JP2003527831A - Endozepine-like polypeptides and polynucleotides encoding endozepine-like polypeptides - Google Patents

Abstract

SUMMARY A novel human nucleic acid sequence encoding an endozepine-like polypeptide is disclosed herein. Also disclosed are polypeptides encoded by these nucleic acid sequences, and antibodies that immunospecifically bind to the polypeptides, as well as derivatives, variants, variants, or fragments of the above-described polypeptides, polynucleotides, or antibodies. You. The invention further discloses therapeutic, diagnostic, and research methods for the diagnosis, treatment, and prevention of disorders, including the novel human endozepine-like nucleic acids and proteins.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates generally to nucleic acids and their encoded polypeptides. More particularly, the present invention relates to nucleic acids encoding endozepine-like polypeptides, and vectors, host cells, antibodies, and recombinant methods for producing these nucleic acids and polypeptides.

BACKGROUND OF THE INVENTION In developed countries, there are over 250 million individuals suffering from disorders related to overnutrition. Obesity, type II diabetes, and metabolic syndrome X have reached epidemic proportions. Problems associated with these disorders (eg, hyperlipidemia, hypertension, vascular disease,
(Strokes and end organ damage) impose tremendous financial burden on affected individuals and large societies. Therefore, new pharmacological approaches to regulating energy metabolism would be of significant benefit in medicine. The present invention comprises a novel family of endozepine polypeptides, which offer new therapeutic opportunities to regulate metabolism by regulating its insulin secretion, insulin sensitivity, glucose and fatty acid utilization and other endocrine functions.

Endozepines are a family of proteins whose members have been reported to have various biological effects. These effects include γ-aminobutyric acid (G
ABA) receptor regulation, insulin homeostasis, and regulation of mitochondrial steroidogenesis may be mentioned. Modulation of the GABA receptor, which resides in the brain, is believed to regulate pathological anxiety.

As members of the endozepine family, diazepam-binding inhibitors (
DBI). DBI or a derivative of DBI is believed to down regulate the effects of GABA. DBI has also been reported to inhibit glucose-induced insulin release from isolated perfused rat spleens both early and late.

Several mammalian DBI polypeptides have been described. Mammalian DBI tends to be highly conserved at its carboxy terminus. A human DBI polypeptide of about 11 kilodaltons has been described. This polypeptide was shown to replace β-carboline and benzodiazepine bound to brain membrane fractions in vitro.

[0006] DBI polypeptides have been reported to be present in both brain and non-brain tissues, including the intestine. DBI is a hormone mechanism, neurocrine (neurocr).
It has been proposed that it may belong to a new family of intestinal polypeptides that inhibit glucose-mediated insulin release by the ine) mechanism, or both.

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery of novel nucleic acid sequences encoding polypeptides associated with endozepines. Nucleic acids encoding endozepine-like polypeptides and derivatives and fragments thereof are collectively referred to hereafter as "END.
OX ".

In one aspect, the invention provides an isolated ENDOX nucleic acid molecule encoding an ENDOX polypeptide, the nucleic acid molecule comprising human endozepine mRNA.
Nucleic acid sequences having identity to the nucleic acid sequences of. In some embodiments, the ENDOX nucleic acid molecule is capable of hybridizing under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule comprising a protein coding sequence for an endozepine nucleic acid sequence. The invention also includes an isolated nucleic acid encoding an ENDOX polypeptide, or a fragment, homologue, analog or derivative thereof. For example, this nucleic acid is SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 2.
0, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 4
It may encode a polypeptide that is at least 85% identical to a polypeptide comprising the 7 and 49 amino acid sequences. This nucleic acid has, for example, SEQ ID NOS: 1, 3, 5, 7, 9
, 22, 25, 28, 31, 34, 46, and 48 can be a genomic DNA fragment or a cDNA molecule.

Oligonucleotides, such as ENDOX nucleic acids (eg, SEQ ID NOs: 1, 3, 5
, 7, 9, 22, 25, 28, 31, 34, 46, and 48).
Oligonucleotides containing three consecutive nucleotides or the complement of this oligonucleotide are also included in the invention.

A substantially purified ENDOX polypeptide (SEQ ID NOs: 2, 4, 6, 8, 10
, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29
, 30, 32, 33, 35-45, 47, and 49) are also included in the present invention. In some embodiments, the ENDOX polypeptide comprises an amino acid sequence that is substantially identical to the amino acid sequence of human endozepine polypeptide.

The invention also features an antibody that immunoselectively binds to the ENDOX polypeptide.

In another aspect, the invention includes a pharmaceutical composition comprising a therapeutically effective amount or a prophylactically effective amount of a therapeutic agent and a pharmaceutically acceptable carrier. The therapeutic agent can be, for example, an ENDOX nucleic acid, an ENDOX polypeptide, or an antibody specific for an ENDOX polypeptide. In a further aspect, the invention comprises a therapeutically or prophylactically effective amount of the pharmaceutical composition in one or more containers.

In a further aspect, the invention provides the ENDOZ encoded by this DNA.
Included is a method of producing a polypeptide by culturing cells containing the ENDOX nucleic acid under conditions that allow expression of the polypeptide. If desired, the ENDOX polypeptide can then be recovered.

In another aspect, the invention includes a method of detecting the presence of ENDOX polypeptide in a sample. In this method, a sample is contacted with a compound that selectively binds to the polypeptide under conditions that allow the formation of a complex between the polypeptide and the compound. This complex, if present, is detected, thereby identifying the ENDOX polypeptide in the sample.

The invention also includes a method of identifying a particular cell or tissue type based on its expression of ENDOX.

By contacting the sample with an ENDOX nucleic acid probe or primer and detecting whether the nucleic acid probe or primer binds to the ENDOX nucleic acid molecule in the sample, the ENDOX nucleic acid molecule in the sample is detected. Methods for detecting the presence are also included.

In a further aspect, the present invention provides a ENDOX polypeptide by contacting a cell sample containing the ENDOX polypeptide with a compound that binds to the ENDOX polypeptide in an amount sufficient to modulate the activity of the polypeptide. A method for modulating the activity of a. The compound may be a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein. .

The use of therapeutic agents in the manufacture of a medicament for treating or preventing a disorder or syndrome is also within the scope of the invention, and these disorders or syndromes include, for example:
Metabolic disorders, diabetes, obesity, infectious diseases, eating disorders, cancer-related cachexia, and various dyslipidemias. The therapeutic agent can be, for example, an ENDOX nucleic acid, an ENDOX polypeptide, or an ENDOX-specific antibody, or a biologically active derivative or fragment thereof.

The present invention further includes a method for screening for modulators of a disorder or syndrome, which disorders or syndromes include, for example, metabolic disorders, diabetes,
Includes obesity, infectious diseases, dietary disorders, cancer-related cachexia, and various dyslipidemias. The method involves contacting a test compound with an ENDOX polypeptide and determining whether the test compound binds to the ENDOX polypeptide. Binding of the test compound to the ENDOX polypeptide indicates that the test compound is a modulator of activity, or a modulator of latency or predisposition to the disorders or syndromes described above.

Also provided are methods for screening for modulators of activity, or latency or predisposition to this disorder or syndrome by administering test compounds to test animals at increased risk of the disorder or syndrome described above. Within the scope of the invention: metabolic disorders, diabetes, obesity, infectious diseases, dietary disorders, cancer-associated cachexia, and various dyslipidemias. The test animal expresses the recombinant polypeptide encoded by the ENDOX nucleic acid. The expression or activity of the ENDOX polypeptide is then measured in this test animal, as is the expression or activity of the protein in control animals that recombinantly express the ENDOX polypeptide and are not at increased risk for this disorder or syndrome. To be measured. The expression of ENDOX polypeptide in both test and control animals is then compared. Changes in the activity of the ENDOX polypeptide in the test animals relative to the control animals indicate that the test compound is a modulator of latency of the disorder or syndrome.

[0021] In yet another aspect, the invention provides an E in a subject (eg, a human subject).
Methods for determining the presence or predisposition to a disease associated with altered levels of NDOX polypeptide, ENDOX nucleic acid, or both. This method
Measuring the amount of ENDOX polypeptide in a test sample from this subject, and comparing the amount of this polypeptide in the test sample with the amount of ENDOX polypeptide present in a control sample. Altered levels of ENDOX polypeptide in the test sample as compared to the control sample are indicative of the presence of or predisposition to the disease in the subject. Preferably, this predisposition is, for example, metabolic disorders, diabetes, obesity, infectious diseases, eating disorders,
Includes cancer-related cachexia and various dyslipidemias. Also, the expression levels of the novel endozepines of the present invention can be used in methods for screening for various cancers.

In a further aspect, the invention provides a subject (eg, a human subject) END
Treating or preventing a pathological condition associated with a disorder in a mammal by administering an OX polypeptide, ENDOX nucleic acid or ENDOX-specific antibody in an amount sufficient to reduce or prevent the pathological condition. Including how to do. In a preferred embodiment, the disorder is, for example, a metabolic disorder, diabetes, obesity, infectious disease,
Includes eating disorders, cancer-related cachexia, and various dyslipidemias.

In a further aspect, the invention comprises a method of altering total body energy metabolism or weight loss by altering serum cholesterol, lipids, glucose and insulin.

[0024] In yet another aspect, the present invention regulates weight loss due to specific fat depletion, increased muscle mass associated with some medical procedures, or regulation of lipid volume in some adipocytes. Including the method.

In a still further aspect, the invention may be used in a method of affecting appetite, nutrient uptake and metabolite accumulation in both positive and negative manners. The present invention may also be used in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X, and dietary disorders, chronic disease-related wasting disorders and various cancers by regulating metabolism.

In yet another aspect, the invention can be used in a method of identifying the cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly used in the art. These include, but are not limited to, two-hybrid systems, affinity purification, co-precipitation with antibodies or other specific interacting molecules.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to be limited in any manner. Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based, in part, on the discovery of novel nucleic acid sequences that encode polypeptides related to polypeptides of the endozepine protein family described previously.
Nucleic acids encoding endozepine-like polypeptides based on the discovered sequences are ENDO1, ENDO2, ENDO3, ENDO4, ENDO5, END, respectively.
Called O6, ENDO7, ENDO8, ENDO9 and ENDO10. Nucleic acids and their encoded polypeptides are generically referred to herein as "END.
OX ".

(ENDO1) The ENDO1 nucleic acid of the present invention comprises SEQ ID NOs: 1, 11, 13, 14, 46, and 4.
8 includes the diffusion sequence shown in FIG. SEQ ID NOS: 11, 13 and 14 are combined to provide the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 does not have to be the complete coding sequence as it has no start methionine and no stop codon. Both SEQ ID NOs: 46 and 48 include nucleotide sequences that encode the amino acid sequences also encoded by SEQ ID NO: 1. Each of these nucleotide sequences and the amino acids they encode are described in detail below.

The ENDO1 nucleic acid of the present invention comprises the nucleic acid sequence shown in Table 1 (SEQ ID NO: 11)
. This sequence relates to an expression sequence tag (EST) from a previously described human cDNA clone with database accession number AA877351. AA8
77351EST is reported to be similar to the nucleic acid encoding "diazepam binding inhibitor-like 5".

[0031]   (Table 1)

[0032]

[Table 1] (SEQ ID NO: 11).

The nucleic acid sequences disclosed in Table 1 include an open reading frame (“ORF”) starting at position 1. This ORF encodes a polypeptide sequence of 89 amino acid residues. The sequence of the encoded polypeptide is shown in Table 2 (SEQ ID NO: 12). The translated protein is associated with diazepam binding inhibitor-like 5 from mouse (SWISSPROT-ACC: O09035) (identity 62).
/ 81; 76% and positive = 70/81; 86%). Furthermore, the translated protein is a type 2 human endozepine-like protein mutant (
GENBANK-ID: AF229804 | acc: AF229804) (identity = 89/89 (100%), positive = 89/89 (100
%)).

[0034]   (Table 2)

[0035]

[Table 2] (SEQ ID NO: 12).

The polypeptide encoded by the ORF set forth in SEQ ID NO: 11 does not contain an amino terminal methionine residue. Thus, the disclosed nucleic acid sequences can be part of an open reading frame encoding a larger gene product. Larger gene products can include, for example, at least one signal peptide that is cleaved during protein processing.

In order to identify additional sequences encoding the ENDO1 nucleic acid, the disclosed EN
The DO1 sequence (SEQ ID NO: 11) was used to design primers for use in a PCR reaction to isolate additional sequences encoding the ENDO1 polypeptide shown in Table 2. The amplified sequence was cloned and sequenced. The sequences of the two products were designated 18517852-2 (SEQ ID NO: 13) and 1187175852-3 (SEQ ID NO: 14). These sequences are shown in Tables 3 and 4, respectively.

[0038]   (Table 3)

[0039]

[Table 3] (SEQ ID NO: 13).

[0040]   (Table 4)

[0041]

[Table 4] (SEQ ID NO: 14).

The sequences shown in Tables 1, 3 and 4 may be combined to provide the nucleotide sequence shown in Table 5 (SEQ ID NO: 1).

[0043]   (Table 5)

[0044]

[Table 5] (SEQ ID NO: 1).

The nucleotide residue designated by “X” may be T or G, ie in various embodiments the ENDO1 nucleic acid of the invention comprises a T at the position designated by X in this nucleic acid sequence. In another embodiment, an ENDO1 nucleic acid sequence of the invention comprises a G at the position indicated by X in this disclosed nucleotide sequence.

The polypeptide encoded by the ORF in SEQ ID NO: 1 does not contain an amino terminal methionine residue. Thus, the disclosed nucleic acid sequences can be part of an open reading frame encoding a larger gene product. ENDO1
Nucleic acid database searches were performed to identify additional sequences encoding nucleic acids. Two novel ENDO1 sequences have been identified here and are shown in Table 5A. SEQ ID NO: 46 is GenBank accession number AL121672 (SEQ ID NO: 46
) Has been edited from the previously described human clone. SEQ ID NO: 48 is G
It was edited from a previously described human clone with the enBank clone AC025743 (SEQ ID NO: 48).

[0047]   (Table 5A)

[0048]

[Table 6] The nucleic acid shown in Table 5 (SEQ ID NO: 1) encodes a polypeptide having the amino acid sequence shown in Table 6 (SEQ ID NO: 2).

[0049]   (Table 6)

[0050]

[Table 7] (SEQ ID NO: 2).

The nucleic acids shown in Table 5A (SEQ ID NO: 46 and SEQ ID NO: 48) encode polypeptides having the amino acid sequences shown in Table 6A (SEQ ID NO: 47 and SEQ ID NO: 49, respectively).

[0052]   (Table 6A)

[0053]

[Table 8] The amino acid residue designated by "X" may be C or F, ie, in various embodiments, the polypeptides of the present invention include a C at the position designated by X in this amino acid sequence. In another embodiment, an ENDO1 polypeptide of the invention comprises an F at the position indicated by X in this listed amino acid sequence.

ENDO1 nucleic acids of the invention can include nucleic acids that encode the polypeptides of SEQ ID NOs: 2, 12, 47, or 49, eg, ENDO1 nucleic acids include SEQ ID NOs: 1, 11
, 46, or 48 nucleic acid sequences. The invention also includes mutant or variant nucleic acids, any of which bases may be altered from the corresponding bases shown in Table 1 or Table 5. In some embodiments, the ENDO1 nucleic acid encodes a protein that maintains its endozepine-like activity and physiological function, or is a fragment of such nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments which are complementary to any of the just described nucleic acids. The invention further includes nucleic acids or nucleic acid fragments, or their complementary strands, the structure of which includes chemical modifications. Such modifications include, but are not limited to, modified bases and nucleic acids in which the sugar phosphate backbone is modified or derivatized.
These modifications are made to at least partially enhance the chemical stability of the modified nucleic acid so that they can be used as antisense binding nucleic acids, eg, in therapeutic applications in a subject.

The ENDO1 polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 2, 12, 47 or 49. The invention also provides that any residue thereof is SEQ ID NO: 2,12.
, 47 or 49, which encodes a protein that can be modified from the corresponding residue but still retains its endozepine-like activity and physiological function, or a variant or variant protein, or a functional fragment thereof (eg, The following active peptides (SEQ ID NO: 15)) are included.

[0056]   The metabolic regulatory peptide # 6 (MRP-6) sequence is as follows: QATQGDCDIPGPPASDVRAR (SEQ ID NO: 15) Is.

The multiple sequence alignments of various embodiments of ENDO1 polypeptides (Table 6B) show their relationship to each other.

[0058]   (Table 6B)

[0059]

[Table 9] The invention further includes antibodies and antibody fragments (eg, F _ab or (F _ab ) ₂ ) that immunospecifically bind to ENDO1 polypeptides, and derivatives and fragments thereof.

The ENDO1 sequence is useful for detecting a particular type of tissue. For example, ENDO1 is highly expressed in liver and endothelial cells when tissue panels are assayed for expression. High expression of ENDO1 is also a marker for multiple types of cancer.

The ENDO1 sequence is also useful in regulating overall energy metabolism or body weight by altering serum glucose or fat levels.

The ENDO1 sequences are also useful in methods for determining cellular receptors and downstream factors of the invention by any one of a number of techniques commonly used in the art.

The ENDO1 sequences are useful in the treatment of various cancers by regulating diabetes, metabolic disorders associated with obesity, metabolic syndrome X and wasting disorders and metabolism associated with anorexia and chronic diseases.

(ENDO2) The ENDO2 nucleic acid of the present invention comprises the nucleic acid sequence shown in Table 7 (SEQ ID NO: 3).
The ORF and the putative untranslated region upstream from the start codon of the ORF and the putative untranslated region downstream from the stop codon of the ORF are present in the nucleotide sequences disclosed in Table 7. Untranslated nucleotides are underlined. The start and stop codons of the ORF are shown in bold.

[0065]   (Table 7)

[0066]

[Table 10] (SEQ ID NO: 3).

The nucleic acid sequences in Table 7 are 470 nucleotides of Rana ridibunda diazepam binding inhibitor (DBI) mRNA (GENBANK-ID: RRU0.
9205 | acc: U09205) with 218 (74%) of the 294 bases that are identical and positive. Rana ridbunda DPI
A BLASTN identity search comparing the sequence regions disclosed in Table 7 to mRNA is shown in Table 8. A region of the disclosed sequence is designated as the "Query" sequence,
And the Rana ridbunda DPI mRNA (SEQ ID NO: 11) sequence is shown as “Subject sequence”.

[0068]   (Table 8)

[0069]

[Table 11] The ORF encodes a polypeptide of 85 amino acids (SEQ ID NO: 4). The amino acid sequence of this polypeptide is shown in Table 9 (SEQ ID NO: 4).

[0070]   (Table 9)

[0071]

[Table 12] (SEQ ID NO: 4).

The polypeptide sequences disclosed in Table 9 relate to previously described duck diazepam binding inhibitor polypeptides. This relationship is shown in Table 10. Table 9
The amino acid sequence shown in (SEQ ID NO: 4) is Anas platyrhynch.
os (Ptnr: SWISSPROT-ACC: P45882)
60 amino acid residues (70%) out of 85 amino acid residues that are identical to the 103 amino acid residue-derived acyl-coA binding protein (ACBP) (diazepam binding inhibitor) (DBI) (endozepine) (EP), And 72 of the 85 amino acid residues that are positive for this (84%). The region of the polypeptide sequence shown in Table 9 is designated as the "Query" sequence.
The region of the duck polypeptide sequence is designated the "Sbct" sequence.

[0073]   (Table 10)

[0074]

[Table 13] A multiple sequence alignment between the amino acids of SEQ ID NO: 4 and various acyl coA binding polypeptides is shown in Table 11. Amino acid sequence of Table 7 (“ACBP_novel”), porcine acyl-coA binding protein (SWISSPROT locus ACBP_PIG, accession number PI2026) (“ACBP_pig”), bovine acyl-coA binding protein (SWISSPROT locus ACBP_BOVIN, accession number P07107) (
"ACBP_bovine"), and human acyl-coA binding protein (SWISS)
The alignment between the PROT locus ACBP_HUMAN, accession number P07108) ("ACBP_human") is shown. Full homology regions are shown in black. Regions with conservative amino acid substitutions are shown in grey. Non-conservative amino acid substitutions are shown without shading.

[0075]   (Table 11)

[0076]

[Table 14] ENDO2 nucleic acids of the invention encoding an endozepine-like protein include nucleic acids encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (eg, the nucleic acid whose sequence is provided in SEQ ID NO: 3). The present invention also includes a fragment of SEQ ID NO: 3, or a mutant or variant nucleic acid in which any of its bases can be changed from the corresponding base shown in SEQ ID NO: 3. In some embodiments, the variant or variant nucleic acid encodes a protein that maintains its endozepine-like activity and physiological function, or is a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including nucleic acid fragments which are complementary to any of the just described nucleic acids.
The invention further includes nucleic acids or nucleic acid fragments, or their complementary strands, the structure of which includes chemical modifications. Such modifications include, but are not limited to, modified bases and nucleic acids in which the sugar phosphate backbone is modified or derivatized. These modifications are made to at least partially enhance the chemical stability of the modified nucleic acid so that they can be used as antisense binding nucleic acids, eg, in therapeutic applications in a subject.

ENDO2 polypeptides according to the present invention include polypeptides comprising the amino acid sequence shown in Table 9 (SEQ ID NO: 4). The present invention also provides mutant or variant proteins (proteins in which any of these residues may be altered from the corresponding residues shown in Table 8, but which maintain endozepine-like activity and physiological function. Still code) or their functional fragments (
For example, the following active peptides (SEQ ID NO: 16)) are included.

[0078]   Metabotropic peptide # 5 (MRP-5) sequence:   QAVIGNINIECSEMLELKGK (SEQ ID NO: 16).

The present invention further includes antibodies and antibody fragments (eg, F _ab or (F _ab ) ₂ ), which immunospecifically bind to ENDO2 polypeptides, and their derivatives and fragments.

The ENDO2 sequence is useful for detecting specific types of tissue. For example, ENDO2 is highly expressed in brain cells and spleen cells when a panel of tissues is assayed for expression. Also, high expression of ENDO2 is a marker for colon and lung cancer.

The ENDO2 sequences are also useful in regulating overall energy metabolism or body weight by altering serum glucose or fat levels.

The ENDO2 sequence also includes any one of a number of techniques commonly used in the art.
The present invention is useful in methods of identifying cell receptors and downstream effectors of the present invention.

The ENDO2 sequences are useful in the treatment of diabetes, metabolic disorders associated with obesity, metabolic syndrome X, and anorexia and wasting disorders associated with chronic diseases and various cancers by regulating metabolism.

(ENDO3) The ENDO3 nucleic acid of the present invention comprises the nucleic acid sequence shown in Table 12 (SEQ ID NO: 5). The ORF, as well as the putative untranslated region upstream from the start codon and downstream from the stop codon of this ORF, is shown in Table 12 by underlining. The start and stop codons of this ORF are shown in bold letters. This ORF contains nucleotide 86
Beginning at the atg start codon at ~ 88 and tg at nucleotides 403-405
ends with an a codon. Putative untranslated regions upstream from the start codon and downstream from the stop codon are indicated by underlining in Table 12, while start and stop codons are indicated in bold letters.

[0085]   (Table 12)

[0086]

[Table 15] The disclosed ENDO3 nucleic acid sequence (SEQ ID NO: 5) is a bacterial artificial chromosome 46.
2G18 (LANL) (GENBANK-ID: AC005736 | acc: A
The sequence on human chromosome 16 incorporated into C005736) has 168 (84%) of the same 199 bases.

The ORFs identified in Table 12 encode a polypeptide sequence of 83 residues (SEQ ID NO: 6), which is shown in Table 13.

[0088]   (Table 13)

[0089]

[Table 16] The amino acid sequence of the polypeptide sequence disclosed in Table 13 (SEQ ID NO: 6) is the 86 amino acid residue bovine acyl-coA-binding protein (ACBP) (diazepam binding inhibitor) (DBI) (endozepine) (EP) (ptnr: S
5 out of 82 amino acid residues identical to WISSPROT-ACC: P07107)
It has 5 (67%) and 66 (80%) of this amino acid residue and 82 positive residues. A comparison of these sequences is shown in Table 14. Table 13
The region of the polypeptide of B. cerevisiae is designated as the "Query" sequence and
The region of the P sequence is shown as the "Sbjct" sequence.

[0090]   (Table 14)

[0091]

[Table 17] The amino acid sequence of the polypeptide sequence disclosed in Table 13 (SEQ ID NO: 6) is 91 amino acid residues (gb: GENBA) of human diazepam binding inhibitor (DBI).
NK-ID: HUMDBI | acc: M14200) with 57 of the 91 amino acid residues (62%) identical and with this amino acid residue and 72 of the positive 91 residues (79%). .

A comparison of these sequences is presented in Table 14A. The region of the polypeptide of Table 13 is shown as the "Query" sequence and is a human diazepam binding inhibitor (D
Regions of BI) sequence are indicated as "Sbjct" sequence.

[0093]   (Table 14A)

[0094]

[Table 18] Multiple sequence alignments showing the relevance of the polypeptides disclosed in Table 13 to bovine and human acyl co-A binding proteins are shown in Table 15 as ClustalW analysis. A comparison is made with the polypeptides of Table 12 ("DBI_novel").
, Bovine acyl coA-binding protein (SWISSPROT locus ACBP
_BOVIN, registration P07107) ("ACBP_bovin"), and A
CBP_human SWISSPROT locus ACBP_HUMAN, registration_P07
108 (“ACBP_Human”). Areas of complete homology are shown in black. Regions with conservative amino acid substitutions are shown in grey. Non-conservative amino acid substitutions are shown without shading.

[0095]   (Table 15)

[0096]

[Table 19] ENDO3 nucleic acids of the invention include nucleic acids that encode a polypeptide that includes SEQ ID NO: 6 (eg, the nucleic acid sequence shown in SEQ ID NO: 5). The present invention also includes fragments of the nucleic acid of SEQ ID NO: 5, as well as mutant or variant nucleic acids (any of these bases may be altered from the corresponding bases shown in Table 12, but with endozepine-like activity and physiological Still encodes proteins that maintain function),
Alternatively, it includes fragments of such nucleic acids. The invention further includes nucleic acids of sequence complementary to those just described, including nucleic acid fragments complementary to any of the nucleic acids just described. The invention further includes nucleic acids or nucleic acid fragments, or their complements, the structures of which include chemical modifications. Such modifications include, but are not limited to, modified bases and nucleic acids, the sugar phosphate backbone of which is modified or derivatized. These modifications are made in therapeutic applications in a subject, eg, to at least partially increase the chemical stability of the modified nucleic acid, such that they can be used as antisense-binding nucleic acids.

The ENDO3 protein of the invention comprises the amino acid sequence shown in Table 13 (SEQ ID NO: 6). The present invention also provides mutant or variant proteins (wherein any of these residues may be altered from the corresponding residues shown in Table 13) proteins which retain endozepine-like activity and physiological function. Still code)
Or a functional fragment thereof (for example, the active peptide below).

Metabotropic peptide # 3 (MRP-3,3s) sequence (SEQ ID NO: 17) and :(
Sequence number 18) RATVGNNIKTERPGMVDFKGK (sequence number 17) RATVGNNIKTERPGMVDFK- (sequence number 18).

The present invention further includes antibodies and antibody fragments (eg, F _ab or (F _ab ) ₂ ) that immunospecifically bind to ENDO3 polypeptides, and derivatives and fragments thereof.

The ENDO3 sequence is useful for detecting specific types of tissue. For example, ENDO3 is highly expressed in fat and skeletal muscle when a panel of tissues is assayed for expression. Also, high expression of ENDO3 is a marker for breast cancer.

The ENDO3 sequences are also useful in regulating overall energy metabolism or body weight by altering serum insulin or fat levels.

The ENDO3 sequence also includes any of the many techniques commonly used in the art.
The present invention is useful in methods of identifying cell receptors and downstream effectors of the present invention.

The ENDO3 sequences are useful in the treatment of diabetes, metabolic disorders associated with obesity, metabolic syndrome X, and anorexia and wasting disorders associated with chronic diseases and various cancers by regulating metabolism.

(ENDO4) The ENDO4 nucleic acid of the present invention comprises the nucleic acid sequence shown in Table 16 (SEQ ID NO: 7). The sequences shown in Table 16 include the ORF and putative untranslated regions upstream and downstream from this ORF. This ORF begins at the atg start codon at nucleotides 11-13 and ends at the tga codon at nucleotides 299-301. The putative upstream and downstream untranslated regions are indicated by underlining in Table 16.

[0105]   (Table 16)

[0106]

[Table 20] The disclosed nucleic acid sequence (SEQ ID NO: 7) is Rana ridibunda endozepine mRNA (GENBANK-ID: RRU09205 | acc: U0).
It has 200 out of 256 bases (78%) identical to 9205). The relationships between the sequences in Table 16 and the Rana ridibunda sequences are shown in Table 17.
The regions of the sequences shown in Table 16 are listed as "Query" sequences. Ran
The region of the aridibunda endozepine mRNA sequence is "Subject
"As an array.

[0107]   (Table 17)

[0108]

[Table 21] The disclosed nucleic acid sequence (SEQ ID NO: 7) is also the human diazepam binding inhibitor mRNA (GENBANK-ID: HUMDBI | acc: M14200).
Related to. The disclosed sequence is 179 of 259 residues (69%) identical to the human diazepam inhibitor mRNA. The relationships between this disclosed sequence and human sequences are shown in Table 18. The regions of the sequences shown in Table 16 are listed as "Query" sequences. The region of the human mRNA sequence is "Sub
Ject ”array.

[0109]   (Table 18)

[0110]

[Table 22] The ORF identified in Table 16 encodes an amino acid sequence of 89 amino acids (SEQ ID NO: 8). The amino acid sequence of the encoded protein (SEQ ID NO: 8) is
It is shown in Table 19.

[0111]   (Table 19)

[0112]

[Table 23] The signal peptide is present in the polypeptide sequence shown in Table 19. The site most likely to be cleaved is the sequence DRA-AE, between residues 18 and 19. The program PSORT predicts a moderate likelihood of extracellular secretion for ENDO4 protein.

The polypeptides shown in Table 19 are related to previously described acyl-coA binding proteins and diazepam binding inhibitor proteins. Table 20 shows that the amino acid sequence of Table 19 is a protein of 103 amino acid residues derived from Ana splatyrhynchos (ptnr: SWISSPROT-ACC: P4588).
It is shown to have 71 of the same 89 amino acid residues (79%) as 2) and 78 of this 89 amino acid residues and positive 89 residues (87%).
The region of the polypeptide sequence shown in Table 19 is designated as the "Query" sequence. The region of the Anas platyrhynchos sequence is designated as the "Sbjct" sequence.

[0114]   (Table 20)

[0115]

[Table 24] Table 20A shows that the amino acid sequences of Table 19 have homology to Rana ridibunda diazepam binding inhibitor (DBI). The region of the polypeptide sequence shown in Table 19 is designated as the "Query" sequence. Ra
The region of the na ridibunda diazepam binding inhibitor (DBI) sequence is designated as the "Sbjct" sequence.

[0116]   (Table 20A)

[0117]

[Table 25] An alignment between the polypeptide sequences shown in Table 18 and the diazepam binding inhibitors or acyl-coA binding polypeptides described above is shown in Table 21. The amino acid sequence shown in Table 19 is expressed as "ba271m1_A". 8
Eight amino acid frog acyl co-A binding protein amino acid sequence (PIR-ID:
A57711) is indicated by "A57711_ACBP_Frog":
88 amino acid human acyl co-A binding polypeptide (PIR-ID: NZHU)
The sequence is indicated by "NZHU_ACBP_Human". 103 amino acid duck endozepine amino acid sequence (SWISSPROT-ACC: _4588)
2) is indicated by "P45882_endozepine_Duck". Regions containing acid substitutions in conservative amino acids are shown in grey. Non-conservative amino acid substitutions are represented unshaded.

[0118]

[Table 26] ENDO4 nucleic acids of the invention include nucleic acids that encode a polypeptide that includes the amino acids of SEQ ID NO: 8. For example, the ENDO4 nucleic acid can include the sequence disclosed in Table 16 (SEQ ID NO: 7). The invention also includes fragments of nucleic acids that encode a polypeptide that includes the amino acid sequence of SEQ ID NO: 8. The present invention also includes variant or variant nucleic acids or fragments of such nucleic acids, wherein any of the bases of these variant or variant nucleic acids may be changed from the corresponding bases shown in Table 16. However, the mutant or variant nucleic acid encodes a protein that retains its endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences include nucleic acids complementary to any of the just described nucleic acids, and nucleic acid fragments complementary to any of the just described nucleic acids. The invention further comprises
It includes nucleic acids or nucleic acid fragments, or their complements, the structure of which includes chemical modifications. Such modifications include, but are not limited to, modified bases, and nucleic acids, which nucleic acids are modified or derivatized in their sugar phosphate backbone. These modifications are performed, at least in part, to enhance the chemical stability of the modified nucleic acid so that they are, for example,
It can be used as an antisense binding nucleic acid in therapeutic applications in a subject.

The amino acid sequence of the ENDO4 protein of the present invention is shown in Table 19 (SEQ ID NO: 8).
Including the proteins shown in. The present invention also includes functional fragments such as mutant or variant proteins or active peptides such as:
Either of these variant or variant protein residues are listed in Table 16
The variant or variant protein, which may be altered from the corresponding base shown in, still encodes a protein that maintains its endozepine-like activity and and physiological function: Metabotropic Regulatory Peptide # 4 (MRP-4 , 4s) Sequence:

[0120]

[Chemical 1] The invention further includes antibodies and antibody fragments that immunospecifically bind to ENDO4 polypeptides, and derivatives and fragments thereof, such as F _ab or (F _ab ) ₂ .

The ENDO4 sequence is useful for detecting specific types of tissue. For example, if a panel of tissues is assayed for expression, ENDO4 is highly expressed in hematopoietic tissues.

The ENDO4 sequence is also useful for regulating overall energy metabolism or weight by altering serum insulin and glucose.

The END04 sequence also includes any one of a number of techniques commonly used in the art.
By way of example, it is useful in methods for identifying cell receptors and downstream effectors of the invention.

The ENDO4 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disease and various cancers associated with chronic diseases by regulating metabolism.

(ENDO5) The ENDO5 nucleic acid according to the present invention comprises the nucleic acid sequence shown in Table 25 (SEQ ID NO: 9). The ORF, as well as the putative untranslated region upstream and downstream of the ORF, are present in the disclosed sequences. This ORF begins at the atg start codon at nucleotides 7-9 and ends at the tag codon at nucleotides 265-267. This putative upstream and downstream untranslated region is indicated by the underline in Table 22.

[0126]

[Table 27] The ENDO5 nucleic acid sequence (SEQ ID NO: 9) is Rana ridibunda endozepine mRNA (GENBANK-ID: RRU09205 | acc: U092).
It has 199 of 274 nucleotides (72%) identical to 05). A comparison of these nucleotide sequences is shown in Table 23. The sequences disclosed in Table 22 are "Q
ury "sequence and the Rana ridibunda endozepine mRNA sequence is represented as the" Sbjct "sequence.

[0127]

[Table 28] The ENDO5 nucleic acid sequence (SEQ ID NO: 9) is also used as a Homo sapiens endozepine mRNA (GENBANK-ID: HUMEDZ | acc: M15887).
) With 173 of 262 nucleotides (66%). A comparison of these nucleotide sequences is shown in Table 24. The sequences disclosed in Table 22 are "Que
The human endozepine mRNA sequence is represented by "Sbjc".
Represented as a "t" array.

[0128]

[Table 29] The ORFs identified in Table 22 are polypeptides of 86 amino acid residues (SEQ ID NO: 1
Code 0). The amino acid sequence of the encoded polypeptide is shown in Table 25.

[0129]

[Table 30] The encoded polypeptide shown in Table 25 (SEQ ID NO: 10) is Rana
5 of 86 amino acid residues, identical to the 88 amino acid diazepan binding inhibitor protein from ridibunda (ptnr: PIR-ID: A57711)
7 (66%) and 67 (77%) of the 88 amino acid diazepan binding inhibitor protein and 86 positive residues. The alignment of these sequences is shown in Table 26. A comparison of these amino acid sequences is shown in Table 26. The sequences disclosed in Table 25 are represented as "Query" sequences and the Rana ridibunda endozepine polypeptide sequences are represented as "Sbjct" sequences.

[0130]

[Table 31] An alignment showing the relevance of the polypeptide sequences shown in Table 25 to the Endozepine sequences described above is shown in Table 27. The 86 amino acid polypeptides in Table 25 are “ci
tb_el — 2540m10_A ”. Other endozepine polypeptide sequences present in the table are the frog diazepine binding inhibitor DBI (PIR-
ID: A57711 ("A57711") amino acid sequence, 103 amino acid sequence duck polypeptide (SWISSPROT-ACC: P45882) (P45
882_Duck_DB1), and 87 amino acid human polypeptide (NZHU
_Human_DBI). Regions with conservative amino acid substitutions are shown in gray. Non-conservative amino acid substitutions are shown shaded.

[0131]

[Table 32] Using the PSORT program, the disclosed ENDO5 proteins were labeled with 0.
It is expected to localize to the cytoplasm with a certainty of 6500. In analyzes using the SIGNALP program, the ENDO5 protein is predicted to have no signal peptide.

The invention encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 10.
A DO5 nucleic acid (eg, a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 9 or a fragment thereof). The invention also includes variant or variant nucleic acids or fragments of such nucleic acids, wherein any of the bases of these variant or variant nucleic acids may be changed from the corresponding bases shown in Table 22. However, the mutant or variant nucleic acid encodes a protein that retains its endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences include nucleic acids complementary to the nucleic acids described above, and nucleic acid fragments complementary to any of the nucleic acids described above. The invention further includes nucleic acids or nucleic acid fragments, or their complements, the structure of which includes chemical modifications. Such modifications include, by way of non-limiting example, the following: modified bases, and nucleic acids, where the sugar phosphate backbone is modified or derivatized. These modifications are performed, at least in part, to enhance the chemical stability of the modified nucleic acids so that they can be used as antisense binding nucleic acids, eg, in therapeutic applications.

The ENDO5 protein of the present invention comprises the polypeptide the sequence of which is shown in Table 25 (SEQ ID NO: 10). The invention also includes variant or variant proteins, or functional fragments such as the following active peptides, any of the bases of these variant or variant proteins corresponding to those shown in Table 25. Residues can be varied, but they still encode proteins that maintain their endozepine-like activity and physiological function: Metabolic Regulatory Peptide # 7 (MRP-7) Sequence:

[0134]

[Chemical 2] The invention further encompasses antibodies and antibody fragments that immunospecifically bind to ENDO5 polypeptides, and derivatives and fragments thereof, such as F _ab or (F _ab ) ₂ .

The ENDO5 sequence is useful for detecting specific types of tissue. For example, when a panel of tissues is assayed for expression, ENDO5 is highly expressed in adipose and hematopoietic tissues.

The ENDO5 sequence is also useful for regulating overall energy metabolism or weight by altering serum cholesterol and glucose.

The ENDO5 sequence also includes any one of a number of techniques commonly used in the art.
By way of example, it is useful in methods for identifying cell receptors and downstream effectors of the invention.

The ENDO5 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disease and various cancers associated with chronic diseases by regulating metabolism.

(ENDO6) The ENDO6 nucleic acid of the present invention comprises the nucleic acid sequence shown in Table 27A (SEQ ID NO: 22).

[0140]

[Table 33] The nucleic acid sequences shown in Table 27A include an open reading frame ("ORF") beginning at position 1 with the start and stop codons shown in bold.
This ORD encodes a polypeptide sequence of 530 amino acid residues. The sequence of this encoded polypeptide (SEQ ID NO: 23) is shown in Table 27B. Translated protein and bovine endozepine (putative ligand of benzodiazepine receptor) related protein (gb: GENBANK-ID: BOVEDZR | acc: M15
888) is shown in Table 27.

[0141]

[Table 34] Translated protein-frame: 1-nucleotides 1-1590

[0142]

[Table 35] The ENDO6 nucleic acid of the invention can include a nucleic acid that encodes the polypeptide of SEQ ID NO: 23 (eg, the ENDO6 nucleic acid can include the nucleic acid sequence of SEQ ID NO: 22).
The present invention also includes mutant or variant nucleic acids, any of their bases being
It can be varied from the corresponding bases shown in Table 27A. In some embodiments, the ENDO6 nucleic acids, or fragments of such nucleic acids, encode proteins that maintain their endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences are complementary to the nucleic acids described herein (including nucleic acid fragments which are complementary to any of the nucleic acids described herein). The present invention further includes nucleic acids or nucleic acid fragments, or their complementary strands, whose structures include chemical alterations. Such changes include base and nucleic acid changes in which the sugar phosphate backbone is altered or induced,
It is not limited to these. These alterations are performed to enhance the chemical stability of the at least partially altered nucleic acid, such that, for example, those nucleic acids are
It can be used as an antisense binding nucleic acid in therapeutic applications in a subject.

The ENDO6 polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 23. The present invention also includes variant or variant proteins, any of those residues of which may be altered from the corresponding residue set forth in SEQ ID NO: 23, while its endozepine-like activity and physiological Proteins that maintain function,
Alternatively, it still encodes a functional fragment thereof (eg active peptide (SEQ ID NO: 24)).

[0144]   Metabotropic peptide number 8 (MRP-8) sequence:   QATEGPCKLSRPGFWDP (SEQ ID NO: 24).

The invention further encompasses antibodies and antibody fragments (eg, F _ab or (F _ab ) ₂ ) that immunospecifically bind to ENDO6 polypeptides, and derivatives and fragments thereof.

The ENDO6 sequence is useful for detecting specific types of tissue. For example, ENDO6 is highly expressed in skeletal muscle when a population of tissues is assayed for expression. High expression of ENDO6 is also a marker for many types of cancer.

The ENDO6 sequence is also useful for regulating overall energy metabolism or body weight by altering serum cholesterol and insulin.

The ENDO6 sequences are also useful in methods of identifying the cellular receptors and downstream effectors of the present invention by any one of a number of techniques commonly used in the art.

The ENDO6 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disorders associated with chronic diseases and various cancers by regulating metabolism.

(ENDO7) The ENDO7 nucleic acid of the present invention has the nucleic acid sequence shown in Table 27D (SEQ ID NO: 2).
5) is included.

[0151]

[Table 36] The nucleic acid sequences disclosed in Table 27D include an open reading frame ("ORF") starting at position 6 (with the start and stop codons shown in bold). This ORF encodes a polypeptide sequence of 86 amino acid residues. The sequence of this encoded polypeptide is shown in Table 27E (SEQ ID NO: 26). Translated protein and bovine endozepine (putative ligand of benzodiazepine receptor) (gb: GENBANK-ID: BOVEDZ | ac
Homology with c: M15886) is shown in Table 27F.

[0152]

[Table 37]

[0153]

[Table 38] An ENDO7 nucleic acid of the invention can include a nucleic acid that encodes the polypeptide of SEQ ID NO: 26 (eg, the ENDO7 nucleic acid can include the nucleic acid sequence of SEQ ID NO: 25).
The present invention also includes mutant or variant nucleic acids, any of their bases being
It can be varied from the corresponding bases shown in Table 27D. In some embodiments, the ENDO7 nucleic acid, or a fragment of such nucleic acid, encodes a protein that maintains its endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences are complementary to the nucleic acids described herein (including nucleic acid fragments which are complementary to any of the nucleic acids described herein).
. The invention further provides a nucleic acid or nucleic acid fragment, the structure of which comprises a chemical alteration,
Alternatively, it includes a complementary strand thereto. Such changes include, but are not limited to, base and nucleic acid changes in which the sugar phosphate backbone is changed or induced. These alterations are performed to enhance the chemical stability of the at least partially altered nucleic acid so that, for example, those nucleic acids can be used as antisense binding nucleic acids in therapeutic applications in a subject. .

The ENDO7 polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 26. The invention also includes variant or variant proteins, any of those residues of which may be changed from the corresponding residue set forth in SEQ ID NO: 26, while its endozepine-like activity and physiological Proteins that maintain function,
Alternatively, a functional fragment thereof (for example, the following active peptide (SEQ ID NO: 27)
)) Is still coded.

[0155]   Metabotropic peptide number 9 (MRP-9) sequence:   QATVHDLNTEWPRMLDLKGK (SEQ ID NO: 27).

The invention further encompasses antibodies and antibody fragments (eg, F _ab or (F _ab ) ₂ ) that immunospecifically bind to ENDO7 polypeptides, and derivatives and fragments thereof.

The ENDO7 sequence is useful for detecting specific types of tissue. For example, ENDO7 is highly expressed in adipose tissue when a population of tissues is assayed for expression. High expression of ENDO7 is also a marker for liver cancer.

The ENDO7 sequence is also useful for regulating overall energy metabolism or body weight by altering serum cholesterol.

The ENDO7 sequences are also useful in methods of identifying the cellular receptors and downstream effectors of the present invention by any one of a number of techniques commonly used in the art.

The ENDO7 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disorders associated with chronic diseases and various cancers by regulating metabolism.

(ENDO8) The ENDO8 nucleic acid of the present invention has the nucleic acid sequence shown in Table 27G (SEQ ID NO: 2).
8) is included.

[0162]

[Table 39] The nucleic acid sequences disclosed in Table 27G have an open reading frame ("ORF") starting at position 1 (with bold start and stop codons).
including. This ORF encodes a polypeptide sequence of 104 amino acid residues.
The sequence of this encoded polypeptide is shown in Table 27H (SEQ ID NO: 29). Translated protein and human diazepam binding inhibitor (DBI) (
Homology with gb: GENBANK-ID: HUMDBI | acc: M14200) is shown in Table 27I.

[0163]

[Table 40]

[0164]

[Table 41] ENDO8 nucleic acids of the invention can include nucleic acid encoding the polypeptide of SEQ ID NO: 29 (eg, ENDO8 nucleic acid can include the nucleic acid sequence of SEQ ID NO: 28).
The present invention also includes mutant or variant nucleic acids, any of their bases being
It can be varied from the corresponding bases shown in Table 27G. In some embodiments, the ENDO8 nucleic acids, or fragments of such nucleic acids, encode proteins that maintain their endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences are complementary to the nucleic acids described herein (including nucleic acid fragments which are complementary to any of the nucleic acids described herein). The present invention further includes nucleic acids or nucleic acid fragments, or their complementary strands, whose structures include chemical alterations. Such changes include base and nucleic acid changes in which the sugar phosphate backbone is altered or induced,
It is not limited to these. These alterations are performed to enhance the chemical stability of the at least partially altered nucleic acid, such that, for example, those nucleic acids are
It can be used as an antisense binding nucleic acid in therapeutic applications in a subject.

The ENDO8 polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 29. The present invention also includes variant or variant proteins, any of those residues of which may be altered from the corresponding residue set forth in SEQ ID NO: 29, while its endozepine-like activity and physiological Proteins that maintain function,
Alternatively, a functional fragment thereof (for example, the following active peptide (SEQ ID NO: 30)
)) Is still coded.

[0166]   Metabotropic peptide number 1 (MRP-1) sequence:   1 QATVGDINTERPGMLDFTGK (SEQ ID NO: 30).

The invention further includes antibodies and antibody fragments (eg, F _ab or (F _ab ) ₂ ) that immunospecifically bind to ENDO8 polypeptides, and derivatives and fragments thereof.

The ENDO8 sequence is useful for detecting specific types of tissue. For example, when a population of tissues is assayed for expression, ENDO8 is highly expressed in cardioskeletal muscle, liver and endothelial tissues. High expression of ENDO8 is also a marker for breast and colon cancer and melanoma.

The ENDO8 sequence is also useful for regulating overall energy metabolism or body weight by altering serum cholesterol, insulin, or glucose. The ENDO8 sequence is also useful in regulating muscle mass or fat levels.

The ENDO8 sequences are also useful in methods of identifying the cellular receptors and downstream effectors of the present invention by any one of a number of techniques commonly used in the art.

The ENDO8 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disorders associated with chronic diseases and various cancers by regulating metabolism.

(ENDO9) The ENDO9 nucleic acid of the present invention has the nucleic acid sequence shown in Table 27J (SEQ ID NO: 3).
Including 1).

[0173]

[Table 42] The nucleic acid sequences disclosed in Table 27J have an open reading frame ("ORF") starting at position 1 (with bold start and stop codons).
including. This ORF encodes a polypeptide sequence of 359 amino acid residues.
The sequence of this encoded polypeptide is shown in Table 27K (SEQ ID NO: 32). Translated protein and human peroxisome D3, D2-enoyl-
CoA isomerase (PECI) (gb: GENBANK-ID: AF1536)
12 | acc: AF153612) is shown in Table 27L.

[0174]

[Table 43]

[0175]

[Table 44] An ENDO9 nucleic acid of the invention can include a nucleic acid that encodes the polypeptide of SEQ ID NO: 32 (eg, the ENDO9 nucleic acid can include the nucleic acid sequence of SEQ ID NO: 31).
The present invention also includes mutant or variant nucleic acids, any of their bases being
It can be varied from the corresponding bases shown in Table 27J. In some embodiments, the ENDO9 nucleic acid, or a fragment of such a nucleic acid encodes a protein that maintains its endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences are complementary to the nucleic acids described herein (including nucleic acid fragments which are complementary to any of the nucleic acids described herein).
. The invention further provides a nucleic acid or nucleic acid fragment, the structure of which comprises a chemical alteration,
Alternatively, it includes a complementary strand thereto. Such changes include, but are not limited to, base and nucleic acid changes in which the sugar phosphate backbone is changed or induced. These alterations are performed to enhance the chemical stability of the at least partially altered nucleic acid so that, for example, those nucleic acids can be used as antisense binding nucleic acids in therapeutic applications in a subject. .

The ENDO9 polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 32. The present invention also includes variant or variant proteins, any of those residues of which may be altered from the corresponding residue set forth in SEQ ID NO: 32, while its endozepine-like activity and physiological Proteins that maintain function,
Alternatively, a functional fragment thereof (for example, the following active peptide (SEQ ID NO: 33
)) Is still coded.

[0177]   Metabotropic peptide number 2 (MRP-2) sequence:   QATEPGPCNMPKPGVFFDLINK (SEQ ID NO: 33).

The invention further encompasses antibodies and antibody fragments such as F _ab or (F _ab ) ₂ that immunospecifically bind to ENDO9 polypeptides, and derivatives and fragments thereof.

The ENDO9 sequence is also useful for regulating overall energy metabolism or body weight by altering serum cholesterol, insulin, or glucose.

The ENDO9 sequences are also useful in methods for identifying cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly used in the art.

The ENDO9 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disease associated with chronic diseases and various cancers by regulating metabolism.

(ENDO10) The ENDO10 nucleic acids of the present invention are shown in Table 27M (SEQ ID NO :).
34).

[0183]   (Table 27M)

[0184]

[Table 45] The nucleic acid sequences disclosed in Table 27M include an open reading frame ("ORF") beginning at position 573 with bold start and stop codons. O
RF encodes a polypeptide sequence of 282 amino acid residues. The sequence of this encoded polypeptide is shown in Table 27N (SEQ ID NO: 35). Homology between the translated protein and (human DBI / ACBP-like protein US Pat. No. 5,734,038-A2 March 31, 1998) is shown in Table 27O.

[0185]   (Table 27N)

[0186]

[Table 46] (Table 27O)

[0187]

[Table 47] The ENDO10 nucleic acid of the present invention may include a nucleic acid encoding the polypeptide of SEQ ID NO: 35. For example, the ENDO10 nucleic acid can include the nucleic acid sequence of SEQ ID NO: 34. The invention also includes variant or variant nucleic acids, where any of these bases may be altered from the corresponding bases shown in Table 27M. In some embodiments, the ENDO10 nucleic acid, or a fragment of such a nucleic acid, encodes a protein that retains its endozepine-like activity and physiological function. The invention further includes nucleic acids whose sequences are complementary to the sequences just described, which include nucleic acid fragments complementary to any of the just described nucleic acids. The invention further includes nucleic acids or nucleic acid fragments, or their complements, the structures of which include chemical modifications. Such modifications include, but are not limited to, modified bases and nucleic acids with modified or derivatized sugar phosphate backbones. These modifications are performed, at least in part, to enhance the chemical stability of the modified nucleic acid. As a result, they can be used, for example, as antisense binding nucleic acids in therapeutic applications in a subject.

The ENDO10 polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 35. The present invention also includes variant or variant proteins, wherein any of these residues may be changed from the corresponding residue set forth in SEQ ID NO: 35,
It still encodes a protein that retains its endozepsin-like activity and physiological function, or a functional fragment thereof (eg, active peptide below (SEQ ID NO: 36)).

[0189]   Metabolism-Regulating Peptide # 10 (MRP-10) Sequence:

[0190]

[Chemical 3] The present invention, that immunospecifically bind to ENDO10 polypeptide, F _ab or (
Antibodies and antibody fragments such as F _{ab) 2,} and further including derivatives and fragments.

The ENDO10 sequence is also useful for regulating overall energy metabolism or body weight by altering serum insulin, or glucose. EN
DO10 sequences are also useful for regulating muscle mass or fat levels.

The ENDO10 sequences are also useful in methods for identifying cellular receptors and downstream effectors of the invention by any one of a number of techniques commonly used in the art.

The ENDO10 sequences are useful in the treatment of diabetes, obesity-related metabolic disorders, metabolic syndrome X and anorexia and wasting disease associated with chronic diseases and various cancers by regulating metabolism.

The ENDOX nucleic acid encodes a polypeptide that is a novel member of the endozepine family. Endozepines have been reported for diverse biologically important functions. Related endozepine polypeptides (eg, diazepam binding inhibitors) bind GABA receptors. In addition, the new ENDOX polypeptides or fragments or variants thereof can be used, for example, in screening assays to identify new agonists or antagonists for GABA receptors. The new ENDOX polypeptides can also be used to modulate GABA receptor activity.

ENDOX polypeptides, nucleic acids, antibodies and other compositions according to the invention also include
It has utility based on other known functions of endozepine family members. For example, diazepam binding inhibitors also have acyl-CoA binding proteins (A
Known as CBP). Acyl-CoA binding proteins are medium chain acyl-C
It can bind oA esters and long chain acyl-CoA esters with high affinity and serve as an intracellular carrier for acyl-CoA esters.

The ACBP gene has also been cloned in yeast. This yeast family,
It was termed the acyl-CoA bond (ACB) (Rose et al., Proc. Natl.
Acad. Sci. USA 89: 11287-11291). This yeast gene encodes a polypeptide of 87 amino acid residues (including the initiation methionine) that is the same length as the human gene product. The yeast polypeptide contains human amino acid residues and 4
8% saved. The most highly conserved yeast domain was found to contain a total of 7 consecutive amino acid residues that are identical in all known protein species from yeast, avian and mammalian. This domain builds a hydrophobic binding site for the acyl-CoA ester and is located within the second helix region of the molecule. The presence of such a highly conserved gene in lower organisms (eg yeast) supports its basic biological role as an acyl-CoA binding protein and is also ascribed to it in higher organisms. Many biological functions are acyl-
It may result from its ability to interact with CoA.

Various endozepine family members, or derivatives of these polypeptides have also been identified as antibacterial peptides. For example, cecropin (ce
cropin) P1 and PR-39. PR-39 is a proline and arginine rich polypeptide of 39 amino acid residues isolated from the upper portion of the porcine small intestine. Amino acid sequence analysis in combination with mass spectrometry showed that gastric inhibitory polypeptide (7-42) (GIP (7-42)) and diazepam binding inhibitor (32-86) (DBI ( 32-86)) and identified 2 or 3 polypeptides. The third polypeptide assembled a previously unknown structure and was referred to as peptide 3910 in connection with its molecular weight.
All three polypeptides show antibacterial activity against Bacillus megaterium. GIP (7-42) is also Streptococcus
12 shows some activity against Escherichia coli mutants lacking pyogenes and outer membrane.

A summary of ENDOX nucleic acid sequences, encoded ENDOX polypeptides, and sequence identification numbers (SEQ ID NOs) corresponding to various disclosed sequences and clones containing these nucleic acids is provided in Table 28 below.

Table 28: Disclosed Sequences of ENDOX Polypeptides and Corresponding SEQ ID NOs:

[0200]

[Table 48] ENDOX Nucleic Acids and Polypeptides One aspect of the invention pertains to isolated nucleic acid molecules encoding ENDOX polypeptides or biologically active portions thereof. Also, a sufficient nucleic acid fragment for use as a hybridization probe to identify a nucleic acid encoding ENDOX (eg, ENDOX mRNA), and ENDO
Also included in the invention are fragments for use as PCR primers for the amplification and / or mutation of X nucleic acid molecules. As used herein,
The term "nucleic acid molecule" refers to a DNA molecule (eg, cDNA or genomic DNA), R
NA molecules (eg, mRNA), D produced using nucleotide analogs
It is intended to include analogs of NA or RNA, and their derivatives, fragments and homologs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably comprises double-stranded DNA.

The ENDOX nucleic acid can encode a mature ENDOX polypeptide. As used herein, the "mature" form of a polypeptide or protein disclosed in the present invention is a naturally occurring polypeptide or precursor form or product of a proprotein. Naturally occurring polypeptides, precursors or proproteins include, by way of non-limiting example, the full length gene product encoded by the corresponding gene. Alternatively, it is defined as a polypeptide, precursor or proprotein encoded by the open reading frame described herein. The product "mature" form also occurs, as a non-limiting example, as a result of one or more naturally occurring processing steps that can occur in the cell or host cell in which the gene product is produced. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include cleavage of the N-terminal methionine residue, or proteolysis of the signal peptide or leader sequence, encoded by the start codon of the open reading frame. Examples include sexual amputation. Thus, the mature form resulting from a precursor polypeptide or protein with residues 1-N, where residue 1 is the N-terminal methionine, is the residue 2 that remains after removal of the N-terminal methionine. Has an N-terminus. Alternatively, the mature form resulting from a precursor polypeptide or protein having residues 1-N (where the N-terminal signal sequence of residues 1-residue M is cleaved) is the remaining residues M + 1-residues N
Have. As further used herein, the "mature" form of a polypeptide or protein may result from a post-translational employment process other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation, or phosphorylation. In general, a mature polypeptide or protein may result from the action of only one of these processes, or any combination thereof.

As used herein, the term "probe" refers to nucleic acid sequences of various lengths, preferably at least about 10 nucleotides (depending on the particular use).
nt), 100 nt, or as large as about 6,000 nt, for example. The probe is used in the detection of identical, similar or complementary nucleic acid sequences.
Longer probes are usually obtained from natural or recombinant sources, are highly specific, and hybridize much slower than shorter oligomer probes. The probe can be single-stranded or double-stranded and is designed to have specificity in such techniques as PCR, membrane-based hybridization techniques or ELISA.

The term “isolated” nucleic acid molecule, as used herein, is one that is separated from other nucleic acid molecules that are present in the natural source of this nucleic acid. Preferably, an "isolated" nucleic acid has the sequences that naturally flank the nucleic acid in the genomic DNA of the organism from which it is derived (ie, the sequences located at the 5'and 3'ends of the nucleic acid). Not included. For example, in various embodiments, isolated ENDOX
A nucleic acid molecule is about 5 kb, 4 kb, 3 kb naturally flanking this nucleic acid in the genomic DNA of the cell / tissue from which it is derived (eg, brain, heart, liver, spleen, etc.).
It may contain less than 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence. Further, an "isolated" nucleic acid molecule, eg, a cDNA molecule, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or chemically synthesized, It may be substantially free of chemical precursors or other chemicals.

Nucleic acid molecules of the invention, eg SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28.
, 31, 34, 46 and 48 nucleotide sequences, or the complements of these nucleotide sequences, have been isolated using standard molecular biology techniques and the sequence information provided herein. obtain. Using all or part of the nucleic acids of SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 and 48 as hybridization probes, the ENDOX molecule can be labeled with standard high levels. Hybridization technology and cloning technology (for example, Sambrook et al. (Ed.), MOLECULAR CLONING: A LABORATORY
MANUAL 2nd Edition, Cold Spring Harbor Labora
tory Press, Cold Spring Harbor, NY, 198.
9; and Ausubel et al., (Eds.), CURRENT PROTOCOLS.
IN MOLECULAR BIOLOGY, John Wiley & Sons
, New York, NY, 1993)).

Nucleic acids of the invention are labeled as templates according to standard PCR amplification techniques with cDNA as template.
It can be amplified using NA, mRNA or genomic DNA, and appropriate oligonucleotide primers. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, E
Oligonucleotides corresponding to NDOX nucleotide sequences can be prepared by using standard synthetic techniques, such as automated DNA synthesizers.

As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases that can be used in a PCR reaction. Short oligonucleotide sequences can be based on, or designed from, genomic or cDNA sequences, and amplify, confirm, or confirm the presence, in a particular cell or tissue, of identical, similar or complementary DNA or RNA. Used to indicate. Oligonucleotides include portions of nucleic acid sequences having a length of about 10 nt, 50 nt, or 100 nt, preferably about 15 nt to 30 nt. In one embodiment of the invention, the oligonucleotide comprising a nucleic acid molecule of less than 100 nt has
Furthermore, it comprises at least 6 consecutive nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23, or the complement thereof.
Oligonucleotides can be chemically synthesized and used as probes.

In another embodiment, the isolated nucleic acid molecule of the invention is SEQ ID NO: 1, 3, 5, 7
, 9, 11, 13, 15, 17, 17, 19, 21 or 23, or a nucleic acid molecule that is the complement of a portion of this nucleotide sequence (
For example, a fragment that can be used as a probe or primer, or a fragment that encodes a biologically active site of an ENDOX polypeptide).
SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 and 48
Nucleic acid molecules that are complementary to the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5,
7, 9, 22, 25, 28, 31, 34, 46 and 48 are nucleic acid molecules that are sufficiently complementary to the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 22.
, 25, 28, 31, 34, 46 and 48 can hydrogen bond with little or no mismatches, thereby forming a stable duplex.

As used herein, the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between the nucleotide units of nucleic acid molecules, and the term “binding” refers to two polypeptides or compounds or related By physical or chemical interaction between polypeptides or compounds or combinations thereof. Binding includes ionic, nonionic, van der Waals, hydrophobic interactions and the like. Physical interactions can be either direct or indirect. The indirect interaction may be via another polypeptide or compound or due to its effect. Direct binding refers to interactions that do not occur through or due to the effects of another polypeptide or compound, but without other substantial chemical intermediates.

The fragments provided herein include at least 6 (consecutive) nucleic acid sequences or at least 4 (consecutive) amino acid sequences (respectively for specific hybridization in the case of nucleic acids). Enablement, or in the case of amino acids, sufficient length to allow specific recognition of the epitope), and at most some portion less than the full length sequence. Fragments can be derived from any contiguous portion of the selected nucleic acid or amino acid sequences. Derivatives are nucleic acid or amino acid sequences formed from a native compound either directly or by modification or partial substitution. An analog is a nucleic acid sequence or amino acid sequence that has a structure (but not identical) similar to that of the native compound, but differs with respect to a particular component or side chain. Analogs can be synthetic or they can be derived from evolutionarily different sources and have similar or opposite metabolic activity as compared to wild type. Homologues are nucleic acid or amino acid sequences of a particular gene that are derived from different species.

Derivatives and analogs can be full length, or other than full length, if the derivative or analog comprises a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention, in various embodiments, when compared to nucleic acids or proteins of the invention, over a nucleic acid or amino acid sequence of the same size, or to an aligned sequence (the alignment is Performed by computer homology programs known in the art), at least about 70%, 80%, or 95% identity (preferred identity is 80-9).
5%) or is substantially homologous to the above, or its encoding nucleic acid hybridizes to the complement of a sequence encoding the above protein under stringent, moderately stringent, or low stringent conditions. Includes, but is not limited to, molecules that include regions. For example, Ausubel et al., CUR
REN PROTOCOLS IN MOLECULAR BIOLOGY,
See John Wiley & Sons, New York, NY, 1993, and below.

“Homologous nucleic acid sequence” or “homologous amino acid sequence”, or variants thereof, refer to sequences characterized by homology at the nucleotide or amino acid level, as discussed above. The homologous nucleotide sequence is ENDO
Encodes a sequence encoding an isoform of the X polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, the isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include species other than humans, including but not limited to vertebrates, and thus, for example, frogs, mice, rats, rabbits, dogs, cats, cows, horses and other Organisms may be included). Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variants and variants of the nucleotide sequences described herein. But,
Homologous nucleotide sequences do not include nucleotide sequences that encode human ENDOX protein. Homologous nucleic acid sequences are SEQ ID NOs: 2, 4, 6, 8, 10, 15, 1
6, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 3
2, 33, 35 to 45, 47 and 49 conservative amino acid substitutions (see below), and nucleic acid sequences encoding polypeptides having ENDOX biological activity. The various biological activities of the ENDOX protein are described below.

The ENDOX polypeptide is an open reading frame of ENDOX nucleic acid (
"ORF"). The ORF corresponds to a nucleotide sequence that can potentially be translated into a polypeptide. The stretch of nucleic acid containing the ORF is not interrupted by the stop codon. The ORF showing the coding sequence of the complete protein is ATG
Starts with a "start" codon and three stop codons, namely TAA, TAG
, Or stop at one of the TGAs. For the purposes of the present invention, the ORF is a start codon,
It can be any part of the coding sequence with or without a stop codon, or both. ORFs that should be considered as good candidates for encoding true cellular proteins are often set with a minimum size requirement (eg, a stretch of DNA encoding a protein of 50 amino acids or more).

Nucleotide sequences determined from cloning of the human ENDOX gene are useful in identifying and / or cloning ENDOX homologs in other cell types (eg, from other tissues), as well as ENDOX homologs from other vertebrates. Allows the production of probes and primers designed for use. The probe / primer typically comprises a substantially purified oligonucleotide. This oligonucleotide is typically represented by SEQ ID NOs: 1, 3, 5, 7, 9,
22, 25, 28, 31, 34, 46 and 48 at least about 12, about 25
, About 50, about 100, about 150, about 200, about 250, about 300, about 350 or about 400 contiguous sense strand nucleotide sequences; or SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 and 48 antisense strand nucleotide sequences; or SEQ ID NOs: 1, 3, 5, 7, 9, 22
, 25, 28, 31, 34, 46 and 48 naturally occurring variants of at least about 12, about 25, about 50, about 100, about 150, about 200, about 2
A region of nucleotide sequence that hybridizes under stringent conditions to 50, about 300, about 350 or about 400 contiguous sense strand nucleotide sequences.

Probes based on human ENDOX nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a labeling group attached to the probe, eg, the labeling group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes may, for example, measure the level of nucleic acid encoding ENDOX in a sample of cells from a subject as part of a diagnostic test kit for identifying cells or tissues that misexpress the ENDOX protein. thing(
For example, detecting ENDOX mRNA levels, or genomic ENDOX
Determining whether a gene is mutated or deleted).

“Polypeptide having a biologically active portion of an ENDOX polypeptide” refers to a polypeptide that exhibits an activity that is similar but not necessarily identical to that of the polypeptide of the invention, with or without dose dependence. Regardless, it includes the mature form as measured in a particular biological assay. "Biologically active part of ENDOX"
A nucleic acid fragment encoding SEQ ID NO: 1, 3, 5, 7, 9, 22, which encodes a polypeptide having the biological activity of ENDOX (the biological activity of the ENDOX protein is described below). , 25, 28, 31, 34, 46 and 48, expressing the encoded portion of the ENDOX protein (eg, by recombinant expression in vitro) and the activity of the encoded portion of ENDOX. Can be prepared by assessing

Variants of ENDOX Nucleic Acids and Polypeptides The present invention further comprises SEQ ID NOs: 1, 3, 5, 7, 9 due to the degeneracy of the genetic code.
, 22, 25, 28, 31, 34, 46 and 48, and nucleic acid molecules that differ from the nucleotide sequences shown. Thereby, these nucleic acids are
It encodes the same ENDOX protein as the protein encoded by the nucleotide sequences shown in 5, 7, 9, 22, 25, 28, 31, 34, 46 and 48. In another embodiment, the isolated nucleic acid molecule of the invention is SEQ ID NO: 2.
4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24
, 26, 27, 29, 30, 32, 33, 35 to 45, 47 and 49 having a nucleotide sequence encoding a protein having an amino acid sequence.

SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 and 4
It will be appreciated by those skilled in the art that in addition to the human ENDOX nucleotide sequence shown in 8, DNA sequence polymorphisms leading to changes in the amino acid sequence of the ENDOX polypeptide may be present in the population (eg, human population). Such genetic polymorphisms in the ENDOX gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to an ENDOX protein, preferably a vertebrate ENDOX.
A nucleic acid molecule containing an open reading frame (ORF) that encodes a protein. Such natural allelic variations are typically found in EN.
Variability in the nucleotide sequence of the DOX gene can occur from 1 to 5%. Any and all such nucleotide variations within the ENDOX polypeptide and resulting amino acid polymorphisms that are the result of natural allelic variation and do not alter the functional activity of the ENDOX polypeptide are within the scope of the invention. Is intended to be.

In addition, it encodes an ENDOX protein from other species, and is therefore SEQ ID NO: 1.
Nucleic acid molecules having nucleotide sequences that differ from the human sequences of 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 and 48 are intended to be within the scope of the present invention. Nucleic acid molecules corresponding to naturally occurring allelic variants and homologs of the ENDOX cDNA of the present invention are based on their homology to the human ENDOX nucleic acids disclosed herein under standard stringent hybridization conditions. Can be isolated using human cDNA or a portion thereof as a hybridization probe according to conventional hybridization techniques.

Thus, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and, under stringent conditions, SEQ ID NOs: 1, 3, 5, 7,
, 9, 22, 25, 28, 31, 34, 46 and 48 nucleotide sequences. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500 or 2000 or more nucleotides in length. In another embodiment, the isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term “hybridize under stringent conditions” refers to conditions under which hybridization, and washing, nucleotide sequences that are at least 60% homologous to one another typically remain hybridized to each other. Intended to be listed.

Homologs (ie, nucleic acids encoding ENDOX proteins from species other than human) or other related sequences (eg, paralogs) are probed with all or part of a particular human sequence and are labeled with nucleic acid hybrids. It can be obtained by low, medium, or high stringency hybridization using methods well known in the art for hybridization and cloning.

As used herein, the phrase “stringent hybridization conditions” refers to under which a probe, primer or oligonucleotide is
It is a condition that hybridizes to the target sequence but does not hybridize to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5 ° C below the thermal melting point (Tm) of a particular sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm 50% of the probe is occupied in equilibrium. Typically, stringent conditions are pH 7.0-8.3 and salt concentrations less than about 1.0 M sodium ion, typically about 0.01-1.0.
M sodium ion (or other salt), and temperature at least about 30 ° C. for short probes, primers or oligonucleotides (eg 10 nt to 50 nt) and at least about 60 ° C. for longer probes, primers and oligonucleotides. It is a condition. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Stringent conditions are known to those of skill in the art and are described by Ausubel et al. (Eds.), CURRENT PROTOCOLS IN MOLECULAR BIO.
LOGY, John Wiley & Sons, N.M. Y. (1989), 6.
It can be found in 3.1-6.3.6. Preferably, this condition is such that sequences that are at least about 65%, about 70%, about 75%, about 85%, about 90%, about 95%, about 98% or about 99% homologous to each other, typically to each other. Conditions are such that they will remain hybridized. A non-limiting example of stringent hybridization conditions is 6 × SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDT.
Hybridization at 65 ° C. in high salt buffer containing A, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg / ml denatured salmon sperm DNA. For this hybridization, 0.2 x SSC
Followed by one or more washes in 0.01% BSA at 50 ° C. SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 under stringent conditions
An isolated nucleic acid molecule of the present invention that hybridizes to sequences 48 and 48 corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally occurring” nucleic acid molecule is a naturally occurring (eg, encodes a naturally occurring protein).
An RNA or DNA molecule having a nucleotide sequence.

In a second embodiment, SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31.
, 34, 46 and 48, or fragments, analogs or derivatives thereof, are provided with nucleic acid sequences capable of hybridizing under moderate stringency conditions to nucleic acid molecules. A non-limiting example of moderate stringency hybridization conditions is hybridization at 55 ° C. in 6 × SSC, 5 × Denhardt's solution, 0.5% SDS and 100 mg / ml denatured salmon sperm DNA, followed by One or more washes at 37 ° C. in 1 × SSC, 0.1% SDS. Other conditions of moderate stringency that can be used are well known in the art. For example, Ausubel et al. (Eds.), 1993, CUR.
REN PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, NY and Kriegler, 1990.
, GENE TRANSFER AND EXPRESSION, A LABO
See RATORY MANUAL, Stockton Press, NY.

In a third embodiment, SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31.
, 34, 46 and 48, or fragments, analogs or derivatives thereof, are provided with nucleic acids capable of hybridizing under low stringency conditions to a nucleic acid molecule. A non-limiting example of low stringency hybridization conditions is 35% formamide, 5 × SSC, 50 mM Tr.
is-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.0
2% Ficoll, 0.2% BSA, 100 mg / ml denatured salmon sperm DN
A. Hybridization in 10% (weight / volume) dextran sulphate at 40 ° C., followed by 2 × SSC, 25 mM Tris-HCl (pH 7.4), 5 m.
One or more washes at 50 ° C. in M EDTA and 0.1% SDS.
Other conditions of low stringency that can be used are well known in the art (eg, as used for cross-species hybridization). For example, Au
subel et al. (ed.), 1993, CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley & Sons,
NY and Kriegler, 1990, GENE TRANSFER AN
D EXPRESSION, A LABORATORY MANUAL, Sto
ckton Press, NY; Shilo and Weinberg, 1981.
, Proc Natl Acad Sci USA 78: 6789-6792.
checking ...

Conservative Variations In addition to naturally occurring allelic variants of ENDOX sequences that may be present in the population, one of skill in the art would appreciate that SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34
It is further understood that mutations to the nucleotide sequences of C., 46 and 48 may introduce changes that result in changes in the amino acid sequence of the encoded ENDOX protein without altering the functional capacity of the ENDOX protein. . For example, nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues are SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 2.
It can be performed in the 0, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45, 47 and 49 sequences. "Non-essential" amino acid residues are those residues that can be altered from the wild-type sequence of the ENDOX protein without altering biological activity, while "essential" amino acid residues are responsible for biological activity. Needed. For example, amino acid residues conserved among the ENDOX proteins of the invention are predicted to be particularly amenable to alteration. Amino acids for which conservative amino acid substitutions are made are well known in the art.

Another aspect of the invention comprises changes in amino acid residues that are not essential for activity,
It relates to a nucleic acid molecule encoding the ENDOX protein. Such ENDOX protein has an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 15, 16, 1.
7, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 3
3, 35 to 45, different from 47 and 49, but still retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence that encodes a protein, wherein the protein is SEQ ID NO: 2,4,6,8,
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27,
29, 30, 32, 33, 35 to 45, including amino acid sequences that are at least about 45% homologous to the amino acid sequences of 47 and 49. Preferably, the protein encoded by this nucleic acid molecule is SEQ ID NO: 2, 4, 6, 8, 10, 15.
, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30
, 32, 33, 35 to 45, 47 and 49 with at least about 60% homology; more preferably SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 17.
8, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 3
5 to 45, at least about 70% homologous to 47 and 49; and even more preferably SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19,
20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45
Up to at least about 80% homology to 47 and 49; and even more preferably SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 2.
1, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45, 4
7 and 49 with at least about 90% homology; and most preferably SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23.
, 24, 26, 27, 29, 30, 32, 33, 35 to 45, 47 and 49 with at least about 95% homology.

SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21
, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45, 47
Isolated nucleic acid molecules encoding ENDOX proteins that are homologous to the proteins of SEQ ID NOs: 49, 49
6 and 48 nucleotide sequences can be made by introducing one or more nucleotide substitutions, additions or deletions, such that one or more amino acid substitutions, additions or deletions are introduced in the encoded protein. It

Mutations are standard techniques (eg, site-directed mutagenesis and PCR-mediated mutagenesis).
By SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20
, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 45, 47 and 49. Preferably, conservative amino acid substitutions are made at one or more putative nonessential amino acid residues. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain.
A family of amino acid residues with similar side chains is defined in the art.
These families include: amino acids with basic side chains (eg lysine, arginine, histidine), amino acids with acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains. Amino acids (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine,
Cysteine), amino acids with non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (eg threonine, valine, isoleucine) and aromatics. Amino acids with side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in the ENDOX protein is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, the mutations are along all or part of the ENDOX coding sequence (eg, saturation mutagenesis).
can be randomly introduced) and the resulting mutant is E
NDOX can be screened for biological activity to identify variants that maintain the activity. SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19
, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35 to 4
Up to 5, following mutagenesis of 47 and 49, the encoded protein can be expressed by any recombinant technique known in the art, and the activity of this protein can be determined.

In one embodiment, mutant ENDOX proteins can be assayed for: (i) other ENDOX proteins, other cell surface proteins, or biologically active portions thereof, and protein: protein interactions. The ability to form an effect, (ii) the formation of a complex between a mutant ENDOX protein and an ENDOX ligand; or (iii) the ability of the mutant ENDOX protein to bind to an intracellular target protein or biologically active portion thereof; (Eg avidin protein)
.

In yet another embodiment, mutant ENDOX proteins can be assayed for their ability to modulate a particular biological function (eg, modulation of insulin release).

(Antisense Nucleic Acid) Another aspect of the present invention is SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31.
, 34, 46 and 48 or fragments, analogs or derivatives thereof, capable of hybridising to or complementary to a nucleic acid molecule comprising a nucleotide sequence.
It relates to an isolated antisense nucleic acid molecule. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid that encodes a protein (eg, complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence). . In a particular aspect, an antisense nucleic acid molecule comprising a sequence complementary to at least about 10, about 25, about 50, about 100, about 250 or about 500 nucleotides or the entire ENDOX coding strand, or only a portion thereof, is provided. Provided.
SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21,
23, 24, 26, 27, 29, 30, 32, 33, 35 to 45, 47 and 49 fragments, homologues, derivatives and analogs of the ENDOX proteins, or SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25,
Further provided are antisense nucleic acids complementary to the nucleic acid sequences of 28, 31, 34, 46 and 48 ENDOX.

[0232] In one embodiment, the antisense nucleic acid molecule is antisense to the "coding region" of the coding strand of a nucleotide sequence encoding an ENDOX protein. The term "coding region" refers to the region of nucleotide sequence that contains codons translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is E
It is antisense to the "non-coding region" of the coding strand of the nucleotide sequence encoding the NDOX protein. The term “non-coding region” refers to the 5 ′ and 3 ′ sequences that are flanked by coding regions and are not translated into amino acids (ie, 5 ').
Also called'untranslated region and 3'untranslated region).

Given the coding strand sequences encoding the ENDOX proteins disclosed herein, the antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of ENDOX mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or non-coding region of ENDOX mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ENDOX mRNA. Antisense oligonucleotides have, for example, lengths of about 5, about 10,
It can be about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50 nucleotides. The antisense nucleic acids of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) are formed to naturally occur nucleotides, or to increase the biological stability of this molecule, or between antisense and sense nucleic acids. It can be chemically synthesized with variously modified nucleotides designed to increase the physical stability of the duplex (eg phosphorothioate derivatives and acridine substituted nucleotides can be used).

Examples of modified nucleotides that can be used to make antisense nucleic acids include: 5-fluorouracil, 5-bromouracil, 5-
Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β- D-galacto silk eosin (galacto
sylqueosine), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6.
-Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyleosin (man
nosylqueosine), 5'-methoxycarboxymethyluracil, 5
-Methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wibutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2,
-Thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N- 2-carboxypropyl) uracil,
(Acp3) w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be biologically produced using an expression vector in which the nucleic acid has been subcloned in the antisense orientation (ie, RNA transcribed from the inserted nucleic acid is further described in the subsection below). Antisense orientation to the target nucleic acid of interest).

The antisense nucleic acid molecules of the invention are typically administered to a subject or produced in situ so that they hybridize to the cellular mRNA and / or genomic DNA encoding the ENDOX protein. Soybeans or binds thereto, thereby inhibiting the expression of this protein, for example by inhibiting transcription and / or translation. Hybridization can be by conventional nucleotide complementation to form a stable duplex, or in the case of antisense nucleic acid molecules that bind to a DNA duplex, for example, the major groove of the double helix.
via specific interactions in r groove). Examples of routes of administration of the antisense nucleic acid molecules of the invention include direct injection at the tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to a receptor or antigen expressed on the surface of selected cells. This is, for example, by linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. The antisense nucleic acid molecule can also be delivered to cells using the vectors described herein. In order to achieve a sufficient amount of nucleic acid molecule, the antisense nucleic acid molecule has a strong pol II promoter or pol
Vector constructs that are under the control of the III promoter are preferred.

[0236] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with complementary RNA. Here, in contrast to the normal β-unit, the chains run parallel to each other (eg Gaultier et al. (1987) Nucl. Ac).
ids Res 15: 6625-6641). The antisense nucleic acid molecule also includes 2'-o-methyl ribonucleotides (eg, Inoue et al.
(1987) Nucl. Acids Res 15: 6131-6148) or chimeric RNA-DNA analogs (eg, Inoue et al. (19).
87) FEBS Lett 215: 327-330).

(Ribozyme and PNA moieties) As non-limiting examples, nucleic acid modifications include modified bases and nucleic acids with modified or derivatized sugar phosphate backbones. These modifications are made to at least partially enhance the chemical stability of the modified nucleic acids so that they can be used as antisense binding nucleic acids, eg, in therapeutic applications in a subject.

[0238] In one embodiment, the antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules having ribonuclease activity that can cleave single-stranded nucleic acids (eg, mRNA), and these have a complementary region to the single-stranded nucleic acid. Therefore, ribozymes (for example, hammerhead ribozyme (Ha
selhoff and Gerlach (1988) Nature 334: 58.
5-591))) can be used to catalytically cleave ENDOX mRNA transcripts, thereby inhibiting translation of ENDOX mRNA. ENDOX
Ribozymes having specificity for nucleic acids encoding the EN are disclosed in EN herein.
The nucleotide sequence of the DOX cDNA (ie, SEQ ID NOs: 1, 3, 5, 7, 9
, 22, 25, 28, 31, 34, 46, and 48). For example, the active site nucleotide sequence is complementary to a nucleotide sequence that is cleaved within the mRNA encoding ENDOX, Tetrahymena L-.
Derivatives of 19 IVS RNA can be constructed. See, eg, Cech et al., US Pat. No. 4,987,071; and Cech et al., US Pat. No. 5,116,742. ENDOX mRNA may also be used to select catalytic RNAs with specific ribonuclease activity from a pool of RNA molecules. For example, B
See artel et al. (1993) Science 261: 1411-1418.

Alternatively, ENDOX gene expression is regulated by a regulatory region of an ENDOX nucleic acid (eg, E
It can be inhibited by targeting a nucleotide sequence complementary to the promoter and / or enhancer of NDOX) and forming a triple helix structure that interferes with transcription of the ENDOX gene in target cells. For example, Helene (1991) A
nt cancer Drug Des. 6: 569-84; Helene et al.
1992) Ann. N. Y. Acad. Sci. 660: 27-36; and M
See aher (1992) Bioassays 14: 807-15.

In various embodiments, the ENDOX nucleic acid can be modified at the base, sugar, or phosphate backbones to improve, for example, the stability, hybridization or solubility of the molecule. For example, by modifying the deoxyribose phosphate backbone of nucleic acids,
Peptide nucleic acids can be produced (Hyrup et al. (1996) Bioorg Med.
See Chem 4: 5-23). As used herein, the term "
“Peptide nucleic acid” or “PNA” refers to the deoxyribose phosphate backbone being replaced by a pseudo-peptide backbone, and the four natural nucleobases.
A nucleic acid mimic in which only is retained (eg, a DNA mimic). The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers is described by Hyrup et al. (1996); Perry-O'Keefe et al. (199), supra.
6) Proc. Natl. Acad. Sci. USA 93: 14670-14.
Standard solid phase peptide synthesis protocol as described at 675.

ENDOX PNAs can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for sequence-specific regulation of gene expression, for example by inducing transcription or translation arrest or inhibiting replication. ENDOX PNAs can also be engineered when used in combination with other enzymes (eg, S ₁ nuclease), eg, in the analysis of single base pair mutations in genes (eg, PNA-directed PCR clamping). As a restriction enzyme (Hyrup et al. (1
996) supra); or as a probe or primer for DNA sequences and hybridization (Hyrup et al. (1996) supra; Per.
ry-O'Keefe et al. (1996), supra), can be used.

In another embodiment, ENDOX PNAs are conjugated to PNA-DNA chimeras, eg, by attaching a lipophilic group or other helper group to the PNAs to enhance their stability or cellular uptake. Or by the use of liposomes or other techniques of drug delivery known in the art. For example, END, which may combine the advantageous properties of PNA and DNA
OX PNA-DNA chimeras can be generated. While the PNA moiety provides high binding affinity and specificity, such chimeras have DNA recognition enzymes (eg, R
Nase H and DNA polymerase) to interact with the DNA portion. The PNA-DNA chimera is composed of base stacking,
It may be ligated using a linker of appropriate length chosen with regard to the number and orientation of the bonds between the nucleobases (see Hyrup et al. (1996) supra). PNA-
The synthesis of DNA chimeras is described by Hyrup et al. (1996) supra and Finn et al. (19).
96) can be performed as described in Nucl Acids Res 24: 3357-63. For example, DNA strands can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs (eg, 5 '-(4-methoxytrityl) amino-5'-. Deoxy-thymidine phosphoramidite) can be used between the PNA and the 5'end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-598).
8). The PNA monomer is then coupled in a stepwise fashion, 5
A chimeric molecule having a'PNA segment and a 3'DNA segment is generated (see, eg, Finn et al. (1996) supra). Alternatively, 5'DNA
Chimeric molecules can be synthesized using the segment and the 3'PNA segment.
For example, Petersen et al. (1975) Bioorg Med Chem L.
See ett 5: 1119-11124.

In other embodiments, the oligonucleotide may include other accessory groups such as: peptides (eg, for targeting host cell receptors in vivo), or to facilitate transport across cell membranes. Factors (eg Letsinger
Et al., 1989, Proc. Natl. Acad. Sci. U. S. A. 86: 6
553-6556; Lemaitre et al., 1987, Proc. Natl. Ac
ad. Sci. 84: 648-652; PCT Publication No. WO88 / 09810), or the blood-brain barrier (eg PCT Publication No. WO89 / 1013).
4). In addition, the oligonucleotide may be conjugated to a hybridization-triggered cleavage agent (eg, Krol et al., 1988, BioTechniques 6:
958-976), or an intercalating agent (eg, Zon,
1988, Pharm. Res. 5: 539-549)). To this end, the oligonucleotide may be linked to another molecule, such as a peptide, hybridization-triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, and the like.

(ENDOX Polypeptide) The polypeptide of the present invention has a sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 15,
16, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30,
32, 33, 35-45, 47, and 49, including proteins comprising the amino acid sequence of the ENDOX polypeptide. The present invention also relates to the ENDOX.
Encodes a protein that retains activity and physiological function, or a functional fragment thereof, where any residue is SEQ ID NO: 2, 4, 6, 8, 10, 15, 16
, 17, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32
, 33, 35-45, 47, and 49, including mutant or variant proteins that can be altered from the corresponding residues.

In general, ENDOX variants that retain ENDOX-like function have a residue at a particular position in the sequence replaced by another amino acid, and further, insert an additional residue between the two residues of the parent protein. It includes any variant, including the possibility, and the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion or deletion is included in the present invention. In the preferred situation, this substitution is a conservative substitution as defined above.

One aspect of the present invention relates to an isolated ENDOX protein, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Polypeptide fragments suitable for use as immunogens to raise anti-ENDOX antibodies are also provided. In one embodiment, the native E
NDOX protein can be isolated from cell or tissue sources by a suitable purification scheme using standard protein purification techniques. In another embodiment, END
OX protein is produced by recombinant DNA techniques. As an alternative to recombinant expression, the ENDOX protein or polypeptide can be chemically synthesized using standard peptide synthesis techniques.

An "isolated" or "purified" polypeptide or protein or biologically active portion thereof is a cellular material or other contaminating protein derived from the cell or tissue source from which the ENDOX protein is derived. Or substantially no chemical precursors or other chemicals when chemically synthesized. The term "substantially free of cellular material" includes a preparation of ENDOX protein in which the protein is separated from the cellular components of cells in which the protein is isolated or recombinantly produced. . In one embodiment, the term "substantially free of cellular material" refers to less than about 30% (by dry weight) of non-ENDOX protein (also referred to herein as "contaminant protein"), more preferably. Less than about 20% non-ENDOX protein, even more preferably non-E
Included are preparations of ENDOX protein having less than about 10% NDOX protein, and most preferably less than about 5% non-ENDOX protein. If the ENDOX protein or biologically active portion thereof is recombinantly produced, preferably the preparation is also substantially free of culture medium. That is, the culture medium is the EN
Less than about 20% of the volume of the DOX protein preparation, more preferably less than about 10%,
And most preferably less than about 5%.

The term “substantially free of chemical precursors or other chemicals” refers to a preparation of ENDOX proteins in which the protein is separated from the chemical precursors or other chemicals involved in the synthesis of that protein. including. In one embodiment, the term "
“Substantially free of chemical precursors or other chemicals” refers to chemical precursors or non-E
Less than about 30% NDOX chemical (by dry weight), more preferably less than about 20% chemical precursor or non-ENDOX chemical, and even more preferably less than about 10% chemical precursor or non-ENDOX chemical. , And most preferably, a preparation of ENDOX protein having less than about 5% chemical precursors or non-ENDOX chemicals.

The biologically active portion of the ENDOX protein comprises fewer amino acids than the full-length ENDOX protein and exhibits at least one activity of the ENDOX protein (eg, SEQ ID NOs: 2, 4, 6).
, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26,
27, 29, 30, 32, 33, 35-45, 47, and 49), or a peptide containing an amino acid sequence derived from the amino acid sequence of the ENDOX protein. Typically, the biologically active portion comprises at least one active domain or motif of the ENDOX protein. The biologically active portion of the ENDOX protein can be, for example, a polypeptide that is 10, 25, 50, 100 or more amino acid residues in length.

In addition, other biologically active moieties in which other regions of the protein are deleted are:
It can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native ENDOX protein.

[0251] In one embodiment, the ENDOX protein is SEQ ID NO: 2, 4, 6, 8.
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27
, 29, 30, 32, 33, 35-45, 47, and 49. In another embodiment, the ENDOX protein is SEQ ID NO: 2,
4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 23, 24,
26, 27, 29, 30, 32, 33, 35-45, 47, and 49, and is due to natural allelic modification or mutagenesis, as discussed in detail below. Amino acid sequence is different, but SEQ ID NOs: 2, 4, 6, 8
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27
, 29, 30, 32, 33, 35-45, 47, and 49 proteins retain the functional activity. Thus, in another embodiment, the ENDOX protein is
SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21,
23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47, and 49 amino acid sequences that are at least about 45% homologous to the amino acid sequences, and
SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21,
23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47, and 49 are proteins that retain the functional activity of the ENDOX proteins.

Determination of Homology between Two or More Sequences To determine the% homology of two amino acid sequences or two nucleic acids, the sequences are aligned for the purpose of optimal comparison. (For example, a gap may be introduced in the sequence of the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. The position in the first array is
A molecule is homologous at that position if it is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence (ie, as used herein, "homology" of amino acids or nucleic acids). Is equivalent to "identity" of amino acids or nucleic acids).

Nucleic acid sequence homology can be determined as the degree of identity between two sequences. Homology can be determined using computer programs known in the art, such as GAP software provided in the GCG program package. Needleman and Wunsch 1970 J Mol Biol
48: 443-453. The following settings for comparing nucleic acid sequences (GA
Using the GCG GAP software with a P production penalty of 5.0, and a GAP extension penalty of 0.3), the coding regions for similar nucleic acid sequences referred to above are SEQ ID NOs: 1, 3, 5, 7 , 9, 22, 25, 28, 31, 34
, 46, and 48 with the CDS (coding) portion of the DNA sequence, preferably at least 70%, at least 75%, at least 80%, at least 85%
, At least 90%, at least 95%, at least 98% or at least 9
Shows a degree of identity of 9%.

The term “sequence identity” refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term “percentage of sequence identity” is calculated by: comparing two sequences that are optimally aligned over this region of comparison, the same nucleobase in both sequences (eg A in the case of nucleic acids). , T, C, G, U, or I) determines the number of positions that are present and derives the number of matched positions;
Divide by the total number of positions within the comparison region (ie, window size) and multiply the result by 100 to derive the percentage sequence identity. The term "substantial identity" as used herein refers to a characteristic of a polynucleotide sequence, where the polynucleotide has at least 80% of the sequence compared to the reference sequence over the comparison region. Includes sequences that have identity, preferably at least 85% identity, and often 90-95% sequence identity, more usually at least 99% sequence identity.

Chimeric and Fusion Proteins The present invention also provides ENDOX chimeric or fusion proteins. As used herein, ENDOX "chimeric protein" or END
An OX "fusion protein" comprises an ENDOX polypeptide operably linked to a non-ENDOX polypeptide. "ENDOX polypeptide" is ENDOX
Proteins (SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19,
20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45,
47 and 49), which refers to a polypeptide having an amino acid sequence corresponding to
A "non-ENDOX polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the ENDOX protein (eg, a protein that is different from the ENDOX protein and is derived from the same or a different organism). . In the ENDOX fusion protein, the ENDOX polypeptide may correspond to all or a portion of the ENDOX protein. In one embodiment, the ENDOX fusion protein comprises at least one biologically active portion of the ENDOX protein. In another embodiment, the ENDOX fusion protein comprises at least two biologically active portions of the ENDOX protein. In yet another embodiment, the ENDOX fusion protein comprises at least 3 biologically active portions of the ENDOX protein. In the fusion protein, the term "
"Operably linked" is intended to indicate that the ENDOX polypeptide and the non-ENDOX polypeptide are fused to each other in frame.
The non-ENDOX polypeptide can be fused to the N-terminus or C-terminus of the ENDOX polypeptide.

In one embodiment, the fusion protein is a GST-ENDOX fusion protein, wherein the ENDOX sequence is fused to the C-terminus of the GST (ie glutathione S-transferase) sequence. Such fusion proteins may facilitate the purification of recombinant ENDOX polypeptide.

In another embodiment, the fusion protein is an ENDOX protein containing a heterologous signal sequence at its N-terminus. In certain host cells, such as mammalian host cells, ENDOX expression and / or secretion can be increased through the use of heterologous signal sequences.

In yet another embodiment, the fusion protein is an ENDOX-immunoglobulin fusion protein, wherein the ENDOX sequences are fused to sequences derived from a member of the immunoglobulin protein family. This EN of the present invention
The DOX-immunoglobulin fusion protein is incorporated into a pharmaceutical composition and administered to a subject to inhibit the interaction between ENDOX ligand and ENDOX protein on the surface of cells, thereby in vivo. It may suppress ENDOX-mediated signaling. This ENDOX-immunoglobulin fusion protein can be used to influence the bioavailability of ENDOX cognate ligands. Inhibition of ENDOX Ligand / ENDOX Interaction Modulates (eg, Promotes or Inhibits) Treatment of Both Proliferative and Differentiation Disorders, and Cell Survival
For, may be therapeutically useful. In addition, this ENDOX-immunoglobulin fusion protein of the invention can be used as an immunogen to produce anti-ENDOX antibodies in a subject, to purify ENDOX ligand and inhibit ENDOX interaction with ENDOX ligand. Can be used in a screening assay to identify the molecule that is to be processed.

The ENDOX chimera or fusion proteins of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences may be labeled according to conventional techniques, eg, blunt or staggered ends for ligation, restriction enzyme digestion to provide suitable ends, and optionally sticky ends. Incorporation is accomplished by employing filling-in at the (cohesive) ends, alkaline phosphatase treatment to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed with anchor primers that result in complementary overhangs between two consecutive gene fragments, which can then be annealed and reamplified to generate chimeric gene sequences (eg, Ausbel et al. (Edit) CURRENT PROTOCOLS IN MOLECULAR BI
See OLOGY, John Wiley & Sons, 1992). In addition, many expression vectors are commercially available that already encode a fusion component (eg, a GST polypeptide). In the nucleic acid encoding ENDOX, this fusion component is EN
It can be cloned into such an expression vector so that it is linked in-frame to the DOX protein.

ENDOX Agonists and Antagonists The present invention also relates to variants of ENDOX proteins that function as ENDOX agonists (mimetics) or as ENDOX antagonists. ENDO
Variants of the X protein, such as discrete point mutations or truncations of the ENDOX protein, can be generated by mutagenesis. An agonist of an ENDOX protein has substantially the same biological activity as the naturally occurring form of ENDOX protein,
Or it may retain a subset of its biological activity. Antagonists of ENDOX proteins may inhibit one or more activities of a naturally occurring form of ENDOX protein, eg, by competitively binding to a downstream or upstream member of a cell signaling cascade that includes the ENDOX protein. Therefore, specific biological effects can be elicited by treatment with variants of limited function. In one embodiment, treatment of the subject with a variant having a subset of biological activities of the naturally occurring form of the protein is performed on the subject relative to treatment with the naturally occurring form of the ENDOX protein. Have less side effects in.

A variant of the ENDOX protein that functions as either an ENDOX agonist (mimetic) or as an ENDOX antagonist is a variant of the ENDOX protein for ENDOX protein agonist or antagonist activity (eg, a truncation variant). It can be identified by screening combinatorial libraries. In one embodiment, a variegated library of ENDOX variants is generated by nucleic acid level combinatorial mutagenesis and is encoded by a variegated gene library. A diverse library of ENDOX variants, for example, a mixture of synthetic oligonucleotides and
The degenerate set of sequences may be ENDO as an individual polypeptide or in
It can be produced by enzymatically linking so that it can be expressed as a larger set of fusion proteins (eg, for phage display) containing a set of X sequences. There are a variety of methods that can be used to generate a library of potential ENDOX variants from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed in an automated DNA synthesizer, which synthetic gene is then ligated into an appropriate expression vector. The use of a degenerate set of genes allows for the full supply of sequences encoding the desired set of potential ENDOX sequences in one mixture. Methods of synthesizing degenerate oligonucleotides are known in the art (eg, Narang (1983) Tetrahedron 39).
: 3; Itakura et al. (1984) Annu Rev Biochem 53.
: 323; Itakura et al. (1984) Science 198: 1056;
Ike et al. (1983) Nucl Acid Res 11: 477).

Polypeptide Libraries In addition, a library of fragments of ENDOX protein coding sequences can be used to generate a diverse population of ENDOX fragments for screening and subsequent selection of variants of ENDOX protein. In one embodiment,
The library of coding sequence fragments is a double-stranded PC of ENDOX coding sequences.
Treating the R fragment with a nuclease under conditions that produce only about one nick per molecule, denaturing the double-stranded DNA, a duplex that may contain sense / antisense pairs from different nick products. Regenerating this DNA to form, removing the single-stranded portion from the reconstituted double-stranded by treatment with S ₁ nuclease, and ligating the resulting fragment library into an expression vector Can be generated by By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the ENDOX protein.

Several techniques are known in the art for screening gene products of combinatorial libraries generated by point mutations or truncations, and for screening cDNA libraries of gene products having selected properties. . Such techniques are applicable to the rapid screening of gene libraries generated by combinatorial mutagenesis of ENDOX proteins. The most widely used technique suitable for high-throughput analysis for screening large gene libraries is typically cloning the gene library into a replicable expression vector, in which the resulting library of vectors is Transforming appropriate cells, and expressing the combinatorial gene under conditions under which detection of the desired activity facilitates isolation of the vector encoding the gene for which the product was detected. A novel technique for increasing the frequency of functional variants in libraries, Recruitable Ensemble Mutagenesis (REM), can be used in combination with screening assays to identify ENDOX variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA
89: 7811-7815; Delgrave et al. (1993) Protein.
Engineering 6: 327-331).

Anti-ENDOX Antibodies The present invention includes antibodies or antibody fragments such as F _ab or (F _ab ) ₂ that immunospecifically bind to a PNDOX polypeptide of the present invention.

The isolated ENDOX protein, or portion or fragment thereof, is
It can be used as an immunogen to generate antibodies that bind ENDOX using standard techniques for polyclonal and monoclonal antibody preparation. The full length ENDOX protein can be used, but alternatively the invention provides antigenic peptide fragments of ENDOX for use as an immunogen. EN
The antigenic peptide of DOX is SEQ ID NO: 2, 4, 6, 8, 10, 15, 16, 17.
, 18, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33
Antibodies comprising at least 4 amino acid residues of the amino acid sequences shown in 35-45 (inclusive), 47 and 49, and raised against this peptide are EN
It contains an epitope of ENDOX so that it forms a specific immune complex with DOX. Preferably, the antigenic peptide is at least 6,8,10,15,20,
Or it contains 30 amino acid residues. Longer antigenic peptides are often preferred over shorter antigenic peptides, depending on the use and methods well known to those skilled in the art.

In certain embodiments of the invention at least one encompassed by an antigenic peptide.
One epitope is a region located on the surface of the ENDOX protein, eg, a hydrophilic region. As a means of targeting antibody production, hydropathic plots showing hydrophilic and hydrophobic regions, for example with or without a Fourier transform,
It may be produced by any method known in the art, including the Kyte Doolittle or Hopp Woods method. For example, Hopp and Woods, 1981, Proc., Which are incorporated herein by reference in their entirety. Nat.
Acad. Sci. USA 78: 3824-3828; Kyte and Doo.
little 1982, J. Mol. Biol. 157: 105-142, which are hereby incorporated by reference in their entireties.

As disclosed herein, ENDOX proteins of SEQ ID NOs: 2, 4, 6, 8, 10 or derivatives, fragments, analogs or homologues thereof are immunospecifically bound to these protein components. Can be used as an immunogen in the generation of antibodies. As used herein, the term "antibody" includes immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, antigen binding sites that specifically bind (immunoreact) with an antigen such as ENDOX. Refers to a molecule. Such antibodies include, without limitation, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, _Fab and F _{(ab ') 2} fragments, and _Fab expression libraries. In certain embodiments, antibodies to human ENDOX protein are disclosed.
Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies against the ENDOX protein sequences of SEQ ID NOs: 2, 4, 6, 8, 10 or derivatives, fragments, analogs or homologues thereof. Some of these proteins are discussed below.

For production of polyclonal antibodies, injection of a variety of suitable host animals (eg, rabbit, goat, mouse or other mammal) with native protein, or synthetic variants thereof, or derivatives thereof as described above. Can be immunized with. Suitable immunogenic preparations can contain, for example, recombinantly expressed ENDOX protein or a chemically synthesized polypeptide. The preparation may further include an adjuvant. Various adjuvants used to increase the immunological response include Freund's complete and incomplete adjuvants, mineral gels (eg aluminum hydroxide), surface-active substances (eg lysolecithin, pluronic polyols, polyanions, peptides, Oil emulsion, dinitrophenol, etc.), Bacille Calmette-Guerin and Coryneba
Examples include, but are not limited to, human adjuvants such as C. parvum, or similar immunostimulants. If desired, antibody molecules raised against ENDOX can be isolated from a mammal (eg, blood) and further purified by well-known techniques, such as protein A chromatography to obtain IgG fractions. Can be done.

As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to a population of antibody molecules that contains only one particular antigen binding site capable of immunoreacting with a particular epitope of ENDOX. . Thus, a monoclonal antibody composition typically displays a single binding affinity for a particular ENDOX protein with which it immunoreacts. A specific ENDOX protein sequence, or derivative thereof,
For preparation of monoclonal antibodies raised against fragments, analogs or homologues, any technique provided for the production of antibody molecules by continuous cell line cultures can be utilized. Such techniques include, without limitation, hybridoma techniques (
Kohler & Milstein, 1975 Nature 256: 495-
497); trioma technique; human B cell hybridoma technique (Ko
Zbor et al., 1983 Immunol Today 4:72) and the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., 1).
985 MONOCLONAL ANTIBODIES AND CANCER
THERAPY, Alan R .; Liss, Inc. Pp. 77-96)). Human monoclonal antibodies may be utilized in the practice of the present invention, and by using human hybridomas (Cote et al., 1983. Proc.
Natl Acad Sci USA 80: 2026-2030) or by transforming human B cells with Epstein-Barr virus in vitro (Cote et al., 1985 MONOCLONAL ANTIBO.
DIES AND CANCER THERAPY, Alan R.A. Liss,
Inc. Pp. 77-96). Each of the above citations is incorporated herein by reference in its entirety.

According to the present invention, the technique may be adapted for the production of single chain antibodies specific for the ENDOX protein (see, eg, US Pat. No. 4,946,778).
. In addition, the method can be adapted for the construction of _Fab expression libraries (eg,
Huse et al., 1989 Science 246: 1275-1281 see), ENDOX protein sequence, or derivatives, fragments, to an analog or homologue, rapid and effective identification of monoclonal F _ab fragments with the desired specificity to enable. Non-human antibodies can be "humanized" by techniques well known in the art. See, eg, US Pat. No. 5,225,539. An antibody fragment containing the idiotype to the ENDOX protein
Can be produced by techniques known in the art, including but not limited to: (i) F _{(ab ') 2} fragments produced by pepsin digestion of antibody molecules; (ii) F _{(ab') 2} fragments. F _ab fragments produced by reducing disulfide bridges; (iii) F _ab fragments and (iv) F _v fragments produced by treatment of antibody molecules with papain and a reducing agent.

In addition, recombinant anti-ENDOX antibodies, such as chimeric antibodies and humanized monoclonal antibodies that include both human and non-human portions, which can be produced using standard recombinant DNA techniques, can be used according to the invention. Within the range of. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art. This technique uses, for example, the method described below: International Application No. PC
T / US86 / 02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. W
O 86/01533; U.S. Pat. No. 4,816,567; U.S. Pat. No. 5,22.
5,539; European Patent Application No. 125,023; Better et al. (1988).
) Science 240: 1041-1043; Liu et al. (1987) PNA.
S 84: 3439-3443; Liu et al. (1987) J Immunol. 1
39: 3521-3526; Sun et al. (1987) PNAS 84: 214-2.
18; Nishimura et al. (1987) Cancer Res 47: 999.
-1005; Wood et al. (1985) Nature 314: 446-449;
Shaw et al. (1988) J Natl Cancer Inst 80: 155.
3-1559); Morrison (1985) Science 229: 12.
02-1207; Oi et al. (1986) BioTechniques 4: 214.
Jones et al. (1986) Nature 321: 552-525; Verh.
Oeyan et al. (1988) Science 239: 1534; and Bei.
dler et al. (1988) J Immunol 141: 4053-4060. Each of the above citations is incorporated herein by reference in their entirety.

In one embodiment, the methodology for screening antibodies that possess the desired specificity is determined by enzyme-linked antibody immunosorbent assay (ELISA) known in the art.
) And other immunologically mediated techniques. In certain embodiments, selection of antibodies that are specific for particular domains of the ENDOX protein is facilitated by the production of hybridomas that bind to fragments of the ENDOX protein that harbor such domains. Therefore, EN
Antibodies that are specific for the desired domain within the DOX protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.

Anti-ENDOX antibodies can be used in methods known in the art for localization and / or quantification of ENDOX protein (eg, for use in measuring the level of ENDOX protein in a suitable physiological sample). For use in diagnostic methods, for use in protein imaging, etc.).
In certain embodiments, the ENDOX protein, or derivative, fragment, analog or homolog antibody thereof (including the binding domain from the antibody) is a pharmacologically active compound [hereinbelow referred to as "therapeutic agent"]. ]] Is used.

Anti-ENDOX antibodies (eg, monoclonal antibodies) can be used to isolate ENDOX polypeptides by standard techniques (eg affinity chromatography or immunoprecipitation). Anti-ENDOX antibody is the natural EN from cells.
Purification of DOX polypeptide, and recombinantly produced ENDOX polypeptide expressed in host cells, can be facilitated. Furthermore, anti-ENDOX antibody
It can be used to detect ENDOX protein (eg, in cell lysates or cell supernatants) and evaluate the amount and pattern of ENDOX protein expression. Anti-ENDOX antibodies can be used, for example, to diagnostically monitor protein levels in tissues as part of a clinical trial procedure to determine the efficacy of a given therapeutic regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent materials, bioluminescent materials and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin /
Examples include biotin and avidin / biotin; examples of suitable fluorophores are umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine.
amine) fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; and examples of suitable radioactive substances. ^{Includes 125} I, ¹³¹ I, ³⁵ S or ³ H.

(ENDOX Recombinant Expression Vector and Host Cell) Another aspect of the present invention relates to a vector, preferably an expression vector, containing a nucleic acid encoding the ENDOX protein, or a derivative, fragment, analog or homolog thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid." This refers to a circular double stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, integrate into the host cell's genome upon introduction into the host cell, and thereby replicate with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." In general, useful expression vectors in recombinant DNA technology are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functions.

The recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for expression of the nucleic acid in host cells. This means that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected on the basis of the host cell to be used for expression. . In the recombinant expression vector,
"Operably linked" allows the nucleotide sequence of interest to be expressed (eg, in an in vitro transcription / translation system or, if the vector is introduced into a host cell, in the host cell). It is intended to be linked in a fashion to regulatory sequences.

The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, by Goeddel; GENE EXPRESSION TEC.
HNOLOGY: METHODS IN ENZYMOLOGY 185, Ac
acoustic Press, San Diego, Calif. (1990). Regulatory sequences include sequences that direct constitutive expression of nucleotide sequences in many types of host cells, and sequences that direct expression of nucleotide sequences only in particular host cells (eg, tissue-specific regulatory sequences). It will be apparent to those skilled in the art that the design of the expression vector may depend on such factors as the choice of host cell to be transformed, the level of expression of the desired protein, etc. The expression vector of the invention can be introduced into a host cell, whereby a protein or peptide (including fusion protein or peptide) encoded by the nucleic acids described herein (eg, ENDOX protein, or a mutation of ENDOX). Morphology, fusion proteins, etc.).

Recombinant expression vectors of the invention can be used in prokaryotic or eukaryotic cells,
It can be designed for ENDOX expression. For example, the ENDOX protein can be expressed in bacterial cells (eg, E. coli), insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are Goeddel; GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic P
less, San Diego, Calif. (1990) for further discussion. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. executed in E. coli. Fusion vectors add a number of amino acids to the protein encoded therein, usually at the amino terminus of recombinant proteins. Such fusion vectors are typically
Useful for three purposes: (1) increasing the expression of the recombinant protein; (2) increasing the solubility of the recombinant protein; and (3) acting as a ligand in affinity purification. Assist in purifying the replacement protein. Often, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion moiety, and this recombinant protein allows the recombinant protein to be separated from the fusion moiety after purification of the fusion protein. . Such enzymes, and their cognate recognition sequences, include Factor Xa,
Includes thrombin and enterokinase. As a typical fusion expression vector, glutathione S-transferase (GST), maltose E binding protein, or protein A is fused to the target recombinant protein, respectively.
GEX (Pharmacia Biotech Inc .; Smith and
Johnson (1988) Gene 67: 31-40), pMAL (Ne.
w England Biolabs, Beverly, Mass. ) And p
RIT5 (Pharmacia, Piscataway, NJ) is mentioned.

Suitable inducible unfused E. An example of an E. coli expression vector is pTrc (Amra
nn et al. (1988) Gene 69: 301-315) and pET 11d (
Studio et al., GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY 185, Academic P
less, San Diego, Calif. (1990) 60-89).

E. One strategy for maximizing recombinant protein expression in E. coli is to express the protein in a host bacterium with impaired ability to proteolytically cleave the recombinant protein. Gottesman, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZ
YMOLOGY 185, Academic Press, San Diego
, Calif. (1990) 119-128. Another strategy is that the individual codons for each amino acid are to change the nucleic acid sequence of the nucleic acid inserted into the expression vector so that it is the preferentially utilized codon in E. coli (Wada et al. (1992) Nucleic Acids Res. 20).
: 2111-2118). Such nucleic acid sequence alterations of the present invention can be performed using standard DN
A synthesis technique.

[0282] In another embodiment, the ENDOX expression vector is a yeast expression vector. Yeast S. Examples of vectors for expression in S. cerevisiae include pYe
pSec1 (Baldari et al., (1987) EMBO J 6: 229-23.
4), pMFa (Kurjan and Herskowitz, (1982) Ce.
11: 30: 933-943), pJRY88 (Schultz et al., (1987).
) Gene 54: 113-123), pYES2 (Invitrogen C
corporation, San Diego, Calif. ), And picZ
(InVitrogen Corp., San Diego, Calif.).

Alternatively, ENDOX can be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (eg, SF9 cells) include pAc series (Sm
ith et al. (1983) Mol Cell Biol 3: 2156-2165).
And pVL series (Lucklow and Summers (1989) Vi
170: 31-39).

In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. An example of a mammalian expression vector is pCDM8
(Seed (1987) Nature 329: 840) and pMT2PC (
Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often
Provided by the regulatory elements of the virus. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, eg, Sambrook et al., MOLECUL.
AR CLONING: A LABORATORY MANUAL. Second edition, C
old Spring Harbor Laboratory, Cold Sp
ring Harbor Laboratory Press, Cold Sp
ring Harbor, N.M. Y. , 1989, chapters 16 and 17.

In another embodiment, the recombinant mammalian expression vector is capable of directing the expression of the nucleic acid preferentially in a particular cell type (eg, expressing the nucleic acid using tissue-specific regulatory elements). . Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev 1: 268-27.
7), lymphoid-specific promoter (Calame and Eaton (1988)
Adv Immunol 43: 235-275), at a specific promoter of the T cell receptor (Winoto and Baltimore (1989) E.
MBO J 8: 729-733) and immunoglobulins (Banerji et al.
1983) Cell 33: 729-740; Queen and Baltimo.
re (1983) Cell 33: 741-748), neuron-specific promoters (eg, neurofilament promoter; Byrne and Rud).
dle (1989) Proc. Natl. Acad. Sci. USA 86: 5
473-5477), pancreas specific promoter (Edlund et al. (1985) S
Science 230: 912-916), and mammary gland-specific promoters (eg, milk whey promoter; US Pat. No. 4,873,316 and European Patent Publication No. 264,166). Developmentally regulated promoters are also included (eg, the murine hox promoter (Kessel and Gruss (1990) Science 249: 374.
-379) and the α-fetoprotein promoter (Campes and Ti)
lghman (1989) Genes Dev 3: 537-546).

The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in the antisense orientation. That is, the DNA molecule expresses an RNA molecule that is antisense to ENDOX mRNA (D
Operatively linked to regulatory sequences in a manner that allows (by transcription of NA molecules). A regulatory sequence operably linked to the nucleic acid cloned in the antisense orientation can be chosen which directs the continuous expression of the antisense RNA molecule in various cell types. For example, viral promoters and / or enhancers, or regulatory sequences that direct constitutive, tissue-specific, or cell-specific expression of antisense RNA can be selected. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus, where the antisense nucleic acid is produced under the control of a high efficiency regulatory region, the activity of which is introduced into the vector. Cell type. For a discussion of the regulation of gene expression using antisense genes, see Weintraub et al., “Antisen.
se RNA as a molecular tool for gene
ic analysis ”, Reviews--Trends in Gene
See Tics, Volume 1 (1) 1986.

Another aspect of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell, but to the progeny or potential progeny of such a cell. Such progeny may, in fact, not be identical to the parental cell, as the particular modification may be present in the next generation, either due to mutations or environmental influences, but is still referred to herein. Within the scope of the term as used in.

The host cell can be any prokaryotic or eukaryotic cell. For example, E
The NDOX protein can be expressed in bacterial cells (eg, E. coli), insect cells, yeast or mammalian cells (eg, Chinese Hamster Ovary cells (CHO) or COS cells). Other suitable host cells are known to those of skill in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to various art-recognized techniques for introducing exogenous nucleic acid (eg, DNA) into a host cell. As intended, these include calcium phosphate or calcium chloride co-precipitation, DEAE dextran mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are described by Sambrook et al. (MOLECULAR C
LONING: A LABORATORY MANUAL. Second Edition, Cold
Spring Harbor Laboratory, Cold Spring
Harbor Laboratory Press, Cold Spring
Harbor, N.M. Y. , 1989) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that only a fraction of the cells can integrate foreign DNA into their genome, depending on the expression vector and transfection technique used. These embedded bodies (
To identify and select (integrant), a gene encoding a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cell along with the gene of interest. Various selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the vector encoding ENDOX, or it can be introduced on a separate vector. Cells that are stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells that have incorporated the selectable marker gene will survive while other cells die).

Host cells of the invention, such as prokaryotic and eukaryotic host cells in culture, can be used to produce (ie, express) ENDOX protein. Accordingly, the invention further provides methods for producing an ENDOX protein using the host cells of the invention. In one embodiment, the method comprises
The host cell of the invention (in a suitable medium in which the ENDOX protein is produced)
A recombinant expression vector encoding the ENDOX protein was introduced here)
And culturing. In another embodiment, the method further comprises the step of isolating ENDOX protein from the medium or host cells.

Transgenic ENDOX Animals The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, the host cells of the invention are ENDO
A fertilized oocyte or embryonic stem cell into which the X protein coding sequence has been introduced. Such host cells can then be used to produce non-human transgenic animals, wherein the exogenous ENDOX sequences are those genomic or homologous recombinant animals (where the endogenous ENDOX sequences are , Has been changed). Such animals are useful for studying ENDOX protein function and / or activity, and for identifying and / or evaluating modulators of ENDOX protein activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, wherein these animals are One or more cells of which contain the transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. A transgene is exogenous DNA that integrates into the genome of a cell (from which cells a transgenic animal develops) and remains in the genome of a mature animal, thereby producing one or more cells of this transgenic animal. Directs expression of the encoded gene product in the form or tissue. As used herein, "homologous recombinant animal"
Is a non-human animal, preferably a mammal, more preferably a mouse,
Here, the endogenous ENDOX gene is produced by homologous recombination between this endogenous gene and an exogenous DNA molecule introduced into an animal cell (eg, an animal embryo cell) prior to animal development. has been changed.

The transgenic animals of the present invention introduce a nucleic acid encoding ENDOX into the male pronucleus of fertilized oocytes (eg, by microinjection, retroviral infection), and the oocytes are pseudo-nuclei. It can be made by allowing it to develop in a pregnant female foster animal. SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 4
The 6 and 48 human ENDOX cDNA sequences can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human ENDOX gene (
For example, the mouse ENDOX gene) can be isolated based on hybridization to human ENDOX cDNA (described further below) and used as a transgene. Intron sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. Tissue-specific regulatory sequence (s) can be operably linked to the ENDOX transgene to direct expression of the ENDOX protein to specific cells. Methods for producing transgenic animals, particularly animals such as mice, via embryo manipulation and microinjection are conventional in the art and are described, for example, in US Pat. No. 4,736,866. The same No. 4,870,
009; and 4,873,191; and Hogan 1986,
MANIPULATING THE MOUSE EMBRYO, Cold S
pring Harbor Laboratory Press, Cold S
pring Harbor, N.M. Y. It is described in. Similar methods are used for the production of other transgenic animals. Transgenic primary animals can be identified based on the presence of the ENDOX transgene in their genome and / or the expression of ENDOX mRNA in the tissues or cells of the animal. The transgenic founder animal can then be used to breed additional animals carrying the transgene. In addition, transgenic animals carrying a transgene encoding an ENDOX protein can be further bred to other transgenic animals carrying other transgenes.

[0294] Deletions, additions or substitutions have been introduced to produce a homologous recombinant animal, thereby altering (eg, functionally disrupting) the ENDOX gene, at least a portion of the ENDOX gene. A vector containing is prepared. The ENDOX gene is a human gene (eg, SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31).
, 34, 46 and 48 cDNA), but more preferably human EN
It is a non-human homolog of the DOX gene. For example, SEQ ID NOs: 1, 3, 5, 7, 9,
Mouse homologs of the 22, 25, 28, 31, 34, 46 and 48 human ENDOX genes can be used to construct homologous recombination vectors suitable for altering the endogenous ENDOX gene in the mouse genome. In one embodiment, the vector is designed such that upon homologous recombination, the endogenous ENDOX gene is functionally disrupted (ie, no longer encodes a functional protein; also referred to as a "knockout" vector).

Alternatively, the vector is designed for homologous recombination such that the endogenous ENDOX gene is mutated or otherwise altered but still encodes a functional protein (eg, , Upstream regulatory regions may be altered, thereby altering the expression of the endogenous ENDOX protein). In the homologous recombination vector, the altered portion of the ENDOX gene is flanked by additional nucleic acid of the ENDOX gene at its 5 ′ and 3 ′ ends, and homologous recombination is carried out with the exogenous ENDOX gene carried by the vector. Endogenous ENDOX in sex stem cells
Allows to happen with genes. This additional flanking ENDOX nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (at both the 5'and 3'ends) are included in the vector. See, for example, Thomas et al. (1987) Cell 51: 503 for a description of homologous recombination vectors. This vector is introduced into an embryonic stem cell line (for example, by electroporation), and cells in which the introduced ENDOX gene is homologously recombined with an endogenous ENDOX gene are selected (for example, Li et al. 1992) Cell 69: 915).

[0296] The selected cells are then injected into the blastocyst of an animal (eg, mouse) to form an aggregated chimera. For example, Terato from Bradley (1987)
carcinomas and Embryonic Stem Cells:
A Practical Approach, edited by Robertson, IRL,
See Oxford pages 113-152. The chimeric embryo can then be transplanted into a suitable pseudopregnant female foster animal, and the embryo is left for a period of time. Offspring carrying the homologously recombined DNA in their germ cells can be used to breed animals, in which all cells of the animal have undergone this homologous recombination by germline transmission of the transgene. Included DNA. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further below; Bradley (
1991) Curr Opin Biotechnol 2: 823-829;
PCT International Publication Number: WO90 / 11354; WO91 / 01140; WO92
/ 0968; and WO / 93/04169.

In another embodiment, non-human transgenic animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1.
For a description of the cre / loxP recombinase system, see, eg, Lakso et al.
1992) Proc. Natl. Acad. Sci. USA 89: 6232-.
See 6236. Another example of a recombinase system is Saccharomyc.
es cerevisiae FLP recombinase system (O'Gorma
n et al. (1991) Science 251: 1351-1355). cre / l
When the oxP recombinase system is used to regulate transgene expression,
Animals containing transgenes encoding both Cre recombinase and selected proteins are needed. Such animals can be "double-stranded", for example, by crossing two transgenic animals, one containing the transgene encoding the selected protein and the other containing the transgene encoding the recombinase. '' Can be provided by the construction of transgenic animals.

The non-human transgenic animal clones described herein also contain W
can be produced according to the method described in ilmut et al. (1997) Nature 385: 810-813. Briefly, cells from transgenic animals (
Somatic cells) are isolated and induced to exit the growth cycle and enter G ₀ phase. The quiescent cells may then be fused to enucleated oocytes from the same animal from which the quiescent cells are isolated, for example, by use of electric pulses. The reconstituted oocyte is then cultured, whereby the oocyte is
It develops into morula or undifferentiated germ cells and is then transferred into pseudopregnant female foster animals. The progeny of this female foster animal are the cells (eg, somatic cells)
Is a clone of the animal isolated.

Pharmaceutical Compositions ENDOX nucleic acid molecules, ENDOX proteins, and anti-ENDOX antibodies of the invention (also referred to herein as “active compounds”), and derivatives, fragments thereof, Analogs, and homologs can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include a nucleic acid molecule, a protein or antibody, and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents compatible with pharmaceutical administration. And absorption delaying agents and the like. A suitable carrier is Remington's Pharmaceuticals.
l Sciences, a standard reference text in the field, which is incorporated by reference herein. Preferred examples of such carriers or diluents include water, saline, and finger's.
) Solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used.
The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional vehicle or agent is incompatible with the active compound, their use in the composition is considered. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (eg, intravenous, intradermal, subcutaneous) administration, oral (
For example, inhalation), transdermal (topical) administration, transmucosal
) Administration, and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may contain the following components: water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic materials. Sterile diluents such as solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite;
Chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or aqueous dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include saline, bacteriostatic water,
Cremophor EL ^™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases,
The composition should be sterile and should be easy to inject.
It should be fluid to the extent that litty) is present. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion, including, for example: water, ethanol, polyols such as glycerol, propylene glycol, and liquid polyethylene glycols, and suitable mixtures thereof. The proper fluidity is maintained by maintaining the required particle size in the case of dispersions, for example by the use of coatings such as lecithin.
And can be maintained by using a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases,
It is preferable to include an isotonicity agent in the composition (eg, sugar, polyalcohol such as mannitol, sorbitol, sodium chloride). Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound in the required amount, eg, ENDOX protein or anti-ENDOX antibody, in a suitable solvent with one or a combination of ingredients enumerated above, as required. , Followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze drying, whereby
A powder of the active ingredient and any further desired ingredients from the solution which has already been sterile filtered is obtained.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. The oral composition also
It can be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished. Then it is exhaled or swallowed. Pharmaceutically compatible binders, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules,
Lozenges and the like can include any of the following ingredients or compounds with similar properties: binders (eg microcrystalline cellulose, gum tragacanth or gelatin); excipients (eg starch or lactose), disintegrants (eg , Alginic acid),
Primogel, or corn starch; lubricants (eg magnesium stearate or Sterotes); glidants (eg colloidal silicon dioxide); sweeteners (eg sucrose or saccharin)
Or a flavoring agent (eg, peppermint, methyl salicylate, or orange flavor).

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by nasal spray or suppositories. For transdermal administration, the active compound may be an ointment, which is generally known in the art,
Formulated in ointment, gel, or cream.

The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body (eg, controlled release formulations),
This includes implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for the preparation of such formulations will be apparent to those of skill in the art. These materials are also available from Alza Corporation and Nova Pharma.
scientifics, Inc. Commercially available from Liposomal suspensions, including liposomes targeted to infected cells, containing monoclonal antibodies against viral antigens, can also be used as pharmaceutically acceptable carriers. These are, for example, as described in US Pat. No. 4,522,811,
It can be prepared according to methods known to those skilled in the art.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically individual units suitable as unitary dosages for the subject to be treated; each unit being associated with a required pharmaceutical carrier. It contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. Details regarding the dosage unit forms of the invention are given by the unique characteristics of this active compound, and the particular therapeutic effect achieved, as well as the limitations inherent in the art of formulating such active compounds for treatment of individuals. Determined or directly dependent on them.

The nucleic acid molecule of the present invention can be inserted into a vector and used as a gene therapy vector. The gene therapy vector is administered to the subject, for example, by intravenous injection, local administration (see US Pat. No. 5,328,470), or stereotactic injection (see US Pat. No. 5,328,470).
For example, Chen et al., 1994, Proc. Natl. Acad. Sci. U.
S. A. 91: 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, the complete gene delivery vector can be produced intact from recombinant cells (eg, retroviral vectors) and the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

The pharmaceutical composition may be included in a container, package, or dispenser together with instructions for administration.

Screening and Detection Methods The isolated nucleic acid molecules of the invention may be used to express an ENDOX protein (eg, via recombinant expression vectors in host cells during gene therapy applications).
, ENDOX mRNA (eg in a biological sample) or ENDOX
It can be used to detect genetic damage in the X gene and to regulate ENDOX activity as described further below. Further, the ENDOX protein has reduced or abnormal activity for screening for drugs or compounds that modulate ENDOX protein activity or expression, as well as insufficient or excessive production of ENDOX protein or ENDOX wild-type protein. Disorders characterized by the production of various ENDOX protein forms (eg, diabetes (modulates insulin release); obesity (binding to lipids to transport lipids); obesity-related metabolic disorders, metabolic X syndrome and chronic diseases And anorexia and wasting disorders associated with various cancers, as well as infectious diseases (having antimicrobial activity) and various dyslipidemias). In addition, the anti-ENDOX antibodies of the invention can be used to detect and isolate ENDOX protein and modulate ENDOX activity. In a further aspect, the present invention may be used in a method of affecting appetite, nutrient absorption and metabolite waste in both positive and negative ways.

The invention further relates to novel agents identified by the screening assays described herein and their use for the treatments described above.

Screening Assays The present invention relates to modulators, ie, candidate compounds or agents that bind to an ENDOX protein or have a stimulatory or inhibitory effect on, for example, ENDOX protein expression or ENDOX protein activity, Alternatively, a method (also referred to herein as a “screening assay”) for identifying a test compound or agent (eg, peptide, peptidomimetic, small molecule or other drug) is provided. The present invention also includes compounds identified in the screening assays described herein.

In one embodiment, the invention provides a candidate for binding to or modulating the activity of a membrane-bound form of a protein or polypeptide of ENDOX, or a biologically active portion thereof. An assay is provided for screening compounds or test compounds. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including these libraries: biological libraries; spatial libraries. Parallel solid phase or solution phase addressable libraries; synthetic library methods that require deconvolution; "one bead, one compound" library methods; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to compounds in peptide libraries, non-peptide oligomer libraries or small molecule libraries. For example, Lam, 1997.
, Anticancer Drug Design 12: 145.

As used herein, "small molecule" is meant to refer to a composition having a molecular weight of less than about 5 kD, most preferably less than about 4 kD. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptidomimetics, sugars, lipids or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures (eg, fungal, bacterial or algae extracts) are known in the art and can be screened using any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al., 1993, Proc. Natl. Acad.
Sci. U. S. A. 90: 6909; Erb et al., 1994, Proc. Nat
l. Acad. Sci. U. S. A. 91: 11422; Zuckermann
Et al., 1994, J. Am. Med. Chem. 37: 2678; Cho et al., 1993,
Science 261: 1303; Carrell et al., 1994, Angew.
． Chem. Int. Ed. Engl. 33: 2059; Carell et al., 19
94, Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al., 1994, J. Am. Med. Chem. 37: 1233.

Compound libraries are available in solution (see, eg, Houghten, 1992,
Biotechniques 13: 412-421) or on beads (L
Am, 1991, Nature 354: 82-84), on-chip (Fodor)
, 1993, Nature 364: 555-556), bacteria (Ladner
US Pat. No. 5,223,409), spores (Ladner, US Pat. No. 5,23).
3,409), plasmid (Cull et al., 1992, Proc. Natl. A.
cad. Sci. USA 89: 1865-1869) or on phage (Sc
Ott and Smith, 1990, Science 249: 386-390.
Devlin, 1990, Science 249: 404-406; Cwi.
rla et al. (1990) Proc. Natl. Acad. Sci. U. S. A. 8
7: 6378-6382; Felici, 1991, J. Am. Mol. Biol. Two
22: 30-310; Ladner, U.S. Pat. No. 5,233,409).

In one embodiment, the assay is a cell-based assay, wherein:
Cells expressing the membrane-bound form of the ENDOX protein, or a biologically active portion thereof, on the cell surface are contacted with a test compound, which test compound
The ability to bind the DOX protein is determined. For example, the cell can be a cell of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the ENDOX protein can be accomplished, for example, by coupling the test compound with a radioisotope label or an enzyme label, such that the test compound's ENDOX protein or its biologically active label is Binding to the moiety can be achieved by being able to be determined by detecting its labeled compound in the complex. For example, a test compound can be labeled either directly or indirectly with ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H, and its radioisotope can be labeled by direct counting of radiation emission or by scintillation counting. Can be detected by Alternatively, the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determining conversion of the appropriate substrate to product. . In one embodiment, the assay involves contacting cells expressing a membrane-bound form of ENDOX protein, or a biologically active portion thereof on its cell surface with a known compound that binds ENDOX. Forming a mixture, contacting a test compound with the assay mixture, and determining the ability of the test compound to interact with the ENDOX protein, wherein the test compound interacts with the ENDOX protein. Determining the ability includes determining the ability of the test compound to preferentially bind to the ENDOX protein or a biologically active portion thereof as compared to a known compound.

[0319] In another embodiment, the assay is a cell-based assay, which comprises:
Contacting a cell expressing a membrane-bound form of the ENDOX protein or a biologically active portion thereof on the cell surface with a test compound, as well as the test compound comprising: Determining the ability to modulate (eg, stimulate or inhibit) activity. This test compound is
Determining the ability to modulate the activity of NDOX or a biologically active portion thereof can be accomplished, for example, by determining the ability of the ENDOX protein to bind to or interact with ENDOX target molecules. As used herein, a "target molecule" is a molecule with which an ENDOX protein naturally binds or interacts, eg, a molecule on the cell surface that expresses an ENDOX interacting protein, a second A molecule on the cell surface, a molecule in the extracellular environment, a molecule that associates with the inner surface of the cell membrane, or a cytoplasmic molecule. The ENDOX target molecule can be a non-ENDOX molecule or an ENDOX protein or polypeptide of the invention. In one embodiment, the ENDOX target molecule is a component of a signal transduction pathway, which is an extracellular signal (eg,
The signal generated by the compound binding to the membrane-bound ENDOX molecule). The target is, for example, a second intercellular protein having catalytic activity,
Or it may be a protein that facilitates the association of downstream signaling molecules with ENDOX.

Determining the ability of an ENDOX protein to bind to or interact with an ENDOX target molecule can be accomplished by one of the above methods for determining direct binding. In one embodiment, the ENDOX protein is ENDO.
Determining the ability to bind to or interact with the X target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of this target molecule can be detected by detecting the induction of its target cellular second messenger (ie, intracellular Ca ²⁺ , diacylglycerol, IP _3, etc.), the catalytic activity / enzymatic activity of the target to the appropriate substrate. Detecting a reporter gene (ENDOX operably linked to a nucleic acid encoding a detectable marker (eg, luciferase)).
It can be determined by detecting induction of responsive regulatory elements) or by detecting a cellular response such as cell survival, cell differentiation, or cell proliferation.

In yet another embodiment, the assay of the present invention is a cell-free assay, E
Contacting the NDOX protein or biologically active portion thereof with a test compound, as well as determining the ability of the test compound to bind to the ENDOX protein or biologically active portion thereof. END of this test compound
Binding to the OX protein can be determined either directly or indirectly, as described above. In one such embodiment, the assay is ENDO
Contacting the X protein or biologically active portion thereof with a known compound that binds ENDOX to form an assay mixture, contacting the assay mixture with a test compound, as well as the test compound being an ENDOX protein Determining the ability to interact with the test compound, wherein the test compound is ENDO
Determining the ability to interact with the X protein includes determining the ability of the test compound to preferentially bind to ENDOX or a biologically active portion thereof as compared to a known compound. .

In yet another embodiment, the assay is a cell-free assay and ENDO
Contacting the X protein or biologically active portion thereof with a test compound,
As well as determining the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of the ENDOX protein or biologically active portion thereof. Determining the ability of this test compound to modulate the activity of ENDOX is described, for example, in END.
This can be achieved by determining the ability of the OX protein to bind to the ENDOX target molecule by one of the above methods for determining direct binding. In an alternative embodiment, determining the ability of the test compound to modulate the activity of the ENDOX protein can be accomplished by determining the ability of the ENDOX protein to further modulate the ENDOX target molecule. For example, the catalytic / enzymatic activity of the target molecule on the appropriate substrate can be determined as described above.

In yet another embodiment, the cell-free assay comprises contacting ENDOX protein or a biologically active portion thereof with a known compound that binds ENDOX protein to form an assay mixture, the assay mixture Contacting the test compound with a test compound, as well as determining the ability of the test compound to interact with the ENDOX protein, wherein determining the ability of the test compound to interact with the ENDOX protein comprises: The step of determining the ability to bind preferentially to an ENDOX target molecule or to modulate the activity of that target molecule preferentially.

The cell-free assay of the present invention can be used with both soluble or membrane bound forms of ENDOX protein. For cell-free assays that include a membrane-bound form of ENDOX protein, it may be desirable to utilize a solubilizer such that the membrane-bound form of ENDOX protein is maintained in solution. Examples of such solubilizers include nonionic surfactants such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N. -Methylglucamide, Trito
n (registered trademark) X-100, Triton (registered trademark) X-114, Thesi
t (registered trademark), isotridecyl poly (ethylene glycol ether) _n (Is
tridecypoly (ethylenglycol ether) _n
), N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, 3- (3-cholamidopropyl) dimethylammonio-1-propanesulfonate (3- (3-cholamidopropyl) dimethylmethyl).
iniol-1-propane sulphonate (CHAPS), or 3- (3-cholamidopropyl) dimethylammonio-2-hydroxy-1-propane sulfonate (3- (3-cholamidopropyl) dimet
hylamminiol-2-hydroxy-1-propane sulf
onate) (CHAPSO).

In more than one embodiment of the above assay methods of the invention, either the ENDOX protein or its target molecule is immobilized to facilitate separation of the complex form from the uncomplexed form of one or both of the proteins. And adapting the automation of the assay may be desirable. Binding of the test compound to the ENDOX protein, or the interaction of the ENDOX protein with the target molecule in the presence and absence of the candidate compound, is performed in any vessel suitable for housing these reactants. , Can be achieved. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein may be provided that adds a domain that allows one or both of the proteins to bind to the matrix. For example, GST-ENDOX fusion proteins or GST target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test compound. , Or the test compound and either non-adsorbed target protein, or ENDOX protein, and the mixture is
Incubated under conditions that contribute to complex formation (eg, physiological conditions with respect to salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components and, in the case of beads, to immobilize the matrix, direct or indirect binding of the complex, eg as described above. To decide. Alternatively, the complex can be dissociated from the matrix and the level of binding or activity of ENDOX protein determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either the ENDOX protein, or its target molecule, can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated ENDOX protein or target molecule can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Ch.
emicals, Rockford, Ill. ), And streptavidin-coated 96-well plates (Pierce Chemical) wells. Alternatively, an antibody that is reactive with the ENDOX protein or target molecule but does not interfere with the binding of the ENDOX protein to its target molecule can be derivatized to the wells of the plate and the unbound target or ENDOX protein is Can be trapped in the well by conjugation of Methods for detecting such complexes include, in addition to those described above for GST-immobilized complexes, immunodetection of the complexes using an antibody reactive with the ENDOX protein or target molecule, as well as the ENDOX protein. Alternatively, an enzyme-linked assay that relies on the detection of enzyme activity associated with the target molecule can be mentioned.

In another embodiment, modulators of ENDOX protein expression are identified in a method of contacting a cell with a candidate compound and determining expression of ENDOX mRNA or protein in the cell. ENDO in the presence of a candidate compound
The expression level of X mRNA or protein is determined in the absence of candidate compound EN
The expression level of DOX mRNA or protein is compared. The candidate compound can then be identified as a modulator of ENDOX mRNA or protein expression based on this comparison. For example, if expression of ENDOX mRNA or protein is greater (ie, statistically significantly greater) in the presence of the candidate compound than in the absence thereof, the candidate compound is
Or identified as a stimulator of protein expression. Or ENDOX
If the expression of mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in the absence of the candidate compound, then the candidate compound is ENDOX mRN.
A or identified as an inhibitor of protein expression. ENDO in cells
The expression level of X mRNA or protein can be determined by the methods described herein for detecting ENDOX mRNA or protein.

In yet another aspect of the invention, ENDOX proteins are used as “bait proteins” in two-hybrid or three-hybrid assays (eg, US Pat. No. 5,283,317; Zervos et al., 1).
993, Cell 72: 223-232; Madura et al., 1993. J. B
iol. Chem. 268: 12046-12054; Bartel et al., 199.
3. Biotechniques 14: 920-924; Iwabuchi et al., 1993. Oncogene 8: 1693-1696; and Brent.
Other proteins that bind to or interact with ENDOX and modulate ENDOX activity (“ENDOX binding protein” or “ENDOX-bp”) may be identified. END like this
OX-binding proteins also appear to be involved in signal amplification by ENDOX proteins, eg, as upstream or downstream elements of the ENDOX pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, this assay utilizes two different DNA constructs. In one construct, the gene encoding ENDOX is the D of a known transcription factor (eg, GAL-4).
It is fused to the gene encoding the NA binding domain. In the other construct,
A DNA sequence encoding an unidentified protein ("prey" or "sample") from a library of DNA sequences is fused to a gene encoding the activation domain of a known transcription factor. If the "bait" and "prey" proteins can interact in vivo to form an ENDOX-dependent complex, the DN of this transcription factor
The A binding domain and activation domain are in close proximity. The proximity allows transcription of a reporter gene (eg, LacZ) operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and END
It can be used to obtain a cloned gene encoding a protein that interacts with OX.

The present invention further relates to novel agents identified by the screening assays described above and their use for treatment as described herein.

Detection Assays The portions or fragments of the cDNA sequences (and corresponding complete gene sequences) identified herein can be used in many ways as polynucleotide reagents. For example (but not limited to), these sequences can be used (i) to map their respective genes to chromosomes; thus localizing regions of genes associated with genetic disorders; (ii) trace amounts An individual may be identified from a biological sample (tissue typing); and (iii) it may aid in forensic identification of the biological sample. Some of these applications are described in the subsections below.

Chromosomal Mapping Once a gene sequence (or part of a sequence) has been isolated, this sequence can be used to map the position of the gene on the chromosome. This process is called chromosome mapping. Thus, using a portion or fragment of the ENDOX sequence, SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46 and 48, or fragments or derivatives thereof, respectively, the position of the ENDOX gene may be used. Can be mapped on the chromosome. Mapping the ENDOX sequences to the chromosome is an important first step in correlating these sequences with genes associated with disease.

Briefly, the ENDOX gene can be mapped to the chromosome by preparing PCR primers (preferably 15-25 bp in length) from the ENDOX sequence. 1 in genomic DNA using computer analysis of ENDOX sequences
Primers that do not span more exons can be quickly selected, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. ENDO
Only hybrids containing the human gene corresponding to the X sequence will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from different mammals (eg, human and mouse cells). As the hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in a random order, but retain mouse chromosomes. Mouse cells are unable to grow because of the absence of specific enzymes, whereas the use of media that allows human cells to grow maintains one human chromosome containing the gene encoding the required enzyme. By using different media, a panel of hybrid cell lines can be established. Each cell line in the panel contains either a single human chromosome or a few human chromosomes and a complete set of mouse chromosomes, which allows easy mapping of individual genes to specific human chromosomes. Become. For example, D'Eustachio et al., 19
83, Science 220: 919-924. Somatic cell hybrids containing only fragments of the human chromosome can also be generated by using human chromosomes with translocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific sequences to specific chromosomes. Three or more sequences can be assigned per day using one thermal cycler. Designing an oligonucleotide primer using the ENDOX sequence results in sublocalization.
Can be achieved with a population of fragments from a particular chromosome.

Fluorescence in situ hybridization (FISH) of DNA sequences to metaphase chromosome spreads can further be used to provide precise chromosomal location in one step. Chromosome spreads can be made using cells whose division was blocked in metaphase by chemicals such as colcemid (which disrupts the spindle). This chromosome can be briefly treated with trypsin and then Giemsa stained. A pattern of light and dark bands develops on each chromosome, resulting in this chromosome
Can be individually identified. FISH technology can be used with DNA sequences as short as 500 or 600 bases. However, clones larger than 1,000 bases are more likely to bind to unique chromosomal locations with sufficient signal strength for easy detection. Preferably 1,000 bases, and more preferably 2,000.
The base is sufficient to give good results in a reasonable amount of time. For a review of this technology, see Verma et al., HUMAN CHROMOSOMES: A MANUA.
L OF BASIC TECHNIQUES (Pergamon Press
, New York 1988).

Chromosomal mapping reagents can be used individually to mark a single chromosome or a single site on that chromosome, or a population of reagents can be multisite and / or
Or it can be used to mark multiple chromosomes. Reagents corresponding to the non-coding regions of the gene are, in fact, preferred for mapping purposes. The coding sequence is conserved within the gene family, thus increasing the chance of cross-hybridization during chromosome mapping.

Once a sequence has been mapped to a precise chromosomal location, the physical location of the sequence on the chromosome can be correlated with genetic map data. Such data is, for example,
McKusick, MENDELIAN INHERITANCE IN MA
N (Johns Hopkins University Welch Med
(available online through icial library). Then the relationship between genes and diseases that map to the same chromosomal region is:
For example, Egeland et al. (1987) Nature, 325: 783-787.
Can be identified by the linkage analysis (co-inheritance of physically adjacent genes) described in.

In addition, differences in DNA sequences between individuals who are afflicted with a disease associated with the ENDOX gene and individuals who are not afflicted with this disease can be determined. If the mutation is found in some or all of the affected individuals but not in any of the unaffected individuals, then the mutation is likely a candidate factor for the particular disease.
A comparison of affected and unaffected individuals generally involves structural alterations in chromosomes (eg,
Initially looking for deletions or translocations that are visible from the chromosomal spread or are detectable using PCR based on their DNA sequences). Finally,
Complete sequencing of genes from some individuals can be performed to confirm the presence of mutations and distinguish mutations from polymorphisms.

Tissue Typing The ENDOX sequences of the invention can also be used to distinguish individuals from small biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes to generate unique bands for discrimination and probed in Southern blots. The sequences of the present invention are based on RFLP (US Pat.
It is useful as an additional DNA marker for "restriction fragment length polymorphism" described in No. 272,057.

In addition, the sequences of the invention may be used to provide an alternative technique for determining the actual base-by-base DNA sequence of a selected portion of an individual's genome. Therefore, using the ENDOX sequence described herein, two PCRs from the 5'and 3'ends of the sequence
Primers can be prepared. These primers are then used to
A can be amplified and then it can be sequenced.

The population of corresponding DNA sequences from the individuals thus prepared may provide a unique individual identification, as each individual has a unique set of such DNA sequences due to allelic differences. . The sequences of the present invention can be used to obtain such discrimination of sequences of individual and tissue origin. The ENDOX sequences of the present invention uniquely represent portions of the human genome. Allelic variation can occur to some extent in the coding regions of these sequences, and to a greater extent in non-coding regions. It is estimated that allelic variation between individual humans occurs approximately once every 500 bases. Many of the allelic variations are due to single nucleotide polymorphisms (SNPs), including restriction fragment length polymorphisms (RFLPs).

Each of the sequences described herein can, to some extent, be used as standards against which DNA from an individual can be compared for purposes of discrimination. Greater numbers of polymorphisms occur in non-coding regions, so less sequence is required to distinguish individuals. The non-coding sequence is probably 10 to 100
A population of 0 primers can be used comfortably to provide positive individual identification. Each of these primers yields 100 bases of amplified non-coding sequence. Putative coding sequence (eg, SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31,
A more suitable number of primers for positive individual identification is between 500 and 2,000 if the coding sequences in 34, 46 and 48) are used.

Predictive Medicine The present invention also provides diagnostic assays, prognostic assays, pharmacogenomics.
Cognomics) and monitoring clinical trials relate to the field of predictive medicine, where prognosis (prediction) is used for the purpose of prophylactic treatment of individuals. Accordingly, one aspect of the invention determines the expression of ENDOX protein and / or nucleic acid, and the activity of ENDOX in the context of a biological sample (eg, blood, serum, cells, tissues), thereby causing abnormalities. Diagnostic assay for determining whether an individual suffers from a disease or disorder associated with the expression or activity of ENDOX or is at risk of developing such a disorder. This disorder includes metabolic disorders, diabetes, obesity, infectious diseases, anorexia, cancer-related cachexia, and various dyslipidemias, metabolic disorders associated with obesity, metabolic syndrome X, and chronic diseases and various diseases. Wasting diseases associated with cancer. The invention also provides a prognostic (or predictive) assay for determining whether an individual is at risk of developing a disorder associated with ENDOX protein, nucleic acid expression or activity. For example, mutations in the gene for ENDOX can be assayed in biological samples. Such an assay
It can be used for prognostic or predictive purposes, which allows the individual to be treated prophylactically prior to the onset of disorders characterized by or related to the expression or activity of ENDOX protein, nucleic acids.

Another aspect of the invention provides a method for determining the expression or activity of ENDOX protein, nucleic acid in an individual, whereby a suitable therapeutic or prophylactic agent for the individual (the present specification) is provided. In the book called "pharmacogenomics"). Pharmacogenomics is a drug for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (eg, the genotype of the individual tested to determine the individual's ability to respond to a particular drug). Allows selection of (eg drug).

Yet another aspect of the invention relates to monitoring the effect of an agent (eg, drug, compound) on ENDOX expression or activity in clinical trials.

[0347]   These and other agents are described in further detail in the sections below.

Diagnostic Assays An exemplary method for detecting the presence or absence of ENDOX in a biological sample is to obtain a biological sample from a test subject, and add the biological sample to ENDOX. Contacting the protein or nucleic acid encoding the ENDOX protein (eg, mRNA, genomic DNA) with a detectable compound or agent, such that the presence of ENDOX is detected in the biological sample. Include. ENDOX mRNA or genomic DNA
An agent for the detection of is a labeled nucleic acid probe capable of hybridizing to ENDOX mRNA or genomic DNA. This nucleic acid probe is, for example, a full-length ENDOX nucleic acid (for example, SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25).
, 28, 31, 35, 46 and 48) or a portion thereof (eg, at least 15, 30, 50, 100, 250 or 500 nucleotides in length with ENDOX mRNA or genomic DNA under stringent conditions). Oligonucleotide, which is sufficient to specifically hybridize. Other suitable probes for use in the diagnostic assays of the invention are described herein.

The agent for detecting the ENDOX protein is an antibody capable of binding to the ENDOX protein, preferably an antibody having a detectable label. The antibody can be polyclonal or, more preferably, a monoclonal antibody. An intact antibody or fragment thereof (eg Fab or F
(Ab ′) ₂ ) can be used. The term "labeled" with respect to a probe or antibody refers to directly labeling the probe or antibody by attaching (ie, physically linking) a detectable substance to the probe or antibody, and It is intended to include indirectly labeling the probe or antibody with its reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of primary antibody with a fluorescently labeled secondary antibody, and DNA with biotin so that it can be detected with fluorescently labeled streptavidin.
The end label of the probe may be mentioned. The term "biological sample" refers to tissues, cells and biological fluids isolated from a subject, as well as tissue present in the subject,
It is intended to include cells and fluids. That is, the detection method of the present invention can be used to detect ENDOX mRNA, protein or genomic DNA in a biological sample in vitro and in vivo. For example, ENDOX mRNA
In vitro techniques for the detection of E. coli include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of ENDOX proteins include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation and immunofluorescence. ENDOX Genome D
In vitro techniques for detecting NA include Southern hybridizations. In addition, in vivo techniques for detection of ENDOX proteins include introducing into a subject a labeled anti-ENDOX antibody. For example, the antibody can be labeled with a radioactive marker and the presence and location of the radioactive marker in a subject can be detected by standard imaging techniques.

[0350] In one embodiment, the biological sample comprises protein molecules from the test subject. Alternatively, the biological sample may include mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.

In another embodiment, the method of the present invention further comprises obtaining a control biological sample from a control subject, the control sample being ENDO.
X protein, mRNA or genomic DNA is contacted with a detectable compound or drug, resulting in ENDOX protein, mRNA or genomic D
The presence of NA is detected in the biological sample, and the ENDOX protein, mRNA or genome D in the process and its control sample
Comparing the presence of NA with the presence of ENDOX protein, mRNA or genomic DNA in the test sample.

The present invention also includes a kit for detecting the presence of ENDOX in a biological sample. For example, the kit may comprise: a labeled compound or agent capable of detecting ENDOX protein or mRNA in a biological sample; a means for determining the amount of ENDOX in the sample;
And means for comparing the amount of ENDOX with the standard in that sample. The compound or agent can be packaged in a suitable container. The kit may further comprise instructions for using the kit to detect ENDOX protein or nucleic acid.

Prognostic Assays The diagnostic methods described herein may be further utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of ENDOX. obtain. For example, the assays described herein (eg,
The diagnostic assays described above or the assays described below) can be utilized to identify subjects who have or are at risk of developing disorders associated with the expression or activity of ENDOX proteins, nucleic acids. Alternatively, this prognostic assay may be utilized to identify subjects who have or are at risk of developing a disease or disorder. Accordingly, the invention provides a method for identifying a disease or disorder associated with aberrant expression or activity of ENDOX, wherein a test sample is obtained from the subject, and E
NDOX protein or nucleic acid (eg, mRNA, genomic DNA) is detected, wherein the presence of ENDOX protein or nucleic acid has or is at risk of developing a disease or disorder associated with aberrant expression or activity of ENDOX. Is a diagnostic indicator for the subject. As used herein, "test sample" refers to a biological sample obtained from a subject of interest. For example, the test sample can be a biological fluid (eg, serum), cell sample, or tissue.

Further, using the prognostic assays described herein, a subject can be tested for drugs (eg, agonists, antagonists, peptidomimetics, proteins, peptides, nucleic acids, small molecules, or other drug candidates). It can be determined whether or not it can be administered to treat a disease or disorder associated with aberrant expression or activity of ENDOX. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. Accordingly, the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant expression or activity of ENDOX, wherein the test sample is Obtained and ENDOX
Of the ENDOX protein or nucleic acid are detected (eg, where the presence of the ENDOX protein or nucleic acid is a diagnostic indicator for a subject that may be administered a drug to treat a disorder associated with aberrant expression or activity of ENDOX). Is).

The methods of the invention also detect genetic damage in the gene for ENDOX, thereby putting a subject carrying the damage gene at risk for a disorder characterized by abnormal cell proliferation and / or differentiation. It can also be used to determine whether or not. In various embodiments, the methods of the invention are characterized by at least one of the alterations affecting the integrity of the gene encoding the ENDOX protein or the misexpression of the ENDOX gene in a sample of cells from the subject. The step of detecting the presence or absence of a genetic lesion that is affixed. For example, such genetic damage can be detected by confirming the presence of at least one of the following: (i) deletion of one or more nucleotides from the gene for ENDOX; (ii) to the gene for ENDOX. Addition of one or more nucleotides; (i
ii) substitution of one or more nucleotides of the gene of ENDOX, (iv) END0
Chromosomal rearrangement of X gene; (v) Messenger RNA of ENDOX gene
Changes in transcript levels, (vi) aberrant modification of the gene for ENDOX (eg, aberrant modification of the methylation pattern of genomic DNA), (vii) the presence of non-wild-type splicing patterns for messenger RNA transcripts of the gene for ENDOX, (
viii) non-wild type levels of the protein of ENDOX, (ix) deletion of alleles of the gene of ENDOX, and (x) inappropriate post-translational modification of the protein of ENDOX. As described herein, there are a number of known assay techniques in the art, which can be used to detect damage in the gene for ENDOX. A preferred biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means. However, any biological sample containing nucleated cells can be used, including, for example, buccal mucosal cells.

In certain embodiments, detection of damage involves the use of probes / primers in the polymerase chain reaction (PCR) (eg, US Pat. No. 4,683).
, 195 and 4,683,202) (for example, anchor P
CR or RACE PCR), or ligation chain reaction (LCR) (eg,
Landegran et al. (1988) Science 241: 1077-108.
0; and Nakazawa et al. (1994) Proc. Natl. Acad. S
ci. USA 91: 360-364). The latter may be particularly useful for detecting point mutations in the gene for ENDOX) (Abravaya).
(1995) Nucl. Acids Res 23: 675-682). The method comprises the steps of collecting a sample of cells from a patient, nucleic acid (eg,
Genome, mRNA or both) from the cells of the sample, E
Contacting one or more primers that specifically hybridize to the NDOX gene with a nucleic acid sample thereof under conditions such that hybridization and amplification of the ENDOX gene (if present) occurs, and the amplification product. Of the presence or absence of, or detecting the length of the amplification product and comparing its size to a control sample. It will be appreciated that PCR and / or LCR may be desired to be used as a preliminary amplification step with any of the techniques used to detect the mutations described herein.

Alternative amplification methods include: self-sustaining sequence replication (Guat).
elli et al., 1990, Proc. Natl. Acad. Sci. USA 87
: 1874-1878), transcription amplification system (Kwoh, et al., 1989, Proc. N.
atl. Acad. Sci. USA 86: 1173-1177), Qβ replicase (Lizardi et al., 1988, BioTechnology 6:11).
97), or any other nucleic acid amplification method, followed by detection of the amplified molecule using techniques well known to those of skill in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very low numbers.

In an alternative embodiment, mutations in the gene for ENDOX from sample cells can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (if necessary), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. To be done. Differences in fragment length size between sample and control DNA indicate mutations in the sample DNA. Furthermore, the use of sequence-specific ribozymes (eg,
(See US Pat. No. 5,493,531) can be used to score for the presence of particular mutations by the occurrence or loss of ribozyme cleavage sites.

In other embodiments, the genetic variation in ENDOX hybridizes sample and control nucleic acids (eg, DNA or RNA) to a high density array containing hundreds or thousands of oligonucleotide probes. (Eg Cronin et al. (1996) Human Mu).
station 7: 244-255; Kozal et al. (1996) Nat. Med
． 2: 753-759). For example, genetic mutations in ENDOX can be identified in a two-dimensional array containing photogenerated DNA probes as described in Cronin et al., Supra. Briefly, a first probe hybridization array was used to scan through a long stretch of DNA in the sample and control to create a linear array of consecutively overlapping probes to create base changes between the sequences. Can be identified. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of a particular mutation using a smaller specialized probe array complementary to all detected variants or variants. . Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

In yet another embodiment, the ENDOX gene can be sequenced directly using any of a variety of sequencing reactions known in the art, and the sample ENDOX gene can be sequenced.
Mutations can be detected by comparing the X sequence to the corresponding wild type (control) sequence. Examples of sequencing reactions are Maxim and Gilbert (
1977) Proc. Natl. Acad. Sci. USA 74: 560 or Sanger (1977) Proc. Natl. Acad. Sci. USA
74: 5463, which is based on the technology developed by It is also contemplated that any of a variety of automated sequencing procedures may be utilized when performing diagnostic assays (eg, Naeve et al., (1995) BioTechnique).
s 19: 448). These include sequencing by mass spectrometry (
For example, PCT International Publication Number WO 94/16101; Cohen et al. (1996).
) Adv. Chromatography 36: 127-162; and Gr.
ifin et al. (1993) Appl. Biochem. Biotechnol.
38: 147-159).

Other methods for detecting mutations in the ENDOX gene include methods for detecting mismatched bases in RNA / RNA or RNA / DNA heteroduplexes using protection from cleavage agents. (For example, Myers et al.
1985) Science 230: 1242). Generally, the art of "mismatch cleavage" is by hybridizing RNA (or DNA) containing (labeled) wild-type ENDOX sequences with a potential mutant RNA or DNA obtained from a tissue sample. Begin by providing the heteroduplex that is formed. This double-stranded double-stranded, single-stranded region of the double-stranded (for example,
Treat with an agent that cleaves the one (which is present due to the base pair mismatch between the control strand and the sample strand). For example, RNA / DNA duplex
It can be treated with RNase, and the DNA / DNA hybrid can be treated with S1 nuclease to enzymatically digest its mismatched regions.
In other embodiments, either DNA / DNA or RNA / DNA duplexes can be treated with hydroxylamine or osmium tetroxide and piperidine to digest mismatched regions. Then, after digesting the mismatched region, the resulting material was size-separated on a denaturing polyacrylamide gel,
Determine the site of mutation. For example, Cotton et al. (1988) Proc. Nat
l. Acad. Sci. USA 85: 4397; Saleeba et al. (1992).
) Methods Enzymol 217: 286-295. 1
In one embodiment, the control DNA or RNA can be labeled for detection.

In yet another embodiment, the mismatch cleavage reaction involves ENDOX obtained from a sample of cells with one or more proteins that recognize mismatched base pairs in double-stranded DNA (so-called “DNA mismatch repair” enzymes). Used in defined systems to detect and map point mutations in cDNA. For example, E. E. coli mutY enzyme cleaves A at a G / A mismatch and He
Thymidine DNA glycosidase from La cells cleaves T at a G / T mismatch (Hsu et al. (1994) Carcinogenesis 15: 1657-.
1662). According to an exemplary embodiment, the sequence of ENDOX (eg, wild type E
Probes based on the sequence of NDOX) are derived from cDNA or other DNs from test cells.
It hybridizes to the A product. The duplex is treated with a DNA mismatch repair enzyme, and its cleavage product, if any, can be detected, such as by electrophoresis protocols. See, eg, US Pat. No. 5,459,039.

In another embodiment, alterations in electrophoretic mobility are used to identify mutations in the gene for ENDOX. For example, single-stranded conformational polymorphism (SSC
P) can be used to detect differences in electrophoretic mobility between mutants and wild type nucleic acids (Orita et al. (1989) Proc Natl Acad.
Sci USA: 86: 2766, Cotton (1993) Mutat R.
es 285: 125-144; Hayashi (1992) Genet An.
al Tech Appl 9: 73-79). Single-stranded DNA fragments of the sample and control ENDOX nucleic acids are denatured and renatured. The secondary structure of single-stranded nucleic acids varies according to sequence, and the resulting changes in electrophoretic mobility can detect even a single base change. The DNA fragment can be labeled or detected with a labeled probe. The sensitivity of the assay can be enhanced by using RNA (rather than DNA) and this secondary structure is more sensitive to changes in sequence. In one embodiment, the method of the invention utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on changes in electrophoretic mobility (Keen et al. (19
91) Trends Genet 7: 5).

In yet another embodiment, migration of mutant or wild type fragments in polyacrylamide gels containing a constant gradient of denaturing agent is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature
e 313: 495). When DGGE is used as a method of analysis, the DNA is modified to ensure that it is not completely denatured, for example by adding a GC clamp of high melting GC rich DNA of about 40 bp by PCR. In a further embodiment, temperature gradients are used instead of denaturant gradients to identify differences in the mobility of control and sample DNA (Rose).
nbaum and Reissner (1987) Biophys Chem 2
65: 12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers can be prepared such that the known mutations are centrally located and then hybridized to the target DNA under conditions that permit hybridization only if a perfect match is found ( Saiki et al. (1986) Nature 324: 163;
Saiki et al. (1989) Proc Natl Acad Sci USA 8
6: 6230). Such allele-specific oligonucleotides hybridize to PCR amplified target DNA or many different mutations when the oligonucleotide is attached to a hybridizing membrane and hybridized to labeled target DNA. To be done.

Alternatively, allele-specific amplification techniques that rely on selective PCR amplification can be used in conjunction with the present invention. Oligonucleotides used as primers for specific amplification are in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucl. Acid.
s Res 17: 2437-2448) or under suitable conditions a mismatch may carry the mutation of interest at the extreme 3'end of one primer (Prossner (1993) Tibtech).
11: 238). Furthermore, introducing a new restriction site in the region of the mutation may be desirable for performing cleavage-based detection (Gasparini et al.
1992) Mol Cell Probes 6: 1). In certain embodiments, it is expected that amplification can also be performed using Taq ligase for amplification (Barany (1991) Proc Natl Acad Sci US.
A 88: 189). In such cases, ligation will only occur if there is an exact match at the 3'end of the 5'sequence, which is searched for the presence or absence of amplification to identify the presence of a known mutation at a particular site. To be able to detect.

The methods described herein can be carried out, for example, by utilizing a prepackaged diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein. , Which can conveniently be used, for example, in a clinical setting for diagnosing patients exhibiting symptoms or family history of a disease or disorder that includes the gene for ENDOX.

In addition, any cell type or tissue in which ENDOX is expressed, preferably peripheral blood leukocytes, can be utilized in the prognosis assay described herein. However, any biological sample containing nucleated cells can be used, including, for example, buccal mucosal cells.

Pharmacogenomics Factors that have a stimulatory or inhibitory effect on the activity of ENDOX (eg, gene expression of ENDOX), or modulators, are determined by screening assays described herein. As identified, disorders associated with metabolic disorders, diabetes, obesity, infectious diseases, loss of appetite, cancer-related malignant fluids, and various dyslipidemias, obesity Can be administered to an individual to treat (prophylactically or therapeutically), including metabolic disorders, metabolic X syndrome and wasting disorders associated with chronic diseases and various cancers. In conjunction with such treatment, an individual's pharmacogenomics, ie, a study of the relationship between an individual's genotype and that individual's response to foreign compounds or drugs, may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relationship between dose and blood concentration of the pharmacologically active drug.
Therefore, the pharmacogenomics of an individual allows for the selection of effective agents (eg, drugs) for prophylactic or therapeutic treatment based on consideration of the individual's genotype. Such pharmacogenomics can further be used to determine the appropriate dosage and therapeutic agent regimen. Therefore, the activity of the ENDOX protein, the expression of the ENDOX nucleic acid, or the mutated content of the ENDOX gene in an individual may be determined, thereby selecting an appropriate agent for therapeutic or prophylactic treatment of the individual.

Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug properties and aberrant effects in affected individuals. For example, Eichelbaum, Clin Exp Pharmacol P
hystol, 1996, 23: 983-985 and Linder, Clin.
Chem, 1997, 43: 254-266. In general, two types of pharmacogenomic states can be distinguished. Genetic status was transmitted as one factor that modifies the way the drug acts on the body (altered drug action), or genetic status is one factor that modifies the way the body acts on the drug. Transmitted (altered drug metabolism). These pharmacogenomic states can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymatic disease, the main clinical complications of which are oxidative drugs (antimalarials, sulfonamides, analgesics, nitrofuran). )
Hemolysis after ingestion and consumption of broad beans.

In an exemplary embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. Drug metabolizing enzymes (eg, N-acetyltransferase 2 (NAT2) and cytochrome P450 enzymes CYP2D6 and C)
The discovery of a genetic polymorphism in YP2C19) indicates that some patients do not get the expected drug effect, or show excessive drug response and severe toxicity after taking standard and safe doses of the drug. Provided an explanation for what is not. These polymorphisms are associated with two phenotypes in the population, those with high metabolic capacity.
(EM) and poor metabolizing capacity (poor metabol)
It is expressed by an (izer) (PM)). The prevalence of PM varies between different populations. For example, the gene encoding CYP2D6 is highly polymorphic, and some mutations have been identified in PM, all leading to the absence of functional CYP2D6. Those with low metabolic capacity for CYP2D6 and CYP2C19 quite often experience exaggerated drug responses and side effects when they receive standard doses. When the metabolite is the active therapeutic moiety, PM does not show a therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-forming metabolite, morphine. The other extreme are the so-called ultra-rapid metabolizers who do not respond to standard doses. Molecules that are the basis for ultrarapid metabolism have recently been identified to be due to CYP2D6 gene amplification.

Accordingly, the activity of the ENDOX protein, the expression of the ENDOX nucleic acid, or the mutation content of the ENDOX gene in an individual can be determined, thereby providing suitable agents for the therapeutic and prophylactic treatment of that individual. You can choose. Further, pharmacogenomic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes for identification of an individual's drug-responsive phenotype. This finding, when applied in dose or drug selection, may avoid adverse reactions or treatment failures,
Thus, enhancing therapeutic or prophylactic efficacy when treating a subject with a modulator of ENDOX (eg, a modulator identified by one of the exemplary screening assays described herein). You can

Monitoring Effects During Clinical Trials Monitoring the effects of agents (eg, drugs, compounds) on ENDOX expression or activity (eg, ability to regulate aberrant cell growth and / or differentiation) , Can be applied not only in basic screening but also in clinical trials. For example, the efficacy of an agent as determined by a screening assay as described herein, to increase ENDOX gene expression, protein levels, or to upregulate ENDOX activity,
It can be monitored in clinical trials in subjects exhibiting reduced ENDOX gene expression, protein levels, or down-regulated ENDOX activity. Alternatively, ENDOX gene expression, decreased protein levels, or ENDOX
Efficacy of agents determined by screening assays to down-regulate the activity of ENDOX can be monitored in clinical trials in subjects who exhibit increased ENDOX gene expression, protein levels, or upregulated ENDOX activity. In such clinical studies, ENDOX expression or activity,
And preferably, other genes involved in, for example, cellular proliferation or immune disorders can be used as "read out" markers or markers of the immune response of particular cells.

For example, but not by way of limitation, a gene containing ENDOX (which is
The activity of X (eg, identified in a screening assay as described herein) is intracellularly modulated by treatment with an agent (eg, compound, drug or small molecule), Can be identified. Thus, to study the effects of agents on cellular proliferative disorders, cells can be isolated and RNA can be prepared and expression of ENDOX and other genes involved in this disorder, eg, in clinical trials. It can be analyzed for levels. The level of gene expression (ie, gene expression pattern) can be determined by Northern blot analysis or RT-PCR, as described herein, or by measuring the amount of protein produced. By one of the methods described in the book,
Alternatively, by measuring the level of activity of ENDOX or other genes,
Can be quantified. In this way, the gene expression pattern can act as a marker that is an indicator of the physiological response of cells to the drug. Thus, the response state can be determined prior to treatment of the individual with the agent and at various times during treatment.

In one embodiment, the invention is a drug (eg, agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other identified by a screening assay described herein). A drug candidate of) and monitoring the efficacy of treatment in a subject using the following steps: (i) a pre-dose sample from the subject prior to administration of the drug. (Ii) In this pre-dose sample, the ENDOX protein, mR
Detecting the level of expression of NA, or genomic DNA; (iii) obtaining one or more post-administration samples from this subject; (iv) in this post-administration sample, ENDOX protein, mRNA, or genomic DNA. (V) the level of expression or activity of ENDOX protein, mRNA or genomic DNA in this pre-administration sample is compared with the level of expression or activity of ENDOX in this post-administration sample.
And (vi) thereby altering the administration of the agent to this subject. For example, increased administration of the agent may be desirable to increase ENDOX expression or activity to levels higher than detected (ie, to increase the efficacy of the agent). Alternatively, reduced administration of the agent may be desirable to reduce ENDOX expression or activity to levels below that detected (ie, to reduce efficacy of the agent).

Methods of Treatment The present invention provides prophylactic and therapeutic treatments for subjects who are at risk (ie, suspected) of a disorder associated with aberrant ENDOX expression or activity, or who have this disorder. To provide both of them. This disorder includes metabolic disorders, diabetes, obesity, infectious diseases, loss of appetite, cancer related malicious fluids, and various dyslipidemias (d
dyslipidemia), metabolic disorders associated with obesity, metabolic X syndrome and wasting disorders associated with chronic diseases and various cancers. The methods of these treatments are discussed more fully below.

Diseases and Disorders Diseases and disorders characterized by increased levels or biological activity (compared to a subject not afflicted with the disease or disorder) antagonize activity (ie, Can be treated with a therapeutic agent (which reduces or inhibits). The therapeutic agents that antagonize activity can be administered in a therapeutic or prophylactic manner. Therapeutic agents that may be utilized include, but are not limited to: (i
) A peptide as described above, or an analog, derivative, fragment or homologue thereof; (ii) an antibody against the above peptide; (iii) a nucleic acid encoding the above peptide; (iv) an antisense nucleic acid and "dysfunctional" ( That is,
Administration of nucleic acids (due to heterologous inserts within the coding sequences of the peptides described above) is utilized to “knock out” the endogenous function of the peptides described above by homologous recombination (eg, Capecchi, 1989, Science 244). : 128
8-1292); or (v) modulators (ie inhibitors, agonists and antagonists (further peptidomimetics of the invention or the present invention) that alter the interaction between the peptide described above and its binding partner. Including antibodies specific for the peptides of the invention)).

Diseases and disorders characterized by decreased levels or biological activity (compared to a subject not afflicted with the disease or disorder) have increased activity (ie, are agonists of the activity). It can be treated with a therapeutic agent. The therapeutic agents that upregulate activity can be administered in a therapeutic or prophylactic manner. Therapeutic agents that may be utilized include, but are not limited to, the peptides described above, or analogs, derivatives, fragments or homologs thereof; or agonists that increase bioavailability.

Increased or decreased levels can be readily detected by quantifying the peptide and / or RNA. This quantification involves obtaining a tissue sample of a patient (eg, from a biopsy tissue) and examining the sample for RNA level or peptide level of the expressed peptide, structure and / or activity (or mRNA of the peptide described above). By assaying in vivo. Methods well known in the art include, but are not limited to:
Immunoassay (eg, Western blot analysis, sodium dodecyl sulfate (
SDS) immunoprecipitation after polyacrylamide gel electrophoresis, by immunocytology, etc.) and / or hybridization assays to detect expression of mRNA (eg Northern assay, dot blot, in situ hybridization, etc.).

(Prophylactic Method) In one aspect, the present invention prevents a disease or condition associated with aberrant ENDOX expression or activity in a subject by administering to the subject an agent that modulates: A method for: expression of ENDOX, or activity of at least one ENDOX. A subject at risk of developing a disease caused by or caused by aberrant ENDOX expression or activity is, for example, by any of the diagnostic or prognostic assays described herein, or a combination thereof. , Can be identified. Administration of a prophylactic drug will prevent this disease or disorder from being delayed or its progression.
Can occur prior to the onset of abnormally characteristic symptoms of. Depending on the type of ENDOX abnormality, for example, an ENDOX agonist agent or an ENDOX antagonist agent can be used to treat the subject. The appropriate agent can be determined based on the screening assays described herein. The prevention methods of the present invention are further discussed in the following subsections.

Method of Treatment Another aspect of the invention relates to a method of modulating the expression or activity of ENDOX for therapeutic purposes. The regulation method of the present invention provides a method for treating cells with END
Contacting with an agent that modulates one or more of the protein activities of OX. The agent that modulates the protein activity of ENDOX can be an agent as described herein. Such agents are, for example, nucleic acids, proteins of ENDOX proteins, naturally occurring cognate ligands of these proteins,
Peptides, peptidomimetics of ENDOX, or other small molecules. In one embodiment, the agent stimulates one or more of the protein activities of ENDOX. Examples of such stimulatory agents include: active ENDOX protein and a nucleic acid molecule encoding ENDOX introduced into the cell. In another embodiment, the agent inhibits one or more of the protein activities of ENDOX. Examples of such inhibitory agents include ENDOX antisense nucleic acid molecules, and anti-ENDOX antibodies. These adjustment methods are
In vitro (eg, by culturing the cells with the agent),
Alternatively, it can be performed in vivo (eg, by administering the agent to a subject). Thus, the invention provides a method of treating an individual suffering from a disease or disorder characterized by aberrant expression or activity of a protein or nucleic acid molecule of ENDOX. In one embodiment, the method comprises
Administer an agent (eg, an agent identified by a screening assay described herein) or a combination of such agents that modulates (eg, upregulates or downregulates) the expression or activity of ENDOX. The step of performing is included. In another embodiment, the method comprises administering an ENDOX, protein or nucleic acid molecule as a treatment to compensate for the reduced or aberrant expression or activity of ENDOX.

Stimulation of ENDOX activity is desirable in situations in which ENDOX is abnormally downregulated, and / or in which increased activity of ENDOX appears to have a beneficial effect. One example of such a situation is when a subject has a disorder characterized by abnormal cell proliferation and / or cell differentiation (eg, cancer or immune related disorders). Another example of such a situation is when the subject has a pregnancy disease (eg, preeclampsia).

Determining Biological Effects of Therapeutic Agents In various embodiments of the invention, suitable in vitro or in vivo assays are performed to indicate the effect of a particular therapeutic agent and its administration is indicative of treatment of diseased tissue. Decide whether or not.

In various specific embodiments, an in vitro assay is performed on a representative cell type involved in the disorder of the patient to determine whether a given therapeutic agent exerted the desired effect on this cell type. You can decide. The compounds used in therapy may be tested in suitable animal model systems before testing in human subjects. These animal model systems include, but are not limited to: rat, mouse, chicken, cow, monkey, rabbit and the like. Similarly, for in vivo testing, any animal model system known in the art can be used prior to administration to human subjects.

Prophylactic and Therapeutic Uses of the Compositions of the Invention The ENDOX nucleic acids and proteins of the invention are potential prophylactic applications and treatments associated with a variety of disorders including, but not limited to: In application,
Useful: metabolic disorders, diabetes, obesity, infectious diseases, loss of appetite, cancer-related malignant fluids, and various dyslipidemias, metabolic disorders associated with obesity, metabolic syndrome X and chronic diseases And wasting disorders associated with various cancers.

As an example, a cDNA encoding an ENDOX protein of the invention may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention have efficacy for the treatment of patients suffering from: metabolic disorders, diabetes, obesity, infectious diseases, loss of appetite, cancer-related malignant fluids, and various Dyslipidemia.

Both the novel nucleic acids encoding ENDOX proteins and the ENDOX proteins of the invention, or fragments thereof, may also be useful in diagnostic applications where the presence or amount of these nucleic acids or proteins is assessed. A further application may be as an antimicrobial molecule (ie some peptides were found to have antimicrobial properties). These materials are further useful in the production of antibodies that immunospecifically bind to the novel agents of the invention for use in therapeutic or diagnostic methods.

[0388]   (Example)   The following examples are intended as non-limiting examples of various aspects of this invention.

A novel family of human genes related to the gene encoding human acyl CoA binding protein (ACBP) / diazepam binding inhibitor (DBI) was identified by analysis of expressed and genomic DNA sequences. See Example 6. The human family consists of seven novel ENDO genes and three known ENDO genes, all containing a highly conserved domain of 20 amino acids. ACBP / DBI is processed to produce a biologically active 18 amino acid peptide (ODN) that affects the energy metabolism of the organism. Biological processing of other family members is expected to lead to other metabolic regulatory peptides.

Synthetic peptides derived from the conserved domain of each human family member were used in metabolic studies and to raise polyclonal antibodies in rabbits. Synthetic peptides from the conserved domains of each of the human and rat family members were injected into various mouse strains. Injected peptides induce unique changes in bioenergy metabolism that result in fat storage, muscle mass, insulin secretion, glucose utilization and serum lipid levels (triglycerides and cholesterol). See Example 1.

Consensus sequences can be derived from full length proteins and individual peptides that have specific metabolic effects. See Examples 2 and 3. These human peptides, peptides derived from non-human species, mutant peptides induced by rational changes based on consensus sequences or combinatorial changes, antibodies and small molecules that interact with full-length proteins, peptides, processing enzymes and / or receptors are , May have important therapeutic value in the treatment of metabolic disorders. See Examples 1 and 4. These disorders include anorexia, cancer-associated cachexia, obesity, type I and type II diabetes and various dyslipidemias. The present invention is also useful as a marker for various cancer types. See Example 5.

Example 1 Peptide-Induced Metabolic Effects in AKR Mice Human and rat endozepine-induced metabolic regulation of metabolic parameters in AKR (obese and diabetic prone) and C57B1 / 6 (control) mice. Studies were conducted to determine the effects of daily ip doses of peptide (MRP) (14 days). In this example, A
The effect of this peptide on KR mice is summarized. Serum cholesterol, triglycerides, insulin and glucose are related to body weight, weight changes, relative organ weights of the liver, reproductive caudal fat pad, pancreas and mesenteric tissue weights, quadriceps weight, food and water. Was monitored with ingestion. Histopathological analysis was performed on selected tissue samples.

Peptides were injected in both AKR and C57B1 / 6 mice. AKR mice are heavier than control C57B1 / 6 mice and have higher serum glucose levels, especially in response to manipulation. Thus, AKR mice
It serves as a polygenic model for the common forms of obesity and type II diabetes in the human population. Table 29 summarizes the metabolic effects induced by each MRP in AKR mice. Peptides with sequences randomized from the amino acids found in MRP and ODN, at the doses indicated in 1% DMSO,
Given by intraperitoneal injection. Control animals are unmanipulated or 1% D
MSO was injected or phosphate buffered saline (PBS) was injected. The data in Table 29, when indicated, are recorded separately for males and females, and are otherwise combined for both genders. Changes from baseline (pre-injection) to serum glucose are reported in mg / dl. Insulin is
Micro U / ml, "relative" measurements of mesenteric (male and female) fat, uterine fat (female) and quadriceps are recorded as percent of body weight. Other findings are
Represents consistent findings for both males and females. Statistically significant findings (p <
0.05) the red (or dark gray) for an increase _{(MRP1 18, MRP2 20, MRP3} 18, MRP4 18, MRP5 20, MRP7 20, and MRP10 ₂₀ glucose increased for; relative the well MRP1 _20, and MRP10 ₂₀ Muscle increase), and bluish green (or light gray) for decrease (MRP1 ₂₀ , MRP4 ₂₀ , and MRP6 ₂₀ for glucose decrease; MRP1 ₂₀ , MRP
_{1 18, MRP2 20, MRP3 18} , MRP4 18, MRP8 20, MRP10 20 and MRP1 ₁₈ (rat) reduction of the final insulin _{for; MRP1 20, MRP5 20, MRP10} 20 and MRP1 ₁₈ (rat) decreased relative fat percentage for ;
And MRP2 ₂₀ and MRP7 ₂₀ ) were shaded.

A separate test group was established for each peptide tested, vehicle control and non-engineered group. Each test group consisted of 5 male and 5 female AKR mice (10 per peptide). The mice were habituated for 2 days after moving. The mice were weighed and divided into groups. Food weight and water volume per cage were measured and recorded.

The mice were acclimated for a further 5 days. On the day before the injection (-2), the glucose measurement was
Glucometer (Johnson & Johnson)
n; One touch Sure Step). Blood samples were first obtained by heating the mice under a heat lamp approximately 5 minutes before obtaining a drop of blood from the tail vein. Dosing of the various peptides started at day (0). Peptides were prepared identically, except as noted in Table 29, at the same concentration (1%
Dosage at 0.05 mg / ml in PBS with DMSO, 10 ml / kg dosing volume). The stock peptide solution was dissolved by sonicating for 1-2 minutes, if necessary, to dissolve 2.5 mg peptide in 0.5 ml DMSO, followed by addition of 0.5 ml sterile PBS to each vial. Prepared. The dosing solution is
Prepared weekly by the addition of 0.5 ml peptide stock solution to 24.5 ml sterile PBS. All dosing solutions were kept refrigerated throughout the study.

Mice were weighed and given daily ip injections of various peptides or vehicles for 14 consecutive days. Each 7-day food weight and water volume was measured and recorded per cage. Blood glucose measurements (by glucometer) were also performed and recorded each 7 days. Blood glucose measurements were taken at 8:00 and 10:00 am and animals were dosed daily at 9:00 and 11:00 am.

On day 14, mice were injected with a final dose of various peptides or vehicle 1 hour before necropsy and blood collection. Blood (heparinized plasma) was obtained by heart or orbital puncture. Plasma compounds, glucose and insulin and final glucometer readings were taken at the same time to calibrate / verify the glucometer readings. Animals were dissected and placed in formalin for histological sample preparation. Mesenteric fat and pancreas, liver, quadriceps and caudal fat pads were removed and weighed for each animal. This study shows that ENDOX peptides can be used to regulate various metabolic functions and to treat metabolic disorders.

[0398]

[Table 49] Example 2 Clustal W Alignment Table 30 shows the Clustal W alignment of all 10 human endozepines. This family consists of two classes of polypeptides, 7 short polypeptides of about 90 amino acids and 3 long polypeptides, containing regions of homology at their N-termini to members of other families. There is no homology between the longer forms at these C-termini. This alignment and phylogenetic distance
It suggests that it could evolve by gene duplication, fusion and independent evolution.

[0399]

[Table 50] Example 3 Metabolic Consensus Sequence-Regulatory Peptides Peptides of all and some subsets of MRP, which have similar metabolic effects in AKR mice, are identified in Table 29. These can be aligned to identify the consensus sequences identified in Table 31. General and preferred consensus sequences are inferred by inspection. Most conserved amino acids at each position in the 20 amino acid motif are printed in bold. If the amino acid is invariant in a particular metabolic subset, it is also underlined. If the amino acid at a particular position is highly variable, it is represented by "X". If a few amino acids are found at a particular position, these are included in [brackets]. A "generic" consensus sequence is possible for most variability in peptide sequences that may have similar metabolic effects. "Preferred" consensus sequences are limited to the amino acids found in the peptide subsets tested. Certain subsets of peptides have been identified in connection with their cholesterol-lowering properties, fat mass loss, insulin lowering, glucose lowering, glucose increasing and muscle mass building properties.

[0400]

[Table 51] Consensus sequences that occur in muscle mass:

[0401]

[Chemical 4] Example 4 Effect on Mesenteric Adipocyte Size FIG. 1 shows six microscopes of mesenteric adipocytes fixed in male AKR mice after treatment with the drugs shown below each panel. It consists of photos. In the upper panels (AC) large adipocytes predominate, while in the lower panels (DF) small adipocytes predominate. This corresponds to a decrease in the mass of specific adipocytes that a particular deposit deposits in response to a particular treatment. There is a subset of MRPs that affect the mass of specific adipocyte deposits, and a subset of MRPs that affect other metabolic parameters.

Example 5: Summary of Gene Expression, Cloning and Antibody Production Table 32 shows the endozepine family (Endo) synthesized from each endozepine.
X) and the corresponding members of the bioactive metabolic control peptide (MRP- #) are listed. Furthermore, the tissues where this gene is most highly expressed are shown by the results from real-time quantitative RTQS-PCR (TaqMan) using total RNA and traditional PCR using ds cDNA as template. PCR products corresponding to the coding regions of various endozepines were amplified for cloning purposes as described. Anti-peptide antibodies were raised in rabbits for each human synthetic 20 amino acid peptide. The determined (ELISA) anti-peptide titer is shown.

[0403]

[Table 52] Example 6: Phylogenetic relationships among new and known endozepines Table 33 shows various full length endozepines and the M encoded within each endozepine.
The relationship of RP is shown. This distance is calculated by the program “Phy
Lip ”(a method for describing the relative relatedness of input sequences).

[0404]

[Table 53] Example 7: Identification of Related Peptides in Other Proteins Tables 34 and 35 show how particular consensus motifs can be used in other animal species to relate to regulation of the same metabolic parameters. Exemplifies whether the polypeptide of can be identified. The GenBank database was searched using two common consensus sequences, the first sequence associated with the muscle mass construct (Table 3
4), and the second sequence is associated with reduced fat mass (Table 35). The consensus sequence identical to each peptide is shown in bold font. Many polypeptides from multiple species were identified with 0, 1 or 2 mismatches with the consensus sequence. The highest degree of similarity was found in ACBP from other species. This is also seen in Figure 1, which illustrates that directed evolutionary sequences can be found for 6 of the 10 members of the human endozepine family.

[0405]

[Table 54]

[0406]

[Table 55] Example 8: Directed evolution gene products in multiple species Table 36 illustrates the high degree of conservation of MRP motifs across diverse species. Human M
The RP sequence was used to search GenBank for related sequences. Eleven other sequences with a high degree of similarity were identified. Additional polypeptides are identified by using a larger peptide centered on the 20 amino acid MRP domain. Eleven entries from other species are indicated by their acquisition number. A set of 21 identified peptides from species ranging from frog to human was tested for their relationship to the set of 10 human peptides by PHYLIP, PILEUP and Clustal W algorithm. Table 3
As can be seen in 6, there are non-human peptides that are more similar to some human sequences than other human sequences. This suggests that the peptide-containing gene followed a divergence in energy metabolism early in evolution and followed.

[0407]

[Table 56] Example 37 also illustrates the high degree of conservation of MRP motifs across diverse species.

[0408]

[Table 57] Example 9: PCR Products of Endozepine Cloning Sequences The cloning sequences of each human endozepine were cloned into a number of expression vectors to test complex methods, where these secreted proteins and biologically active MRP affects energy metabolism. Some of the cDNAs were cloned in bacteria to prepare recombinant proteins. How to express a complete set of proteins in bacterial, yeast and human cells, their individual biochemical activities, protein-protein interactions, and energy metabolism in isolated cells or whole animals I studied how to adjust. Physical DNA molecules for this purpose were prepared from various tissues such as those listed above (Table 29).
Produced by CR and can be found on an ethidium bromide stained agarose gel (Figure 2). The individual PCR products for Endo 1-4 and 6-10 are of the expected size as deduced from the sequence, which now contains 42 bp of flanking cloning sequence.

Other Embodiments The present invention is described in combination with the detailed description thereof, but the above description is intended to exemplify the scope of the present invention defined by the appended claims. And is not intended to be limiting. Other aspects, advantages, and improvements are within the scope of the following claims.

[Brief description of drawings]

FIG. 1 shows mesenteric fat storage in mice in response to treatment (deoict).
ing) shows a micrograph.

[Fig. 2]   Figure 2 shows the PCR product of the endozepine coding sequence.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 3/04 A61P 3/08 4C085 3/06 3/10 4C086 3/08 7/00 4H045 3/10 35 / 00 7/00 43/00 111 35/00 C07K 14/47 43/00 111 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12Q 1 / 02 1/21 1/68 A 5/10 G01N 33/53 D C12Q 1/02 M 1/68 33/566 G01N 33/53 C12P 21/08 C12N 15/00 ZNAA 33/566 5/00 A // C12P 21/08 A61K 37/02 (31) Priority claim number 60 / 174,505 (32) Priority date January 4, 2000 (Jan. 4, 2000) (33) Country of priority claim United States (US) ( 31) Priority claim number 60 / 183,859 (32) Priority date February 22, 2000 (February 22, 2000) (33) Priority claiming United States (US) (31) Priority claiming number 60 / 190,740 (32) Priority date March 20, 2000 (March 20, 2000) (33) Priority claiming United States (US) (31) Priority claim number 60/191, 133 (32) Priority date March 22, 2000 (March 22, 2000) (33) Priority claim country United States (US) (31) Priority claim number 60 / 206,006 (32) Priority date May 19, 2000 (May 19, 2000) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 215,684 (32) Priority Date June 30, 2000 (June 30, 2000) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 219,490 (32) Priority date July 20, 2000 (2000. July 20) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 227,072 (32) Priority date August 22, 2000 (2000.8.22) ( 33) Priority claim country United States (US) (31) Priority claim number 09 / 679,460 (32) Priority date October 4, 2000 (October 2000) (33) Country claiming priority US (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES , FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE) , SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, P, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Majumdale, Kumdo United States Connecticut 06905, Stanford, Silver Hill Lane 140 (72) Inventor Eisen, Andrew United States Maryland 20850, Rockville, Saint James Road 12412 (72) Inventor Barnet, Colleen United States Florida 32060, Gainesville, NW 43 RD Street Pee Number 253 4830 (72) Inventor Spadana, Stephen Kay. United States Connecticut 06037, Berlin, Deerfield Drive 261 F Term (reference) 4B024 AA01 AA11 AA12 CA04 FA02 HA01 HA14 HA15 4B063 QA01 QA18 QA19 QQ08 QQ43 QR55 QR62 QS32 QS33 QS34 4B064 AG27 CA10 DA05B08 CA44 DA02 BAD4 DA06 DA07 DA06 DA07 4 AA02 AA07 AA13 BA01 BA08 BA20 BA21 BA23 CA18 DC50 NA14 ZA512 ZB262 ZC022 ZC032 ZC332 ZC352 4C085 AA13 BB11 4C086 AA01 AA02 AA03 EA16 MA01 MA04 NA14 EA51 EA51 EA51 CA50 A40 ZA51 A50 CA40 ZA33 A50 A40 A40 CA50 ZA33

Claims (49)

[Claims] 1. An isolated polypeptide comprising: (a) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A mature form of the amino acid sequence selected from the group consisting of and 49; (b) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A variant of the mature form of the amino acid sequence selected from the group consisting of and 49, which is one of the variants under the condition that the variant differs from the mature form of the amino acid sequence by not more than 15% of amino acid residues. (C) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, wherein the above amino acid residues are different from the mature amino acid sequence; 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
And an amino acid sequence selected from the group consisting of and 49; and (d) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A variant of an amino acid sequence selected from the group consisting of and 49, wherein one or more amino acid residues in the variant are different under the condition that the variant differs from the amino acid sequence by 15% or less of amino acid residues. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of a variant of the amino acid sequence which differs from the mature form of the amino acid sequence. 2. The polypeptide of claim 1, wherein the polypeptide is SEQ ID NO: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 2.
1, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47, and 49, comprising the amino acid sequence of a naturally occurring allelic variant of the amino acid sequence selected from the group consisting of: Polypeptide. 3. The polypeptide of claim 2, wherein the allelic variant is SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46,
A polypeptide comprising an amino acid sequence that is a translation of a nucleic acid sequence that differs by a single nucleotide from a nucleic acid sequence selected from the group consisting of: 4. The amino acid sequence of the variant contains conservative amino acid substitutions,
The polypeptide according to claim 1. 5. An isolated nucleic acid molecule, which comprises: (a) SEQ ID NO: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A mature form of the amino acid sequence selected from the group consisting of and 49; (b) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A variant of the mature form of the amino acid sequence selected from the group consisting of and 49, which is one of the variants under the condition that the variant differs from the mature form of the amino acid sequence by not more than 15% of amino acid residues. (C) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, wherein the above amino acid residues are different from the mature amino acid sequence; 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
And an amino acid sequence selected from the group consisting of 49; (d) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A variant of an amino acid sequence selected from the group consisting of and 49, wherein one or more amino acid residues in the variant are different under the condition that the variant differs from the amino acid sequence by 15% or less of amino acid residues. An amino acid sequence variant different from the mature form amino acid sequence, (e) SEQ ID NOs: 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20,
21, 23, 24, 26, 27, 29, 30, 32, 33, 35-45, 47,
A nucleic acid fragment encoding at least a part of a polypeptide comprising an amino acid sequence selected from the group consisting of and 49 or a variant of the polypeptide, wherein the variant is 15% of the amino acid sequence and amino acid residues. A nucleic acid fragment (f) (a), (b), (c), (d) or one or more amino acid residues in the variant that differ from the mature form of the amino acid sequence under different conditions below: A nucleic acid molecule comprising the complement of (e),
An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: 6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of a naturally occurring allelic nucleic acid variant. 7. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule encodes a polypeptide that comprises the amino acid sequence of a naturally occurring variant of the polypeptide. 8. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule comprises SEQ ID NOs: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48. A nucleic acid molecule that differs in a single nucleotide from a nucleic acid sequence selected from. 9. The nucleic acid molecule according to claim 5, wherein the nucleic acid molecule is: (a) SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46. A nucleotide sequence selected from the group consisting of:
5,7,9,22,25,28,31,34,46, and 48 under different conditions from the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,
A nucleotide sequence that differs in one or more nucleotides from a nucleotide sequence selected from the group consisting of 22, 25, 28, 31, 34, 46, and 48; (c) a nucleic acid fragment of (a); and (d) ( A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of the nucleic acid fragments of b). 10. The nucleic acid molecule according to claim 5, wherein the nucleic acid molecule is SEQ ID NO: 1, 3, 5, 7, 9, 22, 25, 28, 31, 34, 46, and 48.
A nucleic acid molecule that hybridizes under stringent conditions to a nucleotide sequence selected from the group consisting of or a complement of the nucleotide sequence. 11. The nucleic acid molecule according to claim 5, wherein the nucleic acid molecule is: (a) a first nucleotide sequence, which comprises 20% of the nucleotides of the coding sequence in the first nucleotide sequence. A first nucleotide sequence, wherein the first nucleotide sequence comprises a coding sequence that differs from the coding sequence encoding the amino acid sequence by one or more nucleotide sequences under the following conditions that differ from the coding sequence; (b) A nucleic acid molecule comprising an isolated second polynucleotide that is the complement of the first polynucleotide; and a nucleotide sequence selected from the group consisting of (c) (a) or (b) nucleic acid fragments. 12. A vector comprising the nucleic acid molecule according to claim 11. 13. The vector of claim 12, further comprising a promoter operably linked to the nucleic acid molecule. 14. A cell comprising the vector according to claim 12. 15. A polypeptide that immunospecifically binds to the polypeptide according to claim 1,
antibody. 16. The antibody according to claim 15, wherein the antibody is a monoclonal antibody. 17. The antibody of claim 15, wherein the antibody is a humanized antibody. 18. A method for determining the presence or amount of the polypeptide of claim 1 in a sample, the method comprising: (a) providing the sample; (b) the above. Contacting the sample with an antibody that immunospecifically binds to the polypeptide; and (c) determining the presence or amount of the antibody bound to said polypeptide, whereby the polypeptide in said sample A method of determining the presence or amount of 19. A method for determining the presence or amount of the nucleic acid molecule of claim 5 in a sample, said method comprising: (a) providing said sample; (b) Contacting the sample with a probe that binds to the nucleic acid molecule; and (c) determining the presence or amount of probe bound to the nucleic acid molecule, whereby the presence of the nucleic acid molecule in the sample. Alternatively, a method comprising the step of determining the amount. 20. The method of claim 19, wherein the presence or amount of the nucleic acid molecule is used as a marker for cell or tissue type. 21. The method of claim 20, wherein the cell or tissue type is cancerous. 22. A method for identifying an agent that binds to the polypeptide of claim 1, wherein the method comprises: (a) contacting the agent with the polypeptide; and (b) ) Determining whether or not the factor binds to the polypeptide. 23. The method of claim 22, wherein the factor is a cellular receptor or a downstream effector. 24. A method for identifying a factor that regulates the expression or activity of the polypeptide of claim 1, which method comprises: (a) providing a cell that expresses the polypeptide. (B) contacting the cell with the factor; and (c) determining whether the factor modulates expression or activity of the polypeptide, whereby expression of the peptide. Alternatively, the alteration in activity comprises the step of indicating the factor that regulates expression or activity of the polypeptide. 25. A method for modulating the activity of the polypeptide of claim 1, wherein the method binds to the polypeptide in an amount sufficient to modulate the activity of the polypeptide. And a step of contacting the cell sample expressing the polypeptide according to claim 1 with the method. 26. A method for treating or preventing an ENDOX-related disorder, the method of claim 1 in an amount sufficient to treat or prevent the ENDOX-related disorder in a subject. A method comprising administering a peptide to said subject for whom such treatment or prevention is desired. 27. The method of claim 26, wherein the disorder is diabetes,
A method selected from the group consisting of obesity-related metabolic disorders, metabolic syndrome X, eating disorders, chronic disease-related wasting disorders, cancer, cancer-associated cachexia, and dyslipidemia. 28. The method of claim 26, wherein the disorder is associated with biological energy metabolism leading to fat storage, muscle mass, insulin secretion, glucose utilization and serum lipid levels including triglycerides and cholesterol. ,Method. 29. The method of claim 26, wherein the subject is a human. 30. A method for treating or preventing an ENDOX-related disorder, wherein the method is in an amount sufficient to treat or prevent the ENDOX-related disorder in a subject. Is administered to the subject for which such treatment or prevention is desired. 31. The method of claim 30, wherein the disorder is diabetes,
A method selected from the group consisting of obesity-related metabolic disorders, metabolic syndrome X, eating disorders, chronic wasting-related wasting disorders, cancer, cancer-associated cachexia, and dyslipidemia. 32. The method of claim 30, wherein the disorder is associated with biological energy metabolism leading to fat storage, muscle mass, insulin secretion, glucose utilization and serum lipid levels including triglycerides and cholesterol. ,Method. 33. The method of claim 30, wherein the subject is a human. 34. A method for treating or preventing an ENDOX-related disorder, wherein the method is in an amount sufficient to treat or prevent the ENDOX-related disorder in a subject. Is administered to the subject for which such treatment or prevention is desired. 35. The method of claim 34, wherein the disorder is diabetes,
A method selected from the group consisting of obesity-related metabolic disorders, metabolic syndrome X, eating disorders, chronic wasting-related wasting disorders, cancer, cancer-associated cachexia, and dyslipidemia. 36. The method of claim 34, wherein the disorder is associated with body energy metabolism that results in fat storage, muscle mass, insulin secretion, glucose utilization and serum lipid levels including triglycerides and cholesterol. ,Method. 37. The method of claim 34, wherein the subject is a human. 38. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier. 39. A pharmaceutical composition comprising the nucleic acid molecule of claim 5 and a pharmaceutically acceptable carrier. 40. A pharmaceutical composition comprising the antibody of claim 15 and a pharmaceutically acceptable carrier. 41. A kit comprising the pharmaceutical composition of claim 38 in one or more containers. 42. A kit comprising the pharmaceutical composition of claim 39 in one or more containers. 43. A kit comprising the pharmaceutical composition of claim 40 in one or more containers. 44. A method for determining the presence or predisposition to a disease associated with altered levels of the polypeptide of claim 1 in a first mammalian subject. The following: (a) measuring the level of expression of the polypeptide in the sample from the first mammalian subject; and (b) the amount of the polypeptide in the sample of step (a), Comparing with the amount of said polypeptide present in a control sample from a second mammalian subject known not to have said disease or not predisposed to said disease; Wherein the alteration in the expression level of said polypeptide in said first subject relative to said control sample is indicative of the presence of or predisposition to said disease. 45. The method of claim 44, wherein the predisposition is a predisposition to cancer. 46. A method for determining the presence or predisposition to a disease associated with altered levels of the nucleic acid molecule of claim 5 in a first mammalian subject, the method comprising: The following: (a) measuring the amount of the nucleic acid in a sample from the first mammalian subject;
And (b) the amount of said nucleic acid in said sample of step (a) from a second mammalian subject known not to have said disease or to be predisposed to said disease. Comparing the amount of the nucleic acid present in a control sample of, wherein the alteration in the level of the nucleic acid in the first subject relative to the control sample is the presence of the disease or the A method of indicating the presence of a predisposition to a disease. 47. The method of claim 46, wherein the predisposition is a predisposition to cancer. 48. A method for treating a pathological condition in a mammal, the method comprising administering to the mammal the polypeptide in an amount sufficient to reduce the pathological condition. And the polypeptide is SEQ ID NO: 2, 4, 6, 8,
10, 15, 16, 17, 18, 19, 20, 21, 23, 24, 26, 27,
A polypeptide comprising at least one amino acid sequence of 29, 30, 32, 33, 35-45, 47, and 49, or a polypeptide having an amino acid sequence at least 95% identical to a biologically active fragment thereof. A method that is a peptide. 49. A method for treating a pathological condition in a mammal, wherein the method comprises administering the antibody of claim 15 in an amount sufficient to reduce the pathological condition. A method comprising the step of administering to an animal.