Host Defense Peptides from Asian Frogs as Potential Clinical Therapies

Host defense peptides (HDPs) are currently major focal points of medical research as infectious microbes are gaining resistance to existing drugs. They are effective against multi-drug resistant pathogens due to their unique primary target, biological membranes, and their peculiar mode of action. Even though HDPs from 60 Asian frog species belonging to 15 genera have been characterized, research into these peptides is at a very early stage. The purpose of this review is to showcase the status of peptide research in Asia. Here we provide a summary of HDPs from Asian frogs.


Introduction
The origins of the study into host defense peptides began with the pioneering studies of Vittorio Erspamer, who discovered biogenic amines and peptides secreted by the amphibian skin. The field later diversified when Zasloff isolated magainin from Xenopus laevis [1,2]. In our current era of multi-drug resistant bacterial strains, discovering new and effective treatments to replace traditional antibiotics is a critical area of research. Host defense peptides (HDPs), such as those being found in the skin of Asian OPEN ACCESS frogs, have shown effectiveness in killing both gram-negative and gram-positive bacteria, as well as some viruses and cancers, without any of the drawbacks of antibiotic resistance [3]. These properties make them viable candidates as the next wave of therapies against infectious and non-infectious diseases that are the leading cause of death in developing countries.
Research on how these anti-microbial peptides function is still in the early stages. Most of the HDPs are typically cationic, a feature which allows them to interact with the negatively charged bacterial and cancer cell membranes. This cationic nature allows some peptides to actually penetrate the surface of pathogens as a means of killing them [4]. Asia is well known for its diverse amphibian populations and has been a resource for novel HDPs. Recently, potent HDPs have been isolated from amphibians located in temperate regions (traditionally believed to have low biodiversity) and have proven to be efficient in combating many clinical pathogens, though research into these peptides is still at a very early stage. We hope as we continue to discover new peptides in this region we will also find more potential candidates for novel therapeutics.

Diversity of HDPs Found in the Skin Secretion of Asian Frogs
In the tropics, especially in the rain forests of Asia, effective host defense peptides have helped the frogs to survive within their environment and attain a rich diversity. However with the alarming rate of decline in amphibians globally, many identified and unidentified species have become extinct before researchers ever had a chance to isolate their HDPs for study [5]. Those peptides that have been isolated show a great degree of variability-even within a single genus, different species produce varied repertoires. These differences can be correlated to habitat specific challenges endured by each species, and how these frogs uniquely evolved alongside their environment [6]. Even within the same frog, expression of several members of a particular family of HDP is observed. The variation in these peptide sequences is suggestive evidence for ancestral gene duplication [7]. No two peptides with identical amino acid sequences have been reported even from closely related species. Such diversity provides broad-spectrum protection against the large pool of microbes/predators in their habitat (Figures A1-A4) [8]. Transcriptomic and peptidomic analysis revealed that genera will have HDP families in common, apart from this, some unique peptides, such as neuroendocrine and smooth muscle active, are reported that serve a particular purpose in that habitat e.g., bradykinin related peptides (BRPs) and Trypsin inhibitors from Odorrana genus. Here we review the HDP families reported from Asian frogs. Despite the wealth of biodiversity, attempts to characterize HDPs from Asian frogs have been limited. In the present medical scenario there is an urgent need to uncover the hidden peptides that may lead to developing new and potentially crucial therapeutic agents.
Hainanenins 1-5 were isolated from A. hainanensis [10] and also showed potent antimicrobial function against gram-positive bacteria, gram negative bacteria and fungi in the ranges 4-40 µg/mL, 4-75 µg/mL, and 2-75 µg/mL respectively. Three Bradykinin related peptides (BRPs) named Amolopkinins were found in A. loloesis and A. wuyiensis [16,17]. Amylopkinin from A. loloesis exhibited dose dependent contractile activity in guinea pig ileum [16]. The function of BRPs in frog skin secretion is thought to relate to smooth muscle contraction activity-if a predator swallows the frog, these peptides induce smooth muscle contractions that will result in a vomiting sensation, thereby allowing the frog to escape predation. Two other Amylopkinins from A. wuyiensis inhibited Bradykinin induced contractile effects on isolated rat ileum smooth muscle preparations [17].

Clinotarsus
The Clinotarsus genus is endemic to the Western Ghats of India where new HDPs are being discovered. The peptidomic approach revealed the presence of five novel peptide amides homologous to the Brevinin 1 family from a single species, C. curtipes, that all show promising effectiveness against gram-positive and gram-negative bacteria [81]. The MICs against the tested pathogenic bacteria were reported to be between 6-25 μg/mL (Tables 1 and 2). It was previously reported that bacterial membranes depolarize during pore formation and bacterial killing, but in the case of Brevinin 1 from C. curtipes, it was shown that depolarization and bacterial killing are independent events. We are slowly beginning to understand the methods by which these HDPs effectively fight disease.

Euphlyctis
Ramesh B. et al. [92] reported on the potential of HDPs discovered on the skin of the Indian green frog, Euphlyctis hexadactylus. They showed that the lyophilized crude skin secretion was inhibiting the growth of pathogenic gram-positive and gram-negative bacteria, as well as fungal pathogens-which included plant fungal pathogens. The MIC of the peptide against several human-relevant bacteria ranged from 128 to 512 μg/mL and for fungus it ranged between 32 to 64 μg/mL (Tables 1 and 2). However it has not yet been reported what the composition of these skin secretions might be.

Fejervarya
Two Tigerinin peptides were identified from Fejervarya cancrivora [82], similar to the Tigerinins originally isolated from the Indian frog Hoplobatrachus tigrinus. The effects of a C-terminal amidation on both peptides include a decrease in MIC against pathogens compared to their non-amidated counterparts. MICs of non-amidated Tigerinins against gram-positive bacteria, gram-negative bacteria and fungi ranged from 20-80 µg/mL, 10-40 µg/mL and 80-180 µg/mL respectively (Tables 1 and 2) and that of C terminally amidated peptides were 10-40 µg/mL, 5-40 µg/mL, and 15 µg/mL respectively. They also exhibited 10%-12% hemolysis against rabbit red blood cells (RBCs) at 100 µg/mL [82]. With this data we can better understand the influence of the peptide's sequence on its function.

Hyla
Anntoxin, a peptide that inhibits tetradotoxin-sensitive voltage-gated sodium channels, was identified in the skin secretions of Hyla annectans. This peptide is believed to have a great therapeutic potential as an anti-nociceptive and anti-inflammatory agent [86] (Tables 1 and 2).
H.temporalis is the first among the genus to be reported from Western Ghats, India. Seven novel peptides are reported from H.temporalis, which include one Brevinin 1, two Brevinin 2 peptides, three Esculentin 2 peptides, and one Hylaranakinin peptide [34][35][36]. The MICs of Brevinin and Esculentin peptides ranges from 30 to 150 μg/mL.

Limnonectus
Limnonectins (2 peptides) from L. fujianensis are the only family of HDPs reported from the genus Limnonectus thus far [87] (Tables 1 and 2). These peptides were reported to have no antimicrobial activity towards gram-positive bacteria and fungi, but MICs against gram-negative bacteria range between 35-70 µM. They exhibited no hemolysis up to a concentration of 160 µM.

Nanorana
Genus Nanorana has provided the antimicrobial peptides Japonicin 1 (2 peptides) and a novel family named Parkerin, both isolated from N. parkeri [88] (Tables 1 and 2). Japonicin 1 and Parkerin peptides were reported to be effective only against gram-positive bacteria with MICs in the range 9-37 µg/mL and 37-40 µg/mL respectively. 1%-3% hemolysis was observed for both peptides at 80 µg/mL. Mast cell degranulation, which is thought to mediate antimicrobial activity of HDPs, was exhibited by both the peptides.
Studies of the skin of O. tiannanensis also revealed a set of novel HDP families-Margaretaein (2 peptides), Pleurain, and Tiannanensin [42]. Tiannanensin, an antimicrobial peptide, showed activity against gram-positive bacteria and fungi with MICs 75 and >100 µM, respectively. In addition to these there are other novel HDP families including Iksikawain, Lividin (first report), and Ranateurin, found in O. ishikawae, O. livida, and O. versablis, respectively [41,43,56]. Bradykinin related peptides were also isolated from O. grahamii (3 peptides) and O. schmakeri (7 peptides) [57,58]. Three of the five Bombesin-like peptides from O. grahamii induced contraction of the stomach muscle tissue. The other two BLPs, which have a C terminal octapeptide, antagonized stomach muscle contraction. Inhibition of contraction is thought to be due to the presence of the octapeptide [37].

Rhacophorus
HDPs from two species-Rhacophorus duboisi (previously Polypedates pingbianensis) and Rhacophorus schegelii-have recently been isolated (Tables 1 and 2). Polypedarelaxin from Rhacophorus duboisi effectively relaxes smooth muscle contractions [89]. This peptide exhibited concentration-dependent relaxation effects on isolated rat ileum but did not show antimicrobial and serine protease activity. Cationic antimicrobial peptides do not appear in the dermal immune system of Rhacophorus schegelii [90]. However, Histone 2B was reported from Rhacophorus schegelii, [90] which is evidence for the role of histones as antimicrobials in addition to their ability to remodel chromatin. This was the first report of histones from frog skin secretion.

Sanguirana
HDPs isolated from Sanguirana varians (Tables 1 and 2) of the genus Sanguirana revealed homology to the neuroendocrine peptide Bombesin [91]. These BLPs revealed dose dependent contractile effects of stomach tissue.

Conclusions
With the increasing emergence of multi-drug resistant pathogens, as well as the constant struggle against non-infectious illnesses such as cancer, there is a critical need to develop new therapies where old treatments have failed. Amphibian-derived antimicrobial peptides offer us a potential solution. We are now just beginning to understand the diversity and functions of these peptides, but this review outlines the ways in which we already see the potential clinical uses of these HBPs. The potent antimicrobial peptides in Asian frogs belong to the Brevinin and Esculentin. They are very effective against gram-positive and negative-bacteria as well as fungi. The only problem with these peptides is their high cytotoxicity, which would be eliminated by effective modification. The Temporin family of peptides are potent anticancer agents which are highly toxic against cancer cells, especially breast cancer cell lines and demonstrate very low toxicity towards normal cells (e.g., temporin from R. chensinensis). Hopefully further study of these animals and their skin peptides will reveal novel therapies that may potentially fill the gap left by antibiotic resistance and the failure of many of our current cancer treatments. There is therefore a need to turn our attention to this region and its biodiversity as a key medical resource. 100% similar; 80%-99% similar; 60%-79% similar; Less than 60% similar Figure A3. Multiple sequence alignment of Esculetin-1 from Asian frogs.