JP2000504932A - Therapeutic composition - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to molecules that can regulate apoptosis of animal cells. More specifically, the present invention provides a cell homolog of a virus-derived apoptosis-suppressing molecule useful for regulating apoptosis of animal cells. The molecules according to the invention can be used to promote or suppress cell apoptosis depending on the urgency of the therapeutic situation.

Description

DETAILED DESCRIPTION OF THE INVENTION                               Therapeutic composition   The present invention relates to molecules that can generally modulate apoptosis in animal cells. You. In particular, the present invention is derived from viruses useful for modulating animal cell apoptosis. Provide a cell homolog of an apoptosis-inhibiting molecule. The molecules contemplated by the present invention are therapeutic Can be used to promote or inhibit cell apoptosis as needed in different situations. Will be able to.   Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Sequence identification numbers for nucleotide and amino acid sequences referred to herein The numbers (SEQ ID NOs) are described in the examples below.   Throughout this specification, the term "comprising" or its Variations "including" and "including" refer to the stated element or number or elements. Or groups of numbers, but includes other elements or numbers or groups of elements or numbers. Should be understood to mean no.   Apoptosis is initiated by various stimuli, typically leading to cell-specific changes, Activation of endonucleases, which often catalyze fragmentation and condensation of chromatin, membranes Is a cell death program that causes blebbing and nuclear breakdown.   The mechanism of apoptosis is strongly conserved during evolution (Vaux et al., 1994). Year). For example, a protein similar to Bcl-2 can be used to control nematode, insect and vertebrate cells. Toxosis (Vaux et al., 1992; Alnemri et al., 19 1992), and a cysteine similar to interleukin-1β converting enzyme (ICE). Protease is C.I. elegans and required for mammalian apoptosis (Ellis and Horvitz, 1986; Miura et al., 1993; K. uida et al., 1995). These apoptotic effector proteases Is present as a precursor in many cells, but is cleaved before activation to form a tetramer (Thornberry et al., 1992; Wils on, etc. (1994; Walker et al., 1994; Munday et al., 1995) .   Many apoptosis effector proteases have been discovered and induce apoptosis Cell death stimulation and apoptotic effector mechanism Little is known about the signaling and activation pathways that bind to. An example For example, ligation of the cell surface receptor CD95 (Fas / Apo-1) Apoptosis can be induced in cell types (Nagata and Goldstein, 19 1995; Krammer et al., 1994) and CD in T-lineage cells. ICE is required for 95-induced cell death (Kuida et al., 1995) The molecular pathway leading from D95 to ICE activation is largely undetermined. CD95 The cytoplasmic domain of CD95 with a motif called FADD / MORTI (B, believed to contribute to signal transmission from CD95) oldin et al., 1995; Chinnaiyan et al., 1995) and Other death domain containing proteins such as RIP (Stager et al., 1995). It is known to associate with white matter. Unidentified downstream targets for FADD and RIP It is.   Tumor necrosis factor (TNF) can also initiate apoptosis and its receptor is CD95 (Beutler and van-Huffel, 1994). TNF- Receptor 1 (p55) is known to bind to TRADD (Hsu 1995), TNF-receptor 2 (p75) is a TNF receptor-related factor 1 And 2 (TRAFs) (Rothe et al., 19 1994), but the manner in which these proteins act has not been confirmed.   In the research leading up to the present invention, the inventors have found that the anti-apoptotic proteins of The intermediate stages of cis signaling were studied. Apoptosis protects against viruses Can be used, but probably to keep the host cell alive while the virus replicates Many viruses have genes for anti-apoptotic proteins. Anti-virus virus Some of the potosis proteins are known cellular proteins such as, for example, Bcl-2. Similar (Vaux, 1993 review). For example, baculovirus p No intracellular counterpart is known for other proteins such as 35 protein, but ICE-like Shi For example, nematodes that may act as competitive inhibitors of stain proteases And can function in heterologous systems such as mammals (Clem et al., 1991). Year; Hay et al., 1994; Rabizadeh et al., 1993; Xue and Horvitz, 1995; Bump et al., 1995).   Miller and collaborators have identified a group of proteins in the baculovirus, Named "IAP" because it can inhibit the apoptosis reaction of insect cells due to Rus infection (Birnbaum et al., 1994; Crook et al., 1993). Wooly Rus' IAP protein is typically named baculovirus IAP repeat (BIR) With two terminal repeats and a carboxy-terminal RING finger domain You. Autographa californica nuclear polyhedrosis virus (AcNPW) encodes an IAP protein that does not inhibit apoptosis, AP proteins may perform other functions, such as cytokine regulatory functions. There is a potential.   The present inventors screened a gene fragment that might encode a novel IAP protein. Intracellular IAP homologs and chemical analogues that function in the cell death pathway Derivatives were searched. A full-length cDNA clone was obtained, which was obtained by ICE and The ability to intervene in apoptosis by FADD was tested.   Accordingly, one aspect of the invention is a peptide, polypeptide or protein derived from a virus. Or homologues or chemical analogues of these derivatives, derived from this virus The molecule is capable of inhibiting the apoptotic response of cells to viral infection.   More specifically, the present invention provides for apoptotic response of cells to viral infection. Provide derivatives of isolated proteinaceous molecules or their chemical analogs that can be inhibited But its proteinaceous molecule is a cell-derived cognate of viral apoptosis inhibition (IAP) Including the body.   A virus-derived IAP is a polypeptide or a protein. This IAP is Will Proteins represent a group of sex molecules that inhibit the apoptotic response of cells to bacterial infection You. More specifically, IAPs are responsible for the protection of insect cells against infection by baculovirus. Inhibits the potentiosis reaction. The present invention therefore modulates animal cell apoptosis To provide homologues or chemical analogs of baculovirus IAP useful for: Preferably this apoptosis is ICE and FADD intervening.   Accordingly, another aspect of the present invention relates to baculovirus IAP peptides, polypeptides. Or, homologs or chemical analogs of the protein or their derivatives are contemplated.   Preferably, the homolog is an animal cell-derived molecule. Specific suitable animal cells Examples of humans, livestock animals (e.g., sheep, pigs, horses, donkeys, cows), pets ( (E.g. dogs, cats), laboratory animals (e.g. rabbits, mice, rats, guinea pigs) ), Captive wildlife (eg fox, deer, kangaroo), insects (eg mosquitoes) And from nematodes. Homologues may be from yeast or filamentous fungi.   Particularly preferred homologs are to human, mouse, insect, yeast or nematode cells is there. One example of a nematode is Caenorhabditis elegans.   In a most preferred embodiment, the mammalian homolog is derived from a mouse cell, Named herein MIHA, MIHB, MIHC and MIHE. What is this In another embodiment, the homolog is an insect homolog from Drosophila, termed DIHA. Body. In yet another aspect, the homologs herein are CIA-1 and CIA-2. C. elegans. In yet another aspect, the congener is a book In the specification, it is a homolog of yeast designated as YIA-1. However, the present invention Baculovirus IAP or MIHA, MIHB, MIHC and / or D It extends to human and other mammalian homologs of IHA homologs or chemical analogs.   According to another aspect of the present invention,   Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa   Xaa Trp Pro [Xaa] m [Xaa] n Ala Xaa Ala   Gly Phe [Xaa] o Asp Xaa [Xaa] p Xaa Cys   Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa   Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa   Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys   Xaa [Xaa] q [Xaa] r [where Xaa is an amino acid residue.   [Xaa] m is a system of at least 5, preferably at least 9, amino acids .   [Xaa] n is Met or Leu.   [Xaa] o is a system of at least 3, preferably at least 5, amino acids .   [Xaa] p is Val or Ala.   [Xaa] q is Phe or Tyr.   [Xaa] r is Leu or Val]. Or a homologue, chemical analog or derivative thereof.   More specifically, named MIHA having the amino acid sequence substantially as set forth in SEQ ID NO: 2 Isolated molecules, or their whole or part or their homologues, chemical classes An analog or derivative having at least 40% similarity is provided.   One of the aspects to which the present invention relates has substantially the amino acid sequence shown in SEQ ID NO: 4. An isolated molecule designated MIHB or its whole or part or its cognate Directed to those having at least 40% similarity to the body, chemical analog or derivative I do.   Still another aspect to which the present invention relates is an amino acid substantially as set forth in SEQ ID NO: 6. Isolated molecule named MIHC having the sequence or all or part thereof Has at least 40% similarity to its homolog, chemical analog or derivative About things.   Still another aspect to which the present invention relates is an amino acid substantially as set forth in SEQ ID NO: 8. Isolated molecule named DIHA having the sequence or its whole or part Has at least 40% similarity to its homolog, chemical analog or derivative Provide things.   Yet another aspect to which the present invention relates is an amino acid substantially as set forth in SEQ ID NO: 11. An isolated molecule named DIHA with an acid sequence or all or part thereof Or has at least 40% similarity to a homologue, chemical analog or derivative thereof Offer something.   Still another aspect to which the present invention relates is an amino acid substantially as set forth in SEQ ID NO: 13. An isolated molecule named DIHA with an acid sequence or all or part thereof Or has at least 40% similarity to a homologue, chemical analog or derivative thereof Offer something.   Yet another aspect to which the present invention relates is an amino acid substantially as set forth in SEQ ID NO: 15. An isolated molecule named DIHA with an acid sequence or all or part thereof Or has at least 40% similarity to a homologue, chemical analog or derivative thereof Offer something.   Still another aspect to which the present invention relates is an amino acid substantially as set forth in SEQ ID NO: 17. An isolated molecule named DIHA with an acid sequence or all or part thereof Or has at least 40% similarity to a homologue, chemical analog or derivative thereof Offer something.   The present invention includes, for example, MIHA, MIHB, MIHC, MIHE, CIA-1, Nucleic acids encoding IAP homologs such as CIA-2, YIA-1 and DIHA Molecule.   This IAP homologue may or may not have a RING finger domain You may. For example, MIHA, MIHB and MIHC have this domain But MIHE, CIA-1, CIA-2 or YIAY have this domain Not in.   The invention further provides:   Glu Xaa Xaa Arg Xaa Xaa Thr Phe Xaa   Xaa Trp Pro [Xaa] m [Xaa] n Ala Xaa Ala   Gly Phe [Xaa] o Asp Xaa [Xaa] p Xaa Cys   Phe Xaa Cys Xaa Xaa Xaa Leu Xaa Xaa   Trp Xaa Xaa Xaa Asp Xaa Pro Xaa Xaa   Xaa His Xaa Xaa Xaa Xaa Pro Xaa Cys   Xaa [Xaa] q [Xaa] r [where Xaa is an amino acid residue.   [Xaa] m is a system of at least 5, preferably at least 9, amino acids .   [Xaa] n is Met or Leu.   [Xaa] o is a system of at least 3, preferably at least 5, amino acids .   [Xaa] p is Val or Ala.   [Xaa] q is Phe or Tyr.   [Xaa] r is Leu or Val]. Or a nucleic acid molecule comprising a nucleotide sequence complementary to a sequence encoding an amino acid sequence Or at least 40% similarity to the nucleotide sequence encoding said amino acid sequence Is intended.   In particular, the present invention relates to a sequence substantially encoding the amino acid sequence shown in SEQ ID NO: 2 or Contains a nucleotide sequence that is substantially complementary to the sequence encoding this amino acid sequence A nucleic acid molecule or a nucleotide encoding substantially the amino acid sequence of SEQ ID NO: 2 A nucleic acid molecule having at least 40% similarity to the nucleotide sequence is contemplated.   In a related embodiment, the sequence substantially encodes the amino acid sequence shown in SEQ ID NO: 4. Or a nucleotide sequence substantially complementary to the sequence encoding this amino acid sequence. A nucleic acid molecule comprising the nucleic acid molecule substantially encoding the amino acid sequence shown in SEQ ID NO: 4 A nucleic acid molecule having at least 40% similarity to the otide sequence is provided.   In another related aspect, the amino acid sequence substantially encodes the amino acid sequence set forth in SEQ ID NO: 6. Nucleotides complementary to the sequence or to a sequence substantially encoding this amino acid sequence Encodes a nucleic acid molecule comprising the sequence or the amino acid sequence substantially as set forth in SEQ ID NO: 6 A nucleic acid molecule having at least 40% similarity in nucleotide sequence is contemplated.   In yet another aspect, a sequence substantially encoding the amino acid sequence set forth in SEQ ID NO: 8 Or a nucleotide sequence substantially complementary to the sequence encoding this amino acid sequence Or a nucleic acid encoding the amino acid sequence substantially as set forth in SEQ ID NO: 8 Directs nucleic acid molecules having at least 40% similarity to the leotide sequence.   Another related aspect relates to a sequence encoding the amino acid sequence substantially as set forth in SEQ ID NO: 11. Sequence or a nucleotide sequence substantially complementary to the sequence encoding this amino acid sequence. Encodes a nucleic acid molecule comprising the sequence or substantially the amino acid sequence set forth in SEQ ID NO: 11 Directs nucleic acid molecules with at least 40% similarity to the nucleotide sequence.   Yet another related aspect encodes the amino acid sequence substantially as set forth in SEQ ID NO: 13. To a sequence that is substantially complementary to the sequence that encodes this amino acid sequence A nucleic acid molecule comprising the peptide sequence or the amino acid sequence substantially as set forth in SEQ ID NO: 13. Directing nucleic acid molecules having at least 40% similarity to the nucleotide sequence You.   A further related aspect encodes the amino acid sequence substantially as set forth in SEQ ID NO: 15. Nucleotides complementary to the sequence or to a sequence substantially encoding this amino acid sequence Encoding a nucleic acid molecule comprising the sequence or the amino acid sequence substantially as set forth in SEQ ID NO: 15. To nucleic acid molecules having at least 40% similarity to the nucleotide sequence.   Another related aspect is a sequence encoding the amino acid sequence substantially as set forth in SEQ ID NO: 17. Sequence or a nucleotide sequence substantially complementary to the sequence encoding this amino acid sequence. Encodes a nucleic acid molecule comprising the sequence or substantially the amino acid sequence set forth in SEQ ID NO: 17 Directs nucleic acid molecules with at least 40% similarity to the nucleotide sequence.   Yet another related aspect includes a nucleotide sequence substantially as set forth in SEQ ID NO: 1. At least 40% in the nucleic acid molecule or substantially the nucleotide sequence set forth in SEQ ID NO: 1 Or similar to it under low stringency conditions Nucleic acid molecules that can be   In one aspect related to the present invention, substantially comprises the nucleotide sequence set forth in SEQ ID NO: 3. At least 40 nucleotides in the nucleic acid molecule or substantially the nucleotide sequence set forth in SEQ ID NO: 3. Or similar to it under low stringency conditions Provided are nucleic acid molecules that can be   In yet another aspect related to the present invention, the nucleotide substantially as set forth in SEQ ID NO: 5. Nucleic acid molecule containing the nucleotide sequence or substantially less nucleotide sequence as shown in SEQ ID NO: 5. It has a similarity of at least 40% or has low stringency conditions. Provide a nucleic acid molecule that can be hybridized.   In another aspect related to the invention, a nucleotide sequence substantially as set forth in SEQ ID NO: 7 A nucleic acid molecule comprising at least the nucleotide sequence substantially as set forth in SEQ ID NO: 7. Has 40% similarity or hybridizes thereto under low stringency conditions Provided is a nucleic acid molecule that can be read.   In another aspect related to the present invention, the nucleotide sequence substantially as set forth in SEQ ID NO: 10 is provided. The nucleic acid molecule comprising the sequence or substantially the nucleotide sequence set forth in SEQ ID NO: 10 Both have a similarity of 40% or are high to this under low stringency conditions. Provide a nucleic acid molecule that can be bridged.   In yet another aspect related to the present invention, the nucleotide substantially as set forth in SEQ ID NO: 12. A nucleic acid molecule comprising the nucleotide sequence or substantially the nucleotide sequence set forth in SEQ ID NO: 12. With at least 40% similarity or under low stringency conditions Provide a nucleic acid molecule capable of hybridizing to   In still yet another aspect related to the present invention, the nucleotide sequence substantially as set forth in SEQ ID NO: 14. A nucleic acid molecule comprising a reotide sequence or a nucleotide sequence substantially as set forth in SEQ ID NO: 14. Have at least 40% similarity in the columns or under low stringency conditions A nucleic acid molecule capable of hybridizing thereto is provided.   In yet another aspect related to the present invention, the nucleotide substantially as set forth in SEQ ID NO: 16. A nucleic acid molecule comprising the nucleotide sequence or substantially the nucleotide sequence set forth in SEQ ID NO: 16. Have at least 40% similarity or have low stringency conditions Provide nucleic acid molecules that can be hybridized.   At least about 1% for low stringency conditions at 42 ° C. herein v / v to at least about 15% v / v formamide and at least about 1 M to at least about 2 M of hybridizing salt and at least about 1 M to less than Contains and includes at least about 2 M of washing salt. If necessary, for example at least From about 16% v / v to at least about 30% v / v formamide and at least At least about 0.5M to at least about 0.9M of the hybridizing salt and at least Also comprises about 0.5 M to at least about 0.9 M of washing salt Stringency conditions of at least about 31% v / v to at least about Up to 50% v / v formamide and at least about 0.01 M to at least About 0.15 M of hybridizing salt and at least about 0.01 M to at least Also contain about 0.15M washing salt and contain high stringency conditions. Such other stringency conditions may be applied.   The nucleic acid molecule may be in an isolated form and / or such as an expression vector. What the vector molecule has is preferred.   The IAP homologs of the present invention and their derivatives and chemical analogs are capable of apoptosis. Useful for inhibiting, but not limited to, for example, Alz Debilitating diseases including Hymer's disease, motor neuron disease, neuropathy; seizures and myocardial infarction Ischemic vascular disease including occlusion; infections including acquired immunodeficiency syndrome caused by HIV It is useful for treating diseases characterized by such apoptosis. This specification Some derivatives of this congener are intended to promote apoptosis or Inhibits the apoptotic process, including but not limited to cancer or autoimmune disease Disease in certain cells, including Alzheimer's disease and motor neuron disease It may be useful for treating diseases characterized by insufficiency of tosis. Book Derivatives of the present homologs contemplated herein promote apoptosis or anti-apoptotic Inhibiting the cis process, for example, promoting the death of certain cancer or malignant cells Useful.   Derivatives of homologs of the present invention include variants, parts and fragments of homolog molecules, Contains one or more amino acid substitutions, deletions and / or additions of homologous molecules. No. The nucleic acid derivative is SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 10 or SEQ ID NO: 12 or SEQ ID NO: 12 Or a single or multiple copies of the nucleic acid molecule of SEQ ID NO: 14 or SEQ ID NO: 16 Includes a number of nucleotide substitutions, deletions and / or additions.   Baculovirus IAP analogs or homologs thereof include, but are not limited to, Not modified but peptides of side chain modifications, unnatural amino acids and / or derivatives thereof Introduction during synthesis and steric hindrance to crosslinkers, peptides or analogs Use of other methods.   Examples of side chain modifications according to the invention include αBH after reaction with an aldehyde._Four Of amino group by reductive alkylation, etc .; methylacetoimidide Amidination; acylation with acetic anhydride; to cyanate Carbamoylation of amino group by 2,4,6-trinitrobenzenesulfonic acid (T BS) for the trinitrobenzylation of amino groups; succinic anhydride and tetraanhydride Acylation of amino groups with hydrophthalic acid; and pyridoxal-5'-phosphorus ΑBH after treatment with acid_FourPyridoxylation of lysine to be reduced with   The guanidine group of arginine is 2,3-butanedione, phenylglyoxa The formation of heterocyclic condensation products using reagents such as toluene and glyoxal Can be modified by   Carboxyl groups are activated by carbodiimide by formation of O-acylisourea. And then, for example, by subsequent derivitisation to the corresponding amide Can be modified.   Sulfhydryl groups are carboxylated with iodoacetic acid or iodoacetamide. Methylation; Oxidation of formic acid to cysteic acid; Mixed disulfate with other thiol compounds Formation of sulfide; reaction with maleimide, maleic anhydride or other substituted maleimide 4-chloromercury benzoate, 4-chloromercury phenyl sulfone Acid, phenylmercury chloride, 2-chloromercury-4-nitropheno Of Mercury Derivatives Using Tool and Other Mercury Compounds; Cyanane at Alkaline pH It can be modified by a method such as carbamoylation with a salt.   Tryptophan can be oxidized, for example, by bromosuccinimide or (A) with droxy-5-nitrobenzyl bromide or sulfenyl halide It can be modified by alkylation of the indole ring.   The other tyrosine residue was converted to 3-nitro by nitration with tetranitromethane. It can be changed by forming a tyrosine derivative.   Modification of the imidazole ring of the histidine residue can be achieved by alkylation with an iodoacetic acid derivative. Or N-carbethoxy with diethyl pyrocarbonate Can be.   Examples of the introduction of unnatural amino acids and derivatives during peptide synthesis include: But not limited to, norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy- 5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norva Phosphorus, phenylglycine, norithine, sarcosine, 4-amino- 3-hydroxy-6-methylheptanoic acid, 2-thienylalanine and / or And D-isomers of amino acids. Table 1 shows a list of unnatural amino acids.     Table 1 Unusual amino acid code Unusual amino acid code α-Aminobutyric acid Abu LN-methylalanine Nmala α-Amino-α-methylbutyric acid Mgabu L-N-methylarginine Nmarg Aminocyclopropane-Cpro L-N-methylasparagine Nmasn Carboxylate LN-methylaspartic acid Nmasp Aminoisobutyric acid Aib L-N-methylcysteine Nmcys Amino norbornyl Norb L-N-methylglutamine Nmgln Carboxylate L-N-methylglutamic acid Nmglu Cyclohexylalanine Chexa LN-methylhistidine Nmhis Cyclopentylalanine Cpen LN-methylisoleucine Nmile D-alanine Dal LN-methylleucine Nmleu D-arginine Darg LN-methyllysine Nmlys D-aspartic acid Dasp LN-methylmethionine Nmmet D-cysteine Dcys LN-methylnorleucine Nmnle D-glutamine Dgln LN-methylnorvaline Nmnva D-glutamic acid Dglu LN-methylornithine Nmorn D-histidine Dhis LN-methylphenylalanine Nmphe D-isoleucine Dile LN-methylproline Nmpro D-leucine Dleu LN-methylserine Nmser D-Lysine Dlys LN-methylthreonine Nmthr D-methionine Dmet LN-methyltryptophan Nmtrp D-ornithine Dorn LN-methyltyrosine Nmtyr D-phenylalanine Dphe LN-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-amino-isobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrate Mgabu D-γ-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcyclopentylalanine Mcpen D-α-methyl asparagine Dmasn α-methyl-γ-naphthylalanine Manap D-α-methyl aspartate Dmasp α-methyl penicillamine Mpen D-α-methylcysteine Dmcys N- (4-aminobutyl) glycine Nglu D-α-methylglutamine Dmgln N- (2-aminoethyl) glycine Naeg D-α-methylhistidine Dmhis N- (3-aminopropyl) glycine Norn D-α-methylisoleucine Dmile N-amino-γ-methylbutyrate Nmaabu D-α-Methylleucine Dmleu α-Naphthylalanine Anap D-α-Methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionine Dmmet N- (2-carbamylethyl) glycine Ngln D-α-methylornithine Dmorn N- (carbamylmethyl) glycine Nasl D-α-methylphenylalanine Dmphe N- (2-carboxyethyl) glycine Nglu D-α-methylproline Dmpro N- (carboxymethyl) glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-Methyltryptophan Dmtrp N-Cyclohexylglycine Nchex D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec D-α-methylvaline Dmval N-cyclododecylglycine Nedod D-N-Methylalanine Dnmala N-Cyclooctylglycine Ncoct D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methyl aspartate Dnmasp N- (2,2-diphenylethyl) glycine Nbhm D-N-methylcysteine Dnmcys N- (3,3-diphenylpropyl) glycine Nbhe D-N-methylglutamine Dnmgln N- (3-guanidinopropyl) glycine Narg D-N-methylglutamate Dnmglu N- (1-hydroxyethyl) glycine Nthr D-N-methylhistidine Dnmhis N- (hydroxyethyl) glycine Nser D-N-methylisoleucine Dnmile N- (imidazolylethyl) glycine Nhis D-N-Methylleucine Dnmleu N- (3-Indolylethyl) glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N- (1-methylpropyl) glycine Nile D-N-methylserine Dnmser N- (2-methylpropyl) glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N- (1-methylethyl) glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-naphthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-Aminobutanoic acid Gabu N- (p-hydroxyphenyl) glycine Nhtyr L-t-butylglycine Tbug N- (thiomethyl) glycine Ncys L-Ethylglycine Etg Penicillamine Pen L-Homophenylalanine Hphe L-α-Methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-Methyl Aspartate Masp L-α-Methyl-t-butylglycine Mtbug L-α-Methylcysteine Mcys L-Methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-Methylhistidine Mhis L-α-Methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N- (2-methylthioethyl) glycine Nmet L-α-Methylleucine Mleu L-α-Methyllysine Mlys L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N- (N- (2,2-diphenylethyl) Nnbhm N- (N- (3,3-diphenylpropyl) Nnbhe   Carbamylmethyl) glycine 1-carboxy-1- (2,2-diphenyl Nmbc    Ethylamino) cyclopropane   In order to stabilize the 3D conformation, for example, n = 1 to n = 6 (CH_Two ) Bifunctional imide ester having a spacer group, glutaraldehyde, N- A homo-bifunctional cross-linking linker such as a droxysuccinimide ester, and N -Hydroxysuccinimide, and a maleimide or dithio moiety (SH) or Is an amino reactive moiety such as another specific reactive moiety such as carbodiimide (COOH) A crosslinking linker such as a hetero-bifunctional reagent, which typically contains Further The peptide is, for example, C_αAnd N_α-Of amino acids incorporating methylamic acid C_αAnd C_βIntroduce a double bond between atoms and introduce a covalent bond, eg, Form an amide bond between the C-terminus and between two side chains or between the side chain and the N or C-terminus To form a cyclic peptide, thereby allowing for steric compression .   The invention further provides a method of modulating cell apoptosis in an animal, the method comprising: Baculovirus IAP or a derivative thereof in an animal in a cell apoptosis regulating effective amount Homologs or chemical analogs of the same for a time and under conditions sufficient to regulate the cell apoptosis. Administration.   The term “regulatory” or “modulation” refers to promoting or increasing cell apoptosis, Or means a reduction, inhibition or reduction of cell apoptosis. Cell apoptosis Increased cell size may be important in treating certain cancers and cellular malignancies; Decreased apoptosis is associated with neuropathy and Alzheimer's disease and motor May be important in treating Ron's disease.   Accordingly, the present invention is further useful for regulating cell apoptosis in animals. For therapeutic and pharmaceutical compositions.   Pharmaceutical composition forms are generally known to those of skill in the art and, for convenience, Remington's Ph.D. armaceutical Sciences, 17th end, Mack Publising Co. , Easton, Pencil Vania and the United States can be referenced.   The present invention therefore relates to the aforementioned IAP homologs or chemical analogues or derivatives thereof. Apoptosis-modulating effective amount of one and optionally one or more other active molecules And one or more pharmaceutically acceptable carriers and / or diluents. It is intended to provide a pharmaceutical composition having IAP homologs or derivatives thereof In the present invention, the active ingredient in a pharmaceutical composition containing When administered in an amount that is superior, for example, in regulating apoptosis in animal cells. Demonstrated therapeutic activity. For example, about 0. About 5 mg to about 20 mg / kg body weight / day Can be given. Dosage regimes may be adjusted to provide the optimum therapeutic response. example For example, several doses can be administered daily, weekly or monthly, or if the treatment The dose can be reduced to a fixed rate in an acute manner. Active compound, oral, intravenous Intravenous (if water-soluble), intramuscular, intradermal, intranasal, subcutaneous or suppository route, or transfer Implantation (eg using sustained release molecules), topical administration or post- or intra-operative or other The administration can be made by any convenient method such as a blood technique. Depending on the route of administration , An active ingredient comprising an IAP homolog or a chemical analog or derivative thereof is an enzyme To protect the component from action, acids and other natural conditions that may deactivate the component May need to be coated. For administration methods other than parenteral administration Thus, to administer an IAP homolog, a substance that prevents inactivation of the component may be used. Or with the substance. For example, homologues in adjuvants Can be administered or co-administered with enzyme inhibitors or administered in liposomes it can. Adjuvants contemplated by the present invention include cytokines (eg, Feron), and resorcinols, nonionic surfactants such as polyoxy Sethylene oleyl ester and n-hexadecyl polyethylene ether But not limited to them.   The active compounds according to the invention can also be administered parenterally or intraperitoneally. Dispersant is also grease Serol, liquid polyethylene glycol and their mixtures and prepared in oils Can be manufactured. Under ordinary conditions of storage and use, these preparations will reduce the growth of microorganisms. Contains suppressive preservatives.   Pharmaceutical forms suitable for injection include sterile aqueous solutions (where water soluble) or dispersants And sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. these In all cases, the formulation must be sterile and can be easily syringed It must be as liquid as possible. It is not stable during formulation and storage. Must be protected from the working contamination of microorganisms such as bacteria and fungi. No. Carriers include, for example, water, ethanol, polyol (eg, glycerol) , Propylene glycol and liquid polyethylene glycol). The mixture can be a solvent or dispersion containing the vegetable oil. Suitable Appropriate fluidity, for example, using a coating such as lecithin, in the case of a dispersant, Maintains the required particle size and can be maintained by using surfactants it can. Prevention of the action of microorganisms includes various antibacterial and antifungal agents, for example, parabens. , Chlorobutanol, phenol, sorbic acid, thermerosal, etc. I can. Often contains tonicity agents, such as sugars or sodium chloride Preferably. Prolonged adsorption of injectable compositions can be caused by substances that delay adsorption, for example By using in a composition of aluminum monostearate and gelatin It can be carried out.   The sterile injectable solution is activated if necessary in a suitable solvent containing various other components as described above. It is prepared by putting the required amount of the compound, followed by sterile filtration. Generally, Dispersants can be added to a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. It is prepared by incorporating the various sterilized active ingredients. Prepare sterile injectable solution In the case of sterile powders for preparation, the preferred preparation methods are vacuum drying and freeze drying. This allows the active ingredient and any other desired components to be removed from the previously sterile filtered solution. A powder of the component is obtained.   IAP homologs or chemical analogs or derivatives were protected as described above as appropriate. In this case, the active compound is orally administered, for example, with an inert diluent or an ingestible edible carrier. Or it can be filled into hard or soft gelatin capsules, Alternatively, they may be compressed into tablets or included directly in the diet. For oral therapeutic administration, the active compound may be included with excipients, Tablets, troches, capsules, elixirs, suspensions, syrups, wa It can be used in a dosage form such as a herbal preparation. Such compositions and preparations are rare. It should contain at least 1% by weight of active compound. Of its composition and preparation The percentage may, of course, be varied and, depending on convenience, 〜80%. Activity in such therapeutically useful compositions The amount of compound is such that a suitable dosage will be obtained. According to the present invention, A novel composition or preparation is one that contains an oral dosage unit form of about 0.3 mg. 1 μg to 2000 mg It is prepared to contain the active compound in between.   Tablets, troches, pills, capsules, etc. also contain the following: There are also: like tragacanth gum, acacia, corn starch or gelatin Excipients such as dicalcium phosphate; corn starch, potato starch Disintegrants such as arginic acid; lubricants such as magnesium stearate; And sweeteners such as sucrose, lactose, saccharin or peppermint Fragrances such as lime oil or cherry flavor can also be added. Throw When the dosage unit form is a capsule, it should contain, in addition to materials of the above type, a liquid carrier. Can be. Various other substances may be present as coatings, Or other modifications to the physical dosage form of the dosage unit . For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Can be done. Syrups or elixirs are active compounds, sweeteners As sucrose, as preservative methyl or propylparaben, if colorant And may contain flavoring agents such as cherry and orange flavoring. Rice cake Any substance used in preparing any dosage form is pharmaceutically pure and Should be substantially non-toxic. In addition, sustained release of the active compound (su stai ned-release) preparations and formulations.   The pharmaceutically acceptable carrier and / or diluent may be any and all solvents Agents, diffusion media, coatings, antibacterials, antifungals, isotonic and Includes a retarder. The use of such media for pharmaceutically active substances is well known in the art. It is well known. Such a convenient medium or substance is incompatible with the active ingredient Except where noted, its use in therapeutic compositions is discussed. Also supplement active ingredients It can also be included in the composition.   Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage Is particularly advantageous. Dosage unit form as used herein refers to A physically individual unit suitable for single administration to a mammal to which it is attached; Each unit is calculated to produce the desired therapeutic effect and the determined amount of active substance With the required pharmaceutical carrier.   The description of the novel dosage unit form of the invention relates to (a) the unique properties and achievements of the active substances The particular therapeutic effect to be achieved, and (b) the body as disclosed in detail herein. Such for the treatment of disease in the life of a disease state that is potentially detrimental to health Depends directly on the limitations inherent in the art of compounding the active substance Describe.   Principal active ingredient is pharmaceutically acceptable in an effective amount for convenient and effective administration The dosage unit form as disclosed above, together with the carrier to be administered. For example, unit dosage form If the main active compound is present at about 0. To be contained in an amount ranging from 5 μg to about 2000 mg Can be. In proportion, the active compound is generally present in an amount of about 0,1. 5 μg to about 2000 mg / Present in 1 ml of carrier. In the case of compositions coated with supplementary active ingredients, The dose of is determined by referring to the usual dose and method of administration of the same ingredient. You.   Pharmaceutical compositions can also be transfected into target cells, to which IAP cognate Vectors that carry nucleic acid molecules capable of regulating body expression and IAP homologous activity It may also include genetic molecules such as a vector. The vector is, for example, a virus Sex vector.   Another embodiment of the present invention relates to a degeneration of a baculovirus IAP animal cell homolog. Apoptosis in animals affected by disease, infectious disease, cancer or autoimmune disease It relates to the use of adjusting the power.   Preferably, the cell homolog is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 11, SE Q ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 Or a derivative or chemical analog thereof.   The present invention further contemplates antibodies to the IAP homologs and derivatives thereof of the present invention. You. Antibodies can be isolated from homologous diagnostic assays and from biological fluids or culture media. Useful for purifying or isolating a family. Such antibodies may be monoclonal or Or polyclonal, to select from naturally occurring antibodies of the IAP homolog Or an IAP homolog or a derivative thereof can be made specifically. You. In the latter case, the IAP homologue or derivative thereof will first be contacted with the carrier molecule. It is necessary to make contact. The antibody of the present invention and / or the recombinant IAP The family or derivatives thereof are particularly useful as therapeutic or diagnostic agents.   For example, IAP homologs and derivatives thereof are naturally occurring antibodies to this homolog Can be used for screening. These include, for example, autoimmune diseases. Occurs in some of the cases. Conversely, specific antibodies screen IAP homologs Can be used to Techniques for such assays are well known in the art. , For example, sandwich assays and ELISAs. Know the amount of IAP homologs This may be for the diagnosis of a cancer or its susceptibility to cancer, or for a treatment protocol. Important for monitoring calls.   Antibodies to the IAP homologs of the present invention may be monoclonal or polyclonal. is there. On the other hand, antibody fragments can be used like Fab fragments. In addition, the book The invention extends to recombinant and synthetic and hybrid antibodies. This specification "Synthetic antibodies" are considered herein to include antibody fragments and hybrids. Of the present invention Antibodies of this embodiment are particularly useful for immunotherapy and for assessing apoptosis. Alternatively, it can be used as a diagnostic tool for monitoring treatment programs.   For example, specific antibodies can be used to screen for IAP homologs. The latter, for example, screens for the amount of IAP homologs in cell extracts or other biological fluids. Homologous or IAP homologs prepared by recombination from the culture supernatant It is important as a method for purifying the body. Assay techniques discussed herein Are known in the art and include, for example, sandwich assays and ELISAs.   Any secondary antibody (monoclonal, polyclonal or Or antibody fragments or synthetic antibodies) are also included within the scope of the present invention. Primary and secondary Both antibodies can be used in detection assays, or primary antibodies can be Can be used with the anti-immunoglobulin antibody available at Consider here Antibodies include any antibody specific for any region of an IAP homolog. Antibodies In addition, the sequence must correspond to the amino acid sequence described in SEQ ID NO: 9. There is also.   Both polyclonal and monoclonal antibodies are immune to enzymes or proteins. Obtained by an epidemiological reaction, either type is available for immunoassays. Both Methods for obtaining types of antibody sera are well known in the art. Polyclonal anti Although body serum is less preferred, an appropriate amount of an IAP homolog or Injected the antigen portion, collected serum from the animal, and used known immunosorbent technology. It can be prepared relatively easily by isolation by either method. This Antibodies prepared by the method described in the above are virtually used for any type of immunoassay. Possible but generally less preferred because of the product of potential heterogeneity .   The use of monoclonal antibodies in immunoassays can lead to large-scale production. And the homogeneity of the product. Monoclonal antibody Preparation of production hybridoma cell lines is more efficient than immortal cell lines and immunogenicity. Induced by the fusion of sensitive lymphocytes, this is accomplished by techniques well known to those skilled in the art. Is done.   Another embodiment of the present invention is directed to the identification of an IAP homolog in a biological sample from a subject. A method of detection, under conditions sufficient for a period of time to form an antibody-IAP homolog complex. Specific biological samples for IAP congeners and their derivatives or congeners What Contacting with an antibody and then detecting the complex.   The presence of IAP homologs can be determined in a number of ways, such as Western blotting, It can be confirmed by the ELISA method. US Patent 4,016,043 Nos. 4,424,279 and 4,018,653. As such, a wide range of immunoassay techniques are available. These are, of course, one place And two or both non-competitive "sandwich" assays and transmission Including in a competitive antagonistic binding assay. These assays use labeled antibodies Direct binding to the target.   Sandwich assays are one of the most useful and commonly used assays, Preferred for use in the present invention. Many variations of sandwich asses A exists and all of them are intended to be included in the present invention. In short, typical The above assay immobilizes the unlabeled antibody on a solid material and removes the test sample. Contact with binding molecules. An appropriate time, i.e., sufficient for antibody-antigen complex formation Reporter that can produce a detectable signal after incubation for A secondary antibody specific for this antigen, labeled with a molecule, is added and another antibody- Incubation was for a time sufficient to form the original-labeled antibody complex. React Wash off any material that was not present and the reporter molecule produces the presence of antigen Determined by observing the signal The result is a visible signal Is qualitative by simply observing or contains a known amount of hapten Either quantitative by comparing to a control sample is there. Variations of the above assays include simultaneous assays, in which Simultaneously adds both the sample and the labeled antibody to the bound antibody. these The technique is well known to those skilled in the art and any minor burrs are readily apparent. Including According to the present invention, the sample is a cell extract containing an IAP homolog. Serum, saliva, mucosal secretions, This is a sample containing a damper fluid, a tissue fluid and a respiratory fluid. Therefore this sample Generally a biological sample, including biological fluids, but also from cell culture It extends to the fermentation liquor and supernatant as obtained.   In a typical forward sandwich assay, the IAP homolog or its antibody portion A first antibody having specificity to a solid surface, either covalently or passively; Attach to surface. Solid surfaces are typically glass or polymers, most often Commonly used polymers are cellulose, polyacrylamide, nylon, police Tylene, polyvinyl chloride, or polypropylene. Solid carrier, tube , Beads, microplate discs, or for performing immunoassays Some other solid. Methods of conjugation are well known in the art and generally involve cross-linking Consisting of binding or physical adsorption, the polymer antibody complex Wash in the preparation of Aliquot the sample to be tested into its solid phase complex And for a sufficient time (e.g., 2 to 40 minutes) and under appropriate conditions (e.g., 25 minutes). C) incubate to allow binding of the subunits present in the antibody. I After incubation, the antibody subunit solid phase is washed, dried, and Incubated with a second antibody specific for. The second antibody is directed against a hapten. To a reporter molecule used to indicate binding of the second antibody.   Another method is to immobilize the target molecule in a biological sample and re-immobilize the immobilized target. Exposure to a specific antibody that may or may not be labeled with a porter molecule Including. Depending on the amount of target and the strength of the reporter molecule signal, bound The target is detectable by direct labeling of the antibody. Alternatively, a second antibody specific to the first antibody Exposing the two labeled antibodies to a target-primary antibody complex; -Forming a ternary complex of the second antibody. The complex is Lipo Is detected by the signal emitted by the protein molecule.   As used herein, the term “reporter molecule” refers to, by its chemical nature, an antigen- Means a molecule that gives an analytically identifiable signal that allows the detection of bound antibodies You. Detection may be qualitative or quantitative. This type of assembly The most commonly used reporter molecules in b include enzymes, fluorophores and radionuclides Molecules (eg, radioisotopes) and chemiluminescent molecules. Enzyme im In the case of the assay, the enzyme is conjugated to a second antibody, typically glutaraldehyde or Combined by periodate. However, as will be readily understood, various There are different coupling techniques, which are readily available to one skilled in the art. Generally for Enzymes include horseradish peroxidase, glucose oxidase, β- Lactosidase, alkaline phosphatase and the like. Used for specific enzymes The resulting substrate produces a detectable color change upon hydrolysis of the corresponding enzyme. It is generally selected for Examples of suitable enzymes include alkaline phosphatase and There is peroxidase. Generates a fluorescent product instead of the chromogenic substrate described above It is also possible to use fluorogenic substrates. In all cases, enzyme-labeled anti- The body was added to the first antibody hapten complex, allowed to bind, and the excess reagent was washed away. except. The solution containing the appropriate substrate is then added to the antibody-antigen-antibody complex. You. The substrate reacts with the enzyme bound to the second antibody, producing a qualitative visible signal. The signal is usually further quantified, spectroscopically, and the hapten present in the sample Give an indicator of quantity. "Reporter molecule" refers to red blood cells on latex beads, etc. It also extends to the use of cell aggregation or inhibition of aggregation.   Alternatively, fluorescent compounds such as fluorescein and rhodamine are added to the antibody Chemical binding without altering binding capacity. By irradiating light of a specific wavelength When activated, the fluorochrome-labeled antibody absorbs light energy Induces an excited state in the molecule and emits light in a characteristic color that can be visually detected with a light microscope. Radiate. As in EIA, the fluorescently labeled antibody is the first antibody-hapten comp Can be combined with Rex. After washing away unbound reagents Then, irradiate the remaining ternary complex with light of an appropriate wavelength, and observe the observed fluorescence. Indicates the presence of the title hapten.   Immunofluorescence and EIA techniques are both very well established in the art, Particularly preferred for the method of the invention. However, radioisotopes, chemiluminescent Other reporter molecules such as cent or bioluminescent molecules may be used.   The present invention relates to a PCR assay for detecting an IAP homolog or a derivative thereof. Genetic assays such as are also contemplated. Another method used for conjugation is to use direct Otide sequencing or specific oligonucleotide hybridization, direct Single-chain conformational polymorphisms, such as indirect protein truncation studies Includes mutation scanning such as analysis (SSCP).   The present invention is described in further detail by the following non-limiting Figures and / or Examples. You.   In the figure:   FIG. 1 is a comparison of the deduced amino acid sequence of the IAP protein.   (A) MIHA (SEQ ID NO: 2), MIHB (SEQ ID NO: 4), MIHC (SEQ ID NO: 6) and And DIHA (SEQ ID NO: 8). Amino shared by three or more of the proteins The acid was highlighted. The RING finger domain is indicated by a dashed arrow.   (B) Comparison of BIR of IAP protein. Highly conserved residues are highlighted I did. CPI is derived from Cydia pomonella granulosis virus; OpLAP is O rgyia pseudotsugata PV; CiIAP is Chilo iridescent virus AIP is a candidate gene for SMA; AcIAP is Autograp ha Californica PV; ASV from African swine fever virus From the IAP-like sequence.   (C) Comparison of RING finger motifs of IAP and other proteins. Strongly save The highlighted residues are highlighted. CRAF (TRAF3) and TRAF2 Are members of the TRAF family; BCRA1 and RAG1 C-CBL are two mammalian proteins with a G finger motif; SLI-1 is a cellular oncogene whose C.I. elgans homolog; cpi is Cy dia pomonella granulosis virus; OpIAP is Orgyia pseudotsu gata PV; CiIAP is derived from Chilo irrdesent virus; AcI AP is from Autographa california PV; UKMT is from Drosophila Unkemp EctV is derived from the p28 protein of Ectromelia virus.   FIG. 2 shows that the mammalian IAP homolog is expressed in various tissues. It is a display by a photograph.   (A) Mature mouse tissue total RNA Northern blots were analyzed at high stringency. And probed with the mMIHA cDNA coding region. The northern part of each lane GAPDH was also probed as an indicator of loading.   (BD) Mature mouse tissue poly (A) + RNA Northern blot (CLONT ECH) at low stringency with hMIHB cDNA coding strand (B) and And hMIHC CDNA coding strand (C).   FIG. 3 shows that MIHA and MIHB were caused by overexpression of ICE (but not FADD). 5 is a graphical representation showing protection against induced death.   (A) Induction of apoptosis by transfection with ICE. Column 1-6 (black) Shows the p32ICE-lacZ fusion plasmid and the IAP homolog or control Of cells co-transfected with a plasmid having any of The column (white) shows the amount of cell death due to the transfection process itself.   (B) Induction of apoptosis by transfection with FADD. MIH Tan The plasmid encoding the protein is replaced with the lacZ vector and the FADD coding region. Co-transfected with the construct (columns 1-6). About the ICE experiment Similarly, background death was monitored in parallel sets of cultures (7-12). Column).   FIG. 4 shows a hypothetical, non-limiting, proposed site of action of the IAP protein. It is a schematic display which shows a cell death model. Direct interactions are shown by solid lines and indirect The typical action is indicated by a broken line. Uncertain interactions are indicated by question marks. TRA F and spontaneous self-associating molecules such as proteins with a dead region Activates cell death proteases insufficiently to cause apoptosis . It may give rise to a unique activation signal. IC by transfection Increased amount of E-precursor enables activation of enough enzyme to induce apoptosis I do. IAP proteins vary depending on their affinity for their target To some extent, it may act to reduce spontaneous activation signals. According to this model, OpIAPs have a high affinity for their target, Providing greater protection against FADD than the milk IAP homolog May be able to. However, in cells transfected with FADD, Because of the need to block large unique activation signals, Not as effective against FADD. Crm that directly inhibits active ICE A and p35 discuss ICE media in terms of how to activate ICE. It is an effective inhibitor of mediated apoptosis.   FIG. 5 is a representation of the nucleotide sequence and corresponding amino acid sequence of MIHA. You.   FIG. 6 is a representation of the nucleotide sequence and corresponding amino acid sequence of MIHB. You.   FIG. 7 is a representation of the nucleotide sequence and corresponding amino acid sequence of MIHC. You.   FIG. 8 is a representation of the nucleotide sequence of DIHA and the corresponding amino acid sequence. You.   9A and 9B show deaths induced by overexpression of ICH-1 protease. 1 shows the protection by MIHA, MIHB and MIHC against.   FIG. 10. Western blot of 293T cells transfected with MIHA construct. It is a photographic display of a blot.   The following one-letter and three-letter abbreviations are used for amino acid residues. A summary of the SEQ ID NOs is as follows: Example 1                     cDNA cloning         GCG sequence analysis software package (Madison, WI) Similar to the OpIAP BIR and RING finger amino acid motifs The gene bank database was searched for the translated sequence. Already identified Human X chromosome genome STS (Genbank: L24579) is presumed Used to design PCR primers that harbor the RING finger motif . This region is amplified and a human genomic DNA library (Stratagene) is Used to select. Fragments isolated from this library were Probe the mouse liver cDNA library at high Three types of mouse cDNA clones designated as milk IAP homolog A (MIHA) Generated. Human EST sequence gene bank: R19628 and gene bank: T By translating 96284, the repeat of the BIR in OpIAP is similar I found out. CDNA library of human fetal liver (Strataje Putative BIR domain used to generate the probe used to select Within the range, these sequences were used to design PCR primers. C The hybridized cDNA clones were named MIHB and MIHC, respectively. .         The Drosophila genome sequence (Genebank: DROCCAAT) Primer for amplifying a 900 bp product from Drosophila cDNA Used to design This fragment was subcloned and Lambda Z Oligo (dT) -primed Drosophila larval cDN composed of AP The A library (Stratagene) was used for selection. 8-terminal amino A 2 kb cDNA (DIHA) encoding all but the acid was isolated. Pe Genome sequence for comparison of peptide sequences (FIG. 1) Genbank: DROCCAA These residues inferred from T are added.                             Example 2                           RNA analysis         Radioisotope-labeled mHIHA and GAPDH with high stringency , Northern blot of gene of mouse tissue with 5 μg / lane as total RNA And hybridized. Many mice with 2 μg poly (A) + RNA Heavy tissue Northern blots (CLONTECH) were released at low stringency. Probe with radioisotope labeled hMIHC, strip, low stringency Probe, strip again, and follow the manufacturer's instructions for Lobes were probed with high stringency.                             Example 3                       Yeast two hybrid system         The coding regions of MIHA, MIHB and MIHC were EcoRI at the 5 'end. Part Amplified by PCR using primers that bind to GAL4 DNA It was expressed as a frame fusion with the domain. The PCR product is called p It was ligated to GBT9 vector (CLONTECH). 17 cogs from start ATG On It was cloned into the pGBT9 vector. Mistaken by Taq polymerase The insert from the PCR was sequenced to determine if it had been inserted. T RAF1, TRAF2 and TRAF3 expression vectors have already been described (1994, Rot he et al .; 1994b, by Hu et al.). Cj in GTB9 Detection of protein interaction with vectors with coding regions such as un and fos Was used as a control. Yeast strain HF7c was prepared using the lithium acetate protocol. Transformation with these plasmids (Gietz et al., 1992).                           Example 4               Transient transfection assay         MIHA cDNA of pBIuscript (Stratagene) Generate 1.8 kb SacI-EcoRI from clone to generate pSPMIHA Subcloned in pSP72 (Promega) to obtain PEF or pEF In a derivative of GOS vector (Mizushima and Nagata, 1990) It was recloned as a 1.8 kb BamHI-EcoRV fragment. 1.64 kb BamHI-ScaI fragment from DIHA cDNA Was cloned in pEF. Pfu polymerase (Stratagene) It was used to amplify the coding regions of MIHB and MIHC. These products Specific restriction sites were present at the ends of the primers cloned in pEF .         The p32-lacZ fusion plasmid pβactM11Z is described in Miura et al. (1993). FADD expression construct FADD-AU1 Are described by Chinnaiyan et al. (1995). AcPV -Derived bcl-2, crmA and codes such as p35 and IAP from OpPV The region was inserted into the pEF vector. The truncated OpIAP plasmid is PEF vector coating full-chain IAP is digested with ruI and SmaI and religated It constituted by doing. This encodes the RING finger domain The 3 'sequence of the ruI site in the deleted OpIAP was deleted.         Example 5             Cloning of DIHA, MIHA, MIHB and MIHC       According to the gene bank survey, the Drosophila gene sequence (gene bank) : DROCCAAT) is similar to the baculovirus IAP gene. won. The Drosophila LAP homolog A (DIHA) was designed and Using a PCR-generated probe and an isolated clone encoding The Drosophila cDNA library was screened. Gene bank survey By the way, many mammalian sequences have RINGs of BIRs or viral IAPs. Similarity to the finger domain was also revealed. During transformation Introduces a peptide similar to the baculovirus IAP RING finger motif X HUMSWX59, a PCR-generated probe corresponding to the STS sequence on the chromosome 5, the first mammalian LAP homolog (MIHA) already identified Cloned. Researchers need the human genome to understand the complete coding region. A partial chain of the clone and a MIHA cDNA clone were isolated. More closely A series of human IAP homologs (MIHB and MIHC) were linked to a motif such as BIR. Gene banks corresponding to EST sequences R19628 and T96284 Using a PCR-generated probe.         In FIG. 1, the predicted amino acid sequence of DIHA (predicted molecular weight 55 kD), Compare MIHA (56 kD), MIHB (70 kD) and MIHC (68 kD) I do.   The start codon is most of the 5 'methionine upstream within the frame stop codon. Close. All four amino acids have three BIRs in the terminal half of the amino acids. One RING finger domain with repeats and close to the carboxy terminus Having. MIHB and MIHC are almost adjacent, with 73% amino acids Are independent, with MIHA 43% independent and 62% with MIHB and MIHC Was one. In DIHA, 36% were independent and 57% were identical to MIHC.         The BIR repeats of these proteins are mutually identical to the BIR repeats of AIP. Comparable to the viral IAPs of FIG. 1B. Some B in mammalian IAPs The IRs are BIR analogs from another IAP than the rest of the BIRs in the same protein. More closely resembling the original BIR, which means that a copy of the original BIR It shows that. The four most conserved amino acid residues in BIRs are CX_TwoCX₁₆HX₆The C pattern was three cysteines and one histidine.   These may be associated with metal ions (Birnba, 1994). um et al.). FIG. 1 shows the C3H4RING finger domain of the IAP molecule. 5 shows a comparison of the RING finger with RING fingers from other selected proteins. IAP protein RING fingers from TRAF2 without BIRs (Ro, 1994) the et al.) and CRAF (1994b, Hu et al .; 1995, Cheng et al .; RING fingers from other proteins such as Sato et al., 1995) Similar to each other. o -Identical RING fingers in proteins with BIR are mammalian cancer Gene, c-Cbl (1991, Blake et al.) And its C. Elgans family Present in the body, Sli-1 (1995, Yoon et al.). Baculoyl Like the IAP protein, it has the protein p28 of the Ectomeria virus. RING fingers are essential for poisoning, regardless of growth (1994, Sengevich et al.). However, since p28 has no BIRs, Its function is not yet known.Example 6                         Expression of mammalian homologs         The MIHA message in mice is about 7.5 kb, and the skeletal and cardiac muscles It is expressed in most mouse tissues other than meat. Tested in lung at highest level Issued at intermediate levels in the brain and kidney, and lower levels in the thymus, liver, bone In bone marrow, skin and testis (Fig. 2A).         CDNA that spans the coding region of human MIHB can be mapped with low stringency. 多重 4.0 kb and ５5.5 kb on a mouse multiple tissue Northern blot. Hybridized to the message (FIG. 2B). Less transcripts in upper part are spleen and skeleton It was not expressed in muscle and was analyzed at higher levels in many other tissues. Yo Most ４4.0 kb transcripts are expressed in the spleen at the lowest levels, with intermediate levels In bells, expressed in breast, brain, lung, liver, skeletal muscle and kidney, testis at highest level Was expressed. In addition, 〜9.5 transcripts are detected in testis and not seen in other tissues. won.       The entire strand of the human MIHC cDNA probe was ligated to ~ 3.0 and ~ 4.0 kb. Two messages hybridized with low stringency (FIG. 2C). 4. 0 kb message can be detected in spleen, high level is testis, middle level is other In all tissues. 〜3.0 kb transcripts in lung and skeletal muscle, spleen Found in the gut, liver, kidney and testis. hMIHB and hMIHC probes Hybridized to mouse RNA at low stringency, the upper (4. 0kb) transcription Object can be exactly the same as that detected by MIHB probe 2. .   Some transcripts may also embody other closely related mammalian IAP congeners You.Example 7           Prevention of apoptosis induced by ICE or FADD   Mammalian IAP homologs were expressed in two stimuli, overexpression of p32ICE and And their ability to prevent apoptosis by overexpression of FADD Tested. Using Constructs Expressing Ced-3-Like Cysteine Protease ICE Transfection was previously shown to cause apoptosis (Mi ura et al., 1993). Baculovirus IAPs include bcl-2, crmA, and p3. Prevent this necrosis so that other anti-apoptotic genes, such as 5 (Miura et al., 1993; Xue et al., 1995). We have MIHA , MIHB, and MIHC were tested and caused by ICE overexpression It was determined whether apoptosis could really be stopped. HeLa fine ICE-la together with plasmids encoding IAP homologs or controls Co-transfected with the plasmid carrying the cZ fusion construct. X -Stain with gal to identify transfected cells and survive these The rate was evaluated visually. As shown in FIG. 3A, MIHA, MIHB, And OpIAP significantly reduced the amount of necrosis caused by ICE However, MIHC provided no detectable protection.   Forced expression of FADD, a protein associated with Cd95, also leads to cell necrosis. (Chinnaiyan et al., 1995; Boldin et al., 1995). HeLa cells in three Co-transfected with Rasmid: FADD expression construct, with lacZ gene Encoding a mammalian IAP homolog or OpIAP Vector. OpIAP provided partial protection against FADD, which was E was not as effective as for E (FIG. 3A, lanes 2 and 3B, lane 2 Compare that). The inventors have proposed that MIHA, MIHB) or MIHC No reduction in the amount of DD-induced cell necrosis could be detected.Example 8                 Interaction with members of the TRAF family   Two mammalian IAP homologs, MIHB and MIHC, were Together with the molecules TRAF1 and TRAF2 related to -R2, TNF-R2 ( (R75) isolated as part of a protein complex that binds to the cytoplasmic domain of othe et al., 1994). Yeast two-hybrid system (Fields and Song, 1 989) using the three mammalian IAP homologs and the viral OpI. APs are TRAF1, TRAF2 and TRAF3, ie CD40BP / C That binds a related protein, also known as RAF-1 / CAP, (Hu et al., 1994b; Cheng et al., 1995; Sato et al., 1995). As shown in Table 2, MIHB or MIHC, and TRAF3 But not co-transfected with TRAF1 or TRAF2, his + And lacZ +, an interaction may occur between these proteins in yeast It was shown. In contrast, in this system, the tested TRAF and OpI No interaction between either AP or MIHA was detected. these The result shows that MIHB and MIHC bind to TRAF1 and TRAF2 Shows that OpIAP or MIHA interacts with other proteins I do.Example 9         Identification of MIHE, CIA-1, CIA-2, and YIA-1   The consensus sequence of the baculovirus IAP repeat (BIR) is deduced as follows. Was: [In the array,   Xaa is an amino acid residue;   [Xaa]_mIs a series of at least 5, preferably at least 9 amino acids Yes;   [Xaa]_nIs Met or Leu;   [Xaa]_oIs a series of at least 3, preferably at least 5, amino acids Yes;   [Xaa]_pIs Val or Ala;   [Xaa]_qIs Phe or Tyr;   [Xaa]_rIs Leu or Val Or a homolog, chemical analog, or derivative thereof.   A database search using this consensus sequence revealed that the mouse (MIHE), yeast (Y IA-1), and C.I. elegans (CIA-1, CIA-2) This resulted in the identification of the log. Nucleotides derived from these IAP homologs and And corresponding amino acid sequences are shown in SEQ ID NOs: 10 to 17. Interestingly, MI HE, YIA-1 CIA-1, and CIA-2 are RING finger domains. Do not have                                 Example 10                 Protection by MIHA, MIHB and MIHC   9A and B show deaths induced by overexpression of ICH-1 protease. Figure 2 shows protection against MIHA, MIHB and MIHC.                                 Example 11                             Western blot   Preparation of polyclonal rabbit antibody, MIHA peptide (SEQ ID NO: 18) was affinity purified. This antibody is inter alia In Western blot of MIHA transfected 293T cells used. The result is shown in FIG.   Those skilled in the art will appreciate that the invention described herein is subject to variations other than those specifically described. Or be susceptible to change. The present invention addresses such changes and And that all changes are included. The present invention is also described herein. All of the steps, features, compositions and compounds cited or indicated And individually or collectively, and all two or more of the steps or features Including combinations.                                   Table 2 Binding of TRAF1, TRFA2, TRFA3 and mammalian LAP homologs Yeast Two Hybrid Assay   Yeast strain HF7c is transformed into GAL4 DNA-binding domain and IAP family member Or TRAF protein expressing a fusion protein between Encoding fusion between quality or control and GAL4 activation domain For co-transformation. from his and lacZ reporter genes (indicating interaction) Of double transformants on histidine-deficient medium (column 3) And colonies stained blue with X-gal (column 4), c-jun and c-fos Interacting proteins in a DNA binding vector and an activation vector, respectively Was used as a control gene.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 38/00 C07K 14/39 C07K 14/39 14/435 14/435 14/47 14/47 A61K 37 / 02 (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX , NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN

Claims (1)

[Claims] 1. Derivatives of isolated protein molecules or analogs thereof, which are capable of suppressing the apoptotic response of cells to viral infection and include cell-derived homologs of viral inhibitors of apoptosis (IAP). 2. 2. The isolated protein molecule of claim 1, wherein said molecule is a baculovirus IAP homolog. 3. 3. The isolated protein molecule of claim 2, wherein said baculovirus IAP inhibits apoptosis mediated by interleukin 1β converting enzyme (ICE) protease or cell death inducing region containing protein FADD. 4. 2. The isolated protein molecule of claim 1, wherein said homolog is from an animal cell. 5. 5. The isolated protein molecule according to claim 4, wherein said cell is from a mammal, nematode, yeast or insect. 6. Amino acid sequence: [Where Xaa is an amino acid residue; [Xaa] _m is a sequence of at least 5 amino acids, preferably 9 amino acids; [Xaa] _n is a Met or Leu; [Xaa] _o is a sequence of at least 3 amino acids, preferably 5 amino acids [Xaa] _p is Val or Ala; [Xaa] _q is Phe or Tyr; [Xaa] _r is Leu or Val; or a homologue, analogous compound or derivative thereof). Protein molecule. 7. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 2, or having at least 40% similarity thereto. 8. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 4, or having at least 40% similarity thereto. 9. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 6 or having at least 40% similarity thereto. 10. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 8, or having at least 40% similarity thereto. 11. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 11, or having at least 40% similarity thereto. 12. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 13, or having at least 40% similarity thereto. 13. 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 15, or having at least 40% similarity thereto. 14． 7. The isolated protein molecule of claim 6, having substantially the amino acid sequence set forth in SEQ ID NO: 17, or having at least 40% similarity thereto. 15. An isolated nucleic acid molecule encoding the protein molecule according to any one of claims 1 to 14. 16. It encodes the amino acid sequence shown in SEQ ID NO: 2 or comprises a sequence of nucleotides complementary to the sequence encoding said sequence, or substantially at least 40 nucleotides of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 16. The isolated nucleic acid molecule of claim 15, having a% similarity. 17． It encodes the amino acid sequence shown in SEQ ID NO: 4 or comprises a sequence of nucleotides complementary to the sequence encoding said sequence, or substantially at least 40 nucleotides of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 16. The isolated nucleic acid molecule of claim 15, having a% similarity. 18. At least 40% of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 6 or comprising a sequence of nucleotides complementary to the sequence encoding the sequence, or substantially encoding the amino acid sequence shown in SEQ ID NO: 6 16. The isolated nucleic acid molecule of claim 15, having a% similarity. 19. At least 40% of the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 8 or comprising a sequence of nucleotides complementary to the sequence encoding said sequence, or substantially encoding the amino acid sequence shown in SEQ ID NO: 8 16. The isolated nucleic acid molecule of claim 15, having a% similarity. 20. At least the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 11 or containing a sequence of nucleotides complementary to the sequence encoding the sequence, or substantially encoding the amino acid sequence shown in SEQ ID NO: 11 16. The isolated nucleic acid molecule of claim 15, having 40% similarity. 21. At least the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 13 or comprising a sequence of nucleotides complementary to the sequence encoding the sequence, or substantially encoding the amino acid sequence shown in SEQ ID NO: 13 16. The isolated nucleic acid molecule of claim 15, having 40% similarity. 22. At least the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 15 or comprising a nucleotide sequence complementary to the sequence encoding the sequence, or substantially encoding the amino acid sequence shown in SEQ ID NO: 15 16. The isolated nucleic acid molecule of claim 15, having 40% similarity. 23. At least the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 17 or comprising a nucleotide sequence complementary to the sequence encoding the sequence, or substantially encoding the amino acid sequence shown in SEQ ID NO: 17 16. The isolated nucleic acid molecule of claim 15, having 40% similarity. 24. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 1, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 1 under low stringency conditions. 16. The isolated nucleic acid molecule of claim 15. 25. A nucleic acid molecule comprising substantially the nucleotide sequence shown in SEQ ID NO: 3 or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 3 under conditions of low stringency. 16. The isolated nucleic acid molecule of claim 15. 26. 6. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 5, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 5 under low stringency conditions. 16. The isolated nucleic acid molecule of claim 15. 27. 9. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 7, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 7 under low stringency conditions. 16. The isolated nucleic acid molecule of claim 15. 28. 11. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 10, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 10 under conditions of low stringency. 16. The isolated nucleic acid molecule of claim 15. 29. 12. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 12, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 12 under conditions of low stringency. 16. The isolated nucleic acid molecule of claim 15. 30. 15. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 14, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 14 under low stringency conditions. 16. The isolated nucleic acid molecule of claim 15. 31. 17. A nucleic acid molecule comprising substantially the nucleotide sequence set forth in SEQ ID NO: 16, or having at least 40% similarity thereto, or being capable of hybridizing to SEQ ID NO: 16 under low stringency conditions. 16. The isolated nucleic acid molecule of claim 15. 32. 15. A cell in an animal comprising administering to the animal a cell apoptosis-modulating amount of the protein molecule according to any one of claims 1 to 14 or a derivative or analogous compound thereof for a time and under conditions sufficient to modulate said apoptosis. How to regulate apoptosis. 33. 33. The method of claim 32 for use in treating a degenerative disease, an infectious disease, a cancer or an autoimmune disease. 34. A pharmaceutical composition comprising a protein molecule according to any of claims 1 to 14 or a derivative or analogous compound thereof and one or more pharmaceutical carriers and / or diluents. 35. Use of an animal cell homolog of baculovirus IAP to regulate apoptosis in animals suffering from degenerative diseases, infectious diseases, cancer or autoimmune diseases. 36. 36. Use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 2 or at least 40% similarity. 37. 36. Use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 4 or at least 40% similarity. 38. 36. The use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 6, or at least 40% similarity thereto. 39. 36. The use according to claim 35, wherein the cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 8 or at least 40% similarity. 40. 36. The use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 11, or at least 40% similarity thereto. 41. 36. Use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 13 or at least 40% similarity. 42. 36. Use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 15 or at least 40% similarity. 43. 36. Use according to claim 35, wherein said cell homolog has substantially the amino acid sequence set forth in SEQ ID NO: 17 or at least 40% similarity.