WO2001018549A1 - Therapies et reactifs augmentant la resistance au stress et la longevite - Google Patents

Therapies et reactifs augmentant la resistance au stress et la longevite Download PDF

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WO2001018549A1
WO2001018549A1 PCT/US2000/024487 US0024487W WO0118549A1 WO 2001018549 A1 WO2001018549 A1 WO 2001018549A1 US 0024487 W US0024487 W US 0024487W WO 0118549 A1 WO0118549 A1 WO 0118549A1
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daf
activity
homolog
improved
agent
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Samuel T. Henderson
Thomas E. Johnson
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Neurogenetics, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • AD Alzheimer's Disease
  • cancer cancer
  • diabetes diabetes
  • Parkinson's Disease is a progressive neurodegenerative disorder that primarily affects the elderly.
  • AD is the most common form of dementia in persons over the age of 65, with an estimated 7-10% of the population over 65 years old affected, and up to 40% of the population over 80 (McKhann, G. et al. (1984) Neurology 34:939-44; and Evans, D. et al. (1989) JAMA 262:2551-6).
  • elegans hermaphrodites continue through four molts to become fertile adults that typically live 3 weeks at 20°C. Under conditions of low food and high population density, a decision is made at the second larval stage to suspend development and enter the dauer stage.
  • the dauer stage is a specialized dispersal form that can live many months (five to eight times longer than non-dauers).
  • Dauer larvae upregulate several stress-resistance enzymes and other stress proteins, such as superoxide dismutase (Anderson, G. (1982) Can. J. Zool. 60:288-291; and Larsen, P. (1993) PNAS 90:8905-9), catalase (Vanfleteren, J. et al. (1995) FASEB J.
  • dauer-defective mutations are those that disrupt the ability of the animal to sense its environment, and hence interfere with the ability to respond to crowded conditions.
  • Daf-d mutations also often show defects in chemotaxis or osmotic avoidance, and often have abnormal morphogenesis of sensory neurons. Since a constant signal of abundant food and lack of overcrowding are required to inhibit dauer formation, mutations in the dauer pathway frequently result in a constitutive dauer phenotype. Daf-c mutants will enter the dauer pathway even when conditions are not appropriate, and include daf-1, daf-2, daf-4, daf-7, daf-8, daf-11, daf-14, daf-21 and age-1. All the mutant alleles of daf-1, daf-4, daf-
  • daf-2 and age-1 are temperature sensitive (ts), suggesting that disruption of this arm of the pathway is inherently ts.
  • daf-2 and age-1 are associated with another arm of the pathway, and strong loss-of-function mutations in these genes are either lethal or non-conditional dauer constitutive (Riddle et al. (1997), supra).
  • the extension of life span is not simply inappropriate activation of the dauer pathway. Shifting conditional mutations in most Daf-c genes to restrictive temperature late in life has no effect on life span.
  • mutations in daf-2 and age-1 will increase life span at temperatures that normally would not induce dauer formation (Kenyon, C. et al. (1993) Nature 366:461-4; and Larsen, P.
  • daf-2 activities of daf-2 are not limited to the dauer decision.
  • the strong dqf-2(ts) allele (e979) is 100% embryonic lethal in homozygous animals at the restrictive temperature, implicating a role for daf-2 during early development (Gems, D. et al. (1998) Genetics 150:129-55).
  • daf-2(ts) alleles are shifted to restrictive temperature as adults, the animals adopt a series of characteristic phenotypes.
  • the shifted animals become egg-laying defective, have reduced movement and exhibit body shrinkage, all of which suggest a role for daf-2 in adult nematodes (Gems et al. (1998), supra), age-1 (hx546) mutants have essentially normal growth and development (Friedman, D. et al. (1988) J. Gerontol. 43 :B 102-9) but show a modest effect on fertility (Lithgow, G. et al. (1995) PNAS 92:7540-4).
  • the requirement for daf-2 throughout nematode life suggests that this arm of the dauer pathway functions to promote growth and regulate metabolism throughout the life span, while other arms of the pathway are specific for entry into the dauer stage.
  • daf-2 and age-1 as metabolic regulators are consistent with their molecular identity, age-1 is predicted to encode a phosphatidyl-inositol 3 kinase (PI3K), similar to the pi lOalpha that plays a role in mammalian insulin signaling (Morris, J. et al. (1996) Nature 382:536-9). daf-2 encodes an insulin-like growth factor receptor (Kimura,
  • AKT-1 is homologous to mammalian AKT/PKB (Protein Kinase B)
  • akt-2 was identified in the C elegans genomic sequence by homology to akt-1, and they seem to be at least partially redundant. Inhibition of either akt-1 or akt-2 alone is not sufficient to induce a Daf-c phenotype, while inhibition of both genes results in a constitutive dauer formation (Paradis, S. et al. (1998), supra). This redundancy may explain why these genes were not identified in previous dauer screens.
  • AKT-1/2 may be the major outputs of PI3K (AGE-1) signaling in worms, while DAF-2 is likely to have other outputs (Paradis, S. et al.
  • daf-16 encodes a putative transcription factor with a forkhead, or winged helix, DNA binding domain (Lin, K. et al. (1997) Science 278:1319-22; and Ogg,
  • FKHR had previously been identified as playing a role in alveolar rhabdomyosarcoma, an aggressive pediatric soft tissue tumor. Analysis of tumors detected a translocation that fuses the DNA binding domain of PAX3 or PAX7 to the carboxy-terminus of FKHR (Davis, R. et al. (1994) Cancer Res. 54:2869-72; and
  • FKHRL 1 was identified by homology to FKHR and AFX (Anderson, M. et al. (1998) Genomics 47:187-99).
  • FKHR FKHRLI
  • AFX AFX to DAF-16
  • the endogenous proteins may function in insulin signaling in mammals in a conserved pathway similar to the one identified in C. elegans.
  • Biochemical studies on the mammalian homologues of DAF-16 suggest how growth factor signaling regulates the activity of this family of transcription factors.
  • growth factor signaling activates the serine threonine kinase AKT, which leads to the phosphorylation of three sites on FKHRLI.
  • Phosphorylation of FKHRLI inhibits its entry into the nucleus preventing it from activating transcription of target genes (Brunet, A. et al. (1999) Cell
  • DAF-16 homologs would lead to detrimental effects on cells.
  • the Ruvkun et al. patent (WO 98/51351) describes methods and reagents for lowering the level of DAF-16 homolog activity by either up-regulating the activity of the proteins that negatively regulate DAF-16, or down-regulating DAF-16 itself.
  • the present invention unexpectedly reveals novel beneficial functions of DAF-16.
  • the present invention reveals for the first time that increased dosage of daf-16 greatly increases the resistance to heat.
  • the present invention shows for the first time that increased dosage of daf-16 increases resistance to ultraviolet light.
  • the present invention shows that daf-16 will potentiate the increase in life span induced by a mild heat shock.
  • the present invention demonstrates for the first time that increased activity of daf- 16 leads to increased protein levels of some members of the stress response pathway.
  • the present invention demonstrates for the first time that increased daf-16 dosage will increase resistance to oxidative stress. These cytoprotective effects are not accompanied by phenotypes resulting from defects in the insulin-signaling pathway, e.g., constitutive dauer formation is not observed.
  • the present invention provides for the first time evidence that increasing the activity of DAF-16 homologs may have beneficial therapeutic effects in mammals (e.g., humans) without accompanying detrimental effects on insulin-dependent signaling.
  • the present invention includes methods and reagents that increase the dosage of DAF-16 homologs in human cells.
  • the invention provides isolated DNA encoding DAF-16 or DAF-16 homologs in a vector that is capable of directing the expression of DAF-16 or DAF-16 homologs in human cells.
  • the isolated DNA in such a vector is operatively linked to a promoter that can direct the expression of the polypeptide either in all tissues in the body, or within certain subsets of tissues, or within single cell types.
  • the invention also includes methods for identifying agents that increase the activity of DAF-16 homologs in mammalian (e.g., humans) cells.
  • the invention further includes methods for administering such reagents to humans.
  • the methods of identifying agents that enhance the activity of DAF-16 or a DAF- 16 human homolog have a number of embodiments.
  • the method comprises (a) contacting DAF-16 or a DAF-16 human homolog with candidate agents under assay conditions for DAF-16 activity or DAF-16 human homolog activity; and (b) identifying those agents that enhance the activity of the DAF-16 or DAF-16 human homolog.
  • the method comprises (a) contacting daf-16 or a daf-16 human homolog with candidate agents under conditions permitting expression; (b) conducting an assay for DAF-16 activity or DAF-16 human homolog activity; and (c) identifying those agents that enhance the activity of the DAF-16 or DAF-16 human homolog.
  • Figure 1 illustrates the C. elegans daf-16 dauer pathway.
  • Figure 2 demonstrates the stress resistance of ExR13H8 strains.
  • Figure 2a illustrates the fraction of C. elegans surviving for sterile (Str) and fertile (Fer) animals derived from two independent lines, ExR13H8(l) and ExR13H8(2) at 37.5 °C for 2 hrs.
  • Figure 2b illustrates the fraction of C. elegans surviving for age-1 (hx-546); ⁇ xpRF4 and a sterile ExR13H8 strain at 37.5 °C for 2.5 hrs.
  • Figure 2c illustrates that N2 (wild-type) nematodes with R13H8 extra-chromosomal arrays are more resistant to UV (P ⁇ 0.0002).
  • Figure 2d illustrates that sterile animals derived from age-1 (hx-546);ExR ⁇ 3HS strains are more resistant to UV (PO.0001).
  • Figure 2e illustrates that N2;ExR13H8(Str) animals are more resistant to peroxide treatment than N2;ExpRF4 or age-1 (hx-546);ExpRF4 (PO.0001).
  • Figure 3 demonstrates that heat treatment extends the life span of ExR13H8 strains. N2;ExpRF4 HS (heat shocked) and N2;ExR13H8 (1) HS curves are significantly different (P ⁇ 0.0001). Data shown is from one representative experiment, and has been replicated.
  • Figure 4 illustrates the expression of DAF::GFP in nematodes under various conditions.
  • Figure 4a is a DIC (differential interference contrast) image of ExDAF- 16::GFP strain.
  • Figure 4b (a fluorescence microscopy image, FMI) demonstrate that
  • DAF-16::GFP is widely expressed and largely cytoplasmic. Note the lack of expression in germ line (g) of inset.
  • Figure 4c is a DIC image of ExDAF-16GFP strain after one hour
  • Figure 4d (a FMI) illustrates localization of DAF-16::GFP to the nucleus after heat treatment (arrows).
  • Figure 5 illustrates the stress resistance of ExDAF-
  • FIG. 16 16::GFP strains.
  • Figure 5a shows that N2 nematodes with extrachromosomal arrays of DAF-16::GFP are more thermotolerant. Error bars represent standard error.
  • Figure 5b demonstrates that N2;ExDAF-16::GFP animals are more UV resistant than N2;ExpRF4.
  • Figure 6 illustrates HSP70 levels in aging wild-type C. elegans and daf- 16(mgDf50) animals.
  • Figure 6a is a Western blot of HSP70 levels from five adults of the indicated age.
  • Figure 6b is the optical density corresponding to each of the Figure 6a lanes.
  • Figure 6c illustrates the life span of wild-type and daf-16(mgDf 50) at 25 °C.
  • Figure 7 contains Western analyses for HSP70, HSP90 and ubiquitin in a number of strains.
  • Figure 7a shows unchanged HSP90 levels in wild-type (N2), age-1 (hx-546), N2;ExpRF4, and N2;ExR13H8.
  • Figure 7b shows that HSP70 levels are higher in sterile ExR13H8 animals.
  • Figure 7c shows ubiquitin levels in ExR13H8 strains. Arrows indicate prominent bands.
  • Figure 7d illustrates HSP70 levels in N2;ExpRF4 and N2;ExDAF- 16::GFP strains.
  • Figure 7e shows ubiquitin levels in N2;ExpRF4 and N2;ExDAF-16GFP strains.
  • Figure 8 illustrates routes of DAF-16 activation.
  • Figure 8a depicts insulin-like signaling that activates AKT- 1/2 which phosphorylates DAF-16 enhancing binding to a cytoplasmic retention factor, preventing activation of genes involved in increased stress resistance and life span.
  • Figure 8b illustrates low insulin signaling which results in dephosphorylation of DAF-16, loss of binding to cytoplasmic retention factor and nuclear localization.
  • Figure 8c shows a possible mechanism for heat activation of DAF-16-like molecules. This model is only one of several possible mechanisms for activation of DAF- 16-like proteins.
  • Figure 9 depicts the insulin signaling pathways of mammals.
  • Figure 10 illustrates a construct useful in identifying agents that up-regulate transcription of DAF-16 human homologs.
  • the subject invention comprises methods of identifying agents that can increase the activity of DAF-16 or mammalian (e.g., human) homologs thereof. These agents can be used therapeutically to enhance cytoprotective activities in cells. Discussed in detail herein are methods and assays for screening candidate molecules to identify those agents that can have such therapeutic effects or which can be the subject of further drug discovery methods known in the art to obtain compounds that have therapeutic value in mammals, in particular humans.
  • This invention is also directed to the agents obtained by the assays described therein, and to the therapeutic treatment of mammals (e.g., humans) with such agents to increase life span, improve late life vigor, enhance resistance to environmental stress, and otherwise protect cells from damage. It is also foreseen that the agents obtained using the subject assay methods may be useful in the induction of apoptosis of tumor cells.
  • the subject invention concerns a method for increasing the dosage of one or more DAF-16 homologs in mammalian (e.g., human) cells via gene therapy.
  • mammalian e.g., human
  • agent screening assays, agents and methods of treatment are based on extensive experiments conducted in C. elegans that reveal that activation of DAF-16 can result in cytoprotective effects without the adverse effects associated with the perturbation of insulin signaling pathways observed in some diabetics.
  • Candidadidate agents or “candidate molecules” means agents or molecules that can be tested in screening assays for suitability as agents to enhance DAF-16 or DAF-16 human homolog activity.
  • candidate agents are small molecules, peptides, oligonucleotides and/or derivatives thereof, or other compounds known to be useful as screening candidates in the drug discovery field.
  • Cytoprotective activity means activity that enhances a cell's resistance to environmental stress such as elevated temperature, oxidative stress or ultraviolet irradiation. Through such cytoprotective activity, a cell or organism may exhibit improved
  • the cytoprotective activity may be effected through activation of HSP70, HSP90, catalase and/or superoxide dismutase.
  • “Functional C. elegans DAF-16” refers to a C. elegans DAF-16 that, when activated, performs its wild-type function of inducing cytoprotective activity.
  • Human homolog of DAF-16 refers to the products of the human genes FKHR, FKHRL 1 , AFX or to a human polypeptide containing a region that has at least 50% identity with the winged helix domain of C. elegans DAF-16.
  • the winged helix domain of DAF-16 is the putative DNA binding region and is of the amino acid sequence: KKTTTRRNAWGNMSYAELITTAIMASPEKRLTLAQVYEWMVQNVPYFRDKGDS NSSAGWKNSIRHNLSLHSRFMRIQNEGAGKSSWWV ⁇ NPDAKPGR.
  • the human homolog has at least 60%, 70% 80%, 90% and 95% identity to this region.
  • the human homolog also has a function similar or analogous to that performed by DAF-16. Preferably retains at least 50%, 60%, 70%, 80%, 90%, or 95% of the desired DAF-16 activity.
  • Impaired insulin signaling or “perturbation in insulin signaling” in C. elegans refers to genetic or other alterations that lead to reduced activity in the insulin-like signaling pathway in nematodes. These alterations include, but are not limited to, inhibition of expression or activity of a set of genes defined as homologous to genes involved in insulin-like signaling in mammals. Examples of such genes are daf-2, age-1, pdk-1, akt-1, akt-2, daf-18, and daf-16. Impaired insulin signaling will result from null, loss of function or reduction of function alleles from these as well as other genes in this pathway.
  • Impaired insulin signaling or “perturbation in insulin signaling” in humans refers to alterations that lead to reduced activity of genes or gene products of the insulin signaling pathway in mammals. These alterations include genes and gene products that are generally recognized as being downstream of activation of Insulin receptor or IGF- 1 receptor.
  • Improved target protein activation or “enhanced target protein activation” means increased target protein gene transcription, translation and/or target protein activation.
  • DAF-16 or DAF-16 homologs refers to increased daf-16 or daf-16 human homolog transcription or translation, increased DAF- 16 or D AF- 16 homolog activation and/or increased target protein activation.
  • “Operatively linked” refers to the conjugation of a polypeptide encoding oligonucleotide to a regulatory region or leader containing binding sites or response elements in such a manner to permit the transcription and/or translation of the oligonucleotide. Regulatory or leader regions and methods for assuring proper linkage to oligonucleotides are known in the art.
  • Target protein of DAF-16 or a DAF-16 human homolog refers to any protein that
  • DAF-16 or DAF-16 homolog either binds to directly to activate or whose transcription or translation is activated by binding of DAF-16 or a DAF-16 homolog to the regulatory region of the gene or the mRNA encoding the protein.
  • Target proteins can include, but are not limited to, HSP70, HSP90, catalase, ubiquitin and/or superoxide dismutase.
  • the choice between a reproductive versus endurance life style affects the life span and overall health of the animal, and is regulated by the DAF-16 family of transcription factors.
  • C. elegans The choice of reproductive versus endurance life styles is very apparent in C. elegans.
  • C. elegans high food concentrations and low population density signals the reproductive life style, whereas low food availability and high population density triggers the dauer (endurance) stage.
  • This life style switch is found in many species.
  • Mediterranean fruit flies exhibit "dual modes of aging" characterized by a waiting mode and a reproductive mode.
  • the choice of mode is dictated, at least in part, by diet. Flies fed only sugar exist in a waiting mode, don't reproduce, and have increased life spans compared to animals fed a full diet (Carey, J. et al. (1998) Science 281 :996-8).
  • DAF-16 is a transcription factor that is activated by low levels of insulin signaling and is responsible for both the beneficial and harmful effects of reduced insulin signaling.
  • C. elegans daf-16 is required for the increased stress resistance and longevity phenotypes associated with reduced insulin signaling.
  • Increased dosage of daf-16 containing cosmid R13H8 dramatically increases resistance to heat and ultraviolet stress. Since the transgenes are maintained as extrachromosomal arrays, the animals are likely to be mosaic, and some animals are expected to be more affected then others. Because daf-2(lf) (loss of function) mutations exhibit a sterile phenotype, the inventors predicted that the sterile animals derived from ExR13H8 strains would be the most strongly affected. Consistent with this idea, it was found that the sterile animals were the most stress resistant.
  • thermotolerance has been found to be due to daf-16.
  • Microinjection of dsRNA specific to daf-16a coding region eliminates the increased thermotolerance of these animals. This experiment rules out the possibility that other genes on the cosmid contribute to the effect.
  • Another possibility is that the Itt (increased thermotolerance) phenotype is due to increased amount of genomic sequences on the transgene. For example, extra copies of promoter regions could titrate out a limiting transcription factor for other genes.
  • This model is unlikely, however, since RNAi is thought to inhibit activity of the target genes (see Tabara, H. et al. (1998) Science 282:430-1). Therefore, the increase in thermotolerance is almost certainly due to increased expression of daf-16. Since the effect of daf-16 RNAi on the germ line was weak, other models for this effect cannot be totally ruled out.
  • HSP70 HSP70 protein
  • levels of HSP70 protein were much higher in sterile ExR13H8 animals than in wild type. This is consistent with an increase in HSP70 protein levels seen in age-1 (hx546) versus wild type. Increased dosage of HSP70 has been reported to increase life span of Drosophila (Tatar, M. et al. (1997) Nature 390:30). No increase in HSP90 protein levels was seen, even though dauer larvae have large increases in HSP90 mRNA (Dalley, B. et al. (1992) Dev.
  • Ubiquitin is essential for the stress response in yeast (Finley, D. et al. (1987) Cell 48:1035-
  • HSP70 and ubiquitin levels may be due to increased levels of DAF-16 protein, however it is possible that excessive DAF-16 forms aggregates and thereby stimulates a stress response. Yet, given the role for daf-16 in stress resistance in long-lived nematodes and its central role in insulin signaling, it is likely that the gains in stress resistance are due to increased levels and activation of DAF-16.
  • FKHR a mammalian DAF-16 homolog
  • AKT phosphorylated by AKT
  • AKT phosphorylated by AKT
  • DAF-16 may be excluded from the nucleus by a similar mechanism.
  • a heat treatment may transiently denature the association of DAF-16 from its cytoplasmic partner and allow for nuclear localization.
  • heat treatment may deactivate AKT- 1/2 or activate a phosphatase, which changes the phosphorylation state of DAF-16.
  • DAF-16 may be activated by temperature as a direct method to modulate stress resistance and life span in nematodes based on environmental signals.
  • DAF-16 :GFP fusion protein increased stress resistance and life span (Figure 5).
  • the DAF-16 family of transcription factors may act to direct a set of responses to low food availability, and prepare the organism for stress. Inappropriate activation of DAF-16 in the presence of abundant food, as occurs in age-1 and daf-2 mutations appears to give rise to several beneficial effects. Genetic and biochemical data from invertebrates and vertebrates suggest how DAF-16 is activated. Insulin signaling leads to activation of the serine/threonine kinase AKT. AKT directly phosphorylates the DAF-16 homologue
  • Target genes of the DAF-16 family are likely to be the mediators of the endurance pathway. For example, withdrawal of growth factors increases apoptosis in cell culture, and this is believed to be important in tumor resistance in caloric restricted animals (Mukherjee et al. (1999), supra; and
  • DAF-16 may regulate the expression of sod-3 (a superoxide dismutase) (Furuyama, T. (2000) Biochem. 349:629-634).
  • sod-3 a superoxide dismutase
  • mice over expressing HSP70 incur less myocardial damage from ischemia-reperfusion (Marber, M. et al. (1995) J. Clin. Invest. 95:1446-56; and Plumier, J. et al. (1995) J. Clin. Invest. 95:1854-60).
  • Over-expression of antioxidant enzymes such as superoxide dismutase and catalase also have cytoprotective abilities and can extend life span of Drosophila (Orr, W. (1994) Science 263:1128-30; and
  • DAF-16 family of transcription factors appear to be "master regulators" of a wide variety of stress response enzymes. In this invention it has been shown that activation of DAF-16 is cytoprotective in C. elegans and protects the cells against both thermal, ultraviolet radiation and oxidative damage. The up- regulation or activation of DAF-16 molecules is likely to result in substantial medical benefits.
  • results described herein reveal for the first time that increased activity of DAF- 16 in nematode cells leads to an increase in cytoprotective activity. These results enable the development of a number of screening systems for identifying therapeutic compounds that increase the activity of DAF-16 homologs in human cells. Such compounds will be useful for increasing the cytoprotective abilities of cells in particular, and potentially for increasing individual longevity and late life vigor in general.
  • This section provides a description of several of the possible screens included within the scope of this invention. Many methods are known in the art of drug discovery for obtaining compounds that can be screened for a desirable activity.
  • the compounds can be obtained from natural sources — e.g., plant extracts, fungal extracts etc. — or they can be chemically synthesized.
  • the subject invention comprises a method of identifying an agent that enhances the activity of DAF-16 or a DAF-16 human homolog, comprising (a) contacting DAF-16 or a DAF-16 human homolog with candidate agents under assay conditions for DAF-16 activity or DAF-16 human homolog activity; and (b) identifying those agents that enhance the activity of the DAF-16 or DAF-16 human homolog.
  • a variation on this method comprises (a) contacting daf-16 or a daf-16 human homolog with candidate agents under conditions permitting expression; (b) conducting an assay for
  • the invention includes a method for identifying an agent that increases the activity of DAF-16 in C. elegans cells, whereby cytoprotective activity in the cells may be enhanced, comprising (a) contacting candidate molecules with a C.
  • elegans strain comprising a wild type daf-16 locus; (b) screening for animals that exhibit enhanced DAF-16 activity or cytoprotective activity; and (c) identifying the agent that enhanced the DAF- 16 activity or cytoprotective activity. Detailed methods for performing this screen are provided herein.
  • the invention provides a method for identifying an agent that increases the activity of a DAF-16 human homolog, whereby cytoprotective activity in host or human cells may be enhanced, comprising (a) contacting candidate molecules with a C. elegans strain comprising a daf-16 gene that is mutated or deleted whereby functional
  • C. elegans DAF-16 is not produced, and a daf-16 human homolog gene; (b) screening for animals that exhibit enhanced DAF-16 human homolog activity or cytoprotective activity; and (c) identifying the agent that enhanced the DAF-16 human homolog activity or cytoprotective activity.
  • the daf-16 human homolog can be present as a transgene on an extrachromosomal array.
  • the foregoing embodiment operates on the reasonable presumption that a human DAF-16 homolog can induce the dauer state in C. elegans or otherwise induce cytoprotective effects in C. elegans. It has been found that human FKHRLI can be phosphorylated and inactivated by C. elegans AKT-1/2.
  • the subject invention also includes a method for identifying an agent that increases the activity of DAF-16 in C. elegans cells, whereby cytoprotective activity in the cells may be enhanced, comprising (a) contacting candidate molecules with a C.
  • elegans strain having a gain-of-function daf-2, age-1 or akt-1/2 mutation, whereby DAF-16 is inactivated even in the absence of insulin-signaling; (b) screening for animals that exhibit enhanced DAF-16 activity or cytoprotective activity in the absence of insulin-signaling; and (c) identifying the agent that enhances the DAF-16 activity or cytoprotective activity.
  • insulin-signaling diminishes when there is insufficient food in the environment.
  • low insulin signaling prevents AGE-1 and DAF-2 from activating AKT-1/2 to phosphorylate and inactivate DAF-16.
  • gain-of-function mutations in age-1, daf-2 and/or akt-1/2 will result in consistently inactivated DAF-16, manifested by an inability to form dauers.
  • compounds are screened for their ability to promote the formation of dauer animals under starvation conditions.
  • a method for identifying an agent that increases the activity of a DAF-16 human homolog, whereby cytoprotective activity in host or human cells may be enhanced comprising (a) contacting candidate molecules with a C. elegans strain comprising a daf-16 genes that is mutated or deleted whereby functional C.
  • DAF-16 is not produced, a gain-of-function daf-2, age-1 or akt-1/2 mutated gene, whereby DAF-16 is inactivated even in the absence of insulin-signaling, and a daf-16 human homolog gene; (b) screening for animals that exhibit enhanced DAF- 16 homolog activity or cytoprotective activity under conditions of non-insulin signaling; and (c) identifying the agent that enhanced the DAF-16 homolog activity or cytoprotective activity.
  • the human daf-16 homolog gene is provided on an extrachromosomal array. The screen would be for agents that induce the dauer state under starvation conditions.
  • the assay methods described herein additionally ensure that there is no significant perturbation or impairment in the insulin-signaling pathway (as defined herein).
  • the purpose of the subject assays is to identify agents that act specifically on DAF-16 or human homologs thereof because these agents are very far downstream in the insulin-signaling cascade.
  • agents that activate DAF-16 or its homologs can confer beneficial effects described herein without the detrimental effects of a general reduction or inhibition of insulin signaling. In humans, such perturbation or inhibition of the insulin-signaling pathway can cause diabetes.
  • the subject invention also provides a method for identifying an agent that increases the localization of DAF-16 to the nucleus in C. elegans cells, whereby cytoprotective activity in the cells may be enhanced, comprising (a) contacting candidate molecules with a C. elegans strain comprising a DAF-16::marker fusion protein encoding construct; (b) screening for animals that exhibit localization of DAF-16::marker fusion protein in the nucleus; and (c) identifying the agent that caused localization of the DAF- 16: :marker protein to the nucleus.
  • the marker can be any marker known to persons skilled in the art, including green fluorescent protein (GFP).
  • the subject invention also provides a method for identifying an agent that increases the expression of DAF-16 in host cells, comprising (a) contacting candidate molecules with host cells comprising a construct having a reporter gene under control of a regulatory region of daf-16 or of a human homolog of daf-16; (b) screening for host cells that exhibit increased expression of the reporter; and (c) identifying the agent that caused the increased expression of the reporter.
  • the reporter gene can be any suitable reporter gene known to skilled artisans including those encoding GFP, ⁇ -galactosidase and luciferase.
  • the host cells may be C. elegans cells or cultured mammalian cells that are insulin-responsive.
  • One method for getting the reporter gene constructs into nematodes is by injecting the construct into the germ line of adults, along with a selectable marker (such as the pRF4 plasmid which contains a dominant rol mutation), and then harvesting progeny that express the marker.
  • This method makes it possible to screen for candidate molecules that cause an increase in the expression of the reporter gene and which cause an increase in the cytoprotective activities of DAF-16.
  • the reporter construct when the host cell is human, the reporter construct would comprise the reporter gene operatively linked to a regulatory region binding site for human daf-16 homolog.
  • Candidate molecules would be screened to identify agents that increase the expression of the reporter gene without causing defects in the cell that result from perturbation of the insulin signaling pathway.
  • DAF-16 activation DAF-16 activation.
  • Other assays include those for activation of Insulin Receptor (IR), PI3K and AKT. Suitable assays for activation of these upstream enzymes are known in the art.
  • the subject invention provides an in vitro method of identifying an agent that inhibits AKT-1/2 interaction with DAF-16, comprising (a) contacting candidate molecules with AKT-1/2 or DAF-16; (b) adding to the mixture of step (a), DAF-16 or AKT-1/2 respectively; and (c) identifying those agents that inhibit the interaction of AKT-1/2 and DAF-16.
  • the AKT and DAF-16 or homolog proteins may be purified or recombinant. Inhibition of the interaction between the AKT proteins and the DAF-16 or homolog proteins can be detected by using standard techniques for studying the interaction of proteins, including immunoprecipitation.
  • the subject invention also provides an in vitro method of identifying an agent that inhibits the phosphorylation of DAF-16 by AKT-1/2, comprising (a) contacting candidate molecules with AKT-1/2 or DAF-16; (b) adding to the mixture of step (a), DAF-16 or
  • AKT-2 AKT-2, respectively; and (c) identifying those agents that inhibit the phosphorylation of DAF-16 by AKT-1/2.
  • the phosphorylation of DAF-16 or its homologs can be studied using standard techniques, such as the antibodies to phosphoepitopes.
  • an in vitro method for identifying an agent that promotes the ability of DAF-16 or DAF-16 human homologs to bind to a binding site of a regulatory region of a gene encoding a target protein, comprising (a) contacting candidate molecules with a mixture of DAF- 16 or a D AF- 16 human homolog and a construct comprising a reporter gene operatively linked to the DAF-16 or human homolog gene regulatory region binding site under conditions that permit in vitro expression; (b) detecting reporter gene expression; and (c) identifying those agents that promote DAF-16 or DAF-16 human homolog binding to the mRNA binding site. Reporter gene expression can be detected by assaying for mRNA or reporter protein produced.
  • pools of candidate molecules can be assayed for the desired activity.
  • Each pool contains many different types of compounds.
  • that pool may be fractionated or subdivided into smaller pools, and the new pools may be assayed in the same way as the parent. This cycle can be repeated until the compound with the desired activity is identified.
  • the desired activity may be associated with more than one candidate molecule.
  • some of the agents of the present invention may comprise two or more compounds that function coordinately to increase DAF-16 activity.
  • DAF-16 will cause the coordinate activation of DAF-16.
  • Other compounds may upregulate the expression of the daf-16 gene, leading to increased amounts of DAF-16 protein.
  • Still other compounds may modify endogenous DAF-16 in such a way that it becomes refractory to the inhibitory activity of other gene products.
  • the compound may modify DAF-16 in such a way that it is no longer a substrate for the AKT proteins (akt-1 and akt-2 gene products).
  • the AKT proteins are partially-redundant serine- threonine kinases that phosphorylate DAF- 16, leading to the inactivation of DAF- 16 through its translocation to the cytoplasm.
  • Other compounds may inhibit the 14-3-3 DAF- 16 interaction.
  • the compounds of interest are those that increase DAF-16 activity ⁇ either by increasing expression of the DAF-16 gene or increasing activity of existing DAF-16 — without perturbing other aspects of the insulin signaling pathway.
  • preferred compounds are those that increase the stress resistance of nematodes without giving rise to the dauer-constitutive phenotypes associated with loss-of-function mutations in the genes upstream of daf-16. These compounds can also be identified through the use of Northern and Western analysis of the daf-16 gene.
  • compounds that increase the expression of daf-16 can be identified by showing an increase in daf-16 mRNA on a Northern blot or DAF-16 protein on a Western blot, without development of a dauer state.
  • activation of DAF-16 human homologs without general inhibition of the insulin-signaling pathway can be confirmed by detecting increases in transcription of daf-16 homolog genes or reporter constructs that contain the daf-16 Insulin Response Element (IRE), without inhibition of activation of IR, PI3K or AKT, inhibition of glucose uptake, or inhibition of translocation of GLUT4 to the cell surface (as is discussed above).
  • IRE Insulin Response Element
  • compositions identified by the methods of the instant invention have a variety of uses as therapeutic agents.
  • the compounds exert their beneficial effects by increasing the activity of DAF-16 homologs in cells. In some embodiments, this in turn leads to an increase in the transcription of genes involved in the response to stress.
  • the compounds are useful in any disease state where tissue damage results or in aging itself.
  • the compounds of the instant invention are used to inhibit tumor growth in tumors that are comprised of the subset of cell types that respond to high levels of DAF-16 homolog-activity by inducing apoptosis.
  • the therapeutic agent of the present invention could be a therapeutic agent preventing damage prior to the onset of a disease.
  • the compounds of the invention are used to protect the cells of an organism from damage associated with diseases including, but not limited to, ischemia and reperfusion damages, cardiac hypertrophy, fever, inflammation, metabolic diseases, viral and bacterial infection, cell and tissue trauma, and cancer.
  • the compounds of the invention are used to protect the cells of an organism from environmental stress, including, but not limited to, heat, cytotoxic drugs, toxins, heavy metals, asenite, anoxia, reperfusion or other damage from reactive oxidants and ethanol toxicity.
  • the compounds of the instant invention are used to prevent heat-stroke in aged individuals. It is known that the rate of heat stroke is more than 10-fold higher for persons of 65 years or older as compared to younger individuals (Kilbourne at al. (1982) J. Am. Med. Assoc. 247:3332-3336). This result is paralleled by studies indicating that cells isolated from aged organisms are much more sensitive to heat shock than comparable cells isolated from younger organisms. The increased sensitivity of aged cells is in part due to the decrease in the inducibility of heat shock proteins, including HSP72, in response to thermal stress (Volloch et al. (1998) Cell
  • the compounds of the instant invention are used to treat viral infections. It is known that certain compounds, such as cyclopentenone prostanoids, can endow cells with potent antiviral activity against a number of DNA and RNA viruses, including HIV-1 (Rozera, C. et al. (1996) J. Clin. Invest. 97:1795-1803). The effects of such compounds are mediated through the activation of heat-shock gene transcription, including the HSP70 gene. The heat shock proteins are involved at multiple levels in the viral replication pathway. Because increased DAF-16 homolog activity leads to an increase in HSP70 levels, the compounds of the present invention are useful as novel antiviral agents.
  • the compounds of the invention can act in two different ways to protect cells from viral infection: 1) inhibiting viral replication; and 2) protecting cells from the damage caused by viral activity.
  • such compounds would prevent damage resulting from stress resulting from surgery and/or from anesthetic action or removal or recovery from surgery or anesthesia.
  • the compounds of the instant invention are used to suppress the growth and development of tumors.
  • active DAF-16 homologs such as FRHKLl ⁇ activate the transcription of apoptosis-promoting genes, such as the Fas ligand.
  • DAF-16 homolog activity by inducing apoptosis can be treated with the agents of the instant invention.
  • the compounds of this invention prevent necrosis.
  • the compounds of this invention prevent damage associated with lack of function due to inactivity, coma, etc.
  • Compounds isolated according to the methods of the invention can be administered to patients for the therapeutic purposes outlined above in a pharmaceutically acceptable excipient, diluent, or carrier. Any method for administration is contemplated, including, but not limited to, oral, intravenous, subcutaneous, and intramuscular administration. The compounds can be administered to either treat an existing disease or they can be administered prophylactically. Increasing the Dosage of DAF-16 Homologs in Human Cells
  • the dosage of one or more DAF-16 homologs is increased in human cells.
  • this can be done by isolating DNA that encodes the DAF-16 homolog(s), and splicing the isolated DNA into a vector using any of the techniques well known in the art. Suitable protocols are disclosed, for example, in Ausubel et al., Current Protocols in Molecular Biology, 1996, Wiley & Sons, New York, NY.
  • the vector also contains a promoter sequence located in such a way that the isolated DNA falls under its transcriptional control.
  • the promoter is a general activator of transcription; hence, the vector can direct the expression of the DAF-16 homolog DNA in a large number of different cell types. In other embodiments, the promoter directs the expression of the DAF-16 homolog in a particular subset of tissues, a particular tissue, a small number of cell types, or even in a single cell type.
  • a very wide variety of promoters are known in the art, and the skilled artisan can select and manipulate promoters in order to obtain the desired expression pattern for the operatively-associated DAF-16 homolog
  • DAF-16 homolog as a fusion with another protein.
  • the DAF-16 homologs are expressed at a sufficiently high level that the AKT kinases are not able to phosphorylate ⁇ and hence inactivate — all of the DAF-16 homolog.
  • the constructs express mutated versions of DAF-16 homologs that are unable to respond to AKT phosphorylation. Such mutated DAF-16 homologs would be constitutively active.
  • the DAF-16 homolog constructs are encapsulated within a virus, and the virus is introduced into the human body. Once within the body, the virus can infect cells, and deliver its DAF-16 construct virion to those cells. The virus can be chosen so that it attaches only to specific cells, and so the DAF-16 homolog construct can effectively be targeted to those particular cells.
  • the above methods can be used in any medical application where it would be desirable to increase the level of stress response proteins in a tissue or cell type.
  • one application involves expressing the DAF-16 homolog construct in myocardial cells to protect them from ischemic stress and damage by reperfusion.
  • a patient who requires heart surgery can have the dosage of DAF-16 homologs in the heart increased by the above-mentioned method prior to surgery. Because the heart muscle will have increased expression of stress response proteins, damage sustained by the heart during surgery can be minimized.
  • constructs of the instant invention are used to suppress the growth and development of tumors.
  • the DAF-16 homolog in certain cell types, the DAF-16 homolog
  • FKHRLI when unphosphorylated, and hence active as a transcription factor, can activate the transcription of genes involved in cell death, including the Fas ligand. This activity of
  • FKHRLI is inhibited by the kinase AKT, which phophorylates FKHRLI, and leads to its translocation to the cytoplasm. It has been suggested that the induction of cell death is important in the suppression of tumor development, leading to the death and engulfment of nascent tumor cells in a process known as apoptosis.
  • the constructs of the instant invention although conferring cytoprotective abilities on some cell types, may induce cell death in other types of cell. This difference in response to DAF-16 homologs may result either from intrinsic differences in the way different cells respond to DAF-16 homolog activity.
  • tumors formed from cell types that respond to increased DAF-16 homolog activity by inducing apoptosis can be treated with the constructs of the instant invention.
  • a construct for the over-expression of a DAF-16 homolog can be encapsulated within a virus that binds specifically to the cell types that comprise the tumor. In this way, the over-expression construct is delivered to the tumor.
  • the beneficial effects obtained from the up-regulation or activation of DAF-16 or the production or administration of DAF-16 homologs is useful for the treatment of all diseases or medical conditions where oxidative stress is implicated as a causative or extenuating factor.
  • Nematodes were cultured under standard conditions (Brenner, S. (1974) Genetics 77:71-94). Strains included: N2 (wild type), TJ1052 (age-1 (hx546)), and GR1307 (daf- 16(mgDf50), a strain lacking all daf-16 coding regions). Transgenic nematodes were derived by microinjection of test DNA at 20 ⁇ g/ml and pRF4 (transformation marker) at
  • Cosmid R13H8 was obtained from The Sanger Center (UK). Strains are named as parent strain then extrachromosomal array (Ex). All transgenics carried pRF4. For experiments involving sterile animals, sterility was defined as adult animals that lacked oocytes and eggs. Life span was determined on seeded NGM (nematode growth media) plates at either 20°C or 25°C, as indicated. Animals were considered dead if no pharyngeal pumping was evident and they failed to respond to repeated prodding (Johnson T. et al. (1982) PNAS 79:6603-7).
  • NGM nematode growth media
  • Double stranded RNAi RNA was isolated from mixed stage, wild type (N2) C. elegans using Trizol (Sigma).
  • RNA was used as template for random primed cDNA synthesis using M-MuLV Reverse Transcriptase (New England Biolabs).
  • the single strand cDNA was amplified with daf-16 specific primers; STH14 (atcccgggctagtgatgatggtggtgatggtgcaaatcaaatgaatatgctgccc) and STH17 (tggatccccatgatggagatgctggtagat).
  • the PCR product was cloned into a PCR cloning vector pSTBlue (Novagen). DNA fragments derived from this construct was used in two in vitro transcription reactions, using either T7 or SP6 RNA polymerase (Ambion). Separate single stranded RNAs were mixed and injected into the gonad. FI progeny were examined for phenotypes.
  • thermotolerance was measured as percent of a cohort of staged 3 day old adult worms that survive a near-lethal heat shock. Specifically, 30-40 transgenic adults were placed on seeded NGM plate, and left to lay eggs for 3-4hrs. Adults were removed and eggs were allowed to develop until 3 days past L4 molt. 30-40 rolling adults were placed on a small seeded NGM plate, and the plate was wrapped in parafilm and floated in a circulating water bath set at 37.5°C for 2 hours. Parafilm was then removed and worms were allowed to recover at 20°C for 24hrs, then scored for viability.
  • UV tolerance 30-40 staged three day old adult animals were removed from a seeded plate, washed in lXS-Basal, then transferred to an unseeded NGM plate. Animals were exposed to 400J/m2 in a Stratalinker 2400 (Stratagene). Animals were removed from the unseeded plate and placed on a seeded one. Life span was calculated from the day of UV treatment. UV treatment often led to egg laying defects and bagged adults. These animals were censored from life span calculations. Daily heat shock. Plates containing 30-40 animals were wrapped in parafilm and floated in a circulating water bath at 34°C for lhr, separated by 24 hour periods. Parafilm was removed and animals returned to 20°C. Animals were scored for viability immediately before the heat shock.
  • Oxidative stress Since oxidative stress frequently results in a large number of bagged animals, only purged animals were examined for resistance to oxidative stress.
  • the DAF-16::GFP plasmid was constructed as follows: primers STH6 (ccgcggccgcgaattcaacttgagcatctctttttcttgg) and STH24 (ccactagtgagttctctggaattgg) were used to amplify a six kilobase fragment upstream of the daf-16 gene using cosmid R13H8 as a template. This fragment was digested with Notl and Spel and cloned into Notl/Spel digested pBluescript II SK(-) (Stratagene, Inc.) to create pGPlO. pGPlO was digested with
  • STH17 tggatccccatgatggagatgctggtagat
  • STH32 ctaccggtggcaaatcaaaatgaatatgctgccc
  • Oligonucleotides corresponding to a KPNI to Xhol fragment (ggtaccgagctctaagcaaaacaagccagcaaaacaagtccgcaaaacaagtactcgag , with three consensus insulin response sequences (IRS) (caaaacaa) were hybridized and cut with Kpnl and Xhol and cloned into Kpnl/Xhol digested pGL3 Promoter (Promega).
  • This construct has a minimal SV40 promoter and the luciferase coding region (Luc+). Binding of DAF-16 family member will activate transcription of the luciferase reporter.
  • HSP70 Western analysis used antibodies to Drosophila HSP70 (Sigma, Inc.), which recognizes both constitutive and inducible forms of HSP70 from many species, but does not detect any E. coli proteins. Densitometry measurements were done using Scion Image (Scion, Inc.). EXAMPLE 2 - Multiple copies of R13H8 confer stress resistance on transgenic nematodes.
  • daf-16 Given the central role of daf-16 in long-lived nematode mutants, the effect of multiple copies of daf-16 on stress resistance and life span was tested.
  • the daf-16 gene is contained wholly within the cosmid Rl 3H8, and this cosmid will rescue daf-16(lf) mutations (Ogg, S. et al. (1997) Nature 389:994-9; and Lin, K. et al. (1997) Science
  • EXAMPLE 3 Increased Thermotolerance is due to daf-16.
  • the effect of injection of dsRNA specific to daf-16 coding regions on thermotolerance and frequency of steriles was tested (see Materials and Methods). Injection of dsRNA specifically inhibits the activity of the gene injected, a process called RNAi (for overview see Tabara et al. (1998) Science 282:430-1). The occurrence of steriles and increase in thermotolerance in progeny of injected animals was tested. The age-1 (hx-546) ;ExRl 3H8 strains were injected with dsRNA because of the high frequency of steriles at 20°C.
  • thermotolerance is due to increased levels of daf- 16 transcript or protein, and not another gene on the cosmid.
  • EXAMPLE 4 Increased dosage of daf-16 and life span.
  • EXAMPLE 6 - DAF-16::GFP fusion protein increases life span and stress resistance.
  • Animals transgenic for DAF-16::GFP arrays (ExDAF-16::GFP) were tested to determine if they were longer-lived and more stress resistant. Because ExDAF-16:GFP animals do not segregate animals with weak germ line proliferation (as do ExR13H8 animals), unselected ExDAF-16::GFP animal could be tested. It was found that ExDAF-
  • UV resistant see Materials and Methods, and Figure 5b
  • ExpRF4 animals UV resistant (see Materials and Methods, and Figure 5b) than ExpRF4 animals.
  • a construct that can be useful in identification of agents that up-regulate human homologs of DAF-16, such as FKHR, is illustrated in Figure 10.
  • the construct contains three insulin response sequences (IRS) upstream of a minimal promoter and the luciferase gene (see Materials and Methods).
  • the IRS is a binding site for DAF-16 or its human homologs (Furayama, F. et al. (2000) Biochem. J. 349:629-634).
  • the construct can be transfected into human cell lines and stable transformants selected. The construct is likely to have some low level of background activity.
  • candidate agents can be screened for those that increase transcription of the luciferase. These compounds can be useful in up-regulating activity of FKHR or other DAF-16 human homologs.
  • EXAMPLE 8 Increased dosage of daf-16 leads to high levels of HSP70 and Ubiquitin Generally, as an organism ages, transcripts of several stress inducible genes increase in abundance, which has been interpreted to be a response to accumulated cellular damage (Lee, C. et al. (1999) Science 285:1390-3; and Ly, D. et al. (2000) Science
  • HSP70 proteins are abundant, highly conserved stress proteins induced by the presence of mis-folded proteins. In the present example, it was found that HSP70 protein levels increase in wild-type nematodes as they age (Figure 6). In Figure 6a, each lane contains extracts from five adults of the indicated age. The optical density (Scion
  • RNA was cloned and injected into nematodes as described in Materials and Methods.
  • #Data shown is from one representative experiment at 25 2 C, + SEM.

Abstract

L'invention concerne des procédés d'identification d'agents pouvant activer C. elegans DAF-16 ainsi que des homologues humains de celui-ci ou de leurs gènes correspondants, par lesquels des effets cytoprotecteurs dans des cellules pourraient être entraînés. Ces effets cytoprotecteurs peuvent aboutir à une résistance accrue au stress causé par l'environnement, une longévité rallongée et à une plus grande vigueur en fin de vie, sans inhibition importante des voies métaboliques marquant l'insuline. L'invention concerne également les agents thérapeutiques identifiés et les procédés de traitement employant ces agents.
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Cited By (13)

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Publication number Priority date Publication date Assignee Title
EP1581624A2 (fr) * 2002-08-09 2005-10-05 The Regents Of The University Of California Genes eukaryotes impliques dans la regulation de la duree de vie adulte
EP1581624A4 (fr) * 2002-08-09 2007-01-24 Univ California Genes eukaryotes impliques dans la regulation de la duree de vie adulte
EP1546389A2 (fr) * 2002-09-06 2005-06-29 Elixir Pharmaceuticals, Inc. Regulation de l'axe hormone de croissance/igf-1
EP1546389A4 (fr) * 2002-09-06 2006-07-26 Elixir Pharmaceuticals Inc Regulation de l'axe hormone de croissance/igf-1
WO2004058703A2 (fr) * 2002-12-23 2004-07-15 University Of Massachusetts Procedes pour identifier des modulateurs de longevite et procedes therapeutiques pour les utiliser
WO2004058703A3 (fr) * 2002-12-23 2004-12-09 Univ Massachusetts Procedes pour identifier des modulateurs de longevite et procedes therapeutiques pour les utiliser
US7858327B2 (en) * 2002-12-23 2010-12-28 University Of Massachusetts Methods of identifying longevity modulators and therapeutic methods of use thereof
US7932049B2 (en) * 2002-12-23 2011-04-26 University Of Massachusetts Methods of identifying longevity modulators and therapeutic methods of use thereof
DE10303850A1 (de) * 2003-01-30 2004-08-12 Aventis Pharma Deutschland Gmbh Screening-Assay auf Basis des Forkhead-Transkriptionsfaktor-abhängigen sod-3-Promotors zur Identifizierung von AKT modulierenden Verbindungen oder von stromaufwärts liegenden Regulatoren, wie etwa Insulin/IGF1-Rezeptoren
US7435868B2 (en) 2003-01-30 2008-10-14 Sanofi-Aventis Deutschland Gmbh Screening assay based on the forkhead transcription factor-dependent sod-3 promoter
KR101018910B1 (ko) 2003-08-26 2011-03-02 아우토리브 디벨롭먼트 아베 2개의 텐셔닝 장치를 갖는 벨트 리트랙터
WO2009129424A2 (fr) * 2008-04-17 2009-10-22 Academia Sinica Effets de l'acide acétique et du polysaccharide de reishi favorisant la longévité
WO2009129424A3 (fr) * 2008-04-17 2009-12-23 Academia Sinica Effets de l'acide acétique et du polysaccharide de reishi favorisant la longévité

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