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WO2008027379A2 - Indicateurs d'activité sirtuine et procédés d'utilisation de ceux-ci - Google Patents

Indicateurs d'activité sirtuine et procédés d'utilisation de ceux-ci Download PDF

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Publication number
WO2008027379A2
WO2008027379A2 PCT/US2007/018916 US2007018916W WO2008027379A2 WO 2008027379 A2 WO2008027379 A2 WO 2008027379A2 US 2007018916 W US2007018916 W US 2007018916W WO 2008027379 A2 WO2008027379 A2 WO 2008027379A2
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Prior art keywords
sirtuin
acetylation
level
compound
substrates
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PCT/US2007/018916
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WO2008027379A3 (fr
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Jill Milne
Peggy Romero
Lei Jin
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Sirtris Pharmaceuticals, Inc.
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Publication of WO2008027379A2 publication Critical patent/WO2008027379A2/fr
Publication of WO2008027379A3 publication Critical patent/WO2008027379A3/fr

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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • HATs histone acetyltransferases
  • HDACs histone deacetylases
  • SIR Silent Information Regulator
  • the SIR family of genes represents a highly conserved group of genes present in the genomes of organisms ranging from archaebacteria to a variety of eukaryotes (Frye, 2000).
  • the encoded SIR proteins are involved in diverse processes from regulation of gene silencing to DNA repair.
  • the proteins encoded by members of the SIR gene family show high sequence conservation in a 250 amino acid core domain.
  • a well-characterized gene in this family is 5. cerevisiae SIR2, which is involved in silencing HM loci that contain information specifying yeast mating type, telomere position effects and cell aging (Guarente, 1999; Kaeberlein et al., 1999; Shore, 2000).
  • the yeast Sir2 protein belongs to a family of histone deacetylases (reviewed in Guarente, 2000; Shore, 2000).
  • the Sir2 homolog, CobB in Salmonella typhimu ⁇ um, functions as an NAD (nicotinamide adenine dinucleotide)-dependent ADP-ribosyl transferase (Tsang and Escalante-Semerena, 1998).
  • sirtuins Today, Sir2 genes are believed to have evolved to enhance an organism's health and stress resistance to increase its chance of surviving adversity. Accordingly, it would be highly desirable to further understand the role of sirtuins in the cell and to elucidate the proteins that sirtuins act upon.
  • SIRTl substrate recognition by SIRTl does not depend on the amino acid sequence proximate to the acetylated lysine.
  • Garske et al. (“SIRTl top 40 hits: use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity.” Biochemistry 45(l):94-101, 2006) also described a novel, high-throughput method for determining deacetylase substrate specificity using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging strategy and automated bead-sorting.
  • OBOC one-compound
  • a 5-mer OBOC peptide library of 104,907 unique sequences was constructed around a central epsilon-amino acetylated lysine.
  • the library was screened using the human NAD+-dependent deacetylase SIRTl for the most efficiently deacetylated peptide sequences.
  • Beads preferentially deacetylated by SIRTl were biotinylated and labeled with streptavidin-coated quantum dots. After fluorescent bead-sorting, the top 39 brightest beads were sequenced by mass spectrometry.
  • In-solution deacetylase assays on randomly chosen hit and nonhit sequences revealed that hits correlated with increased catalytic activity by as much as 20-fold. They found that SIRTl can discriminate peptide substrates in a context- dependent fashion.
  • novel sirtuin substrates in a cellular environment will help to further elucidate the role of sirtuins in the cell and will facilitate the design of diagnostic assays, therapeutic monitoring for patients being treated with sirtuin modulators, and screens for identifying novel sirtuin modualting compounds.
  • a subject for example, during therapeutic treatment with a sirtuin modulating compound.
  • the invention provides a method for monitoring the progress of therapeutic treatment with a sirtuin modulator, comprising determining the level of acetylation of one or more sirtuin substrates shown in
  • Table 1 in a biological sample from a subject treated with a sirtuin modulator, wherein a change in the level of acetylation of the one or more sirtuin substrates as compared to a control is indicative of therapeutic sirtuin modulation in said subject.
  • the subject may be a mammal, such as, for example, a human.
  • the biological sample may comprise blood, urine, serum, saliva, cells, tissue, and/or hair.
  • the sirtuin modulator is a sirtuin activating compound. In such embodiments, a decrease in the acetylation level of the one or more sirtuin substrates as compared to a control is indicative of therapeutic sirtuin modulation in said subject.
  • the subject receiving therapeutic administration of the sirtuin modulator may be suffering from a disease or disorder related to aging or stress, diabetes, obesity, a neurodegenerative disease, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, an ocular disease or disorder, cardiovascular disease, a blood clotting disorder, inflammation, or flushing.
  • the sirtuin modulator may be a sirtuin inhibiting compound.
  • an increase in the acetylation level of the one or more sirtuin substrates as compared to a control is indicative of therapeutic sirtuin modulation in said subject.
  • the sirtuin modulator is a sirtuin inhibiting compound
  • the subject receiving therapeutic administration of the sirtuin modulator may be suffering from cancer or may require appetite stimulation or weight gain.
  • control may be an untreated individual, the subject prior to treatment, the subject at an earlier time point during treatment, or a database reference.
  • the level of acetylation of at least two, three, four, five, ten, or more, sirtuin substrates shown in Table 1 are determined.
  • the level of acetylation is determined using an antibody (e.g., immunoblotting) and/or mass spectrometry.
  • the method may further comprise adjusting the dose of the sirtuin modulator administered to the subject, e.g., based on the acetylation level of one or more sirtuin substrates shown
  • the invention provides a method for monitoring the progress of therapeutic treatment with a sirtuin modulator, comprising: (i) administering a sirtuin modulator to a subject, (ii) obtaining a biological sample from said subject, and (iii) determining the level of acetylation of one or more sirtuin substrates shown in Table 1 in said sample, wherein a change in the level of acetylation of the one or more sirtuin substrates as compared to a control is indicative of therapeutic sirtuin modulation in said subject.
  • the sirtuin modulator may be administered to a subject at least twice over time and the level of acetylation of one or more sirtuin substrates is determined at two or more time points during the course of administration.
  • the invention provides a method for detecting modulation of a sirtuin protein in a mammal, comprising: (i) obtaining a biological sample from a subject that has received a sirtuin modulator, and (ii) determining the level of acetylation of one or more sirtuin substrates shown in Table 1 in said sample, wherein a change in the level of acetylation of the one or more sirtuin substrates as compared to a control is indicative of sirtuin modulation in said mammal.
  • the invention provides a method of identifying a subject in need of treatment with a sirtuin activating compound, comprising determining the level of acetylation of one or more sirtuin substrates shown in Table 1 in a biological sample from said subject, wherein a higher level of acetylation in said one or more sirtuin substrates as compared to a control is indicative of a subject in need of treatment with a sirtuin activating compound.
  • the invention provides a method for identifying a compound that modulates a sirtuin protein, comprising: (i) contacting a sirtuin protein with a test compound in the presence of a sirtuin substrate, wherein the sirtuin substrate is at least one of the proteins shown in Table 1, or a fragment thereof, and (ii) determining the level of acetylation of the sirtuin substrate, wherein a change in the level of acetylation of the sirtuin substrate in the presence of the test compound as compared to a control is indicative of a compound that modulates the sirtuin protein.
  • a test compound that activates a sirtuin protein may be identified.
  • a test compound that inhibits a sirtuin protein may be identified.
  • the test compound is a small molecule.
  • a modulator of a human sirtuin protein such as SIRTl
  • the level of acetylation of the sirtuin substrate may be determined using mass spectrometry.
  • the sirtuin substrate further comprises a fluorophore.
  • the level of acetylation of the sirtuin substrate is determined using a fluorescence readout or a fluorescence polarization readout.
  • the invention provides a method for identifying a compound that modulates a sirtuin protein, comprising: (i) contacting a cell that expresses a sirtuin protein with a test compound, and (ii) determining the level of acetylation of one or more sirtuin substrates shown in Table 1 , wherein a change in the level of acetylation of the one or more sirtuin substrates in the presence of the test compound as compared to a control is indicative of a compound that modulates the sirtuin protein.
  • the level of acetylation of at least two, three, four, five, ten, or more, sirtuin substrates shown in Table 1 are determined.
  • the cell is a mammalian cell, such as a human cell.
  • the cell may be an isolated cell, suspended in culture, or may be present in a whole organism, such as a non-human organism.
  • a test compound that activates a sirtuin protein may be identified.
  • a test compound that inhibits a sirtuin protein may be identified.
  • the test compound may be a small molecule.
  • a method for identifying a compound that modulates a sirtuin protein may further comprise one or more of the following: (i) preparing a quantity of the compound, or an analog thereof, (ii) conducting therapeutic profiling of the compound, or an analog thereof, for efficacy and toxicity in animals, (iii) formulating the compound, or analog thereof, in a pharmaceutical formulation, (iv) manufacturing a pharmaceutical preparation of a compound, or an analog thereof, having a suitable animal toxicity profile, or (v) marketing a pharmaceutical preparation of a compound, or an analog thereof, having a suitable animal toxicity profile to healthcare providers.
  • FIGURE 1 shows Table 1 which provides a list of sirtuin inhibitor sensitive peptides.
  • FIGURE 2 shows Table 2 which provides an alignment of the twenty amino acid residues to the amino terminal side of the acetylated lysine residue for the peptides identified in Table 1.
  • FIGURE 3 shows Table 3 which provides an alignment of the twenty amino acid residues to the carboxy terminal side of the acetylated lysine residue for the peptides identified in Table 1.
  • FIGURE 4 shows a schematic of the experimental protocol used for identification of sirtuin inhibitor sensitive peptides.
  • FIGURE 5 shows a western blot of lysates prepared from a number of cell lines as a way to compare endogenous SIRTl protein levels.
  • FIGURE 6 shows a whole cell western blot of HEK293 cells treated for 24 hours with either a sirtuin inhibitor (50 uM in DMSO) or DMSO alone and probed with an acetyl-lysine polyclonal antibody.
  • FIGURE 7 shows a whole cell western of HEK293 cells treated with or without SIRTl siRNA for 48 hours and probed with an acetyl-lysine polyclonal antibody.
  • FIGURE 8 shows a schematic of a procedure for large scale treatment of HEK293 cells with and without a sirtuin inhibitor for preparation of cell lysates for mass spectrometric analysis.
  • FIGURE 9 shows a schematic of a procedure for large scale treatment of HEK293 cells with and without sirtuin inhibitors for preparation of cell lysates for SILAC based mass spectrometric analysis.
  • FIGURE 10 shows a schematic of a procedure for determination of acetylation levels of an endogenous sirtuin substrate.
  • the terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included.
  • the term “conserved residue” refers to an amino acid that is a member of a group of amino acids having certain common properties.
  • the term “conservative amino acid substitution” refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid from the same group.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure, Springer- Verlag).
  • groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer- Verlag).
  • One example of a set of amino acid groups defined in this manner include: (i) a charged group, consisting of GIu and Asp, Lys, Arg and His, (ii) a positively-charged group, consisting of Lys, Arg and His, (iii) a negatively-charged group, consisting of GIu and Asp, (iv) an aromatic group, consisting of Phe, Tyr and Trp, (v) a nitrogen ring group, consisting of His and Trp, (vi) a large aliphatic nonpolar group, consisting of VaI, Leu and He, (vii) a slightly-polar group, consisting of Met and Cys, (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, GIy, Ala, GIu, GIn and Pro, (ix) an aliphatic group consisting of VaI, Leu, He, Met and Cys, and (x) a small hydroxyl group consisting of Ser and
  • mammals include humans, primates, livestock animals (including bovines, porcines, etc.), companion animals (e.g., canines, felines, etc.) and rodents (e.g., mice and rats).
  • modulate when used in reference to the activity of an acetyltransferase or deacetylase, refers to the up regulation (e.g., activation or stimulation), down regulation (e.g., inhibition or suppression), or other change in a quality of such acetyltransferase or deacetylase activity.
  • sirtuin-activating compound refers to a compound that increases the level of a sirtuin protein and/or increases at least one activity of a sirtuin protein.
  • a sirtuin-activating compound may increase at least one biological activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%, 100%, or more.
  • sirtuin activating compounds increase deacetylase activity of a sirtuin protein, e.g., increased deacteylation of one or more substrates shown in Table 1.
  • Exemplary sirtuin activating compounds include flavones, stilbenes, flavanones, isoflavanones, catechins, chal cones, tannins and anthocyanidins.
  • Exemplary stilbenes include hydroxystilbenes, such as trihydroxystilbenes, e.g., 3,5,4'-trihydroxystilbene ("resveratrol"). Resveratrol is also known as 3,4',5-stilbenetriol. Tetrahydroxystilbenes, e.g., piceatannol, are also encompassed. Hydroxychalones including trihydroxychalones, such as isoliquiritigenin, and tetrahydroxychalones, such as butein, can also be used.
  • Hydroxyflavones including tetrahydroxyflavones, such as fisetin, and pentahydroxyflavones, such as quercetin, can also be used.
  • Other sirtuin activating compounds are described in U.S. Patent Application Publication No. 2005/0096256 and PCT Application Nos. PCT/US06/002092, PCT/US06/007746, PCT/US06/007744, PCT/US06/007745, PCT/US06/007778, PCT/US06/007656, PCT/US06/007655 and PCT/US06/007773.
  • sirtuin-inhibiting compound refers to a compound that decreases the level of a sirtuin protein and/or decreases at least one activity of a sirtuin protein.
  • a sirtuin-inhibiting compound may decrease at least one biological activity of a sirtuin protein by at least about 10%, 25%, 50%, 75%, 100%, or more.
  • sirtuin inhibiting compounds decrease deacetylase activity of a sirtuin protein, e.g., decreased deacteylation of one or more substrates shown in Table 1.
  • sirtuin inhibitors include, for example, sirtinol and analogs thereof (see e.g., Napper et al., J. Med. Chem. 48: 8045-54 (2005)), nicotinamide (NAD + ) and suramin and analogs thereof.
  • sirtuin inhibiting compounds are described in U.S. Patent Application Publication No. 2005/0096256, PCT Publication No. WO2005/002527, and PCT Application Nos.
  • PCT/US06/007746 PCT/US06/007744, PCT/US06/007745, PCT/US06/007778, PCT/US06/007656, PCT/US06/007655, PCT/US06/007773 and PCT/US06/007742.
  • sirtuin-modulating compound refers to a compound that may either up regulate (e.g., activate or stimulate), down regulate (e.g., inhibit or suppress) or otherwise change a functional property or biological activity of a sirtuin protein.
  • Sirtuin-modulating compounds may act to modulate a sirtuin protein either directly or indirectly.
  • a sirtuin-modulating compound may be a sirtuin-activating compound or a sirtuin-inhibiting compound.
  • Novel Sirtuin Substrate Polypeptides and Methods of Use Thereof Provided herein are a variety of sirtuin substrates as well as an identification of the lysine residues that are deacetylated by the sirtuin. These substrates may be used as biomarkers for sirtuin activity in vivo which is useful in a wide variety of applications including diagnostic applications, therapeutic monitoring applications and drug screening assays.
  • the invention provides a method for monitoring sirtuin activity in vivo. The methods involve determining the acetylation state of one or more sirtuin substrates, for example, one or more of the substrate shown in Table 1 ( Figure 1).
  • monitoring sirtuin activity in vivo may be useful for diagnostic purposes, e.g., by identifying individuals in need of treatment with a sirtuin modulator.
  • Individuals having an acetylation state for one or more sirtuin substrates that differs from a control sample maybe in need of treatment with a sirtuin modulator.
  • individuals having a higher level of acetylation of one or more sirtuin substrates as compared to a control may be in need of treatment with a sirtuin activating compound.
  • individuals having a lower level of acetylation of one or more of sirtuin substrates as compared to a control may be in need of treatment with a sirtuin inhibiting compound.
  • determination of sirtuin activity in vivo may be useful for monitoring the course of treatment with a sirtuin modulating compound.
  • the acteylation state of one or more sirtuin substrates maybe determined at one or more time points during the course of treatment with a sirtuin therapeutic to monitor sirtuin activity in response to the treatment.
  • Administration of a sirtuin activator may result in a decrease in the level of acetylation of one or more sirtuin substrates while administration of a sirtuin inhibitor may! result in an increase in the level of acetylation of one or more sirtuin substrates.
  • a subject is administered a sirtuin therapeutic over time, for example, at least once a day, once a week, once a month, etc. for at least a week, two weeks, one month, two months, six months, one year, or chronically.
  • Acetylation levels of one or more sirtuin substrates may be monitored on a regular or sporadic basis during the course of treatment, for example, sirtuin substrate acetylation levels maybe measured on a daily, weekly, biweekly, monthly, or bimonthly basis, or once every six months, or once a year.
  • the frequency of sirtuin substrate acetylation level monitoring may differ over time, for example, after an optimal treatment regime (including dosage and/or frequency of administration) is determined, the frequency of monitoring may decrease.
  • the methods described herein may involve monitoring the acetylation level of one or more sirtuin substrates at least once a day or at least once a week until an optimized dosage regime is determined. Subsequently, monitoring of the acetylation level of one or more sirtuin substrates is reduce to no more than once per week or no more than once per month.
  • the subject being treated. with a sirtuin modulating compound may be suffering from one or more of a variety of disorders.
  • subjects being treated with a sirtuin activating compound may be suffering from a disease or disorder related to aging or stress, diabetes, obesity, a neurodegenerative disease, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, an ocular disease or disorder, cardiovascular disease, a blood clotting disorder, inflammation, or flushing.
  • Subjects being treated with a sirtuin inhibiting compound may be suffering from cancer or may be individuals being treated to stimulate appetite or weight gain.
  • the methods described herein involve detection of the acetylation level of one or more of the sirtuin substrates shown in Table 1.
  • the methods described herein may involve detection of the acetylation level of one or more of the following sirtuin substrates: PARPl, VDAC, BBSl, AKRlBl, MIF, MYST2, MYST3, TAFl, TRP1,,RAD51L3, DNA-PK, Ebpl, HB-EFG, NuMAl, CacyBP, Ku70, SFRS8, or TRRAP.
  • the methods described herein do not involve detection of the acetylation level of Ku70 (ATP-dependent DNA helicase II, 70 kDa subunit).
  • control may be a measure of the acetylation level of one or more sirtuin substrates in a quantitative form (e.g., a number, ratio, percentage, graph, etc.) or a qualitative form (e.g., band intensity on a gel or blot, etc.).
  • a quantitative form e.g., a number, ratio, percentage, graph, etc.
  • a qualitative form e.g., band intensity on a gel or blot, etc.
  • a variety of controls may be used.
  • the acetylation level of one or more sirtuin substrates from an individual not being treated with a sirtuin modulator may be used.
  • Levels of sirtuin substrate acetylation from a healthy individual may also be used as a control, e.g., an individual not suffering from a disease or disorder that is present in the individual being treated with a sirtuin modulating compound.
  • the control may be acetylation levels of one or more sirtuin substrates from the individual being treated at a time prior to treatment with the sirtuin modulator or at a time period earlier during the course of treatment with the sirtuin modulator.
  • Still other controls may include acetylation levels present in a database (e.g., a table, electronic database, spreadsheet, etc.).
  • Acetylation levels of sirtuin substrates may be determined in a biological sample from a subject.
  • exemplary biological samples include samples comprising blood, urine, serum, saliva, cells, tissue, and/or hair. Samples may be obtained from a subject using standard techniques. Preferably, biological samples are obtained using minimally invasive, non-surgical procedures, such as, a needle biopsy for obtaining a tissue sample, etc.
  • Acetylation levels of sirtuin substrates may be determined by a variety of methods. Exemplary methods for determining acetylation levels of sirtuin substrates are provided in the exemplification section herein. Other suitable methods include, for example, immunoblotting, immunoprecipitation, mass spectrometry and combinations thereof.
  • sirtuin substrates may be immunoprecipitated from a biological sample (e.g., directly from urine or serum or from a lysate of cells, etc.) using an antibody specific for the substrate. The isolated proteins may then be run on an SDS-PAGE gel and blotted (e.g., to nitrocellulose or other suitable material) using standard procedures.
  • the blot may then be probed with an anti-acetyl specific antibody to determine the level of acetylation of the sirtuin substrates.
  • acetylated proteins may be immunoprecipitated from the biological sample as described using an anti-acetyl specific antibody. These proteins may then be separated, blotted and probed with the sirtuin substrate specific antibodies.
  • the sirtuin substrates may be immunoprecipitated from a biological sample followed by separation using mass spectrometry. In certain embodiments, it may be desirable to cleave the sirtuin substrates with a chemical or enzymatic cleavage reagent prior to conducting the mass spectrometry analysis.
  • mass spectrometry When using mass spectrometry as a means to determine acetylation levels of sirtuin substrates it may be useful to have acetylated and/or non-acetylated proteins or peptides that can be used as standards for the acetylated or non-acetylated polypeptide samples.
  • Gel electrophoresis, immunoprecipitation and mass spectrometry may be carried out using standard techniques, for example, such as those described in Molecular Cloning A Laboratory Manual, 2 nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989), Harlow and Lane, Antibodies: A Laboratory Manual (1988 Cold Spring Harbor Laboratory), G. Suizdak, Mass
  • Antibodies suitable for isolation and detection of sirtuin substrates may be purchased commercially from a variety of sources, such as, for example, Santa Cruz Biotechnology, Inc., Santa Cruz, CA or Abeam Inc., Cambridge, MA
  • Antibodies specific for acetylated lysine residues include the N-epsilon acetyl lysine antibodies ab409 and ab 17324 from Abeam Inc. (Cambridge, MA) and the pan-Acetyl sc-8649 antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
  • Antibodies specific for sirtuin substrates may also be produced using standard techniques. Exemplary techniques for the production of antibodies to sirtuin targets are described below. 3. Production of Sirtuin Substrate Specific Antibodies
  • the methods described herein utilize antibodies specific for sirtuin substrates, for example, the substrates listed in Table 1.
  • the term antibody is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab, F(ab')2 and Fv) so long as they exhibit the desired biological activity.
  • the sirtuin substrate specific antibodies may be polyclonal antibodies.
  • Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c.) or intraperitoneal (i.p.) injections of the relevant antigen and an adjuvant.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intraderrnally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • the sirtuin substrate specific antibodies may be monoclonal antibodies.
  • Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope except for possible variants that arise during production of the monoclonal antibody, such variants generally being present in minor amounts.
  • the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete or polyclonal antibodies.
  • the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred myeloma cells are those that fuse efficiently, support stable high- level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the SaIk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immuno absorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immuno absorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI- 1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-SEPHAROSETM, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • sirtuin substrate specific antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. MoI. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • the DNA encoding the antibodies also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81 :6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • the sirtuin substrate specific antibodies may be chimeric antibodies, e.g., antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567); Morrison et al. Proc. Natl. Acad. Sd. 81, 6851-6855 (1984).
  • chimeric antibodies e.g., antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies
  • the sirtuin substrate specific antibodies may be humanized antibodies.
  • Humanized forms of non-human (e.g. murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementarity determining region
  • humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the sirtuin substrate specific antibodies may be antibody fragments.
  • Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments.
  • antibody fragments Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)).
  • F(ab') 2 fragments can be isolated directly from recombinant host cell culture.
  • the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458.
  • the antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870 for example. Such linear antibody fragments may be monospecific or bispecific.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab') 2 fragments.
  • An "Fv” fragment is the minimum antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer).
  • variable domain interacts to define an antigen binding site on the surface of the V H -V L dimer.
  • the six CDRs confer antigen binding specificity to the antibody.
  • a single variable domain or half of an Fv comprising only three GDRs specific for an antigen has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the Fab fragment also designated as F(ab) also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab 1 fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which, the cysteine residue(s) of the constant domains have a free thiol group.
  • F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab') 2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.
  • Single-chain Fv or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to a small antibody fragments with two antigen- binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH- VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH- VL polypeptide chain
  • Sirtuin substrate specific antibodies may be purified using standard techniques. When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio/Technology 10:163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli.
  • cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
  • PMSF phenylmethylsulfonylfluoride
  • Cell debris can be removed by centrifugation.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • a protease inhibitor such as PMSF maybe included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography is the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc region that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human ⁇ l, ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., 1983, J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al., 1986, EMBO J 5:15671575).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a C H3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • the glycoprotein may be purified using adsorption onto a lectin substrate (e.g. a lectin affinity column) to remove fucose-containing glycoprotein from the preparation and thereby enrich for fucose-free glycoprotein.
  • a lectin substrate e.g. a lectin affinity column
  • the diagnostic, monitoring and screening methods described herein utilize one or more sirtuin substrates shown in Table 1.
  • the diagnostic, monitoring and screening methods described herein utilize one or more of the following sirtuin substrates: PARPl, VDAC, BBSl, AKRlBl, MIF, MYST2, MYST3, TAFl, TRPl, RAD51L3, DNA-PK, Ebpl, HB- EFG, NuMAl, CacyBP, Ku70, SFRS8, or TRRAP.
  • sirtuin substrates include a description of each of these substrates.
  • PARPl Poly (ADP -r ⁇ bose) polymerase 1
  • PARPs The involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-I, in mammalian longevity is reviewed in Burkle et al. (Int J Biochem Cell Biol. 37(5): 1043-53, 2005).
  • PARPs catalyse poly-ADP-ribosylation, a covalent post-translational protein modification in eukaryotic cells.
  • PARP-I and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents.
  • BER DNA base-excision repair
  • PARP-I is the main catalyst of poly-ADP-ribosylation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly( ADP -ribose). DNA-damage- induced poly-ADP-ribosylation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly-ADP-ribosylation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-I physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-I and WRN functionally cooperate in preventing carcinogenesis in vivo.
  • telomere-binding proteins TRF-I and TRF-2 Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity.
  • tankyrase-1, tankyrase-2, P ARP-2 as well as PARP-I have been found in association with telomeric DNA and are able to poly-ADP-ribosylate the telomere-binding proteins TRF-I and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.
  • Hassa et al. JBC, 280(49); 40450-40464, 2005
  • PARP-I directly interacts with p300/CREB-binding Protein and both subunits of NF- kappaB (p65 and p50) and synergistically coactivates NF-kappaB-dependent transcription.
  • PARP-I is acetylated in vivo at specific lysine residues by p300/CREB-binding protein upon stimulation.
  • acetylation of PARP-I at these residues is required for the interaction of PARP-I withp50 and synergistic coactivation of NF-kappaB by p300 and the Mediator complex in response to inflammatory stimuli.
  • VDAC or mitochondrial Porin
  • the VDAC protein is thought to form the major pores through which adenine nucleotides cross the outer mitochondrial membrane.
  • VDAC has also been implicated in the formation of the mitochondrial permeability transition pore complex in apoptotic cells.
  • This complex formed by VDAC, adenine nucleotide translocator (ANT) and cyclophilin D (CypD) is thought to allow the mitochondria to undergo metabolic uncoupling and irreversible morphologic changes that ultimately destroy the mitochondria during apoptosis. Further confirmation of the importance of VDAC can be found in Lemasters et al. (Biochim Biophys Acta. 1762(2): 181-90, 2006). Despite a detailed understanding of their metabolism, mitochondria often behave anomalously.
  • VDAC voltage- dependent anion channels
  • hexokinase In cancer cells, highly expressed hexokinase binds to and inhibits VDAC to suppress mitochondrial function while stimulating glycolysis, but an escape mechanism intervenes when glucose-6-phosphate accumulates and dissociates hexokinase from VDAC. Similarly, glucokinase binds mitochondria of insulin- secreting beta cells, possibly blocking VDAC and suppressing mitochondrial function. Lemasters et al. goes on to propose that glucose metabolism leads to glucose-6-phosphate-dependent unbinding of glucokinase, relief of VDAC inhibition, release of ATP from mitochondria and ATP-dependent insulin release.
  • Bardet— Biedl syndrome is a rare developmental disorder that exhibits significant clinical and genetic heterogeneity. Although modeled initially as a purely recessive trait, recent data have unmasked an oligogenic mode of disease transmission, in which mutations at different BBS loci can interact genetically in some families to cause and/or modify the phenotype. There have been several recent advances in elucidating both genetic and cellular aspects of this phenotype and their potential application in understanding the genetic basis of phenotypic variability and oligogenic inheritance. Aldo-keto reductase family 1, member Bl (AKRlBl) and the aldo-keto reductase superfamily.
  • Aldehyde reductase [EC 1.1.1.2] and aldose reductase [EC 1.1.1.21] are monomeric NADPH-dependent oxidoreductases having wide substrate specificities for carbonyl compounds. These enzymes are implicated in the development of diabetic complications by catalyzing the. reduction of glucose to sorbitol. Enzyme inhibition as a direct pharmacokinetic approach to the prevention of diabetic complications resulting from the hyperglycemia, of diabetes has not been effective because of nonspecificity of the inhibitors and some appreciable side effects.
  • Aldehyde reductase and aldose reductase are monomeric NADPH-dependent oxidoreductases having wide substrate specificities for carbonyl compounds. These enzymes are implicated in the development of diabetic complications by catalyzing the reduction of glucose to sorbitol. Enzyme inhibition as a direct pharmacokinetic approach to the prevention of diabetic complications resulting from the hyperglycemia of diabetes has not been effective because of nonspecificity of the inhibitors and some appreciable side effects. To understand the structural and evolutionary relationship of these enzymes, the cDNAs coding for aldose and aldehyde reductases from human liver and placental cDN A libraries were cloned and sequenced.
  • Human placental aldose reductase has a 65% identity to human liver and placental aldehyde reductase. The two sequences have significant identity to 2,5- diketogluconic acid reductase from corynebacterium, frog rho-crystallin, and bovine lung prostaglandin F synthase (reductase). Southern hybridization analysis of human genomic DNA indicates a multigene system for aldose reductase, suggesting the existence of additional proteins. Thus, the aldo-keto reductase superfamily of proteins may have a more significant and hitherto not fully appreciated role in general cellular metabolism. Macrophage migration inhibitory factor (MIF).
  • MIF Macrophage migration inhibitory factor
  • MIF cytokine macrophage migration inhibitory factor
  • Polymorphisms of the human MIF gene have been associated with increased susceptibility to or severity of juvenile idiopathic and adult rheumatoid arthritis, ulcerative colitis, atopy, or sarcoidosis. Whether these MIF polymorphisms affect the susceptibility to and outcome of sepsis has not yet been examined. Analyses of MIF genotypes in patients with sepsis may help to classify patients into risk categories and to identify those patients who may benefit from anti- MIF therapeutic strategies.
  • the MYST family ofhistone acetyltransferases (MYST2 andMYST3). Multiple chromatin modifying proteins and multisubunit complexes have been characterized in recent years. Histone acetyltransferase (HAT) activities have been the most thoroughly studied, both biochemically and functionally. More is being learned about the current knowledge on a specific group of proteins that is extremely well conserved throughout evolution, the MYST family ofhistone acetyltransferases. These proteins play critical roles in various nuclear functions and the control of cell proliferation.
  • TAFl The therapeutic potential of TAFl is reviewed in Kaji et al. (J Med Invest. 52 Suppl:280-3, 2005).
  • Pathological findings in dystonia have been unclear.
  • X-linked recessive dystonia-parkinsonism XDP, DYT3
  • XDP X-linked recessive dystonia-parkinsonism
  • striosomal neurons inhibit nigrostriatal dopaminergic neurons via GAB Aergic innervation
  • the striosomal lesion could account for dopamine excess in the striatum, which in turn causes a hyperkinetic state or dystonia.
  • Kaji et al. also identified the causative gene as one of the general transcription factor genes, TAFl .
  • XDP has certain similarities to Huntington disease not only in pathological and clinical findings, but also the molecular mechanism, which disturbs expression of genes essential for striatal neurons, such as DRD2. Therapeutic intervention may become possible through pharmacological measures that affect gene expression. TPR (or TPRl) or translocated promoter region (to activated MET oncogene).
  • TRP 1 functions as a component of the cytoplasmic fibrils of the nuclear pore complex implicated in nuclear protein import. Its N-terminus is involved in activation of oncogenic kinases. It is localized to the cytoplasmic surface of the nuclear pore complex (NPC). The assembly of the NPC is a stepwise process in which Trp- containing peripheral structures assemble after other components, including TPRl . TRPl is highly expressed in testis, lung, thymus, spleen and brain, with lower levels in heart, liver and kidney. It is involved in tumorigenic rearrangements with the MET, TRK or RAF genes.
  • RAD51-like 3 (RAD51L3).
  • the importance of RAD51 and related proteins is discussed in Tarsounas et al. (Cell Cycle, 4(5):672-4, 2005).
  • HR homologous recombination
  • HR also plays a role in the maintenance of eukaryotic telomeres; cells defective in the recombinational repair proteins RAD51D or RAD54 exhibit telomere shortening and end-to-end chromosome fusions.
  • Tarsounas et al. goes on to discuss the way in which HR contributes to telomere protection and elongation in mammalian cells. Understanding the mechanisms by which HR promotes telomere maintenance has important implications for genomic stability and tumorigenesis.
  • DNA-PK protein kinase, DNA-activated.
  • DNA-PK DNA-dependent protein kinase
  • the DNA-dependent protein kinase (DNA-PK) is a trimeric factor originally identified as an enzyme that becomes activated upon incubation with DNA. Genetic defects in either the catalytic subunit (DNA-PK(CS)) or the two Ku components of DNA-PK result in immunodeficiency, radiosensitivity, and premature aging. This combined phenotype is generally attributed to the requirement for DNA-PK in the repair of DNA double strand breaks during various biological processes.
  • DNA- PK(CS) a member of the growing family of phosphatidylinositol 3-kinases, participates in signal transduction cascades related to appptotic cell death, telomere maintenance and other pathways of genome surveillance.
  • DNA-PK(CS) a member of the growing family of phosphatidylinositol 3-kinases, participates in signal transduction cascades related to appptotic cell death, telomere maintenance and other pathways of genome surveillance.
  • These manifold functions of DNA-PK(CS) have been associated with an increasing number of protein interaction partners and phosphorylation targets.
  • Estrogen receptor binding protein or Ebpl The importance of Ebpl is described in Zhang et al. (PNAS, 102(28):9890-5, 2005). Down-regulation of the androgen receptor (AR) is being evaluated as an effective therapy for the advanced stages of prostate cancer. It is reported that Ebpl, a protein identified by its interactions with the ErbB3 receptor, down-regulates expression of AR and AR- regulated genes in the LNCaP prostate cancer cell line. Using microarray analysis, Zhang et al. identified six endogenous AR target genes, including the AR itself, that are down-regulated by ebpl overexpression.
  • Chromatin immunoprecipitation assays revealed that Ebpl was recruited to the prostate-specific antigen gene promoter in response to the androgen antagonist bicalutamide, suggesting that Ebpl directly affected the expression of AR-regulated genes in response to androgen antagonists. Ebpl expression was reduced in cells that had become androgen-independent.
  • HB-EFG Heparin-binding epidermal growth factor-like growth factor (HB- EGF) regulates survival of midbrain dopaminergic neurons.
  • Heparin-binding epidermal growth factor-like growth factor is a member of the EGF-family of ligands signaling via the EGF-receptor tyrosine kinase.
  • HB-EGF which is expressed in close proximity of developing mesencephalic dopaminergic neurons, promotes the survival of TH -positive neurons in vitro.
  • the survival promoting effect of HB-EGF is mediated via astroglial cells and utilizes the MAPK as well as the Akt- signaling pathway.
  • Most notably endogenous HB-EGF significantly contributes to the survival of TH-+ neurons in control cultures, suggesting a relevant developmental role of HB-EGF for dopaminergic neurons.
  • NuMAl Sun et al. describes the role of NuMAl in vertebrate cells as an interesting multifunctional protein (Front Biosci. 1 1 :1137-46. 2006).
  • the 236 kDa large coiled-coil protein NuMA (nuclear mitotic apparatus protein) plays diverse important roles in vertebrate cells. It is an important component of the nuclear matrix in interphase cells, and is possibly involved in nuclear re-assembly after mitosis. NuMA's function in spindle microtubule organization is regulated by RanGTP and Pins-related protein LGN.
  • NuMA becomes dephosphorylated, loses its association with dynein/dynactin, and releases from spindle poles after anaphase onset to allow spindle disassembly and reformation of interphase daughter nuclei.
  • the cell-cycle- dependent phosphorylation of NuMA is regulated by the balanced activities of protein kinases and phosphatases. It has been shown that phosphorylation of NuMA by cyclin B/cdc2 kinase allows NuMA to release from the nucleus and to associate with centrosomes and/or microtubules at the spindle poles, while NuMA's dephosphorylation due to the cyclin B degradation allows NuMA to dissociate from the spindle poles after anaphase onset.
  • NuMA interferes with spindle-associated dynein localization and promotes multipolar spindle formation and cancer.
  • NuMA is absent in many kinds of non-proliferating cells and highly differentiated cells.
  • NuMA also functions during meiotic spindle organization in male and female germ cells. Degradation, of NuMA results in the breakdown of normal nuclear structure, and has been used as a marker of cell apoptosis.
  • CacyBP Calcyclin binding protein
  • CacyBP Calcyclin binding protein
  • CacyBP/SIP calcyclin- binding protein/Siah-interacting protein
  • CacyBP/SIP may play a role in cardiomyogenic differentiation and possibly protection of cardiomyocytes during hypoxia/reoxygenation injury.
  • Ku 70 or ATP-dependent DNA helicase II, 70 kDa subunit The biology of Ku and its potential oncogenic role in cancer is described in Gullo et al. (Biochim Biophys Acta. 2006 Jan 25).
  • Ku is a heterodimeric protein made up of two subunits, Ku70 and Ku80. It was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. It is a highly versatile regulatory protein that has been implicated in multiple nuclear processes, e.g., DNA repair, telomere maintenance and apoptosis. Accordingly, Ku is thought to play a crucial role in maintenance of chromosomal integrity and cell survival.
  • SFRS8 (Splicing Factor Arginine/Serine-rich 8). The importance of splicing factors in disease is discussed in Wang et al. (J Neurochem. 88(5): 1078-90, 2004).
  • Tau exon 10 whose missplicing causes frontotemporal dementia, is regulated by an intricate interplay of cis elements and trans factors.
  • Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system.
  • exon 10 of the gene is an alternatively spliced cassette which is adult-specific and which codes for a microtubule binding domain. Mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP).
  • FTDP frontotemporal dementia
  • TRRAP TBP-free TAF Il-containing-type HAT complex subclasses, which contain hGCN5 HAT and TRRAP, appear to act as common coactivator complexes for nuclear receptors. However, their physiological significance with respect to each nuclear receptor remains to be established. To address this issue, hepatic cell lines (HepG2) have been generated with reduced endogenous TRRAP expression through antisense RNA expression or with overexpressed TRRAP or other major coactivators. The ligand-induced transactivation function of liver X receptor alpha (LXRalpha) and farnesoid X receptor/bile acid receptor reflected TRRAP expression levels, while that of PPARgamma did not.
  • LXRalpha liver X receptor alpha
  • farnesoid X receptor/bile acid receptor reflected TRRAP expression levels, while that of PPARgamma did not.
  • TRRAP contains two potential LXRalpha-interacting domains in the C-terminal and central domains.
  • Liver X receptor alpha (LXRalpha) and liver X receptor beta (LXRbeta) are oxysterol receptors that regulate multiple target genes involved in cholesterol homeostasis.
  • the methods may involve, for example, contacting at least one acetylated sirtuin substrate from Table 1 with a sirtuin protein and determining the level of acetylation of the sirtuin substrate.
  • the methods may involve, for example, contacting at least one acetylated sirtuin substrate from Table 1 with a sirtuin protein, cleaving the sirtuin substrate, and determining the fluorescence polarization value of the substrate pool.
  • the methods may involve, for example, contacting at least one acetylated sirtuin substrate from Table 1 with a sirtuin protein and determining the level of acetylation of the sirtuin substrate using mass spectrometry.
  • the invention provides methods for identifying compounds that modulate the activity of a sirtuin protein. The methods may involve, for example, contacting at least one acetylated sirtuin substrate from Table 1 with a sirtuin protein in the presence of a test compound and determining the acetylation level of the sirtuin substrate.
  • the methods may involve, for example, contacting at least one acetylated sirtuin substrate from Table 1 with a sirtuin protein in the presence of a test compound, cleaving the sirtuin substrate, and determining the fluorescence polarization value of the substrate pool.
  • the methods may involve, for example, contacting at least one acetylated sirtuin substrate from Table 1 with a sirtuin protein in the presence of a test compound and determining the level of acetylation of the sirtuin substrate using mass spectrometry.
  • the sirtuin substrates shown in Table 1 , or fragments thereof may be used in association with various types of deacetylation assays to determine sirtuin activity and/or to identify compounds that modulate sirtuin activity.
  • the sirtuin substrates shown in Table 1 , or fragments thereof may be used in association with a fluorescence based assay such as the assay commercially available from Biomol, e.g., the SIRTl Fluorimetric Drug Discovery Kit (AK-555), SIRT2 Fluorimetric Drug Discovery Kit (AK-556), or SIRT3 Fluorimetric Drug Discovery Kit (AK-557)
  • sirtuin substrates shown in Table 1, or fragments thereof include radioimmunoassays (RIA), scintillation proximity assays, HPLC based assays, and reporter gene assays (e.g., for transcription factor targets).
  • RIA radioimmunoassays
  • a sirtuin substrate shown in Table 1, or fragments thereof may be substituted for the substrate used in the referenced assays.
  • the sirtuin substrates shown in Table 1, or fragments thereof may be used in association with a fluorescence polarization assay. Examples of fluorescence polarization assays are described herein and are also described in PCT Application No. PCT/US06/007748.
  • the sirtuin substrates shown in Table 1 , or fragments thereof may be used in association with mass spectrometry based assays. Examples of mass spectrometry based assays are. described herein and are also described in U.S. Provisional Application No. 60/792,126.
  • a sirtuin protein refers to a member of the sirtuin deacetylase protein family, or preferably to the sir2 family, which include yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP_501912), and human SIRTl (GenBank Accession No. NMJM2238 and NP_036370 (or AF083106)) and SIRT2 (GenBank Accession No. NMJ)12237, NM_030593, NPJB6369, NP_085096, and AF083107) proteins.
  • HST genes additional yeast Sir2-like genes termed "HST genes” (homologues of Sn; two) HSTl, HST2, HST3 and HST4, and the five other human homologues hSIRT3, hSIRT4, hSIRT5, hSIRT ⁇ and hSIRT7 (Brachmann et al. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC 260:273).
  • Homologs e.g., orthologs and paralogs, domains, fragments, variants and derivatives of the foregoing may also be used in accordance with the methods described herein.
  • a SIRTl protein refers to a member of the sir2 family of sirtuin deacetylases.
  • a SIRTl protein includes yeast Sir2 (GenBank Accession No. P53685), C. elegans Sir-2.1 (GenBank Accession No. NP_501912), human SIRTl (GenBank Accession No. NM_012238 or NPJ)36370 (or AF083106)), and human SIRT2 (GenBank Accession No.
  • a SIRTl protein includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in
  • SIRTl proteins include polypeptides comprising all or a portion of the amino acid sequence set forth in GenBank Accession Nos. NP_036370., NP_501912, NP_085096, NP 036369, or P53685; the amino acid sequence set forth in GenBank Accession Nos.
  • SIRTl proteins also include homologs (e.g., orthologs and paralogs), variants, or fragments, of GenBank Accession Nos. NP_036370, NP_501912, NP_085096, NP_036369, or P53685.
  • the methods described herein may be used to determine the activity of a SIRT3 protein and/or identify compounds that modulate a SIRT3 protein.
  • a S1RT3 protein refers to a member of the sirtuin deacetylase protein family and/or to a homolog of a SIRTl protein.
  • a S1RT3 protein includes human SIRT3 (GenBank Accession No. AAH01042, NP_036371, or NP_001017524) and mouse SIRT3 (GenBank Accession No. NP_071878) proteins, and equivalents and fragments thereof.
  • a SIRT3 protein in another embodiment, includes a polypeptide comprising a sequence consisting of, or consisting essentially of, the amino acid sequence set forth in GenBank Accession Nos. AAHOl 042, NP_036371, NP_001017524, or NP_071878.
  • SIRT3 proteins include polypeptides comprising all or a portion of the amino acid sequence set forth in GenBank
  • SIRT3 proteins also include homologs (e.g., orthologs and paralogs), variants, or fragments, of GenBank Accession Nos. AAHOl 042, NP_036371, NP_001017524, or NP_071878.
  • a biologically active portion of a sirtuin may be used in accordance with the methods described herein.
  • a biologically active portion of a sirtuin refers to a portion of a sirtuin protein having a biological activity, such as the ability to deacetylate.
  • Biologically active portions of sirtuins may comprise the core domain of a sirtuin.
  • Biologically active portions of SIRTl having GenBank Accession No. NP 036370 that encompass the NAD+ binding domain and the substrate binding domain may include without limitation, amino acids 62-293 of GenBank Accession No. NP_036370, which are encoded by nucleotides 237 to 932 of GenBank Accession No. NM_012238. Therefore, this region is sometimes referred to as the core domain.
  • Other biologically active portions of SIRTl also sometimes referred to as core domains, include about amino acids 261 to 447 of GenBank Accession No.
  • NP_036370 which are encoded by nucleotides 834 to 1394 of GenBank Accession No. NM_012238; about amino acids 242 to 493 of GenBank Accession No. NP_036370, which are encoded by nucleotides 777 to 1532 of GenBank Accession No. NM_012238; or about amino acids 254 to 495 of GenBank Accession No. NP_036370, which are encoded by nucleotides 813 to 1538 of GenBank Accession No. NM_012238.
  • a biologically active portion of a sirtuin may be a fragment of a SIRT3 protein that is produced by cleavage with a mitochondrial matrix processing peptidase (MPP) and/or a mitochondrial intermediate peptidase (MIP).
  • MPP mitochondrial matrix processing peptidase
  • MIP mitochondrial intermediate peptidase
  • Sirtuin proteins for use in the assays described herein may be purchased commercially or purified using standard procedures.
  • human SIRTl Catalog #SE-239
  • human SIRT2 Catalog #SE-251
  • human SIRT3 Catalog #SE-270
  • Methods for expression and purification of human SIRTl and human SIRT3 are described, for example, in PCT Application No. PCT/US06/007748.
  • the concentration of sirtuin protein used in an in vitro assays described herein may be about 0.01 nM to about 1 ⁇ M, or from about 0.1 nM to about 100 nM sirtuin protein.
  • the concentration of sirtuin substrate used in an in vitro assay described herein may be a concentration around the determined Km of the substrate, e.g., which maybe determined empirically for a given substrate/enzyme combination.
  • Sirtuin substrates that may be used in accordance with the methods described herein include any of the polypeptides shown in Table 1, or fragments thereof.
  • the substrates utilized in the reaction will comprise at least one acetylated lysine residue.
  • combinations of two or more sirtuin substrates shown in Table 1, or fragments thereof, may be used in accordance with the methods described herein.
  • the sirtuin substrate is not Ku70.
  • sirtuin substrates for use in accordance with the methods described herein may be fragments of the polypeptides listed in Table 1. Suitable fragments of the sirtuin substrates shown in Table 1 typically contain at least about 5, 8, 10, 15, 20, 25, 30, 40, 50, or more, consecutive amino acid residues of the proteins listed in Table 1. In certain embodiments, fragments of the sirtuin substrates shown in Table 1 may comprise about 5-100, 5-50, 5-25, 5-10, 10-50, 10-25, or 25-50 consecutive amino acid residues of the proteins listed in Table 1. Fragments of the sirtuin substrates shown in Table 1 will comprise at least one lysine residue that may be deacetylated by a sirtuin protein.
  • Table 1 specifies exemplary lysines for each sirtuin substrate protein that may be deacetylated by a sirtuin protein.
  • fragments of a sirtuin substrate protein comprise a fragment of a protein shown in Table 1 that comprises at least one of the acetylated lysine residues specified in column 5 of Table 1 and at least , 8, 10, 15, 20, 25, 30, 40, 50, or more, flanking amino acid residues from the sirtuin substrate protein sequence.
  • the flanking amino acid residues may be from residues to the N-terminus of the acetylated lysine, the C-terminus of the acetylated lysine, or a combination thereof.
  • a sirtuin substrate may be desirable to modify the sequence of a sirtuin substrate, or a fragment thereof, to remove one or more lysine and/or arginine residues.
  • a sirtuin substrate that comprises lysine residues but no other positively charged residues (such as arginine) such that upon cleavage with a protease, such as trypsin, cleavage of the sirtuin substrate will be dependent upon the acetylation state of the lysine residues in the substrate and independent of arginine residues.
  • sirtuin substrate that contains one or more lysine residues located only in desired locations within the substrate, e.g., between a fluorophore and a high molecular weight or bulky group.
  • sirtuin substrate that contains only a single lysine residue and is free of any arginine residues.
  • a lysine residue may be located in the sirtuin substrate such that upon cleavage of the sirtuin substrate at or near the site of the lysine residue, the size differential between an uncleaved sirtuin substrate and the portion of a cleaved sirtuin substrate containing the fluorophore is sufficient to provide a change in fluorescence polarization.
  • One or more lysine and/or arginine residues may be removed from a sirtuin substrate sequence by replacing the amino acid residue with a different amino acid residue or by deleting the amino acid residue from the sequence without substitution of a different amino acid.
  • one or more lysine and/or arginine residues may be replaced using a conservative amino acid substitution wherein said substitution is not lysine or arginine.
  • Sirtuin substrates that may be used in accordance with the methods described herein can be synthesized according to conventional methods.
  • the sirtuin substrates may include naturally occurring polypeptides, polypeptides prepared by genetic recombination techniques, and polypeptides prepared by synthetic techniques.
  • the sirtuin substrates may be fused with other polypeptides (for example, glutathione-S- transferase, HA tag, FLAG tag, etc.) for convenience of purification, etc.
  • the sirtuin substrates may comprise structural units other than amino acids so long as it serves as a substrate for a sirtuin.
  • sirtuin substrate polypeptides typically, synthesis of full length sirtuin substrate polypeptides is carried out by expressing the polypeptide in a host cell (either prokaryotic or eukaryotic) and purifying the polypeptide using standard techniques. Synthesis of fragments of sirtuin substrates may be achieved by adding amino acids, residue by residue, from the carboxyl terminus of the amino acid sequence of interest. Further, some of the peptide fragments synthesized in that way may be linked together to from a larger peptide molecule. For measuring sirtuin deacetylase activity, the sirtuin substrate needs to be acetylated before the reaction is conducted.
  • An exemplary method of amino acid acetylation includes acetylation of amino acids, whose ⁇ -amino groups and side-chain amino groups are blocked with protecting groups, with acetic anhydride, N-hydroxysuccinimide acetate, or similar reagents. These acetylated amino acids are then used to synthesize peptides comprising acetylated lysine residues, for example, using the solid-phase method. Generally, acetylated polypeptides can be synthesized using a peptide synthesizer according to the Fmoc method.
  • acetylated sirtuin substrates may be produced by contacting the substrate polypeptides with an acetylase.
  • the sirtuin substrates may comprise at least one fluorophore. Exemplary fluorophores that may be used in accordance with the methods described herein are provided below in Table 1. Methods for labeling a peptide with a fluorophore are known in the art, and thus, can be conducted according to conventional methods.
  • the fluorophore may be covalently linked or conjugated to the sirtuin substrate so as not to interfere with emission of fluorescence from the label, deacetylation of the lysine residue(s), and/or cleavage of the sirtuin substrate with a protease.
  • Table 1 Commercially available fluorophores and their excitation and emission maxima.
  • the sirtuin substrate may comprise a fluorophore and a high molecular weight group or bulky group.
  • the high molecular weight or bulky group is separated from the fluorophore by at least one lysine residue.
  • the sirtuin substrate is susceptible to cleavage at or near the lysine residue by a cleavage reagent, such as a protease.
  • a cleavage reagent such as a protease.
  • the sirtuin substrate is resistant to cleavage and remains intact upon contact with a cleavage reagent.
  • the fluorophore is separated from the high molecular weight or bulky group thereby increasing the fluorescent polarization value of the sample.
  • Fluorescence polarization is based upon the theory that when a fluorescently labeled molecule is excited with plane-polarized light of the correct wavelength it will emit polarized light after its characteristic emission lifetime (usually nanoseconds). During the time between excitation and emission
  • fluorescence lifetime the molecule tumbles randomly with respect to the original plane of excitation.
  • the rate of tumbling is directly proportional to its molecular volume or size. Small molecules tumble rapidly, while large molecules tumble much more slowly. If the molecule tumbles rapidly during its fluorescence lifetime, the fluorescence emission is depolarized relative to the excitation. If the fluorophore is associated with a much bigger molecule, the tumbling is slow and the observed emission remains more polarized relative to the excitation. Thus by measuring the extent of fluorescence polarization, one can tell if a fluorophore is associated with a larger molecule.
  • FP is useful for high throughput screening (HTS) assays.
  • HTS high throughput screening
  • FP is also amenable to performing assays in real-time, directly in solution and without the need for an immobilized phase.
  • Polarization values can be measured repeatedly both before and after the addition of reagents since measuring the samples is rapid and does not destroy the sample.
  • the polarization value (P) for a given molecule is proportional to the molecule's rotational relaxation time, or the amount of time it takes the molecule to rotate through an angle of 68.5°.
  • the smaller the rotational correlation time the faster the molecule rotates, and the less polarization will be observed.
  • the larger the rotational correlation time the slower the molecule rotates, and the more polarization will be observed.
  • Rotational relaxation time is related to viscosity (i), absolute temperature (T), molecular volume (V), and the gas constant (R). The rotational relaxation time is generally calculated according to the following formula:
  • the methods described herein utilize fluorescence polarization to determine acetylation level of a sirtuin substrate.
  • the starting sirtuin substrate has a slow rotational correlation time.
  • the fluorophore is attached to a portion of the sirtuin substrate that, upon cleavage at the lysine residue, is released and exhibits a relatively fast rotational correlation time (e.g., in comparison to the rotational correlation time of the uncleaved sirtuin substrate).
  • Cleavage of the sirtuin substrate is indirectly proportional to the acetylation state of the sirtuin substrate and directly proportional to the fluorescence polarization of the sirtuin substrate.
  • the cleavage of the peptide substrate is increased and the fluorescence polarization value of the sirtuin substrate will decrease (e.g., the amount of rotation of the fluorophore increases upon release from the larger sirtuin substrate molecule therefore decreasing the rotational correlation time).
  • the fluorescence polarization level is calculated using the following formula:
  • /(H) is the fluorescence detected in the plane parallel to the excitation light
  • /(-L) is the fluorescence detected in the plane perpendicular to the excitation light.
  • the fluorescence polarization as compared to a control will increase, as a greater proportion of the sirtuin substrate pool will remain acetylated and therefore will not be cleaved so that the fluorophore remains associated with the larger sirtuin substrate molecule.
  • the fluorescence polarization as compared to a control will decrease, as a greater proportion of the sirtuin substrate pool will become deacetylated and therefore can be cleaved at the non-acetylated lysine residue to release the fluorophore from the larger sirtuin substrate molecule.
  • Fluorescence polarization may be monitored in any of three different states: steady state, transient state, or dynamic state.
  • transient state FP the excitation light source is flashed on the sample and polarization of the emitted light is monitored by turning on the photomultiplier tube after the excitation light source is turned off. This reduces interference from light scatter, as fluorescence lasts longer than light scatter, but some fluorescence intensity is lost.
  • steady state FP excitation light and emission monitoring are continuous (i.e., the excitation source and photomultiplier tube are on continuously). This results in measurement of an average tumbling time over the monitoring period and includes the effects of scattered light.
  • Dynamic FP may be monitored in either the time- or frequency-domain.
  • Dynamic fluorescence techniques involve determining the lifetime of the fluorescent molecule in nanoseconds.
  • the theory of dynamic fluorescence monitoring is described in "Principles of Fluorescence Spectroscopy” (Lakowicz, Plenum Press, 1983). Whereas steady state FP provides an average or “snapshot" of the fluorescence phenomena, dynamic FP allows one to observe the individual contributions of the fluorescent components in the system being studied.
  • Use of these three fluorescence techniques is described by Kumke, et al. (1995. Anal. Chem. 67, 3945-3951), and Devlin, et al. (1993. Clin. Chem. 39, 1939-1943).
  • Methodology for carrying out fluorescence polarization is set forth in "Fluorescence Polarization. Technical Resource Guide, Third Edition", available from PanVera Corporation, Madison, Wis., USA.
  • High molecular weight groups or bulky groups tliat may be used in association with the sirtuin substrates described herein include anything that is sufficient to permit a differential in the fluorescence polarization value of the fluorophore when it is associated with the uncleaved sirtuin substrate as opposed to when it has been cleaved, or released, from the sirtuin substrate molecule.
  • Examples of high molecular weight groups or bulky groups include, for example, polypeptides, nucleic acids, carbohydrates, etc.
  • the high molecular weight or bulky group may simply be a portion of the sirtuin substrate itself (e.g., the lysine residue is placed such that a very small portion of a larger sirtuin substrate molecule is released upon cleavage).
  • the high molecular weight or bulky group may be covalently attached or non-covalently bound to the sirtuin substrate.
  • the sirtuin substrate may comprise a binding site and the high molecular weight group or bulky group may be a binding moiety that is non- covalently bound to the binding site.
  • association between the binding site and the binding moiety may be carried out either before or after cleavage of the sirtuin substrate with a cleavage reagent.
  • Various binding site/binding moiety pairs may be used in association with the sirtuin substrate molecules, such as for example, a biotin/avidin complex, a biotin/streptavidin complex, an Fc region (or portion thereofyprotein A or protein G, an antigen (e.g., a peptide sequence)/antibody, a ligand/receptor molecule, an aptamer/aptamer binding pair, etc.
  • the antigen When using an antigen as the binding site, the antigen may be a portion of the sirtuin substrate itself, or may be a sequence that is added on, such as, glutathione S-transferase (GST), or an HA, myc, or FLAG tag, for the purpose of interaction with a given antibody.
  • GST glutathione S-transferase
  • Methods for producing antibodies are well known in the art and are described herein.
  • Antibodies specific for a variety of sequences, such as, for example, GST, HA, Myc or FLAG tags, are commercially available.
  • the methods described herein utilize a cleavage reagent that cleaves sirtuin substrates containing non-acetylated lysine residues but will not cleave sirtuin substrates containing acetylated lysine residues.
  • the cleavage reagent may be a chemical or enzymatic reagent.
  • the cleavage reagent is a protease, such as, for example, a protease that cleaves at or near a lysine residue.
  • proteases included, for example, lysylendopeptidase, endoproteinase, Lys-C, plasmin, calpain, or trypsin.
  • the methods described herein are carried out under conditions which permit deacetylation of the peptide substrate by a sirtuin deacetylase.
  • the methods described herein for determining sirtuin activity and/or for identifying a compound that modulates sirtuin activity utilize mass spectrometry for determining the level of acetylation of a sirtuin substrate.
  • the presence of an acetyl group on a polypeptide may be determined by a +42 Da molecular weight shift (per acetyl group) as compared to the unmodified polypeptide.
  • Mass spectrometry or simply MS encompasses any spectrometric technique or process in which molecules are ionized and separated and/or analyzed based on their respective molecular weights.
  • mass spectrometry and MS encompass any type of ionization method, including without limitation electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI) and other forms of atmospheric pressure ionization (API), and laser irradiation.
  • Mass spectrometers may be combined with separation methods such as gas chromatography (GC) and liquid chromatography (LC). GC or LC separates the components in a mixture, and the components are then individually introduced into the mass spectrometer; such techniques are generally called GC/MS and LC/MS, respectively.
  • MS/MS is an analogous technique where the first-stage separation device is another mass spectrometer. In LC/MS/MS, the separation methods comprise liquid chromatography and MS.
  • MS can refer to any form of mass spectrometry; by way of non-limiting example, LC/MS encompasses LC/ESI MS and LC/M ALDI-TOF MS.
  • mass spectrometry and MS include without limitation APCI MS; ESI MS; GC MS; MALDI-TOF MS; LC/MS combinations; LC/MS/MS combinations; MS/MS combinations; etc.
  • MS include, for example, M ALDI-TOF-TOF MS, MALDI Quadrupole-time-of-flight (Q-TOF) MS, electrospray ionization (ESI)- TOF MS, ESI-Q-TOF 5 ESI-TOF-TOF, ESI-ion trap MS, ESI Triple quadrupole MS, ESI Fourier Transform Mass Spectrometry (FTMS), MALDI-FTMS, MALDI-Ion Trap-TOF, ESI-ion Trap TOF, surface-enhanced laser desorption/ionization (SELDI), MS/MS/MS, ESI-MS/MS, quadrupole time-of-flight mass spectrometer QqTOF MS, MALDI-QqTOFMS, ESI-QqTOF MS, and chip capillary electrophoresis (chip-CE)- QqTOF MS, etc.
  • FTMS Fourier Transform Mass Spectrometry
  • MS MS-chromatography
  • High-pressure liquid chromatography is a separative and quantitative analytical tool that is generally robust, reliable and flexible.
  • Reverse-phase is a commonly used stationary phase that is characterized by alkyl chains of specific length immobilized to a silica bead support.
  • RP-HPLC is suitable for the separation and analysis of various types of compounds including without limitation biomolecules, (e.g., glycoconjugates, proteins, peptides, and nucleic acids, and, with mobile phase supplements, oligonucleotides).
  • biomolecules e.g., glycoconjugates, proteins, peptides, and nucleic acids, and, with mobile phase supplements, oligonucleotides.
  • EI electrospray ionization
  • liquid samples can be introduced into a mass spectrometer by a process that creates multiple charged ions (WiIm et al., Anal. Chem. 68:1, 1996).
  • multiple ions can result in complex spectra and reduced sensitivity.
  • peptides and proteins are injected into a column, typically silica based Cl 8.
  • An aqueous buffer is used to elute the salts, while the peptides and proteins are eluted with a mixture of aqueous solvent (water) and organic solvent
  • the aqueous phase is generally HPLC grade water with 0.1 % acid and the organic solvent phase is generally an HPLC grade acetonitrile or methanol with 0.1% acid.
  • the acid is used to improve the chromatographic peak shape and to provide a source of protons in reverse phase LC/MS.
  • the acids most commonly used are formic acid, triflouroacetic acid, and acetic acid.
  • RP HPLC compounds are separated based on their hydrophobic character.
  • MALDI-TOF MS is a technique in which a UV-light absorbing matrix and a molecule of interest (analyte) are mixed and co-precipitated, thus forming analyte:matrix crystals.
  • the crystals are irradiated by a nanosecond laser pulse. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. Nevertheless, matrix molecules transfer their energy to analyte molecules, causing them to vaporize and ionize.
  • the ionized molecules are accelerated in an electric field and enter the flight tube. During their flight in this tube, different molecules are separated according to their mass to charge (m/z) ratio and reach the detector at different times. Each molecule yields a distinct signal.
  • the method may be used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides', with molecular masses between about 400 and about 500,000 Da, or higher.
  • MALDI-MS is a sensitive technique that allows the detection of low (10 ⁇ 15 to 10 "18 mole) quantities of analyte in a sample.
  • Electrospray ionization may be used for both very large and small molecules.
  • the electrospray process produces multiply charged ana ⁇ ytes, making it somewhat easier to detect larger analytes such as proteins.
  • small molecules can be measured readily in the absence of matrix.
  • the MALDI process requires a matrix, which may make it more difficult to analyze small molecules, for example, with molecular weights of less than about 700 daltons.
  • sensitivity decreases as the molecular weight of a molecule increases.
  • the detection sensitivity of molecules with molecular weights in the range of about 10,000 daltons may be an order of magnitude or more lower than detection sensitivity of molecules with molecular weights in the range of about 1,000 daltons.
  • Use and detection of a coding moiety and/or labels with a different, for example lower, molecular weight than the analyte can therefore enhance the sensitivity of the assay.
  • Sensitivity can also be increased by using a coding moiety and/or that is very amenable to ionization.
  • a mass spectrometer such as a quadropole, an ion trap, a TOF, a FTICR, or a tandem mass spectrometer
  • the higher molecular weight compounds for example, proteins are observed as ions having a variable number of charge states. While the multiple charge phenomenon increases sensitivity, the spectra are more complex and difficult to interpret. Use and detection of a coding moiety with a less complex mass spectrum than the analyte can therefore enhance the resolution of the assay.
  • mass spectrometers may be used in accordance with the sirtuin assays and other methods described herein.
  • Representative examples include: triple quadrupole mass spectrometers, magnetic sector instruments (magnetic tandem mass spectrometer, JEOL, Peabody, Mass.), ionspray mass spectrometers (Bruins et al., Anal Chem. 59:2642-2647, 1987), electrospray mass spectrometers (including tandem, nano- and nano-electrospray tandem) (Fenn et al., Science 246:64-71, 1989), laser desorption time-of-flight mass spectrometers (Karas and Hillenkamp, Anal. Chem. 60:2299-2301 , 1988), and a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (Extrel Corp., Pittsburgh, Mass.).
  • the methods described herein that utilize mass spectrometry for determination of acetylation levels are conducted in a high throughput manner as described in CC. Ozbal, et al., Assay and Drug Development Technologies 2: 373-381 (2004).
  • the high throughput mass spectrometry based methods described herein utilize an integrated microfluidic system which uses an atmospheric pressure ionization triple quadrupole mass spectrometer as the detection system with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI).
  • the assays described herein utilize a sirtuin substrate pool that comprises a plurality of copies of one or more sirtuin substrate polypeptides.
  • a sirtuin substrate pool comprises a plurality of copies of the same polypeptide substrate.
  • Such sirtuin substrate pools may comprise the sirtuin substrate free floating in solution or attached to a solid surface such as a plate, bead, filter, etc. Combinations of free floating and anchored sirtuin substrate molecules may also be used in accordance with the methods described herein.
  • the sirtuin assays described herein may be carried out in a single reaction vessel without the need to remove reagents from the reaction mixture (e.g., a homogenous assay).
  • the components of the reactions described herein may be added sequentially or simultaneously. For example, it is possible to add a cleavage reagent concurrently with, or subsequent to, exposure of the sirtuin substrate to the sirtuin deacetylase.
  • the invention provides a method for identifying a compound that modulates the activity of a sirtuin deacetylase. The methods may involve comparing the activity of a sirtuin protein in the presence of a test compound as compared to a control.
  • the control may be the activity of a sirtuin protein in a control reaction or a value in a database.
  • a control reaction may simply be a duplicate reaction in which the test compound is not included.
  • the control reaction may be a duplicate reaction in the presence of a compound having a known effect on the sirtuin protein activity (e.g., an activator, an inhibitor, or a compound having no effect on enzyme activity).
  • the invention provides methods for screening for compounds that modulate activity of a sirtuin deacetylases.
  • the methods described herein may be used to identify a test compound that decreases or increases sirtuin activity by at least about 10%, 25%, 50%, 75%, 80%, 90%, or 100%, or more, relative to the activity in the absence of the test compound.
  • Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity.
  • the compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound” library method, and synthetic library methods using affinity chromatography selection.
  • Test compounds can be screened for the ability to modulate acetyltransferase or deacetylase activity using high throughput screening.
  • free format assays or assays that have no physical barrier between samples, can be used.
  • Assays involving free formats are described, for example, in Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994); Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995); and Salmon et al., Molecular Diversity 2, 57-63 (1996).
  • Another high throughput screening method is described in Beutel et al., U.S. Pat. No. 5,976,813.
  • Compounds that activate or inhibit the acetyltransferase or deacetylase activity are useful as candidate compounds for antimicrobial substances, anticancer agents, and a variety of other uses.
  • compounds that activate a sirtuin deacetylase protein may be useful for increasing the lifespan of a cell, and treating and/or preventing a wide variety of diseases and disorders including, for example, diseases or disorders related to aging or stress, diabetes, obesity, neurodegenerative diseases, chemotherapeutic induced neuropathy, neuropathy associated with an ischemic event, ocular diseases and/or disorders, cardiovascular disease, blood clotting disorders, inflammation, and/or flushing, etc.
  • sirtuin deacetylase inhibitors may be useful for a variety of therapeutic applications including, for example, increasing cellular sensitivity to stress, increasing apoptosis, treatment of cancer, stimulation of appetite, and/or stimulation of weight gain, etc. 6.
  • the invention provides substrates for use in determining the activity of a sirtuin protein.
  • the 5 substrates comprise at least a fragment of the proteins listed in Table 1 having an acetylated lysine at the specified position(s).
  • the substrates may comprise a fragment of the proteins listed in Table 1 that are at least about 5, 10, 15, 20, 25, 30, 40, 50, 100, or more, amino acids in length but less than the full length polypeptide.
  • the fragments comprise at least one of the acetylated lysine0 residues specified in column 5 of Table 1 in the specified protein substrates.
  • the substrates may comprise two or more acetylated lysines as specified in column 5 of Table 1.
  • the substrate fragments comprise at least one acetylated lysine residue as specified in Table 1 and at least 5, 10, 15, 20, 30, 40, 50, 100, or more, flanking amino 5 acid residues from the sequence of the full length polypeptide.
  • flanking residues may be to the N-terminus of the acetylated lysine, the C-terminus of the acetylated lysine residue, or a combination thereof.
  • the sirtuin substrate polypeptides may comprise one or more modifications, such as, for example, at least one 0 fluorophore and/or at least one bulky group, high molecular weight group, or binding site for a bulky group or high molecular weight group, as described further herein.
  • the sirtuin substrate polypeptides may be modified versions of the proteins listed in Table 1, or fragments thereof.
  • a sirtuin substrate protein may be modified to remove all lysines other than a lysine that is deacetylated by the sirtuin protein.
  • a sirtuin substrate protein may be modified to remove all arginine residues from the sequence.
  • the sirtuin substrate protein is modified to remove all arginine residues and all lysine
  • the sirtuin substrate provided herein does not include Ku70 or a fragment thereof.
  • the invention provides cell based methods for identifying a compound that modulates sirtuin activity.
  • the assays may comprise contacting a cell that expresses a sirtuin protein with a test compound and determining the level of acetylation of one or more sirtuin substrates shown in Table 1.
  • the cell based assays described herein may involve determining the acetylation levels of 1 , 2, 3, 4, 5, 10, 15, 20, 25, or more, of the sirtuin substrates shown in Table 1.
  • the assays may further comprise conducting an in vitro assay to further evaluate the sirtuin modulating activity of a compound.
  • Acetylation levels of sirtuin substrates may be determined by a variety of methods. Exemplary methods for determining acetylation levels of sirtuin substrates are provided in the exemplification section herein. Other suitable methods include, for example, immunoblotting, immunoprecipitation, mass spectrometry and combinations thereof.
  • a sirtuin substrate may be immunoprecipitated from a cell lysate using an antibody specific for the substrate. The isolated proteins may then be run on an SDS-PAGE gel and blotted (e.g., to nitrocellulose or other suitable material) using standard procedures.
  • the blot may then be probed with an anti-acetyl specific antibody to determine the level of acetylation of the sirtuin substrates.
  • acetylated proteins may be immunoprecipitated from the biological sample as described using an anti-acetyl specific antibody. These proteins may then be separated, blotted and probed with the sirtuin substrate specific antibodies.
  • the sirtuin substrates may be immunoprecipitated from a biological sample followed by separation using mass spectrometry. In certain embodiments, it may be desirable to cleave the sirtuin substrates with a chemical or enzymatic cleavage reagent prior to conducting the mass spectrometry analysis.
  • mass spectrometry When using mass spectrometry as a means to determine acetylation levels of sirtuin substrates it may be useful to have acetylated and/or non-acetylated proteins or peptides that can be used as standards for the acetylated or non-acetylated polypeptide samples.
  • Gel electrophoresis, immunoprecipitation and mass spectrometry may be carried out using standard techniques, for example, such as those described in Molecular Cloning A Laboratory Manual, 2 nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989), Harlow and Lane, Antibodies: A Laboratory Manual (1988 Cold Spring Harbor Laboratory), G. Suizdak, Mass Spectrometry for Biotechnology (Academic Press 1996), as well as other references cited herein. Additionally, methods for conducting mass spectrometry are described in more detail below.
  • Antibodies suitable for isolation and detection of sirtuin substrates may be purchased commercially from a variety of sources, such as, for example, Santa Cruz Biotechnology, Inc., Santa Cruz, CA or Abeam Inc., Cambridge, MA
  • Antibodies specific for acetylated lysine residues include the N-epsilon acetyl lysine antibodies ab409 and ab 17324 from Abeam Inc. (Cambridge, MA) and the pan-Acetyl sc-8649 antibody from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
  • Antibodies specific for sirtuin substrates may also be produced using standard techniques. Exemplary techniques for the production of antibodies to sirtuin targets are described above.
  • the cell based assays described herein may utilize a cell that endogenously expresses a sirtuin protein.
  • cells may be engineered so as to express a sirtuin (e.g., integration of a sirtuin gene into the genome of the host cell, expression from a plasmid containing a sirtuin sequence, etc.).
  • cells useful in the assays described herein endogenously express at least one sirtuin substrate listed in Table 1 (or a homolog thereof).
  • cells may be engineered so as to express one or more sirtuin substrates listed in Table 1.
  • Cells may be either prokaryotic or eukaryotic.
  • host cells are cultured mammalian cells, preferably human cells, that endogenous express a sirtuin protein and one or more of the sirtuin substrates listed in Table 1.
  • the cell based methods for determining sirtuin activity described herein may utilize a sirtuin activatable cell line.
  • a sirtuin activatable cell line is a cell line that is suitable for use in the cell based sirtuin activating assays described herein.
  • a sirtuin activatable cell line comprises a relatively low endogenous level of one or more sirtuin proteins (e.g., the amount of sirtuin activity in the cell is not saturating and an increase in activity is observable) and a relatively low level of mitochondria and/or oxidative phosphorylation capacity (e.g., the amount of mitochondria and/or oxidative phosphorylation in the cell is not saturating and an increase in ATP levels is observable).
  • Exemplary sirtuin activatable cell lines include, for example, NCI-H358 and MCS7.
  • the cells may be suspended in culture or may be contained within a non-human animal.
  • sirtuin activity assays may be carried out by administering a putative sirtuin modulating compound to a non-human animal, obtaining a cell sample from said animal, and determining the level of acetylation of one or more sirtuin substrates in the cell sample.
  • the methods involve comparing the sirtuin activity in the presence of a test compound to a control.
  • the control may be a duplicate assay conducted in the absence of a test compound or a duplicate assay conducted in the presence of a test compound having known sirtuin modulating activity (e.gi, an activator, inhibitor, or a compound having no sirtuin modulating activity).
  • a control may be a reference number in a database.
  • the cell based assays described herein may be used as a secondary screen to further characterize a putative sirtuin modulating compound.
  • the cell based assays may be used to confirm that a sirtuin modulating compound identified in vitro has sirtuin modulating activity in a cellular environment, provide information about cell membrane permeability and/or cellular toxicity.
  • Compounds that show a lower level of sirtuin modulating activity in a cell based assay as compared to an in vitro assay may be indicative of compounds that have low cell membrane permeability or compounds that are cell membrane impermeable.
  • compounds that show sirtuin activating activity in an in vitro assay but show sirtuin inhibiting activity in a cell based assay may be indicative of compounds that are cytotoxic. Accordingly, such cell based assays will provide useful information for developing therapeutic agents.
  • the cell based methods described herein may be used to determine the effect of a putative sirtuin modulating compound on mitochondrial biogenesis.
  • the cell based methods for determining sirtuin activity described herein involve determination of cell viability and/or ATP levels in a sample of cells.
  • Cell viability can be determined by any method known in the art or any method yet to be discovered. Exemplary methods for determining cell viability include, for example, Alamar Blue, BrdU, MTT, Trypan Blue exclusion, 3 H- thymidine incorporation, and XTT assays. Kits for determining cell viability are commercially available from a variety of sources.
  • kits for determining ATP levels are commercially available from a variety of sources, including, for example, ATP Assay Kit (Calbiochem, San Diego, CA), ATP Determination Kit (Molecular Probes (Invitrogen), Eugene, OR), ENLITEN ATP Assay System (Promega, Madison, WI), ATP Bioluminescence Assay Kit (Roche Applied Science, Indianapolis, IN), Adenosine 5 '-triphosphate (ATP) Bioluminescent Assay Kit (Sigma-Aldrich, St. Louis, MO), ATP Assay Kit (Thermo Electron Corporation, Milford, MA).
  • the cell based assays described herein may comprise determination of ATP levels and cell viability at one or more fixed time points after contacting the cells with a potential sirtuin modulating compound.
  • ATP level and cell viability are determined at about 12-84 hours, about 24-72 hours, about 36-60 hours, or at about 48 hours after exposure of the cells to a potential sirtuin modulating compound.
  • the cell based assays described herein may comprise determination of ATP level and cell viability in a sample of cells that are growing logrhythmically (e.g., log phase growth).
  • kits for measuring the activity of a sirtuin deacetylase and screening for compounds that inhibit or enhance sirtuin deacetylase activity as described above may be useful for research purposes, drug discovery, diagnostic purposes, etc.
  • a kit may comprise a sirtuin substrate (as described above) and one or more of the following: a cleavage reagent, a sirtuin protein, a binding moiety, one or more test compounds, a positive control, a negative control, instructions for use, a reaction vessel, buffers, a MALDI matrix, etc.
  • Kits for determination of sirtuin activity may comprise a previously acetylated sirtuin substrate.
  • the sirtuin substrate may also comprise a fluorophore.
  • the sirtuin substrate may also comprise a binding site, a bulky group or a high molecular weight group.
  • each component e.g., the sirtuin substrate, the cleavage reagent, the sirtuin protein, and/or test compound, may be packaged separately.
  • a kit may comprise a cell expressing at least one sirtuin protein and at least one sirtuin substrate (as described above) and one or more of the following: one or more test compounds, a positive control, a negative control, instructions for use, a reaction vessel, buffers, an antibody, a MALDI matrix, etc. Respective components of the kit may be combined so as to realize a final concentration that is suitable for the reaction. Further, in addition to these components, the kit may comprise a buffer that gives a condition suitable for the reaction.
  • the sirtuin enzyme preparation and the sirtuin substrate may be combined with other components that stabilize proteins.
  • the kit components may be stored and/or shipped in the presence of about 1% BSA and about 1% polyols (e.g., sucrose or fructose) to prevent protein denaturation after lyophilization.
  • Each component of the kit can be provided in liquid form or dried form.
  • Detergents, preservatives, buffers, and so on, commonly used in the art may be added to the components so long as they do not inhibit the measurement of the sirtuin deacetylase activity.
  • FIG. 4 A schematic of the experimental protocol used to identify sirtuin inhibitor sensitive peptides is shown in Figure 4. This protocol was adapted from previous work done to identify novel phosphorylated peptides (Rush et al. Nature Biotechnology, 23(1), 94-101, 2005). As a starting point, cell lines were screened for SIRTl expression to find cells either expressing high or low endogenous levels of SIRTl expression. As shown in Figure 5, equal amounts of cell lysates were analyzed by western analysis for expression of endogenous SIRTl . Blots were probed with a primary rabbit polyclonal antibody to SIRTl (Abeam, Cat.
  • SIRTl expression varies widely across different cell lines with maximum expression observed in HEK293 and minimal expression observed in IMR90 and H358 ( Figure 5). Based on this analysis, HEK293 cells with high endogenous SIRTl expression were picked for further studies. The rationale was that untreated HEK293 cells would be hypoacetylated on the majority of SIRTl targets.
  • HEK293 cells with sirtuin inhibitors would then lead to hyperacetylation of SIRTl targets.
  • Figure 6 shows a whole cell western of HEK293 cells treated for 24 hours with either a sirtuin inhibitor dissolved in DMSO (50 uM final concentration) or DMSO alone.
  • the sirtuin inhibitor that was used is 6-Chloro- 2,3,4,9-tetrahydro-lH-carbazole-l-carboxamide as described in Napper et al. (J. Med. Chem., 48(25):8045-54, 2005).
  • the reported IC 50 of this compound against SIRTl in a fluorimetric assay is 98 nM.
  • this inhibitor demonstrates a greater than two order of magnitude selectivity for SIRTl versus SIRT2, SIRT3 or Class I/II HDACs.
  • Equal amounts of cell lysates were analyzed by western analysis with a primary polyclonal antibody specific for acetyl-lysine residues (Cell Signaling, Inc., Cat. #944 IL) and anti rabbit HRP conjugated IgG secondary antibody (Santa Cruz Biotechnologies, Cat. #SC-2054).
  • a second experiment was done using siRNA specific for SIRTl .
  • Figure 7 shows a whole cell western of HEK293 treated with SIRTl siRNA for 48 hours and processed in the same way as done for Figure 6.
  • Figure 8 details a large scale treatment of HEK293 ceils with and without the sirtuin inhibitor (6-Chloro-2,3,4,9-tetrahydro-l/7-carbazole- 1 -carboxamide) for preparation of cell lysates for mass spectrometric analysis.
  • cells were only exposed to vehicle or compound (10 uM) for 6 hours as a way of looking for direct targets of SIRTl deacetylation.
  • Lysates were prepared and processed and analyzed at Cell Signaling, Inc. (Beverly, Mass) in a protocol adapted from Rush et al. ⁇ Nature Biotechnology, 23(1 ), 94-101 , 2005).
  • acetylated peptides present in the sirtuin inhibited sample and not found or found at a significantly reduced level in the control sample are given in Table 1 (shown in Figure 1). Seventy-two peptide sequences were identified (SEQ ID NOs: 1 through 72) with a total of 82 identified acetylated lysine residues. All of the acetylated peptides were present in the sirtuin inhibitor sample and not found in the control samples. Based on the specificity of the immuno-isolation step employed to enrich for acetyl -lysine containing peptides, all of these peptides represent cellular protein targets of sirtuins and presumably of SIRTl .
  • peptides had single acetylated lysine residues, while a few had multiple acetylated lysine residues.
  • the peptide corresponding to SEQ ID NO: 34 had four acetylated lysine residues.
  • Tables 2 and 3 are alignments of the peptides found in Table 1, using each of the 82 identified acetyl lysine residue as the Position 0 register.
  • Table 2 ( Figure 2) is an alignment of the 20 amino acids on the N- terminal side of the acetylated lysine residue.
  • Table 3 ( Figure 3) is an alignment of the 20 amino acids on the Carboxy-terminal side of the acetylated lysine residue. Based on these alignments, no strong pattern was found in the N-terminal residues. However, on the C-terminal side, there are some significant preferences for certain residues in at least two positions. Position 2 (ie.
  • two amino acids on the carboxy- terminal side of the acetylated lysine position is either a negatively charged residue 26% of the time (and most commonly a glutamate residue 24% of the time) or a hydrophobic residue 58% of the time (and most commonly isoleucine 37% of the time).
  • a glutamate or isoleucine is found more than half the time at position 2 in the 82 sequences that were analyzed.
  • Position 7 was the other significant position, with a hydrophobic residue 45% of the time (and most commonly tryptophan 30% of the time).
  • EXAMPLE 2 Quantitation of Acetylated Peptides Using SILAC
  • Stable isotope labeling with amino acids in cell culture is a simple and straightforward approach for in vivo incorporation of a label into proteins for mass spectrometric (MS)-based quantitative proteomics.
  • SILAC relies on metabolic incorporation of a given "light” or "heavy” form of the amino acid into the proteins.
  • the method relies on the incorporation of amino acids with substituted stable isotopic nuclei (e.g. deuterium, 13C, 15N).
  • Inhibitors to be used include SIRTl specific siRNA (as done in Figure 7) or the SIRTl selective inhibitor 6-Chloro-2,3,4,9- tetrahydro-l//-carbazole-l-carboxamide (as done in Example 1).
  • SIRTl specific siRNA as done in Figure 7
  • SIRTl selective inhibitor 6-Chloro-2,3,4,9- tetrahydro-l//-carbazole-l-carboxamide as done in Example 1.
  • an siRNA pool to all the human sirtuins or nicotinamide would be used. Lysates would then be prepared from the four treated samples.
  • Lysate from the larger control treated sample labeled with heavy amino acids would be split into four samples and combined pair wise with the four treated samples as described in Example 1.
  • SILAC then allows differentiation and quantitation of the ratio of the heavy labeled peptides arising from control samples versus the light or normal labeled peptides arising from the inhibitor treated samples.
  • EXAMPLE 3 Quantitation of Acetylation Levels of Endogenous Sirtuin Substrates Acetylation levels of endogenous proteins, such as those described in Table 1 , may be determined using western blot techniques.
  • Acetylation status of a target protein can be determined in the presence and absence of test compounds as a way of determining the ability of the test compound to modulate sirtuin activity, to determine the basal level acetylation status in a given sample, or to monitor the in vivo effects of a sirtiun modulator, such as during clinical treatment.
  • SIRTl catalyzes the NAD + - dependent deacetylation of protein substrates such as p53, PGCl ⁇ , and the FOXO transcription factors.
  • the acetylation status of a target protein can be measured by resolving cellular proteins from a tissue or cellular lysates on an SDS-PAGE gel followed by western blotting using a primary antibody specific to the acetylated- lysine residue in the protein of interest. It is also possible to immunoprecipitate a protein of interest from a cell lysate (e.g., using a protein specific antibody) followed by SDS-PAGE and western blotting with an anti-acetyl antibody.
  • This example provides a method for determining the relative acetylation status of p53 (defined as percentage of acetylated p53 to total p53) in cell culture using commercially available reagents.
  • p53 is a known substrate of SIRTl (Luo et al., Cell.107: 137-48, 2001 ).
  • SIRTl Lio et al., Cell.107: 137-48, 2001.
  • a similar protocol could be used to determine the relative acetylation status of the proteins described in Table 1.
  • the SIRTl substrate, p53 is hyperacetylated at its C-terminus when cells are exposed to DNA damaging agents such as a chemical insult (e.g. etoposide) or ionizing radiation.
  • etoposide is used to induce p53 hyperacetylation.
  • U2OS cells human osteosarcoma cells
  • the ability of test compounds to modulate the acetylation status of p53 is determined by western blot analysis of crude cellular lysates prepared from compound treated cells.
  • Figure 10 is a flow diagram of the steps used in this assay.
  • U2OS cells are cultured and treated with varying concentrations of test compounds for 12 to 16 hours. Cells are then treated for an additional 6 hours with 20 uM etoposide to induce hyper acetylation of p53.
  • the relative etoposide inducible acetylated p53 is reduced in a dose dependent manner.
  • Cell lysates are prepared using a lysis buffer consisting of 50 mM Tris HCl pH 7.5, 250 mM NaCl, 0.1% Triton-XlOO, 1 mM EDTA, 50 mM NaF, 100 mM DTT, 50 mM PMSF, and 1 mM Sodium vanadate. Lysates are prepared by incubating harvested cells in lysis buffer for 30 minutes at 4°C. Protein concentration of cellular lysates are determined and equal amounts of total cell protein are run on denaturing 4- 25% SDS PAGE gradient denaturing gels. Following transfer to nitrocellulose membranes, total p53 and acetylated p53 is detected on the same membrane using the following antibodies.
  • Anti-Acetylated-p53Lys382 rabbit polyclonal antibody is purchased from Cell Signaling Technology (Cat.# 2525L), diluted 1 :500 in TBST containing 5% BSA just prior to use; and Anti-p53 DO-I mouse monoclonal antibody is purchased from Santa Cruz Biotechnology (Cat.# SC126), diluted 1 :2000 in TBST containing 5% BSA just prior to use.
  • Secondary antibodies used include IR800 Goat anti-rabbit IgG is purchased from Rockland (Cat.# 61 1-132-122), diluted 1:10,000 in TBST containing 5% BSA just prior to use; and Alexa-Fluor 680 goat anti-mouse IgG is purchased from Invitrogen (Cat.# A21057), diluted 1 :10 5 OOO in TBST containing 5% BSA just prior to use.
  • the LICOR Odyssey System (LI-COR Biosciences, Lincoln, NE) is used to visualize and quantitate the levels of acetylated and total p53.
  • the LICOR software can be used to mark a rectangle over the band of interest and quantify the p53 band for total levels in the red channel (700) and for acetylation levels in the green channel (800).
  • the ratio of acetylated p53 to total p53 can then be calculated.
  • a compound that activates SIRTl deacetylation activity will result in a decrease in the acetylated p53:total p53 ratio, while a SIRTl inhibitor would result in the opposite effect.
  • the present invention provides among other things indicators of sirtuin activity and methods of use thereof. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) (www.tigr.org) and/or the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov).
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information

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Abstract

La présente invention concerne des procédés pour suivre la modulation de la sirtuine chez un sujet, par exemple, durant le traitement thérapeutique avec un composé modulateur de sirtuine. Les procédés comprennent la détermination du taux d'acétylation d'un ou de plusieurs nouveaux substrats de sirtuine dans un échantillon biologique provenant dudit sujet. La présente invention concerne en outre des procédés pour identifier des composés qui modulent l'activité d'une protéine sirtuine en utilisant un ou plusieurs nouveaux substrats de sirtuine.
PCT/US2007/018916 2006-08-29 2007-08-28 Indicateurs d'activité sirtuine et procédés d'utilisation de ceux-ci WO2008027379A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010042868A3 (fr) * 2008-10-10 2010-08-12 University Of Washington Procédés pour traiter l'obésité
US8685970B2 (en) 2008-05-01 2014-04-01 GlaxoSmithKline, LLC Quinolines and related analogs as sirtuin modulators
US8846947B2 (en) 2008-07-03 2014-09-30 Glaxosmithkline Llc Benzimidazoles and related analogs as sirtuin modulators
US8916528B2 (en) 2011-11-16 2014-12-23 Resveratrol Partners, Llc Compositions containing resveratrol and nucleotides
US8987258B2 (en) 2008-09-29 2015-03-24 Christopher Oalmann Chromenone analogs as sirtuin modulators
US9556201B2 (en) 2009-10-29 2017-01-31 Glaxosmithkline Llc Bicyclic pyridines and analogs as sirtuin modulators

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* Cited by examiner, † Cited by third party
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US20060025337A1 (en) * 2003-07-01 2006-02-02 President And Fellows Of Harvard College Sirtuin related therapeutics and diagnostics for neurodegenerative diseases
WO2006076681A2 (fr) * 2005-01-13 2006-07-20 Sirtris Pharmaceuticals, Inc. Compositions nouvelles pour le traitement des troubles de la neurodegenerescence et de la coagulation du sang

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8685970B2 (en) 2008-05-01 2014-04-01 GlaxoSmithKline, LLC Quinolines and related analogs as sirtuin modulators
US8846947B2 (en) 2008-07-03 2014-09-30 Glaxosmithkline Llc Benzimidazoles and related analogs as sirtuin modulators
US8987258B2 (en) 2008-09-29 2015-03-24 Christopher Oalmann Chromenone analogs as sirtuin modulators
US9326986B2 (en) 2008-09-29 2016-05-03 Glaxosmithkline Llc Quinazolinone, quinolone and related analogs as sirtuin modulators
WO2010042868A3 (fr) * 2008-10-10 2010-08-12 University Of Washington Procédés pour traiter l'obésité
US9556201B2 (en) 2009-10-29 2017-01-31 Glaxosmithkline Llc Bicyclic pyridines and analogs as sirtuin modulators
US8916528B2 (en) 2011-11-16 2014-12-23 Resveratrol Partners, Llc Compositions containing resveratrol and nucleotides
US9226937B2 (en) 2011-11-16 2016-01-05 Resveratrol Partners, Llc Compositions containing resveratrol and nucleotides

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