WO2005082398A2 - Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de façon differentielle dans les cellules musculaires - Google Patents

Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de façon differentielle dans les cellules musculaires Download PDF

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WO2005082398A2
WO2005082398A2 PCT/US2005/005596 US2005005596W WO2005082398A2 WO 2005082398 A2 WO2005082398 A2 WO 2005082398A2 US 2005005596 W US2005005596 W US 2005005596W WO 2005082398 A2 WO2005082398 A2 WO 2005082398A2
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protein
human
actin
mouse
gene
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WO2005082398A3 (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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Publication of WO2005082398A3 publication Critical patent/WO2005082398A3/fr

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    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the human molecules, or antagonists thereof, could be used for protection against faster-than-normal biological aging, or5 to achieve slower-than-normal biological aging. It was also taught that the human molecules may also be used as markers of biological aging.
  • provisional application Ser. No. 60/474,606, filed June 2, 2003 (our docket Kopchick7-USA) ⁇ our research group0 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 di ferentially., expressed in mice, depending upon their age. The level of5- gene expression of approximately 10,000 mouse.
  • mice- from . the Amersham Codelink UniSet Mouse I Bioarray, produc . ' code: 300013 in. the liver of mice with average' ages of 35, -, 49, 56, 77, 118, 133, 207, .403, 558 and 725 days was * determined.
  • complementary. ?NA derived from mice0 of different ages was screened for hybridization ' with ' , •• oligonucleotide probes each specific to a particular mouse ge e, each gene in turn representative of a particular mouse ' gene cluster (Unigene) .
  • 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
  • the invention relates to various nucleic acid molecules and proteins, and their use in (1) diagnosing hyperinsulinemia and type II diabetes, or conditions associated with their development, and (2) protecting mammals (including humans) against them.
  • diabetes mellitus 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. As more glucose concentrates in the urine, more water is excreted, resulting in .extreme thirst, rapid weight loss, drowsiness, fatigue, and possibly dehydration. Because 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.
  • acidosis acid
  • 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.
  • 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 hyperinsulinemia.
  • 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 ⁇ cell minus that which is taken up by the liver. Therefore, peripheral hyperinsulinemia can be caused by increased ⁇ 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. ' ' ,. ' ,. '
  • Obesi ty and Diabetes are a serious and growing ' ⁇ problem in the United States. Obesity-related health risks include high blood pressure, hardening of the arteries, cardiovascular disease, and Type II diabetes (also known as non-insulin-dependent diabetes mellitus, Type II diabetes) (9,10,11). Recent studies show that 85% of the individuals with Type II diabetes are obese (12) .
  • Metformin insulin therapy for Type I and oral sulfonylureas and/or insulin therapy for Type II.
  • Metformin (glucophage) was the ⁇ first antidiabetic drug approved by FDA (May 1995) for the treatment of Type II diabetes since the oral sulfonylureas were introduced in 1984. Metformin promotes the use of insulin already in the blood. This May 1995 approval was followed by the September 1995 approval of another ' antidiabetic drug, Acarbose (precose) . It slows down the digestion and absorption of complex sugars, which reduces blood sugar levels after meals. Before 1982, insulin was purified from beef or pork pancreas. This was a problem for those diabetics allergic to animal insulin.
  • Complications of diabetes include retinopathy, neuropathy, and nephropathy (traditionally designated as microvascular complications) as well as atherosclerosis (a macrovascular complication) .
  • mice Animal Models Transgenic Mouse Models of Diabetes or Diabetes Resistance. McGrane, et al . , J. Biol. Chem. 263:11443-51 (1988) and Chen, et al . , J.. Biol. Chem.-, 269:15892-7 (1994) describe the genetic engineering of mice to express bovine growth hormone (bGH) or human growth hormone (hGH) , respectively. These mice exhibited an enhanced growth phenotype . They also developed kidney lesions similar to those seen in diabetic glomerulosclerosis, see Yang, et al . , Lab. Invest., 68:62-70 (1993). Ogueta, et al., J.
  • bGH bovine growth hormone
  • hGH human growth hormone
  • Endocrinol., 165: 321-8 (2000) reported that transgenic mice expressing bovine GH develop arthritic disorder and self- antibodies .
  • Growth hormone has many roles, ranging from regulation of protein, fat and carbohydrate metabolism to growth promotion.
  • GH is produced in the somatrophic cells of the anterior pituitary and exerts its effects either through the GH-induced action of IGF-I, in the case of growth promotion, or by direct interaction with the GHR on target cells including liver, muscle, adipose, and kidney cells.
  • Hyposecretion of GH during development leads to dwarfism, ⁇ and hypersecretion before puberty leads to gigantism.
  • GH hypersecretion of GH results in acromegaly, a clinical condition characterized by enlarged facial bones, hands, feet, fatigue and an increase in weight. Of those individuals with acromegaly, 25% develop type II diabetes. This may be due to. insulin resistance caused by the high circulating levels of GH leading to high circulating levels of insulin (Kopchick et.al., Annual Rev. Nutrition 1999. 19:437-61) . ' ' ' , ' ! A further mode of GH. action may be ' through the transcriptional regulation of a number of genes contributing ' . to the' physiological effects of GH.
  • mice have been made that express the GH antagonists bGH-G119R or hGH G120R, and which exhibit a dwarf phenotype., Chen, et al . , J. Biol. Chem., 263:15892-7 (1994); Chen, et al . , Mol. Endocrinol, 5:1845-52 (1991); Chen, et al . , Proc . Nat. Acad. Sci.- USA 87:5061-5 (1990) . .These mice did not develop kidney lesions. See Yang (1993), supra.
  • mice phenotype.-, - GHR/BP-KO mice, made diabetic by streptozotocin treatment, are protected from the development of dia?betes- associated nephropathy. Bellush, et al .,' Endocrinol . , ' 141:163-8 (2000) . ⁇ ,
  • High-Fat Diets have been shown to induce both obesity and Type II diabetes ' in laboratory animals (13) .
  • high-fat fed animals had significantly 5 elevated fasting blood-glucose and insulin levels and also demonstrated a decrease in insulin sensitivity (14) .
  • Ahren and colleagues (15) reported evidence of insulin resistance as well as diminished glucose-stimulated insulin release, after feeding with a high-fat diet for 12 weeks.
  • 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
  • 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
  • fascia fibrous connective-tissue membranes
  • 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.
  • 35 muscles are attached either directly to a bone, or ' indirectly through a tendon.
  • the individual muscle fibers ' (cells) comprise threadlike protein structures , called 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.
  • 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. At least three steps-those mediated by glycogen synthase, hexokinase, and GLUT4-have been reported to be defective in patients with type 2 diabetes .
  • 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 .or ⁇ /iointandmuscleproblems.html [Last modified June 12, 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 . (USA), 100(14): 8466 (July 8, 2003) 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. , Of 7,129 sequences represented on, the microarray, 187*. were differentially expressed between ' control, and diabetic subjects.
  • the top-ranked cellular component terms were mitochondrion, mitochondrial membrane, mitochondrial inner membrane, and ribosome, and the top- ranked process term was ATP biosynthesis.
  • the over-represented groups were energy generation, protein biosynthesis/ribosomal proteins, RNA binding, ribosomal structural protein, and ATP synthase complex.
  • 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.
  • 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 .
  • 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 .
  • 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. In contrast, membrane rupture and release of cellular components during necrosis often leads to tissue inflammation.
  • Caspases are a family of serine proteases that are synthesized as inactive proenzymes. Their activation by 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. Yoon, J-H. Gores G.J. (2002) Death receptor-mediated apoptosis and - the liver. J. Hepatology 37:400-410. These pa.thways are not ⁇ mutually exclusive and some cell types require the activation of both pathways for maximal apoptotic signaling.
  • DR cell surface death receptors
  • type-I cells death receptor' activation, leads to the recruitment and activation of caspases-8/10 and the rapid cleavage and activation of. caspase-3 in a mitochondrial-independent manner, i Hepatocytes are members of the Type-II cells in which mitochondria are essential for DR-mediated apoptosis
  • Bid a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.
  • 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 .
  • the 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc. Natl. Acad. Sci. USA. 95:8461-8466; Halenbeck, R., MacDonald, H.
  • CIDEs cell-death-inducing DFF45-li?ke effectors '
  • CIDE-3 a novel member of the cell-death-inducing DNA- fragmentatio -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 brown adipose tissue
  • BAT brown adipose tissue
  • CIDE-A can interact and inhibit • UCP1 in BAT and may therefore play a role in regulating energy ibalance, see Zhou supra.
  • CIDE-A is not expressed. in either adult human o mouse liver tissue, see Inohara supra, Zhou supra. . , 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.
  • mice that are differentially expressed in the muscle (gastrocnemius) of mice, depending upon their development of hyperinsulinemia or type II diabetes.
  • complementary ?RNA derived from normal mice, or mouse models of hyperinsulinemia or type II diabetes 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 database DNA sequences whether full-length or partial, and whether cDNA or genomic DNA, are referred to herein as "mouse genes". When only the genomic sequence is intended, we will refer specifically to "genomic DNA” or "gDNA” .
  • mouse proteins regardless of whether* they are in fact full length , sequences .
  • mouse genes which were differentially expressed (normal vs. hyperinsulinemic, hyperinsulinemic vs . diabetic, or normal vs. diabetic), as measured by .different levels of hybridization of the respective c?RNA samples with the : particular probe ⁇ corresponding to that mouse gene) were identified.
  • normal and "control” are used interchangeably in thiis specification, unless expressly stated otherwise.
  • the control or normal subject is a mouse which is normal vis-a-vis fasting insulin and fasting glucose levels.
  • normal means normal relative to tlr ⁇ ose parameters, and does not necessitate that the mouse be normal in every respect .
  • a mouse gene is said to have exhibited a favorable behavior if, for a particular mouse age of observation, its average level of expression in mice which are in a more favored state is hig?t ⁇ er than that in mice which are in a less favored state.
  • A. mouse gene is said to have exhibited an unfavorable behavior if, for a particular mouse age of observation, its average level of expression in mice which are in a more favored state is lower than that in mice which are in a less favored state.
  • mice gene were observed at an age other than one of the ages noted in the Examples, we would have observed a still stronger differential expression behavior. Nonetheless, we must classify the mouse genes on the basis of the behavior which we actually observed, not the behavior which might have been observed at some other age.
  • mice genes which exhibit strongly favorable or unfavorable differential expression behaviors.
  • a behavior is considered strong if the ratio of the higher level to the lower level is at least two-fold.
  • a mouse gene may still be identified as favorable or unfavora?ble even if none of its observed behaviors are strong as defined above.
  • tissue than iri either normal or type IT diabetic ; tissue (i.e., C ⁇ HI , HI>D) will be deemed both ! ⁇ ⁇ unfavorable", by virtue of the control :hyperinsulinemic and "favorable", by virtue of , the ' ' - hyperinsulinemic:diabetic comparison.
  • This is one of several possible "mixed” expression patterns.
  • the genes/proteins with "mixed" expression patterns are, by definition, both partially favorable and partially unfavorable.
  • 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.
  • they are used in conjunction with other agents that affect their balance of favorable and unfavorable behavior.
  • mice gene is classified on ' the basis of the strongest C-HI behavior among the ages tested, the strongest HI-D behavior among the ages tested, and the strongest C-D behavior among the ages tested. If at least one of these three behaviors is significantly favorable, and none of the others of these : three behaviors is significantly unfavorable, the mouse gene will be classified as wholly favorable • and listed in subtable lA of Master Table 1. However, that does not mean that it may not have exhibited a ' weaker but unfavorable expression behavior at some tested age.
  • the "favorable”, “unfavorable” and “mixed” mouse proteins of the present invention include the mouse database proteins listed in the Master Table in the same row as a particular "favorable” , ' “unfavorable” or “mixed 7 ' mouse gene, respectively. These proteins may be the exact translation ⁇ product of the identified mouse gene (database DNA) . However, if they were sequenced directly, they could be shorter or longer than that translation product. They could also differ in sequence from the exact translation product as a result of post-translational modifications.
  • mouse proteins of interest also include mouse proteins which, while not listed in the table, correspond to (i.e., homologous to, i.e., which could be aligned in a statistically significant manner to) such mouse proteins or genes, and mouse proteins which are at least substantially identical or conservatively identical to the listed mouse 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 analogox ⁇ s 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 homologus 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 connept the mouse cDNA to the human protein or gene,' measured by an E value),- the more close the correspondence.
  • the connection may be ?direct (mouse gene to human protein) or indirect ' (e.g., mouse gene to human gene, human.gene to human protein) .By "mouse gene”, we mean the mouse gene from which the gene chip DNA in question was derived.: .
  • the human genes/proteins which ,most closely correspond, directly or indirectly; to the mouse genes 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 . Note that it is possible to identify 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 1 probes intended to identify .. the full length gene or protein sequence.
  • a human protein For the sake of convenience, we refer to a human protein as favorable if (1) it is listed in Master Table 1 as corresponding to a favorable mouse gene, or (2) it is at least substantially identical or conservatively identical to a listed protein per (1), or (3) it is a member of a human protein class listed in Master Table 2 (if provided) as corresponding to a favorable mouse gene.
  • a human protein We define a human protein as unfavorable in an analogous manner.
  • a human gene which encodes a. particular human protein may be classified in the same way- as the human ⁇ protein which it encodes. However, it should be noted that this classification is not based on the direct study of the expression of the human gene/protein. of course, the human genes/proteins of ultimate interest will be the ones whose change in level of 5 expression is, in fact, correlated, directly or inversely, with the change of state (normal, hyperinsulinemic, diabetic) of the subject.
  • one 10 may formulate agents useful in screening humans at risk for - progression toward hyperinsulinemia or toward type II diabetes, or protecting humans at risk thereof from progression from a normoinsulinemic state to a hyperinsulinemic state, or from either to a type II diabetic '15 state.
  • Agents which bind the "favora le” and “unfavorable" nucleic acids e.g., the agent is a substantially complementary nucleic acid hybridization probe) , or the
  • corresponding proteins may be used to evaluate whether a human subject is at increased or decreased risk for progression toward type II diabetes.
  • a subject with one or more elevated “unfavorable” and/or one or more depressed “favorable” genes/proteins is
  • the assay may be used as a preliminary screening assay to select subjects for further analysis, or as a formal diagnostic assay.
  • 35 use of the corresponding -mouse or human proteins, in diagnostic agents, to measure progression toward .hyperinsulinemia or type II diabetes, or protection against ⁇ the disorder(s), or to estimate related end ⁇ organ damage such as kidney damage; • • ' (5) use of the corresponding mouse or human proteins in assays to determine whether a substance binds to (and hence may neutralize) the protein; and (6) use of the neutralizing substance to protect 5 against the disorder (s) .
  • 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 10 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 , 15 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 1A or IC. Finally, they include DHSTA sequences which encode peptide (including antibody) antagonists of the 20 proteins of Master Table 1, subtables IB or IC.
  • the related human DNAs may be identified by comparing the mouse sequence (or its AA translation product) to known human DNAs (and their AA translation products) . 25.. Related human 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 30 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.
  • ⁇ • 7 ⁇ is possible that the genes > found- to be unfavorable act indirectly by accentuating obesity. Consequently, it is ⁇ within the compass of the present invention to use the favorable genes and proteins, or to use antagonists of the unfavorable genes and proteins, to protect against obesity, as well as against sequelae of obesity such as hyperinsulinemia and diabetes. Since type II diabetes is an age-related disease, the agents of the present invention may be used in conunction with known anti-aging or anti-age-related disease agents.
  • FIG. la Body weight gain [Fig. la] , fasting glucose [Fig. lb] and fasting insulin [Fig. Ic] levels of mice on the HF or Std diets.
  • Figure 2 Expression levels of Actin, alpha, cardiac (Actcl, NM_009608) using RNA isolated from gastrocnemius muscle of individual diabetic HF mice and corresponding Std mice at different time points.
  • Figure 3 Data shown are expression levels for additional actin-related and actin-binding genes exhibiting a consistent decrease in expression in the HF mice in -comparison to Std mice at all four time points (Fig. 3(a)) or at three of the four time points (Fig. 3(b)) .
  • 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 codon0 (when introns, if any, are ignored) , and thereby encod.es 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,5 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 mature0 proteins, proproteins, preproteins and preproproteins . It also includes substitution and extension mutants of such naturally occurring proteins.
  • a mouse is considered to be a diabetic subject if, regardless of its fasting plasma insulin level, it has a fasting plasma glucose level of at least 190 mg/dL.
  • a mouse is considered to 'be a hyperinsulinemic . subject if its fasting plasma insulin level is at' least 0.67 ng/mL and it0 , does, not qualify as a- diabetic subject.
  • a mouse is considered to be "normal” if it is neither 'diabetic nor - ⁇ hyperinsulinemic. Thus, normality is defined in a very limited manner.
  • a ' mouse is considered “obese” if its weight is at least'5 ' 15% in excess of the -.mean weight for mice of its age and , sex.
  • a mouse which does • n'ot satisfy 'this standard may be characterized as "nonrobese" , the term “normal” being reserved for use in reference to glucose and insulin levels '1' as previously -described.
  • a human is considered a diabetic subject if, regardless of his or her fasting plasma insulin level, the fasting plasma glucose level -is at least 126 mg/dL.
  • a human is considered a hyperinsulinemic subject if the fasting plasma insulin level is more than 26 micro International Units/mL (it is believed that this is equivalent to 1.08 ng/mL) , and does not qualify as a diabetic subject.
  • a human is considered to be "normal” if it is neither diabetic nor hyperinsulinemic.
  • NIDDK Non-overweight
  • HDL cholesterol level >35 mg/dL (0. 90 mmol/L)
  • the diagnostic and protective methods of the present invention are applied to human subjects exhibiting one or more of the aforementioned risk factors. Likewise, in a preferred embodiment, they are applied to human subjects who, while not diabetic, exhibit impaired glucose homeostasis (110 to ⁇ 126 mg/dL) .
  • the age of the subjects is at least 45, at least 50, at least 55, att least 60, at least 65, at least 70, and at least 75.
  • NEDDK says that "The relative risk of diabetes increases by approximately 25 percent for each additional unit of BMI over 22.”
  • the BlV-IIs of- the human subjects is at least 23, at least 24, at least 25 (i.e., overweight by our criterion), at least 26, at least 27, at least 28, at. least 29, at least 30 (i.e. , obese), at least 31, at least 32, at least 33, at least 3-4, at least 35, at least 36, at least 37, at least 38, at heast 39, at least 40, or over 40.
  • Age-related (senescent) diseases i ⁇ clude certain cancers, atherosclerosis, diabetes (type 2) , osteoporosis, hypertension, depression, Alzheimer's, Parkinson's, glaucoma, certain immune system defects, kidney failure, and liver steatosis. In general, 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.
  • the 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, it encodes an entire, functional, naturally occurring protein, then it may be used in the expression of that protein. Likewise, if the corresponding human gene is known in full-length, it may be used to express the human protein.
  • the disclosed genes (gDNA or cDNA)have significant similarities to known DNAs (and their translated AA sequences to known proteins)
  • the results of several such searches are set forth in the Examples. Such 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.
  • the 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 .
  • the 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.
  • an antagonist of a protein or other molecule may be obtained by preparing a combinatorial library, as described belqw, 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 identified 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.
  • the possession of ''one DNA greatly .facilitates -the isolation of homologous DNAs. If only a partial DNA is known, 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 genes .
  • 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 8 bases and the human genomic DNA library is about 10 10 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.
  • the Tm of a perfect duplex of starting DNA is determined. " One may then select a ' ' - hybridization temperature which is sufficiently 'lower than the perfect duplex Tm to allow hybridization?
  • 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 NaCl.
  • 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. : In turn, it is identifiable as corresponding (homologous) to said identified mouse gene, if
  • 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 to a mouse protein, the latter being encoded by said mouse gene, or aligned to said mouse gene BlastX, where 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) l 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 protein with a score worse (i.e., higher) than e-50 may appear in Master Table 1: If the manufacturer of the gene chip identifies the gene chip DNA as corresponding to an EST, or other DNA which is not. a full-length mouse gene or cDNA, a longer (possibly full length) mouse gene. or cDNA may be identified by a BlastN search of the mouse DNA database.
  • the identified DNA 1 may be used to conduct ; 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 1 ) .
  • 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%.
  • .' • ⁇
  • 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- he 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 ⁇
  • a human protein may be said to be functionally homologous to the mo ⁇ se 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.
  • 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) s required for activity, or post-translationally modify the protein so as to inactivate it.
  • 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), may be used to inhibit its activity.
  • This antagonist could be an antibody, a peptide, a peptoid, a nucleic acid, a peptide nucleic acid (PNA) 5 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
  • an agent which degrades, or abets the degradation of, that messenger RNA, its protein product or a downstream product which mediates its activity e.g., a
  • 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
  • RNA product the messenger RNA product, the ⁇ equivalent cDNA, protein product, . or a binding molecule specific for those products, or a downstream product, or a signaling
  • 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., 10 8 ) of related mutants may, be evaluated simultaneously. _ , ' Hence, the mutations are preferably conservative modifications as defined below. ' ;
  • Substantially Identical 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 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%, at least • 66%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 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 "Conservative 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 nonconservative modifications ; Semi- conservative substitutions are preferred to other semi- conservative modifications. Non-conservative substitutions are preferred to other non-conservative modifications.
  • 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 5 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- 10 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
  • Cys belongs to both I and IV. 20 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 a. helices. These residues may be essential in certain regions of the polypeptide, but 25 substitutable elsewhere. One, .two or three conservative substitutions are more likely to be tolerated than a larger number.
  • substitutions are defined herein as being substitutions within supergroup I/II/III or within 30 supergroup IV/V, but not within a single one of groups I-V., They also include replacement of any other amino acid with alanine. If a substitution is not conservative, it preferably is semi-conservative. . ' “Non-conservative substitutions” are substitutions 35 which are not “conservative” or “semi-conservative”.
  • “Highly conservative substitutions” are a subset of conservative substitutions, and are exchanges of ami ⁇ o 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 modifications, 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 wl ⁇ ich 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 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. lternatively or additionally, it ' might"be to cells of a particular- tissue or. organ. Or it • could be cells exposed to particular pharmacological, environmental, or pathogenic conditions.- Or the library could be of chemicals, or a particular class of chemicals, produced by such cells. In a ""controlled structure" library, 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 , j 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.
  • LI and L2 interact to associate the first fusion with the second fusion and hence, indirectly, the library . member with the surface of the cell or virus..
  • 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.
  • Synthetic DNA 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 he smaller than the average number of members in each simple library, as- otherwise ' the advantage of a library over individual , synthesis is small.
  • 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.
  • 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 initurn is a function of the strength of the signal -to be screened. , ⁇ ,
  • ⁇ There are more complex measures of diversity than simple diversity. These attempt to take into account the degree of structural difference between the various unique sequences.- These more comple 'measures are usually used in ' the context of small organic compound libraries, see below.
  • 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.
  • the advantage of an immobilized library is that it is easier to identify the structure of the members which were positive.
  • 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. For examples of nucleotides modified to increase nuclease resistance and chemical stability of aptamers, see Chart 1 in Osborne and Ellington, Chem. Rev., 97: 349-70 (1997).
  • RNA For screening' of RNA, see Ellington and Szostak, Nature, 346: .818-22 (1990). There is no formal minimum or maximum size for these oligonucleotides. However, the number of conformations which an oligonucleotide can assume increases exponentially with its length in bases. Hence, a longer oligonucleotide is more likely to be able to fold to adapt itself to a protein surface: On the other hand, while ver 'long molecules can be ..synthesized and screened, unless they provide a much ⁇ superior affinity to that of shorter molecules, they -are not likely to be found in the selected population, for ,the , ⁇ reasons explained by Osborne and Ellington (1997) .
  • 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.
  • the 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) .
  • PCR polymerase chain reaction
  • nucleic acid molecules can have very high specificity and affinity to targets .
  • this invention prepares and screens Oligonucleotide libraries by the SEL ⁇ X method, as described in King and Famulok, Molec. Biol.
  • 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' phosphodies er 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, - for acetals, 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 ISIH a -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,- 1
  • 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 ater.
  • 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 .
  • the main chain moiety of each amino acid consists of the -NH and -CO linking unctionalities and a core main chain moiety.- Usually, the latter is a single carbon atom.
  • 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 C-l, C-2 and N-2 of each residue form the repeating unit of the main chain, and the word "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. Only alpha amino acids occur in proteins. Proline is a special case of an alpha amino acid; its side chain also binds to the peptide bond nitrogen. For beta and higher order amino acids, there is a choice as to which 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. It ' is also possible ' for more than one main chain atom to carry ' a' side chain ⁇ other than H.
  • a main chain carbon 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 oligopeptide 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 varia?ble.
  • 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 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). Thus, 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) . This is simply because, for a given range of variation at each position, the sequence space (simple diversity) grows exponentially with the number of residue positions, so at some point it becomes inconvenient for all residues of a peptide to be variable positions. Since proteins are usually larger than oligopeptides, it is more common for protein libraries than oligopeptide libraries to feature variable positions.- In the case of a protein library, it is desirable to focus the mutations at those sites which are tolerant, of mutation.
  • variable domains of an antibody possess hypervariable regions and hence, in some embodiments, 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.
  • VH and VL chains may be covaleiitly 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.
  • 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 ⁇ -C0-O- Hydroxyethylene ⁇ -CHOH-CH 2 - Ketomethylene ⁇ -C0-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.
  • the side chains attached 5 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-CO-, where at least 10 one of RI 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 15 ' 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) .
  • a simple combinatorial library may include both peptides and peptoids .
  • a 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.
  • a PNA monomer which comprises a side chain comprising a nucleobase.
  • the classic PNA oligomer is composed of (2- 30 aminoethyl) glycine units, with nucleobases attached by methylene carbonyl linkers. That is, it has the structure
  • 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.
  • a side chain is attached to one of the three main chain carbons not participating in the peptide bond (either instead or in addition to the side chain attached to the N of the classic PNA).; and/or (3) the peptide bonds ' are replaced by pseudopeptide bonds as disclosed previously in the context of peptoids.
  • PNA oligomer libraries have been made; see e.g. Cook, 6,204,326.
  • Small Organic Compound Library 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 .
  • disjunction in- which one moiety is, replaced by another which may be similar or different, but which is not in effect a disjunction or conjunction.
  • 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.
  • the use of the terms "disjunction”, “conjunction” and “alteration” is intended only to connote the structural relationship of the end product to the original leads, and not how the new drugs are actually synthesized, although it is possible that the two are the same.
  • the process of disjunction is illustrated by the evolution of neostigmine (1931) and edrophonium (1952) from physostigmine (1925) . Subsequent conjunction is illustrated by demecarium (1956) and ambenonium (1956) .
  • Alterations may modify the size, polarity, or electron distribution of an original moiety. 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. conj ⁇ gative (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.
  • a library / a compound, or a family of compounds having one or more pharmacological activities (which need not be related to the known or suspected activities of the target protein) , may be disjoined into two or more known orr potential pharmacophoric moieties. Analogues of each of these moieties may be identified, and mixtures of these a-nalogues 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 moi&ties analogous to all of the moieties of the lead compound.
  • benzodiazepines have widespread biological activities; derivatives have been reported to a ct not only as anxiolytics, but also as anticonvul sants ; cholecystokinin (CCK) receptor subtype A or B, kapp.a 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.
  • 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 25 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 characterise those members of the library which are found to be active, based on the characteristic spectroscopic indicia of the various -35 -- building blocks. .Solid phase synthesis permits greater controH ⁇ over , . which derivatives are formed. However, the' solid phase . could interfere with activity.
  • 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.
  • Primata e.g., apes and monkeys
  • Artiodactyla or Perissodactyla e.g., cows, pigs, sheep, horses, goats
  • Carnivora e.g., cats, dogs
  • prevention 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.
  • 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 epidemiologically associated with that disease.
  • a subject who is immunologically compromised e.g., by radiation treatment, HIV infection, drug use,, etc.
  • membership in a particular group e.
  • 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. However, 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. 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.
  • Typical pharmaceutical doses for adult humans, are in the range of 1 ng to lOg per day, more often 1 mg to lg per day.
  • 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.
  • 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 angiospeirms.
  • 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.
  • the microorganism may be human or other animal or plant pathogen, or it may be nonpathogenic. It may be a soil or water organism, 1 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. Among mammals, the target animal preferably belongs to the order
  • 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 Clupeif ⁇ rmes (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. ' .
  • Screening Assays 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 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 ste s 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, 3 ⁇ S, 14 C, 32 P and 33 P. 125 I is preferred for antibody labeling.
  • the label may also be a fluorophore.
  • the • fluorescently labeled reagent When the • fluorescently labeled reagent is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
  • 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 chemiluminescently labeled reagent is then . - determined by detecting the presence of luminescence that' arises during the course of a chemical reaction.
  • 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.
  • 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 5 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
  • 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
  • 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.
  • a label may be conjugated, directly or indirectly
  • 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
  • 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.
  • Biological Assays 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. For example, 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.
  • 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. In that event, 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 models the ultimate entity under some conditions. Or it may be non-naturally occurring, with modifications that increase its resemblance to the ultimate entity.
  • Transgenic animals such as transgenic mice, rats, and rabbits, have been 1 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.
  • a signal biological marker
  • 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 ligard 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.
  • a chimera receptor a hybrid of the target protein and an endogenous receptor
  • 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 USE 3 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.
  • Components A and B may naturally occur, or be substantially identical to moieties which naturally occur, as components of a single naturally occurring biomolecule, or they may naturally occur, or be substantially identical to moieties which naturally occur, as separate naturally occurring biomolecules which interact in nature.
  • 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")
  • the other is expressed as a fusion to a transactivation domain (TAD) (this fusion protein is called the "fish", the "prey”, or the "catch”)
  • 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.
  • 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.--.
  • nonselective screen Various screening and selection systems are discussed in Ladner, USP 5,198,346. 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);
  • 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.
  • 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-labeled ABM in the target organ at a discrete point in time.
  • images called frames
  • 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. ;
  • Various factors should be taken into consideration in ⁇ selecting an- appropriate radioisotope .
  • the radioisotope ⁇ must be 1 ,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 monoiodinatecl 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, U1 ln, 113ra In, 123 I, 186 He, 188 Re or 211 At .
  • the radio-labeled ABM may be prepared by various methods. These include radio-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. ' ⁇ ⁇ There are a number of different methods of delivering the radio-labeled ABM to -the end-user. It may, be administered by any means that enables the active agent- to reach the agent's site of action in the body of a mammal.
  • parenteral administration i.e.,' intravenous, • subcutaneous, intramuscular, would ordinarily be used to optimize absorption of an ABM, such as an antibody, which is a protein.
  • Obesity and subsequent hyperinsulinemia and hyperglycemia were induced by feeding a group of 3 week old mice (50 C57BL/6 males) a high-fat diet (Bio-Serve , Frenchtown, NJ, #F1850 High Carbohydrate-High Fat; 56% of ⁇ calories from fat, 16% from protein and 27% from carbohydrates) : Another group of 3 week old mice (20 C57B1/6 males) were fed the normal control diet (P1 I Nutrition International Inc., Brentwood, MO, Prolab ?RMH3000; 14% of calories from fat, 16% from protein and 60% from carbohydrates) . The mice were placed onto the respective ' diets immediately following weaning. Animal weights were determined weekly.
  • Results reflect mean ⁇ SE of 50 mice on the HF diet and 20 mice on the Std diet. Normal weight, normal fasting blood glucose and normal fasting plasma insulin levels are defined as the respective mean values of the animals fed the control diet. Two of the "most typical" animals were selected for each group (Control, hyperinsulinemic and Diabetic) at each time point ( 2,4, 8, and 16 weeks after commencement of diet) for sacrifice. The selected mice were sacrificed and muscle tissue obtained and immediately processed for RNA- isolation.
  • Plasma insulin measurements were measured from a drop of blood taken from the tip of the tail of fasted (8 hr) mice using a Lifescan Genuine One Touch glucometer. All measurements occurred between 2:00 pm and 5:00 pm. Plasma insulin measurements. . Blood was collected from the tail of, fasted (8 hr) mice into a heparinized capillary tube and stored on ice. All collections occurred between 2:00 pm and 5:00 pm. Plasma was separated from red blood cells by centrifugation for 10 minutes at 8000 x g and then stored at -20'C. Insulin concentrations were determined using the Rat Insulin ELISA kit and rat insulin standards (ALPCO) .essentially as instructed by the manufacturer. Values were adjusted by a factor of 1.23 as determined by the manufacturer to correct for the species difference in cross-reactivity with the antibody.- ' . '
  • RNA isolation Total RNA was isolated from muscle (skeletal muscle , specifically, gastrocnemius) of two mice at each time point ' during the progression of HF diet-induced type 2 diabetes, , as well as age-matched controls on the/Std, diet, /using the ? RNA" STAT-60 Total RNA/mRNA Isolation Reagent .according to : the manufacturer's, instructions (Tel-Test, Friendswood, TX) . Sample Quantification and Quality Assessment Total RNA was quantified and assessed for quality on a Bioanalyzer ?RNA 6000 Nano chip (Agilent) . Each chip contained an interconnected set o f gel-filled channels that allowed for molecular sieving of xiucleic acids.
  • Pin- electrodes in the chip were used to create electrokinetic forces capable of driving molecules through these micro- channels to perform electrophoret c 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 7 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) + UJMA (within the total RNA • population) is primed for reverse transcription by a DNA oligonucleotide containing a T7 RKFA polymerase promoter 5' to a (dT) 24 sequence.
  • the cDNA serves as the template in an in vi tro transcription (IVT) reaction to produce the target cRNA.
  • IVT in vi tro transcription
  • the IVT is performed in the presence of bioti nylated nucleotides to label the target c?NA. This procedure results in. a 50-200 fold linear amplification of the imput poly (A) + RNA.
  • Hybridization Probes The oligonucleotide probes we e provided by the Codelink Uniset Mouse I Bioarray (Amersham,- product code 300013) . Amine-terminated oligonucleotide probes are attached to a three-dimensional' poHyacrylamide gel matrix. There - are 10,000 oligonucleotide pirobes, ' each specific to-a well-characterized mouse gene.' Eac?h 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.
  • 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 f rth in the Motorola instructions. Hybridization to the microarray is detected with an avidinated fluorescent reagent, Streptavidin-Alexa Fluor ® 647 (Amersham) .
  • mice Normal mice compared to hyperinsulinemic mice at 2, 4, 8 and 16 weeks on normal vs. high -fat diet.
  • Hyperinsulinemic compared to hyperinsulinemic/hyperglycemic mice at 2 , 4, 8 and.16 weeks on high-fat diets.
  • 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 sequences were displayed using ABI prism Edit View 1.0.1 (PE Applied Biosystems, Foster City, CA) . Nucleotide database searches were conducted with the then, current version of BIASTN 2.0.12, see Alts ' chul, et al . , "Gapped BIAST 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.
  • 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 http: //www.ncbi .nlm.nih.gov/LocusLink/refseq.html It will be appreciated by those in the art that the exact results of a database search will change from day to day, as new sequences are added. Also, if you query with a longer version of the original sequence, the results will change. The results given here were obtained at one time and no guarantee is made that the exact same hits would be obtained in a • search on the filing date. However, if an alignment between a particular query sequence and a particular database sequence is discussed, that alignment should not change (if the parameters and sequences rremain unchanged) . '
  • 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 hybrid!zedL 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 digoxigenin (Roche Molecular, Biochemicals, Indianapolis , IN) , according to the manufacturer' s instructions .
  • 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 (control, vs. hyperinsulinemic, hyperinsulinemic vs. diabetic, or control vs. diabetic) wi!Ll be evaluated. Confirmation using several independent animals is desirable.
  • si tu hybridizations on selected human (obtained by Tissue Informatics) and mouse tissues using cRNA probes generated from mouse genes found to be up- or down-regulated during the disease progression.
  • si tu hybridizations may also be performed on mouse tissues using cRNA probes generated from differentially eixpressed 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 .
  • tissue sections can also be analyzed using Tissuelnformatics .
  • TissueAnalyticsTM software A single represe tative 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 ana ⁇ ysis may be performed on the resulting images: First a digital image analysis can identify and annotate structural o jects in a tissue using machine vision. These objects, which 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 hyper- ' quantitative 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' etween gene expression and hyper-quantitative tissue data. may be linear or non-linear, in synchronous or asynchronous arrangements .
  • Example 1 Obesity is increasing at an alarming ' rate in the United States. In parallel, the incidence of type II diabetes is also rising. We are interested in defining alterations in gene expression that correlate with the development of these conditions in the hopes of reversing these dangerous trends . Insulin plays a major role in regulating blood glucose levels. It stimulates the uptake of glucose in adipose tissue and striated muscle for storage as intracellular triglycerides and glycogen. Insulin also inhibits the release of glucose from the liver. Normally, this wo ⁇ ld prevent the rise in blood sugar concentration that occurs after eating. However, in the early stages of type 2 diabetes, resistance to insulin is seen. Muscle plays a major role in glucose metabolism.
  • type 2 diabetes In normal situations, muscle cells respond to increasing levels of insulin by increasing glucose uptake from the bloodstream. However, during the very early stages of type 2 diabetes, muscle tissue becomes resistant to insulin, requiring the pancreatic beta cells to increase insulin secretion. Eventually, the beta cells become unable to compensate for this increasing insulin resistance from muscle and other cells, and insulin production drops. 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' (Petersen and Shulman, 2002) .
  • microarray analysis In order to identify additional muscle genes involved in the development of type 2 diabetes, we used microarray analysis to compare RNA expression levels of 10,000 genes in muscle of high fat diet fed and control diet fed mice at various time points, in .the progression of type 2 diabetes. Microarray analysis provides a more global picture, of gene regulation, allowing the identification of families or groups of genes showing similar expression patterns that potentially imply similar or coordinated roles in disease progression.. , , Consumption of the HF diet resulted in significant, progressive increases in body weight- and fasting insulin levels in comparison to consumption of the 'Std diet. Fasting glucose levels of mice on the HF diet were dramatically increased at the first time point assayed (2 weeks) and remained high through the duration of ⁇ the experiment (16 weeks) ., .
  • Actin, alpha, cardiac (Actcl, ?NM_009608) was one of the most down-regulated genes when comparing HF to Std mice. It was consistently expressed at lower levels in the HF. diabetic mice in comparison to the Std mice and also steadily decreased over the 16 week study.
  • the master tables reflect applicants 7 analysis of the gene chip data.
  • Col. 1 The mouse gene (upper) and mouse protein (lower) database accession #s .
  • Col. 2 The corresponding mouse Unigene Cluster, as of the 4 th Quarter 2001 build.
  • Col. 4 A related human protein, identified by its database accession number. Usually, several such proteins are identified relative to each mouse gene. These proteins have been identified by BLAST searches, as explained in cols. 6-
  • Col. 5 The name of the related human protein.
  • Col. 6 The score (in bits) for the alignment performed by the BIAST program.
  • Col. 7 The E-value for the alignment performed by the BLAST program., It is worth noting that Unigene considers a Blastx E Value of less than le-6 to be a "match" to the reference sequence of a cluster.
  • bit score and E-value ' for the alignment is with respect to the alignment of the mouse DNA of col . 1 to the human protein of col . 4 by BlastX, according to the .default parameters.
  • Master Table, 1 is divided into three subtables on the basis of the behavior in col. 3. If a gene ' has at least ' one significantly favorable behavior, and no significantly unfavorable ones, it is put into Subtable 1A. In the opposite case, it is put into Subtable IB. If its ' behavior is mixed, i.e., at least one significantly favorable and at least one significantly unfavorable, it is put into Subtable IC. Note that this classification is based - on the strongest observed differential expression behaviors for each of the three subject comparisons, C-HI, HI-D and C-D.
  • Unigene record link Additional information of interest may be accessed by searching with the mouse gene accession # in the Mouse Gene
  • Subtable 1 A Wholly Favorable Genes and Proteins
  • M12866 F (C-D) AAA37164.1 Mm.214950 -1.69 NP_001091.1 alpha 1 actin precursor; alpha skeletal muscle actin 765 NP_005150.1 cardiac muscle alpha actin proprotein; smooth muscle actin 759 NP 001604.1 alpha 2 actin; alpha-cardiac actin 753
  • Actin-related protein 2 3582e- AAP37280.1 actin alpha 1 skeletal muscle protein 3327e- XP_208204.1 similar to actin-related protein 2 331 2e- XP_377904.1 similar to cytoplasmic beta-actin 3234e- AAH36253.1 ACTR2 protein 321 2e- AAHl 0417.2 ACTG1 protein 321 2e- NP_006678.1 actin-like 7A; actin-like 7-alpha 321 2e NP 06677.1 actin-like 7B; act ⁇ n-like 31036- AAH09544.1- Unknown (protein for IMAGE:3897065) 3105e- NP_848620.1 actin-like -. - ⁇ ' ⁇ • • ' . 3003e AAP20052.1 HSD21 ... . 2999e- . .- • ; "" " XP_377631.1 similar to beta actin " 2999e-
  • beta actin beta actin 724 0
  • XP_293924.1 similar to RIKEN cDNA 4732495G21 gene 689 0
  • XP_377904.1 similar to cytoplasmic beta-actin 3252e-88 AAP37280.1 actin alpha 1 skeletal muscle protein 3236e-88 AAH10417.2 ACTG1 protein 3238e-88 - AAH3G253.1 ACTR2 protein 318 1e-86 NP_006677.1 actin-like 7B; actin-like 7-beta 3169e-86 .
  • AAH09544.1 Unknown (protein for IMAGE:3897065) 311 2e-84 BAB71690.1 unnamed protein product 3036e-82 NP_848620.1 actin-like 3038e-82 AAP20052.1 HSD21 301 2e-81
  • NP_003118.1 spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) 259 2e-68 plectin 1 isoform 1; hemidesmosomal protein 1; epidermolysis bullosa simplex 1
  • PLE1_HUMA hi Plectin 1 (PLTN) (PGN) (Hemidesmosomal protein 1) (HD1) 241 4e « 63
  • BPA Hemidesmosomal plaque protein
  • NP_899236.1 230/240kD
  • dystonin hemidesmosomal plaque protein 231 4e-60
  • actin-binding LIM protein 1 isoform m; LIM actin-binding protein 1; limatin; 111 NP_006710.2 actin-binding LIM protein 3 actin-binding LIM protein 1 isoform s; LIM actin-binding protein 1; limatin; NP_006711.2 actin-binding LIM protein 756 BAA74866.2.
  • NM_016860 F (C-D) protein 1 , yeast) homolog A (centractin alpha); centractin alpha; actin-RPV;
  • beta-actin (beta'-actin) 423 e-118
  • XP_293924.1 similar to RIKEN cDNA 4732495G21 gene 417 e-116
  • XP_292982.4 similar to pote protein; Expressed in prostate, ovary, testis, and placenta 404 e-112
  • NP_536356.3 actin-related protein M2; actin-related protein hArpM2; actin-related protein T2 309 1e-83
  • XP_208204.1 similar to actin-related protein 2 296 1e-79
  • coronin actin binding protein, 2A; coronin, actin-binding protein, 2A; coronin 2A; NP_438171.1 coronin-like protein B; WD-repeat protein 2; WD protein IR10 408 e-113 coronin, actin binding protein, 2A; coronin, actin-binding protein, 2A; coronin 2A; NP_003380.2.
  • coronin-iike protein B coronin-iike protein B; WD-repeat protein2; WD protein IR10 408 e-113 AAB47807.1 - WD protein IR10 404 ⁇ 3-112 T47174 hypothetical protein DKFZp762l 166.1 - human (fragment) 389 3-107 AAS48630.1 unknown 3147e-85 NP_116243.1 hypothetical protein FLJ 14871 311 5e-84 . . . AAQ04659.1 Unknown 311 6e-84 NP_078811.1 hypothetical protein FLJ22021 234 ⁇ ⁇ -61
  • AA118546 F (C-D) ARP3 actin-related protein 3 homolog; ARP3 (actin-related protein 3, yeast)
  • NP_005712.1 homolog 850 0 actin-related protein 3-beta; actin-related protein 3-beta; actin-related protein NP_065178.1 Arp11 ; actin-related protein Arp11 793 0 AAP97150.1 actin related protein 662 0 AAH15207.1 ARP3BETA protein 597 e-170 XP_374583.1 similar to actin-related protein Arp11 348 3e-95 JC7580 actin-related protein Arp11 - human _ 344 4e-94 AAK31778.1 FKSG74 . 253 8e-67 AAK31776.1 FKSG72 .
  • beta-actin (beta'-actin) 247 6e-65 AAH08633.1 actin, beta . . . ... 247 8e-65 NP_005150.1 cardiac muscle alpha actin proprotein; smooth muscle actin 247 8e-65 XP_293924.1 similar to RIKEN cDNA 4732495G21 gene " 246 1e-64 ATHUS actin alpha 2, aortic smooth muscle - human 246 1e-64 NP_001604.1 . alpha 2 actin; alpha-cardiac actin .
  • NP_001606.1 actin gamma 2 propeptide
  • actin alpha-3 245 3e-64
  • ARP1 actin-related protein 1 homolog B centractin beta; centractin beta; ARP1 (actin-related protein 1 , yeast) homolog B (centractin beta); PC3; ARP1 , yeast NP 005726.1 homolog B 236 1e-61
  • NP 14084.1 Mm.2l772 -1.21 NP_003068.2 complex 60 kDa subunit B 828 AAC50696.1 SWI/SNF complex 60 KDa subunit 745 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin d3; Rsc ⁇ p; mammalian chromatin remodeling complex BRG1 -associated factor 60C; Swp73-like protein; chromatin remodeling complex BAF60C subunit; SWI/SNF NP_003069.2 complex 60 kDa subunit C 622 e-178 AAR88510.1 60kDa BRG-1/Brm associated factor subunit c isoform 2 619 e-177 AAC50697.1 SWi/SNF complex 60 KDa subunit 596 e-170 AAH09368.2 SMARCD1 protein 569 e-168
  • SWI/SNF-related matrix-assoGiated actin-dependent regulator of chromatin d1 isoform a Rsc6p; mammalian chromatin remodeling complex BRG1 -associated factor 60A; chromatin remodeling complex BAF60A subunit; Swp73-like protein; NP_003067.2 SWI/SNF complex 60 kDa subunit A " 589 e-168 AAD23390.1 SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin D1 582 e-165 SWI/SNF-related matrix-associated actin-dependent regulator of chromatin d1 - isoform b; Rsc ⁇ p; mammalian chromatin remodeling complex BRG1 -associated factor 60A; chromatin remodeling complex BAF60A subunit; Swp73-Iike protein; NP ⁇ .620710.1 SWI/SNF complex 60 kDa subunit A 505 e-142 AAC50695.1 SWI/SNF complex 60 KDa
  • Actin-reiated protein 2/3 complex subunit 1 A (SOP2-like protein) 723 0 actin related protein 2/3 complex subunit 1 B; ARP2/3 protein complex subunit NP_005711.1 p41; actin related protein 2/3 complex, subunit 1A (41 kD) 533 e-151
  • NM 011418 - F (C-D) subfamily b, member 1; sucrose nonfermenting, yeast, homolog-like 1; integrase
  • NP 035548.1 Mm.279751 -1.14 NP_003064.2 interactor 1 754 0 SNF5_HUMA SWI/SNF related, matrix associated, actin dependent regulator of chromatin N subfamily B member 1 (Integrase interactor 1 protein) (hSNF5) (BAF47) 749 0 CAA09759.1 Inilb 728 0 BAB14784.1 . unnamed protein product . 710 0 CAA76639.1 SNF5/INI1 protein 685 0
  • Subtable IB Wholly Unfavorable Genes and Proteins
  • Subtable IC Mixed Genes and Proteins
  • Ci tation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents is considered material to the patentability of any of the claims of the present application . All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents . The appended claims are to be treated as a non-limi ting recitation of preferred embodiments.
  • references ci ted herein including journal articles or abstracts, published, corresponding, prior or otherwise related U. S. or foreign patent applications, issued U. S . or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and ex presented in the ci ted references . Additionally, the entire contents of the references ci ted wi thin the references ci ted herein are also entirely incorporated by reference . Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art .

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Abstract

L'invention concerne l'identification de gènes murins exprimés de façon différentielle dans la comparaison d'un muscle de patient normal à un muscle de patient souffrant d'hyperinsulinémie, d'un muscle de patient souffrant d'hyperinsulinémie à un muscle de patient souffrant de diabète de type 2, et d'un muscle de patient normal à un muscle de patient souffrant de diabète de type 2 par analyse à puce à ADN, ainsi que les gènes humains et protéines humaines correspondants. Les molécules humaines, ou les antagonistes de celles-ci, peuvent être utilisés dans la protection contre l'hyperinsulinémie ou le diabète de type 2, ou leurs séquelles.
PCT/US2005/005596 2004-02-26 2005-02-24 Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de façon differentielle dans les cellules musculaires WO2005082398A2 (fr)

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CA002557181A CA2557181A1 (fr) 2004-02-26 2005-02-24 Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de facon differentielle dans les cellules musculaires
AU2005216922A AU2005216922A1 (en) 2004-02-26 2005-02-24 Diagnosis of hyperinsulinemia and type II diabetes and protection against same based on genes differentially expressed in muscle cells
EP05713932A EP1732582A2 (fr) 2004-02-26 2005-02-24 Diagnostic d'hyperinsulinemie et du diabete de type ii et protection contre lesdits etats pathologique grace aux genes exprimes de fa on differentielle dans les cellules musculaires

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WO2010096875A1 (fr) * 2009-02-27 2010-09-02 Verva Pharmaceuticals Ltd Protocole d'identification de médicaments pour le diabète de type 2 basé sur des signatures d'expression génique
EP2293074A3 (fr) * 2005-06-17 2011-05-04 Randox Laboratories Ltd. Procédé pour le diagnostic de maladies neuro-dégénératives
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US9512232B2 (en) 2012-05-09 2016-12-06 Ganymed Pharmaceuticals Ag Antibodies against Claudin 18.2 useful in cancer diagnosis
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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10414824B2 (en) 2002-11-22 2019-09-17 Ganymed Pharmaceuticals Ag Genetic products differentially expressed in tumors and the use thereof
US9775785B2 (en) 2004-05-18 2017-10-03 Ganymed Pharmaceuticals Ag Antibody to genetic products differentially expressed in tumors and the use thereof
EP1824518A2 (fr) * 2004-12-07 2007-08-29 Ohio University Diagnostic de l'hyperinsulinemie et du diabete de type ii et protection contre ceux-ci sur la base de proteines exprimees differemment dans le serum
EP1824518A4 (fr) * 2004-12-07 2009-10-28 Univ Ohio Diagnostic de l'hyperinsulinemie et du diabete de type ii et protection contre ceux-ci sur la base de proteines exprimees differemment dans le serum
EP2293074A3 (fr) * 2005-06-17 2011-05-04 Randox Laboratories Ltd. Procédé pour le diagnostic de maladies neuro-dégénératives
EP2293075A3 (fr) * 2005-06-17 2011-05-04 Randox Laboratories Ltd. Procédé pour le diagnostic de maladies neuro-dégénératives
US9751934B2 (en) 2005-11-24 2017-09-05 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US9499609B2 (en) 2005-11-24 2016-11-22 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US9212228B2 (en) 2005-11-24 2015-12-15 Ganymed Pharmaceuticals Ag Monoclonal antibodies against claudin-18 for treatment of cancer
US10017564B2 (en) 2005-11-24 2018-07-10 Ganymed Pharmaceuticals Gmbh Monoclonal antibodies against claudin-18 for treatment of cancer
US10174104B2 (en) 2005-11-24 2019-01-08 Ganymed Pharmaceuticals Gmbh Monoclonal antibodies against claudin-18 for treatment of cancer
US10738108B2 (en) 2005-11-24 2020-08-11 Astellas Pharma Inc. Monoclonal antibodies against claudin-18 for treatment of cancer
US11739139B2 (en) 2005-11-24 2023-08-29 Astellas Pharma Inc. Monoclonal antibodies against Claudin-18 for treatment of cancer
WO2010096875A1 (fr) * 2009-02-27 2010-09-02 Verva Pharmaceuticals Ltd Protocole d'identification de médicaments pour le diabète de type 2 basé sur des signatures d'expression génique
US9512232B2 (en) 2012-05-09 2016-12-06 Ganymed Pharmaceuticals Ag Antibodies against Claudin 18.2 useful in cancer diagnosis
US10053512B2 (en) 2012-05-09 2018-08-21 Ganymed Pharmaceuticals Ag Antibodies against claudin 18.2 useful in cancer diagnosis
US11976130B2 (en) 2012-05-09 2024-05-07 Astellas Pharma Inc. Antibodies against claudin 18.2 useful in cancer diagnosis

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