EP1222199A1 - Complexe de recepteurs peptidiques n-formyle et d'agents de modification du mecanisme de signalisation de la proteine kinase g - Google Patents

Complexe de recepteurs peptidiques n-formyle et d'agents de modification du mecanisme de signalisation de la proteine kinase g

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Publication number
EP1222199A1
EP1222199A1 EP00970817A EP00970817A EP1222199A1 EP 1222199 A1 EP1222199 A1 EP 1222199A1 EP 00970817 A EP00970817 A EP 00970817A EP 00970817 A EP00970817 A EP 00970817A EP 1222199 A1 EP1222199 A1 EP 1222199A1
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Prior art keywords
cell
protein
pro
inflammatory
cells
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EP00970817A
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German (de)
English (en)
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EP1222199A4 (fr
Inventor
James A. Clagett
Craig Palmer
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Mowycal Lending LLC
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Histatek LLC
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Publication of EP1222199A1 publication Critical patent/EP1222199A1/fr
Publication of EP1222199A4 publication Critical patent/EP1222199A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/081—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser

Definitions

  • This invention relates to N-formyl peptide receptors, which are found on the surfaces of peripheral blood cells, and particularly to complexes of such receptors with agents that alter or disrupt the G-protein signal pathways, particularly certain N-formyl peptides, and to methods for altering signal transduction due to co- stimulation by pro-inflammatory agents.
  • the human body has evolved to develop defense mechanisms to bacterial infections by using bacterially-generated N-formylmethionyl peptides as chemoattractants for phagocytes, in particular, neutrophils and monocytes.
  • N-formyl peptides f-Met-Leu-Phe (FMLP) was identified as the most potent in its ability to recruit phagocytes and to stimulate release of lysosomal enzymes by neutrophils (Showell et al., J. Exp. Med. 143: 1 154-1 169, 1976).
  • Synthetic tetrapeptides particularly f-Met-Ile-Phe-Leu and f-Met-Leu-Phe-Ile, have also subsequently been shown to evoke neutrophil responses (Rot et al.,
  • N-formyl peptide receptor (FRP) cDNA Cloning of the N-formyl peptide receptor (FRP) cDNA followed by the delineation of the primary structure of the FPR protein provided a major breakthrough in understanding the mechanism of action of N-formyl peptides (Boulay et al., Biochemistry 29: 11 123-11133, 1990; Boulay et al., Biochem. Biophys. Res. Commun.168: 1103- 1 109, 1990). FPR was initially found on neutrophils and monocytes but have subsequently been shown to be expressed in the human brain, hepatocytes, dendritic cells, and astrocytes (Lacy et al., J. Neuroimmunol.
  • FPR2 also known as FPRL1 and FPRH1
  • FPR2 also known as FPRL1 and FPRH1
  • FPRL1 and FPRH1 Two other FPR genes were later isolated, FPR2 (also known as FPRL1 and FPRH1)
  • FPRL2 also known as FPRL1 and FPRH1
  • Genomics 13: 437-440 1992
  • Murphy et al. J. Biol. Chem. 267: 7637-7643, 1992
  • FPRL2 Boo et al., supra
  • FPRL2 shares 56% identity with FPR and 83% identity with FPRL1.
  • FPRL1 which is expressed in both monocytes and neutrophils
  • FPRL2 was found to be expressed in monocytes but not in neutrophils (Durstin et al., Biochem. Biophys. Res. Commun. 201: 174- 179, 1994).
  • the FPR contains seven hydrophobic domains spanning the plasma membrane, connected by hydrophilic sequences exposed to either the extracellular space or the intracellular space (Murphy, Annu. Rev. Immunol. 12: 593-633, 1994).
  • the first and third intracellular loops are relatively small, consisting of 5 and 16 amino acids, respectively.
  • the carboxy terminal is exposed to the intracellular space, while the N-terminal is exposed to the extracellular space.
  • the intracellular sequences further contain a G-protein - coupling domain (a domain essential for function of the receptor as discussed below) and a potential phosphorylation domain.
  • the FMLP binding domain appears to be located in the first and third extracellular domains of FPR (Quehenberger et al., J. Biol. Chem. 268: 18167- 18175, 1993).
  • FPR Freth Generation Partnership Project
  • G-protein is thought to regulate such interconvertible states. FPR alone, without association with the G-protein, represents a low- affinity state, whereas FPR bound to the G-protein exhibits a high affinity binding state. The model further dissects the G-protein-bound FPR into three different states that account for the different potencies observed by different peptides.
  • State I represents the low affinity binding state in which FPR is not associated with the G-protein but can be converted to the high affinity state of State II upon binding to the G-protein.
  • State III is a high affinity intermediate step in which the receptor binds the ligand, while releasing the G ⁇ subunit of the G-protein.
  • State IV is a transient, low affinity state, in which the remaining G-protein subunits (G ⁇ and G ⁇ ) disassociate from the receptor.
  • the disassociation of the G-protein subunit G ⁇ leads to the effector functions of the G-protein, causing cellular activation.
  • a similar model was proposed by Sklar et al. (J. Biol. Chem. 264:
  • potency of a peptide is determined by the residence time at State III (Kermode et al., supra) .
  • the authors propose that most potent formyl peptides such as fMet-Leu-Phe-Phe and fMet-Leu-Phe-
  • NHBzl temporarily stabilize and maintain State III, which triggers an immediate degranulation response but the duration at State III allows for a sustained signal for chemotactic responses, allowing maximal migration.
  • Less potent formyl peptides mediate a rapid conversion from the high affinity State III to the low affinity State IV.
  • the initial degranulation response at State III is unaffected, the chemotactic signaling ability is minimized and thus cellular migration is limited. Therefore, in general, a highly potent ligand can be said to effect chemotactic responses by binding to the high affinity state of FPR, while a less potent ligand effects the degranulation response by binding to the low affinity state of FPR.
  • Agonist activity and antagonistic activity can also be defined using this model.
  • An agonist stabilizes the activated receptor State III and the degree of stabilization is reflected in its potency.
  • An antagonist on the other hand, binds to the receptor but destabilizes the activated State III and inactivates the receptor.
  • Such a model is not limited to FPR.
  • desensitization is said to occur when a ligand binds to the receptor and the same or different receptor becomes refractory to subsequent stimulation by the same or different ligand. This desensitized state can form after receptor occupancy in the normal course of cell activation, resulting in destabilization of State III, or prior to receptor occupancy, in which State III is never achieved.
  • the refractory state or receptor inactivation is induced by one stimulant and affects multiple nonliganded receptors, this situation is called heterologous desensitization.
  • N-formyl peptide is f-Met-Leu-Phe (FMLP or fMLP).
  • fMet-Leu-Phe-Phe fMet-Leu-Phe-NHBzl (fMet-Leu-Phe benzylamide)
  • fNle-Leu-Phe-Tyr N-formyl-L-norleucyl- Leu-Phe-Tyr
  • Nonformylated peptides may also bind to FPR and can act as potent activators of neutrophil function.
  • Met-Met- Trp-Leu-Leu is a potent pentapeptide and is comparable in activity to FMLP (Chen et al., J. Biol. Chem. 270: 23398-23401 , 1995). Conversion of the pentapeptide to an N-formylated form boosted its potency 100-500 fold, demonstrating that N-formylation still plays a significant role in the potency of a peptide, although bioactivity does not appear to be strictly determined by N- formylation.
  • N-formyl peptides Even prior to stimulation by N-formyl peptides, neutrophils transiently bind to P-selectin expressed on endothelial cells. Activation of neutrophils mediated by N-formyl peptides generated at the site of inflammation lead to neutrophil accumulation at this site. N-formyl peptides upregulate L- selectin on neutrophils and direct rolling of neutrophils along the endothelium, followed by upregulation of integrins on the surface of neutrophils.
  • Integrals mediate cell-cell and cell-extracellular matrix interactions and bind to laminin, fibronectin, victronectin, as well as to ICAM (intracellular cell adhesion molecule) and VCAM (vascular cell adhesion molecule) found on the endothelium.
  • ICAM intracellular cell adhesion molecule
  • VCAM vascular cell adhesion molecule
  • Stimulated neutrophils rapidly activate respiratory burst oxidase, which catalyzes the generation of the superoxide radical
  • the superoxide radical reacts with other molecules to produce hydrogen peroxides and hypochlorous acid, both of which are highly reactive agents and are therefore effective in interfering with microbial functions.
  • Degranulation is also an effective means for destroying foreign microbes. However, degranulation can also damage host tissue. Phagocytosis is another mechanism by which neutrophils eliminate foreign microbes. Many of these functions are stimulated via the G-protein, using phospholipases as second messengers, three of which have been characterized.
  • the phospholipase C, PLC p2 generates two second messengers, 1,4,5- ionsitol triphosphate (IP 3 ) and diacylglycerol (DG) .
  • IP 3 1,4,5- ionsitol triphosphate
  • DG diacylglycerol
  • IP 3 binds to certain calcium channels to stimulate the release of calcium from intracellular storage, resulting in an increase in the cytosolic concentration of calcium that is observed during stimulation by chemoattractants.
  • DG in concert with released calcium, activates protein kinase C (PKC).
  • G-protein activated PLC kinase has recently been reported in the literature (Beaven, et al, J.of Immunology 160:5136-5144, 1998) as a major pathway for mast cell degranulation in rat peritoneal cells in vitro, associated with Ca ⁇ * increases.
  • Phospholipase A 2 (PLA 2 ) generates arachidonic acid from the phospholipids of the inner face of the plasma membrane.
  • Arachidonic acid provides the precursors for the inflammatory mediators such as leukotrienes and prostaglan dins.
  • PLA 2 is activated upon phosphorylation by the mitogen- activated protein (MAP) kinase.
  • MAP mitogen- activated protein
  • the third phospholipase is phospholipase D (PLD), which generates phosphatidic acid and choline from phosphatidylcholine.
  • Phosphatidic acid may be involved in activation of respiratory burst oxidase in addition to playing a role in the production of DG, which activates PKC.
  • activation of PLD requires calcium, and FMLP cannot stimulate PLD in calcium- depleted cells (Kessels et al., J. Biol. Chem. 266: 23152-23156, 1991).
  • G-protein Arf and G-protein Rho regulate PLD activity (Brown et al., Cell 75: 1137- 1 144, 1993; Cockcroft et al., Science 263: 523-526, 1994;
  • Protein phosphorylation plays a central role in signal transduction initiated by FMLP.
  • Three major protein kinases are involved in the phosphorylation of proteins as a result of FMLP stimulation.
  • PKC is activated by DG, which is generated by PLC.
  • PKC act to phosphorylate serine and threonine residues.
  • PKC consists of six different isoforms, three of which are sensitive to intracellular calcium ( ⁇ , ⁇ , and ⁇ forms) and three that are not ( ⁇ , ⁇ , and ⁇ forms) .
  • Neutrophils contain the ⁇ , ⁇ , and ⁇ forms but not the ⁇ form.
  • the calcium-dependent and DG-dependent PKC (PKC- ⁇ ) responds to FMLP and phorbol ester stimulation by translocating from the cytosol to the membrane. It then phosphoiylates a number of cytosolic proteins, such as those involved in the respiratory burst oxidase system.
  • FMLP can also activate the calcium-independent, DG-dependent and phosphatidyl serine-dependent PKC form but their function is unclear.
  • MAP kinase reportedly is activated by the ⁇ subunits of the G-proteins by the activities of Ras and Raf.
  • Recent literature suggests the involvement of high-intensity Ras signaling in inducing apoptosis (Bar-Sagi, et al, J. Mol. Cell Biol. 19(9):5892-901 , 1999) as well as in promoting endothelial cell adherence (Finkel, et al, ).
  • Raf is now posited with a central role in growth signals, including cell survival, growth and differentiation
  • MAPK pathways are responsible for cytokine production; however, the activation of both TH- 1 and TH-2 cytokines, as well as other pro-inflammatory molecules, such as C5a, IL-8 and FMLP, is dependent upon the trimeric G- protein signal transduction.
  • Phosphatidylinositol 3-kinase is responsible for the formation of PI triphosphate (PIP 3 ) that is observed upon stimulation by FMLP. Elevated PIP 3 levels apparently contribute to the activation of the respiratory burst oxidase system and to actin polymerization in neutrophils, which is considered important in regulating cytoskeletal changes and cell migration. Recent literature (Rankin, et al, J. Exp. Med. 188(9): 1621-32, 1998) has reported that elevated PI3 kinase levels also can promote degranulation of eosinophils, based upon G-protein signaling based activation of IL-5.
  • G-protein signaling through the activation of PKC and resulting uptake in Ca2+, also leads to secretion and degranulation of mast cells.
  • G-protein may be essential for the down-stream activation of the
  • FC ⁇ Rl upon IgE antigen challenge and the corresponding ability to interfere with G-protein signaling, can be an important basis for down-stream inhibition of the activation of the FC ⁇ R receptor.
  • Cyclosporin H a cyclic undecapeptide and an analogue of cyclosporin A
  • Cyclosporin H has also been shown to possess antagonist activity to the formyl peptide receptor (Wenzel-Seifert et al., J. Immunol. 150: 4591-4599, 1993).
  • a naturally occurring formyl peptide receptor antagonist has also been identified, a retrovirus-derived hexapeptide (Oostendorp et al., J. Immunol. 149: 1010, 1992).
  • none of these antagonists bind to the formyl peptide receptors with high affinity and are therefore not of practical use as investigative tools or as anti-inflammatory drugs.
  • G- protein kinase signal pathway modification agent after stimulation of such cells with a pro-inflammatory agent inhibits the conventional pro-inflammatory response, particularly down-stream pro-inflammatory responses induced by pro-inflammatory agents such as, for example, C5a, FMLP, IL-4, IL-6, IL-8, II- 10, IL- 13 and TNF ⁇ or by the FC ⁇ receptor.
  • pro-inflammatory agents such as, for example, C5a, FMLP, IL-4, IL-6, IL-8, II- 10, IL- 13 and TNF ⁇ or by the FC ⁇ receptor.
  • the alteration of the cellular production of G-protein inhibits inflammatory response signal transduction pathways mediated by G protein.
  • G -protein kinase signal pathway modification agents are N-formyl-methionyl-leucyl ("f-Met-Leu") peptides having the formula f-Met-Leu X where X is selected from the group consisting of Tyr, Tyr-Phe, Phe-Phe and Phe-Tyr, most preferably f-Met-Leu- Phe-Phe.
  • the human peripheral blood mononuclear or polymorphonuclear cells or fixed tissue cells can be lymphocytes and granulocytes, preferably, eosinophils, basophils, and activated T-cells, and mast cells.
  • the G-protein kinase signal pathway modification agent forms a complex with a cell surface receptor, preferably the formyl peptide receptor ("FPR"), that is present on these cells that, in the presence of pro-inflammatory agents, inhibits phosphorylation of the G-protein subunit ⁇ in preferred embodiments of the present invention. Further, by inhibiting or blocking the down-stream phosphorylation of the G-protein, various pathways dependent upon the trimeric G-protein components block or down-regulate the response of the cell to the pro-inflammatory agents.
  • FPR formyl peptide receptor
  • this G-protein kinase signal pathway modification agent in the presence of IgE, inhibits the activation of the FC ⁇ R by IgE.
  • the pro-inflammatory agents used to stimulate the human peripheral blood mononuclear and polymorphonuclear cells, or fixed tissue cells preferably are cytokines, chemotaxins, or mitogens.
  • cytokines such as FMLP, activated complement fragment (C5a), leukotriene B4 (LTB4), platelet activating factor (PAF), and cytokines such as TNF ⁇ , IL-4, IL-6, IL-8, IL- 10 and
  • IL- 13 plus antigen cross-linked IgE, IgG or IgA.
  • a method for inhibiting a pro-inflammatory response of a human peripheral blood mononuclear or polymorphonuclear cell comprising contacting the cell with a G- protein kinase signal pathway modification agent, and binding of the agent to a receptor on the cell.
  • the pro-inflammatory mediating cell can be a lymphocyte or a granulocyte.
  • the cell is first stimulated by a pro- inflammatory agent such as set forth above.
  • the ligand-bound receptor is a formyl peptide receptor.
  • the down-stream blocking of G-protein activation of the FC ⁇ R receptor provides a similar therapeutic benefit.
  • the present invention further provides a method for identifying a G protein kinase signal pathway modification agent, the method comprising the steps of: a) contacting a pro-inflammatory mediating cell selected from the group consisting of activated T-lymphocyte, mast cell, eosinophil and basophil with a known pro-inflammatory mediator selected from the group consisting of a cytokine, chemotaxin, and mitogen and eliciting a pro-inflammatory response; b) adding to step a) a candidate G- protein signal pathway modification agent; and c) detecting a decrease in amount of a G protein kinase or failure of the G protein kinase to disassociate in (i) a pro-inflammatory cell simultaneously contacted with said candidate agent and said pro-inflammatory mediator compared to (ii)
  • a receptor complexed with a G-protein kinase signal pathway modification agent is provided on the surface of a pro-inflammatory mediating cell selected from the group consisting of activated T-lymphocyte, mast cell, eosinophil and basophil by contacting the pro-inflammatory mediating cell with the G-protein kinase signal pathway modification agent, thereby inhibiting a pro-inflammatory response.
  • a method for identifying a G protein kinase signal pathway modification agent comprises the steps of: contacting a pro-inflammatory mediating cell selected from the group consisting of activated T-lymphocyte, mast cell, eosinophil and basophil with a candidate G- protein signal pathway modification agent; and determining the distribution of protein kinases produced by said cell, wherein compared to cells not contacted by the candidate G- protein signal pathway modification agent there is no change in the amount of PLC ⁇ , Pp60 Src, and ERK- 1 , there is an increase in the amount of PI3 (102 Kd) and PI3 (83 Kd), and there is a decrease in the amount of Raf, Ras, G-protein ⁇ and G-protein ⁇ kinases.
  • the present invention also provides a new receptor complex of a human peripheral blood mononuclear cell or a polymorphonuclear cell comprising a cell surface receptor and a G- protein signal pathway modification agent wherein, compared to cells not exhibiting said receptor complex, the distribution of protein kinases in the cells is such that there is no change in the amount of PLC ⁇ , Pp60 Src, and ERK- 1 , there is an increase in the amount of PI3
  • the cell surface receptor is a FPR receptor. Secondary blocking of G-protein activation of the FC ⁇ R receptor is also preferred.
  • Stimulation by IL-4 of a human peripheral blood mononuclear cell or a polymorphonuclear cell having a cell surface receptor complexed with a G- protein signal pathway modification agent provides a distribution of protein kinases produced by said cell wherein, compared to stimulated cells not exhibiting said receptor complex, there is an increase in the amount of PLC ⁇ ,
  • Stimulation by IL-6 of a human peripheral blood mononuclear cell or a polymorphonuclear cell having a cell surface receptor complexed with a G- protein signal pathway modification agent provides a distribution of protein kinases produced by said cell wherein, compared to stimulated cells not exhibiting said receptor complex, there is an increase in the amount of PLC y , PI3 ( 102 Kd), Raf, Pp60 Src, ERK- 1 and G-protein ⁇ , and a decrease in the amount of PI3 (83 Kd), G-protein ⁇ and G-protein ⁇ kinases.
  • Stimulation by IL- 10 of a human peripheral blood mononuclear cell or a polymorphonuclear cell having a cell surface receptor complexed with a G- protein signal pathway modification agent provides a distribution of protein kinases produced by said cell wherein, compared to stimulated cells not exhibiting said receptor complex, there is an increase in the amount of PI3 (102 Kd), ERK- 1 and G-protein ⁇ , and a decrease in the amount of PLC ⁇ , PI3 (83 Kd), Ras and kinases.
  • Stimulation by the pro-inflammatory agent, IL-13, of a human peripheral blood mononuclear cell or a polymorphonuclear cell having a cell surface receptor complexed with a G- protein signal pathway modification agent provides a distribution of protein kinases produced by said cell wherein, compared to stimulated cells not exhibiting said receptor complex, there is an increase in the amount of PLC ⁇ , PI3 (102 Kd), PI3 (83 Kd), ERK- 1 and Raf, and a decrease in the amount of Ras, Pp60 Src, G-protein ⁇ , G-protein ⁇ and G- protein ⁇ kinases.
  • Stimulation by the pro-inflammatory agent, C5a, of a human peripheral blood mononuclear cell or a polymorphonuclear cell having a cell surface receptor complexed with a G- protein signal pathway modification agent provides a distribution of protein kinases produced by said cell wherein, compared to stimulated cells not exhibiting said receptor complex, there is an increase in the amount of Pp60 Src and Raf, and a decrease in the amount of PLC ⁇ , PI3 (102 Kd), G-protein ⁇ , G-protein ⁇ and G-protein ⁇ kinases.
  • Stimulation by the pro-inflammatory agent, TNF ⁇ , of a human peripheral blood mononuclear cell or a polymorphonuclear cell having a cell surface receptor complexed with a G- protein signal pathway modification agent provides a distribution of protein kinases produced by said cell wherein, compared to stimulated cells not exhibiting said receptor complex, there is an increase in the amount of Raf, Ras, and Pp60 Src, and a decrease in the amount of PLC ⁇ , PI3 (83 Kd) G-protein ⁇ , G-protein ⁇ and G-protein ⁇ kinases.
  • FIG. 1 is a graph showing binding of FITC-labeled HK-X (f-Met-Leu-Phe- Phe) to human peripheral blood nucleated cells.
  • FIG. 2A- FIG. 2C are dot plots of FITC-labeled HK-X binding to activated lymphocytes.
  • FIG. 2A shows lymphocytes stimulated with 6 ⁇ g Concanavalin A (ConA) at 24 hours after placed in culture in addition with lOOnM FITC-labeled HK-X;
  • FIG. 2B shows lymphocytes stimulated with 6 ⁇ g ConA at 120 hours after placed in culture but with no FITC-labeled HK-X;
  • FIG. 2C shows lymphocytes stimulated with 6 ⁇ g ConA at 120 hours after placed in culture in addition with lOOnM FITC-labeled HK-X.
  • FIG. 3A- FIG. 3B are histograms of the DNA content of lymphocytes from FIG. 2 A-C.
  • FIG. 3A is a histogram of ceUs from FIG. 2A and
  • FIG. 3B is a histogram of cells from FIG. 2B and FIG. 2C.
  • FIG. 4 is an autoradiograph of 35 S-methionyl labeled proteins recovered from whole human neutrophil cell lysates and various resins.
  • FIG. 5A- FIG. 5B show the presence of phosphorylated proteins in mononuclear cells treated with vehicle (0.3% DMSO) as detected by monoclonal antibodies to phosphotyrosine.
  • FIG. 5A is a densitometry of SDS-PAGE of normal cells and
  • FIG. 5B is a photograph of the SDS-PAGE gel of normal cells treated with vehicle (DMSO) only.
  • FIG. 6A- FIG. 6B show the presence of phosphorylated proteins in mononuclear cells treated with HK-X as detected by monoclonal antibodies to phosphotyrosine.
  • FIG. 6A is a densitometry of SDS-PAGE of HK-X treated cells and FIG. 6B is a photograph of the SDS-PAGE of normal cells treated with HK- X only.
  • FIG. 7A- FIG. 7B show the presence of phosphorylated proteins in mononuclear cells treated with IL-8 as detected by monoclonal antibodies to phosphotyrosine.
  • FIG. 7A is a densitometry of SDS-PAGE of IL-8 treated cells and
  • FIG. 7B is a photograph of the SDS-PAGE of normal cells treated with IL-8 only.
  • FIG. 8A- FIG. 8B show the presence of phosphorylated proteins in mononuclear cells treated with HK-X and IL-8 as detected by monoclonal antibodies to phosphotyrosine.
  • FIG. 8A is a densitometry of SDS-PAGE of HK-X and IL-8 treated cells and
  • FIG. 8B is a photograph of the SDS-PAGE of normal cells treated with HK-X and IL-8.
  • FIG. 9A- FIG. 9B show the presence of phosphorylated proteins in mononuclear cells freshly collected from peripheral blood as detected by monoclonal antibodies to phosphotyrosine.
  • FIG. 9A is a densitometry of SDS- PAGE of freshly collected cells and
  • FIG. 9B is a photograph of the SDS-PAGE gel of freshly collected cells.
  • G-protein kinase signal pathway modification agents have been found to inactivate certain pro-inflammatory responses of human peripheral blood cells that have been stimulated by pro- inflammatory agents or molecules.
  • Preferred G-protein kinase signal pathway modification agents in accord with the present invention, can bind to receptors found on pro-inflammatory mediating cells such as lymphocytes, particularly activated T-cells, granulocytes such as eosinophils, basophils, and fixed tissue cells such as mast cells.
  • pro-inflammatory mediating cells such as lymphocytes, particularly activated T-cells, granulocytes such as eosinophils, basophils, and fixed tissue cells such as mast cells.
  • pro-inflammatory responses Upon binding of the G protein kinase signal pathway modification agent to its receptor, pro-inflammatory responses are inhibited.
  • the G-protein subunits ⁇ , ⁇ , and ⁇ are modified and also phosphorylation of these subunits is inhibited.
  • Pro-inflammatory responses that can be inhibited by the agent-receptor complex are secretion, degranulation and migration of the receptor-bearing cell, as well as synthesis and secretion of other pro-inflammatory molecules.
  • the G protein kinase signal pathway modification agents of the present invention can inactivate the receptor previously stimulated by a pro-inflammatory molecule but have no effect on non- stimulated cells.
  • pro-inflammatory agents useful for stimulating the cells are IL-8, N-formyl peptides, activated complement fragment (C5a), leukotriene B4 (LTB4) and platelet activating factor (PAF).
  • the agent-receptor complex of the present invention also can desensitize the receptor from further stimulation by the same pro-inflammatory agent or by a different pro-inflammatory agent.
  • the observed effect of the G protein kinase signal pathway modification agent-receptor complex on the receptor-bearing cell can be used to identify G protein kinase signal pathway modification agents. Screening for such agents can be carried out by contacting a pro-inflammatory mediating cell with a known pro-inflammatory mediator to elicit a pro-inflammatory response, followed by the addition of a candidate agent and by detecting a decrease in the amount of a G protein kinase in a pro-inflammatory cell contacted simultaneously with the candidate agent and the pro-inflammatory molecule compared to that amount in a pro-inflammatory mediating cell contacted with the pro-inflammatory molecule alone.
  • a particualrly useful G protein kinase signal pathway modification agent is f-Met-Leu-Phe-Phe.
  • Other useful agents can be identified by routine testing using one of the above procedures.
  • pro-inflammatory responses include secretion or degranulation of pro-inflammatory mediating cells and release of leukotrienes, hista ines, and other cytokines. Such responses also include infiltration of eosinophils, basophils and mast cells into inflammatory tissues as a result of chemotactic adhesion, migration and aggregation of lymphocytes, eosinophils, basophils, mast cells, and neutrophils. Vascular permeability at the site of inflammation and increased production of IgE, IgG and IgA, and their respective FC receptors, also can be associated with pro-inflammatory responses.
  • Inhibition of pro-inflammatory responses can thus include decrease of degranulation and release of leukotrienes, histamines and other cytokines by pro-inflammatory mediating cells, or complete cessation in preferred embodiments, following peptide-receptor binding according to the present invention.
  • Infiltration and migration of pro-inflammatory mediating cells can also be greatly reduced, or completely inhibited.
  • Vascular permeability at the site of inflammation and IgE levels also can be reduced.
  • EXAMPLE 1 Binding of Labeled HK-X to Peripheral Blood Nucleated Cells
  • H-KX binds to activated T-cells, mast cells, eosinophils, and basophils, as well as the known binding to neutrophils.
  • FIGs. 2A-2C show the relationship between activated lymphocytes by ConA and the appearance of binding sites for FITC-labeled HK-X.
  • the four quadrants reveal the following characteristics: the upper left quadrants denote cells with greater than In content of DNA and increased levels of FITC HK-X binding above background levels [ established by using the profile in FIG.
  • the upper right quadrants denote cells that contain greater than In DNA content and have FITC-ligand binding greater than background; the lower right quandrants contain cells that have In DNA content but have bound FITC-ligand above background levels; and the lower left quadrants contain cells with In DNA content and background levels of FITC-ligand.
  • FIG. 3A shows the same samples contained in Fig. 2A and FIG. 3B shows those contained in Figs. 2B and 2C.
  • FIG. 3B shows those contained in Figs. 2B and 2C.
  • FC ⁇ R receptor Receptors that bind HK-X and other N-formyl peptides were isolated and characterized from murine peritoneal mast cells, human polymorphonuclear and mononuclear cell populations. Further, G-protein activation of the FC ⁇ R receptor as a down-stream consequence of increases in PKC, PI3 and mobilization of Ca 2+ , make the FC ⁇ receptor a down-stream effector receptor of
  • HK-X Cells that bind HK-X include mast cells, basophils and eosinophils as evidenced by changed biological reactivity after exposure to HK-X and by binding of a fluorescent or radioactive labelled HK-X. Receptors for HK-X have not been identified. In contrast, neutrophils express formyl peptide receptors on their surface. However, blood lymphocytes and monocytes appear to bind less HK-X than neutrophils. DETAILED MATERIALS AND METHODS:
  • Human peripheral blood mononuclear and polymorphonuclear cells were isolated from peripheral blood obtained from normal donors. The blood was collected in heparin. The various cell types were isolated by centrifugation over Ficoll-Hypaque at 500-x g for 60 min at room temperature. Each fraction was harvested, pooled separately and washed lx in RPMI 1640 with antibiotics.
  • Aliquot B was bound to a HK-X substituted column in the presence of soluble HK-X and the proteins eluted. Each fraction was concentrated and Iyophilized.
  • the approach undertaken in this step involved binding of HK-X to a Sepharose resin to make a HK-X substituted resin. Prior to exposure to HK-X substituted resin, the labeled cellular protein mixture was passed over a resin not substituted with HK-X to remove any protein species reacting with the native resin. Thus, when the cellular proteins including the receptor proteins were passed through the HK-X substituted resin under proper ionic environments, the receptor proteins (for the HK-X receptor) among the other proteins bound tightly with the HK-X.
  • the resin was washed with a gentle agent, such as phosphate buffer at neutral pH, to remove any low affinity binding proteins. Subsequently, the resin was exposed to an excess amount of free HK-X to competitively elute receptor proteins bound to the resin.
  • the radioactive proteins released at each of these steps was concentrated and analyzed on a 12% SDS-PAGE system as detailed in the following step.
  • FIG. 4 shows the result of a representative experiment. All proteins present in the cell lysate are shown in Lane A. In Lane B, the unbound material from the Sepharose column without HK-X substitution shows a pattern of protein band distribution similar to the entire cell lysate. Lane C contains the pre-elution material. Lane D is a blank lane.
  • Lane E three protein bands are visible when the column was eluted in the presence of 1 mg of HK-X (competitor) .
  • the molecular weights are estimated to be -94,000, -68,000 and -40,000 Daltons, respectively. This experimental condition established the specificity of the binding.
  • the corrected ratios revealed a vastly different binding and/ or recovery profile between the values reported by Goetzl and the values obtained in this Example.
  • One possibility is that the detergent disassociated receptor complex binds to FMLP- substituted Sepharose in a selective manner different from that of HK-X substituted Sepharose.
  • HK-X was used to selectively recover the binding proteins in a cell lysate recovered after protein labeling with 35 S- methionine in vitro.
  • Three molecular weight species were isolated which agree with previously reports on the size of peptides associated with the FMLP receptor complex.
  • HK-X binds to receptors on human polymorphonuclear and mononuclear cells, particularly activated T-cells, mast cells, eosinophils, and basophils.
  • Leukocytes respond to a large number of chemoattractants and other proinflammatory mediators. Some mediators cause chemotaxis, activation of enzyme systems and release of pathologically significant mediators.
  • the typical N-formyl peptides (the archetypal one — FMLP), activated complement fragment (C5a), leukotriene B4 (LTB4), platelet activating factor (PAF), and some chemotactic cytokines (such as IL-8) are well-recognized chemotactic and pro- inflammatory agents. These agents bind to G-protein-coupled receptors (GPCRs) with subsequent generation of multiple signal transduction mediated by protein kinase systems.
  • GPCRs G-protein-coupled receptors
  • the cascades resulting for the initial events are complex and interrelated, yet are responsible for the entire behavior of all nucleated cells.
  • Programmed cell death apoptosis
  • generation of immune responses removing of self-recognizing T cells, and control of synthesis of extracellular matrices are just a few examples of the action of signal transduction pathways.
  • Protein kinases were identified by their capacity to transfer a phosphate group from a phosphate donor onto an acceptor amino acid located within a protein. Usually the y phosphate of ATP is the donor. The three major acceptor amino residues within proteins are tyrosine, serine and threonine. As of 1999, over 1 15 protein kinases have been identified and described in the literature.
  • FMLP binding to phagocytes stimulates phosphorylation, which correlates with cellular functions.
  • FMLP and other chemoattractants stimulate phosphatidylinositol 3-kinase (P13K) which in turn activates protein kinase (PKC).
  • P13K phosphatidylinositol 3-kinase
  • PPC protein kinase
  • FMLP binding initiates phosphorylation of an extracellular regulated kinase, (ERK- 1) which belongs to a general family of kinases termed mitogen-activated protein kinases (MAP kinases).
  • MAP kinases mitogen-activated protein kinases
  • phosphotyrosine proteins can be detected from the entire mass of intracellular proteins by monoclonal antibodies which recognize only the phosphotyrosine epitope (Ross et al., Nature (London) 294: 654, 1981; Frackleton et al., Mol. Cell Biol. 3: 1343, 1983).
  • HK-X contained 20 ug HK-X .
  • FMLP contained 0. 1 ug FMLP .
  • IL-8 contained 0.1 ug IL-8 (recombinant human IL-8) .
  • HK-X contained 20 ug HK-X plus 100 uL of IL-8 contained 0.1 ug IL-8.
  • FIGs. 5 through 9 the chemiluminescence patterns of phosphoproteins detected by monoclonal anti-phosphotyrosine antibody are shown. A negative of the chemiluminescence images was made by a professional photographer in order to obtain the highest degree of resolution of the bands and their corresponding intensity of chemiluminescence.
  • FIG. 5A the protein kinases present in cells exposed to vehicle for 30 minutes revealed 9 distinct protein species as shown by peaks in a densitometry analysis of the gel.
  • peaks in the densitometry analysis are marked by arrows and the corresponding molecular species in the gel are also marked by the same arrows. (The origin of the gel is on the right side of the gel where the larger molecular weight species are located).
  • FIG. 6 the protein kinase responses of cells to HK-X are depicted.
  • the protein kinase content and distribution of fresh mononuclear cells is shown in FIG. 9.
  • kinases of these cells are strikingly similar to that of vehicle treated cells (FIG. 5) and HK-X treated cells (FIG. 6).
  • the kinase response patterns to FMLP stimulation alone and to HK-X plus FMLP stimulation is not shown. However, the patterns of each of these were substantially identical to that observed with IL-8 alone and HK-X plus IL-8, respectively.
  • the pattern and distribution of protein kinases for peripheral blood polymorphonuclear cells was essentially the same as that for the mononuclear cells.
  • the primary difference between the two cell types was that mononuclear cells were more metabolicaUy active than the polymorphonuclear cells.
  • Table 3 shows the quantitative change in the distribution of protein kinases from human peripheral blood cells after exposure to HK-X.
  • G-protein ⁇ 9 Kd NC Table 4 shows the quantitative amount of G protein y kinase for various experiments, which confirmed and extended the previous qualitative data. Although it was attempted to use the same number of cells were used for each treatment and approximately the same amount of intracellular protein recovered was applied to each lane, the total amount of kinases differed as seen by the areas observed.
  • Table 5 illustrates the results of another experiment that- shows the change in the distribution of protein kinases from human peripheral blood cells after exposure to HK-X compared to co stimulatory exposure to (1) HK-X and (2) fMLP or IL-8.
  • Tables 6and 7 illustrate the results of a further experiment that shows the change in the distribution of protein kinases from human peripheral blood cells after exposure to HK-X compared to co stimulatory exposure to (1) HK-X and (2) Ca5, TNF ⁇ , IL-4, IL-6, IL-10 or IL-13.
  • the FPR Upon binding FMLP or other analogues, the FPR interacts with the G-protein pool that is common to other chemoattractant receptors such as the LTB4 and C5a receptors (Jacobs et al., J. Leukoc. Biol. 57: 679-686, 1995;
  • C. IL-8, C5a, and FMLP desensitized each other's receptors. These studies were extended to include PAF and LTB4 receptors. The mechanisms by which this process may be initiated may involve receptor phosphorylation by PKC. However, the FPR does not contain intracellular domains capable of being phosphorylated by PKC, but another kinase appears to be responsible.
  • HK-X treatment alone did not specifically down-regulate kinases.
  • G- protein ⁇ kinase was selectively and significantly decreased after simultaneous stimulation of mononuclear cells with HK-X and IL-8 or with HK-X and FMLP.
  • HK-X and potential pro-inflammatory molecules can be present together.
  • corollaries which can be drawn from the data presented here and in previous reports. They are:
  • HK-X demonstrates specificity for cells capable of responding to pro- inflammatory stimuli mediated by specific pro-inflammatory receptors.
  • HK-X should maintain desensitization of the potentially responding cells.
  • HK-X appears to mediate its therapeutic efficacy through desensitization of pro-inflammatory mediator receptors. Downstream from this event, the inactivation of the inflammatory cell is mediated and maintained by either directly or indirectly through inhibition of phosphorylation of G-protein ⁇ subunit.

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Abstract

L'invention concerne un procédé d'inhibition d'une réponse pro-inflammatoire d'une cellule sanguine périphérique, humaine, mononucléaire ou polymorphonucléaire, ou d'une cellule tissulaire fixe. Ce procédé consiste à mettre en contact la cellule avec un agent pro-inflammatoire, afin de stimuler une réponse pro-inflammatoire, puis à mettre la cellule en contact avec un agent de modification du mécanisme de signalisation de la protéine kinase G, de manière à inhiber les mécanismes de transduction du signal de réponse inflammatoire, induits par la protéine G. L'invention concerne un complexe de récepteurs, dans lequel un agent de modification du mécanisme de signalisation de la protéine G kinase se lie à un récepteur de surface cellulaire d'une cellule sanguine périphérique humaine, mononucléaire ou polymorphonucléaire, qui a été stimulée par un agent pro-inflammatoire.
EP00970817A 1999-10-15 2000-10-12 Complexe de recepteurs peptidiques n-formyle et d'agents de modification du mecanisme de signalisation de la proteine kinase g Withdrawn EP1222199A4 (fr)

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US20030220258A1 (en) 2001-12-21 2003-11-27 Robbert Benner Treatment of ischemic events
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US7786084B2 (en) 2001-12-21 2010-08-31 Biotempt B.V. Treatment of burns
WO2004100978A1 (fr) * 2003-05-13 2004-11-25 Medvet Science Pty. Ltd. Procede de modulation de la transmigration cellulaire et agents utilises a cet effet
CN101217971B (zh) 2005-07-05 2011-11-09 比奥滕普特公司 肿瘤的治疗
EP1864692A1 (fr) 2006-06-07 2007-12-12 Biotempt B.V. Utilisation de peptides pour la protection contre les radiolésions
WO2011036308A1 (fr) * 2009-09-28 2011-03-31 Theramab Gmbh Procédé d'essai préclinique de médicaments immunomodulateurs
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