Linear cationic α-helical AMP are a class of small peptides whose charge is imparted by the presence of multiple Lys and Arg, but also with a substantial portion (50% or more) of hydrophobic residues. These peptides are known for their broad-spectrum antimicrobial activity and ability to modulate the innate immune response (Powers and Hancock, 2003). One example is that of melittin, an α-helical cationic peptide from the venom of Apis mellifera bees, composed of 26 AA residues and in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged AA (Raghuraman and Chattopadhyay, 2007). Melittin is a potent antimicrobial that seems to promote membrane permeabilization through pore formation according to the toroidal model (Yang et al., 2001). However, its hemolytic activity is too high for clinical application as a selective AMP, which led to studies addressing synthesis and evaluation of the antimicrobial potential of hybrid peptide constructs where melittin (entire or partial AA sequence) was combined with other non-hemolytic AMP, such as cecropins (Boman et al., 1989; Bastos et al., 2008; López-Rojas et al., 2011). Cecropins constitute a well-known family of α-helical AMP that share a similar structure containing two α-helical domains linked by a flexible region. Insect cecropins are known to induce pore formation in negatively-charged bacterial membranes (Ekengren and Hultmark, 1999; Tanaka et al., 2008). In turn, a positive surface charge or cholesterol present in the membrane bilayer decreases the channel formation potency of cecropins (Christensen et al., 1988), which explains why these have little or no effect on eukaryotic cells (being non-hemolytic) that are richer in zwitterionic phospholipids and contain a high amount of cholesterol as compared to bacteria (Yeaman and Yount, 2003; Beevers and Dixon, 2010; Pretzel et al., 2013).

Other widely studied families of α-helical, linear and cysteine-free AMP are those of magainins and dermaseptins, both naturally occurring in amphibians. Magainins 1 and 2 adopt an α-helical conformation in solution (Zasloff, 1987), and have been proposed to induce toroidal pores in bacterial membranes (Ludtke et al., 1996). The non-hemolytic character of magainin 2 and its protocidal activity underlie its interest as a potential anti-parasitic agent, and also as a template for creation of more potent large spectrum AMP analogs, such as pexiganan (Ge et al., 1999). In what concerns peptides from the dermaseptin super-family, these exhibit a broad range of antimicrobial activity and some of them were found to aggregate on the bacterial membrane surface in a carpet-like manner (Pouny et al., 1992).

A classical example of this group of AMP is that of defensins, peptides mostly found in mammalian phagocytes that usually contain six Cys residues (eight Cys have been found in some insect defensins) stabilizing peptide structure by forming three intramolecular disulfide bridges (Selsted et al., 1985). The mechanism of action of these peptides seems to also involve pore formation inducing membrane permeabilization, which is more extensive on negatively charged phospholipid bilayers (Lehrer et al., 1989; Wimley et al., 1994).

This is a somewhat more heterogeneous group of AMP, as those included are diverse in sequence and tridimensional structure, sharing the feature of having an overrepresentation of certain AA, specifically, Pro, Gly, His, Arg, and Trp. From this follows that AMP of this group seem to also have diverse mechanisms of antimicrobial action, in some cases apparently involving intracellular targets (Otvos, 2005).

A family of Pro-rich AMP is that of apidaecins, short peptides that may adopt a polyproline type II helical structure which could be the structural basis to bind to specific targets underlying its antibacterial activity (Li et al., 2006). In fact, apidaecins do not seem to interact with microbial membranes through formation of pores, but rather by an energy-driven, eventually transporter-mediated, process (Castle et al., 1999).

Gly-rich AMP have been found with variable sizes and without any clear sequence signature, apart from the high proportion (25–50%) of glycine residues. These peptides are in general longer than AMP from other classes, have disordered structure in water, and tend to self-order when in contact with artificial membranes (Bruston et al., 2007). Attacins are family of six Gly-rich AMP that can be divided into four basic (A–D) and two acidic (E–F) peptides, probably derived from two attacin genes (Yi et al., 2014). Attacins inhibit the synthesis of outer membrane proteins of Escherichia coli by blocking transcription of the respective genes (Carlsson et al., 1991), which is presumably achieved by an indirect mechanism, since attacins bind to the bacterial LPS but do not need to enter the cell to exert their action (Carlsson et al., 1998).

Tryptophan-rich AMP contain more than 25% of this amino acid. In what concerns this class of AMP, the archetypical example is that of indolicin, which adopts no particular secondary structure in water, but seems to undergo significant structural changes in the vicinity of lipid bilayers, explaining its strong membrane affinity underlying its antimicrobial activity (Ladokhin and White, 2001). This peptide has the ability to permeate bacterial membranes and, depending of its tridimensional shape, inhibits DNA synthesis by binding to it (Hsu et al., 2005). Other examples of Trp-rich AMP include tritrpticin (Lawyer et al., 1996), lactoferricin B (Bellamy et al., 1992), and Pac-525 (Wei et al., 2006).

His-rich AMP usually have 25% of their AA content represented by His. In general, these peptides show a cationic amphipathic helical structure, and trigger microbial membrane disruption when adopting an alignment parallel to the membrane surface. Still, pore formation is not essential for the high antimicrobial activity of many His-rich AMP (Mason et al., 2009). Clavanin (van Kan et al., 2002), daptomycin (Jeu and Fung, 2004), LAH4 (Bechinger, 1996), or D-HALO-rev (Mason et al., 2009) are a few examples of this class of AMP.

Discovery of antimicrobial activity on natural cyclic peptides that did not fit any of the previous three groups justifies the need to consider a fourth group, dedicated to circular AMP. θ-defensins, for instance, fit this group: they are cyclic octadecamers active against several Gram-positive and Gram-negative bacteria, fungi, and some viruses, which consist of a couple of antiparallel β-sheets linked by three disulfide bonds to produce a very stable structure (Lehrer et al., 2012). Some bacteriocins, which are polypeptide toxins produced by bacteria to inhibit the growth of competing bacterial species or strain(s) (Cotter et al., 2013), are also circular AMP; that is the case of AS-48, a cyclic 70-mer bacteriocin from Enterococcus faecalis, possessing an overall globular structure where five α-helices enclose a dense hydrophobic core (González et al., 2000). Finally, one of the most emblematic families of circular AMP is that of cyclotides (Jagadish and Camarero, 2010): these are plant-derived peptides, with approximately 30 AA, characterized by a head-to-tail cyclic backbone and three or four disulfide bonds forming the so-called cyclic cysteine knot (CCK; Craik et al., 1999), for which they are also known as “knotted peptides.” As a result of their singular structure, these peptides are extremely stable, retaining their biological activity after boiling and being extremely resistant to enzymatic degradation (Vila-Perelló and Andreu, 2005; Craik and Conibear, 2011).

Cecropins, from the moth Hyalophora cecropia, disturb the development of oocysts into sporozoites, with a 50% lethal dose between 0.5 and 1 μg/μL (Gwadz et al., 1989). Synthetic derivatives of cecropins have also been produced and analyzed for their effects against malaria: SB-37, highly similar to cecropin B, and Shiva-1, with 40% homology to the same cecropin from H. cecropia, were significantly lytic to P. falciparum blood stage forms at 50 μM; still, while SB-37 was equipotent to cecropin B, Shiva-1 was twice as active as this cecropin (Jaynes et al., 1988). The effect of another cecropin-like peptide, Shiva-3, on in vitro ookinete development and on the early sporogonic stages of P. berghei in the midgut of Anopheles albimanus mosquitoes was investigated; peptide concentrations of 75 and 100 μM were effective in reducing ookinete production and the number of infected mosquitoes in almost all experiments (Rodriguez et al., 1995).

Hybrids of non-hemolytic cecropins with the potent bee venom toxin melittin have been found to inhibit RBC re-invasion by P. falciparum; an example of one such hybrid is that of CA(1-13)M(1-13), which is an order of magnitude more potent than magainin, cecropin A, and cecropin B, and is active in the 5–10 μM range (Frederik et al., 1989). More recent cecropin-melittin hybrids Vida1 (mainly consisting of α-helices), Vida2 (essentially composed by β-sheets), and Vida3 (a combination of coils and sheets) were developed and tested against P. berghei and P. yoelii gametocytes. The mortality rate of: Vida1 was 65% on young ookinetes (10 h), Vida2 was 60–70% on maturing ookinetes (14 and 24 h), and Vida3 was higher than 60% throughout the entire developmental period (Arrighi et al., 2002). The antimalarial activity of Vida3 was associated to blockage of Plasmodium oocyst development, and the peptide was further found as toxic to Anopheles gambiae cells at 25 μM (Carter et al., 2013).

The exact molecular mechanisms of membrane activity of cecropins have been under debate for more than 30 years, with two general models being proposed: formation of transmembrane pores, and the carpet model (Melo et al., 2009). A recent study demonstrates that cecropins A and B produce well-defined ion channels of different conductance levels in bilayer lipid membranes; further increase in peptide concentration causes destabilization and subsequent breakdown of the bilayer, showing that formation of pores is a first stage of membrane destabilization by these two cecropins, while accumulation of a dense peptide carpet precedes complete bilayer disintegration (Efimova et al., 2014).

Dermaseptins, from the skin of Phyllomedusa frogs, are highly active against intraerythrocytic forms of different P. falciparum strains, with IC₅₀ values between 0.8 and 2.2 μM (Matsuda and Koyasu, 2000). A truncated dermaseptin derivative was found to exert anti-P. falciparum activity within less than 1 min after exposure, involving permeabilization of the host cell membrane (Bell et al., 2006). In order to decrease hemolytic activity of dermaseptins, aminoheptanoyl derivatives were synthesized; a screening against P. falciparum revealed higher activity for the more hydrophobic dermaseptin derivatives, some of which showing significant selectivity between antiplasmodial activity versus hemolytic activity (Efron et al., 2002; Gavigan et al., 2003).

Following discovery of antiplasmodial activity of the 28-residue AMP dermaseptin S4 (Ghosh et al., 1997), a derived 13-residue AMP, K4-S4(1-13), was found to display considerable in vitro efficacy on P. falciparum (Krugliak et al., 2000). The antiplasmodial action of K4-S4(1-13) was fast and shown to be mediated by permeabilization of host cell plasma membrane. Although K4-S4(1-13) was less hemolytic to healthy RBC than to PfRBC, selectivity was not high enough and turned evident the necessity to develop additional derivatives active on parasites but with minimal threat to normal erythrocytes. Recently, acyl derivatives of K4-S4(1-13) were shown to have increased antiplasmodial activity, but the most potent of them was still significantly hemolytic (Dagan et al., 2002).

Antimicrobial peptides of insect origin have been found which display antimalarial properties. Drosomycins, isolated from Drosophila melanogaster, are an example of insect antimalarial peptides. Drosomycins were tested against development of P. berghei ANKA gametocytes, with drosomycin-2 showing 30% inhibition at 20 μM, whereas prototype-peptide drosomycin showed over 70% inhibition at that same concentration (Tian et al., 2008).

The most interesting source of antimalarial AMP concerns insects that are themselves the vectors of parasitic diseases, like anopheline mosquitoes, responsible for malaria transmission. It seems logical that such insects need to be equipped with a considerable defense system against the parasites they carry. In Anopheles mosquitoes, there are different known protection mechanisms, such as (i) upregulation of NO synthase, (ii) melanotic encapsulation in refractory mosquitoes that inhibit parasite development (Collins et al., 1986; Vijay et al., 2011), and production of AMP that might play an important role in refractoriness. In the major vector of P. falciparum in sub-Saharan Africa, A. gambiae, defensin, cecropin, and gambicin AMP have been found (Vizioli et al., 2000; Blandin et al., 2002; Kim et al., 2004).

The A. gambiae cecropin gene is mainly expressed in the mosquito midgut in hemocyte-like cells, and its levels are significantly raised within 2 h of infection (Vizioli et al., 2000). The activity of the A. gambiae cecropin against Plasmodium was studied by creating transgenic mosquitoes with cecA expression under the control of the Aedes aegypti carboxypeptidase promotor. The number of oocysts was reduced by 60% compared to the non-transgenic mosquitoes (Kim et al., 2004).

Gambicin, extracted from two A. gambiae cell lines, is an immune-induced peptide predominantly expressed in the anterior midgut compartment, thorax, and abdomen. The mature gambicin peptide is active against Gram-positive and Gram-negative bacteria, filamentous fungi, and P. berghei ookinetes (Vizioli et al., 2001).

Other mosquito vectors, such as A. aegypti, responsible for transmission of dengue and yellow fever viruses, produce AMP with antimalarial activity. A. aegypti releases three 40-AA long defensins (Def A to C) and cecropin A in response to bacterial infections (Lowenberger et al., 1995, 1999). In transgenic A. aegypti mosquitoes with co-overexpression of A. aegypti cecropin A and defensin A, P. gallinaceum oocyst proliferation was significantly inhibited as compared to wildtype mosquitoes (Lowenberger, 2001; Kokoza et al., 2010).

The strategy to produce transgenic mosquitoes that heterologously express AMP in order to interrupt Plasmodium transmission was also tested for scorpine, an AMP from the venom of Pandinus imperator scorpions. Scorpine belongs to a group of ion channel blockers with high activity against P. berghei ANKA gametes and ookinetes (ED₅₀ of 10 and 0.7 μM, respectively; Conde et al., 2000) that was shown to disrupt the sporogonic development of P. berghei. The scorpine gene was introduced into a vector for generation of transgenic flies resistant to infection by Plasmodia. The final aim of this work was to incorporate this gene under the promoter of proteolytic enzymes of the mosquito digestive tract, for synthesis and release of toxic peptide(s) into the stomach of freshly fed mosquitoes potentially carrying Plasmodium gametes; the presence of recombinant scorpine could be confirmed in transgenic A. gambiae cell supernatants (Possani et al., 2002). At low concentrations, recombinant scorpine reduced the number of ookinetes formed after mosquito feeding on P. berghei-infected mouse blood. Scorpine had highest effects (98% inhibition) when added during gamete formation and fertilization (Carballar-Lejarazu et al., 2008). In view of this, strategies to deliver AMP into the mosquitoes to interrupt the sporogonic cycle are relevant; one such strategy has been reported that uses symbiotic bacteria that live in the mosquito’s midgut: the transgenetic symbiont Pantoea agglomerans, expressing recombinant AMP Shiva-1 and scorpine, completely inhibited P. falciparum sporogonic cycle (Wang et al., 2012).

There is a significant number of cyclic AMP and derived macrocycles that have shown antimalarial properties. The potentially large, but structurally often well-defined conformational space sampled, combined with the variety of AA, renders cyclic peptides ideally suited to interact with many receptors or to interfere with protein/protein interactions (Katoh et al., 2011; Giordanetto and Kihlberg, 2014). In this context, cyclosporin A (CsA, 1 in Figure 2) is a well-characterized immunosuppressant hydrophobic peptide (Borel et al., 1996) that was earlier found to have antimalarial activity against P. berghei and P. yoelii rodent malaria and on cultured P. berghei, P. falciparum, and P. vivax parasites (Nickell et al., 1982). More recently, nine CsA-resistant P. falciparum clones were isolated, of which three had lesions in cyclophilin genes and two in calcineurin-subunit genes (Kumar et al., 2005). The two cyclophilins affected had been identified as cyclosporin-binding proteins in P. falciparum (Gavigan et al., 2003), but CsA may also have other targets in Plasmodia, since, it has a number of different known targets including the mammalian P-glycoprotein transporter. In agreement with this hypothesis, sequence polymorphisms in (and possibly expression levels of) a P. falciparum P-glycoprotein homolog were found to affect susceptibility to CsA (Gavigan et al., 2007).

Thiostrepton (2 in Figure 2), a cyclic thiopeptide, has reported IC₅₀ ranging from 1.8 to 17 μM against parasite growth and protein synthesis of P. falciparum, though this is suggested to be an overestimate since solubility of the compound in culture medium is poor (Clough et al., 1997; McConkey et al., 1997). IC₅₀ values for thiostrepton derivatives were 0.77 μM and above against P. falciparum (Schoof et al., 2010), and other thiopeptides were described as displaying nanomolar IC₅₀ against organellar protein synthesis in the same strain: micrococcin (3 nM), GE2270A (300 nM), and amythiamicin A (10 nM; Clough et al., 1999).

Most recently, three new macro-heterocyclic AMP, balgacyclamides A–C, were isolated from Microcystis aeruginosa EAWAG 251 and thoroughly characterized. Balgacyclamides A (3 in Figure 2) and B (4 in Figure 2) were evaluated for their antiparasitic activity and found to display micromolar IC₅₀ activity against the chloroquine-resistant strain K1 of P. falciparum (9.0 and 8.2 μM, respectively) with good selectivity compared to their cytotoxicity (Portmann et al., 2014).

Another recent report on a cyclic antimalarial peptide concerns the Gly-rich cyclic octapeptide pohlianin C (5 in Figure 2), whose synthesis provided confirmation of the structure of this natural product. Evaluation against P. falciparum showed moderate antiplasmodial activity, consistent with data obtained from the natural sample. In addition, the synthesis of three analogs revealed that the antiplasmodial activity of pohlianin C can be preserved or increased with simplified structures (Lawer et al., 2014).

NK-2 was one of the first synthetic AMP found to have anti-parasitic properties. NK-2 is a shortened version of the mammalian protein NK-lysin, comprising its residues 39–65, and long known to display lytic activity against the fungal pathogen Candida albicans and a variety of Gram-positive and Gram-negative bacteria, while exhibits virtually no hemolytic or cytotoxic activity against human cells (Andrä and Leippe, 1999). In a study aimed at determining the antimicrobial spectrum of both NK-lysin and NK-2, activity against the protozoan parasite Trypanosoma cruzi was observed (Jacobs et al., 2003). Later, NK-2 was found to be hemolytic to PfRBC above ∼1 μM, while harmless for healthy RBC up to 10 μM, along with evidence that parasite membrane also suffered permeabilization at 5 and 10 μM. The selective lytic activity of NK-2 on PfRBC was further demonstrated by observation that fluorescently labeled NK-2 binds to infected erythrocytes and to parasites, but not to healthy erythrocytes (Gelhaus et al., 2008).

Another synthetic AMP with antimalarial activity is D-HALO-rev. This design peptide possesses 26 AA with an even distribution of hydrophobic and charged residues, including non-proteinogenic ornithine (O, Orn), a Lys homolog. D-HALO-rev shows an IC₅₀ value of 0.1 μM against erythrocytic stages of P. falciparum, being able to penetrate PfRBC at sublytic concentrations and kill intraerythrocytic parasites (Mason et al., 2009). Similar results were obtained for an analog resulting from incorporation of Pro, Phe, and D-AA residues (D-Halo-P8F-rev), which showed reduced toxicity toward healthy RBC and fibroblasts (Mason et al., 2009).

A further step forward in this field was recent disclosure of the ability of synthetic peptide IDR-1018 to provide protection against cerebral malaria (Achtman et al., 2012). IDR-1018 is a 12-residue innate defense regulator analog of bactenecin, a cathelicidine from bovine neutrophils. It putatively owes its action to translocation across cell membrane and impairment of an intracellular target (Wieczorek et al., 2010). IDR-1018 was selected for screening in cerebral malaria given its anti-inflammatory capabilities and low toxicity, and found to protect 56% of infected mice from cerebral malaria after prophylactic intravenous administration. When combined with the antimalarial pyrimethamine-chloroquine medicine, IDR-1018 boosted protection from cerebral malaria in 41–68% of infected mice (Achtman et al., 2012).