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  Compositions for manipulating the lifespan and stress response of cells and organismsUSPTO Application #: 20060084135Title: Compositions for manipulating the lifespan and stress response of cells and organisms Abstract: Provided herein are methods and compositions for modulating the activity of sirtuin deacetylase protein family members; p53 activity; apoptosis; lifespan and sensitivity to stress of cells and organisms. Exemplary methods comprise contacting a cell with an activating compound, such as a flavone, stilbene, flavanone, isoflavone, catechin, chalcone, tannin or anthocyanidin; or an inhibitory compound, such as a sphingolipid, e.g., sphingosine. (end of abstract) Agent: Licatla & Tyrrell P.C. - Marlton, NJ, US Inventors: Konrad T. Howitz, Robert E. Zipkin USPTO Applicaton #: 20060084135 - Class: 435029000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Viable Micro-organism The Patent Description & Claims data below is from USPTO Patent Application 20060084135. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This patent application claims the benefit of priority from U.S. Provisional Patent Application No. 60/532,158, filed Dec. 23, 2003 and U.S. Provisional Patent Application No. 60/483,949, filed Jul. 1, 2003, each of which is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION [0002] There is now good evidence from model organisms that the pace of aging can be regulated (Kenyon, C. Cell 105, 165-168 (2001)). Longevity regulatory genes have been identified in many eukaryotes, including rodents, flies, nematode worms and even single-celled organisms such as baker's yeast (reviewed in Sinclair, D. Mech Ageing Dev 123, 857-67 (2002); Hekimi, S. & Guarente, L. Genetics and the specificity of the aging process. Science 299, 1351-4 (2003)). These genes appear to be part of an evolutionarily conserved longevity pathway that evolved to promote survival in response to deteriorating environmental conditions (Kenyon, C. Cell 105, 165-168 (2001); Guarente, L. and Kenyon, C. Nature 408, 255-62. (2000). The yeast S. cerevisiae has proven a particularly useful model in which to study cell autonomous pathways of longevity (Sinclair, D. Mech Ageing Dev 123, 857-67 (2002)). In this organism, replicative lifespan is defined as the number of daughter cells an individual mother cell produces before dying. Yeast lifespan extension is governed by PNC1, a calorie restriction (CR)--and stress-responsive gene that depletes nicotinamide, a potent inhibitor of the longevity protein Sir2. Both PNC1 and SIR2 are required for lifespan extension by CR or mild stress (Lin et al. Science 289, 2126-8 (2000); Anderson et al. Nature 423, 181-5 (2003)) and additional copies of these genes extend lifespan 30-70% (Lin et al. Science 289, 2126-8 (2000); Anderson et al. Nature 423, 181-5 (2003); Kaeberlein et al. Genes Dev 13, 2570-80 (1999). Based on these results we proposed that CR may confer health benefits in a variety of species because it is a mild stress that induces a sirtuin-mediated organismal defense response (Anderson et al. Nature 423, 181-5 (2003). [0003] Sir2, a histone deacetylase (HDAC), is the founding member of the sirtuin deacetylase family, which is characterized by a requirement for NAD.sup.+ as a co-substrate (Landry et al. Proc Natl Acad Sci USA 97, 5807-11 (2000); Imai et al. Nature 403, 795-800 (2000); Smith et al. Proc Natl Acad Sci USA 97, 6658-63 (2000); Tanner et al. Proc Natl Acad Sci USA 97, 14178-82 (2000); Tanny et al. Cell 99, 735-45 (1999); Tanny, J. C. and Moazed, D. Proc Natl Acad Sci USA 98, 415-20 (2001)). SIR2 was originally identified as a gene required for the formation of transcriptionally silent heterochromatin at yeast mating-type loci (Laurenson, P. and Rine, J. Microbiol Rev 56, 543-60. (1992)). Subsequent studies have shown that Sir2 suppresses recombination between repetitive DNA sequences at ribosomal RNA genes (rDNA)(Smith, J. S. and Boeke, J. D. Genes Dev 11, 241-54 (1997); Bryk et al. Genes Dev 11, 255-69 (1997); Gottlieb, S. and Esposito, R. E. Cell 56, 771-6 (1989)). Sir2 has also been implicated in the partitioning of carbonylated proteins to yeast mother cells during budding (Aguilaniu et al. Science (2003). Studies in C. elegans, mammalian cells, and the single-celled parasite Leishmania, indicate that the survival and longevity functions of sirtuins are conserved (Tissenbaum, H. A. and Guarente, L. Nature 410, 227-30 (2001); Vaziri et al. Cell 107, 149-59 (2001); Luo et al. Cell 107, 137-48 (2001); Vergnes et al. Gene 296, 139-50 (2002)). In C. elegans additional copies of sir-2.1 extend lifespan by 50% via the insulin/IGF-1 signalling pathway, the same pathway recently shown to regulate lifespan in rodents (Holzenberger et al. Nature 421, 182-7 (2003); Shimokawa et al. Faseb J 17, 1108-9 (2003); Tatar et al. Science 299, 1346-51 (2003)). SUMMARY OF THE INVENTION [0004] Provided herein are methods for activating a sirtuin deacetylase protein family member. The method may comprise contacting a sirtuin deacetylase protein family member with a compound having a structure selected from the group of formulas 1-25 and 31. Compounds falling within formulas 1-25 and 31 and activating a sirtuin protein are referred to herein as "activating compounds." The activating compound may be a polyphenol compound, such as a plant polyphenol or an analog or derivative thereof. Exemplary compounds are selected from the group consisting of flavones, stilbenes, flavanones, isoflavones, catechins, chalcones, tannins and anthocyanidins or analog or derivative thereof. In illustrative embodiments, compounds are selected from the group of resveratrol, butein, piceatannol, isoliquiritgenin, fisetin, luteolin, 3,6,3',4'-tetrahydroxyflavone, quercetin, and analogs and derivatives thereof. In certain embodiments, if the activating compound is a naturally occurring compound, it may not in a form in which it is naturally occurring. [0005] The sirtuin deacetylase protein family member maybe the human SIRT1 protein or the yeast Sir2 protein. [0006] The sirtuin deacetylase protein family member may be in a cell, in which case the method may comprise contacting the cell with an activating compound or introducing a compound into the cell. The cell may be in vitro. The cell may be a cell of a subject. The cell may be in a subject and the method may comprise administering the activating compound to the subject. Methods may further comprise determining the activity of the sirtuin deacetylase protein family member. [0007] A cell may be contacted with an activating compound at a concentration of 0.1-100 .mu.M. In certain embodiments, a cell is further contacted with an additional activating compound. In other embodiments, a cell is contacted with a least three different activating compounds. [0008] Other methods encompassed herein include methods for inhibiting the activity of p53 in a cell and optionally protecting the cell against apoptosis, e.g., comprising contacting the cell with an activating compound at a concentration of less than about 0.5 .mu.M. Another method comprises stimulating the activity of p53 in a cell and optionally inducing apoptosis in the cell, comprising contacting the cell with an activating compound at a concentration of at least 50 .mu.M. [0009] Also provided herein is a method for extending the lifespan of a eukaryotic cell, such as by increasing its resistance to stress, comprising contacting the cell with a compound selected from the group consisting of stilbene, flavone and chalcone family members. Such compounds are referred to as "lifespan extending compounds." The compound may have the structure set forth in formula 7. Other compounds may be activating compounds having a structure set forth in any of formulas 1-25 and 30, provided they extend lifespan or increase resistance to stress. The compound may be selected from the group consisting of resveratrol, butein and fisetin and analogs and derivatives thereof. In certain embodiments, if the lifespan extending compound is a naturally occurring compound, it is not in a form in which it is naturally occurring. The method may further comprise determining the lifespan of the cell. The method may also further comprise contacting the cell with an additional compound or with at least three compounds selected from the group consisting of stilbene, flavone and chalcone family members or other lifespan extending compound. The cell may be contacted with a compound at a concentration of less than about 10 .mu.M or at a concentration of about 10-100 .mu.M. The cell may be in vitro or in vivo, it may be a yeast cell or a mammalian cell. If the cell is in a subject, the method may comprise administering the compound to the subject. [0010] Methods for inhibiting sirtuins; inhibiting deacetylation of p53; stimulating apoptosis; shorting lifespan and rendering cells and organisms sensitive to stress are also encompassed. One method comprises contacting a sirtuin or cell or organism comprising such with an inhibitory compound having a formula selected from the group of formulas 26-29 and 31. [0011] Also provided herein are compositions comprising, e.g., two compounds each having a formula selected from the group of formulas 1-31. Further provided herein are screening methods for identifying compounds, e.g., small molecules, that modulate sirtuins and/or modulate the life span or resistance to stress of cells. Methods may comprise (i) contacting a cell comprising a SIRT1 protein with a peptide of p53 comprising an acetylated residue 382 in the presence of an inhibitor of class I and class II HDAC under conditions appropriate for SIRT1 to deacetylate the peptide and (ii) determining the level of acetylation of the peptide, wherein a different level of acetylation of the peptide in the presence of the test compound relative to the absence of the test compound indicates that the test compound modulates SIRT1 in vivo. BRIEF DESCRIPTION OF THE DRAWINGS [0012] FIGS. 1a through 1d show the effects of resveratrol on the kinetics of recombinant human SIRT1. FIG. 1a shows resveratrol dose-response of SIRT1 catalytic rate at 25 .mu.M NAD.sup.+, 25 .mu.M p53-382 acetylated peptide. Relative initial rates are the mean of two determinations, each derived from the slopes of fluorescence (arbitrary fluorescence units, AFU) vs. time plots with data obtained at 0, 5, 10 and 20 min. of deacetylation. FIG. 1b shows the SIRT1 initial rate at 3 mM NAD.sup.+, as a function of p53-382 acetylated peptide concentration in the presence (.DELTA.) or absence (.box-solid.) of 100 .mu.M resveratrol. Lines represent non-linear least-squares fits to the Michaelis-Menten equation. Kinetic constants: K.sub.m(control, .box-solid.)=64 .mu.M, K.sub.m(+resveratrol, .DELTA.)=1.8 .mu.M; V.sub.max(control, .box-solid.)=1107 AFU/min., V.sub.max(+resveratrol, .DELTA.)=926 AFU/min. FIG. 1c shows the SIRT1 initial rate at 1 mM p53-382 acetylated peptide, as a function of NAD.sup.+ concentration, in the presence (.DELTA.) or absence (.box-solid.) of 100 .mu.M resveratrol. Lines represent non-linear least-squares fits to the Michaelis-Menten equation. Kinetic constants: K.sub.m(control, .box-solid.)=558 .mu.M, K.sub.m(+resveratrol, .DELTA.)=101 .mu.M; V.sub.max(control, .box-solid.)=1863 AFU/min., V.sub.max(+resveratrol, .DELTA.)=1749 AFU/min. FIG. 1d shows effects of resveratrol on nicotinamide inhibition of SIRT1. Kinetic constants are shown relative to those of the control (no nicotinamide, no resveratrol) and represent the mean of two determinations. Error bars are standard errors of the mean. The variable substrate in each experiment (N=NAD.sup.+, P=p53 acetylated peptide), the presence/absence of nicotinamide (.+-.) and the resveratrol concentration (.mu.M) are indicated beneath each pair of K.sub.m-V.sub.max bars. [0013] FIG. 2a through 2d show the effects of polyphenols on Sir2 and S. cerevisiae lifespan. FIG. 2a shows the initial deacetylation rate of recombinant GST-Sir2 as a function of resveratrol concentration. Rates were determined at the indicated resveratrol concentrations, either with 100 .mu.M `Fluor de Lys` acetylated lysine substrate (FdL) plus 3 mM NAD.sup.+ (.DELTA.) or with 200 .mu.M p53-382 acetylated peptide substrate plus 200 .mu.M NAD.sup.+ (.box-solid.). FIG. 2b shows lifespan analyses determined by micro-manipulating individual yeast cells as described Sinclair, D. A. and Guarente (Cell 91, 1033-42 (1997)) in complete 2% glucose medium with 10 .mu.M of each compound, unless otherwise stated. Average lifespan was determined for wild type untreated (.quadrature.), quercetin (.largecircle.) and piceatannol (.circle-solid.). FIG. 2c shows the average lifespan for wild type untreated (.quadrature.), fisetin (.largecircle.), butein (), or resveratrol (.DELTA.). FIG. 2d shows average lifespan for wild type untreated (.quadrature.), and growth with resveratrol at 10 .mu.M (.DELTA.), 100 .mu.M (.circle-solid.), or 500 .mu.M (.largecircle.). [0014] FIGS. 3a through 3f show resveratrol extending lifespan by mimicking CR and suppressing rDNA recombination. Yeast lifespans were determined as in FIG. 2. FIG. 3a shows average lifespan for wild type (wt) untreated (.quadrature.), wild type+resveratrol (wt+R; .circle-solid.) and glucose-restricted+resveratrol (CR+R; .largecircle.). FIG. 3b shows average lifespans for wild type (.quadrature.), sir2(.DELTA.) sir2+resveratrol (sir2+R; .tangle-solidup.), pnc1 (.largecircle.), and pnc1+resveratrol (pnc1+R; .circle-solid.). FIG. 3c shows resveratrol suppressing the frequency of ribosomal DNA recombination in the presence and absence of nicotinamide (NAM). Frequencies were determined by loss of the ADE2 marker gene from the rDNA locus (RDN1). FIG. 3d shows that resveratrol does not suppress rDNA recombination in a sir2 strain. FIG. 3e show that resveratrol and other sirtuin activators do not significantly increase rDNA silencing compared to a 2.times.SIR2 strain. Pre-treated cells (RDN1::URA3) were harvested and spotted as 10-fold serial dilutions on either SC or SC with 5-fluororotic acid (5-FOA). In this assay, increased rDNA silencing results in increased survival on 5-FOA medium. FIG. 3f show quantitation of the effect of resveratrol on rDNA silencing by counting numbers of surviving cells on FOA/total plated. [0015] FIGS. 4a through 4e show resveratrol and other polyphenols stimulating SIRT1 activity in human cells. FIG. 4a shows a method for assaying intracellular deacetylase activity with a fluorogenic, cell-permeable substrate, FdL (`Fluor de Lys`, BIOMOL). FdL (200 .mu.M) is added to growth media and cells are incubated for 1-3 hours to allow FdL to enter the cells and the lysine-deacetylated product (deAc-FdL) to accumulate intracellularly. Cells are lysed with detergent in the presence of 1 .mu.M TSA and 1 mM nicotinamide. Addition of the non-cell-permeable Developer (BIOMOL) releases a fluorophor, specifically from deAc-FdL. FIG. 4b shows SIRT1 activating polyphenols stimulating TSA-insensitive, FdL deacetylation by HeLa S3 cells. Cells were grown adherently in DMEM/10% FCS and treated for 1 hour with 200 .mu.M FdL, 1 .mu.M TSA and either vehicle (0.5% final DMSO, Control) or 500 .mu.M of the indicated compound. Intracellular accumulation of deAc-FdL was then determined as described briefly in FIG. 4a. The intracellular deAc-FdL level for each compound (mean of six replicates) are plotted against the ratios to the control rate obtained in the in vitro SIRT1 polyphenol screen (see Table 1, Supplementary Tables 1 and 3). FIG. 4c shows U2OS osteosarcoma cells grown to .gtoreq.90% confluence in DMEM/10% FCS exposed to 0 or 10 grays of gamma irradiation (IR). Whole cell lysates were prepared 4 hours post-irradiation and were probed by Western blotting with indicated antibodies. FIG. 4d shows U2OS cells cultured as above and pre-treated with the indicated amounts of resveratrol or a 0.5% DMSO blank for 4 hours after which cells were exposed to 0 or 50 J/cm.sup.2 of UV radiation. Lysates were prepared and analyzed by Western blot as in FIG. 4c. FIG. 4e shows human embryonic kidney cells (HEK 293) expressing wild type SIRT1 or dominant negative SIRT1-H363Y (SIRT1-HY) protein cultured as described above, pre-treated with the indicated amounts of resveratrol or a 0.5% DMSO blank for 4 hours, and exposed to 50 J/cm.sup.2 of UV radiation as above. Lysates were prepared and analyzed as above. [0016] FIG. 5 shows the deacetylation site preferences of recombinant SIRT1. Initial rates of deacetylation were determined for a series of fluorogenic acetylated peptide substrates based on short stretches of human histone H3, H4 and p53 sequence. Substrates examined include: H3-4-9 with the sequence K(Ac)QTARK(Ac) (SEQ ID NO:1); H3-9-14 with the sequence K(Ac)STGGK(Ac) (SEQ ID NO:2); H3-9-14/pS with the sequence K(Ac)--S(PO3)-TGGK(Ac) (SEQ ID NO:3); H3-14-18 with the sequence K(Ac)APRK(Ac) (SEQ ID NO:4); H4-1-5 with the sequence SGRGK(Ac)(SEQ ID NO:5); H4-12-16(Fluor de Lys-H4-AcK16) with the sequence KGGAK(Ac) (SEQ ID NO:6); H4-12-16/diAc with the sequence K(Ac)GGAK(Ac)(SEQ ID NO:7); p53-320 (Fluor de Lys-SIRT2) with the sequence QPKK(Ac)(SEQ ID NO:8); p53-373 with the sequence K(Ac)SKK(Ac)(SEQ ID NO:9); p53-382(Fluor de Lys-SIRT1 with the sequence RHKK(Ac) (SEQ ID NO:10); p53-382/di-Ac (Fluor de Lys-HDAC8) with the sequence RHK(Ac)K(Ac)(SEQ ID NO:11); and .epsilon.-acetyl lysine (Fluor de Lys, Fdl) wit the sequence K(Ac). All substrate were obtained from BIOMOL, Plymouth Meeting, Pa.). Recombinant human SIRT1 (1 .mu.g, BIOMOL), was incubated for 10 minutes at 37.degree. C. with 25 .mu.M of the indicated fluorogenic acetylated peptide substrate and 500 .mu.M NAD.sup.+. Reactions were stopped by the addition of 1 mM nicotinamide and the deacetylation-dependent fluorescent signal was determined. [0017] FIG. 6a through 6c show intracellular deacetylation activity measured with a cell-permeable, fluorogenic HDAC and sirtuin substrate. HeLa S3 cells were grown to confluence in DMEM/10% FCS and then incubated with fresh medium containing 200 .mu.M FdL for the indicated times at 37.degree. C. Intracellular and medium levels of deacetylated substrate (deAc-FdL) were determined according to the manufacturer's instructions (HDAC assay kit, BIOMOL). All data points represent the mean of two determinations. FIG. 6a shows the concentration ratio of intracellular ([deAc-FdL].sub.i) to medium ([deAc-FdL].sub.o) concentrations in the presence (.DELTA.) or absence (.box-solid.) of 1 .mu.M trichostatin A (TSA). FIG. 6b shows total accumulation of deacetylated substrate (deAc-FdL) in the presence (.DELTA.) or absence (.box-solid.) of 1 .mu.M TSA. FIG. 6c shows intracellular accumulation of deacetylated substrate (deAc-FdL) in the presence (.DELTA.) or absence (.box-solid.) of 1 .mu.M TSA. DETAILED DESCRIPTION OF THE INVENTION Definitions [0018] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. [0019] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. "Activating a sirtuin protein" refers to the action of producing an activated sirtuin protein, i.e., a sirtuin protein that is capable of performing at least one of its biological activities to at least some extent, e.g., with an increase of activity of at least about 10%, 50%, 2 fold or more. Biological activities of sirtuin proteins include deacetylation, e.g., of histones and p53; extending lifespan; increasing genomic stability; silencing transcription; and controlling the segregation of oxidized proteins between mother and daughter cells. Continue reading... Full patent description for Compositions for manipulating the lifespan and stress response of cells and organisms Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Compositions for manipulating the lifespan and stress response of cells and organisms patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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