Loading...

METHODS OF MICROBIOLOGY AND MOLECULAR BIOLOGY (ISSN:2517-7435)

Antimicrobial Peptides in Drug Development

Daud Faran Asif 

Department of Biochemistry and Biotechnology,  University of Gujrat, Gujrat, Pakistan

CitationCitation COPIED

Asif DF. Antimicrobial peptides in Drug Development. Methods Microbiol Mol Biol. 2020 Apr;3(1):110.

© 2020 Asif DF. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 international License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Antimicrobial peptides also known as HDP host defense peptides, have been considered as source of antibiotics in future due to their broader activities and mechanism of action different than conventional antibiotics. AMP can kill both gram positive and gram negative bacteria as well as fungi and viruses. AMP can also target cancerous cells. Some AMPs also function as immune modulators. They have certain limitations e.g susceptibility to proteases, toxicity and high cost. In order to minimize these limitation certain efforts have been carried. For example to avoid degradation of peptide by proteases peptide mimics or certain unusual amino acids are introduced into peptides. For economical production short peptides have been designed retaining anti microbial activities.

Keywords

Antimicrobial peptides (AMPs); Antibiotics; SAR structure activity relationship

Introduction

AMPs are polypeptides produced by multicellular organisms. These are produced to protect the host from activity of certain microbes. As AMP provide defense against microbes they are also known as host defense peptides HDPs. AMPs play key role in developing innate immunity [1-4]. AMPs re composed of linear amino acid chain ranging in length up to 50 aminoacids. AMPs have typically cationic amphipathic properties. One side of AMPs is positively charged and other side is hydrophobic in nature. Arginine along with lysine provides positive charge to AMPs [1,2,5,6].

AMPs show activity against a broader range of micro organisms including Gram positive and Gram negative bacteria as well as fungi and viruses [1]. AMPs are effective for bacteria that are resistant to traditional antibiotics i.e MDR bacteria multidrug resistance bacteria [7-9]. Moreover there is less chance that bacteria would get resistant to AMPs. Resistance in bacteria against antibiotics is achieved by inhibition of drug target interaction and modification of drug binding site in target proteins. Genetic pattern of microbes can also be altered by a complex system called sensor transducer response. For example bacteria can alter their gene expression in the presence of AMPs [10]. AMPs possess low propensity to develop resistance due to their unique mode of action. AMPs act directly act on membrane of pathogen. AMPs develope peptide-lipid interactions by their positive side and hydrophobic side [3,11-16].

This characteristic mechanism called membranolytic effect enable AMPS to avoid common resistance mechanism of general antibiotics. AMPs thus receiving great attention as alternative of conventional antiobiotics.

Structure

AMPs are divided into sub groups on the basis of their amino acid in peptide and their structure. These are usually consist of 12-50 amino acid. These AMPs include arginine and lysine as positive charged amino acids and a large proportion of hydrophobic residues. AMPs show antimicrobial activity by targeting membranes or action to cytoplasm (Table 1).

Immunomodulation

In addition to kill bacteria directly AMPs induce innate immunity. It may involve

  • Clearance of infection
  • Ability to change host-gene expression
  • Inhibiting production of cytokine induced by lipopolysaccharide.
  • Modulating the response shown by dendritic cells and the cells of adaptive immune response

Animal models indicate that host defence peptides play key role in clearance and prevention of infection (Figure 1).

Structure activity relationships (SAR) of AMPs

AMPs are of four types β-sheet, extended, α-helical and loop peptides [1-3,6]. The α-helical are well studied in structure activity relationships. It includes magainin, cecropin, and pexiganan. This group of peptides usually loss its structure in aqueous medium. These AMPs form barrel like bundles in bacterial cell membranes and these transmembrane aggregate line amphipathic pores. Many α-helical AMPs can target bacterial membranes by forming carpet like aggregates of peptides. Some α-helical form toroidal pores to distort the bacterial membranes [1-7,9,11].

The β-sheet AMPS are stabilized by S-S bridges and usually form rigid structure. These include β-defensins, and protegrin. Most of β-sheet AMPs disrupt bacterial membranes by tilting or inserting into lipid bilayer to form toroidal pores and hydrophobic regions of peptides interact with polar head groups of membranes [3].

The extended AMPs contain some aminoacids in more amount such as proline, arginine, tryptophan and histidine. Indolicidin is a tryptophan and proline rich peptide that falls in extended type category. Bac5 and Bac7 are proline and arginine rich peptides these peptides target the cell membrane [17,18]. Many extended AMPs are not effective against membranes of microbes but they can show antimicrobial activity by interacting with bacterial proteins inside [3]. Some extended AMPs e.g indolicidin show membrane activity and act directly on membranes of pathogens. 

The loop AMPs form a loop formation with the help of I sulphide bonds. For example bactenecin [17,18].

Understanding structure activity relationships SAR of AMPs is crucial for development of novel peptides that have better properties than natural peptides.Pexiganan also called as MSI-78 , a synthetic derivative of magainin is one of best understood peptide in terms of drug development [19,20]. Pexiganan is currently on clinical trials for the treatment of diabetic foot ulcer infections [4,20,21]. 3D structure of pexiganan determined by NMR (nuclear magnetic resonance) revealed that peptide forms dimeric anti parallel structure in the presence of membrane mimetics [22-30]. Structure of peptide and the orientation of peptide in membrane plays key role in understanding the mode of action of peptide [22].

Antimicrobial peptides as therapeutic agents

AMPs got attention to study as new generation antibiotics because of their broad spectrum activity against microbes and MDR bacteria. Since the isolation of megainins from frog skin their have been many attempts to synthesis antibiotics from natural AMPs [31]. Despite the efforts currently there is no AMP agent approved by FDA [4,32,33-37]. Although AMPs have certain advantages over conventional antibiotics they have certain limitations. The natural AMPs are liable depending upon surrounding conditions such as presence of proteases. change in pH and potential toxicity [2]. Moreover high cost of peptide production make it less economical. In order to overcome these problems many methods have been presented. For instance introduction on unusual amino acids in peptides to prevent its degradation by proteases. Stability of peptides can also be improved by introduction of amide or acetyl groups at terminal regions of peptides. Use of efficient drug delivery vehicles such a liposomes can reduce the potential toxicity [38,39]. High production cost is one of major issues for peptide production roughly cost estimated is $50-$400 per 1 gram of amino acid [7]. In order to sort the cost issue short peptides can by synthesized that retain the potential to act as antimicrobial agent. There have been several examples of peptide engineering to reduce the peptide size with retained anti microbial activity. For example thegaegurin 5 (GGN5N11) can retain the potential to kill microbes just by a single substitution of tryptophanylat amphipathic [29,30].

Classification of AMPs

Based on mode of action and target AMPs can be divided into following catagories (Table 2) 

Important Physiochemical Properties of AMPs

Following are important properties of AMPs

  • Solubility
  • Hydrophobicity
  • Amphipathicity
  • Net Charge
  • Helicity
  • Length

AMP Modifications

Most of AMPs are produced directly in their active form in order to enhance their stability certain modifications can be done. These are as follow

  • Modification of AMPs with Covalent Bonds: Covalent bond modification can effect the structure and funuction of AMP. Even a small change in S-S bond can influence the antimicrobial activity of AMP.
  • Modification of AMPs by Changing Amino Acid Content: By changing the amino acid content we can change the ability of AMP to interact with microbial cell wall.
  • Modification of AMPs by Amidation: Kim et al. in 2011 developed this method of introducing unusual group into AMP to enhance its antimicrobial activity.
  • Modification of AMPs with Unnatural Amino Acids: Introduction of unnatural amino acids in AMP can provide better folding properties.
  • Modification of AMPs with Computer-Assisted Methods: Various bioinformatics tools can used for this purpose.

Small Peptides in Drug Development

hLF1-11 (Human Lactoferrin 1-11): Lactoferin is an iron binding glycoprotein that plays a role in innate defense system. LF can not only bind Fe3+ but target the bacterial membrane that enable LF to act against microbes [40-43]. The synthetic hLF-11 (GRRRRSVQWCA) is LF derivative. It shows activity against microbes including Gram positive as well as Gram negative bacteria. The synthetic peptide is also effective against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii strains [43–45]. Its also effective against fluconazoleresistant C. albicans when used in combination with fluconazole [46].

3D structure of several LF derived peptides in membrane mimetic environment have been studied by NMP spectroscopy [47-50]. The confirmation of hLF1-11 varied depending upon environmental conditions [43]. Molecular dynamics MD results suggested that this peptide should be categorized as loop peptide.

The safety of hFL1-11 have been tested in haemato poietic stem cell transplantation (HSCT) recipients and healthy persons [43,51]. Intra venous injection of hFL1-11 were safe and tolerable in both healthy and HSCT recipient. While some adverse events were reported.

(CKPV)2 Peptide (α-MSH Derivative, also Named CZEN-002): The α-melanocyte stimulating hormone (SYSMEHFRWGKPV) is a neuro peptide showing antimicrobial and anti inflammatory activity [52-54]. It’s studied that α-MSH peptides are effective against Candida albicans and their mode of action is different from other natural AMPs. This activity of peptide is caused by increased cyclic adenosine monophosphate (cAMP) in the C. albicans cells [55], whereas most of AMPs kills microbes by direct interaction with their membranes. It is suggested that cAMP-mediated modulation is essential for gene expression in C. albicans and the cAMP activating effect of α-MSH interferes with the cAMP-mediated signaling pathway [56,57].

The C-terminal tripeptide (α-MSH11-13; KPV) also has antiinflammatory and antimicrobial activities same as shown by α-MSH [55,58]. The synthetic peptide (CKPV)2, also called as CZEN002, was designed by modifying KPV (α-MSH11-13) peptide. This peptide is classified as loop peptide, it is a dimeric octamer consisting of two KPV units connected by cysteine-cysteine linker. The CKPV2 peptide show effective candidacidal activity against C. krusei and C. glabrata that are MDR strains [59-62]. This peptide also exerts anti inflammatory as well as candidacidal effect [60-62]. CKPV2 due to smaller size and simple sequence is regarded as effective agent for development of candidacidal and anti-inflammatory drugs. CKPV2 is being currently studied in clinical trials for treatment of vulvovaginal candidiasis [4]. CKPV2 adopts a symmetric dimer with an extended backbone structure, which resembles the α-MSH peptide [63]. The overall conformation of CKPV2 showed a β-turn like fold, which may be related to the higher activity of CKPV2 than KPV monomer [59].

Table 1: AMPs show antimicrobial activity by targeting membranes or action to cytoplasm

Table 2: Differentiation of peptides based on mode of action and target AMPs

Figure 1: Antimicrobial peptides play a central role in innate and adaptive immunity. A given stimulus by bacteria leads to the release of constitutively expressed AMPs in different cells (here: epidermis). AMPs are released by neutrophils and will activate and recruit macrophages, monocytes, dendritic cells, and T-cells. A direct anti-endotoxin effect of AMPs may decrease the activation of immune cells and thus lead to a decrease in pro-inflammatory cytokine release.

Conclusions

The serious problem caused by MDR bacteria can be overcome by use of these AMPs. These are new generation antiobiotics. There are certain problems with AMPs that can be covered by modification of peptides.

References

  1. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature. 2002 Jan;415:389-395.
  2. Hancock RE, Sahl HG. Antimicrobial and host-defense peptides asnew anti-infective therapeutic strategies. Nat Biotechnol. 2006Dec;24(12):1551-1557.
  3. Nguyen LT, Haney EF, Vogel HJ. The expanding scope of antimicrobial peptide structures and their modes of action. Trends Biotechnol. 2011 Sep;29(9):464-472.
  4. Fjell CD, Hiss JA, Hancock RE, Schneider G. Designing antimicrobialpeptides:Form follows function. Nat Rev Drug Discov. 2011 Dec16;11(1):37-51.
  5. Hancock RE, Peptide antibiotics. Lancet 1997 Feb;349(9049):418-422.
  6. Hancock RE, Lehrer R. Cationic peptides:A new source ofantibiotics. Trends Biotechnol. 1998 Feb;16(2):82-88.
  7. Marr AK, Gooderham WJ, Hancock RE. Antibacterial peptidesfor therapeutic use:Obstacles and realistic outlook. Curr. Opin.Pharmacol. 2006 Oct;6(5):468-472.
  8. Mygind PH, Fischer RL, Schnorr KM, Hansen MT, Sonksen CP, et al.Plectasin is a peptide antibiotic with therapeutic potential from asaprophytic fungus. Nature. 2005 Oct;437:975-980.
  9. van’t Hof W, Veerman EC, Helmerhorst EJ, Amerongen AV.Antimicrobial peptides:Properties and applicability. Biol Chem.2001 May;382(4): 597-619.
  10. Wright GD. Bacterial resistance to antibiotics:Enzymatic degradation and modification. Adv Drug Deliv Rev. 2005 Jul;57(10):1451-1470.
  11. Teixeira V, Feio MJ, Bastos M. Role of lipids in the interaction ofantimicrobial peptides with membranes. Prog. Lipid Res. 2012Apr;51(2):149-177.
  12. Huang Y, Huang J, Chen Y. Alpha-helical cationic antimicrobialpeptides:Relationships of structure and function. Protein Cell2010 Feb;1(2):143-152.
  13. Hancock RE, Chapple DS. Peptide antibiotics. Antimicrob. AgentsChemother. 1999 Jun;43(6):1317-1323.
  14. Shai Y, Oren Z. From “carpet” mechanism to de-novo designeddiastereomeric cell-selective antimicrobial peptides. Peptides.2001 Oct;22(10):1629-1641.
  15. Rotem S, Mor A. Antimicrobial peptide mimics for improvedtherapeutic properties. Biochim Biophys Acta. 2009 Aug,1788(8):1582-1592.
  16. Hancock RE. The bacterial outer membrane as a drug barrier.Trends Microbiol. 1997 Jan;5(1):37-42.
  17. Falla TJ, Karunaratne DN, Hancock RE. Mode of action ofthe antimicrobial peptide indolicidin. J Biol Chem. 1996Aug;271(32):19298-19303.
  18. Frank RW, Gennaro R, Schneider K, Przybylski M, RomeoD. Amino acid sequences of two proline-rich bactenecins.Antimicrobial peptides of bovine neutrophils. J Biol Chem. 1990Nov;265:18871-18874.
  19. Bessalle R, Haas H, Goria A, Shalit I, Fridkin M. Augmentation ofthe antibacterial activity of magainin by positive-charge chainextension. Antimicrob. Agents Chemother. 1992 Feb;36(2):313-317.
  20. Gottler LM, Ramamoorthy A. Structure, Membrane orientation,Mechanism, And function of pexiganan—a highly potentantimicrobial peptide designed from magainin. Biochim. Biophys.Acta 2009 Aug;1788(8):1680-1686.
  21. Maloy WL, Kari UP. Structure-activity studies on magainins andother host defense peptides. Biopolymers 1995;37(2):105-122.
  22. Ramamoorthy A, Thennarasu S, Lee DK, Tan A Maloy L. Solidstate NMR investigation of the membrane-disrupting mechanismof antimicrobial peptides MSI-78 and MSI-594 derived frommagainin 2 and melittin. Biophys J. 2006 Jul;91(1):206-216.
  23. Porcelli F, Buck-Koehntop BA, Thennarasu S, RamamoorthyA, Veglia G. Structures of the dimeric and monomeric variantsof magainin antimicrobial peptides (MSI-78 and MSI-594)in micelles and bilayers, determined by NMR spectroscopy.Biochemistry. 2006 Apr;45(18):5793-5799.
  24. Blondelle SE, Houghten RA. Design of model amphipathicpeptides having potent antimicrobial activities. Biochemistry.1992 Dec;31(50):12688-12694.
  25. Beven L, Castano S, Dufourcq J, Wieslander A, Wroblewski H. Theantibiotic activity of cationic linear amphipathic peptides:Lessonsfrom the action of leucine/lysine copolymers on bacteria of theclass Mollicutes. Eur J Biochem. 2003 Apr;270:2207-2217.
  26. Kang SJ, Won HS, Choi WS, Lee BJ. De novo generation ofantimicrobial LK peptides with a single tryptophan at the criticalamphipathic interface. J Pept Sci. 2009 Sep;15(9):583-588.
  27. Won HS, Kang SJ, Lee BJ. Action mechanism and structural requirements of the antimicrobial peptides, gaegurins. Biochim. Biophys Acta. 2009 Aug;1788(8):1620-1629.
  28. Park SH, Kim HE, Kim CM, Yun HJ, Choi EC, Lee BJ. Role of proline,Cysteine and a disulphide bridge in the structure and activityof the anti-microbial peptide gaegurin 5. Biochem J. 2002Nov;368(Pt 1):171-182.
  29. Won HS, Jung SJ, Kim HE, Seo MD, Lee BJ. Systematic peptideengineering and structural characterization to search for theshortest antimicrobial peptide analogue of gaegurin 5. J BiolChem. 2004. 279;14784-14791.
  30. Won HS, Seo MD, Jung SJ, Lee SJ, Kang SJ, Son WS, Kim HJ, ParkTK, Park SJ, Lee BJ. Structural determinants for the membraneinteraction of novel bioactive undecapeptides derived fromgaegurin 5. J Med Chem. 2006 Jul;49(16):4886-4895.
  31. Zasloff M. Magainins, A class of antimicrobial peptides fromXenopus skin:Isolation, Characterization of two active forms, Andpartial cDNA sequence of a precursor. Proc Natl Acad Sci USA1987 Aug;84(15):5449-5453.
  32. Gordon YJ, Romanowski EG, McDermott AM. A review ofantimicrobial peptides and their therapeutic potential as antiinfective drugs. Curr Eye Res. 2005 Jul;30(7):505-515.
  33. Oyston PC, Fox MA, Richards SJ, Clark GC. Novel peptidetherapeutics for treatment of infections. J Med Microbial. 2009Aug;58(8):977-987.
  34. Rozek A, Powers JP, Friedrich CL, Hancock RE. Structure-baseddesign of an indolicidin peptide analogue with increased proteasestability. Biochemistry. 2003 Nov;42(48):14130-14138.
  35. Lee IH, Cho Y, Lehrer RI. Effects of pH and salinity on theantimicrobial properties of clavanins. Infect. Immun. 1997Jul;65(7):2898-2903.
  36. John H, Maronde E, Forssmann WG, Meyer M, Adermann K.N-terminal acetylation protects glucagon-like peptide GLP-1-(7-34)-amide from DPP-IV-mediated degradation retaining cAMPand insulin-releasing capacity. Eur J Med Res. 2008 Feb;13(2):73-78.
  37. McPhee JB, Scott MG, Hancock RE. Design of host defencepeptides for antimicrobial and immunity enhancing activities.Comb. Chem High. Throughput Screen 2005 May;8(3):257-272.
  38. Khaksa G, D’Souza R, Lewis S, Udupa N. Pharmacokinetic studyof niosome encapsulated insulin. Indian J. Exp. Biol. 2000Sep;38:901-905.
  39. Samad A, Sultana Y, Aqil M. Liposomal drug delivery systems:Anupdate review. Curr Drug Deliv. 2007Oct;4(4):297-305.
  40. Sanchez L, Calvo M, Brock JH. Biological role of lactoferrin. ArchDis Child. 1992 May;67(5):657-661.
  41. Arnold RR, Cole MF, McGhee JR. A bactericidal effect for humanlactoferrin. Science. 1977 Jul;197(4300):263-265.
  42. Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K,Tomita M. Identification of the bactericidal domain of lactoferrin.Biochim Biophys Acta. 1992 May;1121(1-2):130-136.
  43. Brouwer CP, Rahman M, Welling MM. Discovery and developmentof a synthetic peptide derived from lactoferrin for clinical use.Peptides. 2011 Sep;32(9):1953-1963.
  44. Dijkshoorn L, Brouwer CP, Bogaards SJ, Nemec A, van den BroekPJ, Nibbering PH. The synthetic N-terminal peptide of humanlactoferrin, hLF(1-11), Is highly effective against experimentalinfection caused by multidrug-resistant Acinetobacter baumannii.Antimicrob Agents Chemother. 2004 Dec;48(12):4919-4921.
  45. Lupetti A, Paulusma-Annema A, Welling MM, Senesi S, van DisselJT, Nibbering PH. Candidacidal activities of human lactoferrinpeptides derived from the N terminus. Antimicrob. AgentsChemother. 2000 Dec;44(12):3257-3263.
  46. Lupetti A, Paulusma-Annema A, Welling MM, Dogterom-BalleringH, Brouwer CP, Senesi S, Van Dissel JT, Nibbering, PH. Synergisticactivity of the N-terminal peptide of human lactoferrin andfluconazole against Candida species. Antimicrob AgentsChemother. 2003 Jan;47(1):262-267.
  47. Hwang PM, Zhou N, Shan X, Arrowsmith CH, Vogel HJ. Threedimensional solution structure of lactoferricin B, An antimicrobialpeptide derived from bovine lactoferrin. Biochemistry. 1998Mar;37(12):4288-4298.
  48. Japelj B, Pristovsek P, Majerle A, Jerala R. Structural originof endotoxin neutralization and antimicrobial activity of alactoferrin-based peptide. J Biol Chem. 2005;280:16955-16961.
  49. Japelj B, Zorko M, Majerle A, Pristovsek P, Sanchez-Gomez S,et al. The acyl group as the central element of the structuralorganization of antimicrobial lipopeptide. J Am Chem Soc. 2007Jan;129(5):1022-1023.
  50. Nguyen LT, Schibli DJ, Vogel HJ. Structural studies and modelmembrane interactions of two peptides derived from bovinelactoferricin. J Pept Sci. 2005 Dec;11:379-389.
  51. Velden WJ, van Iersel TM, Blijlevens NM, Donnelly JP. Safety andtolerability of the antimicrobial peptide human lactoferrin 1-11(hLF1-11). BMC Med. 2009;7:44.
  52. Rajora N, Ceriani G, Catania A, Star RA, Murphy MT, et al. alphaMSH production, Receptors, And influence on neopterin ina human monocyte/macrophage cell line. J Leuk Biol. 1996Feb;59(2):248-253.
  53. Catania A, Gatti S, Colombo G, Lipton, JM. Targeting melanocortinreceptors as a novel strategy to control inflammation. PharmacolRev. 2004 Mar;56(1):1-29.
  54. Catania A, Colombo G, Rossi C, Carlin A, Sordi A, Lonati C, TurcattiF, Leonardi P, Grieco P, Gatti S. Antimicrobial properties of alphaMSH and related synthetic melanocortins. Scientific WorldJournal 2006;6:1241-1246.
  55. Cutuli M, Cristiani S, Lipton JM, Catania A. Antimicrobial effects ofalpha-MSH peptides. J Leuk Biol. 2000;67:233-239.
  56. Harcus D, Nantel A, Marcil A, Rigby T, Whiteway M. Transcriptionprofiling of cyclic AMP signaling in Candida albicans. Mol BiolCell. 2004;15:4490-4499.
  57. Bhattacharya A, Datta A. Effect of cyclic AMP on RNA and proteinsynthesis in Candida albicans. Biochem. Biophys. Res. Commun.1977;77:1483-1444.
  58. Getting SJ. Melanocortin peptides and their receptors:New targets for anti-inflammatory therapy. Trends Pharmacol. Sci. 2002;23:447-479.
  59. Catania A, Grieco P, Randazzo A, Novellino E, Gatti S, Rossi C,Colombo G, Lipton JM. Three-dimensional structure of thealpha-MSH-derived candidacidal peptide [Ac-CKPV]2. J Pept Res.2005;66:19-26.
  60. Gatti S, Carlin A, Sordi A, Leonardi P, Colombo G, Fassati LR,Lipton JM, Catania A. Inhibitory effects of the peptide (CKPV)2 onendotoxin-induced host reactions. J Surg Res. 2006;131:209-214.
  61. Sanglard D, Odds FC. Resistance of Candida species to antifungalagents:Molecular mechanisms and clinical consequences. LancetInfect. Dis. 2002;2:73-85.
  62. Capsoni F, Ongari A, Colombo G, Turcatti F, Catania A. Thesynthetic melanocortin (CKPV)2 exerts broad anti-inflammatoryeffects in human neutrophils. Peptides. 2007;28:2016-2022.
  63. Prabhu NV, Perkyns JS, Pettitt BM. Modeling of alpha-MSHconformations with implicit solvent. J Pept Res. 1999;54:394-407.
  64. Oppenheim FG, Xu T, McMillian FM, Levitz SM, Diamond RD,Offner GD, Troxler RF. Histatins, A novel family of histidine-richproteins in human parotid secretion. Isolation, Characterization,Primary structure, And fungistatic effects on Candida albicans. JBiol Chem. 1988;263:7472-7477.
  65. Raj PA, Edgerton M, Levine MJ. Salivary histatin 5:Dependence ofsequence, Chain length, And helical conformation for candidacidalactivity. J Biol Chem. 1990;265:3898-3905.
  66. Rothstein DM, Spacciapoli P, Tran LT, Xu T, Roberts FD, Dalla SerraM, Buxton DK, Oppenheim FG, Friden P. Anticandida activityis retained in P-113, A 12-amino-acid fragment of histatin 5.Antimicrob. Agents Chemother. 2001 May;45(5):1367-1373.
  67. Raj PA, Soni SD, Levine MJ. Membrane-induced helicalconformation of an active candidacidal fragment of salivaryhistatins. J. Biol. Chem. 1994 Apr;269(13):9610-9619.
  68. Raj PA, Marcus, E, Sukumaran DK. Structure of human salivaryhistatin 5 in aqueous and nonaqueous solutions. Biopolymers.1998 Dec;45:51-67.
  69. Melino S, Rufini S, Sette M, Morero R, Grottesi A, Paci M, PetruzzelliR. Zn(2+) ions selectively induce antimicrobial salivary peptidehistatin-5 to fuse negatively charged vesicles. Identification andcharacterization of a zinc-binding motif present in the functionaldomain. Biochemistry. 1999 Jul;38(30):9626-9633.
  70. Iovino M, Falconi M, Marcellini A, Desideri A. Molecular dynamicssimulation of the antimicrobial salivary peptide histatin-5 inwater and in trifluoroethanol:A microscopic description of thewater destructuring effect. J Pept Res. 2001;58:45-55.
  71. Porciatti E, Milenkovic M, Gaggelli E, Valensin G, KozlowskiH,Kamysz W, Valensin D. Structural characterization andantimicrobial activity of the Zn(II) complex with P113 (demegen),a derivative of histatin 5. Inorg Chem. 2010 Sep;49(19):8690-8698.