FUTURE PERSPECTIVE

New drugs must meet many criteria such as efficacy, safety, stability and low cost before it can be used clinically. Despite the encouraging outcomes obtained through the studies in vitro, there is still a long way to go towards the ultimate goal of using MUC7 peptides as therapeutic agents for the control of oral infectious diseases.

The outcome of this study leads to the following recommendations for future research.

1) Development of a ȕ-peptide by incorporating synthetic ȕ-substituted amino acids into MUC7 12-mer. MUC7 12-mer D exhibits potent antifungal activities and is

not susceptible to proteases. Unfortunately, this peptide is very costly so that the high cost may limit its therapeutic application. ȕ-peptides contain residues that form a stable helical structure and their unnatural backbone makes them resistant to proteases. ȕ-peptides that mimic natural peptide-based antibiotics, such as magainins, were found to have similar antibacterial activity [19]. Recently, ȕ-peptides were reported to have a potent antifungal activity against planktonic and biofilm C. albicans cells [20]. I hypothesize that ȕ-MUC7 12-mer may exhibit potent antifungal activity against planktonic and biofilm C. albicans cells in saliva and is therefore expected to be a new drug for the treatment and prevention of systemic C. albicans infections.

2) Study the effect of iron and iron chelators on antifungal activity of MUC7 12-mer in saliva. Iron is an important ion in human whole saliva. It also influences the growth

of C. albicans. Iron chelation (deferoxamine) was found to be a promising novel therapeutic strategy for refractory mucormycosis infections [21]. It is interesting to evaluate the efficacy of a combination of MUC7 12-mer and iron-chelating agents.

3) Study the effect of MUC7 12-mer and its derivatives on multi-culture biofilm.

There is a need to set up multi-culture biofilms which include S. mutans, S. gordonii, Fusobacteria and other indigenous bacteria of dental plaque. The rationale is that the multi-culture biofilm is closer to in vivo dental biofilm compared to the single-culture biofilm. Based on the observation that MUC7 12-mer has preference activity against S.

mutans, MUC7 12-mer could be expected to inhibit the formation of multi-culture

biofilm. Moreover, it will be of interests to examine the effects of the combination of MUC712-mer and EDTA on formation or eradication of the biofilm.

4) In vivo study with experimental model. Since the results of previous in vivo

studies were not conclusive [22, 23], further in vivo investigations are recommended on antimicrobial efficacy of MUC7 peptides alone or in combination with other agents against fungal and bacterial infections in animal models. Antifungal activity of MUC7

peptide (16-mer) was found in a murine model of vaginal candidiasis [22]. However, the model of vaginal candidiasis is not applicable to oral candidiasis because of the low pH and high ionic strength in the vaginal environment. An in vivo study with MUC7 12-mer peptide in a model of murine oral candidiasis showed antifungal activity, but consistent results were not obtained in the second trial [23]. These results urge more investigations by making use of modern animal models [24, 25]. Up to now, there are no reports on in vivo antibacterial activity of MUC7 12-mer against mutans streptococci.

I hypothesize that, based on the results of in vitro studies described above, MUC7 12-mer in combination with EDTA and MUC7 12-mer-D isomer or ȕ-MUC7 12-mer will exert effective therapeutic activity in vivo, since these drugs are relative insensitive to proteolysis in saliva.

REFERENCES

1. Hancock RE, Sahl HG: Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 2006, 24(12):1551-1557.

2. Bobek LA, Situ H: MUC7 20-Mer: investigation of antimicrobial activity, secondary structure, and possible mechanism of antifungal action. Antimicrobial agents and

chemotherapy 2003, 47(2):643-652.

3. Sahasrabudhe KS, Kimball JR, Morton TH, Weinberg A, Dale BA: Expression of the antimicrobial peptide, human beta-defensin 1, in duct cells of minor salivary glands and detection in saliva. J Dent Res 2000, 79(9):1669-1674.

4. Sitaram N, Nagaraj R: Interaction of antimicrobial peptides with biological and model membranes: structural and charge requirements for activity. Biochimica et biophysica

acta 1999, 1462(1-2):29-54.

5. Dathe M, Schumann M, Wieprecht T, Winkler A, Beyermann M, Krause E, Matsuzaki K, Murase O, Bienert M: Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 1996, 35(38):12612-12622.

6. Kiyota T, Lee S, Sugihara G: Design and synthesis of amphiphilic alpha-helical model peptides with systematically varied hydrophobic-hydrophilic balance and their interaction with lipid- and bio-membranes. Biochemistry 1996, 35(40):13196-13204. 7. Helmerhorst EJ, Flora B, Troxler RF, Oppenheim FG: Dialysis unmasks the fungicidal

properties of glandular salivary secretions. Infect Immun 2004, 72(5):2703-2709. 8. Helmerhorst EJ, Breeuwer P, van't Hof W, Walgreen-Weterings E, Oomen LC, Veerman EC,

Amerongen AV, Abee T: The cellular target of histatin 5 on Candida albicans is the energized mitochondrion. J Biol Chem 1999, 274(11):7286-7291.

9. Dong J, Vylkova S, Li XS, Edgerton M: Calcium blocks fungicidal activity of human salivary histatin 5 through disruption of binding with Candida albicans. J Dent Res 2003, 82(9):748-752.

10. da Silva A, Jr., Teschke O: Effects of the antimicrobial peptide PGLa on live Escherichia coli. Biochimica et biophysica acta 2003, 1643(1-3):95-103.

11. Thwaite JE, Hibbs S, Titball RW, Atkins TP: Proteolytic degradation of human antimicrobial peptide LL-37 by Bacillus anthracis may contribute to virulence. Antimicrobial agents and

chemotherapy 2006, 50(7):2316-2322.

12. Kauffman CA, Carver PL: Antifungal agents in the 1990s. Current status and future developments. Drugs 1997, 53(4):539-549.

13. van't Hof W, Reijnders IM, Helmerhorst EJ, Walgreen-Weterings E, Simoons-Smit IM, Veerman EC, Amerongen AV: Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity. Antonie Van Leeuwenhoek 2000, 78(2):163-169.

14. Lupetti A, Paulusma-Annema A, Welling MM, Dogterom-Ballering H, Brouwer CP, Senesi S, Van Dissel JT, Nibbering PH: Synergistic activity of the N-terminal peptide of human

lactoferrin and fluconazole against Candida species. Antimicrobial agents and

chemotherapy 2003, 47(1):262-267.

15. Eliopoulos GM, Moellering TC: Antimicrobial combinations. In: Antibiotics in laboratory

medicine. Edited by Lorian V, 4th edn. Baltimore, USA: Williams & Wilkins; 1996: 330-396.

16. Wilson M: Susceptibility of oral bacterial biofilms to antimicrobial agents. J Med Microbiol 1996, 44(2):79-87.

17. Glukhov E, Stark M, Burrows LL, Deber CM: Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes. J Biol Chem 2005,

280(40):33960-33967.

18. Gobbo M, Biondi L, Filira F, Gennaro R, Benincasa M, Scolaro B, Rocchi R: Antimicrobial peptides: synthesis and antibacterial activity of linear and cyclic drosocin and apidaecin 1b analogues. J Med Chem 2002, 45(20):4494-4504.

19. Porter EA, Weisblum B, Gellman SH: Mimicry of host-defense peptides by unnatural oligomers: antimicrobial beta-peptides. J Am Chem Soc 2002, 124(25):7324-7330. 20. Karlsson AJ, Pomerantz WC, Neilsen KJ, Gellman SH, Palecek SP: Design of ȕ-peptides

that inhibit Candida albicans planktonic growth and biofilm formation. In: 107th General

meeting of ASM: May 21-25 2007; Toronto; 2007.

21. Ibrahim AS, Edwards JE, Jr., Fu Y, Spellberg B: Deferiprone iron chelation as a novel therapy for experimental mucormycosis. J Antimicrob Chemother 2006, 58(5):1070-1073. 22. Intini G, Aguirre A, Bobek LA: Efficacy of human salivary mucin MUC7-derived peptide and

histatin 5 in a murine model of candidiasis. Int J Antimicrob Agents 2003, 22(6):594-600. 23. Muralidharan R, Bobek LA: Antifungal activity of human salivary mucin-derived peptide,

MUC7 12-mer, in a murine model of oral candidiasis. J Pept Res 2005, 66 Suppl 1:82-89. 24. Kamai Y, Kubota M, Hosokawa T, Fukuoka T, Filler SG: New model of oropharyngeal

candidiasis in mice. Antimicrobial agents and chemotherapy 2001, 45(11):3195-3197. 25. Takakura N, Sato Y, Ishibashi H, Oshima H, Uchida K, Yamaguchi H, Abe S: A novel murine

model of oral candidiasis with local symptoms characteristic of oral thrush. Microbiol

Samenvatting, discussie, conclusie en