Abstract:
Cyclic analogues of histatin H-5, having from about 7-20 amino acid units exhibiting substantial homology to histatin H-5, and having a cyclic portion of from 5-16 of the amino acid units, have been found to exhibit enhanced bio-activity against a variety of different microorganisms. The cyclic structure is imparted by replacement of naturally occurring amino acids in histatin-5 sequences by, for example, by cysteine units, so as to cause cyclization through formation of disulfide bridges.

Description:
FIELD OF THE INVENTION 
     This invention relates to novel bio chemical compounds and compositions having pharmaceutical and medical activities, and to processes for their preparation and use. More specifically, it relates to novel peptide compounds and compositions of the histatin type, which exhibit antimicrobial properties. 
     BACKGROUND OF THE INVENTION AND PRIOR ART 
     Antimicrobial peptides have been found in a variety of organisms. Among the most studied are the magainins from the skins of amphibians, the cecropins from the moth cecropia, melittin and bombolitin from bee venom. In all cases these are cationic peptides reportedly exerting their antibacterial action by altering the membrane permeability. These peptides have an amphiphatic α-helical structure which is critical for their activity (1). Another class of potent antimicrobial peptides, the defensins, was isolated from mammalian phagocytic cells. Defensins have a broad range of activity against bacteria, fungi, viruses and tumour cells and are considered to be important in the host non-immune defense system. X-ray and NMR analysis has revealed that the defensins assume an antiparallel β-sheet conformation. It has also been shown that defensins form voltage dependent channels in model lipid bilayer. 
     Another class of antimicrobial peptides was recently identified in human saliva. They were originally called histidine-rich peptides (HRP)(2) and later referred to as histatins (3). Twelve members of this family have been characterized (3,4). Histatins are secreted by the parotid and submandibular glands and constitute 3 percent dry weight of saliva (2). The three major forms are histatin-1 (H-1), histatin-3 (H-3), and histatin-5 (H-5) which are 38, 32 and 24 amino acids peptides, respectively and have highly homologous sequence, as depicted in the accompanying Figure. H-1 and H-3 are derived from different genes (5), whereas H-5 is proteolytically derived from H-3. Other members of this family are proteolytically produced from the three main forms although the enzyme(s) responsible for this processing is presently unknown. The smaller members have reduced antimicrobial activities. H-1, H-3 and H-5 contain several basic residues including seven conserved histidines. Homologous histatin sequences have also been isolated from the saliva of other primates. 
     These peptides are highly basic (cationic), non-amphiphilic and show no homology to other known proteins. Histatins have been implicated in several biological functions. Histatin-3 and -5 possess antibacterial and antifungal properties at physiological concentrations and may play an important role in the non-immune oral host defense system. 
     Histatins have been shown to have a number of different biological activities. A phosphorylated member, histatin-1, selectively adsorbs to hydroxylapatite and enamel powder, possibly acting as a precursor of the acquired enamel pellicule (6). More recently, H-5 has been shown to act synergistically with epidermal growth factor (EGF) to promote rabbit chondrocyte proliferation (7). Patents describing the use of histatins for wound healing, and bond and periodontal tissue regeneration have also appeared (8-10). 
     It has been reported that H-1, H-3 and H-5 possess potent antimicrobial properties against bacteria (11) and fungi (12) in a concentration dependent manner at physiological concentrations. The most potent, H-5, has a minimum inhibitory concentration (MIC) of 10 μg/ml against  C. albicans  (12). This value compares favorably with the widely used antifungal agent ketoconazole, which is an inhibitor of lanosterol C-14 demethylase, an important enzyme in the biosynthesis of ergosterol (13). The mode of action of histatin is still unknown. 
     In the one of the earliest structure-activity relationship (SAR) studies of histatins published to date, Raj et al. reported that the C-terminal fragment is most important and that a minimum of 16 amino acids (H-5 8-24 ) were required for activity against  C. albicans  (14). Because of the large number of positive charge on H-5 8-24 , these authors suggested that histatins interact with the polar head groups of the phospholipid membranes, but only weak experimental evidence supports this hypothesis. Raj and co-workers also showed by CD and NMR that H-5 8-24  has no structure in H 2 O but adopts an extended α-helical conformation in trifluoroethanol (TFE) or in DMSO (14). The same group also reported that the H-5 8-24  fragment has α-helical character in the presence of DMPC vesicles, and that the conformation is pH independent (15). More recenty. Oppenheim and co-workers (16) have produced by recombinant methods an H-3 peptide mutant in which the central region of H-3 (13-24-13-24)  was tandemly repeated. At very low concentrations, this H-3 (13-24-13-24)  fusion peptide was apparently more active than H-3 in the candicidal assay (16). 
     The conformation of histatin-5 in water or in the presence of its biological targets has not been firmly established. 
     It is an object of the present invention to provide novel histatin analogs having improved microbiocidal properties. 
     SUMMARY OF THE INVENTION 
     The present invention derives from our discoveries that the most bioactive conformation of histatin peptides involves their adoption of a hairpin loop conformation in the region of positions 8-24 of H-5, likely around E 16 -K 17  and that histatin peptides are reasonably tolerant to amino acid replacement. Accordingly, we have found that the introduction of substantially permanent loop structures into H-5 peptide compounds, to provide cyclic analogs of histatins, leads to compounds having much more potent activity than H-5 against a variety of different microorganisms. Since some amino acid replacement can be tolerated in H-5 without significant loss of activity, the introduction of replacement amino acids at appropriate locations in the chain so as to form chemical bridges between themselves (e.g. disulphide bonds between introduced cysteine residues) can be accomplished. In this way, the loop or cyclic structure is substantially permanently imparted to the compounds, subject only to the normal chemical stability of the loop-forming bonds between the replacement amino acids (mutations). The result is a family of novel cyclic peptides analogous to H-5, but having much greater antimicrobial activity than H-5, as set out in the specific examples below. 
     Thus according to the present invention, from one aspect, there is provided cyclic analogues of histatin H-5 having from about 7-20 amino acid units exhibiting substantial homology to histatin H-5, and having a cyclic portion of from 5-16 of said amino acid units. 
     The bioactive conformation of H-3 is not an extended helix as inferred by previous reports, but a loop or cyclic structure. Although this loop structure motif is unusual for antimicrobial peptides, it has been observed in brevenins from frog skin or of bactenecins from bovine neutrophils (17). 
    
    
     BRIEF REFERENCE TO THE DRAWING 
     The single FIGURE of accompanying drawings illustrates known amino acid sequences of histatins 1 to 12, based upon prior art publication (Ref. 4, Troxler et.al.). In the drawing and throughout the specification and claims, the standard, internationally accepted code letters for individual amino acids are used (Pse=phosphoserine). 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The term “substantial homology” as used herein is intended to mean sequences of amino acids which have at least 60% and preferably at least 80% identity of unit and sequence with the natural product histatin H-5. It has been found that there is considerable tolerance of substitution of individual amino acids in histatins, without substantial loss of activity. 
     Histatins H-5 8-24 , H-5 and H-3 and analogs and mutations thereof an be prepared by standard methods of peptide synthesis. The analogs may comprise D-amino acid analogs, L-amino acid analogs, and analogs incorporating random or preselected mixtures of D- and L-amino acids, in the predetermined, defined sequence. Chemical syntheses for step-by-step assembly of peptides using predetermined sequences of amino acid starting units, with appropriate chemical protection and deprotection steps, are well known and can be adopted in preparation of analogs according to the present invention. Preferred syntheses use solid phase peptide synthesis technology, e.g using 4-alkoxybenzyl alcohol or 2-chlorotrityl chloride solid support resins, on a continuous flow synthesizer. They can also be prepared by the batch mode method as originally described by Merrifield. The peptides can also be made by solution methods or by a combination of liquid phase and solid phase methods. The N-amino group of the amino acid is suitably protected with a base labile fluorenylmethoxycarbonyl (FMOC) group or a t-butyloxycarbonyl (BOC) group during synthesis while its acid group is reacted. Tyrosine, serine, threonine, glutamic acid and aspartic acid side groups are suitably protected with t-butl groups during chain extension. Potential racemization of the imidazole ring of histidine is suitably blocked by use of an N-trityl group. The reactivity of cysteine side chains can also be masked with a trtyl group. Arginine residues are appropriately protected at their N-terminus with pentamethylchroman-6-sulfonyl (PMC) groups. Lysine is suitably protected with BOC groups. 
     Coupling of amino acids is suitably achieved by activating the first amino acid with benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 1-hydroxybenzotriazole, to form the active ester. Activators such as N-methylmorpholine (NMM) or collidine may also be used to assist in the reaction. Other common coupling agents such as DCC, HBTU, HATU etc can be used. All these peptide synthesis techniques, and equivalents and alternatives to them, are within the skill of the art and well known to skilled peptide chemists. 
     The peptides so formed may be cleaved from their solid synthesis supports by standard protocol using trifluoroacetic acid and appropriate scavengers, and the blocking groups by standard procedures. The peptides so formed can be purified by reverse phase HPLC. 
     Peptides containing only L-amino acids can be obtained by chemical synthesis or by DNA recombinant techniques. Other peptides with D-amino acids or with lactam groups as the means for providing cyclization are prepared by chemical synthesis or by semi-synthesis using enzymes. The synthetic protocol (couplings, deprotection and cleavage times) may be optimized for a continuous flow synthesizer (Millipore 9050) using Fmoc-chemistry. Electrospray mass spectrometer (ESMS) is used to detect deletions and other side reactions. Purification to homogeneity is achieved by reverse phase HPLC using a C-18 column. In general, overall yields of pure peptides are greater than 60% (as high as 90%) and quantities &gt;100 mg are routinely obtained. 
     To prepare the cyclic peptides of the present invention, it is necessary to make at least one amino acid substitution, as compared with histatin H-5. The natural product contains within the region forming the active loop structure lysine groups, which in one embodiment of the invention can be used as points of cyclization, utilizing their free amino groups in reaction with free carboxyl of glutamic acid to form a lactam (amide) link and result in ring formation. However, the glutamic acid residue naturally present in histatin H-5, at position 16, should preferably be left intact and not utilized in the cyclization, since it appears to have a role in the activty of the final compounds. Accordingly, it is preferred to introduce a glutamic acid as a substitute amino acid group for purposes of reaction with lysine to form the cyclized form, via a lactam group. Lysine, for participation in the cyclization reaction, can also be introduced as a substitute group, or alternatively one can make use of one of the lysine groups naturally present. 
     An alternative and preferred amino acid substitution into the histatin analogs for cyclization purposes in the present invention is cysteine, which can readily be introduced by known methods, in place of any of the naturally occurring amino acids of histatin. Cysteine residues in the peptide chain readily form disulfide bridges between themselves so as to impart the necessary cyclic structures. While the bonds are chemically reversible, they are sufficiently permanent to meet the criteria for compounds of the present invention. As alternatives to cysteine, there can be used other thiol-containing amino acids e.g. homocysteine or penicillamine. Bicyclic analogues with two disulfide bridges or one disulfide and one lactam bond can also be made. Head-to-tail cyclization can also be made. Cyclization can also be done by other chemical means, e.g. by thioether linkage. 
     Peptides containing cysteine substitutions and cyclized through disulfide groups can be made by air oxidation of the free linear peptide in a 0.1 molar solution of ammonium bicarbonate, with selective protection of the cysteine. Lactam-cyclized peptides can be prepared by the selective removal of the Aloc/Allyl Lys and Glu side chain protecting groups under mild conditions with a Pd(0) catalyst while the peptide is still attached to the resin and the other side chains remain protected. An amide bond can then be formed between the side chains using (benzotriazolyloxy) tris(dimethylamino) phosphonium hexafluorophosphate (pyBOP0 and HOBt as coupling reagents, or with other common coupling reagents. Then the cyclized peptide can be cleaved from the resin and purified by reverse phase HPLC. 
     Potential applications in health care industry include orally administrable formulations (tablets, capsules, lozenges, chewing gums, etc, with standard, known fillers, extenders, excipients and the like), tooth paste, mouth wash products, other dental care products, antiperspirant, coatings on protective device for wound dressing (e.g. BandAid®). Other potential medical applications in medicine include wound healing and tissue regeneration after surgery or accidental trauma. 
     SPECIFIC DESCRIPTION OF MOST PREFERRED EMBODIMENTS 
     EXAMPLE 1 
     COMPOUNDS WITH DISULFIDE BRIDGES 
     Using the standard techniques of peptide synthetic chemistry described above, a series of histatin H-5 analogs according to the present invention was prepared, the amino acid sequences of which are listed below in Table 1, along with that of the naturally occurring H-5. Each possessed substantial homology with H-5 from position 5 to position 19 of the natural H-5, and in some cases beyond. Each contained two cysteine groups C in replacement of two naturally occurring amino acids of H-5, from 5 to 14 amino acids apart, and cyclized as described above to insert disulfide bridges between the two cysteine groups C and create cyclic peptide structures having loops or rings from 7 to 16 amino acids in length. In some cases, e.g.Db2-121, additional deletions were made so that the amino acid chain was shorter than that of either of H-5 or H-3. In some cases also, additional substitutions were made, but leaving the product within the range of substantial homology with H-5. 
     The compounds of Table 1 were tested for fungistatic activity with an assay based on the work of Gordon, Lapa et.al. (18) with the nonpathogenic yeast  S. cerevisiae  in which ketoconazole is used as control. A semisolid is prepared to which is added serial dilutions of peptide. This antifungal agar is distributed in a well plate, and the  S. Cerevisiae  inoculum is then added to each well. Both ketokonoazole and H-5 are used as positive controls and buffer is used as a control blank. Minimum inhibitory concentration MIC, i.e. the concentration of the peptide under test which prevents growth, as observed visually, is recorded for each sample so that low values of MIC indicate potent antifungal compounds. 
     More specifically, stock peptide solutions (600 microliters or 1 ml) were prepared to 10 times the final concentration of the most concentrated test sample in a sodium phosphate buffer (0.01 M0, pH 7.4). Peptide concentrations were determined by UV absorbance at λ=275. Control blanks were also prepared using 1 ml buffer Five serial dilutions were made in sterile culture tubes with buffer to create a range of peptide concentrations. The final volume in each tube was either 300 microliter or 1 ml. Agar medium (2.7 or 4.5 ml) was added to each tube and mixed. Aliquots (1 ml) of each were added to a 24 well plate to give 6 different concentrations of each peptide per plate. Each plate had a row of control blanks and a row of a positive control (ketoconazole). A stock solution (10 mg/mL DMSO) was diluted 1:49 with buffer. A range of final concentrations of 20 to 0.16 micrograms/ml was tested. In addition, in later analog assays, H-5 was included as a positive peptide control. Histatin 5 was tested in a range from 200 micromolar to 6.3 micromolar. Other analogs were tested at concentrations anywhere from 200 μM down to 0.14 μM ubtil the analog with lowest MIC concentration was identified. 
       S. cerevisiae  cells grown overnight were centrifuged and washed with a 0.85% saline solution, three times. Cell concentration was adjusted to 85% transmission at λ=530 with saline solution. Cells were further diluted 1:19. Inoculant (10 microL) was added to the peptide-containing medium. The cells were incubated on this medium for 24 hours at 37° C. The fungistatic MIC for these compounds was defined as the minimum concentration required to achieve 100% inhibition after 24 hours. 
     The results are included in the following Table. 
     
       
         
               
               
               
             
           
               
                   
               
             
             
               
                 Db2-180 
                 Arg His His Gly Tyr Lys Cys Lys Phe His Glu 
                 1.25-2.5 
               
               
                 (14) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                 Db2-179 
                 Arg His His Gly Tyr Cys Arg Lys Phe His Glu 
                 2.5-5 
               
               
                 (15) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                 Db2-178 
                 Arg His His Gly Cys Lys Arg Lys Phe His Glu 
                 2.5-5 
               
               
                 (16) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                 Db3-11 
                 Arg His His Gly Cys Lys Arg Lys Phe His Glu 
                 2.5-5 
               
               
                 (17) 
                 Lys His His Ser Cys Arg Gly Tyr 
               
               
                 Db3-13 
                 Arg His His Cys Tyr Lys Arg Lys His His Glu 
                  2.6-5.1 
               
               
                 (18) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                 Db3-19 
                 Arg His His Cys Tyr Lys Arg Lys Phe His Glu 
                  1.3-2.6 
               
               
                 (19) 
                 Lys His His Cys His Arg Gly 
               
               
                 Db3-15 
                 Arg His His Cys Tyr Lys Arg Lys Phe His Glu 
                  2.6-5.2 
               
               
                 (20) 
                 Lys His His Cys His Arg 
               
               
                 Db3-25 
                 Arg His His Cys Tyr Lys Arg Lys Phe His Glu 
                 &gt;5.2 
               
               
                 (21) 
                 Lys His His Cys His 
               
               
                 Db3-30 
                 Arg His His Cys Tyr Arg Lys Phe His Glu 
                 &gt;5.2 
               
               
                 (22) 
                 Lys His His Cys 
               
               
                 Db3-35 
                 Arg His His Cys Tyr Lys Arg Lys Phe His Glu 
                  1.3-2.5 
               
               
                 (23) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                 Db3-53 
                 Arg His His Cys Tyr Lys Arg Lys Phe His Glu 
                 &gt;5   
               
               
                 (24) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                 Db3-67 
                 Arg His His Cys Trp Lys Arg Lys Phe His Glu 
                 0.67-1.34 
               
               
                 (25) 
                 Lys His His Cys His Arg Gly Tyr 
               
               
                   
                 Ketoconazole 
                  4.7 
               
               
                   
               
             
          
         
       
     
     substantial improvement of activity can be obtained. Db2-121 (Gly 9 −&gt;Cys, Ser 20 −&gt;Cys) was found to be most potent with a MIC of 0.3 μM which is approximately 100 times more potent than H-5 and 10 times more potent than ketoconazole against  S. cerevisiae . Importantly, a similar activity profile for Db2-121 is observed against  C. albicans  and against three different bacteria; IS. Aureus (Gr+).  E. coli  (Gr−) and β. Magatenum (Gr+). From studies of the cysteine scan, it is quite evident that loop stabilization by correct disulfide cyclization is one way to enhance the activity of H-5 and related peptides. Db2-121 is 19 amino acids long and therefore shorter than H-5 or H-3. Additional deletions at the C-terminal (Db2-19 and Db2-15) can be done but with small loss of activity. A disulfide containing peptide with D-amino acids at each end of the chain was prepared and evaluated for biological activty—compound Db3-35. This type of modification often increases the stability of peptides towards proteolytic degradation. As often seen with other peptides, loop stabilization may also increase the proteolytic stability. 
     A hypotonic assay based on the work of Helmerhorst et.al (19) was used to determine the loss of cell viability of  C. albicans  in the presence of peptide compounds of the present invention. This protocol involves incubating the peptide and cells in a low osmolality solution for 1,5 hours with constant mixing. Standard plate count procedure is then followed to determine the concentration of viable cells remaining. By determining the cell concentration levels in negative control wells, the % loss of cell viability due to the presence of the peptide is determined. MICs for H-5 and H-5 8-24  were similar to those reported against  C. albicans.    
     Preliminary toxicity studies indicate that at least one of the cyclic analogs tested has no acute toxicity in mice (5 mice) after injection of a rather massive dose (15 mg/kg) in the intraperitoneal cavity. 
     EXAMPLE 2 
     BICYCLIC PEPTIDE WITH DISULFIDE BRIDGES 
     We also synthesized Db2-100, a peptide which contains two disulfide bonds (giving a bicyclic peptide), by selective protection of the cysteine. In this preparation, the first set of protecting groups, the trityl groups, were removed and oxidized as described above. The second set of protecting groups, the acetamidomethyl (Acm), were removed with iodine, and the resulting peptide purified by RP HPLC and air oxidized to form the second disulfide bridge. 
     Peptide Db2-100 had the following sequence: 
     Arg His His Cys Tyr Lys Arg Lys Cys His Glu Lys His His Cys His Arg Cys Tyr (SEQ ID NO:26) 
     The bicyclic structure is even more conformationally constrained that the other analogs and the compound has retained its biological activity—MIC range determined as described for the compunds of Example 1 is 0.63-1.25. 
     EXAMPLE 3 
     CYCLIC PEPTIDE WITH LACTAM (AMIDE) BOND 
     To demonstrate that the cyclic structure of the peptide, and not the presence of the disulfide itself, is determinant for optimal activity, we prepared Db3-41 a peptide analog of db2-121 in which the cyclization is effected by means of an amide bond (lactam) between the side chain of a glutamic acid and lysine, by methods described above. The glutamic acid is at position 9 and lysine at position 20. In this case, where an orthogonal protection scheme was required, the allyloxycarbonyl (Aloc) protecting group was used for lysine, and the allyl group was used for glutamic acid. 
     The full sequence of Db3-41 is Arg His His  Lys   Tyr Arg Lys Phe His Glu Lys His His  Glu  His Arg Gly Tyr. (SEQ ID NO:27) 
     The fungistatic activity of Db3-41 is comparable to Db2-121, i.e. MIC range (μm) 0.55-1.1. 
     References 
     1. S. E. Blondelle, R. A. Houghten, Annuals Reports in Medicinal Chemistry (1992), 27 159. 
     2. F. G. Oppenheim et al. J. Biol. Chem. (1986) 261 1177. 
     3. F. G. Oppenheim et al. J. Biol. Chem. (1988) 263 7472. 
     4. R. F. Troxler, F. G. Oppenheim et al. J. Dent. Res. (1990) 1 2. 
     5. J. C. VanerSpek et al. Arch. Oral Biol. (1990) 35 137. 
     6. C. F. Richardson et al. Arch. Oral Biol. (1993) 38 997. 
     7. Murakami, Y. et al. BBRC 198 274. 
     8. S. Taniguchi et al. JP 94-76628 940322; CAS:123;350366. 
     9. N. Matsuda et al. JP 93-98530 930331; CAS:122:89439. 
     10. A. Takemura et al. JP 93-45998 930210; CAS:121:308303. 
     11. B. J. MacKay et al. Infect. Immun. (1984) 44 695. 
     12. J. J. Pollock et al. Infect. Immun. (1984) 44 702. 
     13. A. Polaketal. Prog. Drug Res. (1991) 37 181. 
     14. P. A. Raj et al. J. Biol. Chem. (1990) 265 3898. 
     15. P. A. Raj et al. J. Biol. Chem. (1994) 289 9610. 
     16. Y. Zuo. F. G. Oppenheim et al. Gene (1995) 161 87. 
     17. E. R. Hancock The Lancet 39, 418 (1997). 
     18. M. Gordon et al., J. Clinical Microbiol. (1988) 26 1874. 
     19. E. J. Helmerhorst et al., J. Biol. Chem. (1999) 274 7286. 
     
       
         
           
             40 
           
           
             1 
             24 
             PRT 
             Homo sapiens 
           
            1
Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu
1               5                   10                  15
Lys His His Ser His Arg Gly Tyr
            20
 
           
             2 
             23 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            2
Ser His Ala Lys Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys
1               5                   10                  15
His His Ser His Arg Cys Tyr
            20
 
           
             3 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            3
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Ser His
1               5                   10                  15
Arg Cys Tyr
 
           
             4 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            4
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Ser His
1               5                   10                  15
Arg Gly Cys
 
           
             5 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            5
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Ser His
1               5                   10                  15
Arg Cys Tyr
 
           
             6 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            6
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Ser His
1               5                   10                  15
Cys Gly Tyr
 
           
             7 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            7
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Ser Cys
1               5                   10                  15
Arg Gly Tyr
 
           
             8 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            8
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             9 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            9
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His Cys Ser His
1               5                   10                  15
Arg Gly Tyr
 
           
             10 
             17 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            10
Arg His His Cys Tyr Lys Arg Lys Glu Lys Cys His Ser His Arg Gly
1               5                   10                  15
Tyr
 
           
             11 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            11
Arg His His Gly Cys Lys Arg Lys Phe His Glu Lys His His Ser His
1               5                   10                  15
Cys Gly Tyr
 
           
             12 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            12
Arg His His Gly Tyr Lys Arg Lys Cys His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             13 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            13
Arg His His Gly Tyr Lys Arg Cys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             14 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            14
Arg His His Gly Tyr Lys Cys Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             15 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            15
Arg His His Gly Tyr Cys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             16 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            16
Arg His His Gly Cys Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             17 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            17
Arg His His Gly Cys Lys Arg Lys Phe His Glu Lys His His Ser Cys
1               5                   10                  15
Arg Gly Tyr
 
           
             18 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            18
Arg His His Cys Tyr Lys Arg Lys His His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             19 
             18 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            19
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly
 
           
             20 
             17 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            20
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg
 
           
             21 
             16 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            21
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
 
           
             22 
             15 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            22
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys
1               5                   10                  15
 
           
             23 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            23
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             24 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            24
Arg His His Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             25 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            25
Arg His His Cys Trp Lys Arg Lys Phe His Glu Lys His His Cys His
1               5                   10                  15
Arg Gly Tyr
 
           
             26 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            26
Arg His His Cys Tyr Lys Arg Lys Cys His Glu Lys His His Cys His
1               5                   10                  15
Arg Cys Tyr
 
           
             27 
             19 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            27
Arg His His Lys Tyr Lys Arg Lys Phe His Glu Lys His His Glu His
1               5                   10                  15
Arg Gly Tyr
 
           
             28 
             38 
             PRT 
             Homo sapiens 
             
               misc_feature 
               (2)..(2) 
               phosphoserine 
             
           
            28
Asp Xaa His Glu Lys Arg His His Gly Tyr Arg Arg Lys Phe His Glu
1               5                   10                  15
Lys His His Ser His Arg Glu Phe Pro Phe Tyr Gly Asp Tyr Gly Ser
            20                  25                  30
Asn Tyr Leu Tyr Asp Asn
        35
 
           
             29 
             27 
             PRT 
             Homo sapiens 
           
            29
Arg Lys Phe His Glu Lys His His Ser His Arg Glu Phe Pro Phe Tyr
1               5                   10                  15
Gly Asp Tyr Gly Ser Asn Tyr Leu Tyr Asp Asn
            20                  25
 
           
             30 
             32 
             PRT 
             Homo sapiens 
           
            30
Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu
1               5                   10                  15
Lys His His Ser His Arg Gly Tyr Arg Ser Asn Tyr Leu Tyr Asp Asn
            20                  25                  30
 
           
             31 
             21 
             PRT 
             Homo sapiens 
           
            31
Arg Lys Phe His Glu Lys His His Ser His Arg Gly Tyr Arg Ser Asn
1               5                   10                  15
Tyr Leu Tyr Asp Asn
            20
 
           
             32 
             25 
             PRT 
             Homo sapiens 
           
            32
Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu
1               5                   10                  15
Lys His His Ser His Arg Gly Tyr Arg
            20                  25
 
           
             33 
             13 
             PRT 
             Homo sapiens 
           
            33
Arg Lys Phe His Glu Lys His His Ser His Arg Gly Tyr
1               5                   10
 
           
             34 
             12 
             PRT 
             Homo sapiens 
           
            34
Lys Phe His Glu Lys His His Ser His Arg Gly Tyr
1               5                   10
 
           
             35 
             14 
             PRT 
             Homo sapiens 
           
            35
Arg Lys Phe His Glu Lys His His Ser His Arg Gly Tyr Arg
1               5                   10
 
           
             36 
             13 
             PRT 
             Homo sapiens 
           
            36
Lys Phe His Glu Lys His His Ser His Arg Gly Tyr Arg
1               5                   10
 
           
             37 
             8 
             PRT 
             Homo sapiens 
           
            37
Lys Arg His His Gly Tyr Lys Arg
1               5
 
           
             38 
             7 
             PRT 
             Homo sapiens 
           
            38
Lys Arg His His Gly Tyr Lys
1               5
 
           
             39 
             24 
             PRT 
             Homo sapiens 
           
            39
Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu
1               5                   10                  15
Lys His His Ser His Arg Gly Tyr
            20
 
           
             40 
             14 
             PRT 
             Artificial Sequence 
             
               cyclic analogues of histatin 
             
           
            40
Cys Tyr Lys Arg Lys Phe His Glu Lys His His Cys His Arg
1               5                   10