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Antimicrobial Peptides: Mini-Review

Antimicrobial Peptides (AMPs) are found in virtually every living organism—bacteria, fungi, plants, invertebrates, and vertebrates—and play a critical role in the innate and acquired immune responses to pathogenic attacks. AMPs are released by various cells and organelles, such as immune cells (granulocytes and macrophages), epithelial cells (vaginal epithelium, respiratory epithelium, and oral cavity epithelium), and the small intestine. The antimicrobial effects of peptides were first observed in 1966 by Zeya and Spitznagel.[1-4] During their studies of lysosomal cationic proteins and peptides, they noticed many of the peptides contained basic amino acids like arginine and lysine. Soon after, they experimentally demonstrated that ‘lysosomal cationic proteins (LCPs) had antibacterial activity against certain gram-positive and gram-negative bacteria.

One of the primary motivations for the development of peptide antibiotics is the emergence of multi-drug-resistant bacteria from the overuse of antibiotics. According to European Centre for Disease Prevention and Control Antimicrobial Resistance Interactive Database (EARS-NET) in 2013, antimicrobial-resistant infections take a staggering toll of over 50,000 lives each year in the U.S. and Europe alone.[5] For example, in many European countries, methicillin-resistant strains of Staphylococcus aureus account for 10% of the blood infections involving these bacteria.[6] Some countries reportedly have a significantly higher percent of these methicillin-resistant strains of bacteria. Pharmaceutical companies that manufacture small-molecule antibiotics have tried to meet this challenge by modifying the structures of existing antibiotics and developing additional antibiotic classes. Despite some of these heroic efforts, antibiotic resistance continues to emerge as a significant health crisis across the globe.

Structure and Function of AMPs

AMPs have a wide range of structural motifs that are known to adopt secondary structures including alpha-helices, beta-sheets, loops, and extended structures (Figure 1).[7-9] This broad range of structural motifs and properties provide a variety of modes of antimicrobial activity, including acting as membrane disruptors,[10] effector molecules[11-13] to intracellular proteins, immunomodulators,[14] as well as in human contraception.[15,16] Most AMPs are polycationic with charges ranging from +2 to +9 which enables them to be electrostatically attracted to the negatively charged surface of bacterial membranes. In the case of gram negative bacteria, cationic AMPs bind the anionic phosphate groups in the outer membrane. Gram positive bacteria, on the other hand, have neither an outer membrane nor lipopolysaccharides on their surface to bind AMPs. Instead, these bacteria have peptidoglycans on their surface which present anionic teichoic acid to attract AMPs. Lipopolysaccharides (LPS) and peptidoglycans featured on the surface of bacteria enable AMPs to distinguish bacteria from host cells. The ability to differentiate host from bacterial cells make AMPs considerable less toxic than the traditional small molecule antibiotics.

The mode of interaction with the membranes depends on the charge distribution, shape, and secondary structures present in the AMP. Experiments that compare the CD spectra of various AMPs in the presence of liposomes, show that some AMPs can undergo conformational changes in hydrophobic environments. Melittin, for example, behaves as a random coil in solution, but adopts an α-helical conformation when in the membrane surface environment (Table 1). Many other AMPs that contain multiple disulfide bridges like in defensins, have stable β-sheet structures which are important in their modes of action. The modes of action of AMPs involve either the targeting of bacterial cellular membranes (i.e., membrane lysis or disruption) or intracellular proteins and membranes (e.g., endoplasmic reticulum membrane) in order to trigger apoptosis or necrosis.

The insertion or disruption of bacterial cellular membranes by AMPs begins with electrostatic interactions between the membrane and AMP. Following this electrostatic pairing interaction, absorption of AMP occurs in the outer bacterial membrane (in the case of gram-negative bacteria) which involves the formation of hydrogen-bonds between basic amino acid residues in the peptide (e.g., lysine and arginine), and phosphate groups exposed on the membrane surface. This action causes the breakdown of salt bridges between the phosphate groups and neighboring cations to begin the process of membrane destabilization. In addition to the electrostatic interactions, hydrophobic interactions between uncharged residues (leucine, isoleucine, valine, tryptophan, and phenylalanine) and hydrophobic carbon chains of the Lipopolysaccharides (LPS) which further disrupt the close packing structure of the membrane causing more disorganization. Through a process known as “membrane thinning,” when a critical concentration of AMPs is reached around the bacterium, the membrane becomes thin causing lateral expansions and increasing water translocation across the membrane. This disruption causes the membrane to leak and reduce the membrane potentials leading to cell death.

Antimicrobial Peptide Structures

Figure 1. Representative models of common antimicrobial peptides (AMPs) showing alpha-helical, beta-sheet, amphiphilic structural motifs that relate form and function. Molecular graphics images were produced using the Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR-01081).[17]

Antimicrobial Peptide Citations

"(R/K)-RMAD-4 [rhesus myeloid α-defensin 4] [(R1/2/5/7/10/13/14/26/33K)-RMAD-462-94] was custom synthesized by CPC Scientific, Inc. (San Jose, CA) and refolded as described previously…"

1. Llenado, R. Alan, et al. "Electropositive charge in α-defensin bactericidal activity: functional effects of Lys-for-Arg substitutions vary with the peptide primary structure." Infection and Immunity 77.11 (2009): 5035-5043.Learn More »

"LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-NH2) was purchased from CPC Scientific (Sunnyvale, California, USA).."

2. Wuerth, Kelli C., Reza Falsafi, and Robert EW Hancock. "Synthetic host defense peptide IDR-1002 reduces inflammation in Pseudomonas aeruginosa lung infection." PloS One 12, no. 11 (2017): e0187565.Learn More »

"Cryptdin 2 (UniProtKB: P28309, LRDLVCYCRTRGCKRRERMNGTCRKGHLMYTLCCR)... obtained by oxidative refolding of partially purified linear peptides (synthesized by CPC Scientific...) and purifying the correctly folded species by reverse-phase high-pressure liquid chromatography (RP-HPLC). Purity was determined by analytical RP-HPLC, and the mass of the disulfide-bonded peptides was verified by high mass accuracy liquid chromatography-mass spectrometry. "

3. Wilson, Sarah S., et al. "Alpha-defensin-dependent enhancement of enteric viral infection." PLoS Pathogens 13.6 (2017): e1006446.Learn More »

"CATH-2 and LL-37 were synthesized by Fmoc-chemistry at CPC Scientific (Sunnyvale, CA)."

4. Coorens, Maarten, et al. "Cathelicidins Inhibit Escherichia coli–Induced TLR2 and TLR4 Activation in a Viability-Dependent Manner." The Journal of Immunology 199.4 (2017): 1418-1428.Learn More »

"..folded HD5 was generated from a synthesized 80% pure linear peptide (CPC Scientific, Sunnyvale, CA) by thiol disulfide reshuffling overnight at room temperature in the presence of 3 mM reduced and 0.3 mM oxidized glutathione, 2 M guanidine hydrochloride, and 0.25 M sodium bicarbonate, pH 8.3, at a peptide concentration of 0.25 mg/ml, purified to homogeneity by reverse-phase high pressure liquid chromatography, and lyophilized as described previously (17). The synthesis, refolding, purification, and structural validation of the HD5 analogs have been described for R9A, R13A, R25A, R28A, R32A, E21me, and Leu-29 substitutions (12) and R9A/R28A, R9K/R28K, R13A/R32A, and R13K/R32K (9). All α-defensins were quantified by UV absorbance at 280 nm using calculated molar extinction coefficients (18)."

5. Gounder, Anshu P., et al. "Critical determinants of human α-defensin 5 activity against non-enveloped viruses." Journal of Biological Chemistry 287.29 (2012): 24554-24562.Learn More »

"Peptides LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a scrambled LL-37, sLL-37 (RSLEGTDRFPFVRLKNSRKLEFKDIKGIKREQFVKIL) and IG-19 (IGKEFKRIVQRIKDFLRNL-NH2) were obtained from CPC Scientific (Sunnyvale, CA). The peptides were re-suspended in endotoxin-free water and stored at −20° until needed."

6. Choi, Ka-Yee G., Scott Napper, and Neeloffer Mookherjee. "Human cathelicidin LL-37 and its derivative IG-19 regulate interleukin-32-induced inflammation." Immunology 143.1 (2014): 68-80.Learn More »

"The peptides were synthesized as 4-branch MAPS peptides that include a SGGRGG spacer at the C-terminal residue to distance the gp41 sequence from the MAPS (CPC Scientific; San Jose, CA)."

7. Mestecky, Jiri, et al. "Scarcity or absence of humoral immune responses in the plasma and cervicovaginal lavage fluids of heavily HIV-1-exposed but persistently seronegative women." AIDS Research and Human Retroviruses 27.5 (2011): 469-486.Learn More »

"Synthetic peptides of HPV 16 L1 (N′-C-KHTPPAPKEDPLKK-C′; position: 456−471)/E6 (N′-C-RTAMFQDPQERPRK-C′; position: 5−18) and a recombination protein of full-length HPV 16 E7-histag fusion protein (N′-MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP-C′; position: 1−98) were manufactured by CPC Scientific Inc."

8. Huang, Chung-Guei, et al. "Molecular and serologic markers of HPV 16 infection are associated with local recurrence in patients with oral cavity squamous cell carcinoma." Oncotarget 8.21 (2017): 34820-34835Learn More »

bacteria cell surfaces

Figure 2. Comparison of the cell surface architectures of gram-negative and gram-positive bacteria.

TABLE OF COMMON ANTIMICROBIAL PEPTIDES

PEPTIDE NAME SEQUENCE STRUCTURE MECHANISM REFERENCES
Histatin 5 DSHAKRHHGYKRKFHEKHHSHRGY Histidine-Rich ROS generation

M.Nishikata et al., Biochem. Biophys. Res. Commun., 174, 625 (1991)

LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES α-Helix Toroidal pore, carpet

(a) G.H. Gudmundsson et al., Eur. J. Biochem., 238, 325 (1996); (b) Madera, Laurence, and Robert EW Hancock. Journal of Innate Immunity 4.5-6 (2012): 553-568; (c) Choi, Ka Yee G., Scott Napper, and Neeloffer Mookherjee. Immunology 143.1 (2014): 68-80.

Cecropin B KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL-NH2 α-Helix Channel formation

D. Andreau et al., PNAS, 80, 6475 (1983)

Cecropin A, porcine KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK-NH2 α-Helix Channel formation

D. Andreu et al., Proc. 20th Euro. Pept. Symp., 361 (1988)

Cecropin P1, porcine SWLSKTAKKLENSAKKRISEGIAIAIQGGPR α-Helix Channel formation

J.Y. Lee et al., PNAS, 86, 9159 (1989)

Indolicidin ILPWKWPWWPWRR-NH2 Tryptophan-rich, extended Pore

C. Subbalakshmi et al., FEBS Letters, 395, 48 (1996)

Defensin-1 (human) HNP-1 ACYCRIPACIAGERRYGTCIYQGRLWAFCC (3 disulfide bridges) β-Sheet Pore, carpet

M.E. Selsted et al., J. Clin. Invest., 76, 1436 (1985)

beta-Defensin-2, human GIGDPVTCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP (3 disulfide bridges) β-Sheet Pore, carpet

Schröder, Jens-M., and Jürgen Harder. The International Journal of Biochemistry & Cell Biology 31.6 (1999): 645-651.

alpha-Defensin 6 DCYCRIPACIAGERRYGTCIYQGRLWAFCC (3 disulfide bridges) β-Sheet Pore, carpet

P.A.Raj et al., Biochem. J., 347, 633 (2000)

Defensin HNP-3 (human) DCYCRIPACIAGERRYGTCIYQGRLWAFCC (3 disulfide bridges) β-Sheet Pore, carpet

P.A.Raj et al., Biochem. J., 347, 633 (2000)

Defensin (human) HNP-2 CYCRIPACIAGERRYGTCIYQGRLWAFCC (3 disulfide bridges) β-Sheet Pore, carpet

Selsted, M. and S. Harwig, J. Biol. Chem. 264, 4003 (1989)

beta-Defensin-1, human DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (3 disulfide bridges) β-Sheet, α-helix Pore, carpet

Hoover, David M., Oleg Chertov, and Jacek Lubkowski. Journal of Biological Chemistry 276.42 (2001): 39021-39026.

beta-Defensin-4, human ELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRK (3 disulfide bridges) β-Sheet, α-helix Pore, carpet

García, José-Ramón Conejo, et al. The FASEB Journal 15.10 (2001): 1819-1821.

beta-Defensin-3, human GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK (3 disulfide bridges) β-Sheet, α-helix Pore, carpet

(a) J. Harder, J. Bartels, E. Christophers, and J.-M. Schröder, J. Biol. Chem., 276, 5707 (2001) (Original); (b) L.A. Duits, et al., Biochem. Biophys. Res. Commun., 280, 522 (2001); (c) H.P. Jia, et al., Gene, 263, 211 (2001)

Magainin-2 GIGKFLHSAKKFGKAFVGEIMNS α-helix in membrane Toroidal pore

A. Mor et al., Biochemistry, 30, 8824 (1991)

Melittin GIGAVLKVLTTGLPALISWIKRKRQQ-NH2 α-helix Toroidal pore/carpet, ROS generation

Raynor et al., J. Biol. Chem., 266, 2753 (1991) Schweitz, Toxicon, 22, 308 (1984)

Pore formation, unlike membrane thinning, cause intracellular components to be quickly expelled resulting in rapid cell death. The formation of the pores is affected by the structure of the AMP and can be described by either a barrel-stave or toroidal model. In the barrel-stave model, AMPs aggregate and then insert into the membrane bilayer so that hydrophobic regions (i.e., hydrophobic amino acid residues) of the peptide associate and align with the hydrocarbon core of the lipids through a process known as ‘hydrophobic matching’. The hydrophilic regions of the AMP form the interior surface of the pore allowing hydrophilic molecules of appropriate size to move through freely. In the toroidal pore model, AMPs penetrate deeper into the membrane and have less hydrophobic interactions than in the barrel-stave model. As a result of the electrostatic interactions with the polar head groups in the lipid bilayer, AMP’s pull the charged head groups into the bilayer causing membrane curvature and packing disruption. LL-37, melittin, and magainin are good examples of AMPs that induce toroidal pores with the latter forming 2 to 3 nm diameter-sized pores only allow water-sized molecules to leak out. Pore sizes are also concentration dependent, and in the case of melittin only transient pores form at low concentrations, while much larger (3+ nm) more stable pores form with higher AMP concentrations.

The formation of membrane pores often depends on the AMPs ability to first adopt an orientation parallel to the membrane and then reorient itself perpendicularly to ‘drill’ into the lipid bilayer. Not all AMPs undergo a phase transition, but instead remain oriented parallel to the membrane. This permeabilization mechanism has become known as the ‘Carpet Model.’ In this model, the shape and secondary structures are less important and the ability of the AMP to adopt a specific structure is not required to permeabilize membranes. Instead, AMPs aggregate to the surface of the bilayer but bind to the cell surface on their hydrophobic face while projecting their hydrophilic side to the exterior of the cell. As AMPs continue to collect on the outer membrane, eventually charge imbalance and increased surface tension cause the membrane to fragment and collapse. Unlike the pore models, the catastrophic breakup of the bacteria membrane results in all the cytoplasmic contents, including large biomolecules, to be ejected. Therefore, the size of the molecules released can be used to distinguish pore vs carpet models. The carpet model also differs from the pore models in that it requires high enough concentrations of AMPs to cover the entire bacteria cell for high efficiency. The lower concentration requirements for AMPs that utilize the one of pore mechanisms make them more attractive therapeutic targets. Pore-forming AMPs, including defensins, melittin, magainins, and LL-37 have been of particular interest for clinical development.

Intracellular Targets

The mode of action of AMPs in killing microbes doesn’t stop at membrane disrupting and puncturing activities. Instead of simply acting as detergents to membranes, many AMPs translocate across the cellular membranes without disrupting them in order to target intracellular proteins or membranes. AMPs such as indolicidin, HNP-1, dermaseptin, and others, translocate themselves across the cell membrane and inhibit certain critical cellular processes that lead to the cell death. In the case of indolicidin, a 13 amino acid peptide rich in tryptophan residues, upon entering the cytosol of a cell, binds to the calmodulin protein in a calcium-dependent manner. As a result, indolicidin inhibits calmodulin-stimulated phosphodiesterase activity at nanomolar concentrations.[18] Other AMPs like Histatin 5 translocate into cells in a similar fashion, but bind different intracellular targets.[19] Mitochondria are important cellular organelles involved in ATP production and oxygen metabolism and are the likely target responsible for histatin toxicity. While the precise mode of histatin toxicity is unclear, interference in mitochondrial respiration can generate reactive oxygen species (ROS) and lead to cell death or apoptosis.

While many AMPs possess duel mechanisms involving both membrane disruption and apoptosis, some AMPs appear to exert their activity only after entering the cell. Coprisin, for example, an analog of defensin, exhibits broad spectrum antifungal activity without harming human erythrocytes.[20] After completing a number different membrane studies, including calcium leakage measurements, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence assays, and rhodamine-conjugated single giant unilamellar vesicle (GUV) analysis, it could be clearly determined that membrane disruption was not occurring. Like in the case of histatin, reactive oxygen species (hydroxyl radicals in the case of coprisin) contribute to apoptosis. In addition to apoptosis, coprisin also inhibits mitochondrial function and causes cytochrome c release. While not damaging the membrane, coprisin is still able to selectively target membrane LPS.

AMPs in Immunomodulation

Although first investigated for their antimicrobial activity and their direct killing of microbes, AMPs modes of action are considerably more complicated than once thought. AMPs, also known as host defense peptides (HDPs), play key roles in both innate and adaptive immune responses. AMPs have a broad range of immunomodulatory functions that include, but are not limited to, the modulation of (1) chemoattraction, (2) pro- and anti-inflammation responses, cellular differentiation, (3) wound healing, and (4) enhancement of bacteria killing. Human cathelicidin LL-37 is a chemoattractant for a variety of immune cells, including neutrophils, eosinophils,[21] macrophages.[22] The recruitment of immune cells by AMPs occurs through direct and indirect mechanisms. Indirect examples include increasing the expression of chemokines, signaling proteins that direct chemotaxis, in macrophages, mast cells, T cells, and epithelial cells.[23,24] Hancock uncovered the signaling pathway in keratinocytes, a type of epithelial cell located in the outermost layer of the skin, in which LL-37 promotes chemokine production.[21] He demonstrated that the Src family kinases (SFKs) play a key role and that the signaling pathway includes the P2X7 receptor,[25] SFKs, Akt (protein kinase B), and transcription factors CREB and ATF1.

AMPs also play important roles in the immunomodulation of both pro- and anti-inflammatory responses. Pro-inflammation cytokines TNF-α and IL-1 are expressed in human monocytes in the presence of human α-defensins.[26] In vivo studies conducted by Falco and co-workers also found that the injection of HNP1 into rainbow trout (Onccorhynchus mykiss) resulted in the expression of genes that encode for IL-1β, TNF-α1, IL-8 (pro-inflammatory cytokines), CC chemokines (CK5B, CK6 and CK7A), and also genes related to type I interferon (IFN) production.[27] In contrast to α-defensins, β-defensins appear to have the opposite effect on inflammation. Specifically, human β-Defensin-3 downregulates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)-dependent inflammation.

Cell Selectivity in AMPs

The cell surface architectures of mammalian and bacteria cells are very different in composition and electrostatic charge (Figure 2). Drugs or drug carriers that can recognize this difference have the potential to be developed into antimicrobial therapeutic. Gram-negative bacteria (e.g., Escherichia coli, Pseudomonas aeruginosa) contain two lipid bilayer membranes, an outer membrane and an inner cellular (cytoplasmic) membrane, both of which control the passage of material and information. The outer membrane is considered more “leaky” due to the presence of porin, β-barrel proteins that span the outer-membrane and acts as a pore, allowing molecules to passively diffuse.[28] The lipid composition of the outer-membrane is organized asymmetrically, with phospholipids making up much of the inner leaflet and lipopolysaccharides (LPS) consisting of the outer leaflet. LPS, also known as endotoxin, are unique to gram-negative bacteria and are common impurities in peptides and other biologics. The inner cytoplasmic membrane is more symmetric and consists primarily of phosphatidylethanolamine (70-80%), phosphatidylglycerol, and cardiolipin.

AMPs exhibit cell selectivity due to differences in chemical compositions of bacteria and mammalian cell membranes. The ability to selectively kill invading microbes without significantly harming human host cells makes AMPs attractive drug candidates. Bacterial cell membranes are generally more negatively charged compared to mammalian cell membranes. The negatively charged surfaces of bacterial cell surfaces attract the positively charged AMP and in some cases will aggregate around the negative charges. The differences and implied selectivity towards bacteria is due to both the constitution of the membranes as well as the location of the anionic membrane molecules. Membranes consist mainly of phospholipids, membrane proteins, steroids, and others.[29] Bacterial cell membranes are abundant in acidic phospholipids like phosphatidylserine (PS), phosphatidylglycerol (PG), and cardiolipin (CL) (Figure 3). The head groups of these phospholipids are negatively charged at physiological pH’s and when located in the outer “leaflet” of the membrane bilayer, can attract positively charged AMPs. Additionally, gram-positive bacteria have a thick peptidoglycan layer (i.e., cell wall) that is highly functionalized with anionic glycopolymers called wall teichoic acids (WTAs) which can extend beyond the outer membrane of the cell.[30] Gram-negative bacteria have only a thin layer of peptidoglycans and lack WTAs. Instead, gram-negative bacteria gain additional negative charge on their surface from the asymmetric distribution of LPS in the outer leaflet of the outer membrane.


In contrast to bacterial cellular membranes, mammalian cytoplasmic membranes are rich in phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM), all of which are zwitterionic in physiological conditions making the membrane relatively neutral in charge. While mammalian cell membranes contain zwitterionic phospholipids, they also contain phospholipids with negatively charged head groups. But unlike bacterial membranes, the negatively charged phospholipids are asymmetrically distributed to the inner leaflet facing the cell interior.[31] In bovine erythrocytes, for example, 98% of the total PE composition is located in the inner leaflet of the cellular membrane.[32]

mammalian cell membrane molecules

Figure 3a. Comparison of membrane molecules in mammalians. Mammalian cell membranes contain zwitterionic phospholipids PC, PE, and neutral cholesterol.

In addition to locality differences in phospholipids, mammalian cell membranes are also rich in hydrophobic cholesterol and ergosterol molecules.[33] The relative neutrality and hydrophobicity of mammalian cellular membranes allows for only hydrophobic interactions with AMPs, which are much weaker than the electrostatic interactions. Moreover, these electrostatic interactions can be further amplified with increased transmembrane potentials. Bacteria transmembrane potentials are on the order of -140 mV, whereas mammalian membrane potentials are approximately -100 mV. This stronger negative membrane potential may also contribute to cell selectivity among AMPs.[34]

bacteria cell membrane molecules

Figure 3b. Comparison of membrane molecules in bacteria. Bacterial cell membranes have a higher abundance of anionic phospholipids PG, Lysyl PG, LPS, anionic polymer teichoic acid and organic molecule cardiolipin.

AMP’s that target the membranes of cancer cells (i.e., membranolytic ACPs) disrupt the membrane by similar pore-forming and membrane-thinning mechanisms observed in AMP-bacteria interactions. This mode of action should be distinguished from peptides that target specific membrane-bound receptors, like in the case of RGD-containing peptide sequences that target overexpressed integrin receptors in cancer cells. In addition to cellular membrane targets, mitochondrial plasma membranes and other intracellular targets for ACPs exist. A cationic AMP containing a two stranded antiparallel β-sheet structure called gomesin (isolated by Rodrigues and coworkers in 2008 from the Acanthoscurria gomesiana spider)[40] has been shown to have intracellular targets in melanoma cancer cells. Gomesin exerts cytotoxicity well after translocation across the cellular membrane in melanoma cells by targeting the endoplasmic reticulum (ER) membrane.[41] Lysing of the ER membrane significantly increases cytosolic Ca2+ concentrations, overloading the mitochondria and leads to apoptosis.

LL-37 has been well-studied for its anticancer therapeutic value. The interaction among cancer cells and LL-37 are complicated and can have contradictory effects depending on the type of cancer and the tissue affected. In some forms of cancers such as colon and gastric, tumorigenesis is suppressed, which in other forms of cancers, such as ovarian, lung, and breast cancers, tumorigenesis appears to be enhanced. There is some evidence to suggest that the regulation of apoptosis by LL-37 is involved in the pathogenesis of malignant tumors.[42] LL-37 induced apoptosis in tumor cells accounts for anti-tumor activity in colon cancers and hematologic malignancies.[43] Other mechanisms of tumor suppression have been observed in gastric tumor tissues that involve LL-37’s ability to inhibit proteasomes and induce apoptosis by disrupting the regulation of pro-growth cell cycle proteins.[44] However, LL-37 can also promote tumor growth in other tissues. In ovarian cancer tissue, for example, LL-37 assists in the recruitment of mesenchymal stem cells (MSC) to facilitate healing of the cancer cells and promote the pathogenesis of malignant tumors.[45]

Antibiotic Resistance: Traditional vs. AMP Antibiotics

While remarkable at the time, the advent of classical antibiotics drugs and subsequent over use has resulted in antibiotic resistant strains of bacterial pathogens. Specifically, the emergence of multidrug-resistant (MDR) organisms and the infections they cause are associated with high mortality rates and increased economic burden on health care costs throughout the world. The adaptation of bacteria to antibiotics (i.e., resistance) occurs by two general mechanisms: (1) gene mutation or (2) acquisition of genetic material by horizontal gene transfer (HGT). In gene mutation, the mechanism of resistance occurs by either a reduction in antibiotic uptake, lowering of affinity to the compound, active ejection (i.e., efflux) mechanisms, or changes to the cellular metabolism that reduce the harmful action antibiotic agent.

One of the successful paths to resistance in both gram-negative and gram-positive bacteria involves the chemical modification and transformation of the antibiotic into a less lethal or harmless form. The production of enzymes has been described that can acetylate, phosphorylate, or adenylate an antibiotic in order to change the hydrophobicity or other chemical properties. Aminoglycoside modifying enzymes (AMEs), for example, are capable of reacting with amino or hydroxyl groups. Some AMEs have evolved into bifunctional enzymes with both acetylation and phosphotransferase activity.[46] The emergence of bifunctional AMEs is responsible for most of the gentamicin resistant strains found in enterococci and methicillin resistance found in S. aureus.

β-Lactamases offer bacteria another mechanism of resistance by breaking the lactam ring in β-lactam antibiotics such as penicillins, cephalosporins, and cephamycins (Figure 4). Hydrolysis of the lactam ring by β-lactamases and subsequent decarboxylation effectively ‘destroys’ the antibiotic molecule. β-Lactamases were identified in the 1940’s soon after the widespread use of the penicillin began. Penicillinase was the first β-lactamase to be identified and was isolated by Abraham and Chain in 1940 from Gram-negative E. coli.[47] To combat the emergence of penicillin-resistant bacteria due to the presence of β-lactamases, meticillin (known also as methicillin), a narrow-spectrum β-lactam antibiotic of the penicillin class, was developed by Beecham in 1959.[48] The added bulk of the ortho-dimethoxyphenyl substituent makes the antibiotic unable to bind and be cleaved by β-lactamases. Like other β-lactam antibiotics that target the bacterial cell wall biosynthesis by inhibiting cross linking of peptidoglycan polymers, meticillin directly inhibits the transpeptidase enzyme (i.e., penicillin-binding proteins (PBPs)). PBPs are responsible for cross-linking D-alanyl-alanine peptides with peptidoglycan polymers. While metcillin is no longer in use today, replaced by newer analogs such as cloxacillin, it was somewhat ironically termed a penicillinase-resistant β-lactam antibiotic.


AMPs in Cancer Therapeutics Field

In light of the complex immunomodulatory effects of AMPs, it is not surprising that some AMPs have the ability to regulate some forms of cancers. In the realm of cancer therapeutics, AMPs that facilitate cytotoxicity on cancer cells, also termed anti-cancer peptides (ACPs), have been categorized by mechanism as either membranolytic or non-membranolytic.[35] While not all AMPs are ACPs, cancer and bacterial cells share a common characteristic of having negatively charged membranes. The presence of anionic membrane molecules like phosphatidylserines (PS), glycosaminoglycans, heparan sulfate, O-glycosylated mucins, sialylated gangliosides, and glycoproteins in cancer cells creates a negatively charged surface similar to what is observed in bacterial cell membranes. Like with bacteria cells, AMPs that are ACPs can also selectively target cancer over normal mammalian cells. During cell transformation, cancer cells accumulate PS on the outer (presenting) leaflet of the cell membrane, contrary to the typical membrane asymmetry found in normal cells. In addition, normal cells contain a greater concentration of neutral cholesterol molecules that increase membrane stability and resistance to AMPs. Most studies agree that cancer cell membranes are more fluidic than normal cell membranes.[36-38] Like with counter measures presented by bacteria, however, cancer cells, particularly those found in prostate and breast tissues, can in some instances contain higher amounts of cholesterol in their membranes as means of evading AMPs.[39]

AMPs are less likely to promote the type of resistance seen in bacteria with traditional antibiotics because they exert multiple and broad modes of action on bacteria. The mode of action in most conventional antibiotics is very specific and in many cases can be blocked by only a single gene mutation. AMPs target multiple regions of the bacterium that include the cell membrane as well as many intracellular targets that lead to apoptosis. AMPs have the advantage of millions of years of coevolution with bacteria to evolve sequences that are effective antibiotics and resistant to the common modes of resistance. Although some counter measures to AMPs have been reported, resistance is less common and specific compared to traditional antibiotics. A gram-positive bacterium, S. aureus, for example, has evolved a general mechanism of reducing its negatively charged surface by incorporating D-alanine into the teichoic acid polymers.[49] Additionally, S. aureus has the ability to modify the membrane lipid PG by an enzymatic transfer of a L-lysine residue to result in lysyl-PG.[50] This process results in a net charge from -1 to +1 of PG that reduces electrostatic interactions with cationic AMPs.

antibiotic resistant mechanism

Figure 4. (a) Lactam antibiotics penicillins, cephalosporins, and cephamycins. The R groups (R, R1, R2) are variable are not shown for clarity. The red color denotes the 4-membered lactam ring in each derivative. (b) The mechanism of β-lactamase action on penicillin showing two steps starting with a ring-opening hydrolysis of the lactam followed by decarboxylation.

AMPs are less likely to promote the type of resistance seen in bacteria with traditional antibiotics because they exert multiple and broad modes of action on bacteria. The mode of action in most conventional antibiotics is very specific and in many cases can be blocked by only a single gene mutation. AMPs target multiple regions of the bacterium that include the cell membrane as well as many intracellular targets that lead to apoptosis. AMPs have the advantage of millions of years of coevolution with bacteria to evolve sequences that are effective antibiotics and resistant to the common modes of resistance. Although some counter measures to AMPs have been reported, resistance is less common and specific compared to traditional antibiotics. A gram-positive bacterium, S. aureus, for example, has evolved a general mechanism of reducing its negatively charged surface by incorporating D-alanine into the teichoic acid polymers. Additionally, S. aureus has the ability to modify the membrane lipid PG by an enzymatic transfer of a L-lysine residue to result in lysyl-PG. This process results in a net charge from -1 to +1 of PG that reduces electrostatic interactions with cationic AMPs.

AMP Drug Development: Pexiganan and Plectasin

Despite heroic efforts over the past decade, no systemically dosed peptide antibiotics have been approved by the US-FDA. This failure is due, in part, to high peptide manufacturing costs and small market demands for some therapeutic AMPs. The first antimicrobial peptide to enter clinical development was a derivative of magainin 2 called Pexiganan, a 22-amino-acid cationic peptide. Magainin was originally isolated from the skin of African clawed frog (Xenopus laevis) and was first reported in 1987 by the Zasloff group.[51] In the preclinical phase, Pexiganan preformed exceptionally well in vitro against a broad-spectrum of bacteria. Pexiganan was originally designed as topical locally applied antibiotic treatment as an alternative to current systemic dosed antibiotics. Since the peptide is susceptible to proteolytic cleavage in the blood stream, reducing its bioavailability, considerably highly concentrations can be achieved through local applications at the site of infection. As a topical treatment, much less peptide is required compared to the estimated 1.0 gram of intravenous dosing required for typical antibiotics. Early in the development of Pexiganan, the manufacturing cost was $1000/g which made cGMP manufacturing prohibitively expensive for a drug that did not significantly outperform current small molecule antibiotics. As development of Pexiganan reached phase III in clinical trials, however, the FDA halted approval due to inconsistent results and manufacturing concerns.[52] More recently, Pexiganan is being developed by Depexium Pharmaceuticals into a topical cream to treat diabetic foot infections and has successfully completed phase III.

Plectasin was discovered by Mygind in 2005 as novel antimicrobial defensin peptide, the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella.[53] The discovery and clinical development of Plectasin was headed by Dr. Hans-Henrik Kristensen at Novozymes. They discovered the defensin peptide (plectasin) excreted by the fungus had very similar homology to secondary structures to defensins found in insects and mussels. Plectasin exhibited a narrow antibacterial spectrum that was particularly effective against Streptococcus pneumonia. In vivo studies showed that intravenously administered peptide was comparable or exceeded conventional antibiotic therapy. The compact and constrained structure, due in part to the three disulfide bridges, makes defensin-like peptides particularly resistant to degradation by proteolytic enzymes. For this reason, Plectasin can be recovered fully intact from the urine of treated animals. As a narrow spectrum antibiotic, this peptide appeared to be an ideal treatment for penicillin-resistant Pneumococcus. However, due to the small market size of Pneumococcus infected patients combined with the high cost of clinical development and commercial manufacturing, a decision was made to halt development of the peptide in its current form. Additional efforts were made to broaden the antibacterial activity of Plectasin with a new analogy termed NZ2114 that showed systemic activity against S. aureus. In 2010, Novozymes and Sanofi-Aventis entered into an agreement for the clinical development of NZ2114.

Conclusions

While clinical development of AMPs remains active, some challenges and barriers to FDA approval remain. The cost of cGMP peptide manufacturing is high, but with better chemistries to improve yields and lower starting material costs, prices are beginning to erode. The cost of protected amino acids, for example, has been reduced dramatically of the past two decades. Peptides containing all D-form of amino acids,[54] engineered for enhanced protease resistance, have only recently entered into clinical development, due in part, to reduced costs of protected D-forms of amino acids. Peptide stability in general and their ability to resist proteolytic degradation is a key factor in designing drugs intended for intravenous dosing. In the addition to presence of D-amino acids, highly constrained peptides like defensins that utilizes multiple disulfide bridges, makes the sequence less accessible to enzymes. Another method for maintaining peptide stability, particularly for secondary structures like alpha-helices, is the introduction of hydrocarbon-staples.[55-57] Peptide stapling can result in higher resistance to proteolytic degradation compared to the native sequence; however, it does not necessarily increase antimicrobial activity. Other custom peptide modifications[58] that further constrain peptide backbone conformations can be introduced and include macrocyclization,[59-60] (e.g., head-to-tail, sidechain-to-sidechain, etc.), oxidation,[61-62] Click chemistry,[63] N-alkylation,[64] and others.


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