WO2003006048A1 - Combination of chips (chemotaxis inhibiting protein from staphylococcus aureus)-based compounds - Google Patents

Combination of chips (chemotaxis inhibiting protein from staphylococcus aureus)-based compounds Download PDF

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Publication number
WO2003006048A1
WO2003006048A1 PCT/EP2001/008004 EP0108004W WO03006048A1 WO 2003006048 A1 WO2003006048 A1 WO 2003006048A1 EP 0108004 W EP0108004 W EP 0108004W WO 03006048 A1 WO03006048 A1 WO 03006048A1
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Prior art keywords
chips
compounds
combination
phenylalanine
compound
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PCT/EP2001/008004
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French (fr)
Inventor
Cornelis Petrus Maria Van Kessel
Carolina Jacqueline Cornelia Gosselaar-De Haas
Johannes Anna Wilhelminus Kruijtzer
Johannes Antonius Gerardus Van Strijp
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Jari Pharmaceuticals B.V.
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Priority to PCT/EP2001/008004 priority Critical patent/WO2003006048A1/en
Publication of WO2003006048A1 publication Critical patent/WO2003006048A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to combinations of peptides derived from the CHemotaxis Inhibitory Protein from Staphylococcus aureus (CHIPS) having CHIPS activity.
  • the invention in addition relates to the use of non-peptide molecules having peptide structural features and a similar function as the peptides in such combinations.
  • the combination of compounds can be used in the prophylaxis and treatment of various indications, such as inflammation reactions.
  • the peptides and non-peptides can in addition be used for inhibiting activation of leukocytes and endothelial cells.
  • Leukocytes are mainly involved in protecting the body against foreign invaders (e.g. bacteria, viruses, fungi, and cancer cells) .
  • lymphocytes The most important cells are lymphocytes, monocytes and neutrophils. Lymphocytes form the specific immune system and cause immune reactions against invaders. Their most important task is to build up specific memory against the invader, so that the next time the invader enters the body it is recognized, killed and removed rapidly. Sometimes these lymphocytes not only attack invaders, but also react against certain structures and/or molecules (so-called auto-antigens) of the own body, causing auto-immune diseases (e.g. rheumatoid arthritis) . Killing and removal of invaders is mostly done by monocytes and neutrophils, cells of the innate immune system, by direct recognition of the invaders or with the help of specific lymphocytes.
  • cells of the innate system react in a relatively non-specific and aggressive way. Since they are part of the body's first line of defense, their most important task is to kill and remove the invading agent as quickly as possible. This is accomplished through very aggressive substances (e.g. free radicals and enzymes) that are not only lethal to the invader, but also cause damage to host cells in the vicinity. Substances from these damaged cells and the locally activated cells from the innate system itself will further attract increasing numbers of neutrophils and monocytes, causing local inflammation. In most cases, such an aggressive and damaging inflammatory reaction, caused by overactivated neutrophils, is unnecessary.
  • very aggressive substances e.g. free radicals and enzymes
  • this inflammatory response is responsible for serious, sometimes lethal disorders and includes conditions like Adult Respiratory Distress Syndrome (ARDS) , severe tissue damage following thrombotic events such as heart attacks and stroke, inflammatory bowel diseases and rheumatoid arthritis.
  • ARDS Adult Respiratory Distress Syndrome
  • the inflammation will subside once all the invaders have been killed and removed, together with the various cells killed in the process.
  • Healing of the wound, caused by the inflammatory response can then begin.
  • the main task of neutrophils is to attack the invaders and the main task of monocytes is to remove the debris resulting from this attack.
  • neutrophils have another peaceful task in assisting the wound healing process.
  • C5a and fMLP are chemo-attractants : substances that can activate and attract cells from the blood vessels (the migration process) .
  • Neutrophils are responsive to these two substances and also to interleukin-8 (IL-8) .
  • chemokine (the name given to chemo-attractants that are produced by cells of the immune system) is produced mainly by activated monocytes (but also in minute amounts by the activated neutrophils themselves) .
  • Neutrophils interact with these substances, because they have receptors for these substances on the outside of their cell membrane. Activated neutrophils can easily migrate from blood vessels. This is because the chemo-attractants, microbial products and substances from activated monocytes will have increased the permeability of the vessels and stimulated the endothelial cells of the vessel walls to express certain adhesion molecules.
  • Neutrophils express selectins and integrins (e.g. CDllb/CD18) that bind to these adhesion molecules.
  • the neutrophil Once the neutrophil has adhered to the endothelial cells, it is able to migrate through the cells, under the guidance of chemo-attractants/che okines, towards the site of infection, where the concentration of these substances is at its highest. These substances also activate neutrophils to produce a range of other molecules, some of which attract more neutrophils (and subsequently monocytes) , but, mostly, they are responsible for destroying the invading bacteria. Some of these substances (e.g. free radicals, enzymes that break down proteins (proteases) and cell membranes (Upases) ) are so reactive and non-specific that cells from the surrounding tissue (and the neutrophils themselves) are destroyed, causing tissue damage.
  • chemo-attractants/che okines e.g. free radicals, enzymes that break down proteins (proteases) and cell membranes (Upases)
  • monocytes migrate to the scene and become activated. Besides their role in removing bacteria and cell debris, they also produce substances such as tumour necrosis factor (TNF) and IL-8 , which in turn attract more activated neutrophils, causing further local damage. TNF also has a direct stimulatory effect on neutrophils. Once all the invaders have been removed, the inflammatory response will subside and the area will be cleared of the remaining 'casualties'. Then the process of wound healing starts. Although it is known that neutrophils play a pivotal role in wound healing, it is not clear which neutrophil-derived substances are involved and how the neutrophils are active in healing without being aggressive to the surrounding tissue.
  • damaged tissue will be replaced by scar tissue formed mainly of fibroblasts and collagen.
  • scar tissue formed mainly of fibroblasts and collagen.
  • inflammation occurs in areas of the body with an important function, like tissues formed from heart muscle cells, brain cells or lung alveolar cells, normal function will be compromised by the resulting scar formation, causing serious conditions like heart failure, paralysis and emphysema.
  • Intervention to control the acute early phase inflammatory response presents an opportunity to improve the prognosis for a wide range of patients whose symptoms can be traced back to such an event.
  • Such an approach has been advocated for many acute and chronic inflammation- based diseases and shown to have potential based on findings from relevant disease models.
  • Classical anti-inflammatory drugs such as steroids and Non Steroid Anti-Inflammatory Drugs (NSAIDS) do not have the ideal profile of action, either in terms of efficacy or safety.
  • Steroids affect the 'wrong' cell type (monocytes) and their dampening effects are easily bypassed.
  • NSAIDS generally show a relatively mild effect partly because they intervene at a late stage in the inflammatory process.
  • Both classes of drugs produce a range of undesirable side effects resulting from other aspects of their pharmacological activity.
  • Drugs acting directly and specifically to prevent migration and activation of neutrophils may have a number of advantages.
  • Several drugs under early development only interfere with one individual aspect of neutrophil activation (e.g. C5 convertase inhibitors, antibodies against C5a, C5a-receptor blocking drugs) and migration (antibodies against integrins (like CDllb/CD18) and L-selectin on neutrophils and antibodies against adhesion molecules (like ICAM-1 and E-selectin) on endothelial cells) .
  • Antibodies against TNF and IL-8 have effects in chronic inflammation, but only marginal effects in acute inflammation, because of the minimal role monocytes (which are mainly responsible for these substances' production) play in the acute phase.
  • the reduced receptor expression was related to greatly reduced chemotaxis relative to the chemokines, as well as a reduced infection with HIV.
  • the active protein could be further purified, as described in application PCT/NL99/00442. Because this protein is isolated from the supernatant of the Staphylococcus aureus and gives inhibition of chemotaxis, this protein was named "CHIPS": CHemotaxis Inhibitory Protein from Staphylococcus aureus. Isolation of the CHIPS protein out of the supernatant of S . aureus is not very cost-effective.
  • the co-pending application PCT/EP01/00270 describes a nucleic acid molecule comprising a nucleotide sequence encoding a (poly) peptide having CHIPS activity.
  • Figure 1 gives the nucleotide sequence of this nucleic acid molecule. This sequence provides the means for producing the original CHIPS protein or other corresponding (poly) peptides that have CHIPS activity, as well as functional fragments, derivatives or analogues thereof by means of genetic engineering.
  • CHIPS activity is herein defined as the ability to specifically impair at least the responses induced by both fMLP and C5a, including at least impairment of ligand- (C5a or fMLP) binding, and optionally impairment of chemotaxis and cell-activation (e.g. calcium mobilization) .
  • the compounds of this invention may in addition have other biological activities, such as an inhibitory effect on the activation of leukocytes and endothelial cells.
  • the active part of the protein is isolated.
  • Smaller protein or peptide molecules have a reduced risk of inducing an immunological response in a subject receiving the protein or peptide for therapy.
  • this smallest as possible active site within the naturally occurring CHIPS molecule is the N-terminal sequence FTFEPF.
  • This peptide has however an activity that is 10.000 times lower (on molar basis) than the native CHIPS protein. This led the inventors to conclude that one or more further sites are present in the CHIPS molecule that support the CHIPS activity of the peptide.
  • the present invention therefore relates to combinations of compounds, wherein one compound has CHIPS activity (further identified herein as “the compound having CHIPS activity”) and consists of at least 6 amino acids the sequence of which is based on the N-terminal part of the naturally occurring CHIPS molecule, and which sequence optionally comprises one or more substitutions as compared to the naturally occurring sequence, or peptidomimetic derivatives thereof, whereas the one or more other compounds are derived from the amino acids 4- 121 of the naturally occurring CHIPS molecule and support CHIPS activity (further identified herein as the "supporting compound”) .
  • the compound having CHIPS activity consists of at least 6 amino acids the sequence of which is based on the N-terminal part of the naturally occurring CHIPS molecule, and which sequence optionally comprises one or more substitutions as compared to the naturally occurring sequence, or peptidomimetic derivatives thereof, whereas the one or more other compounds are derived from the amino acids 4- 121 of the naturally occurring CHIPS molecule and support CHIPS activity (further identified herein as
  • the invention relates to combinations of compounds, wherein the compound having CHIPS activity, has the sequence:
  • building blocks XI, X2, X3, X4, X5, X6 and X7 are selected from the group consisting of proteinogenic amino acids, non-proteinogenic amino acids, D-amino acids and peptidomimetic building blocks and wherein X7 may or may not be present and may represent either one or more building blocks.
  • XI may be modified (cf.
  • XI may be selected from phenylalanine (F) , cysteine (C) , asparagine (N) , tryptophan (W) ;
  • X2 may be selected from alanine (A) , cysteine (C) , phenylalanine (F) , glycine (G) , histidine (H) , isoleucine (I) , leucine (L) , methionine (M) , asparagine (N) , gluta ine (Q) , serine (S) , threonine (T) , valine (V) , tryptophan (W) , tyrosine (Y) ;
  • X3 may be selected from phenylalanine (F) , tryptophan (W) ;
  • X4 may be selected from alanine (A) , cysteine (C) , aspart
  • XI may be selected from phenylalanine (F) , tryptophan (W) ;
  • X2 may be selected from phenylalanine (F) , isoleucine (I) , serine (S) , threonine (T) ;
  • X3 is phenylalanine (F) ;
  • X4 may be selected from alanine (A) , cysteine (C) , aspartic acid (D) , glutamic acid (E) , asparagine (N) , serine (S) , tryptophan (W) ;
  • X5 may be selected from phenylalanine (F) , isoleucine (I) , leucine (L) , proline (P) , valine (V) tryptophan (W) , tyrosine (Y) ;
  • X6 may be selected from cysteine (C) , phenylalanine (F
  • Particular compounds having CHIPS activity of the invention have the sequence: F-T-F-E-P-F-X7, wherein one or more of the amino acids on positions 1 to 6 are substituted with the building blocks as defined above, and wherein X7 is a stretch of one or more building blocks selected from the group consisting of non-proteinogenic amino acids, D-amino acids and peptidemimetic building blocks and may or may not be present.
  • X7 can be present to further increase the potency (CHIPS activity) of the compound and/or to improve upon its pharmacological and pharmaceutical characteristics (e.g. increase potency, stability, bioavailability, in vivo half-life, decrease side effects, etc. ) .
  • Compounds having CHIPS activity for use in the combinations of the invention in which only one of the original amino acids has been substituted are the following: X1-T-F-E-P-F-X7, F-X2-F-E-P-F-X7, F-T-X3-E-P-F-X7 , F-T-F- X4-P-F-X7, F-T-F-E-X5-F-X7, F-T-F-E-P-X6-X7.
  • XI to X7 are as defined above.
  • a preferred compound having CHIPS activity for use in the combinations of the invention is F-T-F-E-P-F-X7 , wherein X7 may or may not be present.
  • the peptides described in Table 6 are part of the present invention as the compound that has CHIPS activity provided they have CHIPS activity as defined above and as tested in one or more of the CHIPS activity tests as given in the Examples.
  • WTFAFC FFFCFC, FIFCFC, FSFCFC, FTFCFC, WFFCFC WIFCFC,
  • WIFDFC WIFDFC
  • WSFDFC WTFDFC
  • FFFEFC FIFEFC
  • FSFEFC FTFEFC WFFEFC WIFEFC WSFEFC WTFEFC FFFNFC FIFNFC FSFNFC, FTFNFC WFFNFC WIFNFC WSFNFC WTFNFC FFFSFC FIFSFC, FSFSFC FTFSFC WFFSFC WIFSFC WSFSFC WTFSFC FFFWFC, FIFWFC FSFWFC FTFWFC WFFWFC WIFWFC WSFWFC WTFWFC, FFFAIC FIFAIC FSFAIC FTFAIC WFFAIC WIFAIC WSFAIC, WTFAIC FFFCIC FIFCIC FSFCIC FTFCIC WFFCIC WIFCIC, WSFCIC WTFCIC FFFDIC FIFDIC FSFDIC FTFDIC WFFDIC, WIFDIC WSFDIC WTFDIC FFFEIC FIFEIC FSFEIC FTFEIC, WF
  • N-terminal of these peptides may be modified as described above for XI, whereas an X7 group may be present C-terminally .
  • all of these peptides may be used as a starting point for further modification, for example by substitution of one or more of the amino acids with other building blocks.
  • a peptide of the naturally occurring N-terminal sequence of CHIPS i.e. FTFEPF.
  • FTFEPF a "combination of compounds” is to be understood as a mixture of at least one peptide or peptidomimetic that is based on the first six amino acids of the CHIPS protein and has CHIPS activity and one or more other peptides derived from the amino acid sequence of positions 4-121 of CHIPS, wherein the two or more peptides are either present as separate entities (that either stay as separate entities after administration into the body or become attached to each other in the body) or physically attached (either directly or indirectly, such as via a spacer) .
  • the amino acid sequence of the one or more other peptides may be altered as compared to the amino acid sequence occurring in CHIPS in the same manner as described herein for the compound having CHIPS activity.
  • Both types of compounds that are used in the combinations of the invention can be produced by known chemical synthesis.
  • Methods for constructing peptides by synthetic means are known to those skilled in the art. These synthetic peptides, by virtue of sharing primary, secondary and/or tertiary structural and/or confor- ational characteristics with CHIPS will posses an activity in common therewith, meaning CHIPS properties or supportive properties.
  • synthetically produced peptides can be employed as biologically active or immunological substitute for natural purified (poly) peptides having CHIPS activity or supportive properties.
  • the compounds having CHIPS activity provided herein also include peptides characterized by amino acid sequences into which modifications are naturally provided or deliberately engineered.
  • Modifications in the peptide can be made by those skilled in the art using known conventional techniques. Modifications of interest in the CHIPS active peptide sequences may include replacement, insertion or deletion of selected amino acid residues in the coding sequence. The Examples describe such modified synthetic peptides.
  • the potential for use of peptides in drugs may be limited for several reasons. Peptides may for example be too hydrophilic to pass membranes like the cell-membrane and the blood-brain barrier, and may be rapidly excreted from the body by the kidneys and the liver, resulting in a low bioavailability.
  • peptides may suffer from a poor biostability and chemical stability since they may be quickly degraded by proteases, e.g.
  • peptides generally are flexible compounds which can assume thousands of conformations.
  • the bioactive conformation usually is only one of these possibilities, which sometimes might lead to a poor selectivity and affinity for the target receptor.
  • the potency of the peptides may not be sufficient for therapeutical purposes.
  • peptides are sometimes mainly used as sources for designing other drugs, and not as actual drugs themselves. In such case it is desirable to develop compounds in which these drawbacks have been reduced.
  • Alternatives for peptides are the so-called peptidomimetics.
  • Peptidomimetics based on the peptides of the present invention are also part of this application. In that case, one or more of XI to X7 are substituted with peptidomimetic building blocks.
  • peptidomimetics have been described as "chemical structures designed to convert the information contained in peptides into small non-peptide structures", “molecules that mimic the biological activity of peptides but no longer contain any peptide bonds", “structures which serve as appropriate substitutes for peptides in interactions with receptors and enzymes” and as “chemical Trojan horses”.
  • peptidomimetics can be classified into two categories.
  • the first consists of compounds with non-peptidelike structures, often scaffolds onto which pharmacophoric groups have been attached. Thus, they are low molecular-weight compounds and bear no structural resemblance to the native peptides, resulting in an increased stability towards proteolytic enzymes.
  • the second main class of peptidomimetics consists of compounds of a modular construction comparable to that of peptides, i.e. oligomeric peptidomimetics . These compounds can be obtained by modification of either the peptide side chains or the peptide backbone. Peptidomimetics of the latter category can be considered to be derived of peptides by replacement of the amide bond with other moieties.
  • the compounds are expected to be less sensitive to degradation by proteases. Modification of the amide bond also influences other characteristics such as lipophilicity, hydrogen bonding capacity and conformational flexibility, which in favourable cases may result in an overall improved pharmacological and/or pharmaceutical profile of the compound.
  • Oligomeric peptidomimetics can in principle be prepared starting from onomeric building blocks in repeating cycles of reaction steps. Therefore, these compounds may be suitable for automated synthesis analogous to the well-established preparation of peptides in peptide synthesizers.
  • Another application of the monomeric building blocks lies in the preparation of peptide/peptido imetic hybrids, combining natural amino acids and peptidomimetic building blocks to give products in which only some of the amide bonds have been replaced. This may result in compounds which differ sufficiently from the native peptide to obtain an increased biostability, but still possess enough resemblance to the original structure to retain the biological activity.
  • Suitable peptidomimetic building blocks for use in the invention are amide bond surrogates, such as the oligo- ⁇ -peptides (Juaristi, E. Enantioselective Synthesis of b-Amino Acids; Wiley-VCH: New York, 1996) , vinylogous peptides (Hagihari, M. et al . , J. Am. Chem. Soc. 1992,
  • peptoids (Simon, R.J. et al . , Proc. Natl. Acad. Sci. USA 1992, 89, 9367-9371; Zuckermann, R.N. et al., J. Med. Chem. 1994, 37, 2678-2685; Kruijtzer, J.A.W. & Liskamp, R.M.J. Tetrahedron Lett. 1995, 36, 6969-6972) ; Kruijtzer, J.A.W. Thesis; Utrecht University, 1996; Kruijtzer, J.A.W. et al . , Chem. Eur. J.
  • the vinylogous peptides and oligopyrrolinones have been developed in order to be able to form secondary structures ( ⁇ -strand conformations) similar to those of peptides, or mimic secondary structures of peptides. All these oligomeric peptidomimetics are expected to be resistant to proteases and can be assembled in high- yielding coupling reactions from optically active monomers (except the peptoids) .
  • Peptidosulfonamides are composed of ⁇ - or ⁇ - substituted amino ethane sulfonamides containing one or more sulfonamide transition-state isosteres, as an analog of the hydrolysis of the amide bond.
  • Peptide analogs containing a transition-state analog of the hydrolysis of the amide bond have found a widespread use in the development of protease inhibitor e.g. HIV-protease inhibitors.
  • oligomeric peptidomimetics Another approach to develop oligomeric peptidomimetics is to completely modify the peptide backbone by replacement of all amide bonds by nonhydrolyzable surrogates e.g. carbamate, sulfone, urea and sulfonamide groups. Such oligomeric peptidomimetics may have an increased metabolic stability. Recently, an amide-based alternative oligomeric peptidomimetics has been designed viz. N-substituted Glycine-oligopeptides, the so-called peptoids.
  • Peptoids are characterized by the presence of the amino acid side chain on the amide nitrogen as opposed to being present on the ⁇ -C-atom in a peptide, which leads to an increased metabolic stability, as well as removal of the backbone chirality.
  • the absence of the chiral ⁇ -C atom can be considered as an advantage because spatial restrictions which are present in peptides do not exist when dealing with peptoids.
  • the space between the side chain and the carbonyl group in a peptoid is identical to that in a peptide. Despite the differences between peptides and peptoids, they have been shown to give rise to biologically active compounds.
  • the invention thus furthermore relates to combinations of molecules that are not peptides themselves but have a structure and function similar to those of the peptides described herein.
  • Such molecules are the above described peptidomimetics, but also compounds in which one or more of XI to X7 are replaced by non-proteinogenic amino acids or D-amino acids.
  • peptides it is intended to include also such other compounds that have a similar or the same structure and function and as a consequence a similar or the same biological activity as the peptides.
  • non-proteinogenic amino acids selected from the group consisting of 2-naphtylalanine (Nal (2)), ⁇ - cyclohexylalanme (Cha), p-amino-phenylalanine ((Phe(p- NH 2 ) , p-benzoyl-phenylalanine (Bpa) , ornithine (Orn) , norleucine (Nle) , 4-fluoro-phenylalanine (Phe(p-F)), 4- chloro-phenylalanine (Phe(p-Cl)), 4-bromo-phenylalanine (Phe(p-Br)), 4-iodo-phenylalanine (Phe(p-I)), 4-methyl- phenylalanine (Phe(p-Me)), 4-methoxy-phenylalanine (Tyr(Me)), 4-nitro-phenylalanine (Phe(p-N02) )
  • Suitable D-amino acids for substituting the amino acids in the peptides of the invention are for example those that are selected from the group consisting of D-phenylalanine, D-alanine, D-arginine, D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid, D- glutamine, D-histidine, D-isoleucine, D-leucine, D- lysine, D-methionine, D-proline, D-serine, D-threonine, D-tryptophan , D-tyrosine, D-valine, D-2-naphtylalanine (D-Nal(2)), ⁇ -cyclohexyl-D-alanine (D-Cha) , 4-amino-D- phenylalanine (D-Phe(p-NH 2 ) ) , p-benzoyl-D-phenylalanine (D-Bpa
  • N-substituted glycines such as N-benzylglycine (NPhe) , N-methylglycine (NAla) , N-(3- guanidinopropyl) glycine (NArg), N- (Carboxymethyl) glycine (NAsp) , N- (carbamylmethyl) glycine (NAsn) , N- (thioethyl) - glycine (NhCys) , N- (2-carboxyethyl) glycine (NGlu) , N-(2- carbamylethyl) glycine (NGln) , N- (imidazolylethyl) glycine (NhHis) , N-(l-methylpropyl) glycine (Nile), N-(2-methyl- propyl)
  • N-substituted glycines such as N-benzylglycine (NPhe) , N
  • peptides having CHIPS activity may be combined with supportive non-peptides or with supportive peptides, whereas non- peptides having CHIPS activity may be combined with supportive peptides or supportive non-peptides or combinations thereof.
  • both categories may be hybrid compounds (i.e. compounds in which not all proteinogenic amino acids are replaced by non- proteinogenic amino acids, D-amino acids or peptidomimetic building blocks) .
  • the functional activity of compounds of the invention can be assayed by various methods.
  • This CHIPS activity of a compound can be measured by its ability to prevent the binding of fluorescent-fMLP (such as Bodipy-fMLP) or fluorescent-C5a (such as FITC-C5a) to neutrophils as determined by flow cytometry.
  • CHIPS activity is also measured by its ability to prevent migration of neutrophils towards fMLP or C5a as determined by chemotaxis assays, such as the Transwell system.
  • an assay based on the ability of chemokines, including fMLP and C5a, to initiate a rapid and transient rise in intracellular calcium concentration can be employed to screen for CHIPS activity, as described in Materials and Methods.
  • an assay based on the ability of chemokines, including fMLP and C5a, to initiate an excretion of e.g. elastase in cytochalasin B-stimulated neutrophils can be used to screen for CHIPS activity, as described in Materials and Methods.
  • chemokines including fMLP and C5a
  • Various other assays known in the art can be used, including but not limited to the use of various calcium specific fluorescent probes in combination with flow cytometry or fluorometry, or microphysiometry .
  • cells for the screening of CHIPS activity by either method e.g. freshly isolated neutrophils can be used or cells transfected with either FPR or C5aR, wild type or mutated forms of those receptors.
  • the supportive capacity of the other compound can be tested in the same manner as described above by comparing the CHIPS activity of the first compound having CHIPS activity alone with the CHIPS activity of the combination of the two compound.
  • a compound has supportive capacity in case the CHIPS activity of the combination is higher than the CHIPS activity of the first compound alone.
  • Combinations of compounds of the invention may be useful in treating, preventing or ameliorating inflammatory conditions that are involved in many diseases and disorders.
  • the combination of compounds of the invention may for example be used in the treatment or prophylaxis of indications involving the C5a-receptor (C5aR) and/or for ylated peptide receptor (FPR) on neutrophils, monocytes and endothelial cells.
  • C5aR C5a-receptor
  • FPR ylated peptide receptor
  • Such indications often involves acute or chronic inflammation reactions, such as found in cardiovascular diseases, in particular arteriosclerosis, atherosclerosis, vasculitis, ischaemia reperfusion episodes, ischaemic shock, cardiopulmonary bypass, (large) vessel surgery, transplantation, myocardial infarction, diseases of the central nervous system, in particular bacterial meningitis, viral meningitis, multiple sclerosis, stroke, Alzheimer's disease, brain tumour, (traumatic) brain injury, gastrointestinal diseases, in particular pancreatitis, ulcerative colitis, Crohn's disease, alcoholic hepatitis, viral hepatitis, Heliobacter pylori gastritis, gastric carcinoma, peritonitis, skin diseases, in particular psoriasis, contact dermatitis, atopic dermatitis, and other dermatoses, cutaneous T-cell lymphoma, burns, genitourinary diseases, in particular urinary tract infection, glomerulonephritis, Trichomonas vaginalis
  • combination of compounds of the invention may be used in the prophylaxis or treatment of indications involving CXCR4 and/or CCR5 receptors on leukocytes, in particular HIV infection.
  • the combinations of compounds of the invention are also useful in the treatment of indications involving C5aR and/or FPR on cells other than neutrophils, monocytes and endothelial cells, in particular lymphocytes, dendritic cells, eosinophils, basophils, macrophages, microglia cells, astrocytes, Kupfer cells, hepatocytes and epithelial cells.
  • An additional application is the use in prophylactic or therapeutic vaccines (to induce the production of anti-CHIPS antibodies to decrease the in vivo CHIPS activity, thereby stimulating the natural inflammation response against CHIPS producing bacteria) for infections with
  • CHIPS-producing bacteria such as Staphylococcus aureus.
  • Combinations of compounds of the invention may be used themselves as inhibitors of fMLP and C5a binding to their respective receptors FPR and C5aR, or to design inhibitors of CHIPS binding, by screening for competitive inhibition. This can be performed by various approaches, some of which are described hereunder.
  • Inhibitors of CHIPS binding to the putative CHIPS receptor or receptor domains having CHIPS activity (CHIPS agonists) are also useful for treating inflammation conditions and HIV.
  • the invention furthermore relates to the use of the combination of compounds having CHIPS activity for the manufacture of a composition or preparation for prophylaxis or therapy, in particular for the prophylaxis or treatment of acute and chronic inflammation reactions and HIV infection, more in particular for the treatment of the indications listed above.
  • compositions comprising a suitable excipient and one or more of the combinations of compounds having CHIPS activity of the invention.
  • Such composition can be used for the treatments as specified above.
  • the invention further relates to use of the combinations of compounds, wherein the separate compounds, such as peptides, are optionally incorporated in or fused to a larger protein, for various purposes, such as raising antibodies thereto, modulating the CHIPS activity or in a therapeutic preparation.
  • An antibody or biologically active fragment thereof specifically directed to one or both of the compounds in the combinations as claimed is also part of this invention.
  • Such antibodies or biologically active fragments thereof like Fab, scFv fragments etc. can be used in the diagnosis, treatment or prophylaxis of infections of CHIPS producing bacteria, such as Staphylococcus aureus.
  • the information in the compounds of the combinations of the invention and the combinations of compounds themselves can be used to screen for other agents which are capable of interfering (e.g. through binding to CHIPS, thereby blocking the sites that CHIPS uses to bind to and/or activate its receptor or changing the CHIPS conformation such that the binding and/or activating capacity to, respectively of its receptor is impaired) with peptides having CHIPS activity.
  • agents thus may act as inhibitors of CHIPS binding to and/or activation of its putative receptor, thereby improving for instance the neutrophil 's activation and chemotactic capacities in the presence of combinations of compounds having CHIPS activity.
  • Appropriate screening assays may for example use the labeled (e.g.
  • a suitable binding assay may alternatively employ purified CHIPS receptor or receptor domain on a carrier with a form of CHIPS protein as ligand.
  • an assay can be employed that screens for the ability to bind or compete with CHIPS for binding to a specific anti-CHIPS antibody (monoclonal, polyclonal, or single chain antibody) by various immunoassays known in the art, including but not limited to competitive and non-competitive ELISA techniques or Biosensor technology employing a sensor chip coated with either ligand
  • CHIPS CHIPS
  • SPR Surface Plasma Resonance
  • Any (poly) peptide having CHIPS activity other than CHIPS may also be used in the screening assays described. All these methods can be adapted for High Throughput Screening (HTS) .
  • Combinations of compounds interfering with CHIPS or with combinations of compounds of the invention are also part of this application.
  • Such interfering compounds can be used in the diagnosis, treatment or prophylaxis of infections of CHIPS producing bacteria, such as Staphylococcus aureus.
  • the information in the combination of compounds and the combinations of compounds themselves can be used for other agents (including antibodies or fragments thereof) which are capable of interfering (e.g. through binding on the CHIPS receptor or changing the conformation of the CHIPS receptor) with the CHIPS receptor, without having (or only partial) CHIPS activity themselves.
  • Such (partial) CHIPS antagonists may be found using appropriate screening assays. For example using fluorescent labeled purified CHIPS protein or peptide in various competitive CHIPS receptor binding and/or functional inhibiting screening assays, some of which have been mentioned above.
  • CHIPS receptor interfering compounds and such antibodies and such CHIPS interfering compounds that are all based on the combination of compounds of the invention, are for example useful for the prophylaxis or treatment of CHIPS producing bacteria, such as Staphylococcus aureus infection.
  • Both the antibodies and their biologically active fragments and/or the CHIPS interfering compounds and/or the CHIPS receptor interfering compounds are useful for the preparation of a coating composition for use on surfaces of medical devices that are introduced into the human body through the skin or open surgical procedures, such as the surface of a catheter tip.
  • the composition comprising the compounds that inhibit CHIPS activity or combinations thereof is a slow- release composition.
  • the invention further relate to therapeutic, diagnostic or prophylactic compositions comprising a suitable excipient and one or more antibodies and/or biologically active fragments thereof and/or one or more interfering compounds .
  • Also part of the invention are methods for prophylaxis or treatment of a subject suffering from indications involving C5aR and/or FPR on neutrophils, monocytes and endothelial cells comprising administering a therapeutically effective amount of the combinations of compounds of the invention.
  • a further embodiment of the invention relates to a method for the prophylaxis or treatment of a subject suffering from indications involving CXCR4 and/or CCR5 receptors on lymphocytes comprising administering a therapeutically effective amount of the combinations of compounds of the invention.
  • the indication to be treated is for example HIV infection.
  • the invention relates to methods for prophylaxis and treatment of a subject suffering from indications involving C5aR and/or FPR on leukocytes other than neutrophils, monocytes and endothelial cells comprising administering a therapeutically effective amount of the combinations of compounds of the invention.
  • the other leukocytes are for example lymphocytes, dendritic cells, eosinophils, basophils or macrophages.
  • the invention further relates to amino acid sequences that can be identified by so-called "computer cloning".
  • this technique comprises of using the amino acid sequences of peptides used in the combinations of the invention, derivatives, and analogues thereof, as a query for screening protein sequences or protein sequence databases, using search algorithms that can identify regions with homology.
  • search algorithms are known to the person skilled in the art and include, but are not limited to, BLAST searches (Altschul et al . , J. Mol. Biol. 215, 403-410 (1990)).
  • the amino acid sequence database that may be searched include, but are not limited to, the SwissprotTM database.
  • BLAST search or modifications thereof generally subjects that display homology can be identified. Identification is based on the value of the Score or the Smallest Sum Probability P(N).
  • the combination of compounds as claimed can also be used in a diagnostic kit for determining the presence of CHIPS in the body.
  • the invention thus also relates to a diagnostic kit for determining the presence of CHIPS in the body, comprising a combination of compounds of the invention.
  • Such isolation can for instance be performed using the yeast two hybrid system or using tagged molecules of the invention as bait for fishing.
  • All compounds that are used in the combinations of the invention can be labeled in any way.
  • labeling include but are not limited to fluorescence, biotin, radioactive labeling etc.
  • Such labeled molecules can be used for screening of compounds that resemble or overlap with the biological activity of CHIPS, as well as identification of binding sites, both in vivo and in vitro, and for tracing CHIPS protein in an organism.
  • Figure 1 shows the sequence of the chp gene from S. aureus Newman.
  • the chp open reading frame (ORF) is underlined.
  • Figure 2 shows the amino acid sequence deduced from the S . aureus Newman chp gene.
  • Figure 3 shows the effect of the 5 amino acid peptide scan on the fMLP- (A) and PAF-induced (B) calcium mobilization in neutrophils.
  • Figure 4 shows the concentration-dependent inhibition of the fMLP- and PAF-induced calcium mobilization in neutrophils by pepl-15.
  • Figure 5 shows the specific effect of pepl-15 on the fMLP- C5a- and PAF-induced calcium mobilization in neutrophils.
  • Figure 6 shows the effect of different lengths of CHIPS-derived peptides, ranging from pepl-5 to pepl-16 as depicted in Table 2 , on the fMLP-induced elastase release in neutrophils.
  • Figure 7 shows the effect of a one amino acid peptide scan, spanning the amino acids 1 to 22 of CHIPS as depicted in Table 2, on the fMLP-induced calcium mobilization in neutrophils.
  • Figure 8 shows the effect of the addition of small amino acids to the N-terminal phenylalanine of CHIPS-derived pepl-13 on the fMLP-induced calcium mobilization in neutrophils.
  • Figure 9 shows the effect of exchanging the first phenylalanine of pep 1-8 with non-proteinogenic amino acids, D-amino acids, peptidomimetic building blocks, and acetylation of the N-terminus on the fMLP- induced elastase release in neutrophils.
  • Figure IOA shows the effect of rCHIPS 1"121 and rCHIPS 4'121 on fMLP Bodipy binding.
  • Figure 10B shows the competition between rCHIPS 1"121 and rCHIPS 4"121 for CHIPS-FITC binding.
  • Figure 11 shows the structure of the various types of peptidomimetic building blocks.
  • Table 1 shows the sequences of 15 mer synthetic peptides derived from CHIPS that span the entire CHIPS protein.
  • Table 2 shows the sequences of synthetic peptides derived from CHIPS that vary in length from 1 to
  • pepl-5 till pepl-40 that vary in length from 1 to 16 by one amino acid (pepl-5 till pepl-16) , and a one amino acid peptide scan spanning amino acids 1 to 22 of CHIPS, and their effect on the fMLP-induced response in neutrophils (+) .
  • Table 3 (page 1, 2, and 3) shows the sequences of CHIPS-derived peptide 1-10, containing all 20 available natural occurring amino acid exchanges at each position. Substitutions are in gray boxes and the substitutions generating the original pepl-10 are in bold.
  • Table 4A shows the summary of the pepl-10 single amino acids exchanges as determined by their effect on the fMLP-induced elastase release in neutrophils in a 100 ⁇ M concentration.
  • Table 4B shows the summary of the pepl-10 single amino acids exchanges as determined by their effect on the fMLP-induced elastase release in neutrophils in a 10 ⁇ M concentration.
  • Table 5 shows the sequences of cyclic synthetic peptides derived from CHIPS that have two cysteine (C) substitutions in pepl-20 or pepl-10, and their effect on the fMLP-induced elastase release in neutrophils (+) .
  • DNA was sequenced by cycle sequencing on a DNA sequencer 4000 L (LI-COR Inc., Lincoln, Neb., USA) using the Thermo SequenaseTM fluorescent-labeled prime cycle sequencing kit (Amersham, Little Chalfont, UK) . Suitable primers were used to directly sequence genomic DNA which was isolated according to J. Mamur (J. Mol. Biol., 3:208- 218 (1961)). The sequencing method has been described briefly in Peschel et al . (J. Biol. Chem., 274:8405-8410 (1999)). To perform sequence similarity searches, the program BLAST 2.0 with the non-redundant protein database of the NCBI (Bethesda, Md. , USA) was used. Sequence alignments were accomplished using the Higgins-Sharp algorithm of the program MacDNASIS Pro (Hitachi Software Engineering, San Bruno, Calif., USA).
  • the first 35 amino acids of CHIPS have been determined by N-terminal sequencing of the purified protein (PCT/NL99/00442) .
  • the S . aureus DNA is very rich in A and T nucleotides while G and C nucleotides are rare (only about 30% of total bases) .
  • a primer sequence was derived from amino acids 15-24 (GAAAAAGAAAAAGCATATAAAG-AA (SEQ ID NO 1) ) .
  • the primer was used to directly sequence genomic DNA from S . aureus Newman (a commonly used laboratory strain) yielding a sequence of several hundred base pairs.
  • a new primer was derived from this sequence to read toward the binding site of the first primer.
  • the combined DNA sequence contained the binding site of the first primer with two differences (G instead of A in position 3 and T instead of A in position 15) . It encoded an open reading frame of 450 bp preceded by a reasonable Shine Dalgarno sequence for initiation of translation (J. Shine and L. Dalgarno, Proc. Natl. Acad. Sci. USA, 71:1342-1346 (1974)) and followed by three stop codons ( Figure 1 with the gene underlined) . The gene was named chp; it encodes a putative protein of 149 amino acids with no similarities to any protein in the databases.
  • the N-terminal 28 amino acids seem to form a signal peptide for secretion across the cytoplasmic membrane (3 positively charged residues followed by a non-charged region of 22 amino acids and an ALA-X-ALA consensus motive for cleavage by the signal peptidase 1 ; Figure 5) (G. von Heijne, Nucl. Acids Res. 14:4683-4690 (1986)).
  • the signal peptide is followed by a region that matches almost perfectly the N-terminal 35 amino acids of CHIPS.
  • the only exception is a serine in position 33 of the deduced mature protein instead of an asparagine residue predicted by N-terminal sequencing.
  • the deduced mature protein has a size of 121 amino acids and 14.1 kDa and an isoelectric point of 9.32. It thus fulfills all requirements for the CHIPS protein.
  • the derived amino acid sequence is shown in Figure 2.
  • the vector contains an N-terminal polyhistidine (6xHis) tag for rapid purification, a Xpress epitope for easy detection with an anti-Xpress antibody and an Enterokinase cleavage site for removal of fusion tag. This leaves the purified CHIPS protein with an N-terminal phenylalanine (F) .
  • 6xHis polyhistidine
  • F N-terminal phenylalanine
  • the PCR product is digested with EcoRI and the pTrcHIS B vector with BamHI .
  • the 5 ' overhang is removed with Sl-nuclease to make the BamHI site blunt ended exactly where the enterokinase (EK) will digest the protein.
  • EK enterokinase
  • the vector is digested with EcoRI and ligated with the digested PCR product.
  • TOP-10 E.coli is used (InVitroGen) using standard calcium precipitation (F.M. Ausubel et al . , 1990, Current Protocols in Molecular Biology, John Wiley and Sons, Inc. , New York, N.Y.).
  • Clones are screened on Ampicillin containing plates and proper ligation of CHIPS gene is verified by sequencing of the isolated plasmid (clone29) .
  • the E. coli bacteria are lysed and the protein mixture is applied onto a Nickel-ion affinity column (ProBond) . Therefore a culture of clone29 in LB medium + 50 ⁇ g/ml Ampicillin is initiated with 1 mM IPTG for 4 h at 37 °C. Bacteria are centrifuged and the pellet resuspended in cold Phosphate buffer pH 7.8 and stored at in -20 °C. For cell lysis, lysozyme (100 ⁇ g/ml) is added for 15 min on ice, tubes are sonicated, frozen in liquid N 2 and thawed in a 37 °C waterbath.
  • lysozyme 100 ⁇ g/ml
  • This cycle of sonication/freeze/thaw is repeated another 3 times. Thereafter RNase and DNase (5 ⁇ g/ml) is added for 30' on ice. The mixture is centrifuged for at 3000 g for 30' at 4°C and filtered through a 0.45 ⁇ m filter. The final lysate is diluted 1:1 with cold Phosphate buffer pH 7.8 and run through a charged Nickel column (InVitroGen) . The column is washed with Phosphate buffer pH 7.8, with Phosphate buffer pH 6.0 and with Phosphate buffer pH 5.3. The bound CHIPS is eluted with 500 mM imidazole in pH 6.0 Phosphate buffer.
  • the HIS-tag is removed by enterokinase cleavage followed by removal of the protease with an EK-Away enterokinase affinity resin. Therefor the eluate is dialysed overnight in cold digestion buffer (50 mM Tris- HCl, 1 mM CaCl 2 and 0.1% Tween-20, pH 8.0), filtered through a 0.45 ⁇ m filter and digested with 0.175 ⁇ l Enterokinase/ l HIS-CHIPS product. This amount of Enterokinase is batch-dependent and results in a partial digestion to avoid the generation of breakdown products.
  • the digested product is dialyzed against Phosphate buffer pH 7.8 and passed over a fresh Nickel column to eliminate uncleaved HIS-CHIPS; the run through is pure rCHIPS. Undigested HIS-CHIPS can be eluted again from Nickel column for a second digestion round. The Nickel column is finally washed with 50 mM EDTA, 0.5 M NaOH, water, 5 mg/ l NiCl 2 , water and stored in 20% ethanol.
  • HIS-CHIPS All steps in the isolation and digestion of HIS-CHIPS are checked by SDS-PAGE on a 16.5% Tris-Tricine Ready gel using the Mini-Proteanll system (BioRad) .
  • Peptides were prepared by automated simultaneous multiple peptide synthesis, set up by using a standard autosampler (Gilson 221) . Standard 9- fluorenylmethoxycarbonyl chemistry with in situ PyBop/N- methylmorpholine (Novabiochem, Laufelfingen, Switzerland) activation of the amino acids in a fivefold molar excess with respect to 2 ⁇ mol/peptide PAL-PEG-PS resin (Perseptive Biosystems, Framingham, Mass.) was used. Peptides were obtained as C-terminal amides after cleavage with 90 to 95% trifluoroacetic acid-containing scavenger cocktails. Most peptides were dissolved in distilled H 2 0 to a concentration of 1 to 3 mM; others were dissolved in dimethyl sulfoxide. References describing similar methods are:
  • N -Fmoc-protected amino acids were purchased from Alexis (Switzerland) .
  • the following side chain protecting groups were used: Tyr(t-Bu), Lys(Boc), Thr(t-Bu), Ser(t-Bu),
  • the Fmoc-protected N-substituted glycines were synthesized according to Kruijtzer et al. (Kruijtzer, J.A.W., Hofmeyer, L.J.F., Heerma, W. , Versluis, C. , & Liskamp, R.M.J. (1998) Chem. Eur. J. 4, 1570-1580; and Kruijtzer, J.A.W., Synthesis of Peptoid Peptidomimetics, Thesis, Utrecht University, The Netherlands, 1996) .
  • the oligomers were synthesized on a
  • MultiSynTech Syro II Robot Synthesizer MultiSynTech, Witten, Germany
  • HPLC runs were performed on two HPLC systems: a Gilson automated HPLC workstation with UV detector system operating at 214 nm and 278 nm, or a Shimadzu automated HPLC system with a UV detector system operating at the same wavelengths.
  • Analytical HPLC was conducted by using an Alltech Adsorbosphere XL C18 (300 A, 5 ⁇ m, 250 x 4.6 mm) column at a flow rate of 1.0 mL min-1. Elution was effected with a gradient from 0.1% TFA in water/acetonitrile (90/10) to 0.1% TFA in acetonitrile/water (90/10) over 40 minutes.
  • Electrospray ionization mass spectrometry was measured on a Shimadzu LCMS-QP8000 single quadrupole bench-top mass spectrometer operating in a positive ionization mode.
  • MS full scan spectra data were acquired in continuum mode over the range m/z 200 to 1500 in 1 second at unit mass resolution.
  • a deflector voltage of 50 V was set, which produces predominantly (M+H)+ ions.
  • Instrumental control, data acquisition and data processing were carried out using the CLASS 8000 software package (version 1.10). The observed average m/z was compared with the calculated (M+H)+ average mass.
  • the oligomers were synthesized on 0.05 mmol scale in a polypropylene reaction tube equipped with a polypropylene frit.
  • the syntheses were carried out on ArgoGelTM Rink-NH-Fmoc resin (0.32 mmol/g) to obtain C- terminal amides.
  • the synthesis started with 156 mg dry resin.
  • the resin was swollen in DCE (two times with 2 mL for 4 min) followed by NMP (two times with 2 mL for 4 min) .
  • the Fmoc group was removed by a double treatment with 2 mL 20% piperidine in NMP during 8 min. Subsequently, the resin was extensively washed with NMP (five times with 2.5 mL for 2 min).
  • the resin was washed with NMP (five times with 2.5 mL for 2 min) and DCE (three times with 2.5. L for 2 min).
  • the anchored oligomers thus obtained were cleaved from the resin and deprotected by treatment with 2 mL TFA/H 2 0/TIS (95:2.5:2.5, v/v/v) solution for 2 h at room temperature.
  • the oligomers were precipitated with MTBE/n-hexane (1:1, v/v, 50 mL) .
  • the precipitate was collected by centrifugation (3000 rpm, 5 min) , the supernatant was decanted, and the pellet was resuspended in MTBE/n-hexane (1:1, v/v) and centrifuged again. Subsequently, the pellet was washed two times with MTBE (50 mL) . After this, the pellet was dissolved in tert . -BuOH/water (1:1, v/v, 5-10 mL) and lyophilized to obtain the oligomers as a white fluffy solid. The purity was analyzed by analytical HPLC and the hybrids were characterized by mass spectrometry .
  • CHIPS and the synthetic peptides on the intracellular free calcium release induced by fMLP, C5a and PAF (Platelet Activating Factor) in neutrophils was tested as follows. Cells were loaded with 1 ⁇ M of a calcium specific intracellular probe (Fluo-3, acetoxymethyl (AM) ester; Molecular Probes) , washed and incubated with various concentrations of synthetic peptides (1 to 100 ⁇ M) or rCHIPS (1 ⁇ g/ml) for 15 min at room temperature. From each sample the initial fluorescence value was determined in the FACScan by measuring 2000 cells.
  • a calcium specific intracellular probe Fluo-3, acetoxymethyl (AM) ester; Molecular Probes
  • Human neutrophils contain enzymes in their granules, among which elastase. Furthermore, the granules contain the fMLP receptors ready for quick recruitment to the cell surface. Cytochalasin B treatment of neutrophils stimulates the excretion of the granules, which results in an increase of the amount of fMLP receptors expressed on the cell surface of the neutrophils. Upon subsequent stimulation with fMLP, the neutrophils will effectively excrete their granule content into the medium, thereby releasing elastase. CHIPS and CHIPS peptides will inhibit the activation of the neutrophils with fMLP, which can be measured via a decrease in elastase release.
  • the amount of elastase is determined via a specific enzymatic reaction using the fluorescent substrate methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-MAC (elastase substrate V; Calbiochem) .
  • Figure 3 shows the effect of these peptides spanning the CHIPS on the fMLP- (A) and PAF-induced (B) calcium mobilization in neutrophils. It can be seen that pepl-15 and pep61-75 strongly inhibit the fMLP-induced calcium mobilization in neutrophils. The effect of pep61-75 seems to be non-specific for fMLP, as it also inhibits the PAF- induced response. This might be due to a non-specific toxicity of this peptide. Concluding, only pepl-15 shows CHIPS activity.
  • Figure 4 shows the concentration- dependent inhibition of the fMLP-induced calcium mobilization by pepl-15.
  • Figure 5 shows the effect of pepl-15, on the fMLP- ,C5a- and PAF-induced calcium mobilization in neutrophils and confirms the specificity of pepl-15 and rCHIPS for the fMLP- and C5a-induced response.
  • peptides of increasing length spanning amino acid 1 to 40 were chosen all starting with the first phenylalanine (see Table 2) , including newly synthesized pepl-15.
  • Peptides were prepared according to method two (MATERIALS AND METHODS section 2.2).
  • Table 2 shows the effect of different lengths of CHIPS-derived peptides on the fMLP-induced calcium mobilization in neutrophils. This table shows that at least the first 8 amino acids are necessary for CHIPS activity, as pepl-5 shows no CHIPS activity in contrast to pepl-8 and all other depicted longer peptides. No improved activity of longer peptides was observed using different concentrations.
  • Figure 6 shows the effect of different lengths of CHIPS-derived peptides, as depicted in Table 2, and their effect on the fMLP-induced elastase release in neutrophils.
  • This Figure shows that at least the first 6 amino acids are necessary for CHIPS activity, as pepl-5 shows no CHIPS activity in contrast to pepl-6 and all other depicted longer peptides.
  • peptides were constructed spanning the amino acids 1 to 13 of CHIPS with an additional amino acid before the first phenylalanine. Peptides were prepared according to method one (MATERIALS AND METHODS section 2.1).
  • Figure 8 shows the effect of the peptides spanning CHIPS 1 to 13 with additional N-terminal amino acids attached on the fMLP-induced calcium mobilization in neutrophils. It can be seen that blocking the CHIPS N- terminal phenylalanine with the chosen small amino acids Ala (alanine) , Gly (glycine) , Pro (proline) , Ser (serine) , Thr (threonine) or Val (valine) completely abrogated effective inhibition of the fMLP-induced calcium mobilization by the non-blocked pepl-15. The PAF- induced response is not affected by any peptide nor rCHIPS .
  • Peptides were prepared according to method two or three (MATERIALS AND METHODS section 2.2 and 2.3). The peptides were tested in the elastase release assay in a concentration of 100 ⁇ M (optimal) as well 10 ⁇ M (suboptimal) to find the most potent combinations. Individual substitutions also resulted in the original pepl-10 that were indeed positive for each newly synthesized peptide (in bold) .
  • Tables 4A (100 ⁇ M) and 4B (10 ⁇ M) summarizes the positive single amino acid substitutions in pepl-6 (the minimal active part of CHIPS) .
  • 200 individual combinations are synthesized chosen at random from the 2352 possibilities as examples for the total combinations.
  • Non-proteinogenic, natural occurring (meaning non-human) amino acid substitutions were performed on the CHIPS-derived peptide comprising of the first 8 amino acids of CHIPS.
  • Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3).
  • the first phenylalanine (Phe) of pepl-8 was replaced by the structurally related, non-proteinogenic amino acids Nal (2) 2-naphtylalanine) , Cha (cyclohexylalanme) , Phe(p-NH 2 ) (p-amino-phenylalanine) and Bpa (p-benzoyl-phenylalanine) .
  • Figure 9 shows the effect of these exchanges on the fMLP-induced elastase release in neutrophils.
  • D-amino acid substitutions were performed on the CHIPS- derived peptide comprising of the first 8 amino acids of CHIPS.
  • Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3). The first phenylalanine (Phe) of pepl-8 was replaced by D-Phe and tested in the fMLP-induced elastase release in neutrophils. Exchange for D-Phe results in a somewhat less active pepl-8 as shown in Figure 9. 2.4 Peptidomimetic substitutions.
  • pepl-8 The first phenylalanine (Phe) of pepl-8 was replaced by the peptidomimetic oligomer Nphe (N- Benzylglycine) and tested in the fMLP-induced elastase release in neutrophils. Exchange for NPhe results in an equally active pepl-8 as shown in Figure 9.
  • the first phenylalanine (Phe) of pepl-8 was acylated by the introduction of an N-terminal acetyl group (Ac-Phe) .
  • This peptide was tested in the fMLP- induced calcium mobilization in neutrophils and showed diminished CHIPS activity of pepl-8 as shown in Figure 9.
  • Granulocytes are isolated from heparinized blood of healthy volunteers via a Histopaque-Ficoll gradient in accordance with the standard method
  • BODIPY-labeled fMLP (final concentration 0.1 ⁇ M; Molecular Probes) is then added and the sample is incubated for 60 minutes on ice.
  • RPMI/HAS is added to a final volume of 200 ⁇ l and the fluorescent FMLP binding to the granulocytes is analysed with a flow cytometer (FACScan; Becton Dickinson) .
  • the average fluorescence value of 5000 granulocytes is calculated with LysisII software (Becton Dickinson) .
  • Figure IOA shows the effect of CHIPS on the binding of fMLP-Bodipy to the cells.
  • the complete rCHIPS 1" 121 abrogates concentration dependent the binding of fMLP- Bodipy. This is the characteristic feature of CHIPS activity.
  • the mutant rCHIPS 4"121 in the same concentration range tested did not affect the binding of fMLP-Bodipy to the cells, indicating this mutant has no CHIPS activity.
  • CHIPS-FITC 20 ⁇ g/ml
  • various concentrations unlabeled rCHIPS or mutant rCHIPS 4"121 for 15 min at room temperature.
  • 50 ⁇ l granulocytes (5 x 10 6 cells/ml) are added to the tubes and incubated for 60 min on ice.
  • Cells are washed once with medium (RPMI containing 0.05% HSA) and resuspended in 200 ⁇ l fresh medium.
  • Binding of CHIPS- FITC to the leukocyte is measured by flow cytometry (FACScan; Becton Dickinson) .
  • the average fluorescence value of the cells is calculated with LysisII software (Becton Dickinson) .
  • FIG. 10B shows the concentration dependent inhibition of CHIPS-FITC binding by both the complete rCHIPS 1"121 as well as the mutant rCHIPS 4"121 . Both preparations show a similar inhibition pattern with equal effective concentrations. This indicates that the mutant rCHIPS 4"121 still can bind to the cells, but lacks the intrinsic CHIPS activity ( Figure 10A) . These results point to a multi-site model for CHIPS that involves one or more supportive sites (in the 4 to 121 part of CHIPS) and a activity site that comprises the first 6 amino acids as shown with the peptides. Table 1

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Abstract

The invention relates to a combination of compounds, wherein one compound has CHIPS activity and consists of at least 6 building blocks the sequence of which is based on the N-terminal part of the CHIPS molecule, and which sequence may comprise one or more substitutions as compared to the original CHIPS amino acid sequence, whereas the one or more other compounds are derived from the aminoa cids 4-121 of CHIPS and supports CHIPS activity. One or more of the amino acids in the amino acid sequence of the one or more supportive compounds may be replaced by building blocks selected from the group consisting of proteinogenic amino acids, non-proteinogenic amino acids, D-amino acids and peptidomimetic building blocks.

Description

COMBINATION OF CHIPS (CHEMOTAXIS INHIBITING PROTEIN FROM STAPHYLOCOCCUS AU- REUS)-BASED COMPOUNDS
The present invention relates to combinations of peptides derived from the CHemotaxis Inhibitory Protein from Staphylococcus aureus (CHIPS) having CHIPS activity. The invention in addition relates to the use of non-peptide molecules having peptide structural features and a similar function as the peptides in such combinations. The combination of compounds (peptide and non-peptide molecules) can be used in the prophylaxis and treatment of various indications, such as inflammation reactions. The peptides and non-peptides can in addition be used for inhibiting activation of leukocytes and endothelial cells. Leukocytes are mainly involved in protecting the body against foreign invaders (e.g. bacteria, viruses, fungi, and cancer cells) . The most important cells are lymphocytes, monocytes and neutrophils. Lymphocytes form the specific immune system and cause immune reactions against invaders. Their most important task is to build up specific memory against the invader, so that the next time the invader enters the body it is recognized, killed and removed rapidly. Sometimes these lymphocytes not only attack invaders, but also react against certain structures and/or molecules (so-called auto-antigens) of the own body, causing auto-immune diseases (e.g. rheumatoid arthritis) . Killing and removal of invaders is mostly done by monocytes and neutrophils, cells of the innate immune system, by direct recognition of the invaders or with the help of specific lymphocytes.
In contrast to the delicate network of the fine-tuned and controlled reactions of lymphocytes, cells of the innate system react in a relatively non-specific and aggressive way. Since they are part of the body's first line of defense, their most important task is to kill and remove the invading agent as quickly as possible. This is accomplished through very aggressive substances (e.g. free radicals and enzymes) that are not only lethal to the invader, but also cause damage to host cells in the vicinity. Substances from these damaged cells and the locally activated cells from the innate system itself will further attract increasing numbers of neutrophils and monocytes, causing local inflammation. In most cases, such an aggressive and damaging inflammatory reaction, caused by overactivated neutrophils, is unnecessary. In some cases this inflammatory response is responsible for serious, sometimes lethal disorders and includes conditions like Adult Respiratory Distress Syndrome (ARDS) , severe tissue damage following thrombotic events such as heart attacks and stroke, inflammatory bowel diseases and rheumatoid arthritis. The inflammation will subside once all the invaders have been killed and removed, together with the various cells killed in the process. Healing of the wound, caused by the inflammatory response, can then begin. Although there is some overlap in function, the main task of neutrophils is to attack the invaders and the main task of monocytes is to remove the debris resulting from this attack. In addition, neutrophils have another peaceful task in assisting the wound healing process.
When bacteria have invaded the body and, for example, infected the central nervous system (as in meningitis) they start to produce microbial substances, including the for ylated polypeptides (like the fMLP peptide) . Other substances of microbial origin activate the complement factor 5 (C5) convertase enzyme-complex, that converts C5 of the complement system into its activated C5a form. Both C5a and fMLP are chemo-attractants : substances that can activate and attract cells from the blood vessels (the migration process) . Neutrophils are responsive to these two substances and also to interleukin-8 (IL-8) . This
"chemokine" (the name given to chemo-attractants that are produced by cells of the immune system) is produced mainly by activated monocytes (but also in minute amounts by the activated neutrophils themselves) . Neutrophils interact with these substances, because they have receptors for these substances on the outside of their cell membrane. Activated neutrophils can easily migrate from blood vessels. This is because the chemo-attractants, microbial products and substances from activated monocytes will have increased the permeability of the vessels and stimulated the endothelial cells of the vessel walls to express certain adhesion molecules. Neutrophils express selectins and integrins (e.g. CDllb/CD18) that bind to these adhesion molecules. Once the neutrophil has adhered to the endothelial cells, it is able to migrate through the cells, under the guidance of chemo-attractants/che okines, towards the site of infection, where the concentration of these substances is at its highest. These substances also activate neutrophils to produce a range of other molecules, some of which attract more neutrophils (and subsequently monocytes) , but, mostly, they are responsible for destroying the invading bacteria. Some of these substances (e.g. free radicals, enzymes that break down proteins (proteases) and cell membranes (Upases) ) are so reactive and non-specific that cells from the surrounding tissue (and the neutrophils themselves) are destroyed, causing tissue damage. This damage is exacerbated by the presence of blood derived fluid which has transgressed the leaky vessel wall and is responsible for the swelling that always accompanies inflammation (called oedema) . The pressure build up caused by this excess fluid results in further cell damage and death.
Later in the inflammatory process, monocytes migrate to the scene and become activated. Besides their role in removing bacteria and cell debris, they also produce substances such as tumour necrosis factor (TNF) and IL-8 , which in turn attract more activated neutrophils, causing further local damage. TNF also has a direct stimulatory effect on neutrophils. Once all the invaders have been removed, the inflammatory response will subside and the area will be cleared of the remaining 'casualties'. Then the process of wound healing starts. Although it is known that neutrophils play a pivotal role in wound healing, it is not clear which neutrophil-derived substances are involved and how the neutrophils are active in healing without being aggressive to the surrounding tissue. In general, damaged tissue will be replaced by scar tissue formed mainly of fibroblasts and collagen. When inflammation occurs in areas of the body with an important function, like tissues formed from heart muscle cells, brain cells or lung alveolar cells, normal function will be compromised by the resulting scar formation, causing serious conditions like heart failure, paralysis and emphysema. To minimize the debilitating consequences of these conditions, it is important to 'dampen' the inflammatory reaction as quickly as possible.
Intervention to control the acute early phase inflammatory response presents an opportunity to improve the prognosis for a wide range of patients whose symptoms can be traced back to such an event. Such an approach has been advocated for many acute and chronic inflammation- based diseases and shown to have potential based on findings from relevant disease models. Classical anti-inflammatory drugs such as steroids and Non Steroid Anti-Inflammatory Drugs (NSAIDS) do not have the ideal profile of action, either in terms of efficacy or safety. Steroids affect the 'wrong' cell type (monocytes) and their dampening effects are easily bypassed. NSAIDS generally show a relatively mild effect partly because they intervene at a late stage in the inflammatory process. Both classes of drugs produce a range of undesirable side effects resulting from other aspects of their pharmacological activity. Drugs acting directly and specifically to prevent migration and activation of neutrophils may have a number of advantages. Several drugs under early development only interfere with one individual aspect of neutrophil activation (e.g. C5 convertase inhibitors, antibodies against C5a, C5a-receptor blocking drugs) and migration (antibodies against integrins (like CDllb/CD18) and L-selectin on neutrophils and antibodies against adhesion molecules (like ICAM-1 and E-selectin) on endothelial cells) . Antibodies against TNF and IL-8 have effects in chronic inflammation, but only marginal effects in acute inflammation, because of the minimal role monocytes (which are mainly responsible for these substances' production) play in the acute phase.
Sometimes, the cause of the acute inflammation cannot be removed and the inflammation becomes chronic. With the exception of tuberculosis, chronic hepatitis and certain other conditions, this is seldom the case with infections. However, chronic inflammation can also be caused by stimuli other than bacteria, such as auto-immune reactions. Research has shown that in chronic inflammation the role of monocytes is much more prominent, and that neutrophil migration and activation, monocyte migration and activation, tissue damage, removal of dead cells and wound healing are all going on at the same time. This complex cascade of interactions between cells and many different cytokines and chemokines has been the subject of intensive research for many years. It was believed that monocytes and their products were the most important elements that needed to be inhibited to dampen chronic inflammation. This explains why steroids, which specifically interact with monocytes, are generally more effective in chronic as opposed to acute inflammation. Long-term treatment with steroids however, is not a desirable option, because severe and unacceptable side effects can occur at the doses required to produce a clinical effect. Newer treatments using antibodies against TNF or IL-8 have shown good results, and were initially seen as proof of the major role monocytes were thought to play in chronic inflammation. Recent research casts doubts on an exclusive role for monocytes in inflammation and points to a critical role for neutrophils, which are now seen to represent better targets for therapeutic intervention.
The underlying cause of a chronic inflammatory condition is not always clear, and the original cause may not always be responsible for future recurrence. Some scientists believe that in certain chronic inflammatory diseases there is a continuous cycle of events. Their idea is that existing activated neutrophils and monocytes continuously attract and activate new groups of cells, thus perpetuating the inflammatory response even when the initial stimulus is no longer present. This would suggest that an acute or periodic treatment with an effective inhibitor of the neutrophil and monocyte activation would stop the cycle of new cell recruitment, leading in due course to modification of disease progression, or even a complete cure, and not just symptomatic relief.
In the research that led to the present invention a new agent with inflammation-inhibiting properties was found in the extracellular medium of growing Staphylococcus aureus (S . aureus) . This agent is the subject of application PCT/NL99/00442. The agent was found to be capable of directly or indirectly blocking different chemokine receptors. Incubation of different cells with the medium resulted in a greatly reduced expression of a number of the chemokine receptors, both of the expression of receptors of classical chemotactic agents such as fMLP and C5a on neutrophils, monocytes and endothelial cells and of the expression of CXCR4 and CCR5 receptors on lymphocytes, monocytes and macrophages. The reduced receptor expression was related to greatly reduced chemotaxis relative to the chemokines, as well as a reduced infection with HIV. The active protein could be further purified, as described in application PCT/NL99/00442. Because this protein is isolated from the supernatant of the Staphylococcus aureus and gives inhibition of chemotaxis, this protein was named "CHIPS": CHemotaxis Inhibitory Protein from Staphylococcus aureus. Isolation of the CHIPS protein out of the supernatant of S . aureus is not very cost-effective. The co-pending application PCT/EP01/00270 describes a nucleic acid molecule comprising a nucleotide sequence encoding a (poly) peptide having CHIPS activity. Figure 1 gives the nucleotide sequence of this nucleic acid molecule. This sequence provides the means for producing the original CHIPS protein or other corresponding (poly) peptides that have CHIPS activity, as well as functional fragments, derivatives or analogues thereof by means of genetic engineering.
"CHIPS activity" is herein defined as the ability to specifically impair at least the responses induced by both fMLP and C5a, including at least impairment of ligand- (C5a or fMLP) binding, and optionally impairment of chemotaxis and cell-activation (e.g. calcium mobilization) . However, the compounds of this invention may in addition have other biological activities, such as an inhibitory effect on the activation of leukocytes and endothelial cells.
In addition, it is desirable for the practical use of CHIPS in therapy that the active part of the protein is isolated. Smaller protein or peptide molecules have a reduced risk of inducing an immunological response in a subject receiving the protein or peptide for therapy. Furthermore, it is desirable to be able to modify the protein or peptide to further increase the biological activity and/or lower the immunogenicity thereof. It is therefore the object of the present invention to provide the smallest as possible active site within the CHIPS molecule conferring CHIPS activity in order to be able to design individual peptides or non- peptides based on this active site for use in diagnosis, prophylaxis and therapy.
It was found according to the invention that this smallest as possible active site within the naturally occurring CHIPS molecule is the N-terminal sequence FTFEPF. This peptide has however an activity that is 10.000 times lower (on molar basis) than the native CHIPS protein. This led the inventors to conclude that one or more further sites are present in the CHIPS molecule that support the CHIPS activity of the peptide. The present invention therefore relates to combinations of compounds, wherein one compound has CHIPS activity (further identified herein as "the compound having CHIPS activity") and consists of at least 6 amino acids the sequence of which is based on the N-terminal part of the naturally occurring CHIPS molecule, and which sequence optionally comprises one or more substitutions as compared to the naturally occurring sequence, or peptidomimetic derivatives thereof, whereas the one or more other compounds are derived from the amino acids 4- 121 of the naturally occurring CHIPS molecule and support CHIPS activity (further identified herein as the "supporting compound") .
More in particular the invention relates to combinations of compounds, wherein the compound having CHIPS activity, has the sequence:
X1-X2-X3-X4-X5-X6-X7
wherein the building blocks XI, X2, X3, X4, X5, X6 and X7 are selected from the group consisting of proteinogenic amino acids, non-proteinogenic amino acids, D-amino acids and peptidomimetic building blocks and wherein X7 may or may not be present and may represent either one or more building blocks. Optionally XI may be modified (cf.
Theodora W. Greene, Peter G.M. Wuts ; Protective groups in organic synthesis, Third edition 1999, John Wiley & Sons Inc, New York; W.C. Chan, P.D. White (eds) ; Fmoc Solid Phase Peptide Synthesis, a practical approach, 2000, Oxford University Press, New York; and Gunther Jung (ed) ; Combinatorial Peptide and Nonpeptide Libraries, 1996, VCH Verlagsgesellschaft mbH, Weinheim) with a group that is smaller than an amino acid and which is selected from acyl (Greene & Wuts, 1999, supra) moieties, such as acetyl and formyl, and ureum moieties such as carbamoyl, or by reductive alkylation (Chan & White, 2000, supra ; and Jung, 1996, supra) . In case the building blocks of the compound having CHIPS activity are proteinogenic amino acids then XI may be selected from phenylalanine (F) , cysteine (C) , asparagine (N) , tryptophan (W) ; X2 may be selected from alanine (A) , cysteine (C) , phenylalanine (F) , glycine (G) , histidine (H) , isoleucine (I) , leucine (L) , methionine (M) , asparagine (N) , gluta ine (Q) , serine (S) , threonine (T) , valine (V) , tryptophan (W) , tyrosine (Y) ; X3 may be selected from phenylalanine (F) , tryptophan (W) ; X4 may be selected from alanine (A) , cysteine (C) , aspartic acid (D) , gluta ic acid (E) , leucine (L) , asparagine (N) , methionine (M) , serine (S) , tryptophan (W) ; X5 may be selected from phenylalanine (F) , isoleucine (I) , leucine (L) , proline (P) , valine (V) tryptophan (W) , tyrosine (Y) ; X6 is selected from cysteine (C) , phenylalanine (F) , histidine (H) , isoleucine (I) , tryptophan (W) , tyrosine (Y) .
More in particular XI may be selected from phenylalanine (F) , tryptophan (W) ; X2 may be selected from phenylalanine (F) , isoleucine (I) , serine (S) , threonine (T) ; X3 is phenylalanine (F) ; X4 may be selected from alanine (A) , cysteine (C) , aspartic acid (D) , glutamic acid (E) , asparagine (N) , serine (S) , tryptophan (W) ; X5 may be selected from phenylalanine (F) , isoleucine (I) , leucine (L) , proline (P) , valine (V) tryptophan (W) , tyrosine (Y) ; X6 may be selected from cysteine (C) , phenylalanine (F) , histidine (H) , isoleucine (I) , tryptophan (W) , tyrosine (Y) .
Particular compounds having CHIPS activity of the invention have the sequence: F-T-F-E-P-F-X7, wherein one or more of the amino acids on positions 1 to 6 are substituted with the building blocks as defined above, and wherein X7 is a stretch of one or more building blocks selected from the group consisting of non-proteinogenic amino acids, D-amino acids and peptidemimetic building blocks and may or may not be present. X7 can be present to further increase the potency (CHIPS activity) of the compound and/or to improve upon its pharmacological and pharmaceutical characteristics (e.g. increase potency, stability, bioavailability, in vivo half-life, decrease side effects, etc. ) . Compounds having CHIPS activity for use in the combinations of the invention in which only one of the original amino acids has been substituted are the following: X1-T-F-E-P-F-X7, F-X2-F-E-P-F-X7, F-T-X3-E-P-F-X7 , F-T-F- X4-P-F-X7, F-T-F-E-X5-F-X7, F-T-F-E-P-X6-X7. In these XI to X7 are as defined above. A preferred compound having CHIPS activity for use in the combinations of the invention is F-T-F-E-P-F-X7 , wherein X7 may or may not be present. The peptides described in Table 6 are part of the present invention as the compound that has CHIPS activity provided they have CHIPS activity as defined above and as tested in one or more of the CHIPS activity tests as given in the Examples.
Table 6
FTFEPF, WTFEPF, FFFEPF, FIFEPF, FSFEPF, WFFEPF WIFEPF,
WSFEPF, FFFAPF, FIFAPF, FSFAPF, FTFAPF, WFFAPF WIFAPF,
WSFAPF, WTFAPF, FFFCPF, FIFCPF, FSFCPF, FTFCPF WFFCPF, WIFCPF, WSFCPF, WTFCPF, FFFDPF, FIFDPF, FSFDPF FTFDPF,
WFFDPF, WIFDPF, WSFDPF, WTFDPF, FFFNPF, FIFNPF FSFNPF,
FTFNPF, WFFNPF, WIFNPF, WSFNPF, WTFNPF, FFFSPF FIFSPF,
FSFSPF, FTFSPF, WFFSPF, WIFSPF, WSFSPF, WTFSPF FFFWPF,
FIFWPF, FSFWPF, FTFWPF, WFFWPF, WIFWPF, WSFWPF WTFWPF, FFFAFC, FIFAFC, FSFAFC, FTFAFC, WFFAFC, WIFAFC WSFAPC,
WTFAFC, FFFCFC, FIFCFC, FSFCFC, FTFCFC, WFFCFC WIFCFC,
WSFCFC, WTFCFC, FFFDFC, FIFDFC, FSFDFC, FTFDFC WFFDFC,
WIFDFC, WSFDFC, WTFDFC, FFFEFC, FIFEFC, FSFEFC FTFEFC, WFFEFC WIFEFC WSFEFC WTFEFC FFFNFC FIFNFC FSFNFC, FTFNFC WFFNFC WIFNFC WSFNFC WTFNFC FFFSFC FIFSFC, FSFSFC FTFSFC WFFSFC WIFSFC WSFSFC WTFSFC FFFWFC, FIFWFC FSFWFC FTFWFC WFFWFC WIFWFC WSFWFC WTFWFC, FFFAIC FIFAIC FSFAIC FTFAIC WFFAIC WIFAIC WSFAIC, WTFAIC FFFCIC FIFCIC FSFCIC FTFCIC WFFCIC WIFCIC, WSFCIC WTFCIC FFFDIC FIFDIC FSFDIC FTFDIC WFFDIC, WIFDIC WSFDIC WTFDIC FFFEIC FIFEIC FSFEIC FTFEIC, WFFEIC WIFEIC WSFEIC WTFEIC FFFNIC FIFNIC FSFNIC, FTFNIC WFFNIC WIFNIC WSFNIC WTFNIC FFFSIC FIFSIC, FSFSIC FTFSIC WFFSIC WIFSIC WSFSIC WTFSIC FFFWIC, FIFWIC FSFWIC FTFWIC WFFWIC WIFWIC WSFWIC WTFWIC, FFFALC FIFALC FSFALC FTFALC WFFALC WIFALC WSFALC, WTFALC FFFCLC FIFCLC FSFCLC FTFCLC WFFCLC WIFCLC, WSFCLC WTFCLC FFFDLC FIFDLC FSFDLC FTFDLC WFFDLC, WIFDLC WSFDLC WTFDLC FFFELC FIFELC FSFELC FTFELC, WFFELC WIFELC WSFELC WTFELC FFFNLC FIFNLC FSFNLC, FTFNLC WFFNLC WIFNLC WSFNLC WTFNLC FFFSLC FIFSLC, FSFSLC FTFSLC WFFSLC WIFSLC WSFSLC WTFSLC FFFWLC, FIFWLC FSFWLC FTFWLC WFFWLC WIFWLC WSFWLC WTFWLC,
FFFAPC FIFAPC FSFAPC FTFAPC WFFAPC WIFAPC WSFAPC, WTFAPC FFFCPC FIFCPC FSFCPC FTFCPC WFFCPC WIFCPC, WSFCPC WTFCPC FFFDPC FIFDPC FSFDPC FTFDPC WFFDPC, WIFDPC WSFDPC WTFDPC FFFEPC FIFEPC FSFEPC FTFEPC, WFFEPC WIFEPC WSFEPC WTFEPC FFFNPC FIFNPC FSFNPC, FTFNPC WFFNPC WIFNPC WSFNPC WTFNPC FFFSPC FIFSPC, FSFSPC FTFSPC WFFSPC WIFSPC WSFSPC WTFSPC FFFWPC, FIFWPC FSFWPC FTFWPC WFFWPC WIFWPC WSFWPC WTFWPC, FFFAVC FIFAVC FSFAVC FTFAVC WFFAVC WIFAVC WSFAVC, WTFAVC FFFCVC FIFCVC FSFCVC FTFCVC WFFCVC WIFCVC, WSFCVC WTFCVC FFFDVC FIFDVC FSFDVC FTFDVC WFFDVC, WIFDVC WSFDVC WTFDVC FFFEVC FIFEVC FSFEVC FTFEVC, WFFEVC WIFEVC WSFEVC WTFEVC FFFNVC FIFNVC FSFNVC, FTFNVC WFFNVC WIFNVC WSFNVC WTFNVC FFFSVC FIFSVC, FSFSVC FTFSVC WFFSVC WIFSVC WSFSVC WTFSVC FFFWVC,
FIFWVC FSFWVC FTFWVC WFFWVC WIFWVC WSFWVC WTFWVC, FFFAWC FIFAWC FSFAWC FTFAWC WFFAWC WIFAWC WSFAWC, WTFAWC FFFCWC FIFCWC FSFCWC FTFCWC WFFCWC WIFCWC, WSFCWC WTFCWC FFFDWC FIFDWC FSFDWC FTFDWC WFFDWC, WIPDWC WSFDWC WTFDWC FFFEWC FIFEWC FSFEWC FTFEWC, WFFEWC WIFEWC WSFEWC WTFEWC FFFNWC FIFNWC FSFNWC, FTFNWC WFFNWC WIFNWC WSFNWC WTFNWC FFFSWC FIFSWC, FSFSWC FTFSWC WFFSWC WIFSWC WSFSWC WTFSWC FFFWWC, FIFWWC FSFWWC FTFWWC WFFWWC WIFWWC WSFWWC WTFWWC, FFFAYC FIFAYC FSFAYC FTFAYC WFFAYC WIFAYC WSFAYC, WTFAYC FFFCYC FIFCYC FSFCYC FTFCYC WFFCYC WIFCYC, WSFCYC WTFCYC FFFDYC FIFDYC FSFDYC FTFDYC WFFDYC, WIFDYC WSFDYC WTFDYC FFFEYC FIFEYC FSFEYC FTFEYC, WFFEYC WIFEYC WSFEYC WTFEYC FFFNYC FIFNYC FSFNYC, FTFNYC WFFNYC WIFNYC WSFNYC WTFNYC FFFSYC FIFSYC, FSFSYC FTFSYC WFFSYC WIFSYC WSFSYC WTFSYC FFFWYC, FIFWYC FSFWYC FTFWYC WFFWYC WIFWYC WSFWYC WTFWYC, FFFAFF FIFAFF FSFAFF FTFAFF WFFAFF WIFAFF WSFAFF, WTFAFF FFFCFF FIFCFF FSFCFF FTFCFF WFFCFF WIFCFF, WSFCFF WTFCFF FFFDFF FIFDFF FSFDFF FTFDFF WFFDFF, WIFDFF WSFDFF WTFDFF FFFEFF FIFEFF FSFEFF FTFEFF, WFFEFF WIFEFF WSFEFF WTFEFF FFFNFF FIFNFF FSFNFF, FTFNFF WFFNFF WIFNFF WSFNFF WTFNFF FFFSFF FIFSFF, FSFSFF FTFSFF WFFSFF WIFSFF WSFSFF WTFSFF FFFWFF, FIFWFF FSFWFF FTFWFF WFFWFF WIFWFF WSFWFF WTFWFF, FFFAIF FIFAIF FSFAIF FTFAIF WFFAIF WIFAIF WSFAIF, WTFAIF FFFCIF FIFCIF FSFCIF FTFCIF WFFCIF WIFCIF, WSFCIF WTFCIF FFFDIF FIFDIF FSFDIF FTFDIF WFFDIF, WIFDIF WSFDIF WTFDIF FFFEIF FIFEIF FSFEIF FTFEIF, WFFEIF WIFEIF WSFEIF WTFEIF FFFNIF FIFNIF FSFNIF, FTFNIF WFFNIF WIFNIF WSFNIF WTFNIF FFFSIF FIFSIF, FSFSIF FTFSIF WFFSIF WIFSIF WSFSIF WTFSIF FFFWIF, FIFWIF FSFWIF FTFWIF WFFWIF WIFWIF WSFWIF WTFWIF, FFFALF FIFALF FSFALF FTFALF WFFALF WIFALF WSFALF, WTFALF FFFCLF FIFCLF FSFCLF FTFCLF WFFCLF WIFCLF, WSFCLF WTFCLF FFFDLF FIFDLF FSFDLF FTFDLF WFFDLF, WIFDLF WSFDLF WTFDLF FFFELF FIFELF FSFELF FTFELF, WFFELF WIFELF WSFELF WTFELF FFFNLF FIFNLF FSFNLF, FTFNLF WFFNLF WIFNLF WSFNLF WTFNLF FFFSLF FIFSLF, FSFSLF FTFSLF WFFSLF WIFSLF WSFSLF WTFSLF FFFWLF, FIFWLF FSFWLF FTFWLF WFFWLF WIFWLF WSFWLF WTFWLF, FFFAVF FIFAVF FSFAVF FTFAVF WFFAVF WIFAVF WSFAVF WTFAVF FFFCVF FIFCVF FSFCVF FTFCVF WFFCVF WIFCVF WSFCVF WTFCVF FFFDVF FIFDVF FSFDVF FTFDVF WFFDVF WIFDVF WSFDVF WTFDVF FFFEVF FIFEVF FSFEVF FTFEVF WFFEVF WIFEVF WSFEVF WTFEVF FFFNVF FIFNVF FSFNVF FTFNVF WFFNVF WIFNVF WSFNVF WTFNVF FFFSVF FIFSVF FSFSVF FTFSVF WFFSVF WIFSVF WSFSVF WTFSVF FFFWVF FIFWVF FSFWVF FTFWVF WFFWVF WIFWVF WSFWVF WTFWVF FFFAWF FIFAWF FSFAWF FTFAWF WFFAWF WIFAWF WSFAWF WTFAWF FFFCWF FIFCWF FSFCWF FTFCWF WFFCWF WIFCWF WSFCWF WTFCWF FFFDWF FIFDWF FSFDWF FTFDWF WFFDWF WIFDWF WSFDWF WTFDWF FFFEWF FIFEWF FSFEWF FTFEWF WFFEWF WIFEWF WSFEWF WTFEWF FFFNWF FIFNWF FSFNWF FTFNWF WFFNWF WIFNWF WSFNWF WTFNWF FFFSWF FIFSWF FSFSWF FTFSWF WFFSWF WIFSWF WSFSWF WTFSWF FFFWWF FIFWWF FSFWWF FTFWWF WFFWWF WIFWWF WSFWWF WTFWWF FFFAYF FIFAYF FSFAYF FTFAYF WFFAYF WIFAYF WSFAYF WTFAYF FFFCYF FIFCYF FSFCYF FTFCYF WFFCYF WIFCYF WSFCYF WTFCYF FFFDYF FIFDYF FSFDYF FTFDYF WFFDYF WIFDYF WSFDYF WTFDYF FFFEYF FIFEYF FSFEYF FTFEYF WFFEYF WIFEYF WSFEYF WTFEYF FFFNYF FIFNYF FSFNYF FTFNYF WFFNYF WIFNYF WSFNYF WTFNYF FFFSYF FIFSYF FSFSYF FTFSYF WFFSYF WIFSYF WSFSYF WTFSYF FFFWYF FIFWYF FSFWYF FTFWYF WFFWYF WIFWYF WSFWYF WTFWYF FFFAFH FIFAFH FSFAFH FTFAFH WFFAFH WIFAFH WSFAFH WTFAFH FFFCFH FIFCFH FSFCFH FTFCFH WFFCFH WIFCFH WSFCFH WTFCFH FFFDFH FIFDFH FSFDFH FTFDFH WFFDFH WIFDFH WSFDFH WTFDFH FFFEFH FIFEFH FSFEFH FTFEFH WFFEFH WIFEFH WSFEFH WTFEFH FFFNFH FIFNFH FSFNFH FTFNFH WFFNFH WIFNFH WSFNFH WTFNFH FFFSFH FIFSFH FSFSFH FTFSFH WFFSFH WIFSFH WSFSFH WTFSFH FFFWFH FIFWFH FSFWFH FTFWFH WFFWFH WIFWFH WSFWFH WTFWFH FFFAIH FIFAIH FSFAIH FTFAIH WFFAIH WIFAIH WSFAIH WTFAIH FFFCIH FIFCIH FSFCIH FTFCIH WFFCIH WIFCIH WSFCIH WTFCIH FFFDIH FIFDIH FSFDIH FTFDIH WFFDIH WIFDIH WSFDIH WTFDIH FFFEIH FIFEIH FSFEIH FTFEIH WFFEIH WIFEIH WSFEIH WTFEIH FFFNIH FIFNIH FSFNIH FTFNIH WFFNIH WIFNIH WSFNIH WTFNIH FFFSIH FIFSIH FSFSIH FTFSIH WFFSIH WIFSIH WSFSIH WTFSIH FFFWIH, FIFWIH FSFWIH FTFWIH WFFWIH WIFWIH WSFWIH WTFWIH, FFFALH FIFALH FSFALH FTFALH WFFALH WIFALH WSFALH, WTFALH FFFCLH FIFCLH FSFCLH FTFCLH WFFCLH WIFCLH, WSFCLH WTFCLH FFFDLH FIFDLH FSFDLH FTFDLH WFFDLH, WIFDLH WSFDLH WTFDLH FFFELH FIFELH FSFELH FTFELH, WFFELH WIFELH WSFELH WTFELH FFFNLH FIFNLH FSFNLH, FTFNLH WFFNLH WIFNLH WSFNLH WTFNLH FFFSLH FIFSLH, FSFSLH FTFSLH WFFSLH WIFSLH WSFSLH WTFSLH FFFWLH, FIFWLH FSFWLH FTFWLH WFFWLH WIFWLH WSFWLH WTFWLH, FFFAPH FIFAPH FSFAPH FTFAPH WFFAPH WIFAPH WSFAPH, WTFAPH FFFCPH FIFCPH FSFCPH FTFCPH WFFCPH WIFCPH, WSFCPH WTFCPH FFFDPH FIFDPH FSFDPH FTFDPH WFFDPH, WIFDPH WSFDPH WTFDPH FFFEPH FIFEPH FSFEPH FTFEPH, WFFEPH WIFEPH WSFEPH WTFEPH FFFNPH FIFNPH FSFNPH, FTFNPH WFFNPH WIFNPH WSFNPH WTFNPH FFFSPH FIFSPH, FSFSPH FTFSPH WFFSPH WIFSPH WSFSPH WTFSPH FFFWPH, FIFWPH FSFWPH FTFWPH WFFWPH WIFWPH WSFWPH WTFWPH, FFFAVH FIFAVH FSFAVH FTFAVH WFFAVH WIFAVH WSFAVH, WTFAVH FFFCVH FIFCVH FSFCVH FTFCVH WFFCVH WIFCVH,
WSFCVH WTFCVH FFFDVH FIFDVH FSFDVH FTFDVH WFFDVH, WIFDVH WSFDVH WTFDVH FFFEVH FIFEVH FSFEVH FTFEVH, WFFEVH WIFEVH WSFEVH WTFEVH FFFNVH FIFNVH FSFNVH, FTFNVH WFFNVH WIFNVH WSFNVH WTFNVH FFFSVH FIFSVH, FSFSVH FTFSVH WFFSVH WIFSVH WSFSVH WTFSVH FFFWVH, FIFWVH FSFWVH FTFWVH WFFWVH WIFWVH WSFWVH WTFWVH, FFFAWH FIFAWH FSFAWH FTFAWH WFFAWH WIFAWH WSFAWH, WTFAWH FFFCWH FIFCWH FSFCWH FTFCWH WFFCWH WIFCWH, WSFCWH WTFCWH FFFDWH FIFDWH FSFDWH FTFDWH WFFDWH, WIFDWH WSFDWH WTFDWH FFFEWH FIFEWH FSFEWH FTFEWH, WFFEWH WIFEWH WSFEWH WTFEWH FFFNWH FIFNWH FSFNWH, FTFNWH WFFNWH WIFNWH WSFNWH WTFNWH FFFSWH FIFSWH, FSFSWH FTFSWH WFFSWH WIFSWH WSFSWH WTFSWH FFFWWH, FIFWWH FSFWWH FTFWWH WFFWWH WIFWWH WSFWWH WTFWWH, FFAYH, FIFAYH, FSFAYH, FTFAYH, WFFAYH, WIFAYH, WSFAYH,
WTFAYH FFFCYH FIFCYH FSFCYH FTFCYH WFFCYH WIFCYH, WSFCYH WTFCYH FFFDYH FIFDYH FSFDYH FTFDYH WFFDYH, WIFDYH WSFDYH WTFDYH FFFEYH FIFEYH FSFEYH FTFEYH, WFFEYH WIFEYH WSFEYH WTFEYH FFFNYH FIFNYH FSFNYH, FTFNYH WFFNYH WIFNYH WSFNYH WTFNYH FFFSYH FIFSYH, FSFSYH FTFSYH WFFSYH WIFSYH WSFSYH WTFSYH FFFWYH, FIFWYH FSFWYH FTFWYH WFFWYH WIFWYH WSFWYH WTFWYH, FFFAFI FIFAFI FSFAFI FTFAFI WFFAFI WIFAFI WSFAFI , WTFAFI FFFCFI FIFCFI FSFCFI FTFCFI WFFCFI WIFCFI, WSFCFI WTFCFI FFFDFI FIFDFI FSFDFI FTFDFI WFFDFI, WIFDFI WSFDFI WTFDFI FFFEFI FIFEFI FSFEFI FTFEFI , WFFEFI WIFEFI WSFEFI WTFEFI FFFNFI FIFNFI FSFNFI, FTFNFI WFFNFI WIFNFI WSFNFI WTFNFI FFFSFI FIFSFI, FSFSFI FTFSFI WFFSFI WIFSFI WSFSFI WTFSFI FFFWFI , FIFWFI FSFWFI FTFWFI WFFWFI WIFWFI WSFWFI WTFWFI , FFFAII FIFAII FSFAII FTFAII WFFAII WIFAII WSFAII, WTFAII FFFCII FIFCII FSFCII FTFCII WFFCII WIFCII, WSFCII WTFCII FFFDII FIFDII FSFDII FTFDII WFFDII, WIFDII WSFDII WTFDII FFFEII FIFEII FSFEII FTFEII, WFFEII WIFEII WSFEII WTFEII FFFNII FIFNII FSFNII, FTFNII WFFNII WIFNII WSFNII WTFNII FFFSII FIFSII, FSFSII FTFSII WFFSII WIFSII WSFSII WTFSII FFFWII, FIFWII FSFWII FTFWII WFFWII WIFWII WSFWII WTFWII, FFFALI FIFALI FSFALI FTFALI WFFALI WIFALI WSFALI , WTFALI FFFCLI FIFCLI FSFCLI FTFCLI WFFCLI WIFCLI, WSFCLI WTFCLI FFFDLI FIFDLI FSFDLI FTFDLI WFFDLI , WIFDLI WSFDLI WTFDLI FFFELI FIFELI FSFELI FTFELI , WFFELI WIFELI WSFELI WTFELI FFFNLI FIFNLI FSFNLI , FTFNLI WFFNLI WIFNLI WSFNLI WTFNLI FFFSLI FIFSLI, FSFSLI FTFSLI WFFSLI WIFSLI WSFSLI WTFSLI FFFWLI , FIFWLI FSFWLI FTFWLI WFFWLI WIFWLI WSFWLI WTFWLI , FFFAPI FIFAPI FSFAPI FTFAPI WFFAPI WIFAPI WSFAPI , WTFAPI FFFCPI FIFCPI FSFCPI FTFCPI WFFCPI WIFCPI, WSFCPI WTFCPI FFFDPI FIFDPI FSFDPI FTFDPI WFFDPI , WIFDPI WSFDPI WTFDPI FFFEPI FIFEPI FSFEPI FTFEPI, WFFEPI WIFEPI WSFEPI WTFEPI FFFNPI FIFNPI FSFNPI, FTFNPI WFFNPI WIFNPI WSFNPI WTFNPI FFFSPI FIFSPI, FSFSPI FTFSPI WFFSPI WIFSPI WSFSPI WTFSPI FFFWPI , FIFWPI FSFWPI FTFWPI WFFWPI WIFWPI WSFWPI WTFWPI , FFFAVI FIFAVI FSFAVI FTFAVI WFFAVI WIFAVI WSFAVI , WTFAVI FFFCVI FIFCVI FSFCVI FTFCVI WFFCVI WIFCVI, WSFCVI WTFCVI FFFDVI FIFDVI FSFDVI FTFDVI WFFDVI ,
WIFDVI WSFDVI WTFDVI FFFEVI FIFEVI FSFEVI FTFEVI ,
WFFEVI WIFEVI WSFEVI WTFEVI FFFNVI FIFNVI FSFNVI ,
FTFNVI WFFNVI WIFNVI WSFNVI WTFNVI FFFSVI FIFSVI , FSFSVI FTFSVI WFFSVI WIFSVI WSFSVI WTFSVI FFFWVI ,
FIFWVI FSFWVI FTFWVI WFFWVI WIFWVI WSFWVI WTFWVI ,
FFFAWI FIFAWI FSFAWI FTFAWI WFFAWI WIFAWI WSFAWI ,
WTFAWI FFFCWI FIFCWI FSFCWI FTFCWI WFFCWI WIFCWI,
WSFCWI WTFCWI FFFDWI FIFDWI FSFDWI FTFDWI WFFDWI, WIFDWI WSFDWI WTFDWI FFFEWI FIFEWI FSFEWI FTFEWI ,
WFFEWI WIFEWI WSFEWI WTFEWI FFFNWI FIFNWI FSFNWI,
FTFNWI WFFNWI WIFNWI WSFNWI WTFNWI FFFSWI FIFSWI,
FSFSWI FTFSWI WFFSWI WIFSWI WSFSWI WTFSWI FFFWWI,
FIFWWI FSFWWI FTFWWI WFFWWI WIFWWI WSFWWI WTFWWI , FFFAYI FIFAYI FSFAYI FTFAYI WFFAYI WIFAYI WSFAYI,
WTFAYI FFFCYI FIFCYI FSFCYI FTFCYI WFFCYI WIFCYI,
WSFCYI WTFCYI FFFDYI FIFDYI FSFDYI FTFDYI WFFDYI,
WIFDYI WSFDYI WTFDYI FFFEYI FIFEYI FSFEYI FTFEYI ,
WFFEYI WIFEYI WSFEYI WTFEYI FFFNYI FIFNYI FSFNYI, FTFNYI WFFNYI WIFNYI WSFNYI WTFNYI FFFSYI FIFSYI,
FSFSYI FTFSYI WFFSYI WIFSYI WSFSYI WTFSYI FFFWYI ,
FIFWYI FSFWYI FTFWYI WFFWYI WIFWYI WSFWYI WTFWYI ,
FFFAFW FIFAFW FSFAFW FTFAFW WFFAFW WIFAFW WSFAFW,
WTFAFW FFFCFW FIFCFW FSFCFW FTFCFW WFFCFW WIFCFW, WSFCFW WTFCFW FFFDFW FIFDFW FSFDFW FTFDFW WFFDFW,
WIFDFW WSFDFW WTFDFW FFFEFW FIFEFW FSFEFW FTFEFW,
WFFEFW WIFEFW WSFEFW WTFEFW FFFNFW FIFNFW FSFNFW,
FTFNFW WFFNFW WIFNFW WSFNFW WTFNFW FFFSFW FIFSFW,
FSFSFW FTFSFW WFFSFW WIFSFW WSFSFW WTFSFW FFFWFW, FIFWFW FSFWFW FTFWFW WFFWFW WIFWFW WSFWFW WTFWFW,
FFFAIW FIFAIW FSFAIW FTFAIW WFFAIW WIFAIW WSFAIW,
WTFAIW FFFCIW FIFCIW FSFCIW FTFCIW WFFCIW WIFCIW,
WSFCI WTFCIW FFFDIW FIFDIW FSFDIW FTFDIW WFFDIW,
WIFDIW WSFDIW WTFDIW FFFEIW FIFEIW FSFEIW FTFEIW, WFFEIW WIFEIW WSFEIW WTFEIW FFFNIW FIFNIW FSFNIW,
FTFNIW WFFNIW WIFNIW WSFNIW WTFNIW FFFSIW FIFSIW,
FSFSIW FTFSIW WFFSIW WIFSIW WSFSIW WTFSIW FFFWIW,
FIFWIW FSFWIW FTFWIW WFFWIW WIFWIW WSFWIW WTFWIW, FFFALW FIFALW FSFALW FTFALW WFFALW WIFALW WSFALW, WTFALW FFFCLW FIFCLW FSFCLW FTFCLW WFFCLW WIFCLW, WSFCLW WTFCLW FFFDLW FIFDLW FSFDLW FTFDLW WFFDLW, WIFDLW WSFDLW WTFDLW FFFELW FIFELW FSFELW FTFELW, WFFELW WIFELW WSFELW WTFELW FFFNLW FIFNLW FSFNLW, FTFNLW WFFNLW WIFNLW WSFNLW WTFNLW FFFSLW FIFSLW, FSFSLW FTFSLW WFFSLW WIFSLW WSFSLW WTFSLW FFFWLW, FIFWLW FSFWLW FTFWLW WFFWLW WIFWLW WSFWLW WTFWLW, FFFAPW FIFAPW FSFAPW FTFAPW WFFAPW WIFAPW WSFAPW, WTFAPW FFFCPW FIFCPW FSFCPW FTFCPW WFFCPW WIFCPW, WSFCPW WTFCPW FFFDPW FIFDPW FSFDPW FTFDPW WFFDPW, WIFDPW WSFDPW WTFDPW FFFEPW FIFEPW FSFEPW FTFEPW, WFFEPW WIFEPW WSFEPW WTFEPW FFFNPW FIFNPW FSFNPW, FTFNPW WFFNPW WIFNPW WSFNPW WTFNPW FFFSPW FIFSPW, FSFSPW FTFSPW WFFSPW WIFSPW WSFSPW WTFSPW FFFWPW, FIFWPW FSFWPW FTFWPW WFFWPW WIFWPW WSFWPW WTFWPW, FFFAVW FIFAVW FSFAVW FTFAVW WFFAVW WIFAVW WSFAVW, WTFAVW FFFCVW FIFCVW FSFCVW FTFCVW WFFCVW WIFCVW, WSFCVW WTFCVW FFFDVW FIFDVW FSFDVW FTFDVW WFFDVW, WIFDV WSFDVW WTFDVW FFFEVW FIFEVW FSFEVW FTFEV , WFFEVW WIFEVW WSFEVW WTFEVW FFFNVW FIFNVW FSFNVW, FTFNVW WFFNVW WIFNVW WSFNVW WTFNVW FFFSVW FIFSVW, FSFSVW FTFSVW WFFSVW WIFSVW WSFSVW WTFSVW FFFWVW, FIFWVW FSFWVW FTFWVW WFFWVW WIFWVW WSFWVW WTFWVW, FFFAWW FIFAWW FSFAWW FTFAWW WFFAWW WIFAWW WSFAWW, WTFAWW FFFCWW FIFCWW FSFCWW FTFCWW WFFCWW WIFCWW, WSFCWW WTFCWW FFFDWW FIFDWW FSFDWW FTFDWW WFFDWW, WIFDWW WSFDWW WTFDWW FFFEWW FIFEWW FSFEWW FTFEWW, WFFEWW WIFEWW WSFEWW WTFEWW FFFNWW FIFNWW FSFNWW, FTFNWW WFFNWW WIFNWW WSFNWW WTFNWW FFFSWW FIFSWW, FSFSWW FTFSWW WFFSWW WIFSWW WSFSWW WTFSWW FFFWWW, FIFWWW FSFWWW FTFWWW WFFWWW WIFWWW WSFWWW WTFWWW, FFFAYW FIFAYW FSFAYW FTFAYW WFFAYW WIFAYW WSFAYW, WTFAYW FFFCYW FIFCYW FSFCYW FTFCYW WFFCYW WIFCYW, WSFCYW WTFCYW FFFDYW FIFDYW FSFDYW FTFDYW WFFDYW, WIFDYW WSFDYW WTFDYW FFFEYW FIFEYW FSFEYW FTFEYW, WFFEYW WIFEYW WSFEYW WTFEYW FFFNYW FIFNYW FSFNYW, FTFNYW WFFNYW WIFNYW WSFNYW WTFNYW FFFSYW FIFSYW, FSFSYW FTFSYW WFFSYW WIFSYW WSFSYW WTFSYW FFFWYW, FIFWYW FSFWYW FTFWYW WFFWYW WIFWYW WSFWYW WTFWYW, FFFAFY FIFAFY FSFAFY FTFAFY WFFAFY WIFAFY WSFAFY, WTFAFY FFFCFY FIFCFY FSFCFY FTFCFY WFFCFY WIFCFY, WSFCFY WTFCFY FFFDFY FIFDFY FSFDFY FTFDFY WFFDFY, WIFDFY WSFDFY WTFDFY FFFEFY FIFEFY FSFEFY FTFEFY, WFFEFY WIFEFY WSFEFY WTFEFY FFFNFY FIFNFY FSFNFY, FTFNFY WFFNFY WIFNFY WSFNFY WTFNFY FFFSFY FIFSFY, FSFSFY FTFSFY WFFSFY WIFSFY WSFSFY WTFSFY FFFWFY, FIFWFY FSFWFY FTFWFY WFFWFY WIFWFY WSFWFY WTFWFY, FFFAIY FIFAIY FSFAIY FTFAIY WFFAIY WIFAIY WSFAIY, WTFAIY FFFCIY FIFCIY FSFCIY FTFCIY WFFCIY WIFCIY, WSFCIY WTFCIY FFFDIY FIFDIY FSFDIY FTFDIY WFFDIY, WIFDIY WSFDIY WTFDIY FFFEIY FIFEIY FSFEIY FTFEIY, WFFEIY WIFEIY WSFEIY WTFEIY FFFNIY FIFNIY FSFNIY, FTFNIY WFFNIY WIFNIY WSFNIY WTFNIY FFFSIY FIFSIY, FSFSIY FTFSIY WFFSIY WIFSIY WSFSIY WTFSIY FFFWIY, FIFWIY FSFWIY FTFWIY WFFWIY WIFWIY WSFWIY WTFWIY, FFFALY FIFALY FSFALY FTFALY WFFALY WIFALY WSFALY , WTFALY FFFCLY FIFCLY FSFCLY FTFCLY WFFCLY WIFCLY, WSFCLY WTFCLY FFFDLY FIFDLY FSFDLY FTFDLY WFFDLY , WIFDLY WSFDLY WTFDLY FFFELY FIFELY FSFELY FTFELY, WFFELY WIFELY WSFELY WTFELY FFFNLY FIFNLY FSFNLY , FTFNLY WFFNLY WIFNLY WSFNLY WTFNLY FFFSLY FIFSLY, FSFSLY FTFSLY WFFSLY WIFSLY WSFSLY WTFSLY FFFWLY , FIFWLY FSFWLY FTFWLY WFFWLY WIFWLY WSFWLY WTFWLY, FFFAPY FIFAPY FSFAPY FTFAPY WFFAPY WIFAPY WSFAPY, WTFAPY FFFCPY FIFCPY FSFCPY FTFCPY WFFCPY WIFCPY, WSFCPY WTFCPY FFFDPY FIFDPY FSFDPY FTFDPY WFFDPY, WIFDPY WSFDPY WTFDPY FFFEPY FIFEPY FSFEPY FTFEPY, WFFEPY WIFEPY WSFEPY WTFEPY FFFNPY FIFNPY FSFNPY, FTFNPY WFFNPY WIFNPY WSFNPY WTFNPY FFFSPY FIFSPY, FSFSPY FTFSPY WFFSPY WIFSPY WSFSPY WTFSPY FFFWPY , FIFWPY FSFWPY FTFWPY WFFWPY WIFWPY WSFWPY WTFWPY, FFFAVY FIFAVY FSFAVY FTFAVY WFFAVY WIFAVY WSFAVY, WTFAVY FFFCVY FIFCVY FSFCVY FTFCVY WFFCVY WIFCVY, WSFCVY WTFCVY FFFDVY FIFDVY FSFDVY FTFDVY WFFDVY, WIFDVY WSFDVY WTFDVY FFFEVY FIFEVY FSFEVY FTFEVY, WFFEVY, WIFEVY, WSFEVY, WTFEVY, FFFNVY, FIFNVY, FSFNVY, FTFNVY, WFFNVY, WIFNVY, WSFNVY, WTFNVY , FFFSVY, FIFSVY, FSFSVY, FTFSVY, WFFSVY, WIFSVY, WSFSVY, WTFSVY, FFFWVY, FIFWVY, FSFWVY, FTFWVY , WFFWVY, WIFWVY, WSFWVY, WTFWVY, FFFAWY, FIFAWY, FSFAWY, FTFAWY, WFFAWY , WIFAWY, WSFAWY, WTFAWY, FFFCWY, FIFCWY, FSFCWY, FTFCWY , WFFCWY, WIFCWY, WSFCWY, WTFCWY, FFFDWY, FIFDWY, FSFDWY, FTFDWY, WFFDWY, WIFDWY, WSFDWY, WTFDWY, FFFEWY, FIFEWY, FSFEWY, FTFEWY, WFFEWY, WIFEWY, WSFEWY, WTFEWY, FFFNWY, FIFNWY, FSFNWY, FTFNWY, WFFNWY, WIFNWY, WSFNWY, WTFNWY, FFFSWY, FIFSWY, FSFSWY, FTFSWY, WFFSWY, WIFSWY, WSFSWY, WTFSWY, FFFWWY, FIFWWY, FSFWWY, FTFWWY, WFFWWY, WIFWWY, WSFWWY, WTFWWY , FFFAYY, FIFAYY, FSFAYY, FTFAYY , WFFAYY , WIFAYY, WSFAYY, WTFAYY, FFFCYY, FIFCYY, FSFCYY, FTFCYY , WFFCYY, WIFCYY, WSFCYY, WTFCYY, FFFDYY, FIFDYY, FSFDYY, FTFDYY, WFFDYY, WIFDYY, WSFDYY, WTFDYY, FFFEYY, FIFEYY, FSFEYY, FTFEYY , WFFEYY, WIFEYY, WSFEYY, WTFEYY, FFFNYY, FIFNYY, FSFNYY, FTFNYY, WFFNYY, WIFNYY, WSFNYY, WTFNYY , FFFSYY, FIFSYY, FSFSYY, FTFSYY, WFFSYY, WIFSYY, WSFSYY, WTFSYY, FFFWYY , FIFWYY, FSFWYY, FTFWYY , WFFWYY, WIFWYY, WSFWYY, WTFWYY.
The N-terminal of these peptides may be modified as described above for XI, whereas an X7 group may be present C-terminally . In addition, all of these peptides may be used as a starting point for further modification, for example by substitution of one or more of the amino acids with other building blocks.
Particularly preferred for use in combinations of the invention is a peptide of the naturally occurring N-terminal sequence of CHIPS, i.e. FTFEPF. In this application a "combination of compounds" is to be understood as a mixture of at least one peptide or peptidomimetic that is based on the first six amino acids of the CHIPS protein and has CHIPS activity and one or more other peptides derived from the amino acid sequence of positions 4-121 of CHIPS, wherein the two or more peptides are either present as separate entities (that either stay as separate entities after administration into the body or become attached to each other in the body) or physically attached (either directly or indirectly, such as via a spacer) . The amino acid sequence of the one or more other peptides may be altered as compared to the amino acid sequence occurring in CHIPS in the same manner as described herein for the compound having CHIPS activity.
Both types of compounds that are used in the combinations of the invention can be produced by known chemical synthesis. Methods for constructing peptides by synthetic means are known to those skilled in the art. These synthetic peptides, by virtue of sharing primary, secondary and/or tertiary structural and/or confor- ational characteristics with CHIPS will posses an activity in common therewith, meaning CHIPS properties or supportive properties. Thus, such synthetically produced peptides can be employed as biologically active or immunological substitute for natural purified (poly) peptides having CHIPS activity or supportive properties. The compounds having CHIPS activity provided herein also include peptides characterized by amino acid sequences into which modifications are naturally provided or deliberately engineered. Modifications in the peptide can be made by those skilled in the art using known conventional techniques. Modifications of interest in the CHIPS active peptide sequences may include replacement, insertion or deletion of selected amino acid residues in the coding sequence. The Examples describe such modified synthetic peptides. In some cases the potential for use of peptides in drugs may be limited for several reasons. Peptides may for example be too hydrophilic to pass membranes like the cell-membrane and the blood-brain barrier, and may be rapidly excreted from the body by the kidneys and the liver, resulting in a low bioavailability. Furthermore, peptides may suffer from a poor biostability and chemical stability since they may be quickly degraded by proteases, e.g. in the gastro-intestinal tract. Also, peptides generally are flexible compounds which can assume thousands of conformations. The bioactive conformation usually is only one of these possibilities, which sometimes might lead to a poor selectivity and affinity for the target receptor. Finally, the potency of the peptides may not be sufficient for therapeutical purposes.
As a result of the above described drawbacks, peptides are sometimes mainly used as sources for designing other drugs, and not as actual drugs themselves. In such case it is desirable to develop compounds in which these drawbacks have been reduced. Alternatives for peptides are the so-called peptidomimetics. Peptidomimetics based on the peptides of the present invention are also part of this application. In that case, one or more of XI to X7 are substituted with peptidomimetic building blocks.
Various definitions for peptidomimetics have been formulated in literature. Among others, peptidomimetics have been described as "chemical structures designed to convert the information contained in peptides into small non-peptide structures", "molecules that mimic the biological activity of peptides but no longer contain any peptide bonds", "structures which serve as appropriate substitutes for peptides in interactions with receptors and enzymes" and as "chemical Trojan horses".
In general, peptidomimetics can be classified into two categories. The first consists of compounds with non-peptidelike structures, often scaffolds onto which pharmacophoric groups have been attached. Thus, they are low molecular-weight compounds and bear no structural resemblance to the native peptides, resulting in an increased stability towards proteolytic enzymes. The second main class of peptidomimetics consists of compounds of a modular construction comparable to that of peptides, i.e. oligomeric peptidomimetics . These compounds can be obtained by modification of either the peptide side chains or the peptide backbone. Peptidomimetics of the latter category can be considered to be derived of peptides by replacement of the amide bond with other moieties. As a result, the compounds are expected to be less sensitive to degradation by proteases. Modification of the amide bond also influences other characteristics such as lipophilicity, hydrogen bonding capacity and conformational flexibility, which in favourable cases may result in an overall improved pharmacological and/or pharmaceutical profile of the compound.
Oligomeric peptidomimetics can in principle be prepared starting from onomeric building blocks in repeating cycles of reaction steps. Therefore, these compounds may be suitable for automated synthesis analogous to the well-established preparation of peptides in peptide synthesizers. Another application of the monomeric building blocks lies in the preparation of peptide/peptido imetic hybrids, combining natural amino acids and peptidomimetic building blocks to give products in which only some of the amide bonds have been replaced. This may result in compounds which differ sufficiently from the native peptide to obtain an increased biostability, but still possess enough resemblance to the original structure to retain the biological activity.
Suitable peptidomimetic building blocks for use in the invention are amide bond surrogates, such as the oligo-β-peptides (Juaristi, E. Enantioselective Synthesis of b-Amino Acids; Wiley-VCH: New York, 1996) , vinylogous peptides (Hagihari, M. et al . , J. Am. Chem. Soc. 1992,
114, 10672-10674), peptoids (Simon, R.J. et al . , Proc. Natl. Acad. Sci. USA 1992, 89, 9367-9371; Zuckermann, R.N. et al., J. Med. Chem. 1994, 37, 2678-2685; Kruijtzer, J.A.W. & Liskamp, R.M.J. Tetrahedron Lett. 1995, 36, 6969-6972) ; Kruijtzer, J.A.W. Thesis; Utrecht University, 1996; Kruijtzer, J.A.W. et al . , Chem. Eur. J. 1998, 4, 1570-1580), oligosulfones (Sommerfield, T. & Seebach, D. Angew. Chem., Int. Ed. Eng. 1995, 34, 553- 554), phosphodiesters (Lin, P.S.; Ganesan, A. Bioorg. Med. Chem. Lett. 1998, 8, 511-514), oligosulfonamides (Moree, W.J. et al . , Tetrahedron Lett. 1991, 32, 409-412; Moree, W.J. et al . , Tetrahedron Lett. 1992, 33, 6389- 6392; Moree, W.J. et al . , Tetrahedron 1993, 49, 1133-
1150; Moree, W.J. Thesis; Leiden University, 1994; Moree, W.J. et al., J. Org. Chem. 1995, 60, 5157-5169; de Bont, D.B.A. et al., Bioorg. Med. Chem. Lett. 1996, 6, 3035- 3040; de Bont, D.B.A. et al . , Bioorg. Med. Chem. 1996, 4, 667-672; Lόwik, D.W.P.M. Thesis; Utrecht University, 1998) , peptoid sulfonamides (van Ameijde, J. & Liskamp, R.M.J. Tetrahedron Lett. 2000, 41, 1103-1106), vinylogous sulfonamides (Gennari, C. et al., Eur. J. Org. Chem. 1998, 2437-2449), azatides (or hydrazinopeptides) (Han, H. & Janda, K.D. J. Am. Chem. Soc. 1996, 118, 2539-2544), oligocarbamates (Paikoff, S.J. et al . , Tetrahedron Lett. 1996, 37, 5653-5656; Cho, CY. et al . , Science 1993, 261, 1303-1305), ureapeptoids (Kruijtzer, J.A.W. et al . , Tetrahedron Lett. 1997, 38, 5335-5338; Wilson, M.E. & Nowick, J.S. Tetrahedron Lett. 1998, 39, 6613-6616) and oligopyrrolinones (Smith III, A.B. et al., J. Am. Chem. Soc. 1992, 114, 10672-10674). Figure 11 shows the structures of these peptidomimetic building blocks.
The vinylogous peptides and oligopyrrolinones have been developed in order to be able to form secondary structures (β-strand conformations) similar to those of peptides, or mimic secondary structures of peptides. All these oligomeric peptidomimetics are expected to be resistant to proteases and can be assembled in high- yielding coupling reactions from optically active monomers (except the peptoids) .
Peptidosulfonamides are composed of α- or β- substituted amino ethane sulfonamides containing one or more sulfonamide transition-state isosteres, as an analog of the hydrolysis of the amide bond. Peptide analogs containing a transition-state analog of the hydrolysis of the amide bond have found a widespread use in the development of protease inhibitor e.g. HIV-protease inhibitors.
Another approach to develop oligomeric peptidomimetics is to completely modify the peptide backbone by replacement of all amide bonds by nonhydrolyzable surrogates e.g. carbamate, sulfone, urea and sulfonamide groups. Such oligomeric peptidomimetics may have an increased metabolic stability. Recently, an amide-based alternative oligomeric peptidomimetics has been designed viz. N-substituted Glycine-oligopeptides, the so-called peptoids. Peptoids are characterized by the presence of the amino acid side chain on the amide nitrogen as opposed to being present on the α-C-atom in a peptide, which leads to an increased metabolic stability, as well as removal of the backbone chirality. The absence of the chiral α-C atom can be considered as an advantage because spatial restrictions which are present in peptides do not exist when dealing with peptoids. Furthermore, the space between the side chain and the carbonyl group in a peptoid is identical to that in a peptide. Despite the differences between peptides and peptoids, they have been shown to give rise to biologically active compounds.
Translation of a peptide chain into a peptoid peptidomimetic may result in either a peptoid (direct- translation) or a retropeptoid (retro-sequence) . In the latter category the relative orientation of the carbonyl groups to the side chains is maintained leading to a better resemblance to the parent peptide. Review articles about peptidomimetics that are incorporated herein by reference are:
Adang, A.E.P. et al . ; Reel. Trav. Chim. Pays-Bas 1994, 113, 63-78; Giannis, A. & Kolter, T. Angew. Chem. Int. Ed. Engl. 1993, 32,1244-1267; Moos, W.H. et al . , Annu. Rep. Med. Chem. 1993, 28, 315-324; Gallop, M.A. et al . , J. Med. Chem. 1994, 37, 1233-1251; Olson, G.L. et al., J. Med. Chem. 1993, 36, 3039-30304; Liskamp, R.M.J. Reel. Trav. Chim. Pays-Bas 1994, 113, 1-19; Liskamp, R.M.J. Angew. Chem. Int. Ed. Engl . 1994, 33, 305-307; Gante, J. Angew. Chem. Int. Ed. Engl. 1994, 33, 1699-1720; Gordon, E.M. et al., Med. Chem. 1994, 37, 1385-1401; and Liskamp, R.M.J. Angew. Chem. Int. Ed. Engl. 1994, 33, 633-636. The invention thus furthermore relates to combinations of molecules that are not peptides themselves but have a structure and function similar to those of the peptides described herein. Examples of such molecules are the above described peptidomimetics, but also compounds in which one or more of XI to X7 are replaced by non-proteinogenic amino acids or D-amino acids. When reference is made in this application to peptides, it is intended to include also such other compounds that have a similar or the same structure and function and as a consequence a similar or the same biological activity as the peptides.
More in particular substitutions can be made with non-proteinogenic amino acids selected from the group consisting of 2-naphtylalanine (Nal (2)), β- cyclohexylalanme (Cha), p-amino-phenylalanine ((Phe(p- NH2) , p-benzoyl-phenylalanine (Bpa) , ornithine (Orn) , norleucine (Nle) , 4-fluoro-phenylalanine (Phe(p-F)), 4- chloro-phenylalanine (Phe(p-Cl)), 4-bromo-phenylalanine (Phe(p-Br)), 4-iodo-phenylalanine (Phe(p-I)), 4-methyl- phenylalanine (Phe(p-Me)), 4-methoxy-phenylalanine (Tyr(Me)), 4-nitro-phenylalanine (Phe(p-N02) ) .
Suitable D-amino acids for substituting the amino acids in the peptides of the invention are for example those that are selected from the group consisting of D-phenylalanine, D-alanine, D-arginine, D-asparagine, D-aspartic acid, D-cysteine, D-glutamic acid, D- glutamine, D-histidine, D-isoleucine, D-leucine, D- lysine, D-methionine, D-proline, D-serine, D-threonine, D-tryptophan , D-tyrosine, D-valine, D-2-naphtylalanine (D-Nal(2)), β-cyclohexyl-D-alanine (D-Cha) , 4-amino-D- phenylalanine (D-Phe(p-NH2) ) , p-benzoyl-D-phenylalanine (D-Bpa) , D-Ornithine (D-Orn) , D-Norleucine (D-Nle) , 4- fluoro-D-phenylalanine (D-Phe(p-F) ) , 4-chloro-D- phenylalanine (D-Phe(p-Cl) ) , 4-bromo-D-phenylalanine (D- Phe(p-Br)), 4-iodo-D-phenylalanine (D-Phe (p-I) ) , 4- methyl-D-phenylalanine (D-Phe(p-Me) ) , 4-methoxy-D- phenylalanine (D-Tyr(Me)), 4-nitro-D-phenylalanine (D- Phe(p-N02) ) .
One or more of the amino acids in the peptides can be replaced by peptoid building blocks selected from the group consisting of N-substituted glycines, such as N-benzylglycine (NPhe) , N-methylglycine (NAla) , N-(3- guanidinopropyl) glycine (NArg), N- (Carboxymethyl) glycine (NAsp) , N- (carbamylmethyl) glycine (NAsn) , N- (thioethyl) - glycine (NhCys) , N- (2-carboxyethyl) glycine (NGlu) , N-(2- carbamylethyl) glycine (NGln) , N- (imidazolylethyl) glycine (NhHis) , N-(l-methylpropyl) glycine (Nile), N-(2-methyl- propyl) glycine (NLeu), N- (4-aminobutyl) glycine (NLys), N- ( 2- ethylthioethyl) glycine (NMet) , N- (hydroxyethyl) - glycine (NhSer) , N- (2-hydroxypropyl) glycine (NhThr) , N- (3-indolylmethyl) glycine (NTrp) , N- (p-hydroxyphenmethyl) - glycine (NTyr) , N- (1-methylethyl) glycine (NVal) . All compounds of the invention may also be in cyclic form. A cyclic compound may have improved potency, stability, rigidity and/or other pharmaceutical and/or pharmacological characteristics.
In the combinations of the invention peptides having CHIPS activity may be combined with supportive non-peptides or with supportive peptides, whereas non- peptides having CHIPS activity may be combined with supportive peptides or supportive non-peptides or combinations thereof. In addition, both categories may be hybrid compounds (i.e. compounds in which not all proteinogenic amino acids are replaced by non- proteinogenic amino acids, D-amino acids or peptidomimetic building blocks) .
The functional activity of compounds of the invention can be assayed by various methods. This CHIPS activity of a compound can be measured by its ability to prevent the binding of fluorescent-fMLP (such as Bodipy-fMLP) or fluorescent-C5a (such as FITC-C5a) to neutrophils as determined by flow cytometry. CHIPS activity is also measured by its ability to prevent migration of neutrophils towards fMLP or C5a as determined by chemotaxis assays, such as the Transwell system. Furthermore, an assay based on the ability of chemokines, including fMLP and C5a, to initiate a rapid and transient rise in intracellular calcium concentration can be employed to screen for CHIPS activity, as described in Materials and Methods. Alternatively, an assay based on the ability of chemokines, including fMLP and C5a, to initiate an excretion of e.g. elastase in cytochalasin B-stimulated neutrophils, can be used to screen for CHIPS activity, as described in Materials and Methods. Various other assays known in the art can be used, including but not limited to the use of various calcium specific fluorescent probes in combination with flow cytometry or fluorometry, or microphysiometry . As cells for the screening of CHIPS activity by either method, e.g. freshly isolated neutrophils can be used or cells transfected with either FPR or C5aR, wild type or mutated forms of those receptors.
The supportive capacity of the other compound can be tested in the same manner as described above by comparing the CHIPS activity of the first compound having CHIPS activity alone with the CHIPS activity of the combination of the two compound. A compound has supportive capacity in case the CHIPS activity of the combination is higher than the CHIPS activity of the first compound alone. Combinations of compounds of the invention may be useful in treating, preventing or ameliorating inflammatory conditions that are involved in many diseases and disorders.
The combination of compounds of the invention may for example be used in the treatment or prophylaxis of indications involving the C5a-receptor (C5aR) and/or for ylated peptide receptor (FPR) on neutrophils, monocytes and endothelial cells. Such indications often involves acute or chronic inflammation reactions, such as found in cardiovascular diseases, in particular arteriosclerosis, atherosclerosis, vasculitis, ischaemia reperfusion episodes, ischaemic shock, cardiopulmonary bypass, (large) vessel surgery, transplantation, myocardial infarction, diseases of the central nervous system, in particular bacterial meningitis, viral meningitis, multiple sclerosis, stroke, Alzheimer's disease, brain tumour, (traumatic) brain injury, gastrointestinal diseases, in particular pancreatitis, ulcerative colitis, Crohn's disease, alcoholic hepatitis, viral hepatitis, Heliobacter pylori gastritis, gastric carcinoma, peritonitis, skin diseases, in particular psoriasis, contact dermatitis, atopic dermatitis, and other dermatoses, cutaneous T-cell lymphoma, burns, genitourinary diseases, in particular urinary tract infection, glomerulonephritis, Trichomonas vaginalis infection, endometriosis, joint diseases, in particular rheumatoid arthritis, acute reactive arthritis, gout, respiratory diseases, in particular adult respiratory distress syndrome (ARDS) , chronic obstructive pulmonary disease (COPD) , idiopathic pulmonary fibrosis, cystic fibrosis, asthma, pleural emphema, metal fume fever, bacterial pneumonia, chronic bronchitis, hypersensitivity pneumonia, Mycobacterium tuberculosis infection, viral respiratory tract infection, allergic rhinitis, sinusitis, bronchogenic carcinoma, and other diseases and indications, in particular periodontitis, chronic lymph leukemia, acute transplant rejection, glomerulonephritis, frost bite, repetitive strain injury (RSI) , sepsis.
Support for the therapeutical usefulness of the combination of compounds of the invention for treatment of the diseases listed above can be found in the following references: For ARDS: Demling RH (1995). The modern version of adult respiratory distress syndrome.
Ann. Rev. Med. 46:193-202; and Fujishi a S, Aikawa N 1995 Neutrophil-mediated tissue injury and its modulation. Intensive Care Med 21:277-285; For meningitis: Tunkel AR and Scheld WM (1993) . Pathogenesis and pathophysiology of bacterial meningitis. Clin. Microbiol. Rev. 6:118; Tuomanen El (1996) . Molecular and cellular mechanisms of pneumococcal meningitis. Ann. N. Y. Acad. Sci. 797:42-52. For injury after ischaemia/reperfusion: Helier T, et al . (1999) . Selection of a C5a receptor antagonist from phage libraries attenuating the inflammatory response in immune complex disease and ischemia/reperfusion injury. J. Immunol. 163:985-994. For rheumatoid arthritis: Edwards SW and Hallett MB (1997) . Seeing the wood for the trees: the forgotten role of neutrophils in rheumatoid arthritis. Immunology Today 18: 320-324; and Pillinger MH, Abra son SB (1995) . The neutrophil in rheumatoid arthritis. Rheum. Dis. Clin. North Am. 1995 21:691-714. For myocardial infarction: Byrne JG, Smith WJ, Murphy MP, Couper GS, Appleyard RF, Cohn LH (1992). Complete prevention of myocardial stunning, contracture, low-reflow, and edema after heart transplantation by blocking neutrophil adhesion molecules during reperfusion. J. Thorac. Cardiovasc. Surg. 104:1589-96. For COPD: Cox G (1998) . The role of neutrophils in inflammation. Can. Respir. J. 5 Suppl A:37A-40A; and Hiemstra PS, van Wetering S, Stolk J (1998). Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium. Eur. Respir. J. 12:1200-1208. For stroke: Barone FC, Feuerstein GZ (1999) . Inflammatory mediators and stroke: new opportunities for novel therapeutics. J. Cereb. Blood Flow Metab. 19:819-834; and Jean WC, Spellman SR, Nussbaum ES, Low WC (1998). Reperfusion injury after focal cerebral ischemia: the role of inflammation and the therapeutic horizon. Neurosurgery 43:1382-1396.
Furthermore, the combination of compounds of the invention may be used in the prophylaxis or treatment of indications involving CXCR4 and/or CCR5 receptors on leukocytes, in particular HIV infection.
The combinations of compounds of the invention are also useful in the treatment of indications involving C5aR and/or FPR on cells other than neutrophils, monocytes and endothelial cells, in particular lymphocytes, dendritic cells, eosinophils, basophils, macrophages, microglia cells, astrocytes, Kupfer cells, hepatocytes and epithelial cells. An additional application is the use in prophylactic or therapeutic vaccines (to induce the production of anti-CHIPS antibodies to decrease the in vivo CHIPS activity, thereby stimulating the natural inflammation response against CHIPS producing bacteria) for infections with
CHIPS-producing bacteria, such as Staphylococcus aureus.
Combinations of compounds of the invention may be used themselves as inhibitors of fMLP and C5a binding to their respective receptors FPR and C5aR, or to design inhibitors of CHIPS binding, by screening for competitive inhibition. This can be performed by various approaches, some of which are described hereunder. Inhibitors of CHIPS binding (to the putative CHIPS receptor or receptor domains) having CHIPS activity (CHIPS agonists) are also useful for treating inflammation conditions and HIV.
The invention furthermore relates to the use of the combination of compounds having CHIPS activity for the manufacture of a composition or preparation for prophylaxis or therapy, in particular for the prophylaxis or treatment of acute and chronic inflammation reactions and HIV infection, more in particular for the treatment of the indications listed above.
Also part of the present invention are therapeutic compositions comprising a suitable excipient and one or more of the combinations of compounds having CHIPS activity of the invention. Such composition can be used for the treatments as specified above.
The invention further relates to use of the combinations of compounds, wherein the separate compounds, such as peptides, are optionally incorporated in or fused to a larger protein, for various purposes, such as raising antibodies thereto, modulating the CHIPS activity or in a therapeutic preparation. An antibody or biologically active fragment thereof specifically directed to one or both of the compounds in the combinations as claimed is also part of this invention. Such antibodies or biologically active fragments thereof like Fab, scFv fragments etc. can be used in the diagnosis, treatment or prophylaxis of infections of CHIPS producing bacteria, such as Staphylococcus aureus.
The information in the compounds of the combinations of the invention and the combinations of compounds themselves can be used to screen for other agents which are capable of interfering (e.g. through binding to CHIPS, thereby blocking the sites that CHIPS uses to bind to and/or activate its receptor or changing the CHIPS conformation such that the binding and/or activating capacity to, respectively of its receptor is impaired) with peptides having CHIPS activity. Such agents thus may act as inhibitors of CHIPS binding to and/or activation of its putative receptor, thereby improving for instance the neutrophil 's activation and chemotactic capacities in the presence of combinations of compounds having CHIPS activity. Appropriate screening assays may for example use the labeled (e.g. fluorescent) purified CHIPS protein that binds to C5aR/FPR-bearing cells as analyzed by flow cytometry or fluorometry. A suitable binding assay may alternatively employ purified CHIPS receptor or receptor domain on a carrier with a form of CHIPS protein as ligand. Alternatively, an assay can be employed that screens for the ability to bind or compete with CHIPS for binding to a specific anti-CHIPS antibody (monoclonal, polyclonal, or single chain antibody) by various immunoassays known in the art, including but not limited to competitive and non-competitive ELISA techniques or Biosensor technology employing a sensor chip coated with either ligand
(CHIPS) , antibody or putative CHIPS receptor (Surface Plasma Resonance (SPR) technique like the BiaCore) . Any (poly) peptide having CHIPS activity other than CHIPS may also be used in the screening assays described. All these methods can be adapted for High Throughput Screening (HTS) .
Combinations of compounds interfering with CHIPS or with combinations of compounds of the invention are also part of this application. Such interfering compounds can be used in the diagnosis, treatment or prophylaxis of infections of CHIPS producing bacteria, such as Staphylococcus aureus. The information in the combination of compounds and the combinations of compounds themselves can be used for other agents (including antibodies or fragments thereof) which are capable of interfering (e.g. through binding on the CHIPS receptor or changing the conformation of the CHIPS receptor) with the CHIPS receptor, without having (or only partial) CHIPS activity themselves. Such (partial) CHIPS antagonists may be found using appropriate screening assays. For example using fluorescent labeled purified CHIPS protein or peptide in various competitive CHIPS receptor binding and/or functional inhibiting screening assays, some of which have been mentioned above.
Combinations of compounds are claimed that interfere with the CHIPS receptor. Such antagonistic compounds can also be used in the treatment or prophylaxis of infections of CHIPS producing bacteria. The CHIPS interfering compounds, anti-CHIPS antibodies or biologically active fragments thereof and chimerics, single chains, and expression libraries may be used to neutralize the activity of the CHIPS protein or related (poly) peptides in prophylaxis or therapy. Some of these may also be used for diagnostic purposes to bind CHIPS or related (poly) eptides. CHIPS receptor interfering compounds and such antibodies and such CHIPS interfering compounds that are all based on the combination of compounds of the invention, are for example useful for the prophylaxis or treatment of CHIPS producing bacteria, such as Staphylococcus aureus infection.
Both the antibodies and their biologically active fragments and/or the CHIPS interfering compounds and/or the CHIPS receptor interfering compounds are useful for the preparation of a coating composition for use on surfaces of medical devices that are introduced into the human body through the skin or open surgical procedures, such as the surface of a catheter tip. In such cases the composition comprising the compounds that inhibit CHIPS activity or combinations thereof is a slow- release composition.
The invention further relate to therapeutic, diagnostic or prophylactic compositions comprising a suitable excipient and one or more antibodies and/or biologically active fragments thereof and/or one or more interfering compounds .
Also part of the invention are methods for prophylaxis or treatment of a subject suffering from indications involving C5aR and/or FPR on neutrophils, monocytes and endothelial cells comprising administering a therapeutically effective amount of the combinations of compounds of the invention. A further embodiment of the invention relates to a method for the prophylaxis or treatment of a subject suffering from indications involving CXCR4 and/or CCR5 receptors on lymphocytes comprising administering a therapeutically effective amount of the combinations of compounds of the invention. The indication to be treated is for example HIV infection.
Furthermore, the invention relates to methods for prophylaxis and treatment of a subject suffering from indications involving C5aR and/or FPR on leukocytes other than neutrophils, monocytes and endothelial cells comprising administering a therapeutically effective amount of the combinations of compounds of the invention. The other leukocytes are for example lymphocytes, dendritic cells, eosinophils, basophils or macrophages. The invention further relates to amino acid sequences that can be identified by so-called "computer cloning". More specifically, this technique comprises of using the amino acid sequences of peptides used in the combinations of the invention, derivatives, and analogues thereof, as a query for screening protein sequences or protein sequence databases, using search algorithms that can identify regions with homology. Such algorithms are known to the person skilled in the art and include, but are not limited to, BLAST searches (Altschul et al . , J. Mol. Biol. 215, 403-410 (1990)). The amino acid sequence database that may be searched include, but are not limited to, the Swissprot™ database. When using a BLAST search or modifications thereof, generally subjects that display homology can be identified. Identification is based on the value of the Score or the Smallest Sum Probability P(N).
The combination of compounds as claimed can also be used in a diagnostic kit for determining the presence of CHIPS in the body. The invention thus also relates to a diagnostic kit for determining the presence of CHIPS in the body, comprising a combination of compounds of the invention.
All compounds that are used in the combinations of the invention, i.e. peptides and non-peptides, may find various other applications. Such applications include, but are not limited to:
- Isolation of factors that can bind the above mentioned molecules. Examples of such factors being receptors and proteins. Such isolation can for instance be performed using the yeast two hybrid system or using tagged molecules of the invention as bait for fishing.
- Design of further peptidomimetics or small chemical compounds. - Making phage display libraries, which can in turn be used for determining active domains, functional equivalents etc. - Assay for determination of the biological CHIPS activity (chemotaxis inhibition or chemokine receptor expression or calcium flux)
All compounds that are used in the combinations of the invention can be labeled in any way. Examples of labeling include but are not limited to fluorescence, biotin, radioactive labeling etc. Such labeled molecules can be used for screening of compounds that resemble or overlap with the biological activity of CHIPS, as well as identification of binding sites, both in vivo and in vitro, and for tracing CHIPS protein in an organism.
The present invention will be further illustrated in the examples that follows and that is in no way intended to be limiting to this invention. In this description of the example, reference is made to the following figures and tables:
Figure 1 shows the sequence of the chp gene from S. aureus Newman. The chp open reading frame (ORF) is underlined. Figure 2 shows the amino acid sequence deduced from the S . aureus Newman chp gene.
Figure 3 shows the effect of the 5 amino acid peptide scan on the fMLP- (A) and PAF-induced (B) calcium mobilization in neutrophils. Figure 4 shows the concentration-dependent inhibition of the fMLP- and PAF-induced calcium mobilization in neutrophils by pepl-15.
Figure 5 shows the specific effect of pepl-15 on the fMLP- C5a- and PAF-induced calcium mobilization in neutrophils.
Figure 6 shows the effect of different lengths of CHIPS-derived peptides, ranging from pepl-5 to pepl-16 as depicted in Table 2 , on the fMLP-induced elastase release in neutrophils. Figure 7 shows the effect of a one amino acid peptide scan, spanning the amino acids 1 to 22 of CHIPS as depicted in Table 2, on the fMLP-induced calcium mobilization in neutrophils. Figure 8 shows the effect of the addition of small amino acids to the N-terminal phenylalanine of CHIPS-derived pepl-13 on the fMLP-induced calcium mobilization in neutrophils. Figure 9 shows the effect of exchanging the first phenylalanine of pep 1-8 with non-proteinogenic amino acids, D-amino acids, peptidomimetic building blocks, and acetylation of the N-terminus on the fMLP- induced elastase release in neutrophils. Figure IOA shows the effect of rCHIPS1"121 and rCHIPS4'121 on fMLP Bodipy binding.
Figure 10B shows the competition between rCHIPS1"121 and rCHIPS4"121 for CHIPS-FITC binding.
Figure 11 shows the structure of the various types of peptidomimetic building blocks.
Table 1 shows the sequences of 15 mer synthetic peptides derived from CHIPS that span the entire CHIPS protein.
Table 2 shows the sequences of synthetic peptides derived from CHIPS that vary in length from 1 to
40 (pepl-5 till pepl-40) , that vary in length from 1 to 16 by one amino acid (pepl-5 till pepl-16) , and a one amino acid peptide scan spanning amino acids 1 to 22 of CHIPS, and their effect on the fMLP-induced response in neutrophils (+) .
Table 3 (page 1, 2, and 3) shows the sequences of CHIPS-derived peptide 1-10, containing all 20 available natural occurring amino acid exchanges at each position. Substitutions are in gray boxes and the substitutions generating the original pepl-10 are in bold.
Table 4A shows the summary of the pepl-10 single amino acids exchanges as determined by their effect on the fMLP-induced elastase release in neutrophils in a 100 μM concentration.
Table 4B shows the summary of the pepl-10 single amino acids exchanges as determined by their effect on the fMLP-induced elastase release in neutrophils in a 10 μM concentration.
Table 5 shows the sequences of cyclic synthetic peptides derived from CHIPS that have two cysteine (C) substitutions in pepl-20 or pepl-10, and their effect on the fMLP-induced elastase release in neutrophils (+) .
MATERIALS AND METHODS
1. Generating recombinant CHIPS 1.1 Sequence of the CHIPS-encoding gene (chp) of Staphylococcus aureus
DNA was sequenced by cycle sequencing on a DNA sequencer 4000 L (LI-COR Inc., Lincoln, Neb., USA) using the Thermo Sequenase™ fluorescent-labeled prime cycle sequencing kit (Amersham, Little Chalfont, UK) . Suitable primers were used to directly sequence genomic DNA which was isolated according to J. Mamur (J. Mol. Biol., 3:208- 218 (1961)). The sequencing method has been described briefly in Peschel et al . (J. Biol. Chem., 274:8405-8410 (1999)). To perform sequence similarity searches, the program BLAST 2.0 with the non-redundant protein database of the NCBI (Bethesda, Md. , USA) was used. Sequence alignments were accomplished using the Higgins-Sharp algorithm of the program MacDNASIS Pro (Hitachi Software Engineering, San Bruno, Calif., USA).
Previously, the first 35 amino acids of CHIPS have been determined by N-terminal sequencing of the purified protein (PCT/NL99/00442) . The S . aureus DNA is very rich in A and T nucleotides while G and C nucleotides are rare (only about 30% of total bases) .
Thus, for most amino acids, the most A- and T-rich codons are preferred. According to this rule, a primer sequence was derived from amino acids 15-24 (GAAAAAGAAAAAGCATATAAAG-AA (SEQ ID NO 1) ) . The primer was used to directly sequence genomic DNA from S . aureus Newman (a commonly used laboratory strain) yielding a sequence of several hundred base pairs. A new primer was derived from this sequence to read toward the binding site of the first primer.
The combined DNA sequence contained the binding site of the first primer with two differences (G instead of A in position 3 and T instead of A in position 15) . It encoded an open reading frame of 450 bp preceded by a reasonable Shine Dalgarno sequence for initiation of translation (J. Shine and L. Dalgarno, Proc. Natl. Acad. Sci. USA, 71:1342-1346 (1974)) and followed by three stop codons (Figure 1 with the gene underlined) . The gene was named chp; it encodes a putative protein of 149 amino acids with no similarities to any protein in the databases. The N-terminal 28 amino acids seem to form a signal peptide for secretion across the cytoplasmic membrane (3 positively charged residues followed by a non-charged region of 22 amino acids and an ALA-X-ALA consensus motive for cleavage by the signal peptidase 1 ; Figure 5) (G. von Heijne, Nucl. Acids Res. 14:4683-4690 (1986)). The signal peptide is followed by a region that matches almost perfectly the N-terminal 35 amino acids of CHIPS. The only exception is a serine in position 33 of the deduced mature protein instead of an asparagine residue predicted by N-terminal sequencing. The deduced mature protein has a size of 121 amino acids and 14.1 kDa and an isoelectric point of 9.32. It thus fulfills all requirements for the CHIPS protein. The derived amino acid sequence is shown in Figure 2.
1.2. Generating recombinant CHIPS in E.coli The DNA sequence for CHIPS from S . aureus is cloned into a suitable vector that enables efficient expression of CHIPS in competent E.coli host cells using conventional molecular biology techniques. The strategy used enables expression of the complete CHIPS protein linked to a removable HIS-tag at the N-terminus in the cytoplasm of E.coli. The trc Expression System (pTrcHIS B vector; Invitrogen) was used that enables expression of non-toxic proteins in E. coli. This system uses the trc promotor for high-level, regulated expression in any E.coli strain with a ulticloning vector. The vector contains an N-terminal polyhistidine (6xHis) tag for rapid purification, a Xpress epitope for easy detection with an anti-Xpress antibody and an Enterokinase cleavage site for removal of fusion tag. This leaves the purified CHIPS protein with an N-terminal phenylalanine (F) .
S . aureus Newman chromosomal DNA was used as template for the PCR reaction using Pwo-DNA polymerase that results in a blunt ended PCR product. The primers used are CHIPS-TTT (starts exactly with the first amino acid of CHIPS (F) and CHIPS-TAA (containing a stop codon and a EcoRI-site) .
The PCR product is digested with EcoRI and the pTrcHIS B vector with BamHI . The 5 ' overhang is removed with Sl-nuclease to make the BamHI site blunt ended exactly where the enterokinase (EK) will digest the protein. Thereafter the vector is digested with EcoRI and ligated with the digested PCR product. For transformation of the vector, TOP-10 E.coli is used (InVitroGen) using standard calcium precipitation (F.M. Ausubel et al . , 1990, Current Protocols in Molecular Biology, John Wiley and Sons, Inc. , New York, N.Y.). Clones are screened on Ampicillin containing plates and proper ligation of CHIPS gene is verified by sequencing of the isolated plasmid (clone29) .
Once expressed, the E. coli bacteria are lysed and the protein mixture is applied onto a Nickel-ion affinity column (ProBond) . Therefore a culture of clone29 in LB medium + 50 μg/ml Ampicillin is initiated with 1 mM IPTG for 4 h at 37 °C. Bacteria are centrifuged and the pellet resuspended in cold Phosphate buffer pH 7.8 and stored at in -20 °C. For cell lysis, lysozyme (100 μg/ml) is added for 15 min on ice, tubes are sonicated, frozen in liquid N2 and thawed in a 37 °C waterbath. This cycle of sonication/freeze/thaw is repeated another 3 times. Thereafter RNase and DNase (5 μg/ml) is added for 30' on ice. The mixture is centrifuged for at 3000 g for 30' at 4°C and filtered through a 0.45 μm filter. The final lysate is diluted 1:1 with cold Phosphate buffer pH 7.8 and run through a charged Nickel column (InVitroGen) . The column is washed with Phosphate buffer pH 7.8, with Phosphate buffer pH 6.0 and with Phosphate buffer pH 5.3. The bound CHIPS is eluted with 500 mM imidazole in pH 6.0 Phosphate buffer.
The HIS-tag is removed by enterokinase cleavage followed by removal of the protease with an EK-Away enterokinase affinity resin. Therefor the eluate is dialysed overnight in cold digestion buffer (50 mM Tris- HCl, 1 mM CaCl2 and 0.1% Tween-20, pH 8.0), filtered through a 0.45 μm filter and digested with 0.175 μl Enterokinase/ l HIS-CHIPS product. This amount of Enterokinase is batch-dependent and results in a partial digestion to avoid the generation of breakdown products. The digested product is dialyzed against Phosphate buffer pH 7.8 and passed over a fresh Nickel column to eliminate uncleaved HIS-CHIPS; the run through is pure rCHIPS. Undigested HIS-CHIPS can be eluted again from Nickel column for a second digestion round. The Nickel column is finally washed with 50 mM EDTA, 0.5 M NaOH, water, 5 mg/ l NiCl2, water and stored in 20% ethanol.
All steps in the isolation and digestion of HIS-CHIPS are checked by SDS-PAGE on a 16.5% Tris-Tricine Ready gel using the Mini-Proteanll system (BioRad) .
1.3. Production of recombinant CHIPS4"121
When several E.coli colonies containing the plasmid with recombinant CHIPS were analyzed for proper insertion of the chp gene by sequencing, several incomplete insertions were found. One of them that contains the complete HIS-tag, enterokinase cleavage site and the CHIPS protein minus the first three amino acids (CHIPS4"121'*; clone 19) was further propagated and purified as described above for complete CHIPS. 2. Synthesis of CHIPS-derived peptides
2.1 Method one .
Peptides were prepared by automated simultaneous multiple peptide synthesis, set up by using a standard autosampler (Gilson 221) . Standard 9- fluorenylmethoxycarbonyl chemistry with in situ PyBop/N- methylmorpholine (Novabiochem, Laufelfingen, Switzerland) activation of the amino acids in a fivefold molar excess with respect to 2 μmol/peptide PAL-PEG-PS resin (Perseptive Biosystems, Framingham, Mass.) was used. Peptides were obtained as C-terminal amides after cleavage with 90 to 95% trifluoroacetic acid-containing scavenger cocktails. Most peptides were dissolved in distilled H20 to a concentration of 1 to 3 mM; others were dissolved in dimethyl sulfoxide. References describing similar methods are:
Van der Zee, R. , S.M. Anderton, C.A.F. Buskens, E. Alonso de Velasco, and W. Van Eden. 1995. Heat shock protein T cell epitopes as immunogenic carriers in subunit vaccines. H. L. S. Maya (Ed.), Peptides 1994, Proceedings of the Twenty Third European Peptide Symposium, ESCOM Leiden 841-842) .
2.2 Method two Synthesis of CHIPS derived peptides was performed on TGT resin (5 g, 0.3 mmol, NovaBioche ) and transferred to peptide synthesizer, were a solution of piperidine (12 ml) in dimethylformamide (DMF; 18 ml) was added to the resin. The solution was swirled for 1 hour and the resin washed with DMF (3 x 30 ml) followed by dichloromethane (DCM; 3 x 30 ml) and allowed to dry under vacuum for 5 minutes. The remainder of the peptides was sequentially assembled employing standard Fmoc chemistry. Cleavage of the peptides was accomplished by treating the protein resin with a solution of trifluoroacetic acid/triisopropylsilane/H20 [90:8:2 v/v/v] for 2.5 hours. The crude product was isolated by ether precipitation followed by purification by using High Performance Liquid Chromatography. References describing similar methods are:
E Bayer et al . , in: Peptides, Chemistry, Structure and
Biology. Proceedings of the 13th American Peptide symposium. RS Hodeges and JA Smith (eds) ESCOM, Leiden,
(1994) p. 156.
G Grύbler et al . , in: Innovation and perspectives in
Solid Phase Synthesis 3rd International Symposium. RE Pron
(ed) Mayflower Worldwide, Birmingham (1994) p. 517.
2.3 Method three
N -Fmoc-protected amino acids were purchased from Alexis (Switzerland) . For the Nα-Fmoc-protected amino acids, the following side chain protecting groups were used: Tyr(t-Bu), Lys(Boc), Thr(t-Bu), Ser(t-Bu),
Asn(Trt), Glu(Ot-Bu) and Phe(p-NHBoc) . The oligomers were synthesized on ArgoGel™ Rink-NH-Fmoc resin, 0.32 mmol/g (Argonaut Technologies, Muttenz, Switzerland). Peptide grade NMP, DCE, and TFA and HPLC-grade CH3CN, MTBE, and n- hexane were purchased from Biosolve B.V. (Valkenswaard,
The Netherlands) . All other solvents and reagents were acquired from commercial sources and used without further purification.
The Fmoc-protected N-substituted glycines were synthesized according to Kruijtzer et al. (Kruijtzer, J.A.W., Hofmeyer, L.J.F., Heerma, W. , Versluis, C. , & Liskamp, R.M.J. (1998) Chem. Eur. J. 4, 1570-1580; and Kruijtzer, J.A.W., Synthesis of Peptoid Peptidomimetics, Thesis, Utrecht University, The Netherlands, 1996) . The oligomers were synthesized on a
MultiSynTech Syro II Robot Synthesizer (MultiSynTech, Witten, Germany) . HPLC runs were performed on two HPLC systems: a Gilson automated HPLC workstation with UV detector system operating at 214 nm and 278 nm, or a Shimadzu automated HPLC system with a UV detector system operating at the same wavelengths. Analytical HPLC was conducted by using an Alltech Adsorbosphere XL C18 (300 A, 5 μm, 250 x 4.6 mm) column at a flow rate of 1.0 mL min-1. Elution was effected with a gradient from 0.1% TFA in water/acetonitrile (90/10) to 0.1% TFA in acetonitrile/water (90/10) over 40 minutes. Electrospray ionization mass spectrometry (ESI-MS) was measured on a Shimadzu LCMS-QP8000 single quadrupole bench-top mass spectrometer operating in a positive ionization mode. For the MS full scan spectra, data were acquired in continuum mode over the range m/z 200 to 1500 in 1 second at unit mass resolution. A deflector voltage of 50 V was set, which produces predominantly (M+H)+ ions. Instrumental control, data acquisition and data processing were carried out using the CLASS 8000 software package (version 1.10). The observed average m/z was compared with the calculated (M+H)+ average mass. The oligomers were synthesized on 0.05 mmol scale in a polypropylene reaction tube equipped with a polypropylene frit. The syntheses were carried out on ArgoGel™ Rink-NH-Fmoc resin (0.32 mmol/g) to obtain C- terminal amides. The synthesis started with 156 mg dry resin. The resin was swollen in DCE (two times with 2 mL for 4 min) followed by NMP (two times with 2 mL for 4 min) . The Fmoc group was removed by a double treatment with 2 mL 20% piperidine in NMP during 8 min. Subsequently, the resin was extensively washed with NMP (five times with 2.5 mL for 2 min). To this deprotected resin, 1 mL of a 0.2 M Fmoc-protected N-substituted glycine monomer or Fmoc-protected amino acid solution in NMP was added followed by 0.75 mL of a 0.267 M solution of HBTU/HOBt in NMP and 0.5 mL of a 0.4 M solution of DiPEA in NMP. After 45 min, the reaction tube was drained and the resin was washed with NMP (five times with 2.5 mL for 2 min) . During the resin swelling and washing steps, Fmoc deprotection and coupling reactions, the resin suspension was magnetically mixed by levitation stirring for 15 s per min. After the final Fmoc removal the resin was washed with NMP (five times with 2.5 mL for 2 min) and DCE (three times with 2.5. L for 2 min). The anchored oligomers thus obtained were cleaved from the resin and deprotected by treatment with 2 mL TFA/H20/TIS (95:2.5:2.5, v/v/v) solution for 2 h at room temperature. The oligomers were precipitated with MTBE/n-hexane (1:1, v/v, 50 mL) . The precipitate was collected by centrifugation (3000 rpm, 5 min) , the supernatant was decanted, and the pellet was resuspended in MTBE/n-hexane (1:1, v/v) and centrifuged again. Subsequently, the pellet was washed two times with MTBE (50 mL) . After this, the pellet was dissolved in tert . -BuOH/water (1:1, v/v, 5-10 mL) and lyophilized to obtain the oligomers as a white fluffy solid. The purity was analyzed by analytical HPLC and the hybrids were characterized by mass spectrometry .
3. Assays for CHIPS activity. 3.1 Calcium mobilization assay
The effect of CHIPS and the synthetic peptides on the intracellular free calcium release induced by fMLP, C5a and PAF (Platelet Activating Factor) in neutrophils was tested as follows. Cells were loaded with 1 μM of a calcium specific intracellular probe (Fluo-3, acetoxymethyl (AM) ester; Molecular Probes) , washed and incubated with various concentrations of synthetic peptides (1 to 100 μM) or rCHIPS (1 μg/ml) for 15 min at room temperature. From each sample the initial fluorescence value was determined in the FACScan by measuring 2000 cells. Subsequently, stimulus was added (10" M fMLP (Sigma), 10"10 M rC5a (Sigma) or 10"10 M PAF (Calbioche ) ) and the fluorescence intensity from the same sample was determined exactly 15 seconds after administration of the stimulus (the optimal time point for all agonists) . Triggering neutrophils with fMLP, C5a or PAF initiates a rapid and transient increase in free intracellular calcium concentration that is measured by an increase Fluo-3 fluorescence signal. From each activated sample, the initial basal fluorescence value is subtracted. Results are expressed as a percentage of buffer treated cells stimulated with either fMLP, C5a or PAF.
3.2 Elastase release assay. Human neutrophils contain enzymes in their granules, among which elastase. Furthermore, the granules contain the fMLP receptors ready for quick recruitment to the cell surface. Cytochalasin B treatment of neutrophils stimulates the excretion of the granules, which results in an increase of the amount of fMLP receptors expressed on the cell surface of the neutrophils. Upon subsequent stimulation with fMLP, the neutrophils will effectively excrete their granule content into the medium, thereby releasing elastase. CHIPS and CHIPS peptides will inhibit the activation of the neutrophils with fMLP, which can be measured via a decrease in elastase release. The amount of elastase is determined via a specific enzymatic reaction using the fluorescent substrate methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-MAC (elastase substrate V; Calbiochem) .
In a round bottom microtiterplate neutrophils (lxlO4 per well) were incubated for 15 min with 5 μg/ml cytochalasin B together with rCHIPS (1 μg/ml) or CHIPS- derived peptides (0.4 to 100 μM) at room temperature. Subsequently, fMLP (lxl0"8M) was added. After 1 h incubation at 37 °C, the microtiterplate was centrifuged and fluorescent substrate (250 μM) was added to each well. The elastase response was measured for 30 min at 37 °C in a Fluostarll microtiter fluorometer. Results are expressed as a percentage of cytochalasin B- and buffer- treated cells stimulated with fMLP after substraction of the values for non-stimulated cells. EXAMPLES EXAMPLE 1
Identification of the active side within the CHIPS protein 1.1 Scan of the CHIPS protein by overlapping peptides. The sequence of CHIPS (amino acids 1 to 121) was divided into 22 different 15-mer peptides that progressed along the CHIPS sequence by initiating a new peptide every sixth amino acid as shown in Table 1. Peptides were prepared according to method one (MATERIALS AND METHODS section 2.1).
Figure 3 shows the effect of these peptides spanning the CHIPS on the fMLP- (A) and PAF-induced (B) calcium mobilization in neutrophils. It can be seen that pepl-15 and pep61-75 strongly inhibit the fMLP-induced calcium mobilization in neutrophils. The effect of pep61-75 seems to be non-specific for fMLP, as it also inhibits the PAF- induced response. This might be due to a non-specific toxicity of this peptide. Concluding, only pepl-15 shows CHIPS activity. Figure 4 shows the concentration- dependent inhibition of the fMLP-induced calcium mobilization by pepl-15. Figure 5 shows the effect of pepl-15, on the fMLP- ,C5a- and PAF-induced calcium mobilization in neutrophils and confirms the specificity of pepl-15 and rCHIPS for the fMLP- and C5a-induced response.
1.2 Optimal length for the active site.
To verify and optimize the effective length for the active CHIPS peptide, peptides of increasing length spanning amino acid 1 to 40 were chosen all starting with the first phenylalanine (see Table 2) , including newly synthesized pepl-15. Peptides were prepared according to method two (MATERIALS AND METHODS section 2.2). Table 2 shows the effect of different lengths of CHIPS-derived peptides on the fMLP-induced calcium mobilization in neutrophils. This table shows that at least the first 8 amino acids are necessary for CHIPS activity, as pepl-5 shows no CHIPS activity in contrast to pepl-8 and all other depicted longer peptides. No improved activity of longer peptides was observed using different concentrations. To redefine the exact minimal length for the active CHIPS peptide, highly purified (>95% pure) peptides were synthesized starting with pepl-5 with a one amino acid elongation until pepl-12. Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3) .
Figure 6 shows the effect of different lengths of CHIPS-derived peptides, as depicted in Table 2, and their effect on the fMLP-induced elastase release in neutrophils. This Figure shows that at least the first 6 amino acids are necessary for CHIPS activity, as pepl-5 shows no CHIPS activity in contrast to pepl-6 and all other depicted longer peptides.
1.3 Crucial role for the first phenylalanine. The first 22 amino acids of CHIPS were divided into 8 different 15-mer peptides that progressed along the CHIPS sequence by initiating a new peptide every next amino acid. Peptides were prepared according to method one (MATERIALS AND METHODS section 2.1). Figure 7 shows the effect of the one amino acid peptide scan, as depicted in Table 2, spanning the amino acids 1 to 22 of CHIPS, on the fMLP-induced calcium mobilization in neutrophils. It can be seen that the first phenylalanine plays a crucial role in the CHIPS activity since only pepl-15 is active while all others are inactive.
1.4 Requirement for a non-blocked first ' phenylalanine.
To determine the essence for a free available N-terminal phenylalanine, peptides were constructed spanning the amino acids 1 to 13 of CHIPS with an additional amino acid before the first phenylalanine. Peptides were prepared according to method one (MATERIALS AND METHODS section 2.1).
Figure 8 shows the effect of the peptides spanning CHIPS 1 to 13 with additional N-terminal amino acids attached on the fMLP-induced calcium mobilization in neutrophils. It can be seen that blocking the CHIPS N- terminal phenylalanine with the chosen small amino acids Ala (alanine) , Gly (glycine) , Pro (proline) , Ser (serine) , Thr (threonine) or Val (valine) completely abrogated effective inhibition of the fMLP-induced calcium mobilization by the non-blocked pepl-15. The PAF- induced response is not affected by any peptide nor rCHIPS .
EXAMPLE 2
Modifications of the active site
2.1 Proteinogenic amino acid substitutions.
Substitutions with all 20 natural occurring amino acids were done in the CHIPS-derived peptide comprising of the first 10 amino acids of CHIPS. Each of the 10 amino acids were consecutively replaced with all 20 natural occurring amino acids, being A (alanine), C (cysteine) , D (aspartic acid) , E (glutamic acid) , F (phenylalanine) , G (glycine) , H (histidine) , I (isoleucine) , K (lysine) , L (leucine) , M (methionine) , N (asparagine) , P (proline) , Q (glutamine) , R (arginine) , S (serine) , T (threonine) , V (valine) , Y (tyrosine) , W (tryptophan), as shown in Table 3. Peptides were prepared according to method two or three (MATERIALS AND METHODS section 2.2 and 2.3). The peptides were tested in the elastase release assay in a concentration of 100 μM (optimal) as well 10 μM (suboptimal) to find the most potent combinations. Individual substitutions also resulted in the original pepl-10 that were indeed positive for each newly synthesized peptide (in bold) .
Tables 4A (100 μM) and 4B (10 μM) summarizes the positive single amino acid substitutions in pepl-6 (the minimal active part of CHIPS) . The results from the single amino acid substitutions (Table 4B) lead to a new set of possible amino acid substitutions as listed in the description resulting in 2x4x1x7x7x6 = 2352 new combinations. To synthesize and test all those new peptides for CHIPS activity, 200 individual combinations are synthesized chosen at random from the 2352 possibilities as examples for the total combinations.
2.2 Non-proteinogenic amino acid substitutions.
Non-proteinogenic, natural occurring (meaning non-human) amino acid substitutions were performed on the CHIPS-derived peptide comprising of the first 8 amino acids of CHIPS. Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3).
The first phenylalanine (Phe) of pepl-8 was replaced by the structurally related, non-proteinogenic amino acids Nal (2) 2-naphtylalanine) , Cha (cyclohexylalanme) , Phe(p-NH2) (p-amino-phenylalanine) and Bpa (p-benzoyl-phenylalanine) . Figure 9 shows the effect of these exchanges on the fMLP-induced elastase release in neutrophils. These results show that pepl-8, in which the first phenylalanine is exchanged for Cha or Bpa still shows considerable CHIPS activity. Exchange for Nal (2) results in a somewhat less active pep 1-8, whereas exchange for Phe(p-NH2) strongly reduces the CHIPS activity of pep 1-8.
2.3 D-amino acid substitutions. D-amino acid (in contrast to the natural L- isoform) substitutions were performed on the CHIPS- derived peptide comprising of the first 8 amino acids of CHIPS. Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3). The first phenylalanine (Phe) of pepl-8 was replaced by D-Phe and tested in the fMLP-induced elastase release in neutrophils. Exchange for D-Phe results in a somewhat less active pepl-8 as shown in Figure 9. 2.4 Peptidomimetic substitutions.
Peptidomimetic substitutions were performed on the CHIPS-derived peptide comprising of the first 8 amino acids of CHIPS. Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3).
The first phenylalanine (Phe) of pepl-8 was replaced by the peptidomimetic oligomer Nphe (N- Benzylglycine) and tested in the fMLP-induced elastase release in neutrophils. Exchange for NPhe results in an equally active pepl-8 as shown in Figure 9.
2.5 N-terminal modifications.
Chemical modifications (with a small molecular mass) on the N-terminal phenylalanine (Phe) were performed on the CHIPS-derived peptide comprising of the first 8 amino acids of CHIPS. Peptides were prepared according to method three (MATERIALS AND METHODS section 2.3) .
The first phenylalanine (Phe) of pepl-8 was acylated by the introduction of an N-terminal acetyl group (Ac-Phe) . This peptide was tested in the fMLP- induced calcium mobilization in neutrophils and showed diminished CHIPS activity of pepl-8 as shown in Figure 9.
EXAMPLE 3
Cyclisation of the peptides having CHIPS activity 3.1 Cysteine cyclisation.
Two cysteine residues were substituted into the peptide comprising of amino acids 1 to 20 (pepl-20) and 1 to 10 (pepl-10) of CHIPS, to enable these peptides to become cyclic. The position of the 2 cysteine residues varies as is depicted in Table 5. Peptides were prepared according to method two (MATERIALS AND METHODS section 2.2) . Table 5 shows the sequences of the CHIPS-derived peptides 1-20 and 1-10, each containing 2 cysteine residues to enable the formation of cyclic peptides, and their effect on the fMLP-induced elastase release in neutrophils (+) .
EXAMPLE 4 Competition for CHIPS binding to its putative receptor Competition with CHIPS-FITC binding
In Falcon tubes 5 μl serial dilutions of combinations of ompounds of the invention were prepared and mixed with 5 μl CHIPS-FITC (10 μg/ml). Thereafter 50 μl isolated neutrophils at 5 x 106 cells/ml are added and incubated for 30 min on ice. Cells are washed and analyzed for CHIPS-FITC binding by flow cytometry.
EXAMPLE 5 Indication for an additional binding site within the CHIPS protein 5.1 Assay for CHIPS activity: fMLP-Bodipy binding.
Granulocytes are isolated from heparinized blood of healthy volunteers via a Histopaque-Ficoll gradient in accordance with the standard method
(Troelstra et al . , J. Leukocyte Biol. 61, 173-178 (1997) ) . The remaining erythrocytes in the granulocyte fraction are lysed with sterile water (for 30 sec.) and washed after recovery of the isotonicity. The cells are finally resuspended in PRMI (Gibco) with 0.05% Human Serum Albumin (RPMI/HSA) . In Falcon tubes 50 μl cells (5xl06 cells/ml) are incubated with 5 μl CHIPS (10-fold concentrated) for 15 min at room temperature. The cells are placed on ice and washed once with RPMI/HSA (at 4°C) and resuspended in 50 μl fresh medium. 5 μl
BODIPY-labeled fMLP (final concentration 0.1 μM; Molecular Probes) is then added and the sample is incubated for 60 minutes on ice. RPMI/HAS is added to a final volume of 200 μl and the fluorescent FMLP binding to the granulocytes is analysed with a flow cytometer (FACScan; Becton Dickinson) . The average fluorescence value of 5000 granulocytes is calculated with LysisII software (Becton Dickinson) . RESULTS
Figure IOA shows the effect of CHIPS on the binding of fMLP-Bodipy to the cells. The complete rCHIPS1" 121 abrogates concentration dependent the binding of fMLP- Bodipy. This is the characteristic feature of CHIPS activity. In contrast the mutant rCHIPS4"121 in the same concentration range tested did not affect the binding of fMLP-Bodipy to the cells, indicating this mutant has no CHIPS activity.
5.2 Competition for CHIPS-FITC binding.
In Falcon tubes 5 μl CHIPS-FITC (20 μg/ml) is incubated with various concentrations unlabeled rCHIPS or mutant rCHIPS4"121 for 15 min at room temperature. Subsequently, 50 μl granulocytes (5 x 106 cells/ml) are added to the tubes and incubated for 60 min on ice. Cells are washed once with medium (RPMI containing 0.05% HSA) and resuspended in 200 μl fresh medium. Binding of CHIPS- FITC to the leukocyte is measured by flow cytometry (FACScan; Becton Dickinson) . The average fluorescence value of the cells is calculated with LysisII software (Becton Dickinson) .
RESULTS Figure 10B shows the concentration dependent inhibition of CHIPS-FITC binding by both the complete rCHIPS1"121 as well as the mutant rCHIPS4"121. Both preparations show a similar inhibition pattern with equal effective concentrations. This indicates that the mutant rCHIPS4"121 still can bind to the cells, but lacks the intrinsic CHIPS activity (Figure 10A) . These results point to a multi-site model for CHIPS that involves one or more supportive sites (in the 4 to 121 part of CHIPS) and a activity site that comprises the first 6 amino acids as shown with the peptides. Table 1
1 ' 10 20 30 40 50 60 70 80 90 100 110 1 CHIPS FTFEPFPTNEEIESN KMLEKEKAYKESFKNSGLPTTLGKLDEP.LRNYLKKGTKNSAQFEKMVILTENKGYYTVYLNTPLAEDRKNVELLGKMYKTYFFKKGES SSYVINGPGKTNEYAY pepl-15 FTFEPFPTNEEIESN pep6-20 FPTNEEIESN KMLE pepll-25 E1EΞNKKMLEKEKAY pepl6-30 KKM EKEKAYKESFK pep21-35 KEKAYKESFKNSG P pep26- 0 KESFKNSG PTTLGK pep31-45 NSGLPTTLGKLDERL pep36-50 TTLGKLDER RNYLK pep 1-55 DERLRNYLKKGTKN pep46-60 RNYLKKGTKNSAQFE pep51-65 KGTKNSAQFEKMVIL pep56-70 SAQFEKMVI TENKG pepδl-75 KMVILTENKGYYTVY pep66-80 TENKGYYTVYLNTP pep71-85 YYTVY NTP AEDRK pep76-90 NTPLAEDRKNVE L pep81-95 AEDRKNVELLGKMYK pep86-100 NVE LGKMYKTYFFK pap91-105 GKMYKTYFFKKGESK pep96-110 TYFFKKGESKSSYVI peplOl-115 KGESKSSYVINGPGK pepl06-121 ΞSYVINGPGKTNEYAY
Table 2 54
CHIPS 1-4+or-FTFEPFPTNEE I ESNKKMLEKEKAYKESFKNSGLPTTLGK
Varying lengths
P1-40 + FTFEPFPTNEE I ESNKKMLEKEKAYKESFKNSGLPTTLGK
P1-35 ND FTFEPFPTNEE I ESNKK LEKEKAYKESFKNSGLP
P1-30 + FTFEPFPTNEE I ESNKKMLEKEKAYKESFK
P1-25 + FTFEPFPTNEE I ESNKKMLEKEKAY
P1-23 + FTFEPFPTNEE I ESNKKMLEKEK
P1-20 + FTFEPFPTNEE I ESNKKMLE
P1-18 + FTFEPFPTNEE I ESNKKM
P1-15 + FTFEPFPTNEE I ESN
P1-13 + FTFEPFPTNEE I E
P1-10 + FTFEPFPTNE
P1-8 + FTFEPFPT
P1-5 - FTFEP
Varying lengths
P1-5 - FTFEP
P1-6 + FTFEPF
P1-7 + FTFEPFP
P1-8 + FTFEPFPT
P1-9 + FTFEPFPTN
P1-10 + FTFEPFPTNE
P 1-11 + FTFEPFPTNEE
P1-12 + FTFEPFPTNEE I
P1-13 + FTFEPFPTNEE l E
P1-14 + FTFEPFPTNEE I ES
P1-15 + FTFEPFPTNEE I ESN
P1-16 + FTFEPFPTNEE I ESNK
1 aa scan
P1-15 + FTFEPFPTNEE I ESN
P2-16 - TFEPFPTNEE I ESNK
P3-17 - FEPFPTNEE I ESNKK
P4-18 - EPFPTNEE I ESNKKM
P5-19 - PFPTNEE I ESNKKML
P6-20 - FPTNEE I ESNKKMLE
P7-21 - PTNEE I ESNKKMLEK
P8-22 - TNEE I ESNKKMLEKE Table 3 page 1/3
1 23 4567 8 9 # 1 234567 8 9 * 1 23456789* p1-10 ÷orFTFEPFPTN E p 1-10 ÷orFTFEPFPTNE p1-10 + orFTFEPFPTNE p 1-10 subst p1-10A10 FTFEPFPTNA pi-- I0K9 FTFEPFPT.-KE p1-10T8 FTFEPFPTNE p1-10C10 FTFEPFPT N& P1-' !0L9 FTFEPFPTNE p1-10V8 FTFEPFPVNE p1-10D10 FTFEPFPTNE p1-' 10 M9 FTFEPFPTNE p1-10W8 FTFEPFPWNE p1-10E10 FTFEPFPTNΪE p1-' 10 N9 FTFEPFPT§E p1-10Y8 FTFEPFPΫNE P1-10F10 FTFEPFPT N¥ p1-' 10 P9 FTFEPFPTJPΈ p1-10A7 FTFEPFATNE P1-10G10 FTFEPFPT N'ά P1-' 10 Q9 FTFEPFPTQE p1-10 C7 FTFEPFCTNE p1-10H10 FTFEPFPTN& p1-" 10 R9 FTFEPFPTNE p1-10D7 FTFEPFDTNE pi-ιo no FTFEPFPTN p1- 10 S9 FTFEPFPTNE p1-10E7 FTFEPFETNE p1-10K10 FTFEPFPTNE p1- 10 T9 FTFEPFPTNE p1-10 F7 FTFEPFFTNE p1-10L10 FTFE FPTN?U p1- 10 V9 FTFEPFPTNE p1-10G7 FTFEPF GTNE p1-10M10 FTFEPFPT H P1- 10 W9 FTFEPFPTNE p1-10 H7 FTFEPFHTNE p1-10N10 FTFEPFPTNU p1- 10 Y9 FT FEPFPTVE pl-1017 FTFEPF 1 TNE p1-10P10 FTFEPFPTN-B; p1- 10 A8 FTFEPFPANE p1-10K7 FTFEPFKTNE p1-10Q10 FTFEPFPTNE p1- 10 C8 FTFEPFPtNE p1-10 L7 FTFEPFLTNE p1-10R10 FTFEPFPTNE p1- 10 D8 FTFEPFPTNE p1-10M7 FTFEPFMTNE p1-10S10 FTFEPFPTNI p1- 10 E8 FTFEPFPTNE p1-10N7 FTFEP FNTNE p1-10T10 FTFEPFPTNT P1- 10 F8 FTFEPFPTNE p1-10 P7 FTFEPFPTNE p1-10 V10 FTFEPFPTN^ p1- 10 G8 FTFEPFPTNE p1-10Q7 FTFEPFQTNE p1-10W10 FTFEPFPTNIC p1- 10 H8 FTFEPFPTNE p1-10 R7 FTFEPFRTNE p1-10Y10 FTFEPFPTN^ p1- 1018 FTFEPFPTNE p1-10 S7 FTFEPF S TNE p1-10A9 FTFEPFPTNE p1- 10 K8 FTFEPFPTNE p1-10T7 FTFEPFTTNE p1-10C9 FTFEPFPTC'E P1- ■10 L8 FTFEPFPTNE p1-10V7 FTFEPFVTNE p1-10D9 FTFEPFPTNE p1- 10 M8 FTFEPFPTNE p1-10W7 FTFEPFMTNE
P1-10E9 FTFEPFPTNE p1- ■10 N8 FTFEPFPTNE p1-10Y7 FTFEPFVTNE p1-10F9 FTFEPFPTΪE P1- ■10 P8 FTFEPFPTNE p1-10A6 FTFEPAPTNE p1-10 G9 FTFEPFPT&E p1- •10 Q8 FTFEPFPTNE p1-10C6 FTFEPCPTNE p1-10H9 FTFEPFPTME p1- •10 R8 FTFEPFP§NE p1-10 D6 FTFEPDPTNE pl-1019 FTFEPFPTNE p1- •10 S8 FTFEPFPTNE p1-10 E6 FTFEPEP TNE
Table 3 page 2/3
1 23 4567 8 9 * 1 23456789 * 123456789* 1-10 + or FTFEPFPTNE p1-10 + orFTFEPFPTNE p1-10+orFTFEPFPTNE p 1-10 subst. p1-10F6 FTFEPFPTNE p1-10Q5 FTFEQFPTNE P1-10D3 FTDEPFPTNE p1-10G6 FTFEPGPTNE p1-10R5 FTFERFPTNE p1-10E3 FTEEPFPTNE
P1-10H6 FTFEPHPTNE p1-10S5 FTFESFPTNE p1-10F3 FTFEPFPTNE pl-1016 FTFEP I PTNE p1-10T5 FTFETFPTNE P1-10G3 FTGEPFPTNE
P1-10K6 FTFEPKPTNE P1-10V5 FTFEVFPTNE P1-10H3 FTHEPFPTNE p1-10L6 FTFEPLPTNE P1-10W5 FTFEWFPTNE pl-1013 FT1 EPFPTNE
P1-10M6 FTFEPMPTN E P1-10Y5 FTFEYFPTNE P1-10K3 FTKEPFPTNE p1-10N6 FTFEPNPTNE p1-10A4 FTFAPFPTNE P1-10L3 FTLEPFPTNE p1-10P6 FTFEPPPTNE p1-10C4 F T F'CP F P T N E P1-10M3 FTMEPFPTNE p1-10Q6 FTFEPQPTNE p1-10D4 FTFDPFPTNE P1-10N3 FTNEPFPTNE p1-10R6 FTFEPRPTNE p1-10E4 FTFEPFPTNE P1-10P3 FTPEPFPTNE p1-10S6 FTFEPSPTNE p1-10F4 FTFFPFPTNE P1-10Q3 FTQEPFPTNE p1-10T6 FTFEPTPTNE p1-10G4 FTFGPFPTNE P1-10R3 FTREPFPTNE p1-10V6 FTFEPVPTNE P1-10H4 FTFHPFPTNE P1-10S3 FTSEPFPTNE
P1-10W6 FTFEPWPTNE pl-1014 FTF I'PFPTNE P1-10T3 FTTEPFPTNE
P1-10Y6 FTFEPYPTNE P1-10K4 FTFKPFPTNE P1-10V3 FTVEPFPTNE p1-10A5 FTFEAFPTNE p1-10 L4 FTFLPFPTNE P1-10W3 FTWEPFPTNE p1-10C5 FTFECFPTNE p1-10M4 FTFEPFPTNE P1-10Y3 FTYEPFPTNE p1-10D5 FTFEDFPTNE p1-10N4 FTFNPFPTNE P1-10A2 FAFEPFPTNE p1-10E5 FTFEEFPTNE P1-10P4 FTFPPFPTNE p1-10C2 FCF EPFPTNE p1-10F5 FTFEFFPTNE p1-10Q4 FTFQPFPTNE P1-10D2 FDFEPFPTNE p1-10G5 FTFEGFPTNE p1-10R4 FTFRPFPTNE P1-10E2 FEFEPFPTNE p1-10H5 FTFEHFPTNE p1-10S4 FTFSPFPTNE P1-10F2 FFFEPFPTNE pl-1015 FTFE I FPTNE p1-10T4 FTFTPFPTNE P1-10G2 FGFEPFPTNE p1-10K5 FTFEKFPTNE p1-10V4 FTFVPFPTNE P1-10H2 FHFEPFPTNE p1-10L5 FTFELFPTNE p1-10W4 FTFWPFPTNE p1-1012 F I FEPFPTNE p1-10M5 FTFEMFPTNE p1-10Y4 FTFYPFPTNE P1-10K2 FKFEPFPTNE p1-10N5 FTFENFPTNE p1-10A3 FTAEPFPTNE p1-10L2 FLFEPFPTNE p1-10P5 FTFEPFPTNE p1-10C3 FTCEPFPTNE P1-10M2 FMFEPFPTNE
Table 3 page 3/3
1 23 4 567 8 9 * p1-10 + or FTFEPFPTNE p 1-10 subst. p1-10N2 FNFEPFPTNE p1-10P2 FPFEPFPTNE p1-10Q2 FQFEPFPTNE p1-10R2 FRFEPFPTNE
P1-10S2 FSFEPFPTNE
P1-10T2 FTFEPFPTNE
P1-10V2 FVFEPFPTNE p1-10W2 F FEPFPTNE p1-10Y2 FYFEPFPTNE p1-10A1 ATFEPFPTNE p1-10C1 CTFEPFPTNE p1-10D1 DTFEPFPTNE
P1-10E1 ETFEPFPTNE p1-10F1 FTFEPFPTNE p1-10G1 GTFEPFPTNE p1-10H1 HTFEPFPTNE pl-1011 I TFEPFPTNE
P1-10K1 KTFEPFPTNE p1-10L1 LTFEPFPTNE p1-10M1 MTFEPFPTNE p1-10N1 NTFEPFPTNE p1-10P1 PTFEPFPTNE p1-10Q1 QTFEPFPTNE p1-10R1 RTFEPFPTNE p1-10S1 STFEPFPTN E p1-10T1 TTFEPFPTNE p1-10V1 VTFEPFPTNE p1-10W1 WTFEPFPTNE
P1-10Y1 YTFEPFPTNE
Table 4a
Alanine Ala A
Cysteine Cys C
Aspartic Acid Asp D lutamic Acid Glu E
'henylalanine Phe F
Glycine Gly G
Histidine His H
Isoleucine lie 1
Lysine Lys K
Leucine Leu L
Methionine Met M
Asparagine Asn N
Proline Pro P
Glutamine Glu Q
Arganine Arg R
Figure imgf000059_0002
Serine Ser S
Threonine Thr T
Valine Val V
Tryptophan Trp W
Tyrosine Tyr Y
Figure imgf000059_0001
SINGLE amino acid substitutions showing CHIPS activity as tested at 100uM peptide
Table 4b
Alanine Ala A
Cysteine Cys C
Aspartic Acid Asp D
3lutamic Acid Glu E
'henylalanine Phe F
Glycine Gly G
Histidine His H
Figure imgf000060_0002
Isoleucine lie 1
Lysine Lys K
Leucine Leu L
Methionine Met M
Asparagine Asn N
Proline Pro P
Glutamine Glu Q
Arganine Arg R
Serine Ser S
Threonine Thr T
Valine Val V
Tryptophan Trp w
Tyrosine Tyr Y
Figure imgf000060_0001
SINGLE amino acid substitutions showing CHIPS activity as tested at 10 uM peptide
Table 5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
CHIPS 1-20 + or- F T F E P F P T N E E E S N K K M L E
Cyclic peptides
P1-20C1C20 + C T F E P F P T N E E E S N K K M L G
P1-20 C3C20 - F T c E P F P T N E E E S N K K L c
P1-20 C5C20 - F T F E c F P T N E E E S N K K M L c
P1-20 C7C20 + F T F E P F c T N E E E S N K K M L c
P1-20 C9C20 + F T F E P F P T c E E E S N K K M L c
P1-20C11C20 + F T F E P F P T N E C E S N K K M L G
P1-20C13C20 + F T F E P F P T N E E C S N K K M L C
P1-20C15C20 + F T F E P F P T N E E E S C K K M L C
P1-20C17C20 + F T F E P F P T N E E E S N K c M L G
P1-20C1C18 - C T F E P F P T N E E E S N K K c L E
P1-20C1C16 - C T F E P F P T N E E E S N c K M L E
P1-20C1C14 - c T F E P F P T N E E E c N K K M L E
P1-20C1C12 - c T F E P F P T N E E G E s N K K M L E
P1-20C1C10 - c T F E P F P T N C E E s N K K M L E
P1-20C1C8 + c T F E P F P c N E E E s N K K M L E
P1-20C1C6 - c T F E P c P T N E E E s N K K M L E
P1-20C1C4 - c T F C P F P T N E E E s N K K M L E
P1-10C1C10 + c T F E P F P T N c
P1-10C3C10 ND F T C E P F P T N C
P1-10C5C10 + F T F E c F P T N 6
P1-10C7C10 ND F T F E P F c T N c
P1-10C1C8 ND c T F E P F P c N E
P1-10C1C6 + c T F E P C P T N E
P1-10C1C4 ND c T F C P F P T N E

Claims

1. Combination of compounds, wherein one compound has CHIPS activity and consisting of at least 6 building blocks the sequence of which is based on the N- terminal part of the CHIPS molecule, and which sequence may comprise one or more substitutions as compared to the original CHIPS amino acid sequence, whereas the one or more other compounds are derived from the amino acids 4- 121 of CHIPS and supports CHIPS activity.
2. Combination of compounds as claimed in claim 1, wherein one or more of the amino acids in the amino acid sequence of the one or more supportive compounds are replaced by building blocks selected from the group consisting of proteinogenic amino acids, non- proteinogenic amino acids, D-amino acids and peptidomimetic building blocks.
3. Combination of compounds as claimed in claim 1 or 2 , wherein the compound having CHIPS activity has the sequence:
X1-X2-X3-X4-X5-X6-X7
wherein the building blocks XI, X2, X3 , X4 , X5, X6 and X7 are selected from the group consisting of proteinogenic amino acids, non-proteinogenic amino acids, D-amino acids and peptidomimetic building blocks and wherein X7 may or may not be present and may represent either one or more building blocks.
4. Combination of compounds as claimed in claim
3 , wherein in the compound having CHIPS activity XI may be modified with a group that is smaller than an amino acid and which is selected from acyl moieties, such as acetyl and for yl, and ureu moieties such as carbamoyl, or by reductive alkylation.
5. Combination of compounds as claimed in claim 3 and 4 , wherein the building blocks of the compound having CHIPS activity are proteinogenic amino acids and XI is selected from phenylalanine (F) , cysteine (C) , asparagine (N) , tryptophan ( ) ;
X2 is selected from alanine (A) , cysteine (C) , phenylalanine (F) , glycine (G) , histidine (H) , isoleucine (I) , leucine (L) , methionine (M) , asparagine (N) , glutamine (Q) , serine (S) , threonine (T) , valine (V) , tryptophan ( ) , tyrosine (Y) ;
X3 is selected from phenylalanine (F) , tryptophan ( ) ; X4 is selected from alanine (A) , cysteine (C) , aspartic acid (D) , glutamic acid (E) , leucine (L) , asparagine (N) , methionine (M) , serine (S) , tryptophan
(W);
X5 is selected from phenylalanine (F) , isoleucine (I) , leucine (L) , proline (P) , valine (V) tryptophan (W) , tyrosine (Y) ;
X6 is selected from cysteine (C) , phenylalanine (F) , histidine (H) , isoleucine (I) , tryptophan (W) , tyrosine (Y) .
6. Combination of compounds as claimed in claims 3-5, wherein the building blocks of the compound having CHIPS activity are proteinogenic amino acids and
Xl is selected from phenylalanine (F) , tryptophan ( ) ; X2 is selected from phenylalanine (F) , isoleucine (I) , serine (S) , threonine (T) ; X3 is phenylalanine (F) ;
X4 is selected from alanine (A) , cysteine (C) , aspartic acid (D) , glutamic acid (E) , asparagine (N) , serine (S) , tryptophan ( ) ;
X5 is selected from phenylalanine (F) , isoleucine (I) , leucine (L) , proline (P) , valine (V) tryptophan ( ) , tyrosine (Y) ;
X6 is selected from cysteine (C) , phenylalanine (F) , histidine (H) , isoleucine (I) , tryptophan ( ) , tyrosine (Y) .
7. Combination of compounds as claimed in claims 1-6, wherein the compound having CHIPS activity has the sequence:
F-T-F-E-P-F-X7, wherein one or more of the amino acids on positions 1 to 6 are substituted with the building blocks as defined in claim 3, 5 or 6, and wherein X7 is as defined in claim 3 and may or may not be present.
8. Combination of compounds as claimed in claims 1-7, wherein the compound having CHIPS activity has the sequence:
X1-T-F-E-P-F-X7, wherein XI is as defined in claim 3, 4, 5 or 6 and X7 is as defined in claim 3 and may or may not be present.
9. Combination of compounds as claimed in claims 1-7, wherein the compound having CHIPS activity has the sequence:
F-X2-F-E-P-F-X7, wherein X2 is as defined in claim 3 , 5 and 6 and X7 is as defined in claim 3 and may or may not be present.
10. Combination of compounds as claimed in claims 1-7, wherein the compound having CHIPS activity has the sequence:
F-T-X3-E-P-F-X7, wherein X3 is as defined in claim 3, 5 or 6 and X7 is as defined in claim 3 and may or may not be present.
11. Combination of compounds as claimed in claims 1-7 , wherein the compound having CHIPS activity has the sequence: F-T-F-X4-P-F-X7, wherein X4 is as defined in claim 3, 5 or 6 and X7 is as defined in claim 3 and may or may not be present.
12. Combination of compounds as claimed in claims 1-7, wherein the compound having CHIPS activity has the sequence:
F-T-F-E-X5-F-X7, wherein X5 is as defined in claim 3 , 5 or 6 and X7 is as defined in claim 3 and may or may not be present.
13. Combination of compounds as claimed in claims 1-7 , wherein the compound having CHIPS activity has the sequence:
F-T-F-E-P-X6-X7 , wherein X6 is as defined in claim 3 , 5 or 6 and X7 is as defined in claim 3 and may or may not be present.
14. Combination of compounds as claimed in claim 3 , wherein the compound having CHIPS activity has the sequence: F-T-F-E-P-F-X7. wherein X7 is as defined in claim 3 and may or may not be present.
15. Combination of compounds as claimed in claims 1-7, wherein the compound having CHIPS activity belongs to the group listed in Table 6.
16. Combination of compounds as claimed in claim 15, wherein the compound having CHIPS activity further comprises a group X7 at the C-terminal end thereof, wherein X7 is as defined in claim 3.
17. Combination of compounds as claimed in claims 3-16, wherein in the compound having CHIPS activity and/or in the supportive compound one or more of the proteinogenic amino acids is replaced by a non- proteinogenic amino acid.
18. Combination of compounds as claimed in claim 17, wherein the non-proteinogenic amino acid is selected from the group consisting of 2-naphtylalanine (Nal(2)), β-cyclohexylalanine (Cha), p-amino- phenylalanine ( (Phe(p-NH2) , p-benzoyl-phenylalanine (Bpa), ornithine (Orn) , norleucine (Nle) , 4-fluoro-phenylalanine (Phe(p-F)), 4-chloro-phenylalanine (Phe(p-Cl)), 4-bromo- phenylalanine (Phe(p-Br)), 4-iodo-phenylalanine (Phe(p- I)), 4-methyl-phenylalanine (Phe(p-Me)), 4-methoxy- phenylalanine (Tyr(Me)), 4-nitro-phenylalanine (Phe(p- N02) ) .
19. Combination of compounds as claimed in claims 3-18, wherein in the compound having CHIPS activity and/or in the supportive compound one or more of the amino acids is replaced by a D-amino acid.
20. Combination of compounds as claimed in claim 19, wherein the D-amino acid is selected from the group consisting of D-phenylalanine, D-alanine, D- arginine, D-asparagine, D-aspartic acid, D-cysteine, D- glutamic acid, D-glutamine, D-histidine, D-isoleucine, D- leucine, D-lysine, D-methionine, D-proline, D-serine, D- threonine, D-tryptophan, D-tyrosine, D-valine, D-2- naphtylalanine (D-Nal(2)), β-cyclohexyl-D-alanine (D-
Cha) , 4-amino-D-phenylalanine (D-Phe (p-NH2) ) , p-benzoyl-D- phenylalanine (D-Bpa) , D-Ornithine (D-Orn) , D-Norleucine (D-Nle) , 4-fluoro-D-phenylalanine (D-Phe (p-F) ) , 4-chloro- D-phenylalanine (D-Phe (p-Cl) ) , 4-bromo-D-phenylalanine (D-Phe (p-Br) ) , 4-iodo-D-phenylalanine (D-Phe (p-I) ) , 4- methyl-D-phenylalanine (D-Phe (p-Me) ) , 4-methoxy-D- phenylalanine (D-Tyr(Me)), 4-nitro-D-phenylalanine (D- Phe(p-N02) ) .
21. Combination of compounds as claimed in claims 3-20, wherein in the compound having CHIPS activity and/or in the supportive compound one or more of the amino acids is replaced by a peptidomimetic building block selected from the group consisting of oligo-β- peptides, oligosulfonamides, vinylogous sulfonamides, hydrazinepeptide/azatides, oligocarbamates, ureapeptoids, oligourea, phosphodiesters, peptoids, oligosulfones, peptoid sulfonamides, vinylogous peptides.
22. Combination of compounds as claimed in claim 21, wherein the peptidomimetic building block is selected from the group consisting of N-substituted glycines, such as N-benzylglycine (NPhe) , N-methylglycine (NAla) , N-(3-guanidinopropyl) glycine (NArg), N- (Carboxymethyl) glycine (NAsp) , N- (carbamylmethyl) glycine (NAsn) , N-(thioethyl) -glycine (NhCys) , N-(2- carboxyethyl) glycine (NGlu), N-(2-carbamylethyl) glycine (NGln) , N-( imidazolylethyl) glycine (NhHis) , N-(l- methylpropyl) glycine (Nile), N-(2-methylpropyl) glycine (NLeu), N- (4-aminobutyl) glycine (NLys), N-(2- methylthioethyl) glycine (NMet) , N- (hydroxyethyl) glycine (NhSer) , N-(2-hydroxypropyl) glycine (NhThr) , N-(3- indolylmethyl) glycine (NTrp) , N- (p-hydroxyphenmethyl) - glycine (NTyr) , N-(l-methylethyl) glycine (NVal) .
23. Combination of compounds as claimed in claims 1-22, wherein at least one of the compounds is in cyclic form.
24. Combination of compounds as claimed in claims 1-23 for use in prophylaxis or therapy.
25. Combination of compounds as claimed in claims 1-23 for use in the prophylaxis or treatment of indications involving the C5a-receptor (C5aR) and/or for ylated peptide receptor (FPR) on neutrophils, monocytes and endothelial cells.
26. Combination of compounds as claimed in claim 25, wherein the indication involves acute or chronic inflammation reactions.
27. Combination of compounds as claimed in claim 26 wherein the indication is selected from the group consisting of cardiovascular diseases, in particular arteriosclerosis, atherosclerosis, vasculitis, ischaemia reperfusion episodes, ischae ic shock, cardiopulmonary bypass, (large) vessel surgery, transplantation, myocardial infarction, diseases of the central nervous system, in particular bacterial meningitis, viral meningitis, multiple sclerosis, stroke, Alzheimer's disease, brain tumour, (traumatic) brain injury, gastrointestinal diseases, in particular pancreatitis, ulcerative colitis, Crohn's disease, alcoholic hepatitis, viral hepatitis, Heliobacter pylori gastritis, gastric carcinoma, peritonitis, skin diseases, in particular psoriasis, contact dermatitis, atopic dermatitis, and other dermatoses, cutaneous T-cell ly phoma, burns, genitourinary diseases, in particular urinary tract infection, glomerulonephritis, Trichomonas vaginalis infection, endometriosis, joint diseases, in particular rheumatoid arthritis, acute reactive arthritis, gout, respiratory diseases, in particular adult respiratory distress syndrome (ARDS) , chronic obstructive pulmonary disease (COPD) , idiopathic pulmonary fibrosis, cystic fibrosis, asthma, pleural emphema, metal fume fever, bacterial pneumonia, chronic bronchitis, hypersensitivity pneumonia, Mycobacterium tuberculosis infection, viral respiratory tract infection, allergic rhinitis, sinusitis, bronchogenic carcinoma, and other diseases and indications, in particular periodontitis, chronic lymph leukemia, acute trans- plant rejection, glomerulonephritis, frost bite, repetitive strain injury (RSI), sepsis.
28. Combination of compounds as claimed in claims 1-23 for use in the prophylaxis or treatment of indications involving CXCR4 and/or CCR5 receptors on leukocytes.
29. Combination of compounds as claimed in claim 28, wherein the indication is HIV infection.
30. Combination of compounds as claimed in claims 1-23 for use in the prophylaxis or treatment of indications involving C5aR and/or FPR on cells other than neutrophils, monocytes and endothelial cells.
31. Combination of compounds as claimed in claim 30, wherein the other leukocytes are lymphocytes, dendritic cells, eosinophils, basophils, macrophages, microglia cells, astrocytes, Kupfer cells, hepatocytes and epithelial cells.
32. Combination of compounds as claimed in claims 1-23 for use in prophylactic or therapeutic vaccines for infections caused by CHIPS-producing bacteria.
33. Combination of compounds as claimed in claim 32, wherein the CHIPS-producing bacterium is Staphylococcus aureus.
34. Use of the combination of compounds as claimed in claims 1-23 for the manufacture of a therapeutic preparation for prophylaxis or therapy.
35. Use as claimed in claims 34, wherein the therapeutic preparation is for prophylaxis and treatment of indications involving C5aR and/or FPR on neutrophils, monocytes and endothelial cells.
36. Use as claimed in claim 35, wherein the indication involves acute or chronic inflammation reactions.
37. Use as claimed in claim 36 wherein the indication is selected from the group consisting of cardiovascular diseases, in particular arteriosclerosis, atherosclerosis, vasculitis, ischae ia reperfusion episodes, ischaemic shock, cardiopulmonary bypass, (large) vessel surgery, transplantation, myocardial infarction, diseases of the central nervous system, in particular bacterial meningitis, viral meningitis, multiple sclerosis, stroke, Alzheimer's disease, brain tumour, (traumatic) brain injury, gastrointestinal diseases, in particular pancreatitis, ulcerative colitis, Crohn's disease, alcoholic hepatitis, viral hepatitis, Heliobacter pylori gastritis, gastric carcinoma, peritonitis, skin diseases, in particular psoriasis, contact dermatitis, atopic dermatitis, and other dermato- ses, cutaneous T-cell lymphoma, burns, genitourinary diseases, in particular urinary tract infection, glomerulonephritis, Trichomonas vaginalis infection, endometriosis, joint diseases, in particular rheumatoid arthritis, acute reactive arthritis, gout, respiratory diseases, in particular adult respiratory distress syndrome (ARDS) , chronic obstructive pulmonary disease (COPD) , idiopathic pulmonary fibrosis, cystic fibrosis, asthma, pleural emphema, metal fume fever, bacterial pneumonia, chronic bronchitis, hypersensitivity pneumonia, Mycobacterium tuberculosis infection, viral respiratory tract infection, allergic rhinitis, sinusitis, bronchogenic carcinoma, and other diseases and indications, in particular periodontitis, chronic lymph leukemia, acute transplant rejection, glomerulonephritis, frost bite, repetitive strain injury (RSI), sepsis.
38. Use as claimed in claim 34, wherein the therapeutic composition is for prophylaxis and treatment of indications involving CXCR4 and/or CCR5 receptors on leukocytes.
39. Use as claimed in claim 38, wherein the indication is HIV infection.
40. Use as claimed in claim 34, wherein the therapeutic composition is for prophylaxis or treatment of indications involving C5aR and/or FPR on cells other than neutrophils, monocytes and endothelial cells.
41. Use as claimed in claim 40, wherein the other leukocytes are lymphocytes, dendritic cells, eosinophils, basophils, macrophages, microglia cells, astrocytes, Kupfer cells, hepatocytes and epithelial cells.
42. Use as claimed in claim 34, wherein the therapeutic preparation is a prophylactic or therapeutic vaccine that can be used in the prophylaxis or treatment of infections caused by CHIPS-producing bacteria.
43. Use as claimed in claim 42, wherein the CHIPS-producing bacterium is Staphylococcus aureus.
44. A therapeutic composition comprising a suitable excipient and one or more combinations of compounds as claimed in claims 1-23.
45. A prophylactic composition comprising a suitable excipient and one or more combinations of compounds as claimed in claims 1-23.
46. An antibody or biologically active fragment thereof specifically directed to one or more of the compounds of the combination as claimed in claims 1-23.
47. Antibody or biologically active fragment thereof as claimed in claim 46 for use in the diagnosis, treatment or prophylaxis of infections of CHIPS producing bacteria.
48. Antibody or biologically active fragment thereof as claimed in claim 47, wherein the CHIPS producing bacterium is Staphylococcus aureus.
49. A compound interfering with CHIPS or one or more of the compounds of the combination as claimed in claims 1-23.
50. Compound as claimed in claim 49 for use in the diagnosis, treatment or prophylaxis of infections of CHIPS producing bacteria.
51. Compound as claimed in claim 50, wherein 5 the CHIPS producing bacterium is Staphylococcus aureus.
52. A compound interfering with the CHIPS receptor.
53. Compound as claimed in claim 52 for use in the treatment or prophylaxis of infections of CHIPS
10 producing bacteria.
54. Compound as claimed in claim 52, wherein the CHIPS producing bacterium is Staphylococcus aureus.
55. Use of one or more antibodies and/or biologically active fragments thereof as claimed in claim
15 46 and/or one or more CHIPS interfering compounds as claimed in claim 49 and/or one or more CHIPS receptor interfering compounds as claimed in claim 52 for the preparation of a coating composition for use on surfaces of medical devices that are introduced into the human
20 body through the skin, or placed in the body during surgical procedures .
56. Use as claimed in claim 55, wherein the surface is the surface of a catheter tip.
57. Use as claimed in claims 55 and 56, wherein 5 the composition is a slow-release composition.
58. Therapeutic, diagnostic or prophylactic composition comprising a suitable excipient and one or more antibodies and/or biologically active fragments thereof as claimed in claim 46 and/or one or more 0 interfering compounds as claimed in claim 49 or 52.
59. Method for prophylaxis or treatment of a subject suffering from indications involving C5aR and/or FPR on neutrophils, monocytes and endothelial cells comprising administering a prophylactically or 5 therapeutically effective amount of one or more combinations of compounds as claimed in claims 1-23.
60. Method as claimed in claim 59, wherein the indication involves acute or chronic inflammation reactions.
61. Method as claimed in claim 60 wherein the indication is selected from the group consisting of cardiovascular diseases, in particular arteriosclerosis, atherosclerosis, vasculitis, ischaemia reperfusion episodes, ischaemic shock, cardiopulmonary bypass, (large) vessel surgery, transplantation, myocardial infarction, diseases of the central nervous system, in particular bacterial meningitis, viral meningitis, multiple sclerosis, stroke, Alzheimer's disease, brain tumour, (traumatic) brain injury, gastrointestinal diseases, in particular pancreatitis, ulcerative colitis, Crohn's disease, alcoholic hepatitis, viral hepatitis, Heliobacter pylori gastritis, gastric carcinoma, peritonitis, skin diseases, in particular psoriasis, contact dermatitis, atopic dermatitis, and other der ato- ses, cutaneous T-cell ly phoma, burns, genitourinary diseases, in particular urinary tract infection, glomerulonephritis, Trichomonas vaginalis infection, endometriosis, joint diseases, in particular rheumatoid arthritis, acute reactive arthritis, gout, respiratory diseases, in particular adult respiratory distress syndrome (ARDS) , chronic obstructive pulmonary disease (COPD) , idiopathic pulmonary fibrosis, cystic fibrosis, asthma, pleural emphema, metal fume fever, bacterial pneumonia, chronic bronchitis, hypersensitivity pneumonia, Mycobacterium tuberculosis infection, viral respiratory tract infection, allergic rhinitis, sinusitis, bronchogenic carcinoma, and other diseases and indications, in particular periodontitis, chronic lymph leukemia, acute transplant rejection, glomerulonephritis, frost bite, repetitive strain injury (RSI) , sepsis.
62. Method for prophylaxis or treatment of a subject suffering from indications involving CXCR4 and/or CCR5 receptors on lymphocytes comprising administering a prophylactically or therapeutically effective amount of one or more combinations of compounds as claimed in claims 1-23.
63. Method as claimed in claim 62, wherein the indication is HIV infection.
64. Method for prophylaxis or treating a subject suffering from indications involving C5aR and/or FPR on cells other than neutrophils, monocytes and endothelial cells comprising administering a prophylactically or therapeutically effective amount of one or more combinations of compounds as claimed in claims 1-23.
65. Method as claimed in claim 64, wherein the other cells are lymphocytes, dendritic cells, eosinophils, basophils, macrophages, microglia cells, astrocytes, Kupfer cells, hepatocytes and epithelial cells.
66. Method for the prophylactic or therapeutic treatment of a subject against infections with CHIPS- producing bacteria comprising the administration of a prophylactically or therapeutically effective amount of one or more combinations of compounds as claimed in claims 1-23.
67. Combination of compounds as claimed in claims 1-23 for use in the identification of competitors for CHIPS binding.
68. Combination of compounds as claimed in claim 67, wherein the competitor is a CHIPS agonist.
69. Combination of compounds as claimed in claim 67, wherein the competitor is a CHIPS antagonist.
70. Combination of compounds as claimed in claims 1-23 for use in computer modeling to design chemicals having the same function as the combination of compounds as claimed or having an antagonist function.
71. Combination of compounds as claimed in claims 1-23 for use in a diagnostic kit for determining the presence of CHIPS.
72. Diagnostic kit for determining the presence of CHIPS, comprising one or more combinations of compounds as claimed in claim 1-23.
PCT/EP2001/008004 2001-07-11 2001-07-11 Combination of chips (chemotaxis inhibiting protein from staphylococcus aureus)-based compounds WO2003006048A1 (en)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1586583A2 (en) * 2004-04-16 2005-10-19 Alligator Bioscience AB (publ) Compounds that block C5a complement receptor and their use in therapy
US7081513B2 (en) 2000-01-07 2006-07-25 Alligator Bioscience Ab Nucleic acids encoding (poly) peptides having chips activity
US8173774B2 (en) * 2006-04-20 2012-05-08 Alligator Bioscience Ab Polypeptides and use thereof
WO2013064583A1 (en) * 2011-11-04 2013-05-10 Lipotec, S.A. Peptides which inhibit activated receptors and their use in cosmetic or pharmaceutical compositions
US20140155318A1 (en) * 2008-12-29 2014-06-05 Tel Hashomer Medical Research, Infrastructure And Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
AU2015224504B2 (en) * 2008-12-29 2018-02-22 Tel Hashomer Medical Research, Infrastructure And Services Ltd Peptides and compositions for prevention of cell adhesion and methods of using same
CN110066459A (en) * 2019-04-18 2019-07-30 青岛拜士特新材料有限公司 A kind of antibacterial matrices and preparation method of sustained-release antibacterial
US11174299B2 (en) 2008-12-29 2021-11-16 Dispersebio Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2307476A (en) * 1995-06-06 1997-05-28 Procter & Gamble Peptides with anti-adherence activity for use in oral care compositions
WO2000002913A1 (en) * 1998-07-10 2000-01-20 Jari Pharmaceuticals B.V. Chemotaxis-inhibiting protein of staphylococcus (chips) and its use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2307476A (en) * 1995-06-06 1997-05-28 Procter & Gamble Peptides with anti-adherence activity for use in oral care compositions
WO2000002913A1 (en) * 1998-07-10 2000-01-20 Jari Pharmaceuticals B.V. Chemotaxis-inhibiting protein of staphylococcus (chips) and its use

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7081513B2 (en) 2000-01-07 2006-07-25 Alligator Bioscience Ab Nucleic acids encoding (poly) peptides having chips activity
US7388078B2 (en) 2000-01-07 2008-06-17 Alligator Bioscience Ab Nucleic acids encoding polypeptides having chips activity
EP1586583A2 (en) * 2004-04-16 2005-10-19 Alligator Bioscience AB (publ) Compounds that block C5a complement receptor and their use in therapy
WO2005100385A2 (en) * 2004-04-16 2005-10-27 Alligator Bioscience Ab (Publ) Compounds that block the c5a receptor and their use in therapy
EP1586583A3 (en) * 2004-04-16 2005-11-16 Alligator Bioscience AB (publ) Compounds that block C5a complement receptor and their use in therapy
WO2005100385A3 (en) * 2004-04-16 2006-04-06 Alligator Bioscience Ab Publ Compounds that block the c5a receptor and their use in therapy
JP2008509077A (en) * 2004-04-16 2008-03-27 アリゲーター・バイオサイエンス・アーベー Compounds that block the C5A receptor and their use in therapy
US8173774B2 (en) * 2006-04-20 2012-05-08 Alligator Bioscience Ab Polypeptides and use thereof
US9732124B2 (en) 2008-12-29 2017-08-15 Tel Hashomer Medical Research, Infrastructure And Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US20140155318A1 (en) * 2008-12-29 2014-06-05 Tel Hashomer Medical Research, Infrastructure And Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US9045550B2 (en) * 2008-12-29 2015-06-02 Tel Hashomer Medical Research, Infrastructure and Services Ltd, the Technology Transfer Company of Chaim Peptides and compositions for prevention of cell adhesion and methods of using same
AU2015224504B2 (en) * 2008-12-29 2018-02-22 Tel Hashomer Medical Research, Infrastructure And Services Ltd Peptides and compositions for prevention of cell adhesion and methods of using same
US10508136B2 (en) 2008-12-29 2019-12-17 Tel Hashomer Medical Research, Infrastructure And Services Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US11174299B2 (en) 2008-12-29 2021-11-16 Dispersebio Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US11566054B2 (en) 2008-12-29 2023-01-31 Dispersebio Ltd. Peptides and compositions for prevention of cell adhesion and methods of using same
US9333152B2 (en) 2011-11-04 2016-05-10 Lipotec, S.A. Peptides which inhibit activated receptors and their use in cosmetic or pharmaceutical compositions
WO2013064583A1 (en) * 2011-11-04 2013-05-10 Lipotec, S.A. Peptides which inhibit activated receptors and their use in cosmetic or pharmaceutical compositions
CN110066459A (en) * 2019-04-18 2019-07-30 青岛拜士特新材料有限公司 A kind of antibacterial matrices and preparation method of sustained-release antibacterial

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