WO2006060172A2 - Procedes permettant d'identifier des composes suppresseurs de rejets de greffe - Google Patents

Procedes permettant d'identifier des composes suppresseurs de rejets de greffe Download PDF

Info

Publication number
WO2006060172A2
WO2006060172A2 PCT/US2005/041703 US2005041703W WO2006060172A2 WO 2006060172 A2 WO2006060172 A2 WO 2006060172A2 US 2005041703 W US2005041703 W US 2005041703W WO 2006060172 A2 WO2006060172 A2 WO 2006060172A2
Authority
WO
WIPO (PCT)
Prior art keywords
pkcβ
graft
rejection
subject
graft rejection
Prior art date
Application number
PCT/US2005/041703
Other languages
English (en)
Other versions
WO2006060172A3 (fr
Inventor
Karsten Sauer
Jie Li
Original Assignee
Irm Llc
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 Irm Llc filed Critical Irm Llc
Publication of WO2006060172A2 publication Critical patent/WO2006060172A2/fr
Publication of WO2006060172A3 publication Critical patent/WO2006060172A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)

Definitions

  • the present invention generally relates to methods of identifying compounds for treating or preventing graft rejection. More particularly, the invention pertains to methods of screening test compounds for PKC ⁇ inhibitors and to methods of using such inhibitors to suppress or prevent graft rejection in human or non-human subjects.
  • the immune system is well equipped to rapidly identify foreign, diseased or inflamed tissue and rapidly destroys it. This has always been a major barrier to tissue, organ and cell transplantation as well as gene therapy. Graft rejection occurs when the T lymphocytes from the recipient recognize and respond to donor histocompatibility antigens which are present on the surface of donor-derived graft cells and tissues. Graft destruction which occurs within the first few weeks after transplantation is called "acute rejection". Usually, the use of immunosuppressive drugs temporarily prevents this result. Unfortunately, the grafts may eventually be destroyed weeks or months later. This failure of permanent graft acceptance is referred to as "chronic rejection.”
  • the present invention provides methods for identifying agents that prevent or suppress allogeneic graft rejection.
  • the methods entail assaying test compounds for ability to modulate a biological activity or expression of PKC ⁇ . Some of these methods further comprise testing the identified agents for ability to suppress allogeneic graft rejection in a test subject. In some methods, the test subject has received an allogeneic graft with minor histocompatibility antigen mismatches. In some of the screening methods, the identified agents inhibit the biological activity of PKC ⁇ .
  • the biological activity of PKC ⁇ can be its kinase activity. In some of the methods, the identified agents inhibit expression of PKC ⁇ .
  • the invention provides methods for identifying agents that suppress or prevent allogeneic graft rejection. These methods involve (a) assaying a biological activity or expression of PKC ⁇ , or a fragment thereof, in the presence of test compounds to identify one or more modulating agents that modulate the biological activity or expression of the PKC ⁇ molecule; and (b) testing the identified modulating agents for ability to suppress graft rejection. In some of these methods, (b) comprises examining the identified modulating agent for ability to suppress graft rejection in a test subject. In some of the methods, the test subject has received an allogeneic graft with minor histocompatibility antigen mismatches.
  • the PKC ⁇ molecule employed can be a human PCK ⁇ or a murine PKC ⁇ .
  • the identified modulating agents inhibit the biological activity of PKC ⁇ .
  • the biological activity of PKC ⁇ modulated in these methods can be its kinase activity.
  • the identified modulating agents inhibit expression of PKC ⁇ .
  • the invention provides methods for suppressing or preventing graft rejection in a subject. The methods involve administering to the subject a pharmaceutical composition comprising an effective amount of a PKC ⁇ -antagonist. Some of these methods are directed to treating human subjects.
  • the PKC ⁇ antagonist is identified by screening test compounds for ability to inhibit the kinase activity of human PKC ⁇ .
  • the PKC ⁇ antagonist is a known PKC ⁇ inhibitor, e.g., LY333531 or LY379196.
  • Some of the methods are directed to treating subjects with a graft containing minor histocompatibility antigen mismatches. In some of these methods, the subjects have a skin graft.
  • Figures 1A-1C show rejection of skin allografts harboring minor histocompatibility antigen mismatches in PKC ⁇ -deficient and control mice. '
  • GE graft epidermis
  • RD recipient
  • Figures 2A-2B show serum immunoglobulin isotype levels in PKC ⁇ -deficient and control mice.
  • the invention is predicated in part on the discovery by the present inventors of a significant delay of graft rejection in mice lacking protein kinase C, beta isoform (PKC ⁇ ). Specifically, compared to control mice, PKC ⁇ -deficient mice have delayed rejection of minor histocompatibility antigen disparate allografts. In addition, the present inventors also found that the delayed rejection is accompanied by a drastic reduction in IgM and T H I -dependent immunoglobulin isotypes.
  • the present invention provides methods for screening for novel compounds that prevent or suppress graft rejection in transplantation.
  • Test compounds are first examined for their ability to modulate a biological activity of a PKC ⁇ molecule, e.g., its kinase activity.
  • the agents thus identified are then further tested for ability to suppress graft rejection in a test subject.
  • PKC ⁇ molecules can be employed in the screening assays.
  • PKC ⁇ from human, rat or mouse can be used to screen the modulators.
  • a human PKC ⁇ is used.
  • the approach entails administering to a subject in need of treatment a known PKC ⁇ antagonist or one that can be identified in accordance with the present invention.
  • Pharmacological inhibition of PKC ⁇ provides a novel approach for preventing or suppressing various types of graft rejections human and non-human subjects.
  • subjects receiving allogeneic grafts that contain minor histocompatibility mismatches are suitable for treatment with PKC ⁇ -mhibiting compounds.
  • the compounds likely suppress graft rejections by inhibiting T cell activation and T cell mediated immune responses, they are also useful for the treatment of other inflammatory disorders mediated by T cells. Examples include but are not limited to Rheumatoid Arthritis, Diabetes, SLE, Neurodegenerative Disorders, Psoriasis, Dermatitis, Allergy, Asthma, COPD and Anaphylaxis.
  • agent includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms “agent”, “substance”, and “compound” can be used interchangeably.
  • analog is used herein to refer to a molecule that structurally resembles a reference molecule but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs, to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.
  • contacting has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small molecule compounds) or combining agents and cells (e.g., a polypeptide and a cell).
  • agents e.g., polypeptides or small molecule compounds
  • cells e.g., a polypeptide and a cell.
  • Contacting can occur in vitro, e.g., combining two or more agents or combining a test agent and a cell or a cell lysate in a test tube or other container.
  • Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
  • nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology.
  • sequence similarity percentages e.g., BLASTP and BLASTN using default parameters
  • a "host cell,” as used herein, refers to a prokaryotic or eukaryotic cell that contains heterologous DNA that has been introduced into the cell by any means, e.g., electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • sequence identity in the context of two nucleic acid sequences or amino acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
  • the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 95% or 99% or more identical to a reference polypeptide, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters.
  • nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more identical to a reference nucleic acid, as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters.
  • nucleic acid or amino acid sequences means that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, preferably at least 95%, more preferably at least 98% and most preferably at least 99%, compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions.
  • modulate with respect to biological activities of a PKC ⁇ molecule refers to a change in the cellular level or other biological activities (e.g., its kinase activity) of PKC ⁇ .
  • Modulation of PKC ⁇ activities can be up-regulation (i.e., activation or stimulation) or down-regulation (i.e. inhibition or suppression).
  • modulation may cause a change in cellular level of PKC ⁇ , enzymatic modification (e.g., phosphorylation) of PKC ⁇ , binding characteristics (e.g., binding to a substrate or ATP), or any other biological, functional, or immunological properties of PKC ⁇ proteins.
  • the change in activity can arise from, for example, an increase or decrease in expression of the PKC ⁇ gene, the stability of mRNA that encodes the PKC ⁇ protein, translation efficiency, or from a change in other bioactivities of the PKC ⁇ enzymes (e.g., its kinase activity).
  • the mode of action of a PKC ⁇ modulator can be direct, e.g., through binding to the PKC ⁇ protein or to a gene encoding the PKC ⁇ protein.
  • the change can also be indirect, e.g., through binding to and/or modifying (e.g., enzymatically) another molecule which otherwise modulates PKC ⁇ .
  • operably linked refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a PKC ⁇ promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
  • a polylinker provides a convenient location for inserting coding sequences so the genes are operably linked to the PKC ⁇ promoter.
  • Polylinkers are polynucleotide sequences that comprise a series of three or more closely spaced restriction endonuclease recognition sequences.
  • test subject includes mammals, especially humans. It also encompasses other non-human animals such as cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.
  • a test subject is typically a non-human animal that expresses an endogenous PKC ⁇ gene, e.g., a mouse.
  • a transcription regulatory element or sequence include, but is not limited to, a promoter sequence (e.g., the TATA box), an enhancer element, a signal sequence, or an array of transcription factor binding sites. It controls or regulates transcription of a gene operably linked to it.
  • a "variant" of a molecule such as a PKC ⁇ is meant to refer to a molecule substantially similar in structure and biological activity to either the entire molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.
  • PKC ⁇ is an isoform of the PKC family of the serine/threonine protein kinases.
  • PKC kinase family are structurally and enzymatically distinct isoforms. They are divided into the classical ⁇ , ⁇ ( ⁇ l and ⁇ ll) and ⁇ subfamilies, the novel ⁇ , ⁇ , ⁇ and ⁇ subfamilies, and the atypical ⁇ and ⁇ /i subfamilies. Most of the known PKCs are expressed in T lineage cells where they function as sensors of lipid second messengers and/or calcium ion concentration changes caused by engagement of the TCR and its coreceptors.
  • the present invention provides methods of using PKC ⁇ to screen for compounds that suppress graft rejection in human or non-human subjects, especially rejection of grafts with minor histocompatibility antigen mismatches.
  • inhibition of biological activities or cellular level of PKC ⁇ s can lead to suppression of graft rejection.
  • test compounds are first screened for ability to modulate a biological activity of a PKC ⁇ .
  • the biological activity of PKC ⁇ to be monitored in the screening assays is its kinase activity.
  • the biological activity of PKCB to be monitored can also be its expression or its cellular level, as well as a specific binding of PKC ⁇ to a test compound.
  • test compounds that modulate a biological activity of PKC ⁇ After test compounds that modulate a biological activity of PKC ⁇ have been identified, they are typically further examined for ability to suppress or prevent graft rejection in a test subject (e.g., a mouse). This step serves to confirm that by modulating the biological activity of PKC ⁇ , compounds identified in the first step are indeed useful to treat or prevent graft rejection.
  • the compounds can also be examined for ability to modulate other members of the PKC kinase family (e.g., PKC ⁇ , PKC ⁇ , or PKC ⁇ ).
  • compounds that show selective modulation (e.g., inhibition) of PKC ⁇ over the other PKC isoforms are employed.
  • PKC ⁇ - inhibiting compounds which can also inhibit one or more of the other PKC isoforms are used because such compounds could have higher therapeutic efficacy in treating certain diseases and conditions.
  • PKC ⁇ from various species can be employed in screening the PKCB modulators of the present invention. These include PKC ⁇ encoded by polynucleotides with accession numbers BC036472, NM_212535 and NM_002738 (human); NM_012713 (rat); and NM_008855 and BC038148 (mouse). Examples of PKC ⁇ from other species are encoded by polynucleotides with accession numbers NM_200978, BC055154, NM_174587, XM_414868, and AY393849. Any of these PKC ⁇ polynucleotide sequences and their corresponding polypeptides can be used to screen test agents for modulators in the present invention.
  • a human PKC ⁇ molecule is used.
  • Polynucleotide and polypeptide sequences encoding the other PKC isoforms from various species are also know in the art.
  • Molecular structures and biochemical functions of these PKC molecules have all been well characterized in the art, e.g., Coussens et al., Science 233: 859-866, 1986; Kubo et al, FEBS Lett. 223: 138-142, 1987; Greenham et al., Hum. Genet. 103: 483-487, 1998; Feng et al., J. Biol. Chem. 275: 17024-17034, 2000; Ventura et al., Crit Rev Eukaryot Gene Expr. 11 : 243-67, 2001; and Spitaler et al., Nat Immunol. 5:785-90, 2004.
  • a PKC ⁇ fragment, analog, or a functional derivative can also be used.
  • the PKC ⁇ fragments that can be employed in these assays usually retain one or more of the biological activities of the PKC ⁇ molecule (typically, its kinase activity).
  • PKC ⁇ s from the various species have already been sequenced and well characterized. Therefore, their fragments, analogs, derivatives, or fusion proteins can be easily obtained using methods well known in the art.
  • a functional derivative of a PKC ⁇ can be prepared from a naturally occurring or recombinantly expressed protein by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art.
  • the functional derivative can be produced by recombinant DNA technology by expressing only fragments of a PKC ⁇ that retain its kinase activity.
  • Test agents that can be screened with methods of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines, oligocarbamates, polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Some test agents are synthetic molecules, and others natural molecules.
  • Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • Combinatorial libraries can be produced for many types of compound that can be synthesized in a step-by-step fashion.
  • Large combinatorial libraries of compounds can be constructed by the encoded synthetic libraries (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642.
  • Peptide libraries can also be generated by phage display methods (see, e.g., Devlin, WO 91/18980).
  • Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field.
  • Known pharmacological agents can be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • Combinatorial libraries of peptides or other compounds can be fully randomized, with no sequence preferences or constants at any position.
  • the library can be biased, i.e., some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines.
  • the test agents can be naturally occurring proteins or their fragments. Such test agents can be obtained from a natural source, e.g., a cell or tissue lysate. Libraries of polypeptide agents can also be prepared, e.g., from a cDNA library commercially available or generated with routine methods.
  • the test agents can also be peptides, e.g., peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides can be digests of naturally occurring proteins, random peptides, or "biased" random peptides. In some methods, the test agents are polypeptides or proteins.
  • the test agents can also be nucleic acids.
  • Nucleic acid test agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be similarly used as described above for proteins.
  • the test agents are small molecules (e.g., molecules with a molecular weight of not more than about 500 or 1,000).
  • high throughput assays are adapted and used to screen for such small molecules.
  • combinatorial libraries of small molecule test agents as described above can be readily employed to screen for small molecule modulators of PKC ⁇ s.
  • a number of assays are available for such screening, e.g., as described in Schultz (1998) Bioorg Med Chem Lett 8:2409-2414; Weller (1997) MoI Divers. 3:61-70; Fernandes (1998) Curr Opin Chem Biol 2:597-603; and Sittampalam (1997) Curr Opin Chem Biol 1 :384-91.
  • Libraries of test agents to be screened with the claimed methods can also be generated based on structural studies of a PKC ⁇ polypeptide, their fragments or analogs. Such structural studies allow the identification of test agents that are more likely to bind to a PKC ⁇ polypeptide.
  • the three-dimensional structure of a PKC ⁇ polypeptide can be studied in a number of ways, e.g., crystal structure and molecular modeling. Methods of studying protein structures using x-ray crystallography are well known in the literature. See Physical Bio-chemistry, Van Holde, K. E. (Prentice-Hall, New Jersey 1971), pp. 221-239, and Physical Chemistry with Applications to the Life Sciences, D. Eisenberg & D. C.
  • Modulators of the present invention also include antibodies that specifically bind to a PKC ⁇ polypeptide.
  • Such antibodies can be monoclonal or polyclonal.
  • Such antibodies can be generated using methods well known in the art. For example, the production of non-human monoclonal antibodies, e.g., murine or rat, can be accomplished by, for example, immunizing the animal with a PKC ⁇ polypeptide or its fragment (See Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York).
  • Such an immunogen can be obtained from a natural source, by peptides synthesis or by recombinant expression.
  • Humanized forms of mouse antibodies can be generated by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029-10033 (1989) and WO 90/07861. Human antibodies can be obtained using phage-display methods. See, e.g., Dower et al., WO 91/17271; McCafferty et al., WO 92/01047. In these methods, libraries of phage are produced in which members display different antibodies on their outer surfaces. Antibodies are usually displayed as Fv or Fab fragments. Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a PKC ⁇ polypeptide of the present invention.
  • Human antibodies against a PKC ⁇ polypeptide can also be produced from non-human transgenic mammals having transgenes encoding at least a segment of the human immunoglobulin locus and an inactivated endogenous immunoglobulin locus. See, e.g., Lonberg et al., WO93/12227 (1993); Kucherlapati, WO 91/10741 (1991). Human antibodies can be selected by competitive binding experiments, or otherwise, to have the same epitope specificity as a particular mouse antibody. Such antibodies are particularly likely to share the useful functional properties of the mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be concentrated by affinity purification using a PKC ⁇ polypeptide or its fragment.
  • test compounds are first screened for ability to modulate a biological activity of PKC ⁇ as described herein.
  • test compounds are examined for ability to modulate (e.g., inhibit) the kinase activity of PKC ⁇ .
  • Many assays are known in the art which can be used to monitor the kinase activity of PKC ⁇ in the presence of test compounds. These include both cell based assays and non-cell based assays.
  • PKC activity can be measured with commercially available assays kits, e.g., the PKC Enzyme Assay Kits from PanVera/Invitrogen (Carlsbad, California) or Amersham (Bucks, UK).
  • test compounds can be first screened for their ability to bind to a PKC ⁇ polypeptide.
  • Compounds thus identified can be further subject to assay for ability to modulate (e.g., to inhibit) PKC ⁇ kinase activity as described above.
  • Binding of test agents to a PKC ⁇ polypeptide can be assayed by a number of methods including, e.g., labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like. See, e.g., U.S.
  • test agent can be identified by detecting a direct binding to the PKC ⁇ polypeptide, e.g., co- immunoprecipitation with the PKC ⁇ polypeptide by an antibody directed to the PKC ⁇ polypeptide.
  • the test agent can also be identified by detecting a signal that indicates that the agent binds to the PKC ⁇ polypeptide, e.g., fluorescence quenching.
  • test agents are assayed for activity to modulate cellular level of PKC ⁇ , e.g., transcription or translation.
  • the test agent can also be assayed for activities in modulating expression level or stability of the PKC ⁇ polypeptide, e.g., post- translational modification or proteolysis.
  • Various biochemical and molecular biology techniques well known in the art can be employed to practice the present invention. Such techniques are described in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.
  • endogenous levels of a PKC ⁇ can be directly monitored in cells normally expressing this enzyme (e.g., T cells).
  • expression or cellular level of a PKC ⁇ can be examined in an expression system using cloned cDNA or genomic sequence encoding the PKC ⁇ .
  • modulation of expression of a PKC ⁇ gene can be examined in a cell-based system by transient or stable transfection of an expression vector into cultured cell lines.
  • Assay vectors bearing transcription regulatory sequence (e.g., promoter) of a PKC ⁇ gene operably linked to reporter genes can be transfected into any mammalian host cell line for assays of promoter activity.
  • Constructs containing a PKC ⁇ gene (or a transcription regulatory element of a PKC ⁇ gene) operably linked to a reporter gene can be prepared using only routinely practiced techniques and methods of molecular biology (see, e.g., Sambrook et al. and Ausubel et al., supra).
  • reporter genes typically encode polypeptides with an easily assayed enzymatic activity that is naturally absent from the host cell.
  • Typical reporter polypeptides for eukaryotic promoters include, e.g., chloramphenicol acetyltransferase (CAT), firefly or Renilla luciferase, beta-galactosidase, beta-glucuronidase, alkaline phosphatase, and green fluorescent protein (GFP).
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • a PKC ⁇ modulator described above can be further examined to confirm its ability to suppress or prevent graft rejection. This typically involves testing the compounds for ability to suppress or delay graft rejection in a test subject that has received an allogeneic graft.
  • non-human animals to study transplantation and graft rejection is a technique that has long been practiced in the art.
  • General guidance on various techniques of employing small animals (e.g., rats, mice, and rabbits) in transplantation study is provided in Experimental Transplantation Models in Small Animals, Green et al. (Eds.), published by T&F STM (March 1995); Pathology and Immunology of Transplantation and Rejection, Thiru et al.
  • mice were used to study graft rejection, as detailed in the Examples below.
  • mice transplanted with skin grafts with minor histocompatibility antigen mismatches are used.
  • a PKC ⁇ -inhibiting compound can be administered to C57BL/6 recipient mice receiving a skin graft from C57BL/6 donor mice.
  • the donor mice can have a minor histocompatibility antigen mismatch, e.g., by having the male-specific H-Y alloantigen.
  • Administration of the compound to the test subject can take place prior to, simultaneously with, or subsequent to the transplantation.
  • the recipient mice are administered with the compound from the pregrafting stage to the end of the screening process.
  • Detailed guidance of the specific formulations, dosages, and mode of administration for administering PKC ⁇ -inhibiting compounds are provided in the sections below.
  • graft rejection in the treated mice is typically compared to control mice that have not been receiving the PKC ⁇ -inhibiting compound. If administration of a PKC ⁇ -inhibiting compound results in a significant delay or inhibition of graft rejection, it is identified as a novel agent that suppresses or prevents graft rejection.
  • the present invention finds therapeutic applications in treating and preventing graft rejection in subjects that received tissue or organ transplants.
  • compounds of the present invention can be used to suppress immune responses elicited by allogeneic transplantation.
  • the compounds can be identified using the screening methods described above.
  • various other PKC ⁇ -inhibiting agents that are known in the art can also be used in the therapeutic embodiments of the invention (See, e.g., Gordge et al., Cell Signal 6: 871-882, 1994).
  • PKC ⁇ inhibitor (£) ⁇ 13-[(dimethylamino)methyl]- 10,11,14,15-tetrahydro-4,9 : 16,21 -dimetheno- IH, 13H-dibenzo[e,/c]pyrrolo [3 ,4- /z][l,4,13]oxadiazacyclohexadecene-l,3(2H)-dione (LY333531).
  • PKC potent protein kinase C
  • a related compound, LY379196 also selectively inhibits PKC ⁇ over other PKC isoforms (Slosberg et al., MoI Carcinog. 27:166-76, 2000; and Dang et al., Biochem Pharmacol. 67:855-64, 2004).
  • Other PKC ⁇ inhibitors known in the art include, e.g., staurosporine, CGP41251, K252a, UCN-01, Go6976 ( ⁇ ofmann, FASEB J. 11 : 649-669, 1997; and Gordge et al., Cell Signal 6: 871-882, 1994).
  • a cyclo- oxygenase-2 inhibitor, SC-236 also inhibits PKC ⁇ expression and activity (Jiang et al., Oncogene 21: 6113-22, 2002).
  • the PKC ⁇ modulators of the present invention can be directly administered under sterile conditions to the subject to be treated.
  • the modulators can be administered alone or as the active ingredient of a pharmaceutical composition.
  • the PKC ⁇ -inhibitory compounds can also be used alone or in combination with other immune-modulating agents.
  • a PKC ⁇ -modulating compound of the present invention can also be used in conjunction with known immunosuppressive drugs such as cyclosporin.
  • a first PKC ⁇ - inhibiting compound is used in combination with a second PKC ⁇ inhibitor in order to suppress or prevent graft rejection to a more extensive degree than cannot be achieved when one PKC ⁇ modulator is used individually.
  • compositions of the present invention typically comprise at least one active ingredient together with one or more acceptable carriers thereof.
  • Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered ⁇ e.g., nucleic acid, protein, modulatory compounds or transduced cells), as well as by the particular method used to administer the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject.
  • This carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral, sublingual, rectal, nasal, or parenteral.
  • the PKC ⁇ modulator can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties.
  • compositions of the present invention include syrup, water, isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution, oils, glycerin, alcohols, flavoring agents, preservatives, coloring agents starches, sugars, diluents, granulating agents, lubricants, and binders, among others.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • concentration of therapeutically active compound in the formulation may vary from about 0.1-100% by weight.
  • Therapeutic formulations are prepared by any methods well known in the art of pharmacy.
  • the therapeutic formulations can be delivered by any effective means which could be used for treatment.
  • the suitable means include oral, rectal, vaginal, nasal, pulmonary administration, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) infusion into the bloodstream. They can also be administered in eye drops or topical skin application.
  • PKC ⁇ modulators of the present invention may be formulated in a variety of ways.
  • Aqueous solutions of the modulators may be encapsulated in polymeric beads, liposomes, nanoparticles or other injectable depot formulations known to those of skill in the art.
  • the nucleic acids may also be encapsulated in a viral coat.
  • the compounds of the present invention may also be administered encapsulated in liposomes.
  • the compositions depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally te ⁇ ned a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such a diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • compositions may be supplemented by active pharmaceutical ingredients, where desired.
  • Optional antibacterial, antiseptic, and antioxidant agents may also be present in the compositions where they will perform their ordinary functions.
  • the therapeutic formulations can conveniently be presented in unit dosage form and administered in a suitable therapeutic dose.
  • a suitable therapeutic dose can be determined by any of the well known methods such as clinical studies on mammalian species to determine maximum tolerable dose and on normal human subjects to determine safe dosage. Except under certain circumstances when higher dosages may be required, the preferred dosage of a PKC ⁇ modulator usually lies within the range of from about 0.001 to about 1000 mg, more usually from about 0.01 to about 500 mg per day.
  • the preferred dosage and mode of administration of a PKC ⁇ modulator can vary for different subjects, depending upon factors that can be individually reviewed by the treating physician, such as the condition or conditions to be treated, the choice of composition to be administered, including the particular PKC ⁇ modulator, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the chosen route of administration.
  • the quantity of a PKC ⁇ modulator administered is the smallest dosage which effectively and reliably prevents or minimizes the conditions of the subjects. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.
  • Example 1 Material and Methods for Skin Grafting in Mice
  • All mice used in transplant rejection experiments were maintained under specific pathogen free conditions in the GNF barrier facility.
  • C57BL/6J and BALB/cJ mice were obtained from the Jackson labs and intercrossed to generate C57BL/6xBALB/cJ Fl donor animals.
  • the PKC ⁇ -deficient mice (Leitges et al., Science 273: 788-791, 1996) used had been backcrossed onto a C57BL/6 background for more than 10 generations.
  • Skin grafting was performed as follows. Following shaving and disinfection in 70% Ethanol, 1 cm 2 (acute rejection) or 2 cm 2 (minor mismatch rejection) dorsal skin patches were removed from 10-12 weeks old female C57BL/6 or PKC ⁇ - knockout recipients under anesthesia and replaced by equally sized, shaved and disinfected abdominal donor skin patches which were obtained from freshly euthanized female (C57BL/6xBALB/c) Fl (acute rejection) or male C57BL/6 (minor mismatch rejection) donors. Grafts were fixed with 7-0 Prolene sutures and monitored daily for the first 2 weeks post grafting and thereafter weekly for another 5-8 weeks.
  • Graft condition was classified as follows: (1) ++, no signs of rejection and good hair growth; (2) +, no signs of rejection but no hair growth, some mild swelling and erythema may occur; (3) +/-, foci of necrosis and/or a reduction in graft size, significant swelling and erythema; and (4) -, full rejection. Graft size reduced to less than 20%, massive scab formation, no viable graft tissue left.
  • graft survival plots the percentage of un-rejected grafts per group was plotted versus time post grafting, ⁇ -values for statistical significance were calculated in Student's t-tests. Median survival times (MST) were calculated from the observed full rejection times for all animals of each group and are shown +/- standard deviation.
  • serum immunoglobulin isotyping 200 ⁇ l blood per mouse were obtained via retro-orbital bleeding at the indicated time points. Serum Immunoglobulin concentrations were determined via cytometric bead assay (Spherotech) using anti-isotype antibodies from Phamiingen. Heteroscedastic t-tests of statistical differences among groups were performed using Excel.
  • Histological analyses of skin biopsies taken 6 days post grafting revealed a thickened epidermis and massive mononuclear infiltration at the junction of recipient connective tissue and graft as described previously for rejection of MHC-I disparate grafts by C57BL/6 females (Kobayashi, Kawai et al. 1990) to comparable degrees in control and PKC ⁇ -deficient recipients (Fig. IB).
  • histological features reflected progressive rejection with overall similar characteristics to those described in (Kobayashi, Kawai et al. 1990), in particular epidermal thickening and a mononuclear infiltration of the dermis which peaked a few days before full rejection of a given graft was observed.
  • PKC ⁇ -deficient mice on a 129/Sv background showed strongly reduced serum levels of IgM and IgG, but relatively normal levels of other immunoglobulin isotypes compared to 129/Sv mice (Leitges et al., Science 273: 788-791, 1996).
  • serum Ig isotype levels in untreated mice and in allograft recipients 74 days post grafting found that on a C57BL/6 background, PKC ⁇ -deficient mice have strongly reduced basal levels of IgM and also the T H I- dependent isotypes IgG3 and IgG2a compared to controls (Fig. 2A). These differences became larger and much more significant in mice which had undergone allograft rejection (Fig. 2B).
  • PKC ⁇ -deficient mice on a C57BL/6 background show a significant delay in the rejection of minor histocompatibility antigen disparate allografts which is accompanied by a drastic reduction in IgM and T H I -dependent immunoblobulin isotypes compared to control mice.
  • T H I -dependent Ig isotypes were reduced is one observation which suggests that T cell function is impaired in PKC ⁇ -deficient mice and that this T cell defect could contribute to the observed delay in minor histocompatibility antigen mismatched graft rejection.

Abstract

Cette invention concerne de nouveaux procédés permettant d'identifier des agents qui suppriment ou préviennent les rejets de greffes chez un sujet. Ces agents sont identifiés par criblage de composés d'essai pour leur aptitude à moduler une activité biologique de la PKCβ (telle que son activité kinase). Les composés modulateurs de PKCβ identifiés peuvent aussi être examinés pour leur activité de suppression des rejets de greffe chez un sujet soumis au test. Cette invention concerne également des compositions pharmaceutiques renfermant ces composés. Ces compositions pharmaceutiques peuvent être administrées à des receveurs de greffes pour traiter ou prévenir les rejets de greffes, en particulier les rejets de greffes présentant des mésappariements d'histocompatibilité mineurs.
PCT/US2005/041703 2004-11-18 2005-11-16 Procedes permettant d'identifier des composes suppresseurs de rejets de greffe WO2006060172A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US62953804P 2004-11-18 2004-11-18
US60/629,538 2004-11-18

Publications (2)

Publication Number Publication Date
WO2006060172A2 true WO2006060172A2 (fr) 2006-06-08
WO2006060172A3 WO2006060172A3 (fr) 2007-10-04

Family

ID=36565522

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2005/041703 WO2006060172A2 (fr) 2004-11-18 2005-11-16 Procedes permettant d'identifier des composes suppresseurs de rejets de greffe

Country Status (1)

Country Link
WO (1) WO2006060172A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040116388A1 (en) * 1999-10-07 2004-06-17 Amgen Inc. Kinase inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040116388A1 (en) * 1999-10-07 2004-06-17 Amgen Inc. Kinase inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JIANG X.H.: 'Antisense targeting protein kinase C alpha and beta1 inhibits gastric carcinogenesis' CANCER RES. vol. 64, no. 16, 15 August 2004, pages 5787 - 5794 *

Also Published As

Publication number Publication date
WO2006060172A3 (fr) 2007-10-04

Similar Documents

Publication Publication Date Title
Ma et al. γCaMKII shuttles Ca2+/CaM to the nucleus to trigger CREB phosphorylation and gene expression
KR101413005B1 (ko) 세포막 재봉합을 조절하기 위한 조성물 및 방법
Barrios et al. Early life allergen-induced mucus overproduction requires augmented neural stimulation of pulmonary neuroendocrine cell secretion
KR102244872B1 (ko) 당쇄 관련 유전자, 및 그의 이용
US20070020673A1 (en) Model of autoimmune disease and methods for identifying agents against autoimmune disease
Maj et al. Novel insights into the distribution and functional aspects of the calcium binding protein secretagogin from studies on rat brain and primary neuronal cell culture
Shiba-Fukushima et al. A cell-based high-throughput screening identified two compounds that enhance PINK1-Parkin signaling
US20070293579A1 (en) Use of carp inhibitors for the treatment of heart diseases
JP4843487B2 (ja) 関節病を治療するためのｐａｋ阻害剤の使用
EP1896039A2 (fr) Inhibition d'epac, de phospholipase c ''epsilon'', et de phospholipase d en vue de traiter la douleur
US20070054259A1 (en) Modulation of hnRNP H and treatment of DM1
US20050250090A1 (en) Assay systems and methods for detecting molecules that interact with SK2 channels
DE69530261T3 (de) Durch Zelldichte stimulierte Protein-Tyrosin-Phosphatasen
AU2001295457A1 (en) Use of CARP inhibitors for the treatment of heart diseases
CN110812470A (zh) 用于代谢调节的方法和组合物
WO2006060172A2 (fr) Procedes permettant d'identifier des composes suppresseurs de rejets de greffe
DE60105609T2 (de) Metabolismuskontrolle mittels zusamensetzungen des humanen 2-oxoglutarat-trägers
US7595148B2 (en) Methods and compositions for modulating T lymphocytes
US20070111929A1 (en) Methods for the treatment of anxiety and for identification of anxiolytic agents
US20090047259A1 (en) Methods of Using the Calcineurin A Variant CnA-beta 1
EP1876238A1 (fr) Nouveau polypeptide et utilisation
US7122648B2 (en) Ion channel receptor and uses thereof
JP2010506921A (ja) 炎症性疾患における４−１ｂｂリガンド
EP1602666A1 (fr) Nouvelle proteine et son adn
NadaI-Ginard p10? in Rb ‘/‘Muscle Cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05851762

Country of ref document: EP

Kind code of ref document: A2