**9. Advantages and limitations of cationic antimicrobial peptides**

#### **9.1. Bioactivity**

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

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‐

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

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

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‐

breast cancer cells compared to the natural L-form peptide (Hilchie, Hoskin, unpub).

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

ant, cell-permeable peptide that binds its target with increased binding affinity.

resulting lipopeptide, and concurrently the antifungal activity.

stabler than monomeric forms (Pieters et al., 2009).

**8.6. Targeted and hybrid peptides**

**8.3. Peptidomimetics**

**8.4. Acylation**

**8.5. Cyclisation**

CAPs exert rapid, broad spectrum, bactericidal activity at micromolar concentrations and are valuable weapons in the fight against antibiotic-resistant microbes e.g. MRSA. In addi‐ tion, targeted CAPs may be used as probiotics to eradicate pathogenic bacteria while leaving normal flora unaffected. Their ability to kill fungi, viruses and parasites is also beneficial clinically. Many CAPs neutralize endotoxin and can be used to counteract sepsis or to en‐ hance host defenses through immunomodulatory effects. Increased innate clearance of mi‐ crobes results in less bacterial debris (van der Does et al., 2010) and ensuing inflammation (Andra et al., 2006). Their application both in killing tumor cells and in enhancing host antitumor responses is gaining momentum (Yeung et al., 2011).

#### **9.2. Resistance**

CAPs have a low propensity to induce microbial resistance, mainly due to their multiple tar‐ gets and mechanisms of action. CAPs are usually expressed in high concentrations only at sites of infection; continuous exposure of microbes, which often leads to resistance, is there‐ fore minimized. In addition, cocktails of several different CAPs are often produced simulta‐ neously, leading to increased microbial killing.

Despite this, some reports on the emergence of resistance have appeared. These include re‐ duction in bacterial cell surface negative charge, secretion of exoproteases and induction of transporters (see Nizet, 2006; Peschel and Sahl, 2006), all of which require significant expen‐ diture of metabolic energy by the microbe. In most cases, the resistance that arises is very low compared to conventional antibiotics.

In Gram negative organisms such as *Salmonella* and *Pseudomonas*, the PhoP-PhoQ two com‐ ponent sensor system has been shown to control resistance to aminoglycosides, polymyx‐ in B, and cationic antimicrobial peptides (Macfarlane et al., 1999; Macfarlane et al., 2000; Groisman, 2001). In *Pseudomonas*, CAPs and the polymyxins are capable of inducing the pmrA-pmr genes and the putative LPS modification operon, thus increasing resistance to these agents (McPhee et al., 2003). The homologous basR-basS system of *E. coli* is also induced by exposure to sublethal concentrations of the proline-rich CAP, Bac7(1-35), sug‐ gesting that it may also mediate resistance (Tomasinsig et al., 2004). Resistance to Gram positive organisms such as *Staphylococcus* is mediated by the Aps three component sen‐ sor system, and interestingly, some CAPs are inducers of this system (Li et al., 2007). Recently, additional resistance genes in other bacteria such as *Clostridium difficile* (McBride and Sonenshein, 2011) and *Vibrio* (Shen et al., 2010) have been discovered, emphasizing the importance of this phenomenon.

#### **9.3. Cost**

Solid phase peptide synthesis is expensive and companies seeking regulatory approval for peptides in their pipelines require suppliers that adhere to good manufacturing practice (GMP) regulations, which also adds to the cost. However, there have been many advances in synthetic chemistry that have significantly reduced the cost of not only peptides but also non-peptide mimics and peptoids, and the lower operating costs of suppliers in Asia has re‐ sulted in more affordable peptides (Eckert, 2011).

#### **9.4. Stability**

Although CAPs represent a promising class of therapeutics, they have several *in vivo* draw‐ backs such as salt inactivation, protease degradation, and poor bioavailability. As described above, a number of approaches can be used to mitigate these limitations. Serum stability can be overcome by cyclisation or incorporation of amino acid analogs and some CAPs, particu‐ larly those such as pleurocidin that originated from marine sources, are active at physiologi‐ cal salt concentrations (Mai et al., 2011; Patrzykat et al., 2003).

#### **9.5. Side effects**

Some CAPs exhibit *in vivo* toxicity and for others there are unknown toxicity profiles (see section 5.7). Nephrotoxicity has been associated with polymixin cyclic peptides (Mogi and Kita, 2009). Since some CAPs can stimulate growth factor receptors, induction of tumorigen‐ esis must be considered. Similarly, excessive release of histamine from mast cells must be avoided to minimize adverse reactions.
