**7. Production of cationic antimicrobial peptides**

#### **7.1. Peptide synthesis**

**6. Isolation and discovery of cationic antimicrobial peptides**

132 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Standard biochemical techniques for isolation of small peptides have been used to isolate CAPs from tissues, skin secretions and other biological fluids. Briefly, tissue lysates or fluids are diluted in 0.1% trifluoroacetic acid (TFA) and extracted under ice-cold acid conditions. After centrifugation, the acidic supernatant is loaded on a C18 column and the peptide elut‐ ed with increasing acetonitrile (5-80%) in 0.05% TFA. Those fractions with antimicrobial ac‐ tivity are pooled and further purified by reverse-phase HPLC with increasing acetonitrile (5-55%) in 0.05% TFA. The material in absorbance peaks is then pooled, dried under vacuum and reconstituted in MilliQ water for mass and sequence analysis using mass spectrometry

Pleurocidin was originally isolated from homogenates of the skin and mucus secretions of winter flounder using C18 chromatography followed by size fractionation on Sephadex G-50 (Cole et al., 1997). Biologically active fractions were pooled and subjected to strong cat‐

Once even a partial amino acid sequence of a CAP is known, genomic strategies can be em‐ ployed to identify and isolate DNA sequences encoding CAPs. This can involve screening of cDNA and genomic libraries using oligonucleotide probes or amplification of CAP-encod‐ ing sequences from genomic or cDNA by PCR (Iwamuro and Kobayashi, 2010). CAP-encod‐ ing sequences can also be identified in genomic and EST sequencing databases by similarity searching using bioinformatics search engines such as BLAST (Browne et al., 2011; Fer‐ nandes et al., 2010). Full-length cDNA sequences can then be obtained by 5'- and 3'-RACE. The identification of 28 new human and 43 new mouse β-defensin genes was achieved using a computational search tool to find conserved motifs in draft genome sequences (Schutte et

Pleurocidin was originally cloned from cDNA and genomic libraries using degenerate oligo‐ nucleotide probes (Cole et al., 2000; Douglas et al., 2001) or PCR amplicons encoding indi‐ vidual pleurocidin variants (Douglas et al., 2003). Additional pleurocidin variants were amplified by PCR from cDNA of different flatfish species using primers corresponding to the conserved amino-terminal signal peptide and carboxy-terminal anionic propiece (Patr‐

Various *in silico* methods have been used to rationally design CAPs and predict their biolog‐ ical activity (Hadley and Hancock, 2010). These include the use of artificial neural networks, support vector machines and quantitative structure activity relationship (QSAR) modeling (Fjell et al., 2011; Torrent et al., 2011). Combinatorial synthesis and high throughput peptide synthesis using SPOT technology (Hilpert et al., 2007) together with a QSAR-based artificial

zykat et al., 2003). At least 30 variants have now been cloned (Figure 3).

**6.3.** *In silico* **discovery and rational peptide design**

**6.1. Traditional biochemical purification**

and automated Edman degradation.

al., 2002).

ion exchange HPLC and then reversed phase HPLC.

**6.2.Genomics approaches to antimicrobial peptide discovery**

Isolation of CAPs from the native producer is not feasible on the scale necessary for investi‐ gating therapeutic value and pharmaceutical potential. Therefore, CAPs are usually synthe‐ sized by standard solid-phase methods using 9-fluorenyl-methoxycarbonyl (F-moc) protecting groups, cleaved from the resin with 95% trifluoroacetic acid, purified by reversephase high performance liquid chromatography using an acetonitrile gradient, and the mass is verified by mass spectrometry. Although synthesis of linear α-helical peptides is fairly routine, good yields of CAPs that require cysteine oxidation to form the correct disulphide bonding pattern are rarely obtained (Tay et al., 2011). Modifications such as carboxy-termi‐ nal amidation, biotinylation, fluorescent labeling, incorporation of D-amino acids, acylation, etc. are available for synthetic peptides.

#### **7.2. Fermentation**

#### *7.2.1. Bacteria*

Production of CAPs in bacteria using recombinant technology, by definition, is a challenge as they inhibit or kill the host bacteria and bacterial proteases can degrade them. Recombi‐ nant technology, particularly of tagged fusion peptides, has been used successfully to pro‐ duce high yields of pure CAP at relatively low cost (Ingham and Moore, 2007). Factors to be considered include optimization of promoter sequence and codon usage from the source or‐ ganism to bacterial host, signal peptide, tag, fusion partner, ease and cost of cleavage, and cost of subsequent purification steps. A recent approach involving secretion of the tagged SUMO-CAP hybrid into the medium followed by removal of the mature CAP by sumoase protease has been particularly successful in large-scale production of several CAPs (Bom‐ marius et al., 2010; Li et al., 2009). In general, production of α-helical CAPs without disulfide bonds and easily cleavable from their fusion partner has proved to be the most straightfor‐ ward and cost-effective. A recent report of expression of human β-defensin 28 in *E. coli* is encouraging, however (Tay et al., 2011). By using a construct with both a His-tag and mal‐ tose binding protein and including the site for cleavage by TeV, an inexpensive and efficient protease for releasing the mature CAP, large-scale production of correctly folded defensin was achieved.

Pleurocidin has been successfully expressed as a PurF fusion peptide in inclusion bodies, generating milligram quantities of pure peptide (Bryksa et al., 2006). By incorporating ( 15NH4)2-SO4 as the sole nitrogen source in the growth medium, structural elucidation of the uniformly 15N-labelled peptide by NMR was greatly facilitated (Syvitski et al., 2005).

#### *7.2.2. Fungi*

Fungi such as *Saccharomyces cerevisiae* and *Pichia pastoris* represent alternative systems for re‐ combinant expression of CAPs. *P. pastoris* is a methylotrophic yeast that is capable of pro‐ ducing 10-100 times more recombinant protein than *S. cerevisiae.* However, successful production of small proteins less than 10 kDa in *P. pastoris* can be problematic due to low expression, RNA instability or translational blocks. Low amounts (~1 mg/100 mL) of re‐ combinant cecropin were produced in *P. pastoris* (Jin et al., 2006), and recently, high-level production (>200 mg/L) of active recombinant hPAB-β (Chen et al., 2011) and plectasin (Zhang et al., 2011) was achieved in this system.

Production of pleurocidin was attempted using this system; however, no recombinant pep‐ tide was recovered, despite correct integration of the pleurocidin sequence into the yeast ge‐ nome and normal transcription (Burrowes et al., 2005).

#### *7.2.3. Animal cells*

Production of CAPs in animal cells has the advantage of accurate processing of the recombi‐ nant peptide and addition of post-translational modifications. However, there are few re‐ ports of CAP production using this approach. The insect cell/baculovirus system has been used to produce recombinant human β-defensins (Feng et al., 2005) and biologically active hepcidin was successfully expressed, processed and secreted in human embryonic kidney cells (Wallace et al., 2006). The complete pleurocidin pre-pro-peptide gene was cloned into a carp cell line under the control of the carp β-actin promoter. The precursor peptide was ex‐ pressed continuously for over two years and the mature peptide was secreted into the medi‐ um (Brocal et al., 2006).

#### **7.3. Transgenic organisms**

Early attempts were made to express CAPs in the milk of transgenic mice (Yarus et al., 1996), tissues of catfish (Dunham et al., 2002), and leaves of tobacco (Yevtushenko et al., 2005) and rice (Imamura et al., 2010); however, while the recombinant CAPs were able to confer pathogen resistance to the host organism, these systems are not designed for largescale production of CAPs for human applications.

### **8. Modifications to enhance biological activity and stability**

#### **8.1. Amino acid substitutions, additions and deletions**

Modification of the amino acid sequence of CAPs to change the charge, hydrophobicity and bulkiness can have dramatic effects on their ability to interact with membranes and exert their biological activity. Amidation of the carboxy terminus usually results in enhanced ac‐ tivity by increasing the net positive charge but has a negligible effect on protease susceptibil‐ ity. On the other hand, N-terminal acetylation significantly increases resistance to aminopeptidases but decreases antimicrobial activity (Nguyen et al., 2010). In general, high hydrophobicity confers hemolytic and cytotoxic properties on CAPs (McPhee and Hancock, 2005). The effect of amino acid substitutions on antimicrobial vs hemolytic activity has been comprehensively evaluated using a series of rationally designed CAPs of 20 amino acids that differ in charge, polar angle, hydrophobicity and hydrophobic moment (Chou et al., 2008). Increasing the His content of clavanin resulted in an acid-activated CAP that showed enhanced activity against the caries-producing pathogen, *S. mutans,* at low pH (Li et al., 2010). Substitution of Lys by His in a 15-mer CAP resulted in a peptide that was cationic on‐ ly in the acidic microenvironment found in tumors, and exhibited enhanced anti-tumor ac‐ tivity and reduced systemic toxicity (Makovitzki et al., 2009). Addition of hydrophobic amino acids to the termini of CAPs can also increase potency, although reduced solubility and increased production cost present important drawbacks (Hadley and Hancock, 2010). Recent reports of a truncated frog gaegurin (Won et al., 2011b) and the naturally short bee anoplin (Won et al., 2011a) CAPs emphasize the enhanced antimicrobial effects of such mod‐ ifications on bacteria and model membranes.

Naturally occurring pleurocidins have variable amino acid sequences that confer different activities against microbes and cancer cells (Morash et al., 2011; Patrzykat et al., 2003). Align‐ ment of the amino acid sequences of the mature peptides revealed conserved positions that could be of importance in bioactivity. For example, the highly conserved Gly13 residue in the middle hinge region is important for α-helicity (Lim et al., 2004b) and the hydrophobic amino acids in the N-terminal region are more crucial for antifungal activity than those in the C-terminal region (Lee and Lee, 2010). Replacement of Ser14 by His improved the activi‐ ty of a targeted pleurocidin towards *S. mutans* at low pH, which is often found in the oral cavity (Mai et al., 2011).

#### **8.2. Non-natural amino acids and enantiomers**

(

*7.2.2. Fungi*

*7.2.3. Animal cells*

um (Brocal et al., 2006).

**7.3. Transgenic organisms**

(Zhang et al., 2011) was achieved in this system.

nome and normal transcription (Burrowes et al., 2005).

134 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

scale production of CAPs for human applications.

**8.1. Amino acid substitutions, additions and deletions**

15NH4)2-SO4 as the sole nitrogen source in the growth medium, structural elucidation of the

Fungi such as *Saccharomyces cerevisiae* and *Pichia pastoris* represent alternative systems for re‐ combinant expression of CAPs. *P. pastoris* is a methylotrophic yeast that is capable of pro‐ ducing 10-100 times more recombinant protein than *S. cerevisiae.* However, successful production of small proteins less than 10 kDa in *P. pastoris* can be problematic due to low expression, RNA instability or translational blocks. Low amounts (~1 mg/100 mL) of re‐ combinant cecropin were produced in *P. pastoris* (Jin et al., 2006), and recently, high-level production (>200 mg/L) of active recombinant hPAB-β (Chen et al., 2011) and plectasin

Production of pleurocidin was attempted using this system; however, no recombinant pep‐ tide was recovered, despite correct integration of the pleurocidin sequence into the yeast ge‐

Production of CAPs in animal cells has the advantage of accurate processing of the recombi‐ nant peptide and addition of post-translational modifications. However, there are few re‐ ports of CAP production using this approach. The insect cell/baculovirus system has been used to produce recombinant human β-defensins (Feng et al., 2005) and biologically active hepcidin was successfully expressed, processed and secreted in human embryonic kidney cells (Wallace et al., 2006). The complete pleurocidin pre-pro-peptide gene was cloned into a carp cell line under the control of the carp β-actin promoter. The precursor peptide was ex‐ pressed continuously for over two years and the mature peptide was secreted into the medi‐

Early attempts were made to express CAPs in the milk of transgenic mice (Yarus et al., 1996), tissues of catfish (Dunham et al., 2002), and leaves of tobacco (Yevtushenko et al., 2005) and rice (Imamura et al., 2010); however, while the recombinant CAPs were able to confer pathogen resistance to the host organism, these systems are not designed for large-

Modification of the amino acid sequence of CAPs to change the charge, hydrophobicity and bulkiness can have dramatic effects on their ability to interact with membranes and exert

**8. Modifications to enhance biological activity and stability**

uniformly 15N-labelled peptide by NMR was greatly facilitated (Syvitski et al., 2005).

Replacement of L-amino acids by their D counterparts generally does not diminish biologi‐ cal activity but does confer protease resistance on the peptide (Lee and Lee, 2008; Park et al., 2010). Retro-inversion, in which amino acid stereochemistry is changed as well as peptide bond direction yielding isomers with similar side chain topology to the native peptide, has generally met with limited success although partially modified retro-inverso peptides show more promise (Fischer, 2003). Incorporation of non-natural amino acid analogs (Knappe et al., 2010; Marsh et al., 2009) is also effective. Recently, a series of CAPs containing Tic-Oic dipeptide analogues was developed to combat 11 potential bio-terrorism and drug-resistant strains of bacteria (Venugopal, 2010). These CAPs were metabolically stable, potent, and showed selectivity for bacterial relative to host cell membranes.

Replacement of L-Lys and L-Arg residues with D-Lys and D-Arg in pleurocidin conferred resistance to digestion by trypsin but also abolished activity, presumably because the α-heli‐ cal structure was disrupted by the inclusion of just a subset of amino acids (Douglas, un‐ pub.). In support of this, an all D-amino acid analog of pleurocidin showed proteolytic resistance and double the antifungal (Jung et al., 2007) and antibacterial (Lee and Lee, 2008) potency. Similarly, an all D-amino acid analog of pleurocidin NRC-03, showed improved activity against breast cancer cells compared to the natural L-form peptide (Hilchie, Hoskin, unpub).

#### **8.3. Peptidomimetics**

Peptidomimetics such as β-peptides and peptoids have been designed that maintain the amphiphilic structure and antimicrobial activity and are resistant to protease degradation; these abiotic structures exhibit *in vivo* stability and enhanced bioavailability (Rotem and Mor, 2009; Scott et al., 2008). Synthetic mimics of antimicrobial peptides (SMAMPs) that adopt amphiphilic secondary structures and possess potent and selective antimicrobial activity have been inexpensively synthesized from small synthetic oligomers (Lienkamp et al., 2008; Scott et al., 2008). Another novel technique is hydrocarbon stapling (Sa'adedin and Bradshaw, 2010) in which an α-helical peptide is chemically modified to generate a relatively protease resist‐ ant, cell-permeable peptide that binds its target with increased binding affinity.

#### **8.4. Acylation**

Addition of fatty acid chains to the termini of CAPs can increase their antibacterial activity or endow them with antifungal activity. Linear oligomers consisting of alternating un‐ charged and cationic Lys residues displayed varying degrees of antibacterial, antifungal and hemolytic activity when they were N-acylated, depending on the length of the acyl group and the different degrees of oligomerization that were induced (Shai et al., 2006). Addition of fatty acids of increasing lengths to magainin increased the extent of oligomerization of the resulting lipopeptide, and concurrently the antifungal activity.

#### **8.5. Cyclisation**

Cyclisation by, for example, disulfide bridge formation or head-to-tail backbone cyclization, results in a more constrained peptide structure that is less susceptible to protease degrada‐ tion (Nguyen et al., 2010). Cyclisation of two cationic hexapeptides, including the active por‐ tion of lactoferricin, was found to be highly effective for both serum stability and antimicrobial activity. Interestingly, disulfide cyclization resulted in more active peptides while backbone cyclization resulted in more proteolytically stable peptides. The modifica‐ tions did not result in hemolytic activity, thereby making them attractive therapeutic candi‐ dates. Dendrimers of CAPs have enhanced ability to permeabilize membranes and are stabler than monomeric forms (Pieters et al., 2009).

#### **8.6. Targeted and hybrid peptides**

One of the drawbacks of antibiotic use is the deleterious effect broad spectrum antibiotics have on the normal microflora, which often allows opportunistic pathogens such as *C. albi‐ cans* and *S. aureus* to overgrow after treatment. Selectively targeted antimicrobial peptides (STAMPs) can overcome this problem and can selectively kill the target species while leav‐ ing the benign commensals largely unaffected. This effect was first demonstrated using a STAMP consisting of a competence stimulating peptide (CSP) moiety targeting the cariescausing pathogen *Streptococcus mutans* attached to a killing moiety derived from novispirin G10 (Eckert et al., 2006). Incorporation of such a STAMP into an oral rinse resulted in reduc‐ tion in salivary *S. mutans*, plaque, lactic acid and enamel demineralization, suggesting fur‐ ther clinical evaluation is warranted (Sullivan et al., 2011). Targeting of tumor cells has also been achieved and addition of the tumor-homing peptide bombesin to the amino terminus of magainin 2 resulted in tenfold enhanced killing *in vitro* and decrease in the size of tumor xenografts in mice (Liu et al., 2011).

We have used this approach to combine the minimal targeting portion of CSP with the kill‐ ing domain of pleurocidin variant NRC-04 to treat *S. mutans* in both planktonic and biofilm conditions. The hybrid peptide was selectively active against *S. mutans* in the presence of saliva and physiological or higher salt concentration and was non-hemolytic (Mai et al., 2011). Furthermore, activity was augmented by a preventative dose of 1 mM NaF, a com‐ pound commonly used in oral care. The development of such targeted peptides paves the way for their use as a probiotic treatment to prevent dental caries.
