WO2005110460A2 - Méthodes de traitement et diagnostics relatifs au vieillissement, particulièrement celui des muscles (14.1) - Google Patents

Méthodes de traitement et diagnostics relatifs au vieillissement, particulièrement celui des muscles (14.1) Download PDF

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WO2005110460A2
WO2005110460A2 PCT/US2005/014441 US2005014441W WO2005110460A2 WO 2005110460 A2 WO2005110460 A2 WO 2005110460A2 US 2005014441 W US2005014441 W US 2005014441W WO 2005110460 A2 WO2005110460 A2 WO 2005110460A2
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protein
human
age
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biological
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WO2005110460A3 (fr
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John J. Kopchick
Karen T. Coschigano
Keith S. Boyce
Andres Kriete
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Ohio University
Icoria, Inc.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • mice 60/474,606, filed June 2, 2003 (our docket Kopchick7-USA)
  • our research group used a gene chip to study the genetic changes in the liver of C57B1/6J mice that occur at frequent intervals of the aging process.
  • Differential hybridization techniques were used to identify mouse genes that are differentially expressed in mice, depending upon their age.
  • RNA derived from mice of different ages was screened for hybridization with oligonucleotide probes each specific to a particular mouse gene, each gene in turn representative of a particular mouse gene cluster (Unigene) .
  • Related human genes and proteins were identified by sequence comparisons to the mouse gene or protein.
  • Kopchick7A-PCT filed June 2, 2004, we added some additional studies of CIDE-A (see below) .
  • the effect of aging on the expression of genes in mouse skeletal muscle was studied, see provisional application Ser. No. 60/566,068, filed April 29, 2004 (our docket Kopchickl4-USA) .
  • 60/460,415 (our docket: Kopchick6- USA) , filed April 7, 2003, was similar, but complementary RNA, derived from RNA of mouse liver, was screened against a mouse gene chip. See also 60/506,716, filed Sept. 30, 2003 . (Kopchick6.1) . Gene chip analyses have also been used to identify genes differentially expressed in normal vs. hyperinsulinemic, hyperinsulinemic vs. type II diabetic, or normal vs. type II diabetic mouse pancreas, see U.S. Provisional Appl. 60/517,376, filed Nov. 6, 2003 (Kopchickl2) and muscle, see U.S Provisional Appl. 60/547,512, filed Feb.
  • the invention relates to various nucleic acid molecules and proteins, and their use in (1) diagnosing aging, or adverse conditions associated with the aging process, and (2) protecting mammals (including humans) against the aging process or adverse conditions associated with the aging process .
  • mice Since the majority of cellular ROS is produce in the mitochondria during the process of electron transport, it is not surprising that clk-1 mutants have only a moderately extended life-span. C. eleg'ans containing daf- 2/clk-l double mutations, however, exhibit a very long life- span (13) . Decreased IGF-1 signaling may also extend longevity in mice.
  • Four mouse models with deficiencies in pituitary endocrine action have demonstrated retarded aging. In the Propl and Pit! models, pituitary production of growth hormone (GH) , prolactin (PRL) and thyroid stimulating hormone (TSH) are ablated. These mice have reduced growth rates, reduced adult body size and live 40 to 60% longer than normal mice (14,15).
  • GH growth hormone
  • PRL prolactin
  • TSH thyroid stimulating hormone
  • the measured or calculated parameters were total body mass, lean body mass, left leg lean mass (by biopsy) , maximum isometric left knee extension force, left knee extension force/left keg lean mass, Peak V0 2 /lean body mass, and Peak V0 2 /left leg lean mass.
  • There were 1178 "probe sets" (representing 1053 different Unigene clusters) for which differential expression was detected; 550 for which expression was higher in older women, and 628 the inverse effect. The differences ranged from 1.2 to 4 fold; most (78A%) were less than 1.5 fold.
  • Microarrays have also been used in the identification of aging-related genes by virtue of differential expression in other organs and tissues, see, e.g., Miller, J. Gerontol., 56A: B52-57 (2001) (liver) ; Lee et al . , Science, 285 : 1390-93 (1999) and Nature Genetics 25: 294-7 ( 2000) (mouse cerebellum and neocortex); Lee et al . , Proc Natl Acad Sci USA 99:14988-14993 (2002) (Ref. 22) (heart) ; Prolla, Chem Senses 27299-306 (2002) (Ref.
  • Endocrinol., 15 (2): 308-18 (2001) used microarray technology to study the effect of aging and growth hormone treatment on the expression of 3,000 different genes in the rat liver.
  • the proteins which were over-expressed in the older rat were glucose-6- phosphate isomerase (xl.8), pyruvate kinase (x4.8), hepatic product spot 14 (2.4x), fatty acid synthase (1.9x), staryl CoA desaturase (1.7x), enoyl CoA hyydratase (1.7x), peroxisome proliferator activated receptor- ⁇ (1.7x), 3- ketoacyl-CoA thiolase (1.7x), 3-keto-acyl-CoA peroxisomal thiolase (1.9x), CYP4A3 (3.3x), glycerol-3-phosphate dehydrogenase (1.7x), NAPDH-cytochrome P450 oxidoreductase (4.7x) .
  • CUP2C7 (1.9x), CYP3A2 (2.8x), ⁇ -aminoevulinate synthase (2.3x) .
  • the under-expressed proteins were glu ⁇ ose- 6-phosphatase (0.3x), farnesyl pyrophosphate synthase (0.5x), carnitine octanoyltransferase (0.5x), mitochrondrial genome (16S ribosomal RNA) (0.3x), mitochondrial cytochrome c oxidase II (0.4x), mitochondrial NADH dehydrogenase SU 5 (0.3x), mitochondrial cytochrome b (0.4x), mitochondrial NADH dhydrogenase SU 3 (0.5x), NADH-ubiquinone oxidoreductase (SU CI-SGDH and SU 39kDa) (both 0.5x), ubiquinol-cytochrome c reductase (Rieske iron-sulfur protein and core 1) (both 0.5x
  • IGF-1 insulin-like growth factor 1
  • a cyclin genes for two IGF-binding proteins, a cyclin, a heat shock protein, p38 mitogen-activated protein kinase, and an inducible cytochrome P450 were among those implicated by the survey.
  • a simulation study showed that genes with this degree of interanimal variation would often produce false-positive findings when conclusions were based on ratio calculations alone (i.e., without formal significance testing) .
  • Patents of possible interest include the following:
  • Articles of interest include Kayo, et al., Proc. nat . Acad. Sci. (USA) 98:5093-98 (2001); Han, et al . , Mch. Ageing Dev. 115:157-74 (2000); Dozmorov, et al . , J. gerontol . A Biol. Sci. Med. Sci. 56:B72-B80 (2001); Dozmorov, et al . , Id., 57: B99-B108 (2002); Miller, et al . , Mol. Endocrinol., 16: 2657-66 (2002) .
  • PCG-l ⁇ responsive genes involved in oxidative phosphorylation are coordinatively downregulated in human diabetes
  • D A microarrays to detect changes in the expression of sets of related genes, rather than of individual genes. They classified over 22,000 genes into 149 data sets; some of these data sets overlapped. They looked for a statistical correlation between the overall rank order of the genes in differential expression, and the groups to which the genes belonged. Expression was compared pairwise among three groups: males with normal glucose tolerance; males with impaired glucose tolerance; and males with type 2 diabetes.
  • the set with the highest enrichment score (the one whose members ranked highly most often relative to chance expectation) was an internally curated set of 106 genes involved in oxidative phosphorylation. While the average decrease for the individual genes was modest (-20%) , it was also consistent, being observed in 89% (94/106) of the genes in question.
  • This paper is reviewed by Toye and Gauguier, "Genetics and functional genomics of type 2 diabetes mellitus", Genome Biology, 4: 241 (2003). Patti, et al . , “Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and diabetes: Potential role of PGC1 and NRF1", Proc. Nat. Acad. SCi.
  • microarrays used microarrays to analyze skeletal muscle expression of genes in nondiabetic insulin-resistant subjects at high risk for diabetes (based on family hisotry of diabetes and Mexican-American ethnicity) and diabetic Mexican-American subjects.
  • 7,129 sequences represented on the microarray 187 were differentially expressed between control and diabetic subjects.
  • no single gene remained significantly differentially expressed after controlling for multiple comparison false discovery by using the Benjamin!-Hochberg method, see Benjamini, et al . , J. R. Stat . Soc. Sert . B. 57:289-300 (1995); Dudait, et al . , Stat. Sin. 12: 111-139 (2002).
  • MAPP FINDER the top-ranked cellular component terms were mitochondrion, mitochondrial membrane, mitochondrial inner membrane, and ribosome, and the top- ranked process term was ATP biosynthesis.
  • ONTOEXPRESS the over-represented groups were energy generation, protein biosynthesis/ribosomal proteins, RNA binding, ribosomal structural protein, and ATP synthase complex.
  • Kidney androgen-regulated protein gene was used as a positive control, as it is known to be up-regulated by DHT. See also Holland, et al . , Abstract 607, "Identification of Genes Possibly Involved in Nephropathy of Bovine Growth Hormone Transgenic Mice” (Endocrine Society Meeting, June 22, 2000) and Coschigano, et al . , Abstract 333, "Identification of Genes Potentially Involved in Kidney Protection During Diabetes” (Endocrine Society Meeting, June 22, 2000) .
  • the following differential hybridization articles may also be of interest: Wada, et al .
  • Apoptosis and CIDE-A Apoptosis is a form of programmed cell death that occurs in an active and controlled manner to eliminate unwanted cells.
  • Apoptotic cells undergo an orchestrated cascade of morphological changes such as membrane blebbing, nuclear shrinkage, chromatin condensation, and formation of apoptotic bodies which then undergo phagocytosis by neighboring cells.
  • morphological changes such as membrane blebbing, nuclear shrinkage, chromatin condensation, and formation of apoptotic bodies which then undergo phagocytosis by neighboring cells.
  • One of the hallmarks of cellular apoptosis is the cleavage of chromosomal DNA into discrete oligonucleosomal size fragments. This orderly removal of unwanted cells minimizes the release of cellular components that may affect neighboring tissue.
  • membrane rupture and release of cellular components during necrosis often leads to tissue inflammation.
  • the process of apoptosis is highly conserved and involves the activation of the caspase cascade. Cohen, GM.
  • Caspases are a family of serine proteases that are synthesized as inactive proenzymes .
  • Apoptotic signals such as CD95 (Fas) death receptor activation or tumor necrosis factor results in the cleavage of specific target proteins and execution of the apoptotic program.
  • Apoptosis may occur by either an extrinsic pathway involving the activation of cell surface death receptors (DR) or by an intrinsic mitochondrial pathway.
  • DR cell surface death receptors
  • Hepatocytes are members of the Type-II cells in which mitochondria are essential for DR-mediated apoptosis Scaffidi, C. , Fulda, S., Srinivasan, A., Friesen, C. , Li, F., Tomaselli, K.J., Debatin, K.M., Krammer, P.H., Peter, M.E. (1998) Two CD95 (APO-l/Fas) signaling pathways. EMBO J. 17:1675-1687.
  • the pro-apoptotic protein Bid is truncated by activated caspases-8/10 and translocates to the mitochondria.
  • DFF DNA fragmentation factor
  • DFF45 cleavage by activated caspase-3 results in its dissociation from DFF40 and allows the caspase-activated DNAse (CAD) activity of DFF40 to cleave chromosomal DNA into oligonucleosomal size fragments.
  • CAD caspase-activated DNAse
  • CIDEs cell-death-inducing DFF45-like effectors
  • CIDE a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor.
  • CAAT-enhancer-binding protein (C/EBP) and C/EBP-like proteins interact with sequences required for differentiation-dependent expression. J. Biol. Chem. 267:7185-7193; Liang, L. , Zhao, M. , Xu, Z., Yokoyama, K.K., Li, T.
  • CIDE-3 a novel member of the cell-death-inducing DNA- fragmentation-factor (DFF45) -like effector family. Biochem. J. 370:195-203.
  • the CIDEs contain an N-terminal domain that shares homology with the N-terminal region of DFF45 and may represent a regulatory region via protein interaction. See Inohara, supra; Lugovskoy, A.A. , Zhou, P., Chou, J.J., McCarty, J.S., Li, P., Wagner, G.
  • CIDE-A human and mouse CIDE-A are expressed in several tissues such as brown adipose tissue (BAT) and heart and are localized to the mitochondria, Zhou, Z., Yon Toh, S., Chen, Z., Guo, K. , Ng, C.P., Ponniah, S., Lin, S.C, Hong, W. , Li, P. (2003) Cidea-deficient mice have lean phenotype and are resistant to obesity. Nat. Genet. 35:49-56. .
  • CIDE-A can interact and inhibit UCP1 in BAT and may therefore play a role in regulating energy balance, see Zhou supra.
  • Previous reports have indicated that CIDE-A is not expressed in either adult human or mouse liver tissue, see Inohara supra, Zhou supra.
  • CIDE-A The human protein cell death activator CIDE-A is of particular interest because of its highly dramatic change in liver expression with age, first demonstrated in our Kopchick7 application, supra. CIDE-A expression is elevated in older normal mice. CIDE-A expression was studied for normal C57BI/6J mouse ages 35, 49, 77, 133, 207, 403 and 558 days. Expression is low at the first five data points, then rises sharply at 403 days, and again at 558 days. CIDE-A was therefore classified as an "unfavorable protein", i.e., it was taught that an antagonist to CIDE-A could retard biological aging.
  • CIDE-A is also prematurely expressed in hyperinsulinemic and type-II diabetic mouse liver tissue. CIDE-A expression also correlates with liver steatosis in diet-induced obesity, hyperinsulinemia and type-II diabetes. These observations suggest an additional pathway of apoptotic cell death in Non-Alcoholic Fatty Liver Disease (NAFLD) and that CIDE-A may play a role in this serious disease and potentially in liver dysfunction associated with type-II diabetes.
  • NAFLD Non-Alcoholic Fatty Liver Disease
  • RNA derived from mice of different ages was screened for hybridization with oligonucleotide probes each specific to a particular mouse database DNA, the latter being identified, by database accession number, by the gene manufacturer.
  • Each database DNA in turn was also identified by the gene chip manufacturer as representative of a particular mouse gene cluster (Unigene) .
  • this database DNA sequence is a full length genomic DNA or cDNA sequence, and is therefore either identical to, or otherwise encodes the same protein as does, a natural full-length genomic DNA protein coding sequence. Those which don't present at least a partial sequence of a natural gene or its cDNA equivalent .
  • mouse genes all of these mouse database DNA sequences, whether full-length or partial, and whether cDNA or genomic DNA, are referred to herein as "mouse genes".
  • genomic DNA or “gDNA” .
  • the sequences in the protein databases are determined either by directly sequencing the protein or, more commonly, by sequencing a DNA, and then determining the translated amino acid sequence in accordance with the Genetic Code. All of the mouse sequences in the mouse polypeptide database are referred to herein as “mouse proteins” regardless of whether they are in fact full length sequences.
  • a mouse gene is considered to be "favorable" (more precisely, “wholly favorable") for the purpose of Master Table 1 (especially subtable IA) if, for at least one of the time comparisons set forth in the Examples, it exhibited substantially favorable behavior, and if, for all the other comparisons, it at least did not exhibit substantially unfavorable behavior. Note that the classification of a gene as favorable for purpose of the Master Table does not mean that it must have exhibited substantially favorable behavior for all of the comparisons set forth in the Examples .
  • a mouse gene is considered to be "unfavorable” (more precisely, “wholly unfavorable”) for the purpose of the Master Table 1 (especially subtable IB) if, for at least one of the time comparisons set forth in the Examples, it exhibited substantially unfavorable behavior, and if, for all the other comparisons, it at least did not exhibit substantially favorable behavior.
  • a mouse gene is considered to be "mixed” (i.e., partially favorable and partially unfavorable) for the purpose of the Master Table, especially subtable IC, if for at least one of the time comparisons set forth in the Examples it exhibited substantially favorable behavior and if for at least one of the other such comparisons it exhibited substantially unfavorable behavior.
  • the expression of a gene may first rise, then fall, with increasing age. Or it may first fall, and then rise. These are just the two simplest of several possible “mixed” expression patterns . Thus, we can subdivide the "favorables” into wholly and partially favorables. Likewise, we can subdivide the unfavorables into wholly and partially unfavorables .
  • the genes/proteins with "mixed” expression patterns are, by definition, both partially favorable and partially unfavorable. In general, use of the wholly favorable or wholly unfavorable genes/proteins is preferred to use of the partially favorable or partially unfavorable ones.
  • mixed genes/proteins are those exhibiting a combination of favorable and unfavorable behavior.
  • a mixed gene/protein can be used as would a favorable gene/protein if its favorable behavior outweighs the unfavorable. It can be used as would an unfavorable gene/protein if its unfavorable behavior outweighs the favorable. Preferably, they are used in conjunction with other agents that affect their balance of favorable and unfavorable behavior.
  • Use of mixed genes/proteins is, in general, less desirable than use of purely f vorable or purely unfavorable genes/proteins .
  • human genes databases DNAs
  • proteins were identified by searching a database comprising human DNAs or proteins for sequences corresponding to (i.e., homologous to, i.e., which could be aligned in a statistically significant manner to) the mouse gene or protein. More than one human protein may be identified as corresponding to a particular mouse chip probe and to a particular mouse gene.
  • human genes and “human proteins” are used in a manner analogous to that already discussed in the case of "mouse genes” and “mouse proteins”.
  • the term "corresponding” does not mean identical, but rather implies the existence of a statistically significant sequence similarity, such as one sufficient to qualify the human protein or gene as a homologous protein or DNA as defined below.
  • the greater the degree of relationship as thus defined i.e., by the statistical significance of each alignment used to connect the mouse chip DNA, and the corresponding mouse gene/cDNA, to the human protein or gene, measured by an E value
  • the connection may be direct (mouse gene/cDNA to human protein) or indirect (e.g., mouse gene/cDNA to human gene, human gene to human protein) .
  • the human genes/proteins which most closely correspond, directly or indirectly, to the mouse gene/cDNA are preferred, such as the one(s) with the highest, top two highest, top three highest, top four highest, top five highest, and . top ten highest E values for the final alignment in the connection process.
  • the human genes/proteins deemed to correspond to our mouse genes are identified in the Master Tables.
  • homologous full- length human genes and proteins if they are present in the database, even if the query mouse DNA or protein sequence is not a full-length sequence. If there is no homologous full-length human gene or protein in the database, but there is a partial one, the latter may nonetheless be useful.
  • a partial protein may still have biological activity, and a molecule which binds the partial protein may also bind the full- length protein so as to antagonize a biological activity of the full-length protein.
  • a partial human gene may encode a partial protein which has biological activity, or the gene may be useful in the design of a hybridization probe or in the design of a therapeutic antisense DNA.
  • the partial genes and protein sequences may of course also be used in the design of probes intended to identify the full length gene or protein sequence.
  • Agents which bind the "favorable" and “unfavorable” nucleic acids may be used to -estimate the biological age of a human subject, or to predict the rate of biological aging in a human subject (i.e, to evaluate whether a human subject is at increased or decreased risk for faster-than-normal biological aging) .
  • a subject with one or more elevated “unfavorable” and/or one or more depressed “favorable” genes/proteins is at increased risk, and one with one or more elevated “favorable” and/or one or more depressed “unfavorable” genes/proteins is at decreased risk.
  • the assay may be used as a preliminary screening assay to select subjects for further analysis, or as a formal diagnostic assay.
  • the identification of the related genes and proteins may also be useful in protecting humans against faster-than- normal or even normal aging (hereinafter, "the disorders") . They may be used to reduce a rate of biological aging in the subject, and/or delay the time of onset, or reduce the severity, of an undesirable age-related phenotype in said subject, and/or protect against an age-related disease.
  • DNAs of interest include those which specifically hybridize to the aforementioned mouse or human genes, and are thus of interest as hybridization assay reagents or for antisense therapy. They also include synthetic DNA sequences which encode the same polypeptide as is encoded by the database DNA, and thus are useful for producing the polypeptide in cell culture or in situ (i.e., gene therapy) . Moreover, they include DNA sequences which encode polypeptides which are substantially structurally identical or conservatively identical in amino acid sequence to the mouse and human proteins identified in the Master Table 1, subtables IA or IC. Finally, they include DNA sequences which encode peptide (including antibody) antagonists of the proteins of Master Table 1, subtables IB or IC.
  • mouse DNAs also may be identified by screening human cDNA or genomic DNA libraries using the mouse gene of the Master Table, or a fragment thereof, as a probe. If the mouse gene of Master Table 1 is not full-length, and there is no closely corresponding full-length mouse gene in the sequence databank, then the mouse DNA may first be used as a hybridization probe to screen a mouse cDNA library to isolate the corresponding full-length sequence. Alternatively, the mouse DNA may be used as a probe to screen a mouse genomic DNA library.
  • agents of the present invention may be used alone or in conjunction with each other and/or known anti-aging or anti- age-related disease agents. It is of particular interest to use the agents of the present invention in conjunction with an agent disclosed in one of the related applications cited above, in particular, an antagonist to CIDE-A, the latter having been taught in Kopchick7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
  • a "full length" gene is here defined as (1) a naturally occurring DNA sequence which begins with an initiation codon (almost always the Met codon, ATG) , and ends with a stop codon in phase with said initiation codon (when introns, if any, are ignored) , and thereby encodes a naturally occurring polypeptide with biological activity, or a naturally occurring precursor thereof, or (2) a synthetic DNA sequence which encodes the same polypeptide as that which is encoded by (1) .
  • the gene may, but need not, include introns .
  • a "full-length" protein is here defined as a naturally occurring protein encoded by a full-length gene, or a protein derived naturally by post-translational modification of such a protein. Thus, it includes mature proteins, proproteins, preproteins and preproproteins . It also includes substitution and extension mutants of such naturally occurring proteins .
  • Muscle Muscle tissue constitutes about 40% of the body mass. Muscles may be classified by location, i.e., skeletal if attached to bone, cardiac if forming the wall of the heart, and visceral if associated with another body organ. Muscles may also be classified as voluntary or involuntary, depending on how their contractions and relaxations are controlled. Skeletal muscles are voluntary, while cardiac and visceral muscles are involuntary. It is also possible to classify muscles morphologically; skeletal and cardiac muscle cells are striated, whereas visceral muscle cells are not . Each skeletal muscle is composed of many individual muscle cells called muscle fibers. The fibers are held together by fibrous connective-tissue membranes called fascia.
  • the fascium which envelops the entire muscle is the epimysium, and the fascia which penetrate the muscle, separating the fibers into bundles (fasciculi) are called perimysium.
  • Very thin fascia (endomysium) sheath each muscle fiber.
  • Skeletal muscles are attached either directly to a bone, or indirectly through a tendon.
  • the individual muscle fibers (cells) comprise threadlike protein structures called myofibrils.
  • myofibrils There are over 600 muscles in the human body. We will have occasion later to refer to the gastrocnemius . It is a superficial muscle in the posterior compartment of the lower leg, which together with the underlying soleus forms the characteristic bulge of the calf.
  • infancy is defined as the period 0 to 21 days after birth. Sexual maturity is reached, on average, at 42 days after birth. The average lifespan is 832 days. In humans, infancy is defined as the period between birth and two years of age . Sexual maturity in males can occur between 9 and 14 years of age while the average age at first menstrual period for females is 12.6 years. The average human lifespan is 73 years for males and 79 years for females. The maximum verified human lifespan was 122 years, five months and 14 days .
  • the agents of the present invention inhibit aging for at least a subpopulation of mature (post-puberty) adult subjects.
  • healthy aging (sometimes called “successful aging”) refers to post-maturation changes in the body that occur with increasing age even in the absence of an overt disease.
  • total aging includes both the basal effects of healthy aging and the effects of any age-related disease.
  • normal aging uses the term "normal aging” as a synonym for "healthy aging”, but a minority use it to refer to “total aging”. To minimize confusion, we will try to avoid the term “normal aging”, but if we use it, it is as a synonym for "healthy aging”.
  • normal aging changes should be defined as those which are universal, degenerative, progressive and intrinsic.
  • the agents of the present invention inhibit healthy aging for at least a subpopulation of mature (post- puberty) adult subjects.
  • a decline in bone density is considered healthy aging, but when it drops to 2.5 SD below the young adult mean, it is called osteoporosis.
  • the term average (median) "lifespan" is the chronological age to which 50% of a given population survive.
  • the maximum lifespan potential is the maximum age achievable by a member of the population. As a practical matter, it is estimated as the age reached by the longest lived member (or former member) of the population.
  • the (average) life expectancy is the number of remaining years that an individual of a given age can expect to live, based on the average remaining lifespans of a group of matched individuals .
  • the most widely accepted method of measuring the rate of aging is by reference to the average or the maximum lifespan. If a drug treatment achieves a statistically significant improvement in average or maximum lifespan in the treatment group over the control group, then it is inferred that the rate of aging was retarded in the treatment group. Similarly, one can compare long-term survival between the two groups .
  • the agents of the present invention have the effect of increasing the average lifespan and/or the maximum lifespan for at least a subpopulation of mature (post-puberty) adult subjects. This subpopulation may be defined by sex and/or age.
  • age it may be defined by a minimum age (e.g., at least 30, at least 40, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 90, etc.) or by a maximum age (not more than 40, not more than 50, not more than 55, not more than 60, not more than 65, not more than 70, not more than 75, not more than 80, not more than 90, not more than 100, etc.), or by a rational combination of a minimum age and a maximum age so as to define a preferred close-ended age range, e.g., 55-75.
  • a minimum age e.g., at least 30, at least 40, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 90, etc.
  • a maximum age not more than 40, not more than 50, not more than 55, not more than 60, not more than 65, not more than 70, not more than 75, not more than
  • the subpopulation may additionally be defined by race, e.g., Caucasian, negroid or oriental, and/or by ethnic group, and/or by place of residence (e.g., North America, Europe) .
  • the subpopulation may additionally be defined by nonage risk factors for age-associated diseases, e.g., by blood pressure, body mass index, etc.
  • the subpopulation in which an agent of the present invention is reasonably expected to be effective is large, e.g., in the United States, preferably at least 100,000 individuals, more preferably at least 1,000,000 individuals, still more preferably at least 10,000,000, even more preferably at least 20,000,000, most preferably at least 40,000,000.
  • the U.S. population, by age was
  • biological age position in own life span (as fraction in range 0..1) X average life span for species. This simple definition carries with it the implicit assumption that the rate of biological aging is constant. It also has the practical problem of determining one's own life span before death. We will present a more practical definition shortly.
  • Physiological age it is the chronological age at which an average member of the population (or relevant subpopulation) would have the same value of a biomarker of biological aging (or the same value of a composite measure of biomarkers of biological aging) as does the subject. This is the definition that will be used in this disclosure, unless otherwise stated.
  • the effect of aging varies from system to system, organ to organ, etc. For example, between ages 30 and 70 years, nerve conduction velocity decreases by only about 10%, but renal function decreases on average by nearly 40%. Thus, there isn't just one biological age for a subject.
  • biomarker By a suitable choice of biomarker, one may obtain a whole organism, or a system-, organ- or tissue-specific measure of biological aging, e.g., one can say that a person has the nervous system of a 30 year old but the renal system of a 60 year old. Biomarkers may measure changes at the molecular, cellular, tissue, organ, system or whole organism levels. Generally speaking, in the absence of some form of intervention (drugs, diet, exercise, etc.), biological ages will increase with time. The agents of the present invention preferably reduce the time rate of change of a biological age of the subject.
  • a biological age could refer to the overall biological age of the subject, to the biological age of a particular system, organ or tissue of that subject, or to some combination of the foregoing. More preferably, the agents of the present cannot only reduce the rate of increase of a biological age of the subject, but can actually reduce a biological age of the subject .
  • a simple biologic marker is a single biochemical, cellular, structural or functional indicator of an event in a biologic system or sample.
  • a composite biomarker is a mathematical combination of two or more simple biomarkers. (Chronological age may be one of the components of a composite biomarker.)
  • a plausible biomarker of biological age would be a biomarker which shows a cross-sectional and/or longitudinal correlation with chronological age. Nakamura suggests that it is desirable that a biomarker show (a) significant cross- sectional correlation with chronological age, (b) significant longitudinal change in the same direction as the cross-sectional correlation, (c) significant stability of individual differences, and (d) rate of age-related change proportional to differences in life span among related species. Cp.
  • a superior biomarker of biological age would be a better predictor of lifespan than is chronological age (preferably for a chronological age at which 90% of the population is still alive) .
  • the biomarker preferably also satisfies one or more of the following desiderata: a statistically significant age- related change is apparent in humans after a period of at most a few years; not affected dramatically by physical conditioning (e.g., exercise), diet, and drug therapy (unless it is possible to discount these confounding influences, e.g., by reference to a second marker which measures them) ; can be tested repeatedly without harming the subject; works in lab animals as well as humans; simple and inexpensive to use; does not alter the result of subsequent tests for other biomarkers if it is to be used in conjunction with them; monitors a basic process that underlies the aging process, not the effects of disease.
  • biomarker works in lab animals, there is a statistically significant difference in the value of the biomarker between groups of food-restricted and normally-fed animals. It has been shown in some mammalian species that dietary restriction without malnutrition (e.g., caloric decrease of up to 40% from ad libitum feeding) increases lifespan.
  • a biomarker of aging may be used to predict, instead of lifespan, the "Healthy Active Life Expectancy” (HALE) or the "Quality Adjusted Life Years” (QALY) , or a similar measure which takes into account the quality of life before death as well as the time of death itself.
  • HALE Healthy Active Life Expectancy
  • QALY Quality Adjusted Life Years
  • a biomarker of aging may be used to predict, instead of lifespan, the timing and/or severity of a change in one or more age-related phenotypes as described below.
  • a biomarker of aging may be used to estimate, rather than overall biological age for a subject, a biological age for a specific body system or organ. The determination of the biological age of the muscle, and the inhibition of biological aging of the muscle, are of particular interest.
  • Body systems include the nervous system (including the brain, the sensory organs, and the sense receptors of the skin) , the cardiovascular system (includes the heart, the red blood cells and the reticuloendothelial system) , the respiratory system, the gastrointestinal system, the endocrine system (pituitary, thyroid, parathyroid and adrenal glands, gonads, pancreas, ' and parganglia) , the musculoskeletal system, the urinary system (kidneys, bladder, ureters, urethra) , the reproductive system and the immune system (bone marrow, thymus, lymph nodes, spleen, lymphoid tissue, white blood cells, and immunoglobulins) .
  • the nervous system including the brain, the sensory organs, and the sense receptors of the skin
  • the cardiovascular system includes the heart, the red blood cells and the reticuloendothelial system
  • the respiratory system the gastrointestinal system
  • the endocrine system pitu
  • a biomarker may be useful in estimating the biological age of a system because the biomarker is a chemical produced by that system, because it is a chemical whose activity is primarily exerted within that system, because it is indicative of the morphological character or functional activity of that system, etc.
  • a given biomarker may be thus associated with more than one system.
  • a biomarker may be associated with the biological age, and hence the state, of a particular organ or tissue. The prediction of lifespan, or of duration of system or organ function at or above a particular desired level, may require knowledge of the value of at least one biomarker of aging at two or more times, adequately spaced, rather than of the value at a single time.
  • a composite biomarker may be obtained by standard mathematical techniques, such as multiple regression, principal component analysis, cluster analysis, neural net analysis, and so forth.
  • the values may be standardized, e.g., by converting the raw scores into z-scores based on the distributions for each simple biomarker.
  • A/G RATIO is the ratio of albumin to globulin. The relative importance of these five biomarkers was 33.7%, 25.1%, 17.1%, 14.8% and 8.9%, respectively.
  • Biomarkers of aging are characteristics of an organism that correlate in large groups with chronological age and mortality. Of particular value in human applications are biomarkers of aging that also correlate with the quality of life in later life in the sense that they involve functions that are crucial to carrying out the activities of daily living.... A single biomarker of aging is limited by the fact that it measures only one isolated characteristic and is hardly representative of the diversity of functional and structural concomitants of aging.... Biological age, in contrast to chronological age, is an individual's hypothetical age calculated from scores obtained on a battery of tests of biomarkers of aging.
  • the age of which each biomarker score is typical is determined by comparison with scores obtained by a large representative group of persons (or organisms) spanning a range of ages . Then one of a variety of averaging techniques is employed (optionally with standardization steps) to obtain a single index of age, as described in detail by Hochschild. This index varies with, and therefore must be expressed with reference to, the measured biomarkers and the mathematical method of combining scores.” http: //www. longevitvinstituteone.com/
  • Abbo, USP 6,547,729 teaches determining the biological age (he calls it "performance age") of a subject by (1) for a sample population, determining a regression curve relating some set of observed values for an "indicator” of the functionality of a bodily system to the chronological age of the observed individuals, (2) solving the regression equation to obtain a predicted performance age, given the value of the indicator for the subject.
  • the regression can be based on more than one indicator, i.e., it can be a multiple regression.
  • the sample population can be defined by sex, age range, ethnic composition, and geographic location.
  • the bodily system may be a molecular, cellular, tissue or organ system.
  • the following indicators are suggested by Abbo: nervous system (memory tests, reaction time, serial key tapping, digit recall test, letter fluency, category fluency, nerve conduction velocity) , arteries (pulse wave velocity; ankle-brachial index) , skeletal system (bone mineral density) ; lungs (forced vital capacity) , heart (ejection fraction; length of time completed on a treadmill stress test) , kidneys (creatinine clearance) , proteins (glycosylation of hemoglobin) , endocrine glands (load level of bioactive testosterone; level of dehydroepiandrosterone sulfate, ratio of urinary 17-ketosteroids/l7- hydroxycorticosteroids; growth hormone; IGF-1) .
  • the agents of the invention have a favorable effect on the value of at least one simple biomarker of biological aging, such as any of the plausible biomarkers mentioned anywhere in this specification, other than the level of one of the proteins of the present . invention. More preferably, they have a favorable effect on the value of at least two such simple biomarkers of biological aging. Even more preferably, at least one such pair is of markers which are substantially non-correlated (R 2 ⁇ 0.5) .
  • the biomarkers in question reflect different levels of organization, and/or different body components at the same level of organization.
  • a visual reaction time with decision test is on the whole organism level, while a measurement of telomere length is on the cellular level .
  • a biomarker may, but need not, be an indicator related to one of the postulated causes or contributing factors of aging. It may, but need not, be an indicator of the acute health of a particular body system or organ.
  • a biomarker may measure behavior, cognitive or sensory function, or motor activity, or some combination thereof. It may measure the level of a type of cell (e.g., a T cell subset, such as CD4, CD4 memory, CD4 naive, and CD4 cells expressing P-glycoprotein) or of a particular molecule (e.g., growth hormone, IGF-1, insulin, DHEAS, an elongation factor, melatonin) or family of structurally or functionally related molecules in a particular body fluid (especially blood) or tissue. For example, lower serum IGF-1 levels are correlated with increasing age, and IGF-1 is produced by many different tissues . On the other hand, growth hormone is produced by the pituitary gland.
  • a type of cell e.g., a T cell subset, such as CD4, CD4 memory, CD4 naive, and CD4 cells expressing P-glycoprotein
  • a particular molecule e.g., growth hormone, IGF-1, insulin, DHEAS,
  • a biomarker may measure an indicator of stress (particularly oxidative stress) and resistance thereto. It has been theorized that free radicals damage biomolecules, leading to aging.
  • a biomarker may measure protein glycation or other protein modification (e.g., collagen crosslinking) . It has been theorized that such modifications contribute to aging. •
  • the biomarker may measure changes in the lengths of telomeres or in the rate of cell division. It has been theorized that telomere shortening beyond a critical length leads the cell to stop proliferating. Average telomere length therefore provides a biomarker as to how may divisions the cell as previously undergone and how many divisions the cell can undergo in the future. Suggested biomarkers have also included resting heart rate, resting blood pressure, exercise heart rate, percent body fat, flexibility, grip strength, push strength, abdominal strength, body temperature, and skin temperature. The present invention does not require that all of the biomarkers identified above be validated as indicative of biological age, or that they be equally useful as measures of biological age.
  • An indicator of functional status is an indicator that defines a functional ability (e.g., physiological, cognitive or physical function).
  • An indicator of functional status may also be related to the increase in morbidity and mortality with chronological age.
  • Such indicators preferably predict physiological, cognitive and physical function in an age-coherent way, and do so better than chronological age. Preferably, they can predict the years of remaining functionality, and the trajectory toward organ-specific illness in the individual. Also, they are preferably minimally invasive.
  • Suggested indicators include anthropometric data (body mass index, body composition, bone density, etc.), functional challenge tests (glucose tolerance, forced vital capacity) , physiological tests (cholesterol/HDL, glycosylated hemoglobin, homocysteine, etc.) and proteomic tests.
  • mice models for human aging exist. See Troen, supra, Table 3.
  • the drugs identified by the present invention may be further screened in one or more of these models.
  • Age-Related Phenotype An age-related phenotype is an observable change which occurs with age. An age-related phenotype may, but need not, also be a biomarker of biological aging.
  • the agent of the present invention favorably affects at least one age-related phenotype. More preferably, it favorably affects at least two age-related phenotypes, more preferably phenotypes of at least two different body systems .
  • the age-related phenotype may be a system level phenotype, such as a measure of the condition of the nervous system, respiratory system, immune system, circulatory system, endocrine system, reproductive system, gastrointestinal system, or musculoskeletal system.
  • the age-related phenotype may be an organ level phenotype, such as a measure of the condition. of the brain, eyes, ears, lungs, spleen, heart, pancreas, liver, ovaries, testicles, thyroid, prostate, stomach, intestines, or kidney.
  • the age-related phenotype may be a tissue level phenotype, such as a measure of the condition of the muscle, skin, connective tissue, nerves, or bones.
  • the age-related phenotype may be a cellular level phenotype, such as a measure of the condition of the cell wall, mitochondria or chromosomes .
  • the age-related phenotype may be a molecular level phenotype, such as a measure of the condition of nucleic acids, lipids, proteins, oxidants, and anti-oxidants.
  • the age-related phenotype may be manifested in a biological fluid, such as blood, urine, saliva, lymphatic fluid or cerebrospinal fluid.
  • a biological fluid such as blood, urine, saliva, lymphatic fluid or cerebrospinal fluid.
  • the biochemical composition of these fluid may be an overall, system level, organ level, tissue level, etc. phenotype, depending on the specific biochemical and fluid involved.
  • the Aging Liver The aging human liver appears to preserve its morphology and function relatively well. The liver appears to progressively decrease in both mass and volume. It also appears browner (a condition called "brown atrophy") , as a result of accumulation of lipofuscin (ceroid) within hepatocytes . Increases occur in the number of macrohepatocytes, and in polyploidy, especially around the terminal hepatic veins. The number of mitochondria declines, and both the rough and smooth endoplasmic recticulum diminish. The number of lysozymes increase. The liver is the premiere metabolic organ of the body. With regard to metabolism, hepatic glycerides and cholesterol levels increase with age, at least up to age 90.
  • Aging affects human skeletal muscle in a number of ways.
  • One of the principal changes in muscle function is that the force-generating capacity (strength) of the muscles is reduced. This, in turn, can lead to problems in performing normal daily activities.
  • This loss of strength is at least in part attributable to muscle atrophy, and alterations in the percentage of contractile tissue within muscle.
  • the atrophy can be characterized as a decrease in the cross- sectional area of the muscle (sarcopenia) .
  • Sarcopenia can result from reductions in fiber size and/or fiber number; the latter appears to be the more important of the two. Also, it appears that the number of both type I (slow) and type II (fast) fibers is reduced, although the changes in the individual fibers are more pronounced in the case of type II fibers.
  • the effects of aging on skeletal muscle may be determined, inter alia, by measurements on whole muscle, or on individual muscle fibers .
  • Older people have fewer motor units, but this is usually compensated for through increases in the size of the remaining motor units .
  • Muscle mass also decreases with age.
  • the muscle mass is determined by the relative rates of protein synthesis and breakdown, and it appears that with age, the rate of synthesis of at least some muscle proteins declines.
  • the percentage of muscle mass which is contractile tissue also decreases with age. (Non-contractile tissue includes, e.g., connective tissue) .
  • Muscle performance may be a function of changes, not only in the muscle per se, but also other systems, such as the nervous and circulatory systems.
  • Olive et al . did not observe age-related changes in maximal blood flow capacity after exercise, in resting blood flow, or in resting vascular diameter.
  • QOL quality of life
  • Clinicians are interested, not only in simple prolongation of lifespan, but also in maintenance of a high quality of life (QOL) over as much as possible of that lifespan.
  • QOL can be defined subjectively in terms of the subject's satisfaction with life, or objectively in terms of the subject's physical and mental ability (but not necessarily willingness) to engage in "valued activities", such as those which are pleasurable or financially rewarding.
  • Flanagan has defined five domains of QOL, capturing 15 dimensions of life quality.
  • the five domains, and their component dimensions, are physical and material well being (Material well-being and financial security; Health and personal safety) , Relations with other people (relations with spouse; Having and rearing children; Relations with parents, siblings, or other relatives ; Relations with friends) Social, community, civic activities (Helping and encouraging others; Participating in local and governmental affairs ) , Personal development, fulfillment (Intellectual development; Understanding and planning; Occupational role career; Creativity and personal expression) , and recreation (Socializing with others; Passive and observational recreational activities; Participating in active recreation) .
  • Flanagan JC "A research approach to improving our quality of life.” Am Psychol 33:138-147 (1978).
  • Health-related quality of life is an individual's satisfaction or happiness with domains of life insofar as they affect or are affected by “health” .
  • a pharmaceutical agent of the present invention is able to achieve a statistically significant improvement in the expected quality of life, measured according to a commonly accepted measure of QOL, in a treatment group over a control group.
  • QOL Quality of Life
  • a simple approach to measuring subjective QOL is to simply have the subjects rate their overall quality of life on a scale, e.g., of 7 points.
  • Objective QOL can be measured by, e.g., an activities checklist .
  • the Katz Index of Independence in Activities of Daily Living measures adequacy of independent performance of bathing, dressing, toileting, transferring, continence, and feeding. See Katz, S., "Assessing Self-Maintenance : Activities of Daily Living, Mobility and Instrumental Activities of Daily Living, Journal of the American Geriatrics Society, 31(12); 721-726 (1983); Katz S., Down, T.D. , Cash, H.R. et al . Progress in the Development of the Index of ADL. Gerontologist, 10 :20-30 (1970).
  • Performance of a more sophisticated nature is measured by the "Instrumental Activities of Daily Living” (IADL) scale. This inquires into ability to independently use the telephone, shop, prepare food, carry out housekeeping, do laundry, travel locally, take medication and handle finances. See Lawton, MP and Brody, EM, Gerontologist, 9:179-86 (1969) .
  • the 36 question Medical Outcomes Study Short Form (SF-36) (Medical Outcomes Trust, Inc., 20 Park Plaza, Suite 1014, Boston, Massachusetts 02116) assesses eight health concepts: 1) limitations in physical activities because of health problems; 2) limitations in social activities because of physical or emotional problems; 3) limitations in usual role activities because of physical health problems; 4) bodily pain; 5) general mental health (psychological distress and well-being) ; 6) limitations in usual role activities because of emotional problems; 7) vitality (energy and fatigue); and 8) general health perceptions.
  • SF-36 Medical Outcomes Study Short Form
  • a low score on an ADL, IADL or SF-36 test is likely to be associated with a low QOL, but a high score does not guarantee a high QOL because these tests do not explore performance of "valued activities", only of more basic activities. Nonetheless, these tests can be considered commonly accepted measures of QOL for the purpose of this invention.
  • Age-related (senescent) diseases include certain cancers, atherosclerosis, diabetes (type 2) , osteoporosis, hypertension, depression, Alzheimer's, Parkinson's, glaucoma, certain immune system defects, kidney failure, and liver steatosis.
  • they are diseases for which the relative risk (comparing a subpopulation over age 55 to a suitably matched population under age 55) is at least 1.1.
  • the agents of the present invention protect against one or more age-related diseases for at least a subpopulation of mature (post-puberty) adult subjects.
  • Diabetes Type II diabetes is of particular interest.
  • a deficiency of insulin in the body results in diabetes mellitus, which affects about 18 million individuals in the United States. It is characterized by a high blood glucose (sugar) level and glucose spilling into the urine due to a deficiency of insulin.
  • glucose saliva
  • the cells of the diabetic cannot use glucose for fuel, the body uses stored protein and fat for energy, which leads to a buildup of acid (acidosis) in the blood. If this condition is prolonged, the person can fall into a diabetic coma, characterized by deep labored breathing and fruity-odored breath.
  • Type II diabetes is the predominant form found in the Western world; fewer than 8% of diabetic Americans have the type I disease.
  • Type I diabetes In Type I diabetes, formerly called juvenile-onset or insulin-dependent diabetes mellitus, the pancreas cannot produce insulin. People with Type I diabetes must have daily insulin injections. But they need to avoid taking too much insulin because that can lead to insulin shock, which begins with a mild hunger. This is quickly followed by sweating, shallow breathing, dizziness, palpitations, trembling, and mental confusion. As the blood sugar falls, the body tries to compensate by breaking down fat and protein to make more sugar. Eventually, low blood sugar leads to a decrease in the sugar supply to the brain, resulting in a loss of consciousness. Eating a sugary food can prevent insulin shock until appropriate medical measures can be taken.
  • Type I diabetics are often characterized by their low or absent levels of circulating endogenous insulin, i.e., hypoinsulinemia (1) .
  • Islet cell antibodies causing damage to the pancreas are frequently present at diagnosis. Injection of exogenous insulin is required to prevent ketosis and sustain life.
  • Type II diabetes Type II diabetes, formerly called adult-onset or non-insulin-dependent diabetes mellitus (NIDDM) , can occur at any age . The pancreas can produce insulin, but the cells do not respond to it . Type II diabetes is a metabolic disorder that affects approximately 17 million Americans. It is estimated that another 10 million individuals are "prone" to becoming diabetic. These vulnerable individuals can become resistant to insulin, a pancreatic hormone that signals glucose (blood sugar) uptake by fat and muscle. In order to maintain normal glucose levels, the islet cells of the pancreas produce more insulin, resulting in a condition called hyperinsulinemi .
  • NIDDM non-insulin-dependent diabetes mellitus
  • Type II diabetes is a metabolic disorder that is characterized by insulin resistance and impaired glucose-stimulated insulin secretion (2,3,4).
  • Type II diabetes and atherosclerotic disease are viewed as consequences of having the insulin resistance syndrome (IRS) for many years (5) .
  • the current theory of the pathogenesis of Type II diabetes is often referred to as the "insulin resistance/islet cell exhaustion" theory.
  • a condition causing insulin resistance compels the pancreatic islet cells to hypersecrete insulin in order to maintain glucose homeostasis.
  • the islet cells eventually fail and the symptoms of clinical diabetes are manifested. Therefore, this theory implies that, at some point, peripheral hyperinsulinemia will be an antecedent of Type II diabetes.
  • Peripheral hyperinsulinemia can be viewed as the difference between what is produced by the beta cell minus that which is taken up by the liver. Therefore, peripheral hyperinsulinemia can be caused by increased beta cell production, decreased hepatic uptake or some combination of both. It is also important to note that it is not possible to determine the origin of insulin resistance once it is established since the onset of peripheral hyperinsulinemia leads to a condition of global insulin resistance. Multiple environmental and genetic factors are involved in the development of insulin resistance, hyperinsulinemia and type II diabetes. An important risk factor for the development of insulin resistance, hyperinsulinemia and type II diabetes is obesity, particularly visceral obesity (6,7,8). Type II diabetes exists world-wide, but in developed societies, the prevalence has risen as the average age of the population increases and the average individual becomes more obese .
  • Muscle, fat and liver tissues are the major contributors to the development of insulin resistance, hyperinsulinemia, and, ultimately, type II diabetes.
  • Muscle cells respond to insulin by increasing glucose uptake from the bloodstream. Muscle tissue can become resistant to insulin, causing the beta cells to initially increase insulin secretion. Eventually, though, the beta cells become "unable to compensate for this increasing insulin resistance from muscle and other cells, and they fail to respond to elevated blood glucose levels. Thus, clinical type 2 diabetes results from the combination- of insulin resistance and impaired beta cell function. Defects in muscle glycogen synthesis are known to play a role in the development of insulin resistance.
  • Fatty acids can induce insulin resistance, and it has been suggested that this was a consequence of altered insulin signaling through PI3-kinase.
  • PKC-theata has also been implicated. See generally Petersen, et al . , "Pathogenesis of Skeletal muscle insulin resistance in type 2 diabetes mellitus", in "A Symposium: Evolution of type 2 diabetes mellitus management", at Amer. J. Cardiol . , 90 (5A) : 11G-18G, (Sept. 5, 2002) .
  • Myopathy is a general term used to describe any disease of muscles, such as the muscular dystrophies and myopathies associated with thyroid disease. It can be caused by endocrine disorders, including diabetes, metabolic disorders, infection or inflammation of the muscle, certain drugs and mutations in genes. In diabetes, myopathy is thought to be caused by neuropathy, a complication of diabetes . General symptoms of myopathies include muscle weakness of limbs sometimes occurring during exercise although in some cases the symptoms diminish as exercise increases. Depending on the type of myopathy, one muscle group may be more affected than others.” See “Joint and Muscle Problems Associated with Diabetes", www, iddtinternational . org/ ointandmuscleproblems .html [Last modified June 12, 2003] .
  • Diabetic muscle infarction can spontaneously affect patients with a long history of poorly controlled diabetes. "Most affected patients have multiple microvascular complications (neuropathy, nephropathy, and retinopathy) . The clinical presentation is an acute onset of pain and swelling over days to weeks in the affected muscle groups (usually the thigh or calf) , along with varying degrees of tenderness.... Therapy consists of rest and analgesia. Routine daily activities are not deleterious to the condition, but physical therapy may cause exacerbation.
  • the identified mouse or human genes may be used directly. For diagnostic or screening purposes, they (or specific binding fragments thereof) may be labeled and used as hybridization probes. For therapeutic purposes, they (or specific binding fragments thereof) may be used as antisense reagents to inhibit the expression of the corresponding gene, or of a sufficiently homologous gene of another species . If the database DNA appears to be a full-length cDNA or gDNA, that is, that it encodes an entire, functional, naturally occurring protein, then it may be used in the expression of that protein.
  • Such expression may be in cell culture, with the protein subsequently isolated and administered exogenously to subjects who would benefit therefrom, or in vivo, i.e., administration by gene therapy.
  • any DNA encoding the same protein may be used fr the same purpose, and a DNA encoding a protein which a fragment or a mutant of that naturally occurring protein which retains the desired activity, may be used for the purpose of producing the active fragment or mutant.
  • the encoded protein of course has utility therapeutically and, in labeled or immobilized form, diagnostically.
  • the genes may also be used indirectly, that is, to identify other useful DNAs, proteins, or other molecules.
  • the known human protein is known to have additional homologues, then those homologous proteins, and DNAs encoding them, may be used in a similar manner.
  • a human protein homologue of interest can be identified by database searching, including but not limited to:
  • a known human gene Once a known human gene is identified, it may be used in further BlastN or BlastX searches to identify other human genes or proteins .
  • a known human protein Once a known human protein is identified, it may be used in further BlastP searches to identify other human proteins. Searches may also take cognizance, intermediately, of known genes and proteins other than mouse or human ones, e.g., use the mouse sequence to identify a known rat sequence and then the rat sequence to identify a human one.
  • mouse gene encodes a mouse protein which appears similar to a human protein
  • that human protein may be used (especially in humans) for purposes analogous to the proposed use of the mouse protein in mice.
  • a specific binding fragment of an appropriate strand of the corresponding human gene (gDNA or cDNA) could be labeled and used as a hybridization probe (especially against samples of human mRNA or cDNA) .
  • the disclosed genes gDNA or cDNA
  • the disclosed genes have significant similarities to known DNAs (and their translated AA sequences to known proteins)
  • results are dependent, to some degree, on the search parameters.
  • Preferred parameters are set forth in Example 1.
  • the results are also dependent on the content of the database. While the raw similarity score of a particular target (database) sequence will not vary with content (as long as it remains in the database) , its informational value (in bits), expected value, and relative ranking can change. Generally speaking, the changes are small.
  • nucleic acid and protein databases keep growing. Hence a later search may identify high scoring target sequences which were not uncovered by an earlier search because the target sequences were not previously part of a database .
  • cognate DNAs and proteins include not only those set forth in the examples, but those which would have been highly ranked (top ten, more preferably top three, even more preferably top two, most preferably the top one) in a search run with the same parameters on the date of filing of this application.
  • mouse or human database DNA appears to be a partial sequence (that is, partial relative to a cDNA or gDNA encoding the whole naturally occurring protein) , it may be used as a hybridization probe to isolate the full-length DNA. If the partial DNA sequence encodes a biologically functional fragment of the cognate protein, it may be used in a manner similar to the full length DNA, i.e., to produce the functional fragment .
  • an antagonist of a protein or other molecule may be obtained by preparing a combinatorial library, as described below, of potential antagonists, and screening the library members for binding to the protein or other molecule in question. The binding members may then be further screened for the ability to antagonize the biological activity of the target.
  • the antagonists may be used therapeutically, or, in suitably labeled or immobilized form, diagnostically. If the mouse or human database DNA is related to a known protein, then substances known to interact with that protein (e.g., agonists, antagonists, substrates, receptors, second messengers, regulators, and so forth) , and binding molecules which bind them, are also of utility. Such binding molecules can likewise be identified by screening a combinatorial library.
  • a DNA of the present invention is a partial DNA, and the cognate full length DNA is not listed in a sequence database
  • the available DNA may be used as a hybridization probe to isolate the full-length DNA from a suitable DNA library (cDNA or gDNA) .
  • cDNA or gDNA suitable DNA library
  • Stringent hybridization conditions are appropriate, that is, conditions in which the hybridization temperature is 5-10 deg. C. below the Tm of the DNA as a perfect duplex.
  • Identification and Isolation of Homologous Genes Using a DNA Probe It may be that the sequence databases available do not include the sequence of any homologous gene (cDNA or gDNA) , or at least of the homologous gene for a species of interest.
  • this partial DNA may first be used as a probe to isolate the corresponding full length DNA for the same species, and that the latter may be used as the starting DNA in the search for homologous DNAs.
  • the starting DNA, or a fragment thereof is used as a hybridization probe to screen a cDNA or genomic DNA library for clones containing inserts which encode either the entire homologous protein, or a recognizable fragment thereof.
  • the minimum length of the hybridization probe is dictated by the need for specificity.
  • the human cDNA library is about 10 s bases and the human genomic DNA library is about 10 1Q bases.
  • the library is preferably derived from an organism which is known, on biochemical evidence, to produce a homologous protein, and more preferably from the genomic DNA or mRNA of cells of that organism which are likely to be relatively high producers of that protein.
  • a cDNA library (which is derived from an mRNA library) is especially preferred.
  • a synthetic hybridization probe may be used which encodes the same amino acid sequence but whose codon utilization is more similar to that of the DNA of the target organism.
  • the synthetic probe may employ inosine as a substitute for those bases which are most likely to be divergent, or the probe may be a mixed probe which mixes the codons for the source DNA with the preferred codons (encoding the same amino acid) for the target organism.
  • a 1% sequence divergence typically lowers the Tm of a duplex by 1-2 °C, and the DNAs encoding homologous proteins of different species typically have sequence identities of around 50-80%.
  • the library is screened under conditions where the temperature is at least 20°C, more preferably at least 50°C, below the , perfect duplex Tm. Since salt reduces the Tm, one ordinarily would carry out the search for DNAs encoding highly homologous proteins under relatively low salt hybridization conditions, e.g., ⁇ 1M NaCI.
  • DNA library hybridization at 42 °C in 5xSSC.
  • the conditions set forth in these articles may each be considered suitable for the purpose of isolating homologous genes .
  • the manufacturer of the gene chip determines which DNA to place at each position on the chip.
  • This DNA may correspond in sequence to a genomic DNA, a cDNA, or a fragment of genomic or cDNA, and may be natural, synthetic or partially natural and partially synthetic in origin.
  • the manufacturer of the gene chip will normally identify the DNA for a mouse gene chip as corresponding to a particular mouse gene, in which case it will be assumed that the alignments of chip DNA to mouse gene satisfies the homology criteria of the invention.
  • the gene chip manufacturer will provide a' sequence database accession number for the mouse DNA. If so, to identify the corresponding mouse protein, we will first inspect the database record for that mouse DNA.
  • the mouse protein accession number will appear in that record or in a linked record. If it doesn't, the corresponding mouse protein can be identified by performing a BlastX search on a mouse protein database with the mouse database DNA sequence as the query sequence. Even if the protein sequence is not in the database, if the DNA sequence comprises a full-length coding sequence, the corresponding protein can be identified by translating the coding sequence in accordance with the Genetic Code.
  • a human protein can be said to be identifiable as corresponding (homologous) to a gene chip DNA if it is identified as corresponding (homologous) to the mouse gene (gDNA or cDNA, whole or partial) identified by the gene chip manufacturer as corresponding to that gene chip DNA.
  • BlastX it is encoded by a human gene, or can be aligned to a human gene by BlastX, which in turn can be aligned by BlastN to said mouse gene and/or
  • BlastP a mouse protein, the latter being encoded by said mouse gene, or aligned to said mouse gene BlastX,
  • any alignment by BlastN, BlastP or BlastX is in accordance with the default parameters set forth below, and the expected. value (E) of each alignment (the probability that such an alignment would have occurred by chance alone) is less than e-10. (Note that because this is a negative exponent, a value such as e-50 is less than e-10.)
  • a human gene is corresponding (homologous) to a mouse gene chip DNA, and hence to said identified mouse gene (or cDNA) and protein, if it encodes a corresponding (homologous), human protein as defined above, or it can be aligned by BlastN to said mouse gene.
  • the E value is less than e-50, more preferably less than e-60, still more preferably less than e-70, even more preferably less than e-80, considerably more preferably less than e-90, and most preferably less than e-100. Desirably, it is true for two or even all three of these conditions.
  • Master table 1 In constructing Master table 1, we generally used a BlastX (mouse gene vs. human protein) alignment E value cutoff of e-50. However, if there were no human proteins with that good an alignment to the mouse DNA in question, or if there were other reasons for including a particular human protein (e.g., a known functionality supportive of the observed differential cognate mouse protein expression) , then a human protein with a score worse (i.e., higher) than e-50 may appear in Master Table 1.
  • BlastX mouse gene vs. human protein
  • a longer (possibly full length) mouse gene or cDNA may be identified by a BlastN search of the mouse DNA database.
  • the identified DNA may be used to conduct a BlastN search of a human DNA database, or a BlastX search of a mouse or human protein database .
  • a human protein can be said to be identifiable as corresponding (homologous) to a gene chip DNA, or to a DNA identified by the manufacturer as corresponding to that gene chip DNA, if
  • any alignment by BlastN, BlastP, or BlastX is in accordance with the default parameters set forth below, and the expected value (E) of each alignment (the probability that such an alignment would have occurred by chance alone) is less than e-10. (Note that because this is a negative exponent, a value such as e-50 is less than e-10.).
  • the E value is less than e-50, more preferably less than e-60, , still more preferably less than e-70, even more preferably less than e-80, considerably more preferably less than e-90, and most preferably less than e-100.
  • one or more of these standards of preference are met for two, three, four or all five of conditions (1')- (5') .
  • the E value is preferably, so limited for all of said alignments in the connecting chain.
  • a human gene corresponds (is homologous) to a gene chip DNA or manufacturer identified corresponding DNA if it encodes a homologous human protein as defined above, or if it can be aligned either directly to that DNA, or indirectly through a mouse gene which can be aligned to said DNA, according to the conditions set forth above .
  • Master table 1 assembles a list of human protein corresponding to each of the mouse DNAs/proteins identified as related to the chip DNA. These human proteins form a set and can be given a percentile rank, with respect to E value, within that set .
  • the human proteins of the present invention preferably are those scorers with a percentile rank of at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%.
  • human protein which provides the best alignment match as measured by BlastX, i.e., the human protein with the best score (lowest e-value) .
  • These human proteins form a subset of the set above and can be given a percentile rank within that subset, e.g., the human proteins with scores in the top 10% of that subset have a percentile rank of 90% or higher.
  • the human proteins of the present invention preferably are those best scorer subset proteins with a percentile rank within the subset of at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%.
  • BlastN and BlastX report very low expected values as "0.0". This does not truly mean that the expected value is exactly zero (since any alignment could occur by chance) , but merely that it is so infinitesimal that it is not reported.
  • the documentation does not state the cutoff value, but alignments with explicit E values as low as e-178 (624 bits) have been reported as nonzero values, while a score of 636 bits was reported as "0.0".
  • a human protein may be said to be functionally homologous to the mouse gene if the human protein has at least one biological activity in common with the mouse protein encoded by said mouse gene .
  • the human proteins of interest also include those that are substantially and/or conservatively identical (as defined below) to the homologous and/or functionally homologous human proteins defined above.
  • the degree of differential expression may be expressed as the ratio of the higher expression level to the lower expression level. Preferably, this is at least 2-fold, and more preferably, it is higher, such as at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, or at least 10-fold.
  • the human protein of interest corresponds to a mouse gene for which the degree of differential expression places it among the top 10% of the mouse genes in the appropriate subtable .
  • a gene is down-regulated in more favored mammals, or up-regulated in less favored mammals, (i.e., an "unfavorable gene") then several utilities are apparent.
  • the complementary strand of the gene, or a portion thereof may be used in labeled form as a hybridization probe to detect messenger RNA and thereby monitor the level of expression of the gene in a subject. Elevated levels are indicative of progression, or propensity to progression, to a less favored state, and clinicians may take appropriate preventative, curative or ameliorative action.
  • the messenger RNA product (or equivalent cDNA) , the protein product, or a binding molecule specific for that product (e.g., an antibody which binds the product) , or a downstream product which mediates the activity (e.g., a signaling intermediate) or a binding molecule (e.g., an antibody) therefor, may be used, preferably in labeled or immobilized form, as an assay reagent in an assay for said nucleic acid product, protein product, or downstream product (e.g., a signaling intermediate) .
  • elevated levels are indicative of a present or future problem.
  • an agent which down-regulates expression of the gene may be used to reduce levels of the corresponding protein and thereby inhibit further damage.
  • This agent could inhibit transcription of the gene in the subject, or translation of the corresponding messenger RNA.
  • Possible inhibitors of transcription and translation include antisense molecules and repressor molecules.
  • the agent could also inhibit a post-translational modification (e.g., glycosylation, phosphorylation, cleavage, GPI attachment) required for activity, or post-translationally modify the protein so as to inactivate it.
  • a post-translational modification e.g., glycosylation, phosphorylation, cleavage, GPI attachment
  • it could be an agent which down- or up-regulated a positive or negative regulatory gene, respectively.
  • an agent which is an antagonist of the messenger RNA product or protein product of the gene, or of a downstream product through which its activity is manifested e.g., a signaling intermediate
  • This antagonist could be an antibody, a peptide, a peptoid, a nucleic acid, a peptide nucleic acid (PNA) oligomer, a small organic molecule of a .kind for which a combinatorial library exists (e.g., a benzodiazepine) , etc.
  • An antagonist is simply a binding molecule which, by binding, reduces or abolishes the undesired activity of its target.
  • the antagonist if not an oligomeric molecule, is preferably less than 1000 daltons, more preferably less than 500 daltons.
  • an agent which degrades, or abets the degradation of, that messenger RNA, its protein product or a downstream product which mediates its activity may be used to curb the effective period of activity of the protein. If a gene is up-regulated in more favored mammals, or down-regulated in less favored animals then the utilities are converse to those stated above.
  • the complementary strand of the gene, or a portion thereof may be used in labeled form as a hybridization probe to detect messenger RNA and thereby monitor the level of expression of the gene in a subject. Depressed levels are indicative of damage, or possibly of a propensity to damage, and clinicians may take appropriate preventative, curative or ameliorative action.
  • the messenger RNA product the equivalent cDNA, protein product, or a binding molecule specific for those products, or a downstream product, or a signaling intermediate, or a binding molecule therefor, may be used, preferably in labeled or immobilized form, as an assay reagent in an assay for said protein product or downstream product.
  • depressed levels are indicative of a present or future problem.
  • an agent which up-regulates expression of the gene may be used to increase levels of the corresponding protein and thereby inhibit further progression to a less favored state.
  • it could be a vector which carries a copy of the gene, but which expresses the gene at higher levels than does the endogenous expression system.
  • it could be an agent which up- or down-regulates a positive or negative regulatory gene.
  • an agent which is an agonist of the protein product of the gene, or of a downstream product through which its activity (of inhibition of progression to a less favored state) is manifested, or of a signaling intermediate may be used to foster its activity.
  • an agent which inhibits the degradation of that protein .product or of a downstream product or of a signaling intermediate may be used to increase the effective period of activity of the protein.
  • Mutant Proteins The present invention also contemplates mutant proteins (peptides) which are substantially identical (as defined below) to the parental protein (peptide) .
  • the fewer the mutations the more likely the mutant protein is to retain the activity of the parental protein.
  • the effect of mutations is usually (but not always) additive. Certain individual mutations are more likely to be tolerated than others .
  • a protein is more likely to tolerate a mutation which (a) is a substitution rather than an insertion or deletion; (b) is an insertion or deletion at the terminus, rather than internally, or, if internal, is at a domain boundary, or a loop or turn, rather than in an alpha helix or beta strand; (c) affects a surface residue rather than an interior residue; (d) affects a part of the molecule distal to the binding site; (e) is a substitution of one amino acid for another of similar size, charge, and/or hydrophobicity, and does not destroy a disulfide bond or other crosslink; and
  • (f) is at a site which is subject to substantial variation among a family of homologous proteins to which the protein of interest belongs.
  • Binding Si te Residues forming the binding site may be identified by (1) comparing the effects of labeling the surface residues before and after complexing the protein to its target, (2) labeling the binding site directly with affinity ligands, (3) fragmenting the protein and testing the fragments for binding activity, and (4) systematic mutagenesis (e.g., alanine-scanning mutagenesis) to determine which mutants destroy binding. If the binding site of a homologous protein is known, the binding site may be postulated by analogy. Protein libraries may be constructed and screened that a large family (e.g., IO 8 ) of related mutants may be evaluated simultaneously. Hence, the mutations are preferably conservative modifications as defined below.
  • a mutant protein (peptide) is substantially identical to a reference protein (peptide) if (a) it has at least 10% of a specific binding activity or a non-nutritional biological activity of the reference protein, and (b) is at least 50% identical in amino acid sequence to the reference protein (peptide) . It is "substantially structurally identical” if condition (b) applies, regardless of (a) .
  • Percentage amino acid identity is determined by aligning the mutant and reference sequences according to a rigorous dynamic programming algorithm which globally aligns their sequences to maximize their similarity, the similarity being scored as the sum of scores for each aligned pair according to an unbiased PAM250 matrix, and a penalty for each internal gap of -12 for the first null of the gap and - 4 for each additional null of the same gap.
  • the percentage identity is the number of matches expressed as a percentage of the adjusted (i.e., counting inserted nulls) length of the reference sequence .
  • a mutant DNA sequence is substantially identical to a reference DNA sequence if they are structural sequences, and encoding mutant and reference proteins which are substantially identical as described above.
  • mutant sequences are substantially identical if they are regulatory sequences, they are substantially identical if the mutant sequence has at least 10% of the regulatory activity of the reference sequence, and is at least 50% identical in nucleotide sequence to the reference sequence. Percentage identity is determined as for proteins except that matches are scored +5, mismatches - 4, the gap open penalty is -12, and the gap extension penalty (per additional null) is -4. More preferably, the sequence is not merely substantially identical, but rather is at least 51%, 66%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98% or 99% identical in sequence to the reference sequence.
  • DNA sequences may also be considered "substantially identical" if they hybridize to each other under stringent conditions, i.e., conditions at which the Tm of the heteroduplex of the one strand of the mutant DNA and the more complementary strand of the reference DNA is not in excess of 10°C. less than the Tm of the reference DNA homoduplex. Typically this will correspond to a percentage identity of 85-90%.
  • Constant Modifications are defined as (a) conservative substitutions of amino acids as hereafter defined; or (b) single or multiple insertions (extension) or deletions (truncation) of amino acids at the termini . Conservative modifications are preferred to other modifications. Conservative substitutions are preferred to other conservative modifications. "Semi-Conservative Modifications” are modifications which are not conservative, but which are (a) semi- conservative substitutions as hereafter defined; or (b) single or multiple insertions or deletions internally, but at interdomain boundaries, in loops or in other segments of relatively high mobility. Semi-conservative modifications are preferred to non ⁇ onservative modifications. Semi- conservative substitutions are preferred to other semi- conservative modifications.
  • Non-conservative substitutions are preferred to other non-conservative modifications.
  • the term "conservative" is used here in an a priori sense, i.e., modifications which would be expected to preserve 3D structure and activity, based on analysis of the naturally occurring families of homologous proteins and of past experience with the effects of deliberate mutagenesis, rather than post facto, a modification already known to conserve activity.
  • a modification which is conservative a priori may, and usually is, also conservative post facto.
  • no more than about five amino acids are inserted or deleted at a particular locus, and the modifications are outside regions known to contain binding sites important to activity.
  • insertions or deletions are limited to the termini .
  • a conservative substitution is a substitution of one amino acid for another of the same exchange group, the exchange groups being defined as follows. I Gly, Pro, Ser, Ala (Cys) (and any nonbiogenic, neutral amino acid with a hydrophobicity not exceeding that of the aforementioned a.a.'s) II Arg, Lys, His (and any nonbiogenic, positively- charged amino acids) III Asp, Glu, Asn, Gin (and any nonbiogenic negatively-charged amino acids) IV Leu, lie, Met, Val (Cys) (and any nonbiogenic, aliphatic, neutral amino acid with a hydrophobicity too high for I above) V Phe, Trp, Tyr (and any nonbiogenic, aromatic neutral amino acid with a hydrophobicity too high for I above) .
  • Cys belongs to both I and IV. Residues Pro, Gly and Cys have special conformational roles. Cys participates in formation of disulfide bonds. Gly imparts flexibility to the chain. Pro imparts rigidity to the chain and disrupts ⁇ helices. These residues may be essential in certain regions of the polypeptide, but substitutable elsewhere. One, two or three conservative substitutions are more likely to be tolerated than a larger number. "Semi-conservative substitutions" are defined herein as being substitutions within supergroup i/lI/III or within supergroup IV/V, but not within a single one of groups I-V. They also include replacement of any other amino acid with alanine.
  • Non-conservative substitutions are substitutions which are not “conservative” or “semi-conservative”.
  • “Highly conservative substitutions” are a subset of conservative substitutions, and are exchanges of amino acids within the groups Phe/Tyr/Trp, Met/Leu/lle/Val, His/Arg/Lys, Asp/Glu and Ser/Thr/Ala. They are more likely to be tolerated than other conservative substitutions. Again, the smaller the number of substitutions, the more likely they are to be tolerated.
  • a protein (peptide) is conservatively identical to a reference protein (peptide) it differs from the latter, if at all, solely by conservative modi ications, the protein (peptide) remaining at least seven amino acids long if the reference protein (peptide) was at least seven amino acids long.
  • a protein is at least semi-conservatively identical to a reference protein (peptide) if it differs from the latter, if at all, solely by semi-conservative or conservative modifications .
  • a protein (peptide) is nearly conservatively identical to a reference protein (peptide) if it differs from the latter, if at all, solely by one or more conservative modifications and/or a single nonconservative substitution. It is highly conservatively identical if it differs, if at all, solely by highly conservative substitutions. Highly conservatively identical proteins are preferred to those merely conservatively identical. An absolutely identical protein is even more preferred.
  • the core sequence of a reference protein is the largest single fragment which retains at least 10% of a particular specific binding activity, if one is " specified, or otherwise of at least one specific binding activity of the referent. If the referent has more than one specific binding activity, it may have more than one core sequence, and these may overlap or not .
  • a peptide of the present invention may have a particular similarity relationship (e.g., markedly identical) to a reference protein (peptide)
  • preferred peptides are those which comprise a sequence having that relationship to a core sequence of the reference protein (peptide) , but with internal insertions or deletions in either sequence excluded. Even more preferred peptides are those whose entire sequence has that relationship, with the same exclusion, to a core sequence of that reference protein (peptide) .
  • Library generally refers to a collection of chemical or biological entities which are related in origin, structure, and/or function, and which can be screened simultaneously for a property of interest . Libraries may be classified by how they are constructed (natural vs. artificial diversity; combinatorial vs. noncombinatorial) , how they are screened (hybridization, expression, display) , or by the nature of the screened library members (peptides, nucleic acids, etc.). In a "natural diversity” library, essentially all of the diversity arose without human intervention. This would be true, for example, of messenger RNA extracted from a non- engineered cell.
  • a "controlled origin” library the source of the diversity is limited in some way.
  • a limitation might be to cells of a particular individual, to a particular species, or to a particular genus, or, more complexly, to individuals of a particular species who are of a particular age, sex, physical condition, geographical location, occupation and/or familial relationship.
  • it might be to cells of a particular tissue or organ.
  • it could be cells exposed to particular pharmacological, environmental, or pathogenic conditions.
  • the library could be of chemicals, or a particular class of chemicals, produced by such cells.
  • the library members are deliberately limited by the production conditions to particular chemical structures. For example, if they are oligomers, they may be limited in length and monomer composition, e.g. hexapeptides composed of the twenty genetically encoded amino acids.
  • hybridization Library In a hybridization library, the library members are nucleic acids, and are screened using a nucleic acid hybridization probe. Bound nucleic acids may then be amplified, cloned, and/or sequenced.
  • the screened library members are gene expression products, but one may also speak of an underlying library of genes encoding those products.
  • the library is made by subcloning DNA encoding the library members (or portions thereof) into expression vectors (or into cloning vectors which subsequently are used to construct expression vectors) , each vector comprising an expressible gene encoding a particular library member, introducing the expression vectors into suitable cells, and expressing the genes so the expression products are produced.
  • the expression products are secreted, so the library can be screened using an affinity reagent, such as an antibody or receptor.
  • the bound expression products may be sequenced directly, or their sequences inferred by, e.g., sequencing at least the variable portion of the encoding DNA.
  • the cells are lysed, thereby exposing the expression products, and the latter are screened with the affinity reagent .
  • the cells express the library members in such a manner that they are displayed on the surface of the cells, or on the surface of viral particles produced by the cells. (See display libraries, below) .
  • the screening is not for the ability of the expression product to bind to an affinity reagent, but rather for its ability to alter the phenotype of the host cell in a particular detectable manner.
  • the screened library members are transformed cells, but there is a first underlying library of expression products which mediate the behavior of the cells, and a second underlying library of genes which encode those products .
  • the library members are each conjugated to, and displayed upon, a support of some kind.
  • the support may be living (a cell or virus) , or nonliving (e.g., a bead or plate) .
  • display will normally be effectuated by expressing a fusion protein which comprises the library member, a carrier moiety allowing integration of the fusion protein into the surface of the cell or virus, and optionally a lining moiety.
  • the cell coexpresses a first fusion comprising the library member and a linking moiety LI, and a second fusion comprising a linking moiety L2 and the carrier moiety.
  • Soluble Library In a soluble library, the library members are free in solution.
  • a soluble library may be produced directly, or one may first make a display library and then release the library members from their supports.
  • Encapsulated Library In an encapsulated library, the library members are inside cells or liposomes. Generally speaking, encapsulated libraries are used to store the library members for future use; the members are extracted in some way for screening purposes. However, if they differentially affect the phenotype of the cells, they may be screened indirectly by screening the cells.
  • a cDNA library is usually prepared by extracting RNA from cells of particular origin, fractionating the RNA to isolate the messenger RNA (mRNA has a poly (A) tail, so this is usually done by oligo-dT affinity chromatography) , synthesizing complementary DNA (cDNA) using reverse transcriptase, DNA polymerase, and other enzymes, subcloning the cDNA into vectors, and introducing the vectors into cells. Often, only mRNAs or cDNAs of particular sizes will be used, to make it more likely that the cDNA encodes a functional polypeptide.
  • a cDNA library explores the natural diversity of the transcribed DNAs of cells from a particular source. It is not a combinatorial library.
  • a cDNA library may be used to make a hybridization library, or it may be used as an (or to make) expression library.
  • Genomic DNA Library A genomic DNA library is made by extracting DNA from a particular source, fragmenting the DNA, isolating fragments of a particular size range, subcloning the DNA fragments into vectors, and introducing the vectors into cells. Like a cDNA library, a genomic DNA library is a natural diversity library, and not a combinatorial library. A genomic DNA library may be used the same way as a cDNA library.
  • a synthetic DNA library may be screened directly (as a hybridization library) , or used in the creation of an expression or display library of peptides/proteins.
  • combinatorial libraries refers to a library in which the individual members are either systematic or random combinations of a limited set of basic elements, the properties of each member being dependent on the choice and location of the elements incorporated into it. Typically, the members of the library are at least capable of being screened simultaneously. Randomization may be complete or partial; some positions may be randomized and others predetermined, and at random positions, the choices may be limited in a predetermined manner.
  • the members of a combinatorial library may be oligomers or polymers of some kind, in which the variation occurs through the choice of monomeric building block at one or more positions of the oligomer or polymer, and possibly in terms of the connecting linkage, or the length of the oligomer or polymer, too.
  • the members may be nonoligomeric molecules with a standard core structure, like the 1,4-benzodiazepine structure, with the variation being introduced by the choice of substituents at particular variable sites on the core structure.
  • the members may be nonoligomeric molecules assembled like a jigsaw puzzle, but wherein each piece has both one or more variable moieties (contributing to library diversity) and one or more constant moieties (providing the functionalities for coupling the piece in question to other pieces) .
  • each piece has both one or more variable moieties (contributing to library diversity) and one or more constant moieties (providing the functionalities for coupling the piece in question to other pieces) .
  • a "simple combinatorial library” is a mixture of two or more simple libraries, e.g., DNAs and peptides ' , or peptides, peptoids, and PNAs, or benzodiazepines and carbamates.
  • the number of component simple libraries in a composite library will, of course, normally be smaller than the average number of members in each simple library, as otherwise the advantage of a library over individual synthesis is small. Libraries of thousands., even millions, of random oligopeptides have been prepared by chemical synthesis (Houghten et al .
  • the first combinatorial libraries were composed of peptides or proteins, in which all or selected amino acid positions were randomized. Peptides and proteins can exhibit high and specific binding activity, and can act as catalysts. In consequence, they are of great importance in biological systems.
  • nucleic acids have also been used in combinatorial libraries. Their great advantage is the ease with which a nucleic acid with appropriate binding activity can be amplified. As a result, combinatorial libraries composed of nucleic acids can be of low redundancy and hence, of high diversity.
  • the size of a library is the number of molecules in it.
  • the simple diversity of a library is the number of unique structures in it . There is no formal minimum or maximum diversity. If the library has a very low diversity, the library has little advantage over just synthesizing and screening the members individually. If the library is of very high diversity, it may be inconvenient to handle, at least without automatizing the process.
  • the simple diversity of a library is preferably at least 10, 10E2, 10E3, 10E4, 10E6, 10E7, 10E8 or 10E9, the higher the better under most circumstances.
  • the simple diversity is usually not more than 10E15, and more usually not more than 10E10.
  • the average sampling level is the size divided by the simple diversity.
  • the expected average sampling level must be high enough to provide a reasonable assurance that, if a given structure were expected, as a consequence of the library design, to be present, that the actual average sampling level will be high enough so that the structure, if satisfying the screening criteria, will yield a positive result when the library is screened.
  • the preferred average sampling level is a function of the detection limit, which in turn is a function of the strength of the signal to be screened.
  • the library members may be presented as solutes in solution, or immobilized on some form of support.
  • the support may be living (cell, virus) or nonliving (bead, plate, etc.) .
  • the supports may be separable (cells, virus particles, beads) so that binding and nonbinding members can be separated, or nonseparable (plate) .
  • the members will normally be placed on addressable positions on the support.
  • the advantage of a soluble library is that there is no carrier moiety that could interfere with the binding of the members to the support .
  • oligonucleotide libraries An oligonucleotide library is a combinatorial library, at least some of whose members are single-stranded oligonucleotides having three or more nucleotides connected by phosphodiester or analogous bonds.
  • the oligonucleotides may be linear, cyclic or branched, and may include non- nucleic acid moieties.
  • the nucleotides are not limited to the nucleotides normally found in DNA or RNA.
  • nucleotides modified to increase nuclease resistance and chemical stability of aptamers see Chart 1 in Osborne and Ellington, Chem. Rev., 97: 349-70 (1997).
  • Ellington and Szostak Nature, 346: 818-22 (1990).
  • the libraries of the present invention are preferably composed of oligonucleotides having a length of 3 to 100 bases, more preferably 15 to 35 bases.
  • oligonucleotides in a given library may be of the same or of different lengths.
  • Oligonucleotide libraries have the advantage that libraries of very high diversity (e.g., 10 15 ) are feasible, and binding molecules are readily amplified in vitro by polymerase chain reaction (PCR) . Moreover, nucleic acid molecules can have very high specificity and affinity to targets .
  • this invention prepares and screens oligonucleotide libraries by the SELEX method, as described in King and Famulok, Molec. Biol. Repts., 20: 97- 107 (1994) ; L. Gold, C. Tuerk.
  • nucleic acid ligands US#5595877; Oliphant et al . Gene 44:177 (1986) .
  • the term "aptamer” is conferred on those oligonucleotides which bind the target protein. Such aptamers may be used to characterize the target protein, both directly (through identification of the aptamer and the points of contact between the aptamer and the protein) and indirectly (by use of the- aptamer as a ligand to modify the chemical reactivity of the protein) .
  • each nucleotide (monomeric unit) is composed of a phosphate group, a sugar moiety, and either a purine or a pyrimidine base.
  • the sugar is deoxyribose and in RNA it is ribose.
  • the nucleotides are linked by 5' -3' phosphodiester bonds.
  • the deoxyribose phosphate backbone of DNA can be modified to increase resistance to nuclease and to increase penetration of cell membranes.
  • Derivatives such as mono- or dithiophosphates, methyl phosphonates, boranophosphates, formacetals, carbamates, siloxanes, and dimethylenethio- - sulfoxideo- and-sulfono- linked species are known in the art .
  • a peptide is composed of a plurality of amino acid residues joined together by peptidyl (-NHC0-) bonds.
  • a biogenic peptide is a peptide in which the residues are all genetically encoded amino acid residues; it is not necessary that the biogenic peptide actually be produced by gene expression.
  • Amino acids are the basic building blocks with which peptides and proteins are constructed. Amino acids possess both an amino group (-NH 2 ) and a carboxylic acid group (- COOH) . Many amino acids, but not all, have the alpha amino acid structure NH 2 -CHR-COOH, where R is hydrogen, or any of a variety of functional groups.
  • Twenty amino acids are genetically encoded: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamic Acid, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine. Of these, all save Glycine are optically isomeric, however, only the L- form is found in humans. Nevertheless, the D-forms of these amino acids do have biological significance; D-Phe, for example, is a known analgesic.
  • amino acids are also known, including: 2- Aminoadipic acid; 3-Aminoadipic acid; beta-Aminopropionic acid; 2-Aminobutyric acid; 4-Aminobutyric acid (Piperidinic acid) ;6-Aminocaproic acid; 2-Aminoheptanoic acid; 2- Aminoisobutyric acid, 3-Aminoisobutyric acid; 2-Aminopimelic acid; 2,4-Diaminobutyric acid; Desmosine; 2,2'- Diaminopimelic acid; 2, 3-Diaminopropionic acid; N- Ethylglycine; N-Ethylasparagine; Hydroxylysine; allo- Hydroxylysine; 3-Hydroxyproline; 4-Hydroxyproline; Isodesmosine; allo-Isoleucine; N-Methylglycine (Sarcosine) ; N-Methylisoleucine; N-Methyl
  • Peptides are constructed by condensation of amino acids and/or smaller peptides.
  • the amino group of one amino acid (or peptide) reacts with the carboxylic acid group of a second amino acid (or peptide)- to form a peptide (-NHCO-) bond, releasing one molecule of water. Therefore, when an amino acid is incorporated into a peptide, it should, technically speaking, be referred to as an amino acid residue.
  • the core of that residue is the moiety which excludes the -NH and -CO linking functionalities which connect it to other residues. This moiety consists of one or more main chain atoms (see below) and the attached side chains .
  • each amino acid consists of the -NH and -CO linking functionalities and a core main chain moiety.
  • the core main chain moiety may include additional carbon atoms, and may also include nitrogen, oxygen or sulfur atoms, which together form a single chain.
  • the core main chain atoms consist ' solely of carbon atoms .
  • the side chains are attached to the core main chain atoms.
  • the side chain refers to the C-3 and higher numbered carbon atoms and their substituents. It also includes H atoms attached to the main chain atoms .
  • Amino acids may be classified according to the number of carbon atoms which appear in the main chain between the carbonyl carbon and amino nitrogen atoms which participate in the peptide bonds.
  • alpha, beta, gamma and delta amino acids are known. These have 1-4 intermediary carbons.
  • Proline is a special case of an alpha amino acid; its side chain also binds to the peptide bond nitrogen.
  • main chain core carbon a side chain other than H is attached to.
  • the preferred attachment site is the C-2 (alpha) carbon, i.e., the one adjacent to the carboxyl carbon of the -CO linking functionality.
  • a main chain atom may carry either one or two side chains; one is more common.
  • a side chain may be attached to a main chain carbon atom by a single or a double bond; the former is more common.
  • a simple combinatorial peptide library is one whose members are peptides having three or more amino acids connected via peptide bonds.
  • the peptides may be linear, branched, or cyclic, and may covalently or noncovalently include nonpeptidyl moieties.
  • the amino acids are not limited to the naturally occurring or to the genetically encoded amino acids.
  • a biased peptide library is one in which one or more (but not all) residues of the peptides are constant residues .
  • Cyclic Peptides Many naturally occurring peptides are cyclic. Cyclization is a common mechanism for stabilization of peptide conformation thereby achieving improved association of the peptide with its ligand and hence improved biological activity. Cyclization is usually achieved by intra-chain cystine formation, by formation of peptide bond between side chains or between N- and C- terminals. Cyclization was usually achieved by peptides in solution, but several publications have appeared that describe cyclization of peptides on beads. A peptide library may be an oligopeptide library or a protein library.
  • the oligopeptides are at least five, six, seven or eight amino acids in length. Preferably, they are composed of less than 50, more preferably less than 20 amino acids. In the case of an oligopeptide library, all or just some of the residues may be variable.
  • the oligopeptide may be unconstrained, or constrained to a particular conformation by, e.g., the participation of constant cysteine residues in the formation of a constraining disulfide bond.
  • Proteins Proteins like oligopeptides, are composed of a plurality of amino acids, but the term protein is usually reserved for longer peptides, which are able to fold into a stable conformation.
  • a protein may be composed of two or more polypeptide chains, held together by covalent or noncovalent crosslinks. These may occur in a homooligomeric or a heterooligomeric state .
  • a peptide is considered a protein if it (1) is at least 50 amino acids long, or (2) has at least two stabilizing covalent crosslinks (e.g., disulfide bonds).
  • conotoxins are considered proteins .
  • the proteins of a protein library will be characterizable as having both constant residues (the same for all proteins in the library) and variable residues (which vary from member to member) .
  • a protein library it is desirable to focus the mutations at those sites which are tolerant of mutation. These may be determined by alanine scanning mutagenesis or by comparison of the protein sequence to that of homologous proteins of similar activity. It is also more likely that mutation of surface residues will directly affect binding. Surface residues may be determined by inspecting a 3D structure of the protein, or by labeling the surface and then ascertaining which residues have received labels . They may also be inferred by identifying regions of high hydrophilicity within the protein. Because proteins are often altered at some sites but not others, protein libraries can be considered a special case of the biased peptide library.
  • the protein library comprises members which comprise a mutant of VH or VL chain, or a mutant of an antigen-specific binding fragment of such a chain.
  • VH and VL chains are usually each about 110 amino acid residues, and are held in proximity by a disulfide bond between the adjoing CL and CHI regions to form a variable domain. Together, the VH, VL, CL and CHI form an Fab fragment .
  • the hypervariable regions are at 31-35, '49-65, 98-111 and 84-88, but only the first three are involved in antigen binding. There is variation among VH and VL chains at residues outside the hypervariable regions, but to a much lesser degree .
  • a sequence is considered a mutant of a VH or VL chain if it is at least 80% identical to a naturally occurring VH or VL chain at all residues outside the hypervariable region.
  • such antibody library members comprise both at least one VH chain and at least one VL chain, at least one of which is a mutant chain, and which chains may be derived from the same or different antibodies.
  • the VH and VL chains may be covalently joined by a suitable linker moiety, as in a "single chain antibody", or they may be noncovalently joined, as in a naturally occurring variable domain. If the joining is noncovalent, and the library is displayed on cells or virus, then either the VH or the VL chain may be fused to the carrier surface/coat protein.
  • the complementary chain may be co-expressed, or added exogenously to the library.
  • the members may further comprise some or all of an antibody constant heavy and/or constant light chain, or a mutant thereof .
  • a peptoid is an analogue of a peptide in which one or more of the peptide bonds (-NH-CO-) are replaced by pseudopeptide bonds, which may be the same or different. It is not necessary that all of the peptide bonds be replaced, i.e., a peptoid may include one or more conventional amino acid residues, e.g., proline.
  • a peptide bond has two small divalent linker elements, -NH- and -CO-.
  • a preferred class of psuedopeptide bonds are those which consist of two small divalent linker elements.
  • Each may be chosen independently from the group consisting of amine (-NH-) , substituted amine (-NR-) , carbonyl (-CO-) , thiocarbonyl (-CS-) , methylene (-CH2-) , monosubstituted methylene (-CHR-) , disubstituted methylene (-CR1R2-) , ether (-0-) and thioether (-S-) .
  • the more preferred pseudopeptide bonds include: N-modified -NRCO- Carba ⁇ -CH 2 -CH 2 - Depsi ⁇ -CO-0- Hydroxyethylene ⁇ -CH0H-CH 2 - Ketomethylene ⁇ -CO-CH 2 - Methylene-Oxy -CH 2 -0- Reduced -CH 2 -NH- Thiomethylene -CH 2 -S- Thiopeptide -CS-NH- Retro-Inverso -CO-NH-
  • a single peptoid molecule may include more than one kind of pseudopeptide bond.
  • one may vary (1) the side chains attached to the core main chain atoms of the monomers linked by the pseudopeptide bonds, and/or (2) the side chains (e.g., the - R of an -NRC0-) of the pseudopeptide bonds.
  • the monomeric units which are not amino acid residues are of the structure -NR1-CR2-C0- , where at least one of Rl and R2 are not hydrogen. If there is variability in the pseudopeptide bond, this is most conveniently done by using an -NRCO- or other pseudopeptide bond with an R group, and varying the R group.
  • the R group will usually be any of the side chains characterizing the amino acids of peptides, as previously discussed. If the R group of the pseudopeptide bond is not variable, it will usually be small, e.g., not more than 10 atoms (e.g., hydroxyl, amino, carboxyl, methyl, ethyl, propyl) . If the conjugation chemistries are compatible, a simple combinatorial library may include both peptides and peptoids .
  • PNA oligomer is here defined as one comprising a plurality of units, at least one of which is a PNA monomer which comprises a side chain comprising a nucleobase.
  • PNA monomer which comprises a side chain comprising a nucleobase.
  • the classic PNA oligomer is composed of (2- a inoethyl) glycine units, with nucleobases attached by methylene carbonyl linkers. That is, it has the ' structure
  • outer parenthesized substructure is the PNA monomer.
  • nucleobase B is separated from the backbone N by three bonds, and the points of attachment of the side chains are separated by six bonds .
  • the nucleobase may be any of the bases included in the nucleotides discussed in connection with oligonucleotide libraries.
  • the bases of nucleotides A, G, T, C and U are preferred.
  • a PNA oligomer may further comprise one or more amino acid residues, especially glycine and proline.
  • the small organic compound library (“compound library”, for short) is a combinatorial library whose members are suitable for use as drugs if, indeed, they have the ability to mediate a biological activity of the target protein.
  • Peptides have certain disadvantages as drugs . These include susceptibility to degradation by serum proteases, and difficulty in penetrating cell membranes. Preferably, all or most of the compounds of the compound library avoid, or at least do not suffer to the same degree, one or more of the pharmaceutical disadvantages of peptides . In designing a compound library, it is helpful to bear in mind the methods of molecular modification typically used to obtain new drugs .
  • disjunction in which a lead drug is simplified to identify its component pharmacophoric moieties
  • con unction in which two or more known pharmacophoric moieties, which may be the same or different, are associated, covalently or noncovalently, to form a new drug
  • alteration in which one moiety is replaced by another which may be similar or different, but which is not in effect a disjunction or conjunction.
  • disjunction in which a lead drug is simplified to identify its component pharmacophoric moieties
  • con unction in which two or more known pharmacophoric moieties, which may be the same or different, are associated, covalently or noncovalently, to form a new drug
  • alteration in which one moiety is replaced by another which may be similar or different, but which is not in effect a disjunction or conjunction.
  • Alterations include ring closing or opening, formation of lower or higher homologues, introduction or saturation of double bonds, introduction of optically active centers, introduction, removal or replacement of bulky groups, isosteric or bioisosteric substitution, changes in the position or orientation of a group, introduction of alkylating groups, and introduction, removal or replacement of groups with a view toward inhibiting or promoting inductive (electrostatic) or conjugative (resonance) effects.
  • the substituents may include electron acceptors and/or electron donors.
  • Typical electron donors (+1) include -CH 3 , -CH 2 R, -CHR 2 , -CR 3 and -COO " .
  • the substituents may also include those which increase or decrease electronic density in conjugated systems.
  • the former (+R) groups include -CH 3 , -CR 3 , -F, -CI, -Br, -I, -OH, -OR, -0C0R, -SH, -SR, -NH 2 , -NR 2 , and -NHCOR.
  • the later (-R) groups include -N0 2 , -CN, -CHC, -COR, -COOH, -COOR, -CONH 2 , -S0 2 R and -CF 3 . Synthetically speaking, the modifications may be achieved by a variety of unit processes, including nucleophilic and electrophilic substitution, reduction and oxidation, addition elimination, double bond cleavage, and cyclization.
  • a compound, or a family of compounds, having one or more pharmacological activities may be disjoined into two or more known or potential pharmacophoric moieties.
  • Analogues of each of these moieties may be identified, and mixtures of these analogues reacted so as to reassemble compounds which have some similarity to the original lead compound. It is not necessary that all members of the library possess moieties analogous to all of the moieties of the lead compound.
  • the design of a library may be illustrated by the example of the benzodiazepines .
  • benzodiazepine drugs including chlordiazepoxide, diazepam and oxazepam, have been used as anti-anxiety drugs.
  • Derivatives of benzodiazepines have widespread biological activities; derivatives have been reported to act not only as anxiolytics, but also as anticonvulsants; cholecystokinin (CCK) receptor subtype A or B, kappa opioid receptor, platelet activating factor, and HIV transactivator Tat antagonists, and GPIIblla, reverse transcriptase and ras farnesyltransferase inhibitors.
  • CCK cholecystokinin
  • the benzodiazepine structure has been disjoined into a 2-aminobenzophenone, an amino acid, and an alkylating agent. See Bunin, et al . , Proc. Nat. Acad. Sci. USA, 91:4708 (1994) . Since only a few 2-aminobenzophenone derivatives are commercially available, it was later disjoined into 2- aminoarylstannane, an acid chloride, an amino acid, and an alkylating agent. Bunin, et al . , Meth. Enzymol., 267:448 (1996) .
  • the arylstannane may be considered the core structure upon which the other moieties are substituted, or all four may be considered equals which are conjoined to make each library member.
  • a basic library synthesis plan and member structure is shown in Figure 1 of Fowlkes, et al . , U.S. Serial No. 08/740,671, incorporated by reference in its entirety.
  • the acid chloride building block introduces variability at the R 1 site.
  • the R 2 site is introduced by the amino acid, and the R 3 site by the alkylating agent.
  • the R 4 site is inherent in the arylstannane. Bunin, et al .
  • variable elements included both aliphatic and aromatic groups.
  • aliphatic groups both acyclic and cyclic (mono- or poly-) structures, substituted or not, were tested. (although all of the acyclic groups were linear, it would have been feasible to introduce a branched aliphatic) .
  • the aromatic groups featured either single and multiple rings, fused or not, substituted or not, and with heteroatoms or not.
  • the secondary substitutents included - NH 2 , -OH, -OMe, -CN, -CI, -F, and -COOH.
  • spacer moieties such as -0-, -S-, -00-, -CS-, -NH- , and - NR-, could have been incorporated.
  • Bunin et al suggest that instead of using a 1, 4- benzodiazepine as a core structure, one may instead use a 1, 4-benzodiazepine-2, 5-dione structure.
  • the hydantoins were synthesized by first simultaneously deprotecting and then treating each of five amino acid resins with each of eight isocyanates.
  • the benzodiazepines were synthesized by treating each of five deprotected amino acid resins with each of eight 2-amino benzophenone imines. Chen, et al . , J. Am. Chem. Soc, 116:2661-62 (1994) described the preparation of a pilot (9 member) combinatorial library of formate esters.
  • a polymer bead- bound aldehyde preparation was "split" into three aliquots, each reacted with one of three different ylide reagents. The reaction products were combined, and then divided into three new aliquots, each of which was reacted with a different Michael donor. Compound identity was found to be determinable on a single bead basis by gas chromatography/mass spectroscopy analysis. Holmes, USP 5,549,974 (1996) sets forth methodologies for the combinatorial synthesis of libraries of thiazolidinones and metathiazanones . These libraries are made by combination of amines, carbonyl compounds, and thiols under cyclization conditions.
  • each member is synthesized only at a particular coordinate on or in a matrix, or in a particular chamber. This might be, for example, the location of a particular pin, or a particular well on a microtiter plate, or inside a "tea bag".
  • the present invention is not limited to any particular form of identification. However, it is possible to simply characterize those members of the library which are found to be active, based on the characteristic spectroscopic indicia of the various • building blocks . Solid phase synthesis permits greater control over which derivatives are formed. However, the solid phase could interfere with activity. To overcome this problem, some or all of the molecules of each member could be liberated, after synthesis but before screening.
  • Examples of candidate simple libraries which might be evaluated include derivatives of the following: Cyclic Compounds Containing One Hetero Atom Heteronitrogen pyrroles pentasubstituted pyrroles pyrrolidines pyrrolines prolines indoles beta-carbolines pyridines dihydropyridines 1,4-dihydropyridines pyrido [2 , 3 -d] pyri idines tetrahydro-3H-imidazo [4,5-c] pyridines Isoquinolines tetrahydroisoquino1ines quinolones beta-lactams azabicyclo [4.3.0] onen-8-one amino acid
  • Heterooxygen furans tetrahydrofurans 2 5-disubstituted tetrahydrofurans pyrans hydroxypyranones tetrahydroxypyranones gamma-butyrolactones Heterosulfur sulfolenes Cyclic Compounds with Two or More Hetero atoms Multiple heteronitrogens imidazoles pyrazoles piperazines diketopiperazines arylpiperazines benzylpiperazines benzodiazepines 1,4 -benzodiazepine- 2, 5-diones hydantoins 5-alkoxyhydantoins dihydropyrimidines
  • Heteronitrogen and Heterosulfur thiazolidines N-axylthiazolidines dihydrothiazoles 2-methylene-2, 3-dihydrothiazates 2-aminothiazoles thiophenes 3 -amino thiophenes 4-thiazolidinones 4-melathiazanones benzisothiazolones
  • the preferred animal subject of the present invention is a mammal.
  • mammal an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects, although it is intended for veterinary and nutritional uses as well.
  • Preferred nonhuman subjects are of the orders Primata (e.g., apes and monkeys), Artiodactyla or Perissodactyla (e.g., cows, pigs, sheep, horses, ⁇ goats) , Carnivora (e.g., cats, dogs), Rodenta (e.g., rats, mice, guinea pigs, hamsters), Lagomorpha (e.g., rabbits) or other pet, farm or laboratory mammals.
  • the term "protection”, as used herein, is intended to include “prevention,” “suppression” and “treatment.” "Prevention”, strictly speaking, involves administration of the pharmaceutical prior to the induction of the disease (or other adverse clinical condition) .
  • “Suppression” involves administration of the composition prior to the clinical appearance of the disease.
  • “Treatment” involves administration of the protective composition after the appearance of the disease. It will be understood that in human and veterinary medicine, it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, unless, qualified, the term “prevention” will be understood to refer to both prevention in the strict sense, and to suppression.
  • the preventative or prophylactic use of a pharmaceutical usually involves identifying subjects who are at higher risk than the general population of contracting the disease, and administering the pharmaceutical to them in advance of the clinical appearance of the disease.
  • the effectiveness of such use is measured by comparing the subsequent incidence or severity of the disease, or of particular symptoms of the disease, in the treated subjects • against that in untreated subjects of the same high risk group .
  • high risk factors vary from disease to disease, in general, these include (1) prior occurrence of the disease in one or more members of the same family, or, in the case of a contagious disease, in individuals with whom the subject has come into potentially contagious contact at a time when the earlier victim was likely to be contagious, (2) a prior occurrence of the disease in the subject, (3) prior occurrence of a related disease, or a condition known to increase the likelihood of the disease, in the subject; (4) appearance of a suspicious level of a marker of the disease, or a related disease or condition; (5) a subject who is immunologically compromised, e.g., by radiation treatment, HIV infection, drug use,, etc., or (6) membership in a particular group (e.g., a particular age, sex, race, ethnic group, etc.) which has been epidemi
  • prophylaxis for the general population, and not just a high risk group. This is most likely to be the case when essentially all are at risk of contracting the disease, the effects of the disease are serious, the therapeutic index of the prophylactic agent is high, and the cost of the agent is low.
  • a prophylaxis or treatment may be curative, that is, directed at the underlying cause of a disease, or ameliorative, that is, directed at the symptoms of the disease, especially those which reduce the quality of life. It should also be understood that to be useful, the protection provided need not be absolute, provided that it is sufficient to carry clinical value.
  • administration may be systemic or topical.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • administration may be by the oral route.
  • Parenteral administration can be by bolus injection or by gradual perfusion over time.
  • a typical regimen comprises administration of an effective amount of the drug, administered over a period ranging from a single dose, to dosing over a period of hours, days, weeks , months , or years .
  • the suitable dosage of a drug of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in. the art, without undue experimentation. This will typically involve adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight .
  • a drug Prior to use in humans, a drug will first be evaluated for safety and efficacy in laboratory animals.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the protein may be administered alone or in conjunction with other therapeutics directed to the disease or directed to other symptoms thereof .
  • the appropriate dosage form will depend on the disease, the pharmaceutical, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, including all references cited therein.
  • the drug may be administered in the form of an expression vector comprising a nucleic acid encoding the peptide; such a vector, after incorporation into the genetic complement of a cell of the patient, directs synthesis of the peptide.
  • Suitable vectors include genetically engineered poxviruses (vaccinia) , adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses which are or have been rendered nonpathogenic .
  • a pharmaceutical composition may contain suitable pharmaceutically acceptable carriers, such as excipients, carriers and/or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. See, e.g., Berker, supra, Goodman, supra, Avery, supra and Ebadi, supra, which are entirely incorporated herein by reference, included all references cited therein.
  • Target Organism The invention contemplates that it may be appropriate to ascertain or to mediate the biological activity of a substance of this invention in a target organism.
  • the target organism may be a plant, ' animal, or microorganism.
  • the drug may be intended to increase the disease, weather or pest resistance, alter the growth characteristics, or otherwise improve the useful characteristics or mute undesirable characteristics of the plant.
  • it may be a weed, in which case the drug may be intended to kill or otherwise inhibit the growth of the plant, or to alter its characteristics to convert it from a weed to an economic plant.
  • the plant may be a tree, shrub, crop, grass, etc.
  • the plant may be an algae (which are in some cases also microorganisms) , or a vascular plant, especially gymnosperms (particularly conifers) and angiosperms .
  • Angiosperms may be monocots or dicots.
  • the plants of greatest interest are rice, wheat, corn, alfalfa, soybeans, potatoes, peanuts, tomatoes, melons, apples, pears, plums, pineapples, fir, spruce, pine, cedar, and oak.
  • the target organism is a microorganism, it may be algae, bacteria, fungi, or a virus (although the biological activity of a virus must be determined in a virus-infected cell) .
  • the microorganism may be human or other animal or plant pathogen, or it may be nonpathogenic. It may be a soil or water organism, or one which normally lives inside other living things. If the target organism is an animal, it may be a vertebrate or a nonvertebrate animal . Nonvertebrate animals are chiefly of interest when they act as pathogens or parasites, and the drugs are intended to act as biocidic or biostatic agents. Nonvertebrate animals of interest include worms, mollusks, and arthropods. The target organism may also be a vertebrate animal, i.e., a mammal, bird, reptile, fish or amphibian.
  • the target animal preferably belongs to the order Primata (humans, apes and monkeys), Artiodactyla (e.g., cows, pigs, sheep, goats, horses), Rodenta (e.g., mice, rats) Lagomorpha (e.g., rabbits, hares), or Carnivora (e.g., cats, dogs) .
  • the target animals are preferably of the orders Anseriformes (e.g., ducks, geese, swans) or Galliformes (e.g., quails, grouse, pheasants, turkeys and chickens) .
  • the target animal is preferably of the order Clupeiformes (e.g., sardines, shad, anchovies, whitefish, salmon) .
  • Target Tissues refers to any whole animal, physiological system, whole organ, part of organ, miscellaneous tissue, cell, or cell component (e.g., the cell membrane) of a target animal in which biological activity may be measured. Routinely in mammals one would choose to compare and contrast the biological impact on virtually any and all tissues which express the subject receptor protein.
  • the main tissues to use are: brain, heart, lung, kidney, liver, pancreas, skin, intestines, adipose, stomach, skeletal muscle, adrenal glands, breast, prostate, vasculature, retina, cornea, thyroid gland, parathyroid glands, thymus, bone marrow, bone, etc.
  • B cells B cells, T cells, macrophages, neutrophils, eosinophils, mast cells, platelets, megakaryocytes, erythrocytes, bone marrow stomal cells, fibroblasts, neurons, astrocytes, neuroglia, microglia, epithelial cells (from any organ, e.g. skin, breast, prostate, lung, intestines etc) , cardiac muscle cells, smooth muscle cells, striated muscle cells, osteoblasts, osteocytes, chondroblasts, chondrocytes , keratinocytes, melanocytes, etc.
  • target organism and the "target tissue”.
  • Screening Assays intended to determine the binding or the biological activity of a substance are called preliminary screening assays. Screening assays will typically be either in vitro (cell-free) assays (for binding to an immobilized receptor) or cell-based assays (for alterations in the phenotype of the cell) . They will not involve screening of whole multicellular organisms, or isolated organs. The comments on diagnostic biological assays apply mutatis mutandis to screening cell-based assays.
  • in vitro is descriptive of an event, such as binding or enzymatic action, which occurs within a living organism.
  • the organism in question may, however, be genetically modified.
  • the term in vi tro refers to an event which occurs outside a living organism. Parts of an organism (e.g., a membrane, or an isolated biochemical) are used, together with artificial substrates and/or conditions.
  • the term in vitro excludes events occurring inside or on an intact cell, whether of a unicellular or multicellular organism.
  • In vivo assays include both cell-based assays, and organismic assays.
  • the cell-based assays include both assays on unicellular organisms, and assays on isolated cells or cell cultures- derived from multicellular organisms.
  • the cell cultures may be mixed, provided that they are not organized into tissues or organs.
  • organismic assay refers to assays on whole multicellular organisms, and assays on isolated organs or tissues of such organisms.
  • the in vitro assays of the present invention may be applied to any suitable analyte-containing sample, and may be qualitative or quantitative in nature.
  • sample will normally be a biological fluid, such as blood, urine, lymph, semen, milk, or cerebrospinal fluid, or a fraction or derivative thereof, or a biological tissue, in the form of, e.g., a tissue section or homogenate.
  • a biological fluid or tissue it may be taken from a human or other mammal, vertebrate or animal, or from a plant.
  • the preferred sample is blood, or a fraction or derivative thereof.
  • the assay may be a binding assay, in which one step involves the binding of a diagnostic reagent to the analyte, or a reaction assay, which involves the reaction of a reagent with the analyte.
  • the reagents used in a binding assay may be classified as to the nature of their interaction with analyte: (1) analyte analogues, or (2) analyte binding molecules (ABM) . They may be labeled or insolubilized.
  • the assay may look for a direct reaction between the analyte and a reagent which is reactive with the analyte, or if the analyte is an enzyme or enzyme inhibitor, for a reaction catalyzed or inhibited by the analyte.
  • the reagent may be a reactant, a catalyst, or an inhibitor for the reaction.
  • An assay may involve a cascade of steps in which the product of one step acts as the target for the next step. These steps may be binding steps, reaction steps, or a combination thereof .
  • SPS Signal Producing System
  • the assay In order to detect the presence, or measure the amount, of an analyte, the assay must provide for a signal producing system (SPS) in which there is a detectable difference in the signal produced, depending on whether the analyte is present or absent (or, in a quantitative assay, on the amount of the analyte) .
  • the detectable signal may be one which is visually detectable, or one detectable only with instruments. Possible signals include production of colored or luminescent products, alteration of the characteristics (including amplitude or polarization) of absorption or emission of radiation by an assay component or product, and precipitation or agglutination of a component or product.
  • signal is intended to include the discontinuance of an existing signal, or a change in the rate of change of an observable parameter, rather than a change in its absolute value.
  • the signal may be monitored manually or automatically.
  • the signal is often a product of the reaction.
  • a binding assay it is normally provided by a label borne by a labeled reagent .
  • a label may be, e.g., a radioisotope, a fluorophore, an enzyme, a co-enzyme, an enzyme substrate, an electron-dense compound, an agglutinable particle.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention include 3 H, 125 I, 131 I, 35 S, 14 C, 32 P and 33 P. 125 I is preferred for antibody labeling.
  • the label may also be a fluorophore.
  • fluorescent labelling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.
  • fluorescence-emitting metals such as 125 Eu, or others of the lanthanide series, may be incorporated into a diagnostic reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) of ethylenediamine-tetraacetic acid (EDTA) .
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediamine-tetraacetic acid
  • the label may also be a chemiluminescent compound.
  • the presence of the chemilummescently labeled reagent is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isolumino, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used for labeling. Bioluminescence is a type of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Enzyme labels such as horseradish peroxidase and alkaline phosphatase, are preferred.
  • the signal producing system must also include a substrate for the enzyme. If the enzymatic reaction product is not itself detectable, the SPS will include one or more additional reactants so that a detectable product appears.
  • An enzyme analyte may act as its own label if an enzyme inhibitor is used as a diagnostic reagent.
  • Binding assays may be' divided into two basic types, heterogeneous and homogeneous.
  • heterogeneous assays the interaction between the affinity molecule and the analyte does not affect the label, hence, to determine the amount or presence of analyte, bound label must be separated from free label.
  • homogeneous assays the interaction does affect the activity of the label, and therefore analyte levels can be deduced without the need for a separation step.
  • the ABM is insolubilized by coupling it to a macromolecular support, and analyte in the sample is allowed to compete with a known quantity of a labeled or specifically labelable analyte analogue.
  • analyte analogue is a molecule capable of competing with analyte for binding to the ABM, and the term is intended to include analyte itself. It may be labeled already, or it may be labeled subsequently by specifically binding the label to a moiety differentiating the analyte analogue from analyte.
  • the solid and liquid phases are separated, and the labeled analyte analogue in one phase is quantified. The higher the level of analyte analogue in the solid phase, i.e., sticking to the ABM, the lower the level of analyte in the sample .
  • both an insolubilized ABM, and a labeled ABM are employed.
  • the analyte is captured by the insolubilized ABM and is tagged by the labeled ABM, forming a ternary complex.
  • the reagents may be added to the sample in either order, or simultaneously.
  • the ABMs may be the same or different.
  • the amount of labeled ABM in the ternary complex is directly proportional to the amount of analyte in the sample.
  • the two embodiments described above are both heterogeneous assays. However, homogeneous assays are conceivable. The key is that the label be affected by whether or not the complex is formed.
  • a label may be conjugated, directly or indirectly (e.g., through a labeled anti-ABM antibody), covalently (e.g., with SPDP) or noncovalently, to the ABM, to produce a diagnostic reagent.
  • the ABM may be conjugated to a solid phase support to form a solid phase ("capture") diagnostic reagent.
  • Suitable supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite .
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to its target.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • a biological assay measures or detects a biological response of a biological entity to a substance.
  • the biological entity may be a whole organism, an isolated organ or tissue, freshly isolated cells, an immortalized cell line, or a subcellular component (such as a membrane; this term should not be construed as including an isolated receptor) .
  • the entity may be, or may be derived from, an organism which occurs in nature, or which is modified in some way. Modifications may be genetic (including radiation and chemical mutants, and genetic engineering) or somatic (e.g., surgical, chemical, etc.). In the case of a multicellular entity, the modifications may affect some or all cells.
  • the entity need not be the target organism, or a derivative thereof, if there is a reasonable correlation between bioassay activity in the assay entity and biological activity in the target organism.
  • the entity is placed in a particular environment, which may be more or less natural.
  • a culture medium may, but need not, contain serum or serum substitutes, and it may, but need not, include a support matrix of some kind, it may be still, or agitated.
  • It may contain particular biological or chemical agents, or have particular physical parameters (e.g., temperature), that are intended to nourish or challenge the biological entity. There must also be a detectable biological marker for the response.
  • the most common markers are cell survival and proliferation, cell behavior (clustering, motility) , cell morphology (shape, color) , and biochemical activity (overall DNA synthesis, overall protein synthesis, and specific metabolic activities, such as utilization of particular nutrients, e.g., consumption of oxygen, production of C0 2 , production of organic acids, uptake or discharge of ions) .
  • the direct signal produced by the biological marker may be transformed by a signal producing system into a different signal which is more observable, for example, a fluorescent or colorimetric signal.
  • the entity, environment, marker and signal producing system are chosen to achieve a clinically acceptable level of sensitivity, specificity and accuracy.
  • the goal will be to identify substances which mediate the biological activity of a natural biological entity, and the assay is carried out directly with that entity.
  • the biological entity is used simply as a model of some more complex (or otherwise inconvenient to work with) biological entity.
  • the model biological entity is used because activity in the model system is considered more predictive of activity in the ultimate natural biological entity than is simple binding activity in an in vitro system.
  • the model entity is used instead of the ultimate entity because the former is more expensive or slower to work with, or because ethical considerations forbid working with the ultimate entity yet .
  • the model entity may be naturally occurring, if the model entity usefully mpdels the ultimate entity under some conditions.
  • Transgenic . animals such as transgenic mice, rats, and rabbits, have been found useful as model systems.
  • the receptor may be functionally connected to a signal (biological marker) producing system, which may be endogenous or exogenous to the cell.
  • “Zero-Hybrid” Systems In these systems, the binding of a peptide to the target protein results in a screenable or selectable phenotypic change, without resort to fusing the target protein (or a ligand binding moiety thereof) to an endogenous protein. It may be that the target protein is endogenous to the host cell, or is substantially identical to an endogenous receptor so that it can take advantage of the latter' s native signal transduction pathway. Or sufficient elements of the signal transduction pathway normally associated with the target protein may be engineered into the cell so that the cell signals binding to the target protein. "One-Hybrid” Systems In these systems, a chimera receptor, a hybrid of the target protein and an endogenous receptor, is used.
  • the chimeric receptor has the ligand binding characteristics of the target protein and the signal transduction characteristics of the endogenous receptor.
  • the normal signal transduction pathway of the endogenous receptor is subverted.
  • the endogenous receptor is inactivated, or the conditions of the assay avoid activation of the endogenous receptor, to improve the signal-to-noise ratio. See Fowlkes USP 5,789,184 for a yeast system.
  • Another type of "one-hybrid" system combines a peptide: DNA-binding domain fusion with an unfused target receptor that possesses an activation domain.
  • the cell-based assay is a two hybrid system. This term implies that the ligand is incorporated into a first hybrid protein, and the receptor into a second hybrid protein.
  • the first hybrid also comprises component A of a signal generating system, and the second hybrid comprises component B of that system.
  • Components A and B by themselves, are insufficient to generate a signal. However, if the ligand binds the receptor, components A and B are brought into sufficiently close proximity so that they can cooperate to generate a signal .
  • Two-Hybrid System Transcription Factor Type
  • one member of a peptide ligand: receptor binding pair is expressed as a fusion to a DNA-binding domain (DBD) from a transcription factor (this fusion protein is called the "bait"), and the other is expressed as a fusion to a transactivation domain (TAD) (this fusion protein is called the "fish", the “prey”, or the "catch”) .
  • DBD DNA-binding domain
  • TAD transactivation domain
  • the transactivation domain should be complementary to the DNA-binding domain, i.e., it should interact with the latter so as to activate transcription of a specially designed reporter gene that carries a binding site for the DNA-binding domain.
  • the two fusion proteins must likewise be complementary. This complementarity may be achieved by use of the complementary and separable DNA-binding and transcriptional activator domains of a single transcriptional activator protein, or one may use complementary domains derived from different proteins.
  • the domains may be identical to the native domains, or mutants thereof.
  • the assay members may be fused directly to the DBD or TAD, or fused through an intermediated linker.
  • the target DNA operator may be the native operator sequence, or a mutant operator.
  • Mutations in the operator may be coordinated with mutations in the DBD and the TAD.
  • An example of a suitable transcription activation system is one comprising the DNA-binding domain from the bacterial repressor LexA and the activation domain from the yeast transcription factor Gal4, with the reporter gene operably linked to the LexA operator. It is not necessary to employ the intact target receptor; just the ligand-binding moiety is sufficient.
  • the two fusion proteins may be expressed from the same or different vectors.
  • the activatable reporter gene may be expressed from the same vector as either fusion protein (or both proteins) , or from a third vector.
  • Potential DNA-binding domains include Gal4 , LexA, and mutant domains substantially identical to the above. Potential activation domains include E.
  • the assay system will include a signal producing system, too.
  • the first element of this system is a reporter gene operably linked to an operator responsive to the DBD and TAD of choice .
  • the expression of this reporter gene will result, directly or indirectly, in a selectable or screenable phenotype (the signal) .
  • the signal producing system may include, besides the reporter gene, additional genetic or biochemical elements which cooperate in the production of the signal. Such an element could be, for example, a selective agent in the cell growth medium.
  • the system may include more than one reporter gene .
  • the sensitivity of the system may be adjusted by, e.g., use of competitive inhibitors of any step in the activation or signal production process, increasing or decreasing the number of operators, using a stronger or weaker DBD or TAD, etc.
  • the assay is said to be a selection.
  • the signal merely results in a detectable phenotype by which the signaling cell may be differentiated from the same cell in a nonsignaling state (either way being a living cell)
  • the assay is a screen.
  • screening assay may be used in a broader sense to. include a selection. When the narrower sense is intended, we will use the term “nonselective screen” .
  • Screening and selection may be for or against the peptide: target protein or compound: target protein interaction.
  • Preferred assay cells are microbial (bacterial, yeast, algal, protozooal) , invertebrate, vertebrate (esp. mammalian, particularly human) .
  • the best developed two- hybrid assays are yeast and mammalian systems.
  • two hybrid assays are used to determine whether a protein X and a protein Y interact, by virtue of their ability to reconstitute the interaction of the DBD and the TAD.
  • augmented two-hybrid assays have been used to detect interactions that depend on a third, non- protein ligand.
  • two-hybrid assays see Brent and Finley, Jr., Ann. Rev. Genet., 31:663-704 (1997); Fremont- Racine, et al., Nature Genetics, 277-281 (16 July 1997); Allen, et al . , TIBS, 511-16 (Dec. 1995); LeCrenier, et al .
  • reporter Enzyme type In another embodiment, the components A and B reconstitute an enzyme which is not a transcription factor.
  • the effect of the reconstitution of the enzyme is a phenotypic change which may be a screenable change, a selectable change, or both.
  • Radio-labeled ABM may be administered to the human or animal subject. Administration is typically by injection, e.g., intravenous or arterial or other means of administration in a quantity sufficient to permit subsequent dynamic and/or static imaging using suitable radio-detecting devices.
  • the dosage is the smallest amount capable of providing a diagnostically effective image, and may be determined by means conventional in the art, using known radio-imaging agents as a guide. Typically, the imaging is carried out on the whole body of the subject, or on that portion of the body or organ relevant to the condition or disease under study. The amount of radio-labeled ABM accumulated at a given point in time in relevant target organs can then be quantified.
  • a particularly suitable radio-detecting device is a scintillation camera, such as a gamma camera.
  • a scintillation camera is a stationary device that can be used to image distribution of radio-labeled ABM.
  • the detection device in the camera senses the radioactive decay, the distribution of which can be recorded.
  • Data produced by the imaging system can be digitized.
  • the digitized information can be analyzed over time discontinuously or continuously.
  • the digitized data can be processed to produce images, called frames, of the pattern of uptake of the radio- labelled ABM in the target organ at a discrete point in time. In most continuous (dynamic) studies, quantitative data is obtained by observing changes in distributions of radioactive decay in target organs over time.
  • a time-activity analysis of the data will illustrate uptake through clearance of the radio-labeled binding protein by the target organs with time.
  • the radioisotope must be selected with a view to obtaining good quality resolution upon imaging, should be safe for diagnostic use in humans and animals, and should preferably have a short physical half-life so as to decrease the amount of radiation received by the body.
  • the radioisotope used should preferably be pharmacologically inert, and, in the quantities administered, should not have any substantial physiological effect.
  • the ABM may be radio-labeled with different isotopes of iodine, for example 123 I, 125 I, or 131 I (see for example, U.S. Patent 4,609,725).
  • the extent of radio-labeling must, however be monitored, since it will affect the calculations made based on the imaging results (i.e. a diiodinated ABM will result in twice the radiation count of a similar monoiodinated ABM over the same time frame) .
  • radioisotopes other than 125 I for labeling in order to decrease the total dosimetry exposure of the human body and to optimize the detectability of the labeled molecule (though this radioisotope can be used if circumstances require) . Ready availability for clinical use is also a factor. Accordingly, for human applications, preferred radio-labels are for example, 99m Tc, S7 Ga, 68 Ga, 90 Y, 11:L In, 113ra In, 123 I, 18 ⁇ Re, 188 Re or 211 At .
  • the radio-labelled ABM may be prepared by various methods.
  • radio-labeling examples include radi ⁇ -halogenation by the chloramine - T method or the lactoperoxidase method and subsequent purification by HPLC (high pressure liquid chromatography) , for example as described by J. Gutkowska et al in "Endocrinology and Metabolism Clinics of America: (1987) 16 . (1) :183.
  • Other known methods of radio-labeling can be used, such as IODOBEADSTM.
  • IODOBEADSTM IODOBEADSTM.
  • C57B1/6J mice i.e., C57B1/6 mice developed by Jackson Labs
  • a normal diet PMI Nutrition International Inc., Brentwood, MO, Prolab RMH3000
  • mice Two mice were sacrificed at an average of 35, 49, 77, 118, 133, 207, 403, 558 and 725 days of age.
  • RNA isolation Total RNA was isolated from muscle (ga trocnemius) using the RNA STAT-60 Total RNA/mRNA Isolation Reagent according to the manufacturer's instructions (Tel-Test, Friendswood, TX) .
  • Each chip contained an interconnected set of gel-filled channels that allowed for molecular sieving of nucleic acids .
  • Pin- electrodes in the chip were used to create electrokinetic forces capable of driving molecules through these micro- channels to perform electrophoretic separations.
  • Ribosomal peaks were measured by fluorescence signal and displayed in an electropherogram.
  • a successful total RNA sample featured 2 distinct ribosomal peaks (18S and 28S rRNA) .
  • RNA was prepared for use as a hybridization target as described in the manufacturer's instructions for CodeLink Expression Bioarrays (TM) (Amersham Biosciences) .
  • the CodeLink Expression Bioarrays utilize nucleic acid hybridization of a biotin-labeled complementary RNA(cRNA) target with DNA oligonucleotide probes attached to a gel matrix.
  • the biotin-labeled cRNA target is prepared by a linear amplification method.
  • Poly (A) + RNA (within the total RNA population) is primed for reverse transcription by a DNA oligonucleotide containing a T7 RNA polymerase promoter 5 1 to a (dT) 24 sequence.
  • the cDNA serves as the template in an in vitro transcription (IVT) reaction to produce the target cRNA.
  • IVT in vitro transcription
  • the IVT is performed in the presence of biotinylated nucleotides to label the target cRNA. This procedure results in a 50-200 fold linear amplification of the input poly (A) + RNA.
  • the oligonucleotide probes were provided by the Codelink Uniset Mouse I Bioarray (Amersham, product code 300013) . Amine-terminated oligonucleotide probes are attached to a three-dimensional polyacrylamide gel matrix. There are 10,000 oligonucleotide probes, each specific to a well-characterized mouse gene. Each mouse gene is representative of a unique gene cluster from the fourth quarter 2001 Genbank Unigene build. There are also 500 control probes . The sequences of the probes are proprietary to Amersham. However, for each probe, Amersham identifies the corresponding mouse gene by NCBI accession number, OGS, LocusLink, Unigene Cluster ID, and description (name) .
  • Hybridization Using the cRNA target, the hybridization reaction mixture is prepared and loaded into array chambers for bioarray processing as set forth in the manufacturer's instructions for CodeLink Gene Expression BioarraysTM (Amerhsam Biosciences) . Each sample is hybridized to an individual microarray. Hybridization is at 37°C. The hybridization buffer is prepared as set forth in the Motorola instructions. Hybridization to the microarray is detected with an avidinated fluorescent reagent, Streptavidin-Alexa Fluor ® 647 (Amersham) .
  • Nucleotide sequences and predicted amino acid sequences were compared to public domain databases using the Blast 2.0 program (National Center for Biotechnology Information, National Institutes of Health) . Nucleotide database searches were conducted with the then current version of BLASTN 2.0.12, see Altschul, et al . , "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res., 25:3389-3402 (1997) . Searches employed the default parameters, unless otherwise stated. For blastN searches, the default was the blastN matrix (1,-3), with gap penalties of 5 for existence and 2 for extension. Protein database searches were conducted with the then- current version of BLAST X, see Altschul et al .
  • RefSeq is the source database.
  • the identifier that follows is a RefSeq accession number, not a GenBank accession number.
  • RefSeq sequences are derived from GenBank and provide non-redundant curated data representing our current knowledge of known genes. Some records include additional sequence information that was never submitted to an archival database but is available in the literature . A small number of sequences are provided through collaboration; the underlying primary sequence data is available in GenBank, but may not be available in any one GenBank record.
  • RefSeq sequences are not submitted primary sequences. RefSeq records are owned by NCBI and therefore can be updated as needed to maintain current annotation or to incorporate additional sequence information.” See also htt : //www.ncbi .nlm.nih.gov/LocusLink/refseq. html
  • Northern analysis may be used to confirm the results.
  • Favorable and unfavorable genes, identified as described above, or fragments thereof, will be used as probes in Northern hybridization analyses to confirm their differential expression.
  • Total RNA isolated from subject mice will be resolved by agarose gel electrophoresis through a 1% agarose, 1 % formaldehyde denaturing gel, transferred to positively charged nylon membrane, and hybridized to a probe labeled with [32P] dCTP that was generated from the aforementioned gene or fragment using the Random Primed DNA Labeling Kit (Roche, Palo Alto, CA) , or to a probe labeled with digoxygenin according to the manufacturer's instructions (Roche, Palo Alto, CA) .
  • Real-Time RNA Analysis may also be used for confirmation.
  • RNA will be converted to cDNA and then probed with gene-specific primers made for each clone.
  • "Real-time” incorporation of fluorescent dye will be measured to determine the amount of specific transcript present in each sample. Sample differences (older vs. younger) of 2-fold or greater (in either direction) will be considered differentially expressed. Confirmation using several independent animals is desirable.
  • NISH nonisotopic in si tu hybridizations
  • si tu hybridizations may also be performed on mouse tissues using cRNA probes generated from differentially expressed DNAs. These cRNA's will hybridize to their corresponding messenger RNA's present in cells and will provide information regarding the particular cell types within a tissue that is expressing the particular gene as well as the relative level of gene expression.
  • the cRNA probes may be generated by in vi tro transcription of template cDNA by Sp6 or T7 RNA polymerase in the presence of digoxigenin-11-UTP (Roche Molecular Biochemicals, Mannheim, Germany; Pardue, M.L. 1985. In: In situ hybridization, Nucleic acid hybridization, a practical approach: IRL Press, Oxford, 179-202) .
  • Transgenic Animals may be used to confirm the results.
  • a mouse is engineered to overexpress the favorable or unfavorable mouse gene in question.
  • a mouse is engineered to express the corresponding favorable or unfavorable human gene.
  • a nonhuman animal other than a mouse such as a rat, rabbit, goat, sheep or pig, is engineered to express the favorable or unfavorable mouse or human gene.
  • tissue sections can also be analyzed using Tissuelnformatics, Inc's TissueAnalyticsTM software.
  • a single representative section may be cut from each tissue block, placed on a slide, and stained with H&E.
  • Digital images of each slide may be . acquired using an research microscope and digital camera (Olympus E600 microscope and Sony DKC-ST5) . These images may be acquired at 20x magnification with a resolution of 0.64 mm/pixel.
  • a hyperquantitative analysis may be performed on the resulting images : First a digital image analysis can identify and annotate structural objects in a tissue using machine vision. These objects, that are constituents of the tissue, can be annotated because they are visually identifiable and have a biological meaning.
  • Mathematical statistics provides a rich set of additional tools to analyze time resolved data sets of hyperquantitative and gene expression profiles for similarities, including rank correlation, the calculation of regression and correlation coefficients, and clustering. Continuous functions may also be fitted through the data points of individual gene and tissue feature data. Relation between gene expression and hyper-quantitative tissue data may be linear or non-linear, in synchronous or asynchronous arrangements .
  • the related applications may contain reference to "2-16 week old mice" . In the anti-diabetes series of applications, 3 week old mice were put on a diet to induce obesity, hyperinsulinemia and diabetes.
  • mice were more accurately described as mice who had been on that diet for 2-16 weeks, i.e., they were actually 5-19 weeks (35-133 days) old. Even some of the anti-aging series of applications made reference to 2-16 week old mice, even though the mice were in fact 5-19 weeks (35-133 days) old.
  • NM_009242 secreted protein, acidic, cysteine-rich (osteonectin); Osteonectin (secreted protein, NP 033268.1 Mm.35439 F:4.66 NP_003109.1 acidic, cysteine-rich) SPRC_HUMAN SPARC precursor (Secreted protein acidic and rich in cysteine) P09486 (Osteonectin) (ON) (Basement membrane protein BM-40) GEHUN osteonectin precursor CAA68724.1 extracellular matrix protein BM-40 (AA 1 - 303) AAA60570.1 osteonectin AAH04974.1 secreted protein, acidic, cysteine-rich (osteonectin) AAH08011.1 secreted protein, acidic, cysteine-rich (osteonectin) AAA60993.1 osteonectin 1 BMO A Chain A, Bm-40, FsEC DOMAIN PAIR 1BMO B Chain B, Bm-40, FsEC DOMAIN PAIR 1 NUB A Chain A, Helix C
  • IGF2_HUMAN Insulin-like growth factor II precursor (IGF-II) (Somatomedin A) 255
  • AAA52442.1 preproinsulin-like growth factor II, domains A-E 255
  • AAA52443.1 preproinsulin-like growth factor II, domains A-E 250
  • GTP binding protein 1 G-protein 1 984 000178 GTP-binding protein 1 (G-protein 1) (GP-1) (GP1) 984 AAB51273.1 putative G-protein 984 CAB42864.1 dJ508H5.3 (GTP binding protein 1) 984 AAH 14075.1 GTP binding protein 1 984 JC5291 GTP-binding protein GP-1 - human 984 PC7084 GTP-binding protein 2 - human (fragment) 441 AAF78884.1 putative GTP-binding protein 441 CAC36269.1 bA22l24.2.1 (GTP binding protein 2) 441 AAH64968.1 GTPBP2 protein 441 AAH28347.2 GTPBP2 protein 440 CAD38999.1 hypothetical protein 427 NP_061969.2 GTP binding protein 2 424 BAB12431.1 GTP-binding like protein 2 424 AAH20980.2 GTPBP2 protein 380
  • MFA2_HUMAN Microfibrillar-associated protein 2 precursor MFAP-2
  • P56001 Microfibril-associated glycoprotein
  • MAMP Microfibril-associated glycoprotein
  • microfibril-associated glycoprotein 288 dJ37C 0.4 microfibrillar-associated protein 2 (microfibril-associated glycoprotein CAB96824.1 precursor, MGAP1)) 288
  • PCPE Procollagen C-proteinase enhancer protein precursor
  • NM_008788 procollagen COOH-terminal proteinase enhancer (Type 1 procollagen C-proteinase NP 032814.1 Mm.18808 F:2.7 Q15113 enhancer protein) 588 BAA23281.1 type 1 procollagen C-proteinase enhancer protein 588 AAC78800.1 PCOLCE 588 AAD16041.1 procollagen C-proteinase enhancer protein 688 AAH00574.1 procollagen C-endopeptidase enhancer 588 AAH33205.1 procollagen C-endopeptidase enhancer 588 procollagen C-endopeptidase enhancer; procollagen, type 1, COOH-terminal NP_002 ⁇ 84.1 proteinase enhancer 585 A55362 procollagen l C-proteinase enhancer protein precursor 686 AAA61949.1 procollagen C-proteinase enhancer protein 585
  • NP_002014.1 fibromodulin precursor 237
  • osteomodulin 229 OMD_HUMAN Osteomodulin precursor (Osteoadherin) (OSAD) (Keratan sulfate
  • NP_002336.1 lumican 227 LUMJHUMAN Lumican precursor (Keratan sulfate proteoglycan lumican) (KSPG
  • AAH32953.1 Unknown protein for MGC:33476, ficolin 2 isoform a precursor; ficolin (collagen/fibrinogen domain-containing lectin) 2;
  • NPJD04099.1 ficolin (collagen/fibrinogen domain-containing lectin) 2 (hucolin); hucolin FCN2JHUMAN Ficolin 2 precursor (Collagen/fibrinogen domain-containing protein 2) Q15486 (Ficolin-B) (Ficolin B) (Serum lectin p35) (EBP-37) (Hucolin) (L-Ficolin)
  • NP_004902.1 protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) 340
  • AAH00425.1 protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) 340
  • AAH0192 ⁇ .1 protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) 340
  • AAH06344.1 protein disulfide isomerase related protein (calcium-binding protein, intestinal-related) 340 Similar to protein disulfide isomerase related protein (calcium-binding protein,
  • AAH11764.1 intestinal-related 340 procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), beta polypeptide (protein disulfide isomerase; thyroid hormone binding protein p ⁇ );
  • NP_000909.2 v-erb-a avian erythroblastic leukemia viral oncogene homolog 2-like 260
  • Rho guanine nucleotide exchange factor 7 isoform b; SH3 domain-containing proline-rich protein; PAK-interacting NP_663788.1 exchange factor beta 333 e-111 AAH50521.1 Rho guanine nucleotide exchange factor (GEF) 7 333 e-111
  • NM_009075 ribose ⁇ -phosphate isomerase A ribose ⁇ -phosphate epimerase
  • AAF61404.1 NF-E2-reIated factor 3 2993e-080 AAF61415.1 NF-E2-related factor 3 2993e-0 ⁇ 0 AAG43276.1 NF-E2-related factor 3 2993e-030
  • RNA polymerase III DNA-directed RNA polymerase III 47 kDa polypeptide (RNA polymerase C AAH16102.1 Mm.20420 F:2.4 ⁇ P06423 subunit 4) (RPC4) (RPC53) (BN61 protein) 562 e-165 AAH02603.1 POLR3D protein 582 e-165 AAH04434.1 POLR3D protein 582 e-165 AAM16216.1 RNA polymerase III 53 kDa subunit RPC4 678 e-164
  • RNA polymerase III 63 kDa subunit RPC4; temperature sensitive complementation, cell cycle specific, tsBN51; BN61 NP_001713.1 (BHK21) temperature sensitivity complementing 5 ⁇ e-158 A43700 BN51 protein - human 55 ⁇ e-158 AAA51833.1 BN61 protein 558 e-158 AAH03039.1 POLR3D protein 2192e-067
  • NM_018 ⁇ 62 1-acylglycerol-3-phosphate O-acyltransferase 1; lysophosphatidic acid acyltransferase 035083 Mm. ⁇ 6 ⁇ 4 2.44 NP 006402.1 alpha; 1 -AGP acyltransferase 1 ; lysophospholipid acyltransferase 496 e-140 1-acylglycerol-3-phosphate O-acyltransferase 1; lysophosphatidic acid acyltransferase NP 116130.2 alpha; 1-AGP acyltransferase 1 ; lysophospholipid acyltransferase 496 e-140 PLCAJHUMAN 1-acyl-sn-glycerol-3-phosphate acyltransferase alpha (1-AGP acyltransferase 1) (1-AGPAT 1) (Lysophosphatidic acid acyltransferase-alpha) Q
  • Chain A The 2.1 Angstrom Resolution Crystal Structure Of The Heterodimer Of The Human Rxralpha And Ppargamma Ligand Binding Domains Respectively Bound With 9-Cis Retinoic 1 FM9
  • 473 e-133 Chain A The 2.0 Angstrom Resolution Crystal Structure Of The Rxralpha Ligand Binding Domain Tetramer In The Presence 1 G5Y
  • 473 e-133 Chain B The 2.0 Angstrom Resolution Crystal Structure Of The Rxralpha Ligand Binding Domain Tetramer In The Presence 1 G5Y]B Of A Non-Activating Retinoic Acid Isomer.
  • 473 e-133 Chain C The 2.0 Angstrom Resolution Crystal Structure Of The Rxralpha Ligand Binding Domain Tetramer In The Presence 1 G5Y
  • MIP26 Lens fiber major intrinsic protein
  • AAB30268.1 hAQP-CD coliecting duct aquaporin [human, kidney, Peptide, 271 aa] 2151e-055
  • NM_009179 Mm.20038 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2, A54420 8 F:2.38 NP_008858.1 3-sialyltransferase 702 CMP-N-acetylneuraminate-beta-gaIactosamide-alpha-2, 3-sialyltransferase (Beta-galactoside alpha-2,3-sialyltransferase) (Alpha 2,3-ST) (Gal-NAc6S) (Gal-beta-1,3-GalNAc-alpha-2,3-sialyltransferase) Q16842 (ST3GalA.2) (SIAT4-B) (ST3Gal II) 702 0 JC5251 beta-galactoside alpha-2,3-sialyltransferase (EC 2.4.99.4) - human 702 0 CAA65
  • sialyltransferase 7D isoform a; NeuAc-alpha-2,3-Gal-beta-1,3-GalNAc-alpha-2, 6-sialyltransferase alpha2,6-sialyltransferase; sialyltransferase 3C; NeuAc-alpha-2,3-Gal-beta-1 ,3-GalNAc-alpha-2, NP_05521 ⁇ .3 6-sialyltransferase IV 203 1e-053 sialyltransferase 7D isoform a; NeuAc-alpha-2,3-Gal-beta-1,3-GalNAc-alpha-2, 6-sialyltransferase alpha2,6-sialyltransferase; sialyltransferase 3C; NeuAc-alpha-2,3-Gal-beta-1,3-GaiNAc-
  • AK009218 Mm.22809 SKI-interacting protein; nuclear receptor coactivator, 62-kD; BX42, BAB26144.1 5 F:2.36 NPJ336377.1 Drosophila, homolog of 587 e-167 Nuclear protein SkiP (Ski-interacting protein) (SNW1 protein) Q13573 (Nuclear receptor coactivator NCoA-62) 587 e-167 AAC15912.1 nuclear protein Skip 587 e-167 AAC31697.1 nuclear receptor coactivator NCoA-62 587 e-167 AAF23325.1 nuclear receptor coactivator NCoA-62 587 e-167 AAH40112.1 SNW1 protein 587 e-167
  • serine (or cysteine) proteinase inhibitor clade B (ovalbumin), member 8; protease
  • NP_002631.1 inhibitor 8 (ovalbumin type) 207 SPB8_HUMAN Cytoplasmic antiproteinase 2 (CAP2) (CAP-2) (Protease inhibitor 8)
  • cytoplasmic antiproteinase 2 207 serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 10; protease NPJD05015.1 inhibitor 10 (ovalbumin type, bomapin) 179
  • AAH44672.1 solute carrier family 15 H+/peptide transporter
  • member 2 1128 Oligopeptide transporter, kidney isoform (Peptide transporter 2) (Kidney H+/peptide cotransporter) (Solute carrier family Q16348 15, member 2) 1122 152481 PEPT 2 - human 1122 AAB34388.1 PEPT 2 1122 2113193A H/peptide cotransporter 1122 AAC15477.1 Caco-2 oligopeptide transporter 561 solute carrier family 15 (oligopeptide transporter), member 1; NP 005064.1 peptide transporter HPEPT1 561 Oligopeptide transporter, small intestine isoform (Peptide transporter 1) (Intestinal H+/peptide cotransporter) P46059 (Solute carrier family 15, member 1) 561 A56163 peptide transport protein hPEPTI - human 561 AAA63797.1 peptide transporter 561 AAB61693.1 intestinal H+/peptide cotransporter 561 bA5 ⁇ 1 18.
  • NP_77 ⁇ 1 ⁇ 8.1 transcription factor NFAT4 1643 Nuclear factor of activated T-cells, cytoplasmic 3 (T cell Q12968 transcription factor NFAT4) (NF-ATc3) (NF-AT4) (NFATx) 1643 A57377 transcription factor NFATx - human 1643 AAA86308.1 NFATx 1643 AAH01050.1 Cytoplasmic nuclear factor of activated T-cells 3, isoform 1 1643 cytoplasmic nuclear factor of activated T-cells 3 isoform 3; nuclear factor of activated T-cells, cytoplasmic 3; T cell NP_775186.1 transcription factor NFAT4 1598 cytoplasmic nuclear factor of activated T-cells 3 isoform 2; nuclear factor of activated T-cells, cytoplasmic 3; T cell NP_004 ⁇ 46.1 transcription factor NFAT4 1598 AAA79174.1 alternative splicing form 1698
  • NP_003218.2 transferrin receptor 2 545 e-164 Q9UP52 Transferrin receptor protein 2 (TfR2) 545 e-154
  • P42024 4 F:2.27 NP_005727.1 A 756 Alpha-centractin (Centractin) (Centrosome-associated actin homolog) P42024 (Actin-RPV) (ARP1) 755 0 S29089 alpha-centractin - human 75 ⁇ 0 CAA78701.1 actin-related protein 756 0 actin-related protein, actin-RPV dynactin complex major component AAB23391.1 [human, N-Tera teratocarcinoma, Peptide, 376 aa] 766 0 CAA57690.1 alpha-centractin 75 ⁇ 0 CAC08404.1 bA13114.9 (novel protein similar to beta-centracin (ACRTR1B)) 75 ⁇ 0 AAH00693.1 ARP1 actin-related protein 1 homolog A, centractin alpha 756 0 AAH26016.1 ACTR1A protein 755 0 1818368A actin-related protein 753 0 ARP1
  • beta actin beta actin 424 e-' 18
  • CAA26099.1 unnamed protein product 424 e- 18
  • AF378762 tumor endothelial marker 8 isoform 1 precursor; anthrax toxin receptor; tumor AAL11999.1 Mm.29636 F:2.23 NP_115534.1 endothelial marker 8, isoform 3 precursor 881 0 Q9H6X2 ATR_HUMAN Anthrax toxin receptor precursor (Tumor endothelial marker ⁇ ) 8 ⁇ 1 0 AAK52094.1 tumor endothelial marker ⁇ precursor 881 0 tumor endothelial marker 8 isoform 2 precursor; anthrax toxin receptor; tumor NP 444262.1 endothelial marker 8, isoform 3 precursor 600 3-171
  • AAC ⁇ 1735.1 uncoupling protein 3 NP_073714.1 uncoupling protein 3 isoform UCP3S; Uncoupling protein-3 AAC51356.1 UCP3S AAB46411.1 uncoupling protein-2 NP_003346.2 uncoupling protein 2; Uncoupling protein-2 P55851 UCP2_HUMAN Mitochondrial uncoupling protein 2 (UCP 2) (UCPH) AAC51336.1 UCP2 AAC39690.1 uncoupling protein 2 AAD21151.1 uncoupling protein-2 AAH11737.1 uncoupling protein 2 (mitochondrial, proton carrier) AAB53091.1 uncoupling protein homolog CAA11402.1 uncoupling protein 2
  • NP_068605.1 uncoupling protein 1 mitochondrial brown fat uncoupling protein
  • UCP1JHUMAN Mitochondrial brown fat uncoupling protein 1 (Thermogenin)
  • AAH03392.1 Similar to uncoupling protein 3 (mitochondrial, proton carrier) Mm.8033 F:2.21
  • AAK61566.1 AF371328 chondroadherin
  • AAH36360.1 Similar to chondroadherin NP_001253.1 chondroadherin precursor 015335 CHAD_HUMAN Chondroadherin precursor (Cartilage leucine-rich protein)
  • P10253 Lysosomal alpha-glucosidase precursor (Acid maltase) 1559 CAA68763.1 glucan 1 , 4-alpha-glucosidase 1559 CAA68764.1 70 kD alpha-glucosidase 1302 MGAJHUMAN Maltase-glucoamylase, intestinal [Includes: Maltase (Alpha-glucosidase); Glucoamylase (Glucan 043451 1 ,4-alpha-giucosidase)] 747 AAC39568.2 maltase-glucoamylase 747 NP_0046 ⁇ 9.1 maltase-glucoamylase; brush border hydrolase; alpha-glucosidase 745 AAL83560.1 maltase-glucoamylase 724 NP_001032.1 sucrase-isomaltase 717 P14410 Sucrase-isomaltase, intestinal [Contains: Sucrase ; I
  • Potential phospholipid-transporting ATPase IB (ATPase class 1 type ⁇ A Q9NTI2 member 2) (ML-1) 1568 CAD97 ⁇ 48.1 hypothetical protein 1357 BAC86402.1 unnamed protein product 1285 BAC04396.1 unnamed protein product 1062
  • RNA-binding region containing protein 1 isoform a; ssDNA binding S33384 Mm.3865 F:2.2 NP_059965.2 protein SEB4; CLL-associated antigen KW-5 3529e-097 S38382 SEB4D protein - human (fragment) 3273e-0 ⁇ 9 CAA53063.1 SEB4D 3273e-089 RNA-binding region containing protein 1 (HSRNASEB) (ssDNA binding Q9H0Z9 protein SEB4) (CLL-associated antigen KW-5) 3267e-089 CAC21462.1 dJ ⁇ OOJ21.2.1 (ssDNA binding protein SEB4D (HSRNASEB), isoform 1) 3267e-089 AAH18711.1 RNPC1 protein 3267e-089 AAL99924.1 CLL-associated antigen KW-5 3267e-0 ⁇ 9 S3 ⁇ 3 ⁇ 3 SEB4B protein - human (fragment) 3121e-084 CAA53
  • NM_011607 tenascin C hexabrachion
  • Hexabrachion tenascin
  • hexabrachion tenascin
  • Tenascin precursor (TN) (Hexabrachion) (Cytotactin) (Neuronectin) (GMEM) (Jl) (Miotendinous antigen) (Glioma-associated-extracellular matrix antigen) (GP P24621 150-226)
  • Tenascin-C (TN-C) 2595 0 A32160 tenascin-C - human 2595 . 0
  • P17612 cAMP-dependent protein kinase, alpha-catalytic subunit (PKA C-alpha) 661 protein kinase (EC 2.7.1.37), cAMP-dependent, alpha catalytic chain - OKHU2C human 661
  • decorin isoform a preproprotein; dermatan sulphate proteoglycans II; bone NPJD01911.1 proteoglycan II; proteoglycan core protein 395 decorin isoform a preproprotein; dermatan sulphate proteoglycans II; bone NP_59 ⁇ 010.1 proteoglycan II; proteoglycan core protein 395 P07585 PGS2_HUMAN Decorin precursor (Bone proteoglycan II) (PG-S2) (PG40) 395 NBHUC8 decorin precursor 395 AAB00774.1 proteoglycan core protein 395 AAD44713.1 decorin variant A 395 AAH05322.1 decorin 395 AAL92176.1 AF491944 decori 395 AAA52301.1 decorin 375
  • sialyltransferase 4A CMP-N-acetylneuraminate-beta-galactosamide-alpha-2, 3-sialyltransferase; sialyltransferase 4A (beta-galactoside alpha-2,3-sialytransferase); alpha NP_775479.1 2,3-ST; Gal-beta-1 ,3-GalNAc-alpha-2,3-sialyItransferase 562 e-160 CMP-N-acetylneuraminate-beta-galactosamide-aipha-2, 3-sialyltransferase (Beta-galactoside alpha-2,3-sialyltransferase) (Alpha 2,3-ST) (Gal-NAc6S) (Gal-beta-1 ,3-GalNAc-alpha-2,3-sialyltransferase)
  • sialyltransferase 4B (beta-galactoside alpha-2,3-sialytransferase); alpha 2,3-ST; Gal-beta-1 ,3-GalNAc-alpha-2,3-sialyltransferase; CMP-N-acetylneuraminate-beta-galactosamide-alpha-2, NP_00 ⁇ 5 ⁇ .1 3-siaiyltransferase 332 2e-090 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2, 3-sialyltransferase (Beta-galactoside alpha-2,3-sialyltransferase) (Alpha 2,3-ST) (Gal-NAc6S) (Gal-beta-galactoside alpha-2,3-sialyltransferase) (Alpha 2,3-ST) (Gal-NAc
  • AAF05834.1 AF196571 Delta-like-1 protein 226
  • F13A_HUMAN Coagulation factor XIII A chain precursor (Protein-glutamine P00488 gamma-glutamyltransferase A chain) (Transglutaminase A chain) 481 EKHUX protein-glutamine gamma-glutamyltransferase (EC 2.3.2.13), plasma 4 ⁇ 1 1 EVU A Chain A, Human Factor Xiii With Calcium Bound In The Ion Site 431 1EVU B Chain B, Human Factor Xiii With Calcium Bound In The Ion Site 431 AAA524 ⁇ 9.1 factor XIII precursor 431 1FIE A Chain A, Recombinant Human Coagulation Factor Xiii 431 1FIE B Chain B, Recombinant Human Coagulation Factor Xiii 4 ⁇ 1 AAH27963.1 coagulation factor XIII, A1 polypeptide 430
  • AAH32722.1 tumor necrosis factor (ligand) superfamily member 10 345
  • NP_002336.1 lumican 291 LUM HUMAN Lumican precursor (Keratan sulfate proteoglycan lumican) (KSPG P513 ⁇ 4 lumican) 291
  • PRLP_HUMAN Prolargin precursor Proline-arginine-rich end leucine-rich repeat P61388 protein
  • NP_008966.1 keratocan cornea plana 2 (autosomal recessive) 233
  • KTN KERA_HUMAN Keratocan precursor
  • NM_007901 1 ; edg-1 ; G protein-coupled sphingolipid receptor; 006530 Mm .982 F:2.04
  • AAF43420.1 G protein-coupled sphingolipid receptor 683 0
  • NP_005217.2 47 369 e-101 AAP84353.1 endothelial differentiation sphingolipid G-protein-coupled receptor 3 369 e-101 Endothelial differentiation, sphingolipid G-protein-coupled receptor,
  • NP_110387.1 1-phosphate receptor 5 317 1e-035 AAG3 ⁇ 113.1 sphingosine 1-phosphate receptor Edg-8 317 1e-035 AAL57041.1 SPPR 317 1e-0 ⁇ 5 BAB89315.1 putative G-protein coupled receptor 317 1e-085 Endothelial differentiation, sphingolipid G-protein-coupled receptor,
  • AAH34703.1 8 317 1e-0 ⁇ 5 BAC11119.1 unnamed protein product 317 1e-035
  • NP_004941.1 dermatan sulfate proteoglycan 3; Pg-Lb; dermatan sulphate proteoglycan 3 210
  • AAC50945.1 dermatan sulfate proteoglycan 3 210
  • NM_016635 pseudoachondroplasia (epiphyseal dysplasia 1 , multiple); cartilage oligomeric matrix NP 057694.1 Mm.45071 F:2.03 NP_0000 ⁇ 6.1 protein(pseudoachondroplasia, epiphyseal dysplasia 1 , multiple) P49747 COMP_HUMAN Cartilage oligomeric matrix protein precursor (COMP) AAA57253.1 matrix protein BAC533 ⁇ 8.1 cartilage oligomeric matrix protein AAB86501.1 COMP_HUMAN Similar to cartilage oligomeric matrix protein (pseudoachondroplasia, epiphyseal AAH33676.1 dysplasia 1, multiple) NP_003239.1 thrombospondin 4 P35443 TSP4_HUMAN Thrombospondin 4 precursor TSHUP4 thrombospondin 4 precursor CAA79635.1 thrombospondin-4 NP_009043.1 thrombospondin 3 P49746 T
  • NM_009762 Mm.23427 SET and MYND domain containing 1; CD8 beta opposite; zinc finger, NP 033692.1 4 F:2.03 NP_938015.1 MYND domain containing 1 ⁇ 935 0 Q ⁇ NB12 SET and MYND domain containing protein 1 935 0 BAC03732.1 unnamed protein product 935 0 SET and MYND domain containing 2; HSKM-B protein; zinc finger, MYND NP_064532.1 domain containing 14 2437e-064 AAF ⁇ 6953.1 HSKM-B 2437e-064 SET and MYND domain containing protein 3 (Zinc finger MYND domain Q9H7B4 containing protein 1) 2339e-061
  • AAH31010.1 SMYD3 protein 2339e-061 AAH49367.1 SMYD2 protein 2244e-053 SET and MYND domain containing 3; zinc finger protein, subfamily 3A (MYND domain containing), 1 ; zinc finger, MYND domain NP_073530.1 containing 1 2106e-054 BAB149 ⁇ 1.1 unnamed protein product 2106e-054
  • transducin-Iike enhancer protein 4 transducin-like enhancer of split S35681 8 F:2.03 NP_00 ⁇ 936.2 4; enhancer of split groucho 4;
  • B lymphocyte gene 1 1043 0 Q04727
  • Transducin-like enhancer protein 4 1043 0 T47149 hypothetical protein DKFZp547P103.1 - human (fragment) 1043 0 CAB ⁇ 2397.1 hypothetical protein 1043 0 BAA ⁇ 657 ⁇ .1 KIAA1261 protein 1043 0 AAH59406.1 TLE4 protein 1026 0
  • Crystal Form 563 e-160 B Chain B, Crystal Structure Of Recombinant Human Placental Annexin V Complexed 1HAK With K-201 As A Calcium Channel Activity Inhibitor 563 e-160 A Chain A, Crystal Structure Of Recombinant Human Placental Annexin V Complexed 1HAK With K-201 As A Calcium Channel Activity lnhibitor563 563 e-160 CAA30935.1 VAC protein (AA 1-320) 563 e-160 AAA35570.1 anticoagulant precursor (5' end put.); putative 563 e-160 AAA52336.1 endonexin II 563 e-160 AAB59545.1 anticoagulant protein 4 563 e-160 BAA00122.1 blood coagulation inhibitor 563 e-160
  • folate hydrolase prostate-specific membrane antigen 1 ; folate hydrolase 1 NP_004467.1 (prostate-specific membrane antigen) 228 FOH1_HUMAN Glutamate carboxypeptidase II (Membrane glutamate carboxypeptidase) (mGCP) (N-acetylated-alpha-iinked acidic dipeptidase I) (NAALADase I) (Pteroylpoly-gamma-glutamate carboxypeptidase) (Folylpoly-gamma-glutamate carboxypeptidase) (FGCP) (Folate hydrolase 1) Q04609 (Prostate-specific membrane antigen) (PSMA) (PSM) 228
  • T-box 1 isoform A; brachyury; T-box 1 transcription factor C; P70323 4 F:2.02 NP_ ⁇ 42377.1 Testis-specific T-box protein 350 6e-097 T-box transcription factor TBX1 (T-box protein 1 ) (Testis-specific 043435 T-box protein) 3506e-097 AAB9401 ⁇ .1 brachyury 3506e-097 T-box 1 isoform C; brachyury; T-box 1 transcription factor C; NP_542378.1 Testis-specific T-box protein 3506e-097 AAK ⁇ 8955.1 T-box 1 transcription factor C 3506e-097 T-box 1 isoform B; brachyury; T-box 1 transcription factor C; NP_005983.1 Testis-specific T-box protein 3506e-097 AAB94019.1 brachyury 3506e-097 NP_005936.2 T-box 10 3101
  • NM_007484 oncogene RHO H9; Aplysia ras-related homolog 9; RhoC; Q62159 Mm.262 F:2.02 NP_7 ⁇ 6 ⁇ 6.1 RAS homolog gene family, member C (oncogene RHO H9) 394 e-109 P0 ⁇ 134 Transforming protein RhoC (H9) 394 e-109 TVHURC GTP-binding protein rhoC - human 394 e-109
  • NP_001655.1 oncogene RHO H12 369 P06749 Transforming protein RhoA (H12) 369 TVHU12 GTP-binding protein rhoA - human 369 CAA28690.1 unnamed protein product 369 AAC33173.1 GTP-binding protein 369 AAH01360.1 ARHA protein 369 AAH05976.1 ARHA protein 369 AAM21117.1 small GTP binding protein RhoA 369 CAE46190.1 hypothetical protein 369 Chain B, Crystal Structure Of The Dbl And Pleckstrin Homology
  • NP_008966.1 keratocan cornea plana 2 (autosomal recessive) 220
  • KTN KERA_HUMAN Keratocan precursor
  • osteomodulin 211 OMD_HUMAN Osteomodulin precursor (Osteoadherin) (OSAD) (Keratan sulfate
  • CAB90270.1 dJ34M23.3 (gap junction protein, beta 4 (connexin 30.3)) 254
  • CAB90271.1 dJ34M23.4 (gap junction protein, beta 5 (connexin 31.1 )) 241
  • AAC95472.1 connexin 31.1 241 gap junction protein, alpha 8, ⁇ OkDa (connexin 50); gap junction membrane channel protein alpha-8; connexin 50; Gap junction membrane channel protein alpha-8 NP_00 ⁇ 258.1 (connexin ⁇ O); gap junction protein, alpha 8, 50kD (connexin 50) 241
  • IGF-IB Insulin-like growth factor IB precursor
  • Somatomedin C Insulin-like growth factor IB precursor
  • NM_021282 monooxygenase; flavoprotein-linked monooxygenase; cytochrome P450, subfamily
  • NM 009994 hydroxylase cytochrome P450, subfamily l (dioxin-inducible), polypeptide 1 (glaucoma 3, primary infantile); microsomal monooxygenase; xenobiotic
  • Cytochrome P450 1A1 (CYPIA1) (P450-P1) (P450 form 6) (P450-C) 324 8e-08 ⁇ aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.-) cytochrome P450
  • cytochrome P(1)-460 322 5e-037 cytochrome P450, family 1, subfamily A, polypeptide 2; cytochrome P450, subfamily l (aromatic compound-inducible), polypeptide 2; dioxin-inducible P3-450; P450 form 4; xenobiotic monooxygenase; aryl hydrocarbon hydroxylase; microsomal
  • Cytochrome P460 1A2 (CYPIA2) (P460-P3) (P(3)450) (P4604) 310 1e-083

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Abstract

Une expression différente des gènes des souris en comparaison à l’expression des gènes selon l'âge des muscles des souris a été découverte et correspond aux gènes et protéines humaines. Les molécules humaines, ou antagonistes, peuvent être utilisées pour se protéger contre un vieillissement biologique plus rapide que la norme ou pour obtenir un vieillissement biologique plus lent que la norme. Les molécules humaines peuvent aussi être utilisées comme des indicateurs de vieillissement biologique.
PCT/US2005/014441 2004-04-29 2005-04-28 Méthodes de traitement et diagnostics relatifs au vieillissement, particulièrement celui des muscles (14.1) WO2005110460A2 (fr)

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EP2293075A3 (fr) * 2005-06-17 2011-05-04 Randox Laboratories Ltd. Procédé pour le diagnostic de maladies neuro-dégénératives
CN110809718A (zh) * 2017-06-21 2020-02-18 韩国生命工学研究院 利用血液生物标志物诊断肌肉衰弱相关疾病的方法和试剂盒
CN113667739A (zh) * 2021-08-27 2021-11-19 河北医科大学第二医院 冠状动脉疾病的诊断标志物组合

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CN109540890A (zh) * 2018-11-27 2019-03-29 湖南品胜生物技术有限公司 一种基于细胞显微镜图像的dna定量分析方法

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WO2003025122A2 (fr) * 2001-08-13 2003-03-27 University Of Kentucky Research Foundation Biomarqueurs de profil d'expression genique et cibles therapeutiques destines au vieillissement cerebral et a la deficience intellectuelle liee a l'age

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EP2293075A3 (fr) * 2005-06-17 2011-05-04 Randox Laboratories Ltd. Procédé pour le diagnostic de maladies neuro-dégénératives
CN110809718A (zh) * 2017-06-21 2020-02-18 韩国生命工学研究院 利用血液生物标志物诊断肌肉衰弱相关疾病的方法和试剂盒
CN113667739A (zh) * 2021-08-27 2021-11-19 河北医科大学第二医院 冠状动脉疾病的诊断标志物组合

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