[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO1994017095A1 - Analogs of receptor tyrosine activation motifs and therapeutic uses thereof - Google Patents

Analogs of receptor tyrosine activation motifs and therapeutic uses thereof Download PDF

Info

Publication number
WO1994017095A1
WO1994017095A1 PCT/US1994/001025 US9401025W WO9417095A1 WO 1994017095 A1 WO1994017095 A1 WO 1994017095A1 US 9401025 W US9401025 W US 9401025W WO 9417095 A1 WO9417095 A1 WO 9417095A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
seq
tam
amino acid
amino acids
Prior art date
Application number
PCT/US1994/001025
Other languages
French (fr)
Inventor
Manfred Weigele
Guo Tao
Raji Sundaramoorthi
David C. Dalgarno
Lynne D. Zydowsky
Jeremy Green
Oluyinka M. Green
Original Assignee
Ariad Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ariad Pharmaceuticals, Inc. filed Critical Ariad Pharmaceuticals, Inc.
Priority to AU60981/94A priority Critical patent/AU6098194A/en
Publication of WO1994017095A1 publication Critical patent/WO1994017095A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705Receptors; Cell surface antigens; Cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K7/00Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02Linear peptides containing at least one abnormal peptide link
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides and analogs and derivatives thereof that are related to all or a portion of tyrosine activation motifs found in multichain immune recognition receptors.
  • the immune system consists of lymphocytes, macrophages, and other specialized cells.
  • An organism responds to antigen by virtue of having a large number of lymphocytes, each bearing receptors which recognize distinct antigens.
  • B lymphocytes are precursors of antibody-secreting cells, and T lymphocytes are involved in regulatory and effector functions.
  • natural killer cells mediate certain
  • T lymphocytes that have been sensitized to an antigen by virtue of their
  • Immunoglobulins are synthesized by B lymphocytes, and by their progeny, the plasma cells. B cells interact with antigen via the antibody molecules in their plasma membranes. Plasma cells secrete antibodies.
  • IgGs are the major component of serum immunoglobulin, and the predominant antibody involved in a secondary immune response.
  • IgM is the predominant antibody involved in a primary immune response.
  • IgD is a cell surface receptor present on mature B cells.
  • IgA is found in bodily secretions.
  • IgE is found in normal human serum at very low concentrations, and is
  • IgE appears to mediate local and systemic immediate hypersensitivity and anaphylactic reactions. Atopic individuals produce IgE in response to many environmental antigens. The event that initiates immediate hypersensitivity in such patients is the binding of antigen to IgE on the surface of mast cells. Binding of the Fc domain of the allergen-bound IgE to the extracellular domain of the FceRI receptor complex on mast cells and basophils induces
  • Crosslinking of the receptor causes mast cell activation and a cascade of intracellular signaling events which ultimately results in the release of lipid mediators of inflammation (leukotrienes, etc.), proinflammatory cytokines (interleukins, etc.) and of secretory granules (containing histamine, 5-hydroxytryptamine, hexosaminidase, etc.). This release causes the appearance of many of the symptoms of acute allergic response.
  • Cells respond to external stimuli through the binding of extracellular ligands to transmembrane receptor molecules. Binding of protein or peptide ligands to the extracellular domain of such a
  • membrane-spanning receptor results in a conformational change or aggregation of the receptor, and a
  • Intracellular signal transduction occurs via changes in the intermolecular associations of protein molecules arranged in a complex multi-step sequential pathway leading from the intracellular portion of the receptor to proteins involved directly in controlling gene expression, cytoskeletal
  • T and B cells are found on T and B cells, mast cells, macrophages, natural killer cells and platelets.
  • MIRRs constitute a family of immune cell receptors involved in the activation of cells involved in the immune system. MIRRs include the B cell antigen receptor complex (BCR); the T cell antigen receptor complex (TCR); Fc ⁇ RIII (FC7RIIIA in humans), a
  • MIRRs consist of polypeptide chains that bind antigen or Fc, associated with chains whose intracellular domains contain a related short sequence of 18-27 amino acids termed the tyrosine activation motif
  • TAM TAM
  • Figure 1 schematically depicts these MIRRs.
  • the TCR consists of the ⁇ (alpha) and ⁇ (beta) T cell antigen receptor antigen-binding chains (which, in a minority of T cells, are replaced by the ⁇ (gamma) and ⁇ (delta) heterodimer), the ⁇ , ⁇ and s (epsilon) chains of the CD3 complex, and a disulfide linked ⁇ (zeta) dimer or heterodimer of ⁇ and ⁇ (eta) chains.
  • the BCR consists of the heavy and light chains and at least one disulfide-linked heterodimer consisting of Ig- ⁇ (MB-1 gene product) and Iq- ⁇ (B29 gene product).
  • the Fc ⁇ RI receptor is a complex of three proteins, the ⁇ , ⁇ , and ⁇ subunits.
  • the ⁇ subunit of FceRI binds IgE, the ⁇ subunit contains four transmembrane regions, and the 7 subunit consists of a part of disulphide-linked chains (Fig. 2).
  • FceRI- ⁇ subunit residues lie on the cytoplasmic side of the cell membrane. This ⁇ subunit is shared by the FC7RIII MIRR, and contains a TAM motif. FceRI- ⁇ also contains a TAM motif (but not FceRI- ⁇ ), as do the CD3- ⁇ ,CD3- ⁇ and ⁇ components of the TCR (but not the CD3- ⁇ or TCR- ⁇ or TCR- ⁇ ), MB-1 and B29 in the BCR, and the envelope protein (gp30) of bovine leukemia virus (Reth, 1989, Nature 338:383-384; see also Fig. 3 herein).
  • TAM motif in signal transduction has been investigated for the TCR.
  • the consensus sequence shown above occurs in the ⁇ chain of the TCR.
  • Cell lines expressing a chimeric molecule in which the cytoplasmic tail of the ⁇ chain was attached to the unrelated CD8 receptor respond to antibody crosslinking stimuli (Romeo et al., 1992, Cell 68:889-897).
  • the present invention is directed to
  • TAMs tyrosine activation motifs
  • analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions.
  • a hybrid molecule is provided which contains an amino(N)- or carboxy(C)-terminal non- peptide helix inducer.
  • the hybrid molecule contains an internal non-peptide structure that maintains the helical character of the TAM mimic.
  • invention inhibit the activation of various cells with immune system function.
  • TAM Mimics are formulated for pulmonary
  • the invention provides inhalers containing compositions comprising a therapeutically or prophylactically effective amount of a TAM Mimic.
  • FIG. 1 Schematic diagram of four multichain immune recognition receptors (MIRRs): IgMR (the B cell antigen receptor complex), FC7RIII, EceRI, and TCR (the T cell antigen receptor complex).
  • IgMR the B cell antigen receptor complex
  • FC7RIII the B cell antigen receptor complex
  • EceRI the T cell antigen receptor complex
  • TCR the T cell antigen receptor complex
  • FceRI receptors Two FceRI receptors are depicted cross-linked by allergen binding.
  • Two IgMRs are depicted cross-linked by antigen binding.
  • TAM motifs are indicated by cross- hatching.
  • FIG. 1 A schematic view of the MIRR for the Fc region of IgE, called the FceRI receptor (after Keegan and Paul, 1992, Immunology Today 13 (2): 63-68).
  • FIG. 5 Computer-generated drawings.
  • the left side shows an idealized (computer-generated) drawing of an alpha-helical peptide.
  • 7 amino acids i.e., an 11 angstrom stretch
  • spacer molecule like that present in a compound of formula (V).
  • N-terminal and C-terminal helix inducers are depicted.
  • Compound I and Compound III were developed by Paul Bartlett (Bartlett et al., "Intuitive- and Computer- Assisted Approaches to the Design of Conformationally Restrained Peptides and Their Mimics," October 28-29, 1991, reprinted from Proceedings of The Robert A.
  • the present invention is directed to peptides related to all or a portion of tyrosine activation motifs (TAMs), as well as analogs and derivatives thereof (collectively termed herein “TAM Mimics”).
  • TAMs tyrosine activation motifs
  • analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions.
  • a hybrid molecule is provided which contains an amino (N)- or carboxy(C) -terminal non- peptide helix inducer.
  • the hybrid molecule contains an internal non-peptide structure that maintains the size and general shape of the TAM mimic.
  • the TAM Mimics of the invention retain the conformational information of TAM motifs required for biological activity, however, preferably incorporate sequence changes that provide the TAM Mimic with enhanced ability to cross lipid bilayers.
  • the TAM motif plays a critical role in the activation of the immune functions of T cells
  • TAM motif within receptor complexes appears to interact with target proteins that transmit the activation signal within the cytoplasm to bring about cellular immune responses.
  • the TAM Mimics of the invention mimic the structure of these TAM motifs. While Applicants do not intend to be bound by any specific mechanism, Applicants believe that the TAM Mimics function by binding to the target protein, mimicking the interaction with the receptor-associated TAM motif, and thus preventing the natural activation of this target. Stimulation of mast cells, B
  • lymphocytes T lymphocytes, and macrophages is
  • the TAM Mimics of the invention thus act as inhibitors of the signal transduction events mediated by the various MIRRs containing TAM sequences, by competitively inhibiting the interaction of such molecules with an effector or regulatory molecule via their TAM motifs, and thus preventing the natural sequence of activation in MIRRs.
  • TAM Mimics of the invention can also be used to study and elucidate the signal transduction mechanism in cells expressing the MIRRs. 5.1. TAM MIMICS
  • TAM Mimics of the invention include peptides and hybrid molecules, as detailed more fully in the subsections below.
  • the TAM Mimic is a peptide.
  • a TAM Mimic of the invention comprises or consists of all or a portion of the sequences shown in Figure 3 (SEQ ID NOS:2-12).
  • a TAM Mimic comprises or consists of the sequence
  • a TAM mimic comprises or consists of the following sequences:
  • DGGYMTLNPRAPTDDDKNTYLTLP (part of SEQ ID NO: 2)
  • DQLYQPLKDREDDQYSHLQ (part of SEQ ID NO: 5)
  • DQVYQPLRDRDDAQYSHLG (part of SEQ ID NO: 6)
  • ENLYEGLNLDDCSMYEDIS (part of SEQ ID NO: 9, 10)
  • EHTYEGLNIDQTATYEDIV (part of SEQ ID NO: 12)
  • a TAM Mimic peptide comprises or consists of the ⁇ or ⁇ TAM motif, or a portion thereof, present in the human FceRI complex, or one of the three TAM motifs, or a portion thereof, present in the human CD3 zeta chain; such TAM motifs are as follows:
  • CD3 ⁇ TAM motif #1 CD3 ⁇ TAM motif #1:
  • CD3 ⁇ TAM motif #2 CD3 ⁇ TAM motif #2:
  • CD3 ⁇ TAM motif #3 CD3 ⁇ TAM motif #3:
  • TAM motifs from different receptor subunits appear to specifically activate distinct biological responses in different cell types.
  • T cell receptor is not expected to be as active in activating mast cells as in activating T cells
  • the ⁇ subunit TAM motif from the IgE mast cell receptor is not expected to be as active in activating T cells as in activating mast cells.
  • SEQ ID NO:1 other than X are invariant residues that are shared by all members of the TAM family. These shared residues must be important for a common
  • TAM Mimics are also provided which specifically interfere with activation or immune responses in specific immune cells, by virtue of containing sequences of variant residues (e . g . , residue numbers 2-8, 10-11, 13-14, 16-22, and/or 24-25 in SEQ ID N0:1) which are identical or substantially the same as those sequences present in MIRRs (see Fig. 3).
  • TAM Mimics which mimic FceRI 7 or ⁇ TAM motifs.
  • a TAM mimic comprises one of the following sequences, related to the FceRI 7 TAM motifs:
  • dashed lines represent potential salt bridges between the indicated residues; solid lines represent cyclization (covalent linkage) of the indicated residues.
  • SEQ ID NO: 16 is via disulfide bond formation.
  • the envisioned salt bridges and the cyclizations are preferably present in the indicated sequences because they are expected to increase the conformational stability of the peptide.
  • SEQ ID NO: 13 is the actual sequence contained in the C-terminal region of human FceRI- ⁇ .
  • a TAM Mimic comprises or consists of the sequence:
  • TAM Mimics of the invention which have a sequence more homologous to a TAM motif found in an FceRI ⁇ or ⁇ chain than to other TAM motifs are preferred for inhibition of mast cell (or basophil cell) activation.
  • TAM Mimics which have a sequence more homologous to a TAM motif found in FC7RIII (or FC7RIIIA) ⁇ or ⁇ chain are preferred for inhibition of macrophage or natural killer cell activation.
  • TAM Mimics which have a sequence more homologous to a TAM motif found in the B cell antigen receptor complex (MB-1 or B29) are preferred for inhibition of B cell activation.
  • TAM Mimics which have a sequence more homologous to a TAM motif found in the CD3- ⁇ , CD3- ⁇ , or ⁇ chain of the T cell antigen receptor complex are preferred for inhibition of T cell activation.
  • TAM Mimics of the invention can be derived from TAM motifs of proteins of human or other animal origin, including but not limited to mammals such as cows, horses, pigs, sheep, goats, rats, mice, dogs, chickens, rabbits, etc.
  • the peptide TAM Mimics of the invention have a sequence in the range of 15-39 amino acids. In a specific aspect, the peptides have a sequence in the range of 17-25 amino acids. In a preferred aspect, the peptides contain 19 amino acids.
  • the TAM mimic peptides of the invention preferably contain naturally-occurring amino acids.
  • the most common naturally-occurring amino acids are shown in Table I:
  • TAM mimic peptides can contain non-natural amino acids or cyclic peptides.
  • Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ - alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • the amino acid can be the D (dextrorotary) or L
  • the peptide may be prepared by methods that are known in the art.
  • solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
  • the protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
  • the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by
  • TAM Mimics of the invention also include hybrid molecules that comprise peptides bound to amino- or carboxy-terminal helix inducers.
  • such hybrid molecules are TAM Mimic peptides, in which two amino acid residues at the N-terminus and/or C-terminus of the peptide are replaced respectively by an N-terminal or C-terminal helix inducer, or a helix inducer of 0-3 residues can be added.
  • Such helix inducers are nonpeptide organic structures that function as helix nucleating modules. Examples of N-terminal and C-terminal helix inducers, respectively in C-terminal or N-terminal linkage to a peptide amino or carboxyl group respectively, are shown in Figure 6.
  • the invention thus provides a compound of formula (IIe), wherein X is OH or an amino-terminally linked peptide having a
  • X is a hydrogen atom or a carboxy-terminally linked peptide having a sequence in the range of 17-39 amino acids and comprising the sequence V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 13).
  • hybrid peptide (la) is produced, containing helix inducer (lb) replacing the N-terminal DG in the sequence of the TAM Mimic Peptide TAM-1 (see Section 6) having the sequence:
  • the C-terminus of compound (la) is the free acid, an ester (OR, where R is preferably an alkyl of 1-4 carbon atoms), or an amide.
  • R is preferably an alkyl of 1-4 carbon atoms
  • hybrid peptide (la) may be prepared according to Scheme I.
  • 3,5-Dimethoxybenzoic acid is converted to its acid chloride by treatment with oxalyl chloride in
  • the cis-acid (9) is coupled to L-valine benzyl ester using bromo-tris-pyrrolidino- phosphonium hexafluorophosphate and diisopropyl ethylamine in dichloromethane at 0°C to r.t. for 16 h to give a 1:1 mixture of diastereomers 10L and 10D.
  • Hydrogenolysis of the mixture using hydrogen and a catalytic amount Pd/C followed by HPLC on a reverse phase column provides the desired diastereamer 11L along with diastereomer 11D.
  • Coupling of 11L to TAM peptide 16-mer followed by removal of side chain protecting groups gives the desired hybrid peptide (la).
  • the above-described synthetic scheme has been carried out as described to yield the hybrid peptide (la).
  • hybrid peptide (IIa) is produced, containing helix inducer (lIb) replacing the N-terminal DG in the sequence of the TAM Mimic peptide TAM-1 (see Section 6.2).
  • N-terminal helix inducer lib can be synthesized from L-aspartic acid and L-pyroglutamic acid, according to Scheme II.
  • L-pyroglutamic acid was first protected as its methyl ester by treatment with thionyl chloride in methanol at 0°C to room temperature (r.t.) for 20 h and then treated with triethyloxonium
  • TAM Mimics of the invention also include hybrid molecules with helix replacements to achieve gradual reduction of the peptide character of the molecules.
  • peptide hybrids ol the invention are TAM Mimic peptides in which the peptide sequence corresponding to amino acid numbers 16-22 in the consensus sequence (SEQ ID NO:1) (see e.g. Fig. 3) are replaced by a phenothiazine ring structure.
  • Compounds of the invention thus include peptide-like hybrid molecules of the formula (V) where R 1 is a sequence of at least 4, and is preferably 7, amino acids, wherein R 1 comprises a C-terminal leucine, and a tyrosine in the fourth position counting from a C to N-terminal direction.
  • R 1 corresponds to a portion of SEQ ID NO:1, amino-terminal to SEQ ID NO:1 residue number 16, except that the amino acid
  • R 1 has a sequence in the range of 4-25 amino acids.
  • R 2 is a sequence of at least 4 amino acids, and is most preferably 5 amino acids, comprising an amino- terminal Tyr, and Leu or Ile in the fourth position counting in the N- to C-terminal direction.
  • R 2 has a sequence in the range of 4-25 amino acids, and preferably 4-6 amino acids.
  • R 3 is Hydrogen or C 1 -C 6 alkyl. 2 is S, SO, or SO 2 .
  • TAM Mimics of the invention which are peptide hybrids and which mimic the FceRI ⁇ TAM, are of structure (V) in which R 3 is H; Z is S; R 2 is YETLK-NH 2 (part of SEQ ID NO: 17); and R 1 is DGVYTGL (part of SEQ ID NO: 17), QKVYDKL (part of SEQ ID NO:19), or QKVYCKL (part of SEQ ID NO:20).
  • the phthalimide group is removed with hydrazine producing the free benzyl amine which is subsequently protected as its BOC (tert- butoxycarbonyl) derivative (30) by reaction with 2- (tert-butoxycarbonyloxyamino)-2-phenylacetonitrile.
  • BOC tert- butoxycarbonyl
  • Metallation of (30) with n-butyllithium at -78°C followed by quenching with carbon dioxide produces the carboxylic acid (31).
  • Peptide coupling with the carboxyl end of (31) followed by deprotection of the amine followed by a second peptide coupling with the amino end of (31) yields the desired peptide-like hybrid molecules.
  • TAM Mimics are further characterized as described below prior to in vitro and in vivo testing of
  • TAM Mimic peptides The conformational preferences and membrane-penetrating properties of the TAM Mimic peptides can be investigated both
  • Mimic peptide can be predicted based on four
  • amphiphilic moment (iii) the electric dipole of the peptide; and (iv) the net charge of the peptide.
  • the lipid-induced helix-forming potential of a TAM Mimic can be assessed by CD (circular dichroism) spectroscopy in the presence of lipid-mimicking solvents (e.g., trifluorethanol) and lipid vesicles.
  • CD circular dichroism
  • NMR nuclear magnetic resonance
  • NMR has been used extensively to study the three-dimensional structure of biologically active peptides in solution or bound to micelles or
  • TAM Mimics The membrane transport properties of TAM Mimics can be studied by size exclusion
  • Lipid vesicles are prepared,
  • TAM Mimics incubated with TAM Mimics, then chromatographed to separate free TAM Mimic.
  • the vesicle contents are then assayed to determine the amount of TAM Mimic present in the lipid vesicle fraction (determinable by HPLC or amino acid analysis).
  • a fluorescence assay for peptide transport can be used; the assay uses a water-stable fluorogenie reagent that becomes trapped inside the vesicles during formation. This
  • fluorogenie reagent reacts with TAM Mimic molecules that have crossed the lipid bilayer, producing a fluorescent derivative. 5.5. DEMONSTRATION OF THERAPEUTIC UTILITY
  • TAM Mimics are tested in vitro and then preferably in vivo for the desired therapeutic
  • TAM Mimic inhibitory to mast cell
  • histamine release can be detected in in vitro assays as indications of mast cell or basophil activation (see e.g., Stephan et al., 1992, J. Biol. Chem. 267:5434). Histamine release can be assayed, for example, by commercially available radioimmunoassay (e.g., AMAC Inc., Westbrook, Maine, Cat. No. 1302).
  • cytokines e.g., interleukins
  • phosphatidylinositol hydrolysis or tyrosine phosphorylation can be detected in in vitro assays as indications of mast cell or basophil activation (see e.g., Stephan et al., 1992, J. Biol. Chem. 267:5434). Histamine release can be assayed, for example, by commercially available radioimmunoassay (e.g., AMAC Inc., Westbrook, Maine, Cat. No. 1302).
  • Basophilic cell lines such as RBL-2H3 (Stephan et al., supra ) , KU812 (Matsson et al., 1989, Int. Arch. Allergy Appl. Immunol. 88:122-125; Valent et al., 1990, J. Immunol. 145:1885-1889), etc. can be employed in such in vitro assays.
  • the release of histamine from basophils is an in vitro assay of immediate hypersensitivity (Ishizaka & Ishizaka, 1975, Prog. Allergy 19:60).
  • cytotoxicity assays measuring natural killer lysis of target cells such as K562 erythromyeloid leukemia cells. Macrophage activation can be assayed by measuring macrophage phagocytic activity, induction of macrophage cytoxicity, or induction of macrophage Class II MHC cell surface expression, or by observing morphological changes associated with activation (see e . g . , Wright and Meyer, 1985, J. Exp. Med.
  • T cell activation can be assayed by T cell proliferation in vitro, or by measuring expression of cell surface interleukin-2 receptor (IL-2R), which increases upon activation of T cells (Waldman et al., 1984, J. Exp. Med. 160:1450-1466).
  • IL-2R cell surface interleukin-2 receptor
  • B cell activation can be assayed by measuring B cell proliferation in vitro .
  • TAM Mimics demonstrated to have the desired activity in vitro can be tested in vivo for the desired inhibitory activity.
  • such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • suitable model systems are also used to demonstrate therapeutic utility (see infra) .
  • an animal model system for rheumatoid arthritis is that consisting of animals of the autoimmune MRL/l mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease. Rose, N.R., et al . , eds., Elsevier/North- Holland, New York, pp. 207-219), that develop a spontaneous rheumatoid arthritis-like disease (Hang et al., 1982, J. Exp. Med. 155:1690-1701).
  • mice used as animal models for asthma include mice, rats, guinea pigs, rabbits, ponies, dogs, sheep, and primates.
  • TAM Mimics have therapeutic and prophylactic utility in the modulation of functions mediated by MIRRs, in particular in diseases or disorders involving the immune system or inflammation (inflammatory and immune disorders). TAM Mimics which inhibit lymphocytes are important therapeutically, because lymphocytes initiate autoimmune and alloimmune diseases.
  • TAM Mimics which inhibit an immune or inflammatory response and thus are useful according to the invention are most preferably identified by use of known convenient in vitro assays, e .g . , based on their ability to inhibit activation of cells of the immune system assayed in vitro , or in vivo assays (see
  • TAM Mimics which inhibit activation of T cells, B cells, and/or macrophages in vitro are preferred for treatment (or prevention) of immune disorders such as but not limited to autoimmune diseases and transplant rejection.
  • TAM Mimics which inhibit activation of macrophages and/or natural killer cells in vitro are preferred for treatment (or prevention) of immune complex diseases such as but not limited to glomerulonephritis and other autoimmune diseases.
  • TAM Mimics which inhibit activation of mast cells in vitro are preferred for treatment (or
  • the invention provides methods of treating or preventing diseases and disorders associated with undesirable or inappropriate immune system activity or inflammation by administration to a subject of an effective amount of a TAM Mimic of the invention.
  • the subject is preferably an animal
  • animals including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • inflammatory/ immune response include but are not limited to the following:
  • Inflammatory arthritis e.g., rheumatoid arthritis, seronegative spondyloarthritites (Behcets disease, Reiter's syndrome, etc.), juvenile rheumatoid arthritis, vasculitis, psoriatic arthritis,
  • SLE Systemic lupus erythematosus
  • Inflammatory dermatoses e.g., psoriasis, dermatitis herpetiformis, eczema, necrotizing and cutaneous vasculitis, bullous diseases.
  • autoimmune disorders In addition to the autoimmune disorders SLE and rheumatoid arthritis, disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e . g . , rejection of transplanted organs such as kidney, heart, pancreas, bowel, or liver), and graft-versus- host disease can be treated.
  • SLE and rheumatoid arthritis disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e . g . , rejection of transplanted organs such as kidney, heart, pancreas, bowel, or liver), and graft-versus- host disease can be treated.
  • TAM Mimics of the invention including but not limited to those associated with hemolytic anemia, blood transfusion, certain
  • necrotizing enterocolitis necrotizing enterocolitis, granulocyte-transfusion- associated syndromes, Reynaud's syndrome, or other central nervous system inflammatory disorders.
  • a TAM Mimic which inhibits mast cell activation is administered to treat (or prevent) a type I allergic reaction such as one or more of the following listed in Table 1 (see generally, Terr, 1987, in Basic & Clinical Immunology, 6th Ed., ch. 24, Stites et al. (eds), Appleton & Lange, Norwalk,
  • Urticaria or Angioedema increased cutaneous
  • the invention provides methods of treatment
  • the TAM Mimic is purified.
  • subject is preferably an animal, including but not
  • microcapsules expression by recombinant cells
  • TAM Mimic-encoding nucleic acid as part of a retroviral or other vector, etc. Since preferred TAM Mimics of the invention are permeable to the cell membrane, a preferred mode of delivery is via
  • pulmonary administration as detailed more fully in Section 5.7.1 infra .
  • methods of introduction include but are not limited to intradermal,
  • TAM Mimics may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • TAM Mimics which are used for inhibition of mast cell activation, e . g . , for therapy of asthma or allergy, the preferred route of
  • administration is nasal or via a bronchial aerosol.
  • invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., for skin conditions such as
  • psoriasis by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or
  • gelatinous material including membranes, such as sialastic membranes, or fibers.
  • compositions comprise a therapeutically (or prophylactically) effective amount of a TAM Mimic, and a
  • Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile.
  • the formulation should suit the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a
  • solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by:
  • infusion it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the TAM Mimics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartarlc acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the
  • Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of
  • a TAM Mimic is administered by pulmonary
  • bronchial aerosol is employed. This mode of administration is particularly preferred where the TAM Mimic inhibits mast cell activation and thus is useful in treating IgE-related disorders.
  • pulmonary administration is particularly preferred where the TAM Mimic inhibits mast cell activation and thus is useful in treating IgE-related disorders.
  • treatment e.g., of allergy or asthma.
  • Pulmonary administration can be
  • nebulizer Melt, Inc., St. Louis, Missouri
  • Acorn II nebulizer Marquest Medical Products
  • Such devices typically entail the use of formulations suitable for dispensing from such a device, in which a propellant material may be present.
  • Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis,"
  • a nebulizer may be used to produce aerosol particles, or any of various physiologically
  • acceptable inert gases may be used as an aerosolizing agent.
  • Other components such as physiologically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, and diluents may also be included.
  • FmocTyr ('Bu)OH [Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; 'Bu, tert-butyl; Fmoc,
  • NMP N-methylpyrrolidone
  • DMF N,N-dimethylformamide
  • Deprotection of the Fmoc group is effected using ca. 20% piperidine in NMP. At the end of each synthesis the amount of peptide present is assayed by
  • dibenzofulvene-piperidine adduct (formed by cleavage of the N-terminal Fmoc group) is recorded at 301 nm.
  • w is the weight of the peptide-resin sample (in mg).
  • N-terminal Fmoc group is cleaved using 20% piperidine in DMA, then acetylated using acetic anhydride and pyridine in DMA.
  • the peptide resin is thoroughly washed with DMA, CH 2 Cl 2 and finally diethyl ether.
  • the air-dried peptide resin is treated with ethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole (PhSMe) for approximately 20 min. prior to addition of 95% aqueous trifluoracetic acid (TFA).
  • EtSMe ethylmethyl-sulfide
  • EDT ethanedithiol
  • PhSMe thioanisole
  • TAM-1 19-mer TAM Mimic peptide, termed "TAM-1", having the TAM native sequence shown was synthesized:
  • TAM-2 TAM Mimic peptide
  • Non-consensus amino acids were changed relative to the TAM-1 peptide sequence, with a view toward achieving a higher degree of helicity.
  • TAM-4 TAM Mimic peptide
  • the peptide contained two cysteine residues linked via a disulfide bridge to help lock the peptide into the presumed desired conformation.
  • the crude peptide (385 mg) was initially partially purified by gel filtration on Sephadex G-10 eluted with 10% aqueous acetic acid to give 300 mg material.
  • Peptide (73 mg) was dissolved in degassed 50% aqueous acetic acid (2.5 mL) and mercuric acetate (91 mg) in 50% aqueous acetic acid (1.0 mL) was added. The mixture was stirred under N 2 for 3 h, then
  • TAM-5 TAM Mimic peptide
  • TAM-6 TAM Mimic peptide
  • NMR nuclear magnetic resonance
  • the purified TAM-1 was prepared both as a 3 mM solution in 30% (v/v) deuterated trifluoroethanol (30% TFE) and as a 3 mM solution in the presence of deuterated
  • DPC dodecylphosphatidylcholine
  • the NMR analysis of the secondary structure of the TAM-1 peptides was based on an analysis of the relative intensities of interproton NOE data
  • ⁇ -hexosaminidase from basophils is measured as an in vitro assay of basophil activation.
  • a cell line, RBL-2H3 (from the laboratory of Dr. Siraganian, National Institutes of Health), of rat basophilic leukemia (RBL) cells is used in such an in vitro assay, according to the protocol described below (see also Barsumian et al., 1981, Eur. J.
  • BSA bovine serum albumin
  • Beta-hex buffer Solution 1 0.2 M Na 2 PO 4 (sodium phosphate dibasic ANHYDROUS) - 14.2 g/500 ml distilled H 2 O Note: If there is no anhydrous, use the sodium phosphate dibasic HEPTAHYDRATE (7H 2 O), but use 26.7 grams.
  • Beta-hex buffer (from above): 90 ml Distilled H 2 O: 135 ml p-nitrophenyl-N-acetyl-beta-D-glucosaminide
  • Beta-hex STOP solution glycine 15.0 g/ liter bring to pH 10.7 with 10 N NaOH (will need to add -30 ml of NaOH)
  • the mast cell/basophil degranulation assay can also be performed using permeabilized cells, according to the protocol described below (see also Cunha-Melo et al., 1989, J. Immunol. 143:2617-2625; Ali et al., 1989, J. Immunol. 143:2626-2633; Ali et al., 1989, Biochim. Biophys. Acta 1010:88-99).
  • LiCl (10 to 20 mM) for phosphoinositide hydrolysis experiments LiCl (10 to 20 mM) for phosphoinositide hydrolysis experiments.
  • RBL-2H3 cells (0.2 x 10 6 / well, in growth medium) are plated in 24 well tissue culture plate and incubated overnight.
  • the cells are permeabilized by exposure to
  • streptolysin 0 (0.1 to 0.3 units/ml, 200-500 ⁇ l/well) for 5 to 10 min.
  • concentration of streptolysin 0 and the time required for permeabilization depends on the number of passages the cells have been cultured for and may vary with batch of streptolysin 0.
  • a useful starting point is to permeabilize cells with 0.25 I.U. /ml streptolysin for 10 min.
  • Streptolysin O solution is prepared just before permeabilization by dilution of the stock solution (10 I.U. /ml) into prewarmed KG buffer. After the cells are permeabilized, remove buffer by aspiration, add fresh buffer (without toxin) and perform experiment as desired to measure cell
  • activation e.g., measure phosphoinositide hydrolysis, or ⁇ -hexosaminidase or histamine release.
  • Modifications of the permeabilization procedure can be made for measuring degranulation.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention is directed to peptides representing all or a portion of tyrosine activation motifs (TAMs), as well as analogs and derivatives thereof (collectively termed herein 'TAM Mimics'). In particular, such analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions. In a specific embodiment, a hybrid molecule is provided which contains an amino(N)- or carboxy(C)-terminal non-peptide helix inducer. In another specific embodiment, the hybrid molecule contains an internal non-peptide structure that maintains the helical character of the TAM mimic. TAM Mimics of the invention inhibit the activation of various cells with immune system function. Therapeutic methods based on the immune function inhibitory activity of the TAM Mimics, as well as pharmaceutical compositions, are also provided. In a preferred embodiment of the invention, TAM Mimics are formulated for pulmonary administration; thus, for example, the invention provides inhalers containing compositions comprising a therapeutically or prophylactically effective amount of a TAM Mimic.

Description

ANALOGS OF RECEPTOR TYROSINE ACTIVATION
MOTIFS AND THERAPEUTIC USES THEREOF 1. INTRODUCTION
The present invention relates to peptides and analogs and derivatives thereof that are related to all or a portion of tyrosine activation motifs found in multichain immune recognition receptors.
Such peptides, analogs, and derivatives have utility in the treatment of diseases or disorders associated with undesirable or inappropriate immune activity or inflammation. 2. BACKGROUND OF THE INVENTION
2.1. IMMUNOGLOBINS AND THE IMMUNE RESPONSE
The immune system consists of lymphocytes, macrophages, and other specialized cells. An organism responds to antigen by virtue of having a large number of lymphocytes, each bearing receptors which recognize distinct antigens. B lymphocytes are precursors of antibody-secreting cells, and T lymphocytes are involved in regulatory and effector functions. In addition, natural killer cells mediate certain
cytotoxic responses. (See generally, Fundamental Immunology, 1989, 2d Ed., Paul, W.E. (ed.), Raven Press Ltd., New York; Benacerraf & Unanue, 1979,
Textbook of Immunology, Williams and Wilkins,
Baltimore).
Cellular (or cell-mediated) immunity results from the activity of T lymphocytes that have been sensitized to an antigen by virtue of their
recognition of the antigen via their T cell antigen receptor complex (see generally, Hedrick, 1989, in Fundamental Immunology, 2d Ed., ch. 11, Paul, W.E.
(ed.), Raven Press Ltd., New York, pp. 291-313; Weiss, 1989, in Fundamental Immunology, 2d Ed., ch. 13, Paul, W.E. (ed.), Raven Press Ltd., New York, pp. 359-384).
Antibody or humoral immunity is mediated by molecules called antibodies that combine specifically with antigens; immunoglobulins are proteins with antibody activity. There are five classes of human immunoglobulins, differing in the structure of their respective heavy chains: IgG, IgA, IgM, IgD, and IgE. Immunoglobulins are synthesized by B lymphocytes, and by their progeny, the plasma cells. B cells interact with antigen via the antibody molecules in their plasma membranes. Plasma cells secrete antibodies. IgGs are the major component of serum immunoglobulin, and the predominant antibody involved in a secondary immune response. IgM is the predominant antibody involved in a primary immune response. IgD is a cell surface receptor present on mature B cells. IgA is found in bodily secretions. IgE is found in normal human serum at very low concentrations, and is
involved in type I allergic reactions (atopy).
IgE appears to mediate local and systemic immediate hypersensitivity and anaphylactic reactions. Atopic individuals produce IgE in response to many environmental antigens. The event that initiates immediate hypersensitivity in such patients is the binding of antigen to IgE on the surface of mast cells. Binding of the Fc domain of the allergen-bound IgE to the extracellular domain of the FceRI receptor complex on mast cells and basophils induces
crosslinking of the receptor. Crosslinking of IgE receptors (FceRI) causes mast cell activation and a cascade of intracellular signaling events which ultimately results in the release of lipid mediators of inflammation (leukotrienes, etc.), proinflammatory cytokines (interleukins, etc.) and of secretory granules (containing histamine, 5-hydroxytryptamine, hexosaminidase, etc.). This release causes the appearance of many of the symptoms of acute allergic response.
2.2. MULTICHAIN IMMUNE RECOGNITION RECEPTORS
Cells respond to external stimuli through the binding of extracellular ligands to transmembrane receptor molecules. Binding of protein or peptide ligands to the extracellular domain of such a
membrane-spanning receptor results in a conformational change or aggregation of the receptor, and a
subsequent relay of the conformational signal induced by the external stimulus to the intracellular portion of the receptor. Intracellular signal transduction occurs via changes in the intermolecular associations of protein molecules arranged in a complex multi-step sequential pathway leading from the intracellular portion of the receptor to proteins involved directly in controlling gene expression, cytoskeletal
architecture and cell division (Cantley et al., 1991, Cell 64:281-302). Recent research shows that signal transduction is accomplished at a molecular level via protein-protein complex formation, or, more
specifically, by the creation of new protein-protein interaction sites, e.g., by enzymatic modification of tyrosine residues or by conformational changes induced by binding. Specific examples of intracellular signaling events modulated by protein-protein
interactions are receptor-coupled G-protein
activation, immunosuppression via immunophilin- immunosuppressive agent complexes and the release of the contents of secretory vesicles, e.g., histamine release in mast cell degranulation. Several protein- protein interaction sites involved in signaling have been localized to short (less than 20 amino acid) contiguous sequences (see, e.g., Koch et al., 1991, Science 252:668-674).
Multichain immune recognition receptors
(MIRRs) (Keegan and Paul, 1992, Immunology Today
13:63-68) are found on T and B cells, mast cells, macrophages, natural killer cells and platelets.
MIRRs constitute a family of immune cell receptors involved in the activation of cells involved in the immune system. MIRRs include the B cell antigen receptor complex (BCR); the T cell antigen receptor complex (TCR); FcγRIII (FC7RIIIA in humans), a
receptor for the Fc region of IgG antibody molecules, found on mast cells, basophils, natural killer cells, macrophages, and neutrophils; and FceRI, a receptor for the Fc region of IgE antibody molecules. These MIRRs consist of polypeptide chains that bind antigen or Fc, associated with chains whose intracellular domains contain a related short sequence of 18-27 amino acids termed the tyrosine activation motif
("TAM") (Samelson and Klausner, 1992, J. Biol. Chem. 267(35) :24913-24916; Reth, 1989, Nature 338:383-384). The consensus sequence for the TAM is:
(D/E)XXXXXXX(D/E)XXYXXLXXXXXXXYXX(L/I)X (SEQ ID NO:1), where X is any amino acid (Reth, 1989, Nature
338:383-384). Figure 1 schematically depicts these MIRRs.
The TCR consists of the α (alpha) and β (beta) T cell antigen receptor antigen-binding chains (which, in a minority of T cells, are replaced by the γ (gamma) and δ (delta) heterodimer), the γ, δ and s (epsilon) chains of the CD3 complex, and a disulfide linked ζ (zeta) dimer or heterodimer of ζ and η (eta) chains. The BCR consists of the heavy and light chains and at least one disulfide-linked heterodimer consisting of Ig-α (MB-1 gene product) and Iq-β (B29 gene product). The FcεRI receptor is a complex of three proteins, the α, β, and λ subunits. The α subunit of FceRI binds IgE, the β subunit contains four transmembrane regions, and the 7 subunit consists of a part of disulphide-linked chains (Fig. 2).
Most of the FceRI-γ subunit residues lie on the cytoplasmic side of the cell membrane. This γ subunit is shared by the FC7RIII MIRR, and contains a TAM motif. FceRI-β also contains a TAM motif (but not FceRI-α), as do the CD3-γ,CD3-δ and ζ components of the TCR (but not the CD3-ε or TCR-α or TCR-β), MB-1 and B29 in the BCR, and the envelope protein (gp30) of bovine leukemia virus (Reth, 1989, Nature 338:383-384; see also Fig. 3 herein).
The function of the TAM motif in signal transduction has been investigated for the TCR. The consensus sequence shown above occurs in the ζ chain of the TCR. Cell lines expressing a chimeric molecule in which the cytoplasmic tail of the ζ chain was attached to the unrelated CD8 receptor respond to antibody crosslinking stimuli (Romeo et al., 1992, Cell 68:889-897).
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. 3. SUMMARY OF THE INVENTION
The present invention is directed to
peptides representing all or a portion of tyrosine activation motifs (TAMs), as well as analogs and derivatives thereof (collectively termed herein "TAM Mimics"). In particular, such analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions. In a specific embodiment, a hybrid molecule is provided which contains an amino(N)- or carboxy(C)-terminal non- peptide helix inducer. In another specific
embodiment, the hybrid molecule contains an internal non-peptide structure that maintains the helical character of the TAM mimic. TAM Mimics of the
invention inhibit the activation of various cells with immune system function.
Therapeutic methods based on the immune function inhibitory activity of the TAM Mimics, as well as pharmaceutical compositions, are also
provided. In a preferred embodiment of the invention, TAM Mimics are formulated for pulmonary
administration; thus, for example, the invention provides inhalers containing compositions comprising a therapeutically or prophylactically effective amount of a TAM Mimic.
4. DESCRIPTION OF THE FIGURES
Figure 1. Schematic diagram of four multichain immune recognition receptors (MIRRs): IgMR (the B cell antigen receptor complex), FC7RIII, EceRI, and TCR (the T cell antigen receptor complex). Two FceRI receptors are depicted cross-linked by allergen binding. Two IgMRs are depicted cross-linked by antigen binding. TAM motifs are indicated by cross- hatching.
Figure 2. A schematic view of the MIRR for the Fc region of IgE, called the FceRI receptor (after Keegan and Paul, 1992, Immunology Today 13 (2): 63-68).
Figure 3. TAM Motifs in the cytoplasmic tail sequences of various MIRRs. h, human; r, rat; m, mouse. Figure 4. Exemplary TAM Mimic compounds of Formula (V).
Figure 5. Computer-generated drawings. The left side shows an idealized (computer-generated) drawing of an alpha-helical peptide. In the structure on the right, 7 amino acids (i.e., an 11 angstrom stretch) have been replaced by a "spacer molecule" like that present in a compound of formula (V).
Figure 6. Helix nucleating modules.
N-terminal and C-terminal helix inducers are depicted. Compound I and Compound III were developed by Paul Bartlett (Bartlett et al., "Intuitive- and Computer- Assisted Approaches to the Design of Conformationally Restrained Peptides and Their Mimics," October 28-29, 1991, reprinted from Proceedings of The Robert A.
Welch Foundation Conference on Chemical Research, XXXV Chemistry at the Frontiers of Medicine, Houston,
Texas).
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to peptides related to all or a portion of tyrosine activation motifs (TAMs), as well as analogs and derivatives thereof (collectively termed herein "TAM Mimics"). In particular, such analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions. In a specific embodiment, a hybrid molecule is provided which contains an amino (N)- or carboxy(C) -terminal non- peptide helix inducer. In another specific
embodiment, the hybrid molecule contains an internal non-peptide structure that maintains the size and general shape of the TAM mimic. The TAM Mimics of the invention retain the conformational information of TAM motifs required for biological activity, however, preferably incorporate sequence changes that provide the TAM Mimic with enhanced ability to cross lipid bilayers.
The TAM motif plays a critical role in the activation of the immune functions of T cells,
B cells, mast cells, basophils, and natural killer (NK) cells. The TAM motif within receptor complexes appears to interact with target proteins that transmit the activation signal within the cytoplasm to bring about cellular immune responses. The TAM Mimics of the invention mimic the structure of these TAM motifs. While Applicants do not intend to be bound by any specific mechanism, Applicants believe that the TAM Mimics function by binding to the target protein, mimicking the interaction with the receptor-associated TAM motif, and thus preventing the natural activation of this target. Stimulation of mast cells, B
lymphocytes, T lymphocytes, and macrophages is
interfered with at the first stage in the stimulus- response coupling, thereby abrogating the responses of these cells.
The TAM Mimics of the invention thus act as inhibitors of the signal transduction events mediated by the various MIRRs containing TAM sequences, by competitively inhibiting the interaction of such molecules with an effector or regulatory molecule via their TAM motifs, and thus preventing the natural sequence of activation in MIRRs.
Therapeutic methods based on the immune function inhibitory activity of the TAM Mimics, as well as pharmaceutical compositions, are also
provided. The TAM Mimics of the invention can also be used to study and elucidate the signal transduction mechanism in cells expressing the MIRRs. 5.1. TAM MIMICS
The TAM Mimics of the invention include peptides and hybrid molecules, as detailed more fully in the subsections below.
5.1.1. PEPTIDES
In a specific embodiment, the TAM Mimic is a peptide. Such a peptide can comprise all or a portion of a TAM motif actually present in a MIRR. For example, a TAM Mimic of the invention comprises or consists of all or a portion of the sequences shown in Figure 3 (SEQ ID NOS:2-12). In a preferred aspect, a TAM Mimic comprises or consists of the sequence
(D/E)XXXXXXX(D/E)XXYXXLXXXXXXXYXX(L/I)X (SEQ ID NO:1). In another embodiment, a TAM mimic comprises or consists of the following sequences:
(D/E)XXYXXLXXXXXXXYXX(L/I) (part of SEQ ID NO:1)
DGGYMTLNPRAPTDDDKNTYLTLP (part of SEQ ID NO: 2)
DAVYTGLSTRNQETYETLK (part of SEQ ID NO: 3)
DRLYEELHVYSPIYSALE (part of SEQ ID NO: 4)
DQLYQPLKDREDDQYSHLQ (part of SEQ ID NO: 5)
DQVYQPLRDRDDAQYSHLG (part of SEQ ID NO: 6)
DGLYQGLSTATKDTDALH (part of SEQ ID NO: 7, 8)
ENLYEGLNLDDCSMYEDIS (part of SEQ ID NO: 9, 10)
DHTYEGLNIDQTATYEDIV (part of SEQ ID NO: 11)
EHTYEGLNIDQTATYEDIV (part of SEQ ID NO: 12)
In yet another specific embodiment, a TAM Mimic peptide comprises or consists of the β or γ TAM motif, or a portion thereof, present in the human FceRI complex, or one of the three TAM motifs, or a portion thereof, present in the human CD3 zeta chain; such TAM motifs are as follows:
FceRI β TAM motif:
E D R V Y E E L N I L S A T Y S E L E D P G E N
(SEQ ID NO:26) FcεRI γ TAM motif:
S D G V Y T G L S T R N Q E T Y E T L K H E K
(SEQ ID NO: 27)
CD3 ζ TAM motif #1:
Y Q Q G Q N Q L Y N E L N L G R R E E Y D V L D K R R G R D P E M G G K P (SEQ ID NO:28)
CD3 ζ TAM motif #2:
R R K N P Q E G L Y N E L Q K D K M A E A Y S E I G M K G E (SEQ ID NO: 29)
CD3 ζ TAM motif #3:
R R R G K G H D G L Y Q G L S T A T K D T Y D A L H M Q A L P P R (SEQ ID NO:30)
The TAM motifs from different receptor subunits appear to specifically activate distinct biological responses in different cell types. For example, the TAM motif from the ζ subunit of the
T cell receptor is not expected to be as active in activating mast cells as in activating T cells, the γ subunit TAM motif from the IgE mast cell receptor is not expected to be as active in activating T cells as in activating mast cells. The amino acid in
SEQ ID NO:1 other than X are invariant residues that are shared by all members of the TAM family. These shared residues must be important for a common
function of all of these domains, whereas the variant residues provide a structural scaffold or contribute specificity to this domain (i.e., recognition of different target proteins). Thus, TAM Mimics are also provided which specifically interfere with activation or immune responses in specific immune cells, by virtue of containing sequences of variant residues (e . g . , residue numbers 2-8, 10-11, 13-14, 16-22, and/or 24-25 in SEQ ID N0:1) which are identical or substantially the same as those sequences present in MIRRs (see Fig. 3). In a preferred aspect, immune activation of mast cells and basophils via the allergen-IgE receptor complex is interfered with by use of TAM Mimics which mimic FceRI 7 or β TAM motifs. In a preferred aspect, a TAM mimic comprises one of the following sequences, related to the FceRI 7 TAM motifs:
D G V Y T G L S T R N Q E T Y E T L K (SEQ ID NO: 19)
Figure imgf000013_0001
Figure imgf000013_0002
Q K V Y D K L L K R N Q E T Y E T L K (SEQ ID NO: 14)
Figure imgf000013_0003
Figure imgf000013_0004
Q K V Y D K L L K R N Q E L Y E T L K (SEQ ID NO: 15)
Figure imgf000013_0005
Q K V Y C K L L C R N Q E L Y E T L K (SEQ ID NO: 16)
Figure imgf000013_0006
D G V Y T G L S T R N Q K T Y K T L K (SEQ ID NO: 17) N G V Y T G L S T R N Q K T Y K T L K (SEQ ID NO:18)
In the above-presented sequences, dashed lines represent potential salt bridges between the indicated residues; solid lines represent cyclization (covalent linkage) of the indicated residues.
Cyclization in SEQ ID NO: 15 is via an amide linkage between the indicated residues. Cyclization in
SEQ ID NO: 16 is via disulfide bond formation. The envisioned salt bridges and the cyclizations are preferably present in the indicated sequences because they are expected to increase the conformational stability of the peptide.
In a preferred aspect, the TAM Mimics consist of the following 19-mer peptides (Ac = acetyl; solid lines between residues indicates cyclization): Ac-D G V Y T G L S T R N Q E T Y E T L K-NH2 (SEQ ID NO: 19);
Ac-Q K V Y D K L L K R N Q E T Y E T L K-NH2 (SEQ ID NO: 20);
Ac-Q K V Y D K L L K R N Q E L Y E T LL K-NH2 (SEQ ID
Figure imgf000013_0007
NO: 21); Ac-Q K V Y C K L L C R N Q E L Y E T L K-NH2 ( SEQ ID
Figure imgf000014_0001
NO : 22 ) ;
Ac-D G V Y T G L S T R N Q K T Y K T L K-NH2 ( SEQ ID
NO : 23 ) ; and
Ac-N G V Y T G L S T R N Q K T Y K T L K-NH2 ( SEQ ID
NO:24).
SEQ ID NO: 13 is the actual sequence contained in the C-terminal region of human FceRI-γ.
In another embodiment, a TAM Mimic comprises or consists of the sequence:
D X V Y X X L X X R N Q E X Y E T L K (SEQ ID NO:25)
TAM Mimics of the invention which have a sequence more homologous to a TAM motif found in an FceRI γ or β chain than to other TAM motifs are preferred for inhibition of mast cell (or basophil cell) activation. TAM Mimics which have a sequence more homologous to a TAM motif found in FC7RIII (or FC7RIIIA) γ or ζ chain are preferred for inhibition of macrophage or natural killer cell activation. TAM Mimics which have a sequence more homologous to a TAM motif found in the B cell antigen receptor complex (MB-1 or B29) are preferred for inhibition of B cell activation. TAM Mimics which have a sequence more homologous to a TAM motif found in the CD3-γ, CD3-δ, or ζ chain of the T cell antigen receptor complex are preferred for inhibition of T cell activation.
The TAM Mimics of the invention can be derived from TAM motifs of proteins of human or other animal origin, including but not limited to mammals such as cows, horses, pigs, sheep, goats, rats, mice, dogs, chickens, rabbits, etc.
The peptide TAM Mimics of the invention have a sequence in the range of 15-39 amino acids. In a specific aspect, the peptides have a sequence in the range of 17-25 amino acids. In a preferred aspect, the peptides contain 19 amino acids.
The TAM mimic peptides of the invention preferably contain naturally-occurring amino acids. The most common naturally-occurring amino acids are shown in Table I:
Figure imgf000015_0001
However, the peptides of the invention are not limited to containing the 20 natural amino acids. In other embodiments, TAM mimic peptides can contain non-natural amino acids or cyclic peptides. Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β- alanine, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be the D (dextrorotary) or L
(levorotary) amino acid.
The peptide may be prepared by methods that are known in the art. For example, in brief, solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids. The protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed. The coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by
Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178 and in U.S. Patent Nos. 4,305,872 and 4,316,891. In a preferred aspect, an automated peptide synthesizer is employed.
Purification of the synthesized peptides can be carried out by standard methods including
chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In a preferred embodiment, reverse phase HPLC (high
performance liquid chromatography) is employed.
5.2. HYBRID MOLECULES WITH AMINO- OR
CARBOXY-TERMINAL HELIX INDUCERS
TAM Mimics of the invention also include hybrid molecules that comprise peptides bound to amino- or carboxy-terminal helix inducers. In
particular, such hybrid molecules are TAM Mimic peptides, in which two amino acid residues at the N-terminus and/or C-terminus of the peptide are replaced respectively by an N-terminal or C-terminal helix inducer, or a helix inducer of 0-3 residues can be added. Such helix inducers are nonpeptide organic structures that function as helix nucleating modules. Examples of N-terminal and C-terminal helix inducers, respectively in C-terminal or N-terminal linkage to a peptide amino or carboxyl group respectively, are shown in Figure 6.
In a specific embodiment, the invention thus provides a compound of formula (IIe), wherein X is OH or an amino-terminally linked peptide having a
sequence in the range of 17-39 amino acids and
comprising the sequence V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 13).
Figure imgf000017_0001
In another specific embodiment, the
invention provides a compound of formula (IVa), wherein X is a hydrogen atom or a carboxy-terminally linked peptide having a sequence in the range of 17-39 amino acids and comprising the sequence V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 13).
Figure imgf000018_0002
In a preferred aspect of the invention, hybrid peptide (la) is produced, containing helix inducer (lb) replacing the N-terminal DG in the sequence of the TAM Mimic Peptide TAM-1 (see Section 6) having the sequence:
D G V Y T G L S T R N Q E T Y E T L K (SEQ ID NO: 13)
Figure imgf000018_0001
In specific embodiments, the C-terminus of compound (la) is the free acid, an ester (OR, where R is preferably an alkyl of 1-4 carbon atoms), or an amide. By way of example, hybrid peptide (la) may be prepared according to Scheme I.
3,5-Dimethoxybenzoic acid is converted to its acid chloride by treatment with oxalyl chloride in
dichloromethane at 0°C to room temperature (r.t.) for 5 h. The acid chloride is esterified using potassium tert-butoxide in tert-butanol at 30-40°C for 22 h yielding the tert-butyl ester (1). Birch reductive alkylation of (1) with potassium in a mixture of tert- butanol and ammonia with iodomethane as the alkylating reagent at 78°C for 2 h followed by treatment with mercurous nitrate at reflux in aqueous acetonitrile for 14 h affords the di-keto ester (2). Treatment of (2) with nitro ester (4) and triethylamine in methanol at 0°C to r.t. for 2 d gives the hydroxy di-ester (5). Olefin (4) is synthesized from methyl aerylate by reaction with iodine and nitrogen dioxide at 0°C for 1 h and then at r.t. for 4 h yielding (3) followed by elimination of hydrogen iodide with sodium acetate in ether at reflux for 3 h to give (4). Reduction of the nitro group of (5) with hydrogen at 50 psi using Raney nickel in MeOH/EtOH affords the diester (6) via concomitant ring closure of the amine produced by the reduction of the nitro group. Deprotonation of the amine (6) with potassium hydride followed by
protection with 2-tert-Butoxycarbonyl)-2-tert-butyl-3- methyl-4-imidazolidinone yields the protected amine (7). Treatment of (7) with lithium
hexamethyldisilazane and hexamethylphosphoramide in tetrahydrofuran at -78°C leads to the formation of the methyl ester enolate which is subsequently alkylated with iodomethane to yield a mixture of cis/trans (8). Selective hydrolysis of the methyl ester of (8) using lithium hydroxide followed by silica gel
chromatography separation gives the desired cis diastereomer (9). The cis-acid (9) is coupled to L-valine benzyl ester using bromo-tris-pyrrolidino- phosphonium hexafluorophosphate and diisopropyl ethylamine in dichloromethane at 0°C to r.t. for 16 h to give a 1:1 mixture of diastereomers 10L and 10D. Hydrogenolysis of the mixture using hydrogen and a catalytic amount Pd/C followed by HPLC on a reverse phase column provides the desired diastereamer 11L along with diastereomer 11D. Coupling of 11L to TAM peptide 16-mer followed by removal of side chain protecting groups gives the desired hybrid peptide (la). The above-described synthetic scheme has been carried out as described to yield the hybrid peptide (la).
Figure imgf000021_0001
In another aspect of the invention, hybrid peptide (IIa) is produced, containing helix inducer (lIb) replacing the N-terminal DG in the sequence of the TAM Mimic peptide TAM-1 (see Section 6.2).
Figure imgf000022_0001
By way of example, the N-terminal helix inducer lib can be synthesized from L-aspartic acid and L-pyroglutamic acid, according to Scheme II.
Thus, L-pyroglutamic acid was first protected as its methyl ester by treatment with thionyl chloride in methanol at 0°C to room temperature (r.t.) for 20 h and then treated with triethyloxonium
tetrafluoroborate in dichloromethane at r.t. for 2 d to give the imino ether (12). Reaction of the imino ether (12) with a 4-substituted-2-azetidinone (13) at 135°C neat for 4 h provided the desired hexahydro- pyrrolopyrimidine cis-dicarboxylic acid ester (14). The azetidinone (14) was formed from treatment of L-aspartic acid dibenzyl ester with trimethylsilyl chloride and triethylamine at 0°C for 1 h followed by tert-butyl magnesium chloride treatment for 20 h.
Deprotection of the methyl ester of (14) by treatment with 10% aqueous HCl at 50°C is expected to give the free acid (15), which is coupled to TAM peptide 17-mer followed by removal of side chain protecting groups to provide the desired hybrid peptide mimic (IIa).
Figure imgf000023_0001
5.3. HYBRID MOLECULES WITH INTERNAL
HELIX REPLACEMENTS
TAM Mimics of the invention also include hybrid molecules with helix replacements to achieve gradual reduction of the peptide character of the molecules. In a preferred aspect, peptide hybrids ol the invention are TAM Mimic peptides in which the peptide sequence corresponding to amino acid numbers 16-22 in the consensus sequence (SEQ ID NO:1) (see e.g. Fig. 3) are replaced by a phenothiazine ring structure.
Compounds of the invention thus include peptide-like hybrid molecules of the formula (V) where R1 is a sequence of at least 4, and is preferably 7, amino acids, wherein R1 comprises a C-terminal leucine, and a tyrosine in the fourth position counting from a C to N-terminal direction. Thus R1 corresponds to a portion of SEQ ID NO:1, amino-terminal to SEQ ID NO:1 residue number 16, except that the amino acid
corresponding to residue number 9 of SEQ ID NO:1 can be any amino acid. In a specific but non-limiting aspect, R1 has a sequence in the range of 4-25 amino acids. R2 is a sequence of at least 4 amino acids, and is most preferably 5 amino acids, comprising an amino- terminal Tyr, and Leu or Ile in the fourth position counting in the N- to C-terminal direction. In a specific but non-limiting aspect, R2 has a sequence in the range of 4-25 amino acids, and preferably 4-6 amino acids. R3 is Hydrogen or C1-C6 alkyl. 2 is S, SO, or SO2.
Figure imgf000024_0001
A computer-generated drawing of a compound of formula (V) is shown in Figure 5.
A specific example of compound (V) is compound (Va):
Figure imgf000025_0001
In specific embodiments, TAM Mimics of the invention which are peptide hybrids and which mimic the FceRI γ TAM, are of structure (V) in which R3 is H; Z is S; R2 is YETLK-NH2 (part of SEQ ID NO: 17); and R1 is DGVYTGL (part of SEQ ID NO: 17), QKVYDKL (part of SEQ ID NO:19), or QKVYCKL (part of SEQ ID NO:20).
Exemplary compounds of Formula (V) which may be synthesized by the methods described herein are shown in Figure 4.
Compounds of Formula (V) wherein R3 is hydrogen; Z is S, SO, or SO2; and disubstitution occurs at the 3 and 7 positions of the phenothiazine ring may be prepared according to Scheme III. Condensation of 4-methyl aniline (16) (1 equiv.) (R. Adams, 1957, Org. Reactions, 3:240D) with ammonium thiocyanate under standard conditions [NH4SCN (2 equiv.) in HOAc, Br2 in HOAc (1 equiv., 8-10°C, 1 h] yields the corresponding 5-methyl-2-amino benzothiazole (17). Hydrolysis of
(17) using 50% aqueous potassium hydroxide affords the potassium salt of 2-amino-4-methyl thiophenol (18). Protonation of (18) with five normal acetic acid affords the thiol (19) which when condensed with metanitrobromobenzene in ethanol yields the sulfide (20) (Mital, 1969, J. Chem. Soc. (C), 2148). Formylation of the amino function with formic acid or acetylation of the amine with acetic anhydride yields the
formamide or acetamide derivative (21), respectively (Jain et al., 1991, Indian J. Chem. 9:1236). Smiles rearrangement (Jain et al., 1991, Indian J. Chem.
9:1236) of (21) leads to 3-bromo-7-methyl
phenothiazine (22). Transmetallation of (22) with 2 equivalents of n-Butyllithium at -78°C in
tetrahydrofuran followed by quenching with carbon dioxide yields the carboxylic acid (23). Carboxylic acid (23) is converted to its tert-butyl ester (24) via standard conditions (Armstrong et al., 1988, Tet. Lett. 29 (20) :2483). Benzylic bromination of the methyl group of (24) with N-bromosuccinimide at reflux in carbon tetrachloride affords the benzyl bromide which is treated with sodium azide to give the
corresponding azido derivative. Reduction of the azide under standard conditions yields the amino ester (25). Peptide coupling with the amino end of (25) followed by conversion of the tert-butyl ester to a carboxylic acid followed by a second peptide coupling with the carboxyl end of (25) yields the desired peptide-like hybrid molecules.
Figure imgf000027_0001
Compounds wherein R3 is alkyl; Z is S, SO, or SO2; and disubstitution occurs at the 3 and 7 positions of the phenothiazine ring may be prepared according to Scheme IV. Treatment of phenothiazine with
iodomethane in dimethylsulfoxide yields the N- methylated derivative (26). Reaction of (26) with pyridinium bromide perbromide yields the 3-bromo derivative (27) (L. R. Biehl et al., 1974, J.
Heterocyclic Chem. 11:247) which is subsequently formylated with dimethylformamide and phosphorus oxychloride yielding the aldehyde (28). Reduction of the aldehyde with sodium borohydride in methanol affords the corresponding benzyl alcohol. The benzyl alcohol is converted to the phthalimido derivative (29) (Bose et al., 1973, Tet. Lett. 1619) to afford the protected benzyl amine (29) (Bose et al., 1973, Tet. Lett. 1619). The phthalimide group is removed with hydrazine producing the free benzyl amine which is subsequently protected as its BOC (tert- butoxycarbonyl) derivative (30) by reaction with 2- (tert-butoxycarbonyloxyamino)-2-phenylacetonitrile. Metallation of (30) with n-butyllithium at -78°C followed by quenching with carbon dioxide produces the carboxylic acid (31). Peptide coupling with the carboxyl end of (31) followed by deprotection of the amine followed by a second peptide coupling with the amino end of (31) yields the desired peptide-like hybrid molecules.
Figure imgf000029_0001
Compounds where R3 is alkyl; Z is S, SO, or SO2; and disubstitution occurs at the 2 and 8 positions of the phenothiazine ring may be prepared according to Scheme V. 2-Chloro-phenothiazine (32) (available from Aldrich Chemical Co.) is acylated with acetic anhydride and pyridine to give (33). Freidel-Crafts acylation of ester (33) with aluminum chloride and acetyl chloride in carbon disulfide at reflux affords the N-acetyl-2-carbmethoxy-8-acetyl phenothiazine (34). The acetyl moiety of compound (34) is
selectively converted to a carboxylate with sodium hypochlorite in dioxane with concomitant removal of the N-acetyl group (Baltzy et al., 1946, J. Amer.
Chem. Soc. 68:2673) to yield the 2-carbmethoxy-8- carboxy-phenothiazine (35). Esterification of the carboxyl moiety of (35) with tert-butyl 2,2,2- trichloroacetimidate and boron trifluoride etherate produces the diester (36). N-methylation of (36) with iodomethane in dimethylsulfoxide yields the N-methyl phenothiazine (37). (37) is treated with n-butyl lithium at -78°C in tetrahydrofuran to produce a metallated intermediate which is quenched with
α-bromo-α'-phthalimido methane to give (38).
Hydrazinolysis of (38) yields the desired amino ester (39). Peptide coupling with the amino end of (39) followed by conversion of the tert-butyl ester to a carboxylic acid followed by a second peptide coupling with the carboxyl end of (39) yields the desired peptide-like hybrid molecules.
Figure imgf000031_0001
Schemes III, IV, and V, described supra , have been carried out as described. Particular compounds found in Schemes III, IV, and V have been synthesized according to the following protocols, presented by way of example:
Preparation of Compound 23
To a well stirred solution of
3-bromo-7methyl phenothiazine (1 equiv.) in 10-20 ml THF at -78°C, add η-BuLi (2.5 M solution;, 2 equiv.) slowly. After 30 min at -78°C, pass dry CO2 gas through the reaction mixture for 15-20 min. Allow the reaction to come to r.t., quench it with water.
Remove excess THF in vacuo . To the residue add a solution of sodium bicarbonate (1-2 equiv.). Wash the aqueous layer with ether (2 x 10 ml). After cooling the aqueous layer in ice, acidify with 5 N HCl to pH 2. Collect the crystals by filtration. Wash the crystals with water (10 ml). Dry in air.
Recrystallize the acid from hot alcohol.
Preparation of Compound 28
This compound was prepared following the procedure for the preparation of indole 3-aldehyde from Indole Org. Synthesis Coll. Vol. 4, 539 (1972).
To dimethylformamide (DMF) (7.5 ml) cooled in ice was added freshly distilled POCl3 (3.22 g, 21 mmol) and this Vilsmeier complex was stirred at 0°C in an atmosphere of N2 for another 30 minutes. Then the 2-bromo-10-methyl phenothiazine (3.055 g, 10.55 mmol) was added in one lot followed by 2.5 ml DMF to rinse the side of the flask. After stirring at r.t. for 1 h (by which time the solid gets wetted by DMF and assumes a purple color), the flask was filled with a reflux condenser and heated on an oil bath at 90°C for 16 h. After 16 h, the whole mixture was poured over crushed ice (~ 250 g) and stirred for 20 min. Solid ammonium acetate was then added and the pH of the aqueous layer was checked from time to time. When the pH of the solution was ~ 7-8, the aqueous layer was repeatedly extracted with dichloromethane (3 x 75 ml). The dark yellow dichloromethane phase was washed (H2O, 50 ml), dried (Na2SO4) and evaporated to leave a honey colored gum (3.58 g) which when chromatographed on a column of Silica gave yellow crystals of the
bromoaldehyde (3.26 g, 97%) which was crystallized from hexane/EtoAC. M.p. 125°C. Preparation of Compound 29
To a well stirred solution of the bromoaldehyde (28) (4 g; 0.0125 mol) in MeOH (720 ml) was added NaBH4 (400 mg) in small portions in such a way that the reaction was not too vigorous. After 45 min, the reaction mixture was stirred for a further 30 min and the MeOH was evaporated in vacuo . To the residue was added water (25 ml) and repeatedly
extracted with dichloromethane (25 ml x 5). The dichloromethane phase was washed (sat. saline, 25 ml), dried (Na2SO4) and evaporated to give a white solid, m.p. 114°C and was used as such for the next step without further purification and after drying over P2O5 for 24 h (4.011 g; ~ 98%). The next step was carried out as described (Bose et al., 1973, Tet. Lett. 1619; Bose et al., 1973, Tet. Lett. 3937).
Preparation of Compound 30
Part A
To a solution of phthalimido derivative (29) (400 mg; 0.888 mmol) in THF/EtOH (7/14 ml) was added anhydrous hydrazine (0.5 ml, a known excess) and this mix was heated to reflux on an oil bath at 90°C for 16-18 h. The THF and EtOH were removed in vacuo . To the residue was added dichloromethane (30 ml) and filtered to remove the phthaloyl hydrazide. The residue was washed twice (5 ml each) with
dichloromethane. The combined dichloromethane phase was concentrated in vacuo to give a yellow oil
(292 mg).
Part B
To a solution of the amine (300 mg; 0.9375 mmol), BOC2O (245.25 mg; 1.125 mmol), DMAP
(30 mg) in THF (10 ml) in an atmosphere of N2 with stirring was added Et3N (114.5 mg; 1.125 mmol). This reaction mixture was allowed to stir at r.t. for
16-18 h after which THF was removed in vacuo. To the residue was added water (15 ml) and repeatedly
extracted with dichloromethane (15 ml x 3). The combined dichloromethane phase was washed (H2O, 10 ml), dried (Na2SO4) and evaporated to give a pale yellow gum (552 mg) which was purified by column chromatography on Silica gel using hexane-ethyl acetate 5-7% to give a white microcrystalline powder, m.p. 172°C (306 mg; 78%).
Preparation of Compound 31
To a well cooled solution of the bromo derivative (30) (100 mg; 0.2381 mmol) in THF (5 ml) at -78°C was added η-BuLi (2.5 M in hexane; 0.19 ml;
0.4762 mmol; 2 equiv.). The solution turned yellow immediately and this was allowed to stir at -78°C for 10 min (care should be taken to keep the temperature of the anion below -40°C as this will deprotect the BOC group on the amine). Dry CO2 gas was passed through this solution carefully for 15 min and the solution was allowed to come to r.t. The reaction was guenched with crushed ice. THF was removed in vacuo . A saturated solution of NaHCO3 (5 ml) was added to the aqueous residue and washed with ether (2 x 5 ml). The aqueous layer was cooled in ice and acidified with cone. HCl to pH 2. The yellow precipitate was
collected and washed with chilled water (2 x 5 ml), giving a yellow microcrystalline powder (66.17 mg; 72%).
5.4. CHARACTERIZATION OF TAM MIMICS In a preferred although optional aspect, TAM Mimics are further characterized as described below prior to in vitro and in vivo testing of
inhibitory/therapeutic activity. The conformational preferences and membrane-penetrating properties of the TAM Mimic peptides can be investigated both
theoretically and experimentally.
Membrane-penetrating properties of a TAM
Mimic peptide can be predicted based on four
parameters (Schwyzer et al., 1986, Helvetica Chimica Acta 69:1789-1797): (i) the free energy of
hydrophobic association, (ii) the molecular
amphiphilic moment; (iii) the electric dipole of the peptide; and (iv) the net charge of the peptide.
These four factors can be combined to predict the energy of association for a peptide segment in a particular orientation with a lipid bilayer possessing known charge characteristics. By searching for the minimum energy conformation, it is possible to predict the length of helix formed on association, and its orientation with respect to the bilayer surface. This approach can be used to predict the effect of sequence modification on the lipid bilayer binding properties of a series of peptides. These predictions can be tested as described below.
The lipid-induced helix-forming potential of a TAM Mimic can be assessed by CD (circular dichroism) spectroscopy in the presence of lipid-mimicking solvents (e.g., trifluorethanol) and lipid vesicles. NMR (nuclear magnetic resonance) spectroscopy can be used to determine the three-dimensional structure of a TAM Mimic both in helix-promoting solvents and in the presence of liposomes.
NMR has been used extensively to study the three-dimensional structure of biologically active peptides in solution or bound to micelles or
phospholipid vesicles. The use of NMR to study the solution conformation of proteins and peptides is well established (Wuthrich, K., 1986, NMR of Proteins and Nucleic Acids , John Wiley & Sons, Inc.) and similar NMR analyses have been applied to a number of peptide- bilayer systems.
The membrane transport properties of TAM Mimics can be studied by size exclusion
chromatography. Lipid vesicles are prepared,
incubated with TAM Mimics, then chromatographed to separate free TAM Mimic. The vesicle contents are then assayed to determine the amount of TAM Mimic present in the lipid vesicle fraction (determinable by HPLC or amino acid analysis). A fluorescence assay for peptide transport can be used; the assay uses a water-stable fluorogenie reagent that becomes trapped inside the vesicles during formation. This
fluorogenie reagent reacts with TAM Mimic molecules that have crossed the lipid bilayer, producing a fluorescent derivative. 5.5. DEMONSTRATION OF THERAPEUTIC UTILITY
TAM Mimics are tested in vitro and then preferably in vivo for the desired therapeutic
utility.
Any in vitro assay known in the art can be used to detect inhibition by a TAM Mimic of the invention. For example, in a preferred embodiment wherein a TAM Mimic inhibitory to mast cell or
basophil activation is desired, a functional in vitro assay for mast cell or basophil degranulation is employed (see, e .g. , Section 8 infra , and Barsumian et al., 1981, Eur. J. Immunol. 11:317-323; Cunha-Melo et al., 1989, J. Immunol. 143:2617-2625). The release of histamine, the release of beta-hexosaminidase, the release of cytokines (e.g., interleukins) and/or increased phosphatidylinositol hydrolysis or tyrosine phosphorylation can be detected in in vitro assays as indications of mast cell or basophil activation (see e.g., Stephan et al., 1992, J. Biol. Chem. 267:5434). Histamine release can be assayed, for example, by commercially available radioimmunoassay (e.g., AMAC Inc., Westbrook, Maine, Cat. No. 1302). Basophilic cell lines such as RBL-2H3 (Stephan et al., supra ) , KU812 (Matsson et al., 1989, Int. Arch. Allergy Appl. Immunol. 88:122-125; Valent et al., 1990, J. Immunol. 145:1885-1889), etc. can be employed in such in vitro assays. The release of histamine from basophils is an in vitro assay of immediate hypersensitivity (Ishizaka & Ishizaka, 1975, Prog. Allergy 19:60). Other
specific examples of in vitro assays of activation of other cell types are as follows: Natural killer activation can be assayed, e . g . , by in vitro
cytotoxicity assays measuring natural killer lysis of target cells such as K562 erythromyeloid leukemia cells. Macrophage activation can be assayed by measuring macrophage phagocytic activity, induction of macrophage cytoxicity, or induction of macrophage Class II MHC cell surface expression, or by observing morphological changes associated with activation (see e . g . , Wright and Meyer, 1985, J. Exp. Med.
162:762-767; Fidalgo and Najjar, 1967, Biochemistry 6(11) :3386-3392; U.S. Patent No. 5,049,659 dated
September 17, 1991). T cell activation can be assayed by T cell proliferation in vitro, or by measuring expression of cell surface interleukin-2 receptor (IL-2R), which increases upon activation of T cells (Waldman et al., 1984, J. Exp. Med. 160:1450-1466). B cell activation can be assayed by measuring B cell proliferation in vitro .
TAM Mimics demonstrated to have the desired activity in vitro can be tested in vivo for the desired inhibitory activity. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc. Suitable model systems are also used to demonstrate therapeutic utility (see infra) .
For in vivo testing, prior to administration to humans, any animal model system known in the art may be used. For example, an animal model system for rheumatoid arthritis is that consisting of animals of the autoimmune MRL/l mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease. Rose, N.R., et al . , eds., Elsevier/North- Holland, New York, pp. 207-219), that develop a spontaneous rheumatoid arthritis-like disease (Hang et al., 1982, J. Exp. Med. 155:1690-1701). There are numerous animal models of asthma that have been developed and can be used (for reviews, see Larson, 1991, "Experimental models of reversible airway obstruction," in The Lung: Scientific Foundations , Crystal, West et al. (eds.), Raven Press, New York, pp. 953-965; Warner et al., 1990, Am. Rev. Respir.
Dis. 141:253-257). Species used as animal models for asthma include mice, rats, guinea pigs, rabbits, ponies, dogs, sheep, and primates.
Many other in vivo models are available and can be used, e . g. , as described in the following references:
Cross et al. (1990) Homing to central nervous system vasculature by antigen-specific lymphocytes. I.
Localization of 14C-labeled cells during acute,
chronic, and relapsing experimental allergic
encephalomyelitis. Lab. Invest. 63:162-170.
Kakimoto et al. (1992) The effect of anti-adhesion molecule antibody on the development of collagen- induced arthritis. Cell. Immunol. 142:326-337.
Keffer et al. (1991) Transgenic mice expression human tumour necrosis factor: A predictive genetic model of arthritis. EMBO J. 10:4025-4031. Koh et al. (1992) Less mortality but more relapses in experimental allergic encephalomyelitis in CD8-/- mice. Science 256:1210-1213.
Raine, C.S. (1991) Multiple sclerosis: a pivotal role for the T cell in lesion development. Neuropathol. Appl. Neurobiol. 17:265-274.
Shiozawa et al. (1992) Destructive arthritis without lymphocyte infiltration in H2-c-fos transgenic mice. J. Immunol. 148:3100-3104. Thorbecke et al. (1992) Involvement of endogenous tumor necrosis factor α and transforming growth factor β during induction of collagen type II arthritis in mice. Proc. Natl. Acad. Sci. USA 89:7375-7379.
5.6. THERAPEUTIC AND PROPHYLACTIC USES The TAM Mimics have therapeutic and prophylactic utility in the modulation of functions mediated by MIRRs, in particular in diseases or disorders involving the immune system or inflammation (inflammatory and immune disorders). TAM Mimics which inhibit lymphocytes are important therapeutically, because lymphocytes initiate autoimmune and alloimmune diseases.
TAM Mimics which inhibit an immune or inflammatory response and thus are useful according to the invention are most preferably identified by use of known convenient in vitro assays, e .g . , based on their ability to inhibit activation of cells of the immune system assayed in vitro , or in vivo assays (see
Section 5.5 supra) . TAM Mimics which inhibit
activation of T cells, B cells, and/or macrophages in vitro are preferred for treatment (or prevention) of immune disorders such as but not limited to autoimmune diseases and transplant rejection. TAM Mimics which inhibit activation of macrophages and/or natural killer cells in vitro are preferred for treatment (or prevention) of immune complex diseases such as but not limited to glomerulonephritis and other autoimmune diseases. TAM Mimics which inhibit activation of mast cells in vitro are preferred for treatment (or
prevention) of type I allergic (IgE-mediated)
reactions such as but not limited to asthma and allergic rhinitis. Further descriptions of diseases and disorders subject to treatment with TAM Mimics are described below.
The invention provides methods of treating or preventing diseases and disorders associated with undesirable or inappropriate immune system activity or inflammation by administration to a subject of an effective amount of a TAM Mimic of the invention.
The subject is preferably an animal,
including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
Diseases and disorders which can be treated by administration of a therapeutically effective amount of a TAM Mimic which inhibits the
inflammatory/ immune response include but are not limited to the following:
Inflammatory arthritis - e.g., rheumatoid arthritis, seronegative spondyloarthritites (Behcets disease, Reiter's syndrome, etc.), juvenile rheumatoid arthritis, vasculitis, psoriatic arthritis,
polydermatomyositis.
Systemic lupus erythematosus (SLE).
Asthma.
Inflammatory dermatoses - e.g., psoriasis, dermatitis herpetiformis, eczema, necrotizing and cutaneous vasculitis, bullous diseases.
Inflammatory bowel disease - Crohn's disease and ulcerative colitis.
Tissue damage relating to tissue
transplantation.
Other autoimmune disorders. In addition to the autoimmune disorders SLE and rheumatoid arthritis, disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e . g . , rejection of transplanted organs such as kidney, heart, pancreas, bowel, or liver), and graft-versus- host disease can be treated.
In addition, other diseases and clinical correlates of undesirable inflammatory responses can be treated with inhibitor TAM Mimics of the invention, including but not limited to those associated with hemolytic anemia, blood transfusion, certain
hematologic malignancies, scleroderma,
atherosclerosis, cytokine-induced toxicity,
necrotizing enterocolitis, granulocyte-transfusion- associated syndromes, Reynaud's syndrome, or other central nervous system inflammatory disorders.
Furthermore, in a preferred aspect of the invention, a TAM Mimic which inhibits mast cell activation is administered to treat (or prevent) a type I allergic reaction such as one or more of the following listed in Table 1 (see generally, Terr, 1987, in Basic & Clinical Immunology, 6th Ed., ch. 24, Stites et al. (eds), Appleton & Lange, Norwalk,
Connecticut, pp. 435-456).
---------------------------------------------------------------------------------------------------------------------
TABLE 1
Atopic Diseases
• allergic rhinitis (hay fever) - e.g.,
due to pollens, fungal spores, dust,
animal dander
• asthma
• atopic dermatitis
• allergic gastroenteropathy (due to
ingested food)
Anaphylaxis (a systemic immediate
hypersensitivity affecting multiple organs),
e .g . due to drugs (proteins such as in
vaccines, and nonproteins such as
antibiotics, anesthetics, salicylates),
foods, venom of stinging insects)
Urticaria or Angioedema (increased cutaneous
vascular permeability)
---------------------------------------------------------------------------------------------------------------------
5.7. THERAPEUTIC/PROPHYLACTIC
ADMINISTRATION AND COMPOSITIONS
The invention provides methods of treatment
(and prophylaxis) by administration to a subject of an
effective amount of a TAM Mimic of the invention. In
a preferred aspect, the TAM Mimic is purified. The
subject is preferably an animal, including but not
limited to animals such as cows, pigs, chickens, etc.,
and is preferably a mammal, and most preferably human.
Various delivery systems are known and can
be used to administer a TAM Mimic of the invention,
e .g. , encapsulation in liposomes, microparticles,
microcapsules, expression by recombinant cells,
receptor-mediated endocytosis (see, e .g . , Wu and Wu,
1987, J. Biol. Chem. 262:4429-4432), construction of a
TAM Mimic-encoding nucleic acid as part of a retroviral or other vector, etc. Since preferred TAM Mimics of the invention are permeable to the cell membrane, a preferred mode of delivery is via
pulmonary administration, as detailed more fully in Section 5.7.1 infra . However, methods of introduction include but are not limited to intradermal,
intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The TAM Mimics may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. For TAM Mimics which are used for inhibition of mast cell activation, e . g . , for therapy of asthma or allergy, the preferred route of
administration is nasal or via a bronchial aerosol.
In a specific embodiment, it may be desirable to administer the TAM Mimics of the
invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., for skin conditions such as
psoriasis), by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic membranes, or fibers.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically (or prophylactically) effective amount of a TAM Mimic, and a
pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the
ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
Where the composition is to be administered by
infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
The TAM Mimics of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartarlc acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
The amount of the TAM Mimic of the invention which will be effective in the treatment of a
particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the
pharmaceutical compositions of the invention.
Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. 5.7.1. PULMONARY ADMINISTRATION
In a preferred embodiment of the invention, a TAM Mimic is administered by pulmonary
administration. In particular, a bronchial aerosol is employed. This mode of administration is particularly preferred where the TAM Mimic inhibits mast cell activation and thus is useful in treating IgE-related disorders. Thus, pulmonary administration is
preferred for treatment, e.g., of allergy or asthma.
Pulmonary administration can be
accomplished, for example, using any of various delivery devices known in the art (see e.g., Newman, S.P., 1984, in Aerosols and the Lung, Clarke and Davia (eds.), Butterworths, London, England, pp. 197-224;
PCT Publication No. WO 92/16192 dated October 1, 1992; PCT Publication No. WO 91/08760 dated June 27, 1991; NTIS Patent Application 7-504-047 filed April 3, 1990 by Roosdorp and Crystal), including but not limited to nebulizers, metered dose inhalers, and powder
inhalers. Various delivery devices are commercially available and can be employed, e . g . , Ultravent
nebulizer (Mallinckrodt, Inc., St. Louis, Missouri); Acorn II nebulizer (Marquest Medical Products,
Englewood, Colorado), Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina); Spinhaler powder inhaler (Fisons Corp., Bedford,
Massachusetts). Such devices typically entail the use of formulations suitable for dispensing from such a device, in which a propellant material may be present.
Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis,"
Consensus Conference, Clinical Outcomes for Evaluation of New CF Therapies, Rockville, Maryland, December 10-11, 1992, Cystic Fibrosis Foundation).
A nebulizer may be used to produce aerosol particles, or any of various physiologically
acceptable inert gases may be used as an aerosolizing agent. Other components such as physiologically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, and diluents may also be included.
6. SYNTHESIS OF 19-MER TAM MIMICS Five different 19-mer Tarn Mimic peptides were synthesized, as described infra . The five peptides had the following sequences, and were termed respectively TAM-1, TAM-2, TAM-4, TAM-5, and TAM-6: Ac-D G V Y T G L S T R N Q E T Y E T L K-NH2 (SEQ ID
NO: 19);
Ac-Q K V Y D K L L K R N Q E T Y E T L K-NH2 (SEQ ID
NO: 20);
Ac-Q K V Y C K L L C R N Q E L Y E T L K-NH2 (SEQ ID
Figure imgf000048_0001
NO: 22);
Ac-D G V Y T G L S T R N Q K T Y K T L K-NH2 (SEQ ID NO: 23); and
Ac-N G V Y T G L S T R N Q K T Y K T L K-NH2 (SEQ ID
NO:24).
6.1. SYNTHESIS OF PEPTIDES
6.1.1. GENERAL PROCEDURES FOR SOLID PHASE SYNTHESIS
Peptides were synthesized on an Applied Biosystems Inc. ("ABI") model 431A automated peptide synthesizer using the "Fastmoc" synthesis protocol supplied by ABI, which uses 2-(1H-Benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate ("HBTU") (R. Knorr et al., 1989, Tet. Lett., 30:1927) as coupling agent. Syntheses were carried out on 0.25 mmol of commercially
available 4- (2',4'-dimethoxyphenyl-(9-fluorenyl- methoxycarbonyl)-aminomethyl)-phenoxy polystyrene resin ("Rink resin" from Advanced ChemTech) (H. Rink, 1987, Tet. Lett. 28:3787). Fmoc amino acids (1 mmol) were coupled according to the Fastmoc protocol. The following side chain protected Fmoc amino acid
derivatives were used: FmocArg(Pmc)OH;
FmocAsn(Mbh)OH; FmocAsp('Bu)OH; FmocCys(Acm)OH;
FmocGlu('Bu)OH; FmocGln (Mbh) OH; FmocHis (Tr) OH;
FmocLys(Boc)OH; FmocSer ('Bu) OH; FmocThr ('Bu) OH;
FmocTyr ('Bu)OH. [Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; 'Bu, tert-butyl; Fmoc,
9-fluorenylmethoxycarbonyl; Mbh,
4,4'-dimethoxybenzhydry1; Pmc,
2,2,5,7,8-pentamethylchroman-6-sulfonyl; Tr, trityl].
Synthesis is carried out using
N-methylpyrrolidone (NMP) as solvent, with HBTU dissolved in N,N-dimethylformamide (DMF).
Deprotection of the Fmoc group is effected using ca. 20% piperidine in NMP. At the end of each synthesis the amount of peptide present is assayed by
ultraviolet spectroscopy. A sample of dry peptide resin (ca. 3-10 mg) is weighed, then 20% piperidine in DMA (10 mL) is added. After 30 min sonication, the UV (ultraviolet) absorbance of the
dibenzofulvene-piperidine adduct (formed by cleavage of the N-terminal Fmoc group) is recorded at 301 nm.
Peptide substitution (in mmol g-1) can be calculated according to the equation:
A x v
substitution = ------------------ x 1000
7800 x w where A is the absorbance at 301 nm, v is the volume of 20% piperidine in DMA (in mL), 7800 is the
extinction coefficient (in mol-1dm3cm-1) of the
dibenzofulvene-piperidine adduct, and w is the weight of the peptide-resin sample (in mg).
Finally, the N-terminal Fmoc group is cleaved using 20% piperidine in DMA, then acetylated using acetic anhydride and pyridine in DMA. The peptide resin is thoroughly washed with DMA, CH2Cl2 and finally diethyl ether.
6.1.2. CLEAVAGE AND DEPROTECTION — GENERAL PROCEDURE
The air-dried peptide resin is treated with ethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole (PhSMe) for approximately 20 min. prior to addition of 95% aqueous trifluoracetic acid (TFA). A total volume of ca. 50 mL of these reagents are used per gram of peptide-resin. The following ratio is used: TFA : EtSMe : EDT : PhSme (10 : 0.5 : 0.5 :
0.5). The mixture is stirred for 3 h at room
temperature under an atmosphere of N2. The mixture is filtered and the resin washed with TFA (2 x 3 mL).
The combined filtrate is evaporated in vacuo , and anhydrous diethyl ether added to the yellow/orange residue. The resulting white precipitate is isolated by filtration.
6.2. TAM-1 PEPTIDE
The following 19-mer TAM Mimic peptide, termed "TAM-1", having the TAM native sequence shown was synthesized:
Ac-D G V Y T G L S T R N Q E T Y E T L K-NH2
(SEQ ID NO: 19). 6.2.1. SYNTHESIS
Synthesis was carried out according to the procedure detailed in Section 6.1.1. UV analysis following the final coupling of FmocAsp('Bu)OH to the peptide-resin revealed a substitution of 0.147 mmol g-1 (0.194 mmol g-1 is theoretically predicted, based on an initial resin substitution of 0.62 mmol g-1). The peptide-resin was subsequently acetylated (see Section 6.1.1) and 600 mg of the peptide-resin was cleaved and deprotected in accordance with the procedure described in Section 6.1.2.
6.2.2. PURIFICATION OF Ac- (TAM-1) -NH2 The crude Ac-(TAM-1)-NH2 (276 mg) was partially purified by gel filtration in 10% aqueous acetic acid on Sephadex G-10 (column 2.5 cm x 40 cm). Product-containing fractions were pooled and
lyophilized to give 152 mg material which was further purified by reversed phase HPLC on a Vydac C18 column (25 x 250 mm) to give 52 mg of >90% pure peptide.
6.3. TAM-2 PEPTIDE
The following TAM Mimic peptide, termed TAM-2, was synthesized:
Ac-Q K V Y D K L L K R N Q E T Y E T L K-NH2
(SEQ ID NO: 20). Non-consensus amino acids were changed relative to the TAM-1 peptide sequence, with a view toward achieving a higher degree of helicity.
6.3.1. SYNTHESIS
Synthesis was carried out by the methods described in Section 6.1.1. 6.4. TAM-4 PEPTIDE
The following TAM Mimic peptide, termed TAM-4, was synthesized:
Ac-Q K V Y C K L L C R N Q E L Y E T L K-NH2 (SEQ ID
Figure imgf000052_0001
NO:22). The peptide contained two cysteine residues linked via a disulfide bridge to help lock the peptide into the presumed desired conformation.
6.4.1. SYNTHESIS
Synthesis was carried out according to the methods described in Section 6.1.1, then 766 mg of the peptide-resin was cleaved and deprotected as described in Section 6.1.2 to give 385 mg of crude peptide.
6.4.2. PURIFICATION, CYCLIZATION AND
ISOLATION OF TAM-4 PEPTIDE
The crude peptide (385 mg) was initially partially purified by gel filtration on Sephadex G-10 eluted with 10% aqueous acetic acid to give 300 mg material. Peptide (73 mg) was dissolved in degassed 50% aqueous acetic acid (2.5 mL) and mercuric acetate (91 mg) in 50% aqueous acetic acid (1.0 mL) was added. The mixture was stirred under N2 for 3 h, then
β-mercaptoethanol (300 μL) was added and the mixture stirred overnight. The grey precipitate was separated by centrifugation and the supernatant desalted on a Sephadex G-10 column eluted with 10% aqueous acetic acid under an atmosphere of N2. Desired fractions were pooled and lyophilized. The peptide (62 mg) was reduced by dissolution in 6 M urea/0.2 M
tris(trihydroxymethyl)aminomethane-HCl buffer pH 8 (3 mL), followed by addition of dithiothreitol (40 mg) in the same buffer (1 mL). The mixture was stirred in the dark for 3 h under an atmosphere of N2. The sample was desalted on a Sephadex G-10 column eluted with 1 M NH4HCO3 pH 8 buffer under an atmosphere of N2. Relevant column fractions were combined, diluted to a total volume of 620 mL (i.e., 0.1 mg ml-1) with 1 M NH4HCO3 and stirred vigorously in air for 66 h. The sample was then lyophilized and subsequently desalted on Sephadex G-10 eluted with 10% aqueous AcOH to give 35 mg of product. Further purification was carried out by HPLC on a Kromasil C8 100 Å column (20 x 250 mm, eluted with a gradient of 30-50% MeCN in H2O (0.1% TFA). Purified product was 10.5 mg.
6.5. TAM-5 PEPTIDE
The following TAM Mimic peptide, termed TAM-5 was synthesized:
Ac-D G V Y T G L S T R N Q K T Y K T L K-NH2
(SEQ ID NO:23). In this sequence the two glutamate residues of the native sequence (TAM-1) are replaced by lysine.
6.5.1. SYNTHESIS
Synthesis was carried out as described in Section 6.1.1. UV analysis of the peptide-resin following coupling of FmocAsp('Bu) revealed a
substitution of 0.138 mmol g-1 (compared to 0.191 mmol g-1 predicted from an initial resin substitution of 0.62 mmol g-1). The peptide-resin was acetylated
(Section 6.1.1), then 466 mg was cleaved and
deprotected as described in Section 6.1.2 to afford 400 mg crude material. Purification of 49 mg crude peptide on reversed-phase HPLC (Kromasil C8, 100 Å, 10 μ particle size, 20 x 250 mm column) using a gradient of MeCN in H2O (0.1% TFA) gave 21 mg of the desired product. 6.6. TAM-6 PEPTIDE
The following TAM Mimic peptide, termed TAM-6 was synthesized:
Ac-N G V Y T G L S T R N Q K T Y K T L K-NH2
(SEQ ID NO:24). In this sequence the aspartate residue of the native sequence (TAM-1) is replaced by asparagine, and the two glutamate residues are
replaced by lysine; thus all acidic amino acids are substituted.
6.6.1. SYNTHESIS
Synthesis was carried out as described in Section 6.1.1. UV analysis of the peptide resin following coupling of FmocAsn(Mbh)OH revealed a substitution of 0.109 mmol g-1 (compared to a predicted value of 0.185 mmol g-1 from an initial resin
substitution of 0.62 mmol g-1). The peptide-resin was acetylated (Section 6.1.1), then 358 mg peptide-resin was cleaved and deprotected as described in Section
6.1.2 to give 199 mg of crude product. Crude peptide (153 mg) was purified by reversed-phase HPLC on a column packed with Kromasil C8, 100 Å, 10 μ particle size (20 x 250 mm) eluted with a gradient of MeCN in H2O) (0.1% TFA) to give 38 mg of the desired peptide, TAM-6.
7. SECONDARY STRUCTURE DETERMINATION OF TAM-1 The synthesized and purified TAM-1 (see Section 6.1) was subjected to nuclear magnetic
resonance (NMR) spectroscopy, which allowed the determination of its secondary structure and
appropriate templates for helix nucleation in the formation of peptide hybrids (see Section 5.2).
To carry out the NMR spectroscopy, the purified TAM-1 was prepared both as a 3 mM solution in 30% (v/v) deuterated trifluoroethanol (30% TFE) and as a 3 mM solution in the presence of deuterated
dodecylphosphatidylcholine (DPC) micelles. NMR spectra were acquired using 600 MHz NMR spectrometers (Bruker AMX600). A series of two-dimensional spectra were obtained for each sample (DQF-COSY, HOHAHA and NOESY experiments), and the NMR spectra for both samples were assigned using standard sequential assignment methodologies (Wuthrich K., 1986, NMR of Proteins and Nucleic Acids, John Wiley and Sons,
Inc.).
The NMR analysis of the secondary structure of the TAM-1 peptides was based on an analysis of the relative intensities of interproton NOE data
(Wuthrich K., 1986, NMR of Proteins and Nucleic Acids, John Wiley and Sons, Inc.) and a chemical shift index analysis (Wishart et al., 1992, Biochem. 31:1647- 1651).
For both samples of TAM-1 sequential and medium range NOE peak intensities indicated that a helical structure is present in the C-terminal portion of the molecule.
The results of the chemical shift index analysis were as follows:
Figure imgf000056_0001
An extended sequence of -1 scores in the chemical shift index analysis implies that a helical conformation is present in the peptide. The chemical shift index analysis results above are both
self-consistent and consistent with the analysis of the NOE data. Taken together the analyses imply that the peptide exists in a predominantly helical
conformation in both solvent systems. Such a helical conformation contains three turns, with the conserved Tyr and Leu residues from the motif stacked on the same side of the molecule. We propose that the face of the helix that contains the conserved amino acids is the functionally important side and that the other side of the helix can accommodate structural changes.
8. MAST CELL/BASOPHIL DEGRANULATION ASSAYS
8.1. INTACT CELL ASSAYS
The release of β-hexosaminidase from basophils is measured as an in vitro assay of basophil activation. A cell line, RBL-2H3 (from the laboratory of Dr. Siraganian, National Institutes of Health), of rat basophilic leukemia (RBL) cells is used in such an in vitro assay, according to the protocol described below (see also Barsumian et al., 1981, Eur. J.
Immunol. 11:317-323). The percentage of
β-hexosaminidase in cells that is released into the cell medium is determined, and is a measure of the activation of the RBL-2H3 cells. 8.1.1. ACTIVATION OF 2BL-2H3 CELLS
Materials
• RBL medium containing 2% fetal bovine serum (FBS) 100 ml E-MEM
2 ml FBS
1 ml glutamine
1.2 ml penicillin-streptomycin filter sterilize into sterile bottles or tissue culture flasks and store at 4°C
• 10x PIPES buffer 75.6 g PIPES (0.25 M)
69.2 g NaCl (1.2 M) 3.72 g KCl (0.05 M)
43 ml of 10 N NaOH (0.4 M)
Add water to 1000 ml filter sterilize into sterile bottles or tissue culture flasks and store at 4°C
• 100x calcium chloride
1.47 g CaCl2-2H2O (100 mM)
Add water to 100 ml filter sterilize into sterile bottles or tissue culture flasks and store at 4°C
• 2x-IgE (anti DNP-IgE mouse monoclonal
antibody; Zymed) in regular RBL medium containing 15% FBS
Need 0.5 ml at 2-times an appropriate final dilution for each 16 mm well to be seeded with cells • 1x PIPES+glucose+calcium (prepare fresh on second day of assay)
10 ml of 10X PIPES
0.1 g glucose (dextrose) (5.6 Mm)
0.1 g bovine serum albumin (BSA)
1 ml of 100x calcium chloride
Add water to 90 ml; pH to 7.4; milli-Q water to 100 ml Method
• Remove RBL cells from flasks by trypsin-EDTA treatment (see protocol infra ). Plan to use 1 x 105 cells/16 mm well to be seeded;
harvest extra cells to allow for losses during centrifugation.
• Centrifuge the RBL cells and resuspend in 5 ml of regular RBL medium containing 15% FBS. Count the cells and dilute them to 2 x 105 cells/ml in the same medium.
• For each well to be seeded, mix 0.5 ml of cells with 0.5 ml of 2x-IgE.
• Seed 1 ml of IgE-RBL per 16 mm well.
• Incubate plate(s) overnight at 37°C/5% CO2.
• Next day, dilute the DNP-HSA
(dinitrophenol-human serum albumin; Calbiochem) to an appropriate dilution in 1x PIPES+glucose+calcium; 0.5 ml will be needed for each well.
• Remove the plate (s) from the incubator and aspirate the media from the wells.
• Add 2 ml of 1X PIPES+glucose+calcium to each well and aspirate again.
• Repeat the last step. • Add the 0.5 ml of diluted DNP-BSA (or 1x PIPES+glucose+calcium alone as the negative control in triplicate) to wells.
• . Incubate 40 min at 37°C/5% CO2.
• Meanwhile, label 12 x 75 tubes (round or
conical bottom) to receive each supernatant and the cells themselves from the triplicate negative controls; also label 12 x 75 tubes for the beta-hexosaminidase assay.
• After the incubation, carefully transfer
each supernatant from the 16 mm wells to appropriate tubes on ice.
• Add 250 μl of enzyme-free cell dissociation solution (Enzyme Free Cell Dissociation Solution Hank's Balanced Salts Based
Formulation, Specialty Media Inc.,
Lavallette, NJ) to each of the triplicate negative control wells and incubate 5 min at 37°C.
• Transfer the 250 μl aliquots to conical
bottom centrifuge tubes. Add 250 μl of PIPES+glucose+calcium to rinse each well and transfer to the appropriate tubes.
• Spin tubes 5 min at 1000 rpm and remove
supernatants. Resuspend each in 500 μl of PIPES+glucose+calcium. • Sonicate cell suspensions on ice (5 pulses); if no probe sonifier is available, freeze thaw cells 3x using a methanol or ethanoldry ice bath and a 37°C water bath. • Transfer 100 μl of each supernatant or cell lysate to a 12 x 75 tube and proceed with the beta-hexosaminidase ("beta-hex") assay protocol. Protocol for Removing RBL Cells from Flasks
• Prepare trypsin/EDTA by diluting stock 1:10 in HBSS-- ; filter sterilize.
• Aspirate the medium from a flask of cells.
• Add 10 ml of HBSS-- and gently rinse the flask.
• Aspirate the HBSS--.
• Add 3 ml of trypsin/EDTA and incubate 5 min at 37°C.
• Rap the flask several times to loosen the cells.
• Transfer the 3 ml to a tube.
• Rinse flask with 10 ml of the above medium and pool to the tube.
8.1.2. BETA-HEXOSAMINIDASE ASSAY
Materials 1. Beta-hex buffer Solution 1: 0.2 M Na2PO4 (sodium phosphate dibasic ANHYDROUS) - 14.2 g/500 ml distilled H2O Note: If there is no anhydrous, use the sodium phosphate dibasic HEPTAHYDRATE (7H2O), but use 26.7 grams.
Solution 2: 0.4 M citric acid monohydrate - 42.1 g/ 500 ml distilled H2O
Mix approximately 70 ml of solution 1 with approximately 20 ml solution 2 until the pH is 4.5 (use solution 1 to raise the pH and solution 2 to lower it)
2. Beta-hex cocktail
Beta-hex buffer (from above): 90 ml Distilled H2O: 135 ml p-nitrophenyl-N-acetyl-beta-D-glucosaminide
(p-nitrophenyl-beta-D-2-acetamide-2-deoxy-beta-D- gluσopyranoside) (Sigma N-9376): 300 mg
(0.30 g); stored at -20°C
Mix until dissolved Aliquot in 15 ml tubes, label, date, and freeze at -20°C.
3. Beta-hex STOP solution glycine: 15.0 g/ liter bring to pH 10.7 with 10 N NaOH (will need to add -30 ml of NaOH)
Method
1. Place 100 μl of sample in each 12 x 75
polystyrene round-bottom tube.
2. Add 400 μl of beta-hex cocktail to each tube.
3. Cover with tin foil and incubate tubes 30 min at 37°C.
4. Add 1.5 ml of beta-hex stop to each tube.
5. Turn on the spectrophotometer and allow it to
warm up 10 minutes.
6. Turn on the vacuum pump attached to the
spectrophotometer.
7. Set the wavelength for 410 nm.
8. Aspirate any liquid in the spectrophotometer
tubing.
9. Zero the spectrophotometer using beta-hex stop solution.
10. Aspirate the liquid from the tubing; double check reading with more beta-hex stop. 11. Aspirate the liquid from the tubing; read the
absorbance at 410 nm (A410) of each sample within one hour, remembering to aspirate the sample from the tubing between each one.
8.1.3. CALCULATION OF RESULTS Determine the total beta-hex present in the RBL cells by adding the supernatant (S) and cell lysate (C) values for each of the triplicate negative controls and determine the mean [S+C].
Determine the % release for each supernatant by taking the beta-hex value and dividing by (S+C) x 100.
Determine the net % release by subtracting the % release value for the control from each % release value where DNP-BSA was added.
8.2. PERMEABILIZED CELL ASSAYS
The mast cell/basophil degranulation assay can also be performed using permeabilized cells, according to the protocol described below (see also Cunha-Melo et al., 1989, J. Immunol. 143:2617-2625; Ali et al., 1989, J. Immunol. 143:2626-2633; Ali et al., 1989, Biochim. Biophys. Acta 1010:88-99).
8.2.1. PROTOCOL FOR PERMEABILIZATION OF
RBL-2H3 CELLS WITH STREPTOLYSIN O
(1) Buffer:
Potassium glutamate 138.7 mM
Glucose 5 mM
Potassium salt of PIPES 20 mM
Magnesium Acetate 7 mM Make 1 litre of the above buffer, do not adjust pH, sterile filter and store at 4°C until use. At the time of use, remove appropriate volume and add the following:
1 M EGTA to give final concentration of 1 mM.
ATP (Sigma #A2383) to a final concentration of 5 mM. Add CaCl2 (1 M) to a final concentration of 0.213 mM.
In the presence of 1 mM EGTA the free [Ca2+] is 100 nM.
Adjust pH to 7.0.
BSA (1 mg/ml) optional.
LiCl (10 to 20 mM) for phosphoinositide hydrolysis experiments.
( 2 ) Streptolysin O
Streptolysin O reduced (Burrough's Wellcome; catalog #MR 16).
Add 4 ml H2O to 40 I.U. of streptolysin O. (10
units/ml). Make aliquots and freeze immediately.
(3) Permeabilization
RBL-2H3 cells (0.2 x 106/ well, in growth medium) are plated in 24 well tissue culture plate and incubated overnight.
The following day, cells are washed twice with
potassium glutamate (KG) buffer (500 μl).
The cells are permeabilized by exposure to
streptolysin 0 (0.1 to 0.3 units/ml, 200-500 μl/well) for 5 to 10 min. The concentration of streptolysin 0 and the time required for permeabilization depends on the number of passages the cells have been cultured for and may vary with batch of streptolysin 0. A useful starting point is to permeabilize cells with 0.25 I.U. /ml streptolysin for 10 min.
Streptolysin O solution is prepared just before permeabilization by dilution of the stock solution (10 I.U. /ml) into prewarmed KG buffer. After the cells are permeabilized, remove buffer by aspiration, add fresh buffer (without toxin) and perform experiment as desired to measure cell
activation (e.g., measure phosphoinositide hydrolysis, or β-hexosaminidase or histamine release).
Modifications of the permeabilization procedure can be made for measuring degranulation.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become
apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Weigele, Manfred
Guo, Tao
Sundaramoorthi, Raji
Dalgarno, David C.
Zydowsky, Lynne D.
Green, Jeremy
Green, Oluyinka M.
(ii) TITLE OF INVENTION: ANALOGS OF RECEPTOR TYROSINE ACTIVATION
MOTIFS AND THERAPEUTIC USES THEREOF.
(iii) NUMBER OF SEQUENCES: 30
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Pennie & Edmonds
(B) STREET: 1155 Avenue of the Americas
(C) CITY: New York
(D) STATE: New York
(E) COUNTRY: U.S.A.
(F) ZIP: 10036
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US To be assigned
(B) FILING DATE: On even date herewith
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Misrock, S. Leslie
(B) REGISTRATION NUMBER: 18,872
(C) REFERENCE/DOCKET NUMBER: 7337-034-999
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 790-9090
(B) TELEFAX: (212) 69-8864/9741
(C) TELEX: 66141 PENNIE
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 can only be D (Asp) or E (Glu)"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(D) OTHER INFORMATION: /label= note
/note= "Residue 9 can only be D (Asp) or E (Glu)" (ix) FEATURE :
(A) NAME/KEY: Modified-site
(B) LOCATION: 26
(D) OTHER INFORMATION: /label= Note
/note= "Residue 26 can only be L (Leu) or I (lle)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Leu Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa
20 25
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met Thr Leu Asn Pro Arg Ala 1 5 10 15
Pro Thr Asp Asp Asp Lys Asn Thr Tyr Leu Thr Leu Pro Pro Asn Asp
20 25 30
His Val Asn Ser Asn Asn
35
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Cys Arg Leu Lys lle Gln Val Arg Lys Ala Asp lle Ala Ser Arg Glu
1 5 10 15
Lys Ser Asp Ala Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr
20 25 30
Tyr Glu Thr Leu Lys His Glu Lys Pro Pro Gln
35 40
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Tyr Arg lle Gly Gln Glu Phe Glu Arg Ser Lyε Val Pro Asp Asp Arg 1 5 10 15
Leu Tyr Glu Glu Leu His Val Tyr Ser Pro lle Tyr Ser Ala Leu Glu
20 25 30
Asp Thr Arg Glu Ala Ser Ala Pro Val Val Ser
35 40
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Gly Gln Asp Gly Val Arg Gln Ser Arg Ala Ser Asp Lys Gln Thr Leu 1 5 10 15
Leu Pro Asn Asp Gln Leu Tyr Gin Pro Leu Lys Asp Arg Glu Asp Asp
20 25 30
Gln Tyr Ser His Leu Gln Gly Asn Gln Leu Arg Arg Asn
35 40 45
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Gly His Glu Thr Gly Arg Leu Arg Gly Ala Ala Asp Thr Gln Ala Leu 1 5 10 15
Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala
20 25 30
Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Arg
35 40 45
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid
(D ) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 1 5 10 15
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Phe 20 25 30
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ala Asp Ala Tyr Ser Asp lle Gly Thr Lys Gly Glu Arg Arg Arg Gly 1 5 10 15
Lye Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
20 25 30
Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala Pro Arg
35 40 45
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu 1 5 10 15
Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser
20 25 30
Met Tyr Glu Asp lle Ser Arg Gly Leu Gln Gly Arg Tyr Gln Asp Val
35 40 45
Gly Ser Leu Asn lle Ala Gln
50 55
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Arg Lys Arg Trp Gln Asn Glu Lys Phe Gly Val Glu Met Pro Asp Asp 1 5 10 15
Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser
20 25 30
Met Tyr Glu Asp lle Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val 35 40 45
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
Asp Lys Asp Asp Gly Lys Ala Gly Met Glu Glu Asp His Thr Tyr Glu 1 5 10 15
Gly Leu Asn lle Asp Gln Thr Ala Thr Tyr Glu Asp lle Val Thr Leu
20 25 30
Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu His Pro Gly Gln Glu
35 40 45
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Asp Lys Asp Glu Gly Lys Ala Gly Met Glu Glu Glu His Thr Tyr Glu 1 5 10 15
Gly Leu Asn lle Asp Gln Thr Ala Thr Tyr Glu Asp lle Val Thr Leu
20 25 30
Arg Thr Gly Glu Val Lys Trp Ser Val Gly Glu His Pro Gly Gln
35 40 45
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY : unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Gln Lys Val Tyr Asp Lys Leu Leu Lys Arg Asn Gln Glu Thr Tyr Glu 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Cross-links
(B) LOCATION: 5..9
(D) OTHER INFORMATION: /label= NOTE
/note= "Residue 5 and 9 are covalently bonded, which results in cyclization."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Gln Lys Val Tyr Asp Lys Leu Leu Lys Arg Asn Gln Glu Leu Tyr Glu 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE :
(A) NAME/KEY: Cross-links
(B) LOCATION: 5..9
(D) OTHER INFORMATION: /label= note
/note= "Residue 5 and 9 are covalently bonded, which results in cyclization."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Gln Lys Val Tyr Cys Lys Leu Leu Cys Arg Asn Gln Glu Leu Tyr Glu 1 5 10 15
Thr Leu Lys (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Lys Thr Tyr Lys 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Asn Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Lys Thr Tyr Lys 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 has an amine terminal acetyl group."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /label= note
/note= "Residue 19 has a carboxy terminal amide group. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu 1 5 10 15
Thr Leu Lys (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 has an amide terminal acetyl group."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /label= note
/note= "Residue 19 has a carboxy terminal amide group."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gln Lys Val Tyr Asp Lys Leu Leu Lys Arg Asn Gln Glu Thr Tyr Glu 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 has an amide terminal acetyl group."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /label= note
/note= "Residue 19 has a carboxy terminal amide group."
(ix) FEATURE :
(A) NAME/KEY: Cross-links
(B) LOCATION: 5..9
(D) OTHER INFORMATION: /label= NOTE
/note= "Residue 5 and 9 are covalently bonded, which results in cyclization."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
Gln Lys Val Tyr Asp Lys Leu Leu Lys Arg Asn Gln Glu Leu Tyr Glu 1 5 10 15 Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 has an amide terminal acetyl group."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /label= note
/note= "Residue 19 has a carboxy terminal amide group"
(ix) FEATURE:
(A) NAME/KEY: Cross-links
(B) LOCATION: 5..9
(D) OTHER INFORMATION: /label= NOTE
/note= "Residue 5 and 9 are covalently bonded, which results in cyclization."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
Gln Lys Val Tyr Cys Lys Leu Leu Cys Arg Asn Gln Glu Leu Tyr Glu 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 has an amide terminal acetyl group."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /label= note
/note= "Residue 19 has a carboxy terminal amide group."
"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Lys Thr Tyr Lys 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
( ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION: /label= note
/note= "Residue 1 has an amide terminal acetyl group."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 19
(D) OTHER INFORMATION: /label= note
/note= "Residue 19 has a carboxy terminal amide group."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Asn Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Lys Thr Tyr Lys 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
Asp Xaa Val Tyr Xaa Xaa Leu Xaa Xaa Arg Asn Gln Glu Xaa Tyr Glu 1 5 10 15
Thr Leu Lys
(2) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Glu Asp Arg Val Tyr Glu Glu Leu Asn lle Leu Ser Ala Thr Tyr Ser 1 5 10 15
Glu Leu Glu Asp Pro Gly Glu Asn
20
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Ser Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr
1 5 10 15
Glu Thr Leu Lys His Glu Lys
20
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 1 5 10 15
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg
20 25
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 1 5 10 15
Lys Met Ala Glu Ala Tyr Ser Glu lle Gly Met Lys Gly Glu
20 25 30
(2) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 amino acids
(B) TYPE: amino acid
(D ) TOPOLOGY : unknown (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 1 5 10 15
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
20 25 30
Arg

Claims

WHAT IS CLAIMED IS:
1. A peptide having an amino acid sequence in the range of 15-39 amino acids, and comprising the amino acid sequence
(D/E)XXXXXXX(D/E)XXYXXLXXXXXXXYXX(L/I) (SEQ ID NO:1), wherein X is any amino acid.
2. A peptide having an amino acid sequence in the range of 15-39 amino acids, and comprising the amino acid sequence (D/E)XXYXXLXXXXXXXYXX(L/I)X (part of SEQ ID NO:1), wherein X is any amino acid.
3. A peptide having an amino acid sequence in the range of 15-39 amino acids, and comprising an amino acid sequence selected from the group consisting of: D G G Y M T L N P R A P T D D D K N T Y L T L P (part of SEQ ID NO:2); D A V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 3); D R L Y E E L H V Y S P I Y S A L E (part of SEQ ID NO: 4) ; D Q L Y Q P L K D R E D D Q Y S H L Q (part of SEQ ID NO: 5) ; D Q V Y Q P L R D R D D A Q Y S H L G (part of SEQ ID NO: 6) ; D G L Y Q G L S T A T K D T D A L H (part of SEQ ID NO: 7, 8) ; E N L Y E G L N L D D C S M Y E D I S (part of SEQ ID
NO: 9, 10) ; D H T Y E G L N I D Q T A T Y E D I V (part of SEQ ID NO: 11); and E H T Y E G L N I D Q T A T Y E D I V (part of SEQ ID NO: 12).
4. A peptide having an amino acid sequence in the range of 15-39 amino acids, and compriεing an amino acid sequence selected from the group consisting of: D G V Y T G L S T R N Q E T Y E T L K (SEQ ID NO: 13) ; Q K V Y D K L L K R N Q E T Y E T L K (SEQ ID NO: 14) ; Q K V Y D K L L K R N Q E L Y E T L K (SEQ ID NO: 15) ; Q K V Y C K L L C R N Q E L Y E T L K (SEQ ID NO: 16); and D G V Y T G L S T R N Q K T Y K T L K (SEQ ID NO: 17); and N G V Y T G L S T R N Q K T Y K T L K (SEQ ID NO: 18).
5. A peptide having 19 amino acids, and having the following amino acid sequence: D G V Y T G L S T R N Q E T Y E T L K-NH2 (SEQ ID NO: 19).
6. A peptide having 19 amino acids, and having the following amino acid sequence: Q K V Y D K L L K R N Q E T Y E T L K-NH2 (SEQ ID NO: 20).
7. A peptide having 19 amino acids, and having the following amino acid sequence: Q K V Y D K L L K R N Q E L Y E T L K-NH2 (SEQ ID NO: 21).
8. A peptide having 19 amino acids, and having the following sequence: Q K V Y C K L L C R N Q E L Y E T L K-NH2 ( SEQ ID NO : 22 ) .
9. A peptide having 19 amino acids, and having the following sequence: D G V Y T G L S T R N Q K T Y K T L K-NH2 (SEQ ID NO: 23) .
10. A peptide having 19 amino acids, and having the following sequence: N G V Y T G L S T R N Q K T Y K T L K-NH2 (SEQ ID NO: 24).
11. The peptide of claim 5, 6, or 7 which is acetylated at its amino-terminus.
12. The peptide of claim 8, 9, or 10 which is acetylated at its amino-terminus.
13. A peptide having an amino acid sequence in the range of 15-39 amino acids, and comprising the amino acid sequence D X V Y X X L X X R N Q E X Y E T L K (SEQ ID NO:25), wherein X is any amino acid.
14. A compound of formula la or the
carboxy-terminal amide thereof:
Figure imgf000081_0001
15. A compound of formula llc
Figure imgf000081_0002
wherein X is OH or an amino-terminally linked peptide having a sequence in the range of 17-39 amino acids and comprising the sequence V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 13).
16. A compound of formula IVa:
Figure imgf000082_0001
wherein X is a hydrogen atom or a carboxy-terminally linked peptide having a sequence in the range of 17-39 amino acids and comprising the sequence V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 13).
17. A compound of the general formula V:
Figure imgf000082_0002
in which R1 is an amino acid sequence of 4-25 amino acids, having a carboxy-terminal L, and a Y in the fourth position counting from the carboxy- to aminoterminal direction; R2 is an amino acid sequence of 4-6 amino acids, having an amino-terminal Y, and an L or I in the fourth position counting from the amino- to carboxy-terminal direction; C is a hydrogen atom or an alkyl group of 1-6 carbon atoms; Z is S, SO, or SO2.
18. The compound of claim 17 which is of formula Va:
Figure imgf000083_0001
19 . The compound of claim 17 in which R3 is a hydrogen atom; Z is S ; R2 is Y E T L K-NH2 (part of SEQ ID NO : 17 ) ; and R1 is D G V Y T G L (part of SEQ ID NO : 17 ) , or Q K V Y D K L (part of SEQ ID NO : 19 ) .
20. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 1; and a pharmaceutically acceptable carrier.
21. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 2, 3, or 4; and a pharmaceutically acceptable carrier.
22. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 5, 6, or 7; and a pharmaceutically acceptable carrier.
23. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 8, 9, or 10; and a pharmaceutically acceptable carrier.
24. A pharmaceutical composition comprising a therapeutically effective amount of the peptide of claim 13; and a pharmaceutically acceptable carrier.
25. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 14; and a pharmaceutically acceptable carrier.
26. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 15 or 16; and a pharmaceutically acceptable carrier.
27. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 17; and a pharmaceutically acceptable carrier.
28. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 18 or 19; and a pharmaceutically acceptable carrier.
29. The composition of claim 20, 25, or 27 in which the pharmaceutically acceptable carrier is a propellant.
30. An inhaler containing a composition comprising the peptide of claim 1.
31. An inhaler containing a composition comprising the compound of claim 14.
32. An inhaler containing a composition comprising the compound of claim 17.
33. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the peptide of claim 1 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
34. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the peptide of claim 2 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
35. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the peptide of claim 3 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
36. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the peptide of claim 5, 6, or 7 to a subject having or suspected of having a disease or disorder associated with
undesirable or inappropriate immune system activity or inflammation.
37. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the peptide of claim 8, 9, or 10 to a subject having or suspected of having a disease or disorder associated with
undesirable or inappropriate immune system activity or inflammation.
38. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the peptide of claim 12 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
39. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the compound of claim 14 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
40. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the compound of claim 15 or 16 to a subject having or suspected of having a disease or disorder associated with
undesirable or inappropriate immune system activity or inflammation.
41. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the compound of claim 18 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
42. A method of treating or preventing a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation comprising administering a therapeutically or
prophylactically effective amount of the compound of claim 19 to a subject having or suspected of having a disease or disorder associated with undesirable or inappropriate immune system activity or inflammation.
PCT/US1994/001025 1993-01-29 1994-01-28 Analogs of receptor tyrosine activation motifs and therapeutic uses thereof WO1994017095A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60981/94A AU6098194A (en) 1993-01-29 1994-01-28 Analogs of receptor tyrosine activation motifs and therapeutic uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US1341493A 1993-01-29 1993-01-29
US013,414 1993-01-29

Publications (1)

Publication Number Publication Date
WO1994017095A1 true WO1994017095A1 (en) 1994-08-04

Family

ID=21759846

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1994/001025 WO1994017095A1 (en) 1993-01-29 1994-01-28 Analogs of receptor tyrosine activation motifs and therapeutic uses thereof

Country Status (2)

Country Link
AU (1) AU6098194A (en)
WO (1) WO1994017095A1 (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997004010A1 (en) * 1995-07-20 1997-02-06 Syntex (U.S.A) Inc. SIGNAL TRANSDUCTION AGENTS OF T-CELL RECEPTOR Vα-1 GENE LOCUS
EP0769957A1 (en) * 1995-04-13 1997-05-02 Milkhaus Laboratory, Inc. Methods for treating motor deficits
EP0804218A1 (en) * 1994-03-17 1997-11-05 National Jewish Center For Immunology And Respiratory Medicine Product and process for regulating signal transduction pathways
US5723462A (en) * 1996-04-26 1998-03-03 Neurogen Corporation Certain fused pyrrolecarboxamides a new class of GABA brain receptor ligands
US5976819A (en) * 1995-11-21 1999-11-02 National Jewish Medical And Research Center Product and process to regulate actin polymerization in T lymphocytes
US6080873A (en) * 1996-01-19 2000-06-27 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
WO2000063372A1 (en) * 1999-04-16 2000-10-26 Celltech Therapeutics Limited Synthetic signalling molecules
US6211365B1 (en) 1996-01-19 2001-04-03 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
WO2001032709A2 (en) * 1999-11-01 2001-05-10 Celltech R & D Limited Polypeptides with non-natural primary signalling motifs
US6353109B2 (en) 1996-03-22 2002-03-05 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor
US7109351B1 (en) 1999-08-31 2006-09-19 Neurogen Corporation Fused pyrrolecarboxamides; GABA brain receptor ligands
EP2145884A1 (en) * 2007-04-02 2010-01-20 Banyu Pharmaceutical Co., Ltd. Indoledione derivative
US20200190141A1 (en) * 2017-12-28 2020-06-18 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANNUAL REPORTS IN MEDICINAL CHEMISTRY, Volume 24, issued 1989, B.A. MORGAN et al., "Approaches to the Discovery of Non-Peptide Ligands for Peptide Receptors and Peptidases", pages 243-252. *
PROC. NATL. ACAD. SCI. USA, Volume 84, Number 24, issued December 1987, H. SATO et al., "Close Linkage to the Mouse and Human CD3Gamma- and Delta-Chain Genes Suggest that their Tanscription is Controlled by Common Regulatory Elements", pages 9131-9134. *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804218A1 (en) * 1994-03-17 1997-11-05 National Jewish Center For Immunology And Respiratory Medicine Product and process for regulating signal transduction pathways
EP0804218A4 (en) * 1994-03-17 1997-11-12
EP0769957A4 (en) * 1995-04-13 1999-11-03 Milkhaus Lab Inc Methods for treating motor deficits
EP0769957A1 (en) * 1995-04-13 1997-05-02 Milkhaus Laboratory, Inc. Methods for treating motor deficits
WO1997004010A1 (en) * 1995-07-20 1997-02-06 Syntex (U.S.A) Inc. SIGNAL TRANSDUCTION AGENTS OF T-CELL RECEPTOR Vα-1 GENE LOCUS
US5976819A (en) * 1995-11-21 1999-11-02 National Jewish Medical And Research Center Product and process to regulate actin polymerization in T lymphocytes
US6211365B1 (en) 1996-01-19 2001-04-03 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US6080873A (en) * 1996-01-19 2000-06-27 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US6720339B2 (en) 1996-03-22 2004-04-13 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US6353109B2 (en) 1996-03-22 2002-03-05 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor
US6096887A (en) * 1996-04-26 2000-08-01 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US5723462A (en) * 1996-04-26 1998-03-03 Neurogen Corporation Certain fused pyrrolecarboxamides a new class of GABA brain receptor ligands
WO2000063372A1 (en) * 1999-04-16 2000-10-26 Celltech Therapeutics Limited Synthetic signalling molecules
US7109351B1 (en) 1999-08-31 2006-09-19 Neurogen Corporation Fused pyrrolecarboxamides; GABA brain receptor ligands
WO2001032709A2 (en) * 1999-11-01 2001-05-10 Celltech R & D Limited Polypeptides with non-natural primary signalling motifs
WO2001032709A3 (en) * 1999-11-01 2002-05-10 Celltech R&D Ltd Polypeptides with non-natural primary signalling motifs
EP2145884A1 (en) * 2007-04-02 2010-01-20 Banyu Pharmaceutical Co., Ltd. Indoledione derivative
EP2145884A4 (en) * 2007-04-02 2010-11-03 Banyu Pharma Co Ltd Indoledione derivative
US8106086B2 (en) 2007-04-02 2012-01-31 Msd K.K. Indoledione derivative
US20200190141A1 (en) * 2017-12-28 2020-06-18 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same
AU2018353934B2 (en) * 2017-12-28 2021-04-01 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same
EP3708574A4 (en) * 2017-12-28 2021-11-03 Avixgen Inc. Peptide for inhibiting skin inflammation and composition containing same for prevention or treatment of skin inflammation
US11208433B2 (en) 2017-12-28 2021-12-28 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same

Also Published As

Publication number Publication date
AU6098194A (en) 1994-08-15

Similar Documents

Publication Publication Date Title
JP3248584B2 (en) Fibronectin adhesion inhibitor
US5668110A (en) Peptides and compounds that bind to the IL-5 receptor
US5654276A (en) Peptides and compounds that bind to the IL-5 receptor
US5677280A (en) Peptides and compounds that bind to the IL-5 receptor
US5683983A (en) Peptides and compounds that bind to the IL-5 receptor
US20040087013A1 (en) Stable macroscopic membranes formed by self-assembly of amphiphilic peptides and uses therefor
JP2002514053A (en) Peptide inhibitor of nuclear protein transport having nuclear localization sequence and method of using the same
WO1994017095A1 (en) Analogs of receptor tyrosine activation motifs and therapeutic uses thereof
JP2000506166A (en) Receptor-derived peptides involved in modulating the response to ligand binding
JPH09509944A (en) Products and methods for T cell regulation
JP2000516470A (en) Soluble monovalent and multivalent MHC class II fusion proteins and uses thereof
JPH07508025A (en) Insulin-like growth factor (IGF-1) analogs
JPH03504013A (en) Peptide with T cell helper activity
CA2153228A1 (en) Peptide inhibitors of cell adhesion
JPH11500100A (en) Pseudopeptide and non-peptide bradykinin receptor antagonists
JP3468528B2 (en) Peptide derivative
US6057294A (en) Peptide
JPH04500905A (en) DNA sequence, recombinant DNA molecule, and method for producing PI-linked lymphocyte function-related antigen-3
US4223016A (en) Peptides
US5645837A (en) Peptides that inhibit T cell activation and methods of using the same
US6455244B1 (en) Methods for the detection of antibodies associated with autoimmune disorders and infectious agents employing immunoretroid peptides derived from antigens associated with said disorders and agents
US20050032173A1 (en) Fusion proteins with a membrane translocating sequence and methods of using same to inhibit an immune response
JP2023011689A (en) Supramolecular high affinity protein-binding system for purification of biomacromolecules
JPH08503920A (en) Antithrombotic peptide and pseudopeptide derivative
AU627781B2 (en) Peptides having t cell suppressor activity

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR BY CA CN CZ FI HU JP KR KZ LK LV MN MW NO NZ PL RO RU SD SK UA UZ

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

NENP Non-entry into the national phase

Ref country code: CA