Protein and Peptide Letters, Vol. 9, No. 1, pp. 53-57, 2002 Bentham Science Publishers Ltd. 0929-8665/02 $ 35.00 + .00 SYNTHETIC ANTIBIOTIC PEPTIDES DATABASE David Wade1* and Jukka Englund2 1 Peptide Laboratory, Haartman Institute, and 2 Research and Development Unit, National Library of Health Sciences, FIN-00014 Helsinki University, Finland A computerized database for synthetic antibiotic peptides, the SAPD, has been created and is available on the Internet at web address, http://oma.terkko.helsinki.fi:8080/~SAPD. The SAPD is modelled on two pre-existing computer databases for naturally occurring peptide antibiotics, the Antimicrobial Sequences Database and the Peptaibol Database, and it will contain both chemical and biological information on all published synthetic antibiotic peptides. KEYWORDS: peptide antibiotics, synthetic peptides, computer database, world wide web, internet INTRODUCTION During the past two decades, there has developed a serious public health problem caused by antibiotic resistant microorganisms, and this has necessitated a need for the development of new antibiotics. Fortuitously, during the same period, an entirely new category of antibiotics was discovered, the gene-encoded, peptide antibiotics of the animal, plant, and bacterial kingdoms . These new peptide antibiotics are thought to represent a previously unrecognised form of innate immunity, and there is extensive interest in them as a potential partial solution to the need for new antibiotics. The numbers of new peptide antibiotics being discovered, and scientific articles about them, has been growing almost exponentially (Figure 1). This has created a need for a centralized catalog, or database, of information about these new compounds. One such database has been created for naturally occurring peptide antibiotics, the Antimicrobial Sequences Database (AMSDb), and it is available on the Internet (http://www.bbcm.univ.trieste.it/~tossi/pag1.htm) . At the time of this writing, the AMSDb contains over 600 peptide structures for gene-encoded peptides from animal and plant sources; bacterial sources have been excluded. Hopefully, databases will eventually be developed for the new antibiotic peptides from bacterial sources, or the AMSDb will be expanded to include these other antibiotic peptides. The AMSDb contains both chemical and biological information about each peptide, plus a list of literature references. There is also another related database available on the Internet, the Peptaibol Database (http://www.cryst.bbk.ac.uk/peptaibol/welcome.html) and it currently contains about 242 peptaibol structures . The combined number of naturally occurring antibiotics in these two databases is quite substantial. Due to the availability of solid phase synthesis and combinatorial chemistry technologies, the number of possible synthetic variations of the naturally occurring peptide antibiotic structures is in the millions. Thousands of new peptides have already been synthesized based on the naturally occurring structures, and some of these peptides have shown therapeutic potential and are currently 53 54 in clinical trials. Figure 1 gives only a rough indication of the scope of this new field because many of the articles about new synthetic antibiotic peptides contain information on several peptides. For example, a recent paper about synthetic derivatives of a cecropin-melittin hybrid peptide antibiotic, CA(1-7)M(2-9)NH2, contained information on 100 derivative peptides . The discovery of new peptide antibiotics appears to be limited only by the speed with which these new synthetic peptides can be tested for biological activities. The rapid pace of this research activity has created a need for a centralized catalog of chemical and biological information about the new synthetic peptides, and it was in response to this need that the Synthetic Antibiotic Peptides Database (SAPD) was created. It is expected that the information available on the SAPD, the AMSDb, and Peptaibol databases will help to accelerate the pace of antibiotics research by preventing the duplication of research efforts and stimulating new research ideas. 100 90 80 Number of articles 70 60 50 40 30 20 10 0 1968 1972 1976 1980 1984 1988 1992 1996 2000 Year Figure 1. Increase in the number of articles published on antibiotic peptides during the past two decades. Data was obtained by doing a search of Medline from 1966 to 2000 using the search terms, “antibacterial-, antibiotic-, or antimicrobial peptide”. There were no articles containing the search terms in 1966-67. METHODS The data on new synthetic antibiotic peptides is available in the literature in two forms, in printed journals and in journals available in electronic format. Most of the data that is in electronic journals is available only for recent years (e.g., since 1997 for many of the more than 1,500 electronic journals available 55 through Finland’s National Library of Health Sciences). As can be seen from Figure 1, many of the articles on antibiotic peptides in the medical literature were published after 1980. Therefore, much of the collection of data on antibiotic peptides for the SAPD is being done through the use of printed journals. Searching for articles on antibiotic peptides is done through electronic databases, such as Medline, Current Contents, and Chemical Abstracts. Some of these databases are more complete in the range of journals that they cover (e.g., Chemical Abstracts is more complete than Medline and Current Contents for the chemical literature), and since the field of synthetic antibiotic peptides includes both chemical and biological specialities, all of these literature databases are being utilized in developing the SAPD. The SAPD currently consists of a series of files, one for each peptide antibiotic, that were created as Netscape hypertext documents using the Composer function within Netscape Communicator 4.7 . The SAPD website consists of commercially available software, and data files are added to the SAPD by simply posting them on the website. When a user wants to access the SAPD, they must register and create a user identification name and password. This is less convenient than the AMSDb or Peptaibol Database, but it enables the SAPD manager to determine statistics on usage of the database. Once logged onto the database, a user selects the file for the desired peptide, and is then presented with a text document. The SAPD also includes a function for searching text throughout the entire database. Due to the enormous task involved in developing the SAPD, it includes a feature for acknowledging data contributions by users who are not primarily involved in SAPD development. Every contribution of a substantial amount of verifiable data (e.g., all of the chemical, biological, and literature reference data for one peptide antibiotic) is acknowledged in a highly visible location in the beginning of the contributed file. This feature is not available in the AMSDb or Peptaibol Database, and its purpose is to encourage users of the SAPD to make data contributions themselves. One feature of the AMSDb that is not currently available in the SAPD, but may be included at some future date, is that most of the AMSDb entries are correlated to entries found in the SWISS-PROT protein sequences database (http://www.expasy.ch/sprot/), and it includes the SWISS-PROT ID code. The reason for this omission in the SAPD is due to the fact that unlike the proteins in the SWISS-PROT database, relatively few of the peptide sequences listed in the SAPD occur in nature. Additional features that will be included as the size of the database increases are links both within the database and to other relevant sites on the Internet. RESULTS The SAPD currently contains information for about 200 synthetic antibiotic peptides. The format for one representative entry from the SAPD, that of Temporin A (TA), is shown in Table 1. For comparison purposes, the AMSDb entry for the naturally occurring form of TA is shown in Table 2, with font styles and sizes modified to conform to journal requirements. 56 Table 1. Typical format for one entry from the synthetic antibiotic peptides database (SAPD). 1. Temporin A (TA) 2. Literature references: a. Simmaco, M., Mignogna, G., et al. Rana temporaria. Eur. J. Biochem. (1996) 242: 788. b. Harjunpää, I., Kuusela, P., et al., FEBS Letts. (1999) 449: 187. c. Mangoni, M.L., Rinaldi, A.C., et al.. Eur. J. Biochem. (2000) 267: 1447. d. Wade, D., Silberring, J., et al., FEBS Letts. (2000) 479: 6. 3. Contributor of information to database: a. D. Wade, Haartman Institute, Helsinki University, Finland. b. Dates of contribution, verification, entry into database, and updating: June 9, 2001. 4. Structural information: a. The peptide is found in the skin secretions of the European red frog, Rana temporaria. b. AA sequence: FLPLIGRVLSGIL-NH2; amidated carboxyl terminus. c. Both all-L and all-D variants of TA have been synthesized (Refs. a, b, d). d. Secondary structure, methods of determination, and comments: 1) α-Helices present when peptide is in an aqueous solution of trifluoroethanol (TFE). 2) Structure determined by circular dichroism: a) Helical in water plus TFE; max. helicity in 30% (Ref. c) or 80% TFE (Ref. d) b) Antibacterial activity of temporins correlated to increased helix formation. (Ref. a) 5. Bioactivity of peptide: a. Antibacterial activities [LC, lethal concentration (Ref. a) or MIC*, minimal inhibitory concentration (Refs. b and d) values (µM)]: 1) Aerobic bacteria: a) Gram-positive (MRSA, methicillin resistant Staph. aureus; VRE, vancomycin resistant Enterococcus): (1) all-L TA: Bacillus megaterium Bm11: 1.2; Staphylococcus aureus: Cowan I, 2.3; Newman, 6.8*; K35 (MRSA), 2.6-13.7*; K52, 5.2-13.7*; ATCC 25923, 2.6* Enterococcus faecium (VRE): 18578, 10.5-27.3*; 18172, 10.5-54.6* Enterococcus faecalis ATCC 29212: 20.9* Streptococcus pyogenes (β hem. group A): 2 Acinetobacter junii (Rt-4): 7.7; (Bo-2): 24.0 (2) all-D TA: Staphylococcus aureus: K35 (MRSA), 3.1*; K52, 3.1*; ATCC 25923, 2.6* Enterococcus faecium (VRE): 18578, 12.5*; 18172, 12.5* Enterococcus faecalis ATCC 29212: 10.5* b) Gram-negative (all-L TA): Yersinia pseudotuberculosis: 2 Esherichia coli (D21): 11.9 Pseudomonas aeruginosa ATCC15692: >360 Klebsiella pneumoniae (Rt-1): 9.6 Proteus vulgaris (Rt-3): >340 Aeromonas hydrophila (Bo-3): >360; (Bo-4): >20 Enterobacter agglomerans (Bo-1): >50 2) Anaerobic bacteria: Not tested. b. Other cell types or organisms: Eukaryotic cells [Refs. a and c; LC values (µM)]: 1) Fungus: Candida albicans: 3.4 2) Human red blood cells (hemolysis): >120 (agarose method); 50% hemol. at 42 µM (saline method) 6. Physicochemical properties of peptide: a. MW: 1397 (Calc. from ExPASy Molecular Biology server, http://www.expasy.ch/tools/pi_tool.html) b. Electromagnetic radiation absorption characteristics: UV (210, 260 nm) c. Solubility: Soluble in water (≥ 2.5 mg/ml) 57 Table 2. Representative entry from the Antimicrobial Sequences Database (AMSDb) for Temporin A . TMPA_RANTE ID TMPA_RANTE LOCAL PRT; 13 AA. AC DE TEMPORIN A OS RANA TEMPORARIA (RED FROG/RANA SPECIES /AMPHIBIAN). RN  RP SEQUENCE RC TISSUE=SKIN RA SIMMACO M., MIGNOGNA G., CANOFEI S., MIELE R., MANGONI M., BARRA D. RL EUR. J. BIOCHEM. 242:788-792(1996). RN  RP SPECTRUM OF ACTIVITY RA MANGONI M., RINALDI A.C., DI GIULIO A., MIGNOGNA G.,BOZZI A., BARRA D., SIMMACO M., RL EUR. J. BIOCHEM. 267:1447-1454 (2000) CY LINEAR PEPTIDE TY (?) FY TEMPORINS AY ANTIBACT.: G+ (B. MEGATERIUM, S. AUREUS, S. PYOGENES, MIC 1-2 MICROMOLAR) AY ANTIBACTERIAL: G- LESS ACTIVE (E. COLI MIC = 12 MICROMOLAR) AY ANTIFUNGAL: (C. ALBICANS MIC 3.4 MICROMOLAR AY HEMOLYTIC (E50 = 10 MICROM) CC -!- FUNCTION: SHOWS ANTIBACTERIAL ACTIVITY AGAINST REPRESENTATIVE CC GRAM-POSITIVE BACTERIAL SPECIES. POORLY ACTIVE AGAINST G- SPECIES. CC SHOWS ANTIFUNGAL ACTIVITY AGAINST C. ALBICANS. CC -!- SIMILARITY: TO PEPTIDES OF THE TEN-MEMBERED FAMILY OF TEMPORINS CC THE RELATED PEPTIDES TEMPORINS F AND G MAY HAVE A SIMILAR ACTIVITY. CC -!- TISSUE SPECIFICITY: SKIN. CC -!- SUBCELLULAR LOCATION: SECRETED (?). FT MOD_RES 13 13 AMIDATION FT ANALOG 7 7 ARG -> LYS IN TEMPORIN F KW AMPHIBIAN SKIN; ANTIBIOTIC; HEMOLYSIS. MW MATURE PEPTIDE: 1397; SQ SEQUENCE 13 AA; TMPA_RANTE flpligrvlsgil* ACKNOWLEDGEMENTS D.W. thanks Dr. Alessandro Tossi, of the University of Trieste, for encouragement and helpful discussions during the early stages of the creation of the synthetic antibiotic peptides database. D.W. also thanks the Academy of Finland for providing visiting scientist fellowship support during 1998, 1999, and 2001, and the Wade Research Foundation for supplemental support during these periods. REFERENCES  Boman, H.G. (1998) Scand. J. Immunol., 48, 15.  Tossi, A., University of Trieste, Dept. of Biochemistry, Biophysics, and Macromolecular Chemistry, Trieste, Italy.  Whitmore, L., Chugh, J., Snook, C. F., and Wallace B. A. (2000) The Peptaibol Database. Birkbeck College, Crystallography Dept., London, U.K.  Mee, R.P., Anton, T.R. and Morgan, P.J. (1997) J. Peptide Res., 49, 89-102.  Netscape Comunicator 4.76, Netscape Communications Corporation. 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