of the venom gland of the scorpion Mesobuthus eupeus. Meucin-24 and Meucin-25 managed to significantly reduce the density of intraerythrocytic P. falciparum concentrations after 48 h of exposure to 10 ␮M of both peptides and no infected P. falciparum erythrocytes were detected after 72 h of treatment. Additionally both peptides lacked any antimicrobial and antifungal activities at micromolar concentrations against a panel of several microorganisms with no effect on the cell viability of mammalian cells displaying the exclusive selective antimalarial nature of both peptides [22].

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Table 3 A summary of the anticancer activities of scorpion NDBPs. NDBP

Cancer Cell line

IC50 (␮M)

Mauriporin

PC3 LnCAP DU-145

7.7 7.8 4.4

TsAP-1

NCI–H157 NCI–H838 PC3 MCF-7 U251 NCI–H157

55.9 52.5 NA NA NA 4.1

TsAP-2

NCI–H838 PC3 MCF-7 U251

11.0 13.3 6.4 2.9

NA, no activity.

4.6. Anticancer activity Cationic alpha-helical peptides displaying anticancer activities represent a novel therapeutic class of bioactive peptides that have gained a great interest in the past decade [30]. This group of peptides is naturally found in several organisms including bacteria, fungi, plants, and animals and is thought to play a major role in defending the organisms against invading pathogens and seems to constitute one of the major pillars of the innate defense system of host organisms [51]. Named as Anticancer peptides (ACPs), members of this group have been assessed for their ability to kill malignant cells both in vitro and in vivo and a significant number displayed potent antiproliferative activities. ACPs can be defined generally as small peptides having less than 50 amino acid residues in length and carrying a net positive charge ranging from +2 to +7 with a substantial portion of the amino acid residues composing the peptide exhibiting a hydrophobic nature and adopting an ␣-helical secondary structure [15,51]. These structural parameters permit the peptides to fold into an amphipathic or amphiphilic structures once in contact with membranes or membrane mimetic environments. The mechanism of action of ACPs against cancer cell lines can be explained by the unique structural properties of ACPs and the distinctive morphology of cancer cells that makes them susceptible to induction of cell death by these peptides. Cancer cells display a relatively high negative surface charge due to structural abnormalities that accompany the process of cell malignancy and these cells usually contain a higher percentage of phosphophatidylserines and o-glycosylated mucins in their membranes, which could be the major factor behind the ACPs apparent selective activity [5,34]. Another factor that could aid ACPs in targeting cancerous cells is the presence of microvilli or pseudopodia on transformed cancerous cells. These projections tend to increase the total surface area of tumor cells thus enabling the binding of a higher number of ACPs to their cellular membranes and consequently disrupting their structural integrity [55]. Till date, only three scorpion NDBP has been found to display potent anti-proliferative and anticancer activity (Table 3). Mauriporin a 48 amino acid peptide that was identified from the venom derived cDNA library of the scorpion Androctonus mauritanicus was found to exert potent selective cytotoxic and antiproliferative activity against three different prostate cancer cell lines (IC50 4.4–7.8 ␮M) when compared with non-tumorigenic cells as the human umbilical vein cells (HUVECs) and monkey African green monkey kidney epithelial cells (Vero) were resistant to Mauriporin at the IC50 values that were needed to inhibit the proliferation of tumorigenic cell lines [1]. Additionally, Mauriporin displayed diminished hemolytic activity against sheep erythrocytes in the

concentration range needed to kill the cancer cells. Even at a concentration of 80 ␮M, which is 10 times higher than the IC50 values obtained for Mauriporin against the prostate cancer cells, no significant hemolytic activity was observed and the percentage of hemolysis never exceeded 4.8% [1]. The mode of cell death induced by Mauriporin was investigated and the results revealed that Mauriporin is not inducing cell death through an apoptotic mode of cell death and consequently is not acting upon an intracellular target. Mauriporin was not able to induce DNA fragmentation, a major biochemical hallmark indicative of apoptosis. Additionally, the ability of Mauriporin to increase the concentration of caspase-3, one of the major enzymes activated during the cascade of events associated with apoptosis was not detected excluding the possibility of an apoptotic mode of cell death and indicating that Mauriporin is possibly exerting its cytotoxic activity through a necrotic mode of cell death which is consistent with the mechanism of action of other cationic ␣-helical peptides identified from venomous animals [1]. Two anticancer peptides named TsAP-1 and TsAP-2 were also identified recently from the venom derived cDNA library of the Brazilian scorpion T. serrulatus [25]. The antiproliferative activity of both peptides were tested against five different cancer cell lines and these include the non-small cell lung adenocarcinoma (H838), the human oral squamous cell carcinoma (H157), the human breast adenocarcinoma (MCF-7), the human androgenindependent prostate cancer (PC-3) and the human glioblastoma (U251-MG) cell lines. TsAP-1 managed to inhibit the proliferation of two of the five cell lines tested and with the non-small cell lung adenocarcinoma cancer cell line H838 and the human oral squamous cell carcinoma cell line H157.The IC50 values obtained for TsAP-1 against the two cell lines were 55.9 ␮M and 52.5 ␮M, respectively. TsAP-2 displayed significant potency against all five human cancer lines tested with IC50 values of 4.1 ␮M (H157), 11 ␮M (H838), 15.4 ␮M (U251-MG) and 13.3 ␮M (PC3) [25]. The antiproliferative activity of both peptides was not assessed against non-tumorigenic cell lines and consequently no data regarding the selectivity and toxicity of both peptides was obtained. However, the hemolytic activity of both peptides was assessed against horse erythrocytes. TsAP-1 possessed little hemolytic activity and the percentage hemolysis reported for the peptide at the highest concentration employed (160 ␮M) was 6.4% while Peptide TsAP-2 demonstrated no hemolytic activity up to a concentration of 15 ␮M. However at 40 ␮M the peptide caused significant hemolysis of 86% and at concentrations of 80 ␮M and higher, TsAP-2 caused complete hemolysis of the sheep erythrocytes [25].

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4.7. Immune-modulatory activity The majority of scorpion NDBPs and due to their structural and functional properties can be considered as members of the large family of cationic host defense peptides that is widely distributed in nature and has been isolated and identified from plants, insects to highly evolved mammals with more advanced immune systems [41]. These peptides are generally constitutively expressed within their secretory tissue or their expression is induced through external stimuli such as microbial invasion, inflammation and tissue injury [6]. Generally host defense peptides interact with biological membranes and act as pore formers causing cell membrane destabilization which eventually lead to cell lysis and death [45]. However, cationic host defense peptides have been also reported to influence cell function by interacting with cellular cell signaling molecules and consequently modulating the immune response of the cell [41]. Some of the immunemodulatory functions cationic host defenses peptides demonstrate include stimulation of chemotaxis, induction of inflammatory gene expression and regulating the activity of adaptive immunity [3,26]. Several scorpion NDBPs have been assessed for their roles as regulators of the immune system and these include Opsitoporin 1, Parabutoporin and BmKbpp. At high concentrations all peptides behave as membranes destabilizers and display potent lytic activity against target cells while at non-lytic submicromolar concentrations the peptides seem to alter the activity of human neutrophils [64]. Neutrophils have the capability to produce large amounts of superoxide ions such as hydrogen peroxide and hydroxyl radicals that are generated by NADPH oxidase, a membrane bound enzyme complex that can be found in the plasma membranes as well as the membrane of the neutrophil phagosome [56]. As the degranulation of human neutrophil granulocytes is considered to be a Ca2+ mediated process [33], the effects of both Parabutoporin and Opistoporin 1 on the release of intracellular Ca2+ and the involvement of certain receptors in the process was investigated for both peptides. The results of the study revealed that both peptides managed to increases intracellular Ca2+ release from intracellular stores in addition to Ca2+ release as a result of extracellular space leakage in HL-60 cells [40]. Additionally, the increase in Ca2+ release was not mediated through the interaction with the formyl peptide receptor (FPRL1) excluding the possibility of the involvement of this receptor in Ca2+ release. Parabutoporin was also found to be a potent inhibitor of granulocyte NADPH oxidase, as NADPH oxidase system activation is mainly mediated by the generation of active Rac which normally proceeds through the activation of heterotrimeric G-proteins and PKC/PLC and PI3 pathways. It has been proposed that Parabutoprin inhibitory effects on NADPH oxidase could be explained by its ability to act as a substrate for the protein kinase C (PKC) and thus inactivating the assembly of the NADPH oxidase system [63]. Additionally, at submicromolar concentrations Parabutoporin has been shown to stimulate neutrophil motility through the activation of pertussis toxin sensitive signaling pathways through the upregulation of GTP bound Rac2 levels and possibly through interaction of G proteins as proposed earlier [50].

5. Molecular mechanisms for NDBPs diversity Different molecular and genetic evolutionary models have been proposed to explain the genetic and functional diversity of non-disulfide bridged scorpion peptides. The sequence diversification of scorpion NDBPs could be explained by the process of gene duplication. Gene duplication relieves the gene copy from selective pressure and the correctional mechanisms the gene undergoes in case any serious deleterious mutations might

arise and this could lead to significant alterations in the genetic sequence of the original gene [76]. In the case of scorpion NDBPs, multiple sequence alignment studies indicate a high degree of intron divergence and a significant degree of mutation probability when compared to the exons which are usually intact and generally similar [36]. This model could explain genomic mechanisms responsible for peptide diversification of most scorpion NDBPs. Polymorphisms of NDBP genes could also be a major factor responsible for the diversity of this group of peptides. Several gene isoforms identified from the cDNA library of different scorpions have indicated the presence of single base mutations, deletion, and insertions that took place at the genomic DNA level. Additionally, examination of the cDNA library of several scorpion species indicated the presence of different deduced peptides with high sequence homology within the same species which possibly resulted from a genetic polymorphism mechanism. One alternative model for peptide divergence occurs through a trans-splicing event that could effectively join different exons from two pre-mRNA transcripts to produce a natural recombinant transcript. This mechanism has been considered as one of the most powerful tools in the great evolutionary machine that generates protein diversity [75]. An example of such a genetic mechanism within scorpion NDBPs is the BmKbpp cDNA clone that was identified from the venom of the scorpion Buthus martensii Karsch. The C-terminal region of BmKbpp is highly homologous to the bradykinin potentiating peptide K-12 while the N-terminal region of the peptide seems to be derived from a presumed transcript of an unknown antimicrobial peptide. This finding for BmKbpp is highly suggestive of its generation by a recombinant event such as transsplicing and could explain the multifunctional nature of the peptide [70].

6. Classification of NDBPs NDBPs represent a highly divergent and versatile group of peptides in regards to their primary sequence, thus creating a difficulty in classifying these peptides on the principle of their sequence or structural similarity. Zeng et al. introduced a classification system for NDBPs identified from scorpion venoms which is based in principle on the pharmacological activity, peptide length and sequence similarity of the peptides [68]. In this scheme, the peptides were classified into six subfamilies with the suggestion that each peptide should be named NDBP-x·y where x and y stand for the subfamily and number of peptide within the subfamily which should be assigned chronologically. The Classification advised by Zeng et al. proved to be satisfactory at the time when it was introduced as the number of peptides did not exceed 17 peptides in total including the functionally characterized peptides and the peptides with no identified biological activity. Due to the significant increase in the number of NDBPs discovered in the last decade, this classification system has to be modified in order to accommodate the newly discovered peptides and the ones that could be identified in the future. One of the major drawbacks of the Zeng’s classification system is that two subfamilies within the system were temporarily assigned to peptides with no identified biological activity with the assumption that these peptides along with newly discovered ones will be functionally characterized and their biological activity identified in future studies. Unfortunately this prediction was not achieved as these peptides remain without any known biological activities. In order to accommodate the significant growth of the newly characterized scorpion NDBPs, we propose a new modified classification system that includes only functionally characterized peptides and uses the same criteria employed in the old system which is based in

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Table 4 Classification of scorpion NDBPs. Group number

Group name

NDBP number

Group 1 Peptide T K12

Bradykinin potentiating peptides KKDGYPVEYDRAY LRDYANRVINGGPVEAAGPPA

1.1 1.2

Group 2 Hadrurin BmKbpp Pandinin1 Opistoporin1 Parabutoporin Im-1 Vejovine Mauriporin

Long chain multifunctional peptides GILDTIKSIASKVWNSKTVQDLKRKGINWVANKLGVSPQAA FRFGSFLKKVWKSKLAKKLRSKGKQLLKDYANKVLNGPEEEAAAPAE GKVWDWIKSAAKKIWSSEPVSQLKGQVLNAAKNYVAEKIGATPT GKVWDWIKSTAKKLWNSEPVKELKNTALNAAKNLVAEKIGATPS FKLGSFLKKAWKSKLAKKLRAKGKEMLKDYAKGLLEGGSEEVPGQ FSFKRLKGFAKKLWNSKLARKIRTKGLKYVKNFAKDMLSEGEEAPPAAEPPVEAPQ GIWSSIKNLASKAWNSDIGQSLRNKAAGAINKFVADKIGVTPSQAAS FKIGGFIKKLWRSKLAKKLRAKGRELLKDYANRVINGGPEEEAAVPAE

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Group 3 Pandinin2 Css54 HsAP

Medium-length antimicrobial peptides FWGALAKGALKLIPSLFSSFSKKD FFGSLLSLGSKLLPSVFKLFQRKKE SGTSEKERESGRLLGVVKRLIVCFRSPFP

3.1 3.2 3.3

Group 4 IsCT ISCT2 BmKb1 BmKn2 Mucroporin Meucin-13 Imcroporin StCT1 HP1090 Ctriporin AamAP1 AamAP2 VmCT1 VmCT2 StCT2 UyCT1 UyCT2 UyCT3 UyCT5 Pantinin1 Pantinin2 Pantinin3 TsAP-1 TsAP-2

Short antimicrobial peptides ILGKIWEGIKSLF IFGAIWNGIKSLF FLFSLIPSAISGLISAFK FIGAIANLLSKIF LFGLIPSLIGGLVSAFK IFGAIAGLLKNIF FFSLLPSLIGGLVSAIK GFWGSLWEGVKSVV IFKAIWSGIKSLF FLWGLIPGAISAVTSLIKK FLFSLIPHAIGGLISAFK FPFSLIPHAIGGLISAIK FLGALWNVAKSVF FLSTLWNAAKSIF GFWGKLWEGVKSAI GFWGKLWEGVKNAI FWGKLWEGVKNAI ILSAIWSGIKSLF IWSAIWSGIKGLL GILGKLWEGFKSIV IFGAIWKGISSLL FLSTIWNGIKSLL FLSLIPSLVGGSISAFK FLGMIPGLIGGLISAFK

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15 4.16 4.17 4.18 4.19 4.20 4.21 4.22 4.23 4.24

Group 5 Meucin-24 Meucin-25

Antimalarial peptides GRGREFMSNLKEKLSGVKEKMKNS VKLIQIRIWIQYVTVLQMFSMKTKQ

5.1 5.2

principle on the Pharmacological activity, Sequence similarity and peptide length. Using this criteria, all known NDBPs can be classified into 5 different groups (Table 4). The naming of the peptides remain as in the old system with each peptide named NDBP-x·y in each group where NDBP represent the non disulfide bridged class of peptides while x and y represent the group number and the number of peptide within the same group. In each group the peptide numbers are assigned chronologically. As shown in Table 4, the first group remains as in the old system for the bradykinin potentiating peptides with peptide T and peptide K12 as members of this group of NDBPs. The second group is assigned to long-chain multifunctional peptides which include Hadrurin, Bmkbpp, Pandinin1, Opistoporin 1 Parabutoporin, Im-1, Vejovine and Mauriporin. Biological functions reported by members of this group include antimicrobial, bradykinin-potentiating, insecticidal and anticancer activity which clearly highlights the multifunctional nature of this group. Group 3 represents medium-length antimicrobial peptides that include three members and these are Pandinin2, Css54 and HsAP. The 4h group represents the short scorpion antimicrobial peptides. This group includes 24 members with a peptide chain length ranging from 13 to 19 amino acids. Group number 5 has been assigned for antimalarial scorpion peptides and includes Meucin 24 and Meucin

25. Both peptides have been reported to display exclusive antimalarial activities with no effect on bacterial or mammalian cells. This classification could provide researchers in this field a reproducible method for assigning any novel scorpion NDBPs to their corresponding groups in the future upon their discovery and functional characterization. References [1] Almaaytah A, Tarazi S, Mhaidat N, Al-Balas Q, Mukattash T. Mauriporin, a novel cationic ␣-helical peptide with selective cytotoxic activity against prostate cancer cell lines from the venom of the scorpion Androctonus mauritanicus. Int J Peptide Res Ther 2013;19:291–3. [2] Almaaytah A, Zhou M, Wang L, Chen T, Walker B, Shaw C. Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: biochemical and functional characterization of natural peptides and a single site-substituted analog. Peptides 2012;35: 291–9. [3] Auvynet C, Rosenstein C. Multifunctional host defence peptides: antimicrobial peptides, the small yet big players in innate and adaptive immunity. FEBS J 2009;276:6497–508. [4] Bell A. Antimalarial peptides: the long and the short of it. Curr Pharm Des 2011;25:2719–31. [5] Bhutia SK, Maiti TK. Targeting tumors with peptides from natural sources. Trends Biotechnol 2008;26:210–7. [6] Brown KL, Hancock RE. Cationic host defense (antimicrobial) peptides. Curr Opin Immunol 2006;18:24–30.

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