HHS Public Access Author manuscript Author Manuscript
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Expert Rev Anti Infect Ther. 2016 November ; 14(11): 989–991. doi:10.1080/14787210.2016.1236687.
Clinical potential of engineered cationic antimicrobial peptides against drug resistant biofilms Jeffrey A. Melvin, Ronald C. Montelaro, and Jennifer M. Bomberger* Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Author Manuscript
Keywords Biofilm infections; Antimicrobial peptide; Multidrug resistance; Polymicrobial infections; Rational design
Author Manuscript
Biofilms are involved in the pathogenesis of 80% of microbial infections in humans[1]. This prevalence is of major clinical importance as microbial populations growing in biofilms are far more tolerant to traditional antibiotics than when growing planktonically due to altered metabolic programs and increased expression of antimicrobial defenses[2]. Biofilms are a major source of recurrent and chronic infections, leading to tens of millions of infections and hundreds of thousands of deaths in the USA each year[1]. The majority of nosocomial infections are caused by biofilm colonization of medical implants, for which removal of the colonized implant is often the only effective treatment[3]. Additionally, biofilms are often polymicrobial[4], making treatment more complex as well as possibly further modulating antimicrobial susceptibility[5].
Author Manuscript
Many strategies to combat biofilm-associated infections have been proposed[6], focusing on preventing biofilm biogenesis as well as disrupting established biofilms. Towards biofilm prevention, compounds capable of blocking microbial adhesion to host or medical implant structures or inhibiting production of extracellular matrix (ECM) are ideal. For biofilm disruption, compounds capable of destabilizing ECM, interfering with microbial community communication to initiate dispersal, or killing slow-growing/persister cells are desirable. Antimicrobial peptides, especially engineered cationic antimicrobial peptides (eCAPs), are able to perform many of these functions, including killing cells in biofilms, disrupting ECM production and stability, and interfering with microbial communication, and as such have begun to be more recognized for their potential as antibiofilm antimicrobial agents[7].
*
Corresponding author: 416 Bridgeside Point II, 450 Technology Drive, Pittsburgh, PA 15219, T: (412) 624-1963, F: (412) 624-1401, [email protected]. Declaration of interest R Montelaro holds stock in and serves on an advisory board for Peptiologics. The findings included in this publication may not necessarily be related to the interests of Peptiologics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Melvin et al.
Page 2
Author Manuscript Author Manuscript
Cationic antimicrobial peptides (AMPs), or cationic host defense peptides, are an incredibly diverse family of antimicrobial compounds, with approximately 4,000 discovered to date[8], and are widely distributed in nature, constituting a major aspect of the immune defense system[9]. While some of these peptides display effective killing in vitro, efficacy in vivo is often limited. This loss of activity is largely due to the fact that many physiological conditions deactivate or inhibit natural AMPs[10]. Thus, the reason for the diversity of AMPs in nature is perhaps that they have evolved for activity in particular biological environments or for activity against specific microbial species common to those sites. Additionally, many natural AMPs have immunomodulatory functions[9], and this requirement for dual functionality may constrain their ability to maintain microbial killing in diverse physiological environments. eCAPs are synthetic peptides designed based on natural AMP sequences and structures that can overcome many of these limitations, as rational design has yielded peptides capable of maintaining killing activity in a wide range of environments and against a breadth of microbial species and strains[6]. Initial design of an eCAP should include consideration of the potential treatment environment; for instance, an eCAP produced for treatment of biofilms in the lungs of cystic fibrosis patients should maintain activity in low pH, high sodium chloride concentrations, and the presence of mucus that inactive many natural AMPs[11]. Some eCAPs have also been shown to modulate inflammation to increase clearance of microbial pathogens, which can contribute to their antimicrobial efficacy[9].
Author Manuscript Author Manuscript
Most AMPs, including eCAPs, are generally thought to kill microbial pathogens via membrane disruption. This mechanism of action is a key component of the potential efficacy of eCAPs for treatment of biofilm infections. Traditional antibiotics require bacteria to be actively growing in order to kill, making them ineffective against slow-growing or persister cells that are common in mature biofilms[2]. Membrane disruption kills largely regardless of metabolic status, and accordingly eCAPs have been shown to efficiently kill persisters[10]. An additional detrimental effect of the inability of traditional antibiotics to kill slow-growing cells is the potential for highly tolerant microbes to develop resistance to antibiotics that they are exposed to for long durations. eCAPs kill rapidly, on the seconds to minutes timescale[12, 13]. The rates of resistance to eCAPs are generally low while the mutants that do arise display variable cross-resistance development rates for natural AMPs[14], which is a topic under intense investigation[15, 16]. To attempt to limit cross-resistance, an intriguing strategy is to utilize eCAPs in conjunction with traditional antibiotics, as the distinct mechanisms of action often result in additive or synergistic activity[11, 17]. One major benefit of the dual treatment approach is that traditional antibiotics may be able to kill dispersed or planktonic cells, which are more metabolically active, while eCAPs can kill the remaining slow-growing or persister cells[6]. Due to general membrane disruption activity, eCAPs are often able to kill a broad range of microbial species and strains, even multidrug resistant and select agent pathogens[14, 18, 19]. Considering that infection biofilms are often polymicrobial, treatment with eCAPs is potentially beneficial for their broad-spectrum activity. Furthermore, AMPs have been shown to neutralize fungi, parasites, and even viruses[10], potentially also via membrane interactions. Polymicrobial infections can modulate antimicrobial resistance[5], and viral bacterial co-infections of the respiratory tract have been shown to increase bacterial Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
Melvin et al.
Page 3
Author Manuscript
resistance to frontline traditional antibiotics[20]. However, we have recently used eCAPs to concurrently disrupt extraordinarily resistant Pseudomonas aeruginosa biofilms and inactivate respiratory syncytial virus[13], revealing the potential for eCAPs to simultaneously treat infections with pathogens from multiple kingdoms even when the infection displays synergistic antimicrobial resistance. While the ability of eCAPs to treat established biofilms is of major interest, their use in preventing biofilm biogenesis, either by prophylactic treatment or coating of medical implants[11, 18], is of equal importance. There is current interest in tethering eCAPs to implant materials, with some success in maintaining bactericidal activity[21]. Of note, the ability of eCAPs to strongly interact with bacterial membrane structures has led some to attempt to tether eCAPs with the goal of removing lipopolysaccharide (LPS) from the blood stream via hemoperfusion as a potential treatment for septic shock[22].
Author Manuscript Author Manuscript
Some of the major challenges facing development of eCAPs as therapeutics are regulatory. Due to the fact that eCAPs target membranes, safety is a primary concern. However, recent advances in eCAP design have shown promising advances towards low levels of toxicity to host cells and in small animal models[10, 23]. Bioavailability is also a consideration, as peptides may not be as stable in body, though eCAPs are largely more stable than natural CAPs[10]. Many techniques have been employed to increase stability of eCAPs, including the use of crosslinking, introduction of D-amino acids, and amino acid modification to avoid proteolysis, with some success[10]. Antigenicity of eCAPs is also a possibility, though even during covalent linkage to antigenic carrier proteins very little antibody production has been observed[24]. Additionally, modification of peptides has shown promise for reducing peptide antigenicity[25, 26]. Production costs are often raised as an issue for eCAPs, though production costs are lowering due to new technologies and approaches[27]. Additionally, we have optimized eCAPs to be as short as possible, to contain only 2–3 different amino acids arranged in repeat motifs to facilitate higher yield synthesis chemistry[28].
Author Manuscript
One of the largest challenges for developing eCAPs for use as antibiofilm agents is the relative lack of in vivo models for studying treatment efficacy against biofilm infections. Though small animal models have been used to demonstrate the efficacy of eCAPs for acute infections[23], most small animals are not amenable to the chronic or recurrent infection that is critical to the development of relevant model biofilms[29]. Porcine models are more reflective chronic human infections, though the relative lack of ability to manipulate the host, the inability for some human pathogens to cause infection, and the cost associated with such models make this option inaccessible for many studies. Implanted devices in small animals show reasonably good mimicry of human biofilm infections associated with medical implants, but are not reflective of mucosal or wound biofilms. Additionally, the fact that biofilm infections are commonly polymicrobial indicates that model systems should be amenable multiple pathogens. An alternative initial approach is to use in vitro differentiated cell culture systems. We have developed a model of P. aeruginosa biofilm biogenesis in association with the airway epithelium that mimics biofilm morphology observed in patient samples and displays extreme levels of antibiotic resistance that is further increased during viral co-infection[13], similar to clinical findings[20]. Models such as this are readily
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
Melvin et al.
Page 4
Author Manuscript
amenable to relatively high throughput analysis of treatment and pathogen combinations and may serve as useful, relatively low cost systems to initially evaluate novel eCAP candidates.
Conclusions
Author Manuscript
Recent advances in the design of eCAPs and the technology used to produce them have driven a renaissance of interest in their therapeutic potential. Numerous groups have achieved increased efficacy and reduced toxicity with eCAPs and demonstrated their ability to kill multidrug resistant and select agent pathogens. Perhaps most exciting are the recent reports of eCAPs preventing and disrupting biofilms formed by important human pathogens. Recent use of novel animal and differentiated cell culture models and an increased focus on demonstrating the ability of eCAPs to clear regulatory hurdles promises to bring these compounds to clinic sooner rather than later, potentially providing much needed relief for patients and physicians battling chronic antibiotic resistant infections.
Acknowledgments Funding This paper was supported by funding from the National Institutes of Health (grant numbers: R00HL098342, R01HL123771, T32AI49820, P30DK072506) and the Cystic Fibrosis Foundation (grant numbers: BOMBER14G0, MELVIN15F0).
References
Author Manuscript Author Manuscript
1. Mihai MM, Holban AM, Giurcaneanu C, Popa LG, Oanea RM, Lazar V, Chifiriuc MC, Popa M, Popa MI. Microbial biofilms: impact on the pathogenesis of periodontitis, cystic fibrosis, chronic wounds and medical device-related infections. Curr Top Med Chem. 2015; 15:1552–76. [PubMed: 25877092] 2. Olsen I. Biofilm-specific antibiotic tolerance and resistance. Eur J Clin Microbiol Infect Dis. 2015; 34:877–86. [PubMed: 25630538] 3. Wilkins M, Hall-Stoodley L, Allan RN, Faust SN. New approaches to the treatment of biofilmrelated infections. J Infect. 2014; 69(Suppl 1):S47–52. [PubMed: 25240819] 4. Wolcott R, Costerton JW, Raoult D, Cutler SJ. The polymicrobial nature of biofilm infection. Clin Microbiol Infect. 2013; 19:107–12. [PubMed: 22925473] 5. Birger RB, Kouyos RD, Cohen T, Griffiths EC, Huijben S, Mina MJ, Volkova V, Grenfell B, Metcalf CJ. The potential impact of coinfection on antimicrobial chemotherapy and drug resistance. Trends Microbiol. 2015; 23:537–44. [PubMed: 26028590] 6. Ribeiro SM, Felicio MR, Boas EV, Goncalves S, Costa FF, Samy RP, Santos NC, Franco OL. New frontiers for anti-biofilm drug development. Pharmacol Ther. 2016; 160:133–44. [PubMed: 26896562] 7. Batoni G, Maisetta G, Esin S. Antimicrobial peptides and their interaction with biofilms of medically relevant bacteria. Biochim Biophys Acta. 2016; 1858:1044–60. [PubMed: 26525663] 8. Zhao X, Wu H, Lu H, Li G, Huang Q. LAMP: A Database Linking Antimicrobial Peptides. PLoS One. 2013; 8:e66557. [PubMed: 23825543] 9. Hiemstra PS, Amatngalim GD, van der Does AM, Taube C. Antimicrobial Peptides and Innate Lung Defenses: Role in Infectious and Noninfectious Lung Diseases and Therapeutic Applications. Chest. 2016; 149:545–51. [PubMed: 26502035] 10. Bahar AA, Ren D. Antimicrobial peptides. Pharmaceuticals (Basel). 2013; 6:1543–75. [PubMed: 24287494]
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
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Page 5
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11. Lashua LP, Melvin JA, Deslouches B, Pilewski JM, Montelaro RC, Bomberger JM. Engineered cationic antimicrobial peptide (eCAP) prevents Pseudomonas aeruginosa biofilm growth on airway epithelial cells. J Antimicrob Chemother. 2016 12. Deslouches B, Steckbeck JD, Craigo JK, Doi Y, Mietzner TA, Montelaro RC. Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug-resistant pathogens. Antimicrob Agents Chemother. 2013; 57:2511–21. [PubMed: 23507278] 13. Melvin JA, Lashua LP, Kiedrowski MR, Yang G, Deslouches B, Montelaro RC, Bomberger JM. Simultaneous Antibiofilm and Antiviral Activities of an Engineered Antimicrobial Peptide during Virus-Bacterium Coinfection. mSphere. 2016:1. 14. Deslouches B, Steckbeck JD, Craigo JK, Doi Y, Burns JL, Montelaro RC. Engineered cationic antimicrobial peptides to overcome multidrug resistance by ESKAPE pathogens. Antimicrob Agents Chemother. 2015; 59:1329–33. [PubMed: 25421473] 15. Fleitas O, Franco OL. Induced Bacterial Cross-Resistance toward Host Antimicrobial Peptides: A Worrying Phenomenon. Front Microbiol. 2016; 7:381. [PubMed: 27047486] 16. Scott RW, Tew GN. Mimics of Host Defense Proteins; Strategies for Translation to Therapeutic Applications. Curr Top Med Chem. 2016 17. Zemke AC, Shiva S, Burns JL, Moskowitz SM, Pilewski JM, Gladwin MT, Bomberger JM. Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells. Free Radic Biol Med. 2014; 77:307–16. [PubMed: 25229185] 18. de la Fuente-Nunez C, Cardoso MH, de Souza Candido E, Franco OL, Hancock RE. Synthetic antibiofilm peptides. Biochim Biophys Acta. 2016; 1858:1061–9. [PubMed: 26724202] 19. Abdelbaqi S, Deslouches B, Steckbeck J, Montelaro R, Reed DS. Novel engineered cationic antimicrobial peptides display broad-spectrum activity against Francisella tularensis, Yersinia pestis and Burkholderia pseudomallei. J Med Microbiol. 2016; 65:188–94. [PubMed: 26673248] 20. Patel JA, Reisner B, Vizirinia N, Owen M, Chonmaitree T, Howie V. Bacteriologic failure of amoxicillin-clavulanate in treatment of acute otitis media caused by nontypeable Haemophilus influenzae. J Pediatr. 1995; 126:799–806. [PubMed: 7752010] 21. Costa F, Carvalho IF, Montelaro RC, Gomes P, Martins MC. Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces. Acta Biomater. 2011; 7:1431–40. [PubMed: 21056701] 22. Ryder MP, Wu X, McKelvey GR, McGuire J, Schilke KF. Binding interactions of bacterial lipopolysaccharide and the cationic amphiphilic peptides polymyxin B and WLBU2. Colloids Surf B Biointerfaces. 2014; 120:81–7. [PubMed: 24905681] 23. Deslouches B, Islam K, Craigo JK, Paranjape SM, Montelaro RC, Mietzner TA. Activity of the de novo engineered antimicrobial peptide WLBU2 against Pseudomonas aeruginosa in human serum and whole blood: implications for systemic applications. Antimicrob Agents Chemother. 2005; 49:3208–16. [PubMed: 16048927] 24. Koyama Y, Motobu M, Hikosaka K, Yamada M, Nakamura K, Saido-Sakanaka H, Asaoka A, Yamakawa M, Isobe T, Shimura K, Kang CB, Hayashidani H, Nakai Y, Hirota Y. Cytotoxicity and antigenicity of antimicrobial synthesized peptides derived from the beetle Allomyrina dichotoma defensin in mice. Int Immunopharmacol. 2006; 6:1748–53. [PubMed: 16979131] 25. Gregoriadis G, Jain S, Papaioannou I, Laing P. Improving the therapeutic efficacy of peptides and proteins: a role for polysialic acids. Int J Pharm. 2005; 300:125–30. [PubMed: 16046256] 26. Jain A, Jain A, Gulbake A, Shilpi S, Hurkat P, Jain SK. Peptide and protein delivery using new drug delivery systems. Crit Rev Ther Drug Carrier Syst. 2013; 30:293–329. [PubMed: 23662604] 27. Glaser V. Reducing the Cost of Peptide Synthesis. Genetic Engineering & Biotechnology News. 2013 28. Deslouches B, Phadke SM, Lazarevic V, Cascio M, Islam K, Montelaro RC, Mietzner TA. De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity. Antimicrob Agents Chemother. 2005; 49:316–22. [PubMed: 15616311] 29. Roberts AE, Kragh KN, Bjarnsholt T, Diggle SP. The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection. J Mol Biol. 2015; 427:3646–61. [PubMed: 26344834]
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Expert Rev Anti Infect Ther. 2016 November ; 14(11): 989–991. doi:10.1080/14787210.2016.1236687.
Clinical potential of engineered cationic antimicrobial peptides against drug resistant biofilms Jeffrey A. Melvin, Ronald C. Montelaro, and Jennifer M. Bomberger* Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Author Manuscript
Keywords Biofilm infections; Antimicrobial peptide; Multidrug resistance; Polymicrobial infections; Rational design
Author Manuscript
Biofilms are involved in the pathogenesis of 80% of microbial infections in humans[1]. This prevalence is of major clinical importance as microbial populations growing in biofilms are far more tolerant to traditional antibiotics than when growing planktonically due to altered metabolic programs and increased expression of antimicrobial defenses[2]. Biofilms are a major source of recurrent and chronic infections, leading to tens of millions of infections and hundreds of thousands of deaths in the USA each year[1]. The majority of nosocomial infections are caused by biofilm colonization of medical implants, for which removal of the colonized implant is often the only effective treatment[3]. Additionally, biofilms are often polymicrobial[4], making treatment more complex as well as possibly further modulating antimicrobial susceptibility[5].
Author Manuscript
Many strategies to combat biofilm-associated infections have been proposed[6], focusing on preventing biofilm biogenesis as well as disrupting established biofilms. Towards biofilm prevention, compounds capable of blocking microbial adhesion to host or medical implant structures or inhibiting production of extracellular matrix (ECM) are ideal. For biofilm disruption, compounds capable of destabilizing ECM, interfering with microbial community communication to initiate dispersal, or killing slow-growing/persister cells are desirable. Antimicrobial peptides, especially engineered cationic antimicrobial peptides (eCAPs), are able to perform many of these functions, including killing cells in biofilms, disrupting ECM production and stability, and interfering with microbial communication, and as such have begun to be more recognized for their potential as antibiofilm antimicrobial agents[7].
*
Corresponding author: 416 Bridgeside Point II, 450 Technology Drive, Pittsburgh, PA 15219, T: (412) 624-1963, F: (412) 624-1401, [email protected]. Declaration of interest R Montelaro holds stock in and serves on an advisory board for Peptiologics. The findings included in this publication may not necessarily be related to the interests of Peptiologics. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Melvin et al.
Page 2
Author Manuscript Author Manuscript
Cationic antimicrobial peptides (AMPs), or cationic host defense peptides, are an incredibly diverse family of antimicrobial compounds, with approximately 4,000 discovered to date[8], and are widely distributed in nature, constituting a major aspect of the immune defense system[9]. While some of these peptides display effective killing in vitro, efficacy in vivo is often limited. This loss of activity is largely due to the fact that many physiological conditions deactivate or inhibit natural AMPs[10]. Thus, the reason for the diversity of AMPs in nature is perhaps that they have evolved for activity in particular biological environments or for activity against specific microbial species common to those sites. Additionally, many natural AMPs have immunomodulatory functions[9], and this requirement for dual functionality may constrain their ability to maintain microbial killing in diverse physiological environments. eCAPs are synthetic peptides designed based on natural AMP sequences and structures that can overcome many of these limitations, as rational design has yielded peptides capable of maintaining killing activity in a wide range of environments and against a breadth of microbial species and strains[6]. Initial design of an eCAP should include consideration of the potential treatment environment; for instance, an eCAP produced for treatment of biofilms in the lungs of cystic fibrosis patients should maintain activity in low pH, high sodium chloride concentrations, and the presence of mucus that inactive many natural AMPs[11]. Some eCAPs have also been shown to modulate inflammation to increase clearance of microbial pathogens, which can contribute to their antimicrobial efficacy[9].
Author Manuscript Author Manuscript
Most AMPs, including eCAPs, are generally thought to kill microbial pathogens via membrane disruption. This mechanism of action is a key component of the potential efficacy of eCAPs for treatment of biofilm infections. Traditional antibiotics require bacteria to be actively growing in order to kill, making them ineffective against slow-growing or persister cells that are common in mature biofilms[2]. Membrane disruption kills largely regardless of metabolic status, and accordingly eCAPs have been shown to efficiently kill persisters[10]. An additional detrimental effect of the inability of traditional antibiotics to kill slow-growing cells is the potential for highly tolerant microbes to develop resistance to antibiotics that they are exposed to for long durations. eCAPs kill rapidly, on the seconds to minutes timescale[12, 13]. The rates of resistance to eCAPs are generally low while the mutants that do arise display variable cross-resistance development rates for natural AMPs[14], which is a topic under intense investigation[15, 16]. To attempt to limit cross-resistance, an intriguing strategy is to utilize eCAPs in conjunction with traditional antibiotics, as the distinct mechanisms of action often result in additive or synergistic activity[11, 17]. One major benefit of the dual treatment approach is that traditional antibiotics may be able to kill dispersed or planktonic cells, which are more metabolically active, while eCAPs can kill the remaining slow-growing or persister cells[6]. Due to general membrane disruption activity, eCAPs are often able to kill a broad range of microbial species and strains, even multidrug resistant and select agent pathogens[14, 18, 19]. Considering that infection biofilms are often polymicrobial, treatment with eCAPs is potentially beneficial for their broad-spectrum activity. Furthermore, AMPs have been shown to neutralize fungi, parasites, and even viruses[10], potentially also via membrane interactions. Polymicrobial infections can modulate antimicrobial resistance[5], and viral bacterial co-infections of the respiratory tract have been shown to increase bacterial Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
Melvin et al.
Page 3
Author Manuscript
resistance to frontline traditional antibiotics[20]. However, we have recently used eCAPs to concurrently disrupt extraordinarily resistant Pseudomonas aeruginosa biofilms and inactivate respiratory syncytial virus[13], revealing the potential for eCAPs to simultaneously treat infections with pathogens from multiple kingdoms even when the infection displays synergistic antimicrobial resistance. While the ability of eCAPs to treat established biofilms is of major interest, their use in preventing biofilm biogenesis, either by prophylactic treatment or coating of medical implants[11, 18], is of equal importance. There is current interest in tethering eCAPs to implant materials, with some success in maintaining bactericidal activity[21]. Of note, the ability of eCAPs to strongly interact with bacterial membrane structures has led some to attempt to tether eCAPs with the goal of removing lipopolysaccharide (LPS) from the blood stream via hemoperfusion as a potential treatment for septic shock[22].
Author Manuscript Author Manuscript
Some of the major challenges facing development of eCAPs as therapeutics are regulatory. Due to the fact that eCAPs target membranes, safety is a primary concern. However, recent advances in eCAP design have shown promising advances towards low levels of toxicity to host cells and in small animal models[10, 23]. Bioavailability is also a consideration, as peptides may not be as stable in body, though eCAPs are largely more stable than natural CAPs[10]. Many techniques have been employed to increase stability of eCAPs, including the use of crosslinking, introduction of D-amino acids, and amino acid modification to avoid proteolysis, with some success[10]. Antigenicity of eCAPs is also a possibility, though even during covalent linkage to antigenic carrier proteins very little antibody production has been observed[24]. Additionally, modification of peptides has shown promise for reducing peptide antigenicity[25, 26]. Production costs are often raised as an issue for eCAPs, though production costs are lowering due to new technologies and approaches[27]. Additionally, we have optimized eCAPs to be as short as possible, to contain only 2–3 different amino acids arranged in repeat motifs to facilitate higher yield synthesis chemistry[28].
Author Manuscript
One of the largest challenges for developing eCAPs for use as antibiofilm agents is the relative lack of in vivo models for studying treatment efficacy against biofilm infections. Though small animal models have been used to demonstrate the efficacy of eCAPs for acute infections[23], most small animals are not amenable to the chronic or recurrent infection that is critical to the development of relevant model biofilms[29]. Porcine models are more reflective chronic human infections, though the relative lack of ability to manipulate the host, the inability for some human pathogens to cause infection, and the cost associated with such models make this option inaccessible for many studies. Implanted devices in small animals show reasonably good mimicry of human biofilm infections associated with medical implants, but are not reflective of mucosal or wound biofilms. Additionally, the fact that biofilm infections are commonly polymicrobial indicates that model systems should be amenable multiple pathogens. An alternative initial approach is to use in vitro differentiated cell culture systems. We have developed a model of P. aeruginosa biofilm biogenesis in association with the airway epithelium that mimics biofilm morphology observed in patient samples and displays extreme levels of antibiotic resistance that is further increased during viral co-infection[13], similar to clinical findings[20]. Models such as this are readily
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
Melvin et al.
Page 4
Author Manuscript
amenable to relatively high throughput analysis of treatment and pathogen combinations and may serve as useful, relatively low cost systems to initially evaluate novel eCAP candidates.
Conclusions
Author Manuscript
Recent advances in the design of eCAPs and the technology used to produce them have driven a renaissance of interest in their therapeutic potential. Numerous groups have achieved increased efficacy and reduced toxicity with eCAPs and demonstrated their ability to kill multidrug resistant and select agent pathogens. Perhaps most exciting are the recent reports of eCAPs preventing and disrupting biofilms formed by important human pathogens. Recent use of novel animal and differentiated cell culture models and an increased focus on demonstrating the ability of eCAPs to clear regulatory hurdles promises to bring these compounds to clinic sooner rather than later, potentially providing much needed relief for patients and physicians battling chronic antibiotic resistant infections.
Acknowledgments Funding This paper was supported by funding from the National Institutes of Health (grant numbers: R00HL098342, R01HL123771, T32AI49820, P30DK072506) and the Cystic Fibrosis Foundation (grant numbers: BOMBER14G0, MELVIN15F0).
References
Author Manuscript Author Manuscript
1. Mihai MM, Holban AM, Giurcaneanu C, Popa LG, Oanea RM, Lazar V, Chifiriuc MC, Popa M, Popa MI. Microbial biofilms: impact on the pathogenesis of periodontitis, cystic fibrosis, chronic wounds and medical device-related infections. Curr Top Med Chem. 2015; 15:1552–76. [PubMed: 25877092] 2. Olsen I. Biofilm-specific antibiotic tolerance and resistance. Eur J Clin Microbiol Infect Dis. 2015; 34:877–86. [PubMed: 25630538] 3. Wilkins M, Hall-Stoodley L, Allan RN, Faust SN. New approaches to the treatment of biofilmrelated infections. J Infect. 2014; 69(Suppl 1):S47–52. [PubMed: 25240819] 4. Wolcott R, Costerton JW, Raoult D, Cutler SJ. The polymicrobial nature of biofilm infection. Clin Microbiol Infect. 2013; 19:107–12. [PubMed: 22925473] 5. Birger RB, Kouyos RD, Cohen T, Griffiths EC, Huijben S, Mina MJ, Volkova V, Grenfell B, Metcalf CJ. The potential impact of coinfection on antimicrobial chemotherapy and drug resistance. Trends Microbiol. 2015; 23:537–44. [PubMed: 26028590] 6. Ribeiro SM, Felicio MR, Boas EV, Goncalves S, Costa FF, Samy RP, Santos NC, Franco OL. New frontiers for anti-biofilm drug development. Pharmacol Ther. 2016; 160:133–44. [PubMed: 26896562] 7. Batoni G, Maisetta G, Esin S. Antimicrobial peptides and their interaction with biofilms of medically relevant bacteria. Biochim Biophys Acta. 2016; 1858:1044–60. [PubMed: 26525663] 8. Zhao X, Wu H, Lu H, Li G, Huang Q. LAMP: A Database Linking Antimicrobial Peptides. PLoS One. 2013; 8:e66557. [PubMed: 23825543] 9. Hiemstra PS, Amatngalim GD, van der Does AM, Taube C. Antimicrobial Peptides and Innate Lung Defenses: Role in Infectious and Noninfectious Lung Diseases and Therapeutic Applications. Chest. 2016; 149:545–51. [PubMed: 26502035] 10. Bahar AA, Ren D. Antimicrobial peptides. Pharmaceuticals (Basel). 2013; 6:1543–75. [PubMed: 24287494]
Expert Rev Anti Infect Ther. Author manuscript; available in PMC 2017 November 01.
Melvin et al.
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