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WO2008021048A2 - Procédés et compositions fondés sur l'adp-ribosyltransférase - Google Patents

Procédés et compositions fondés sur l'adp-ribosyltransférase Download PDF

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WO2008021048A2
WO2008021048A2 PCT/US2007/017461 US2007017461W WO2008021048A2 WO 2008021048 A2 WO2008021048 A2 WO 2008021048A2 US 2007017461 W US2007017461 W US 2007017461W WO 2008021048 A2 WO2008021048 A2 WO 2008021048A2
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hydrogen
alkyl
aryl
sulfonyl
hydroxyl
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WO2008021048A3 (fr
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David A. Sinclair
Ron Firestein
Basil Hubbard
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Harvard University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91142—Pentosyltransferases (2.4.2)
    • 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)

Definitions

  • Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance either by increasing the rate of cell proliferation or decreasing the rate of cell death can result in the abnormal growth of cells and is thought to be a major event in the development of cancer, as well as other cell proliferative disorders such as restenosis.
  • Prostate cancer is the most common non-skin cancer in America. Predictions estimated that in 2006, over 232,000 men would be diagnosed with prostate cancer, and over 30,000 men would die from it. One new case occurs every 2.5 minutes and a man dies from prostate cancer every 17 minutes.
  • sirtuin ADP- ribosyltransferases referred to herein as "sirtuin ADP- ribosyltransferases” or “sirtuin ribosyltransferases.”
  • Other methods described herein may be used for identifying targets of the sirtuin ribosyltransferases.
  • Agents that modulate the activity of sirtuin ribosyltransferases may be used as therapeutics for treating or preventing cancer, ischemia-reperfusion injury, and other disorders, as well as delaying the aging process.
  • a method may comprise administering to a subject in need thereof a therapeutically effective amount of an agent that decreases the level of protein or activity of a sirtuin ribosyltransferase, e.g., Sirt ⁇ , protein or inhibits a sirtuin ribosyltransferase, e.g., Sirt ⁇ , dependent ribosylation pathways.
  • a hyper-proliferating disease may be cancer, such as prostate cancer or a skin cancer, or benign cancer, or a non-cancerous cellular growth.
  • An agent for use in the therapeutic or prophylactic methods may be a small molecule, e.g., a compound of any of formulas I-XI.
  • An agent may also be an siRNA, antisense molecule, triplex DNA, antibody, aptamer, dominant negative mutant of Sirt6 or a substrate of Sirt ⁇ or functional homolog thereof.
  • Methods may further comprise administering to a subject a second agent, e.g., a second compound of any of formulas I-XI, or other chemotherapeutic agent.
  • a second agent e.g., a second compound of any of formulas I-XI, or other chemotherapeutic agent.
  • Figure l is a diagram of an exemplary method for identifying agents that modulate sirtuin ribosyltransferase activity or for identifying targets of these enzymes.
  • Figure 2 panels A, B and C, shows a strategy for isolation of ribosylation targets.
  • Figure 3 is an exemplary biotin-labeled NAD+ molecule.
  • Figure 4 panels A and B, shows SIRT6 knockdown in TRAMPC2 cells.
  • Figure 5 panels A-D, shows that inhibition of SIRT6 dramatically reduces focus or anchorage dependent colony formation of prostate cancer cells, as assessed by growing a fixed number of cells in culture and subsequently staining with Crystal Violet dye.
  • FIG. 6 panels A-C, shows that inhibition of SIRT6 reduces colony growth of prostate cancer cells in soft agar.
  • Figure 7 is a histogram showing the focus formation assay results of Figure 5.
  • Figure 8 shows the size of tumors resulting from injection into a xenograft model of prostate cancer cells transfected with vector alone or cells in which Sirt ⁇ expression is downregulated.
  • Figure 9 is a histogram showing the percent invasion of RS485 prostate cancer cells in which SIRTl, SIRT6 or SIRT7 is either down-regulated or overexpressed.
  • Figure 10 shows the level of SIRT6 protein via Western Blot (panel A) or mRNA via RT-PCR (panel B) in TRAMP prostate cells in which SIRT6 was stably knocked down with an hsRNA based on the SIRT6 nucleotide sequence shown in panel C.
  • FIG 11 panels A, B and C show that knockdown of SIRT6 suppresses cell migration and invasion (two markers for metastasis).
  • Figure 12 show the results of microarray analysis, which indicate that several genes involved in cellular migration are modified in SIRT6 knockdown cells. The genes are listed in the order in which they were in the cluster analysis. A few fold changes from the original data are provided for reference.
  • Figure 13 show the results of microarray analysis, which indicate that several genes involved in cell motility are modified in SIRT6 knockdown cells. The genes are listed in the order in which they were in the cluster analysis. A few fold changes from the original data are provided for reference.
  • Figure 14 show the results of microarray analysis, which indicate that several genes involved in chemotaxis are modified in SIRT6 knockdown cells. The genes are listed in the order in which they were in the cluster analysis. A few fold changes from the original data are provided for reference.
  • Figure 15 is a histogram showing that several signaling pathways are reduced in SIRT6 knockdown TRAMP cells.
  • Figure 16 is a diagram of an exemplary method for identifying agents that modulate sirtuin ribosyltransferase activity or for identifying targets of these enzymes.
  • FIG 17 panels A and B, is a diagram of an exemplary method for identifying agents that modulate sirtuin ribosyltransferase activity.
  • Figure 18 shows the structure of sirtinol analogues described herein. Detailed description
  • Described herein are methods for identifying agents that modulate ADP- ribosyltransferases, as well as uses of agents that modulate these enzymes.
  • Both poly- and mono- AD ribosyltransferases comprise an important subclass of enzymes which have been linked to cancer, ischemia-reperfusion injury and the aging process.
  • Members of the sirtuin class of enzymes (Sirt 4 and Sirt 6) have recently been identified as ADP-ribosyltransferases.
  • Sirtuins are class III histone/protein deacetylases (Brachmann et al. (1995) Genes Dev. 9:2888 and Frye et al. (1999) BBRC 260:273). Some of the sirtuins, in particular, Sirt4 and Sirt ⁇ are ribosyl transferases, in particular, mono-ADP-ribosyltransferases (see Liszt et al. (2005) J. Biol. Chem. 280:21313 regarding Sirt ⁇ ). Sirt7 is an activator of Poll (Ford et al. (2006) Genes & Dev. 20:1075), however, it is also likely to be a ribosyltransferase.
  • the mono-ADP-ribosyltransferase reaction is set forth in Corda and Girolamo (2003) EMBO J. 22:1953. Briefly, a mono-ADP-ribosyltransferase catalyzes the reaction in which the ribosyl group from ⁇ NAD + is transferred onto an amino acid, e.g., arginine or lysine, residue of a target or acceptor protein, thereby releasing nicotinamide.
  • an amino acid e.g., arginine or lysine
  • a ribosyl may be transferred to a protein or peptide, generally referred to herein as a "target,” “substrate” or “acceptor,” which may be either a peptide, a polypeptide or a protein, or a modified form thereof.
  • Targets of the ribosyltransferase activity of Sirt4 include glutamate dehydrogenase (GDH), the human version of which has the nucleotide and amino acid sequences set forth under GenBank Accession numbers NM 005271 and NP_005262, respectively.
  • Targets of the ribosyltransferase activity of Sirt6 proteins include histones, e.g., core histones (see Liszt et al., infra).
  • Another target protein is nucleoplasmin, also referred to as nucleophosmin, nucleolar phosphoprotein B23, numatrin, and as chromatin decondensation protein.
  • Nucleophosmin 1 isoform 1 is the predominant variant and represents the longest transcript and encodes the longest isoform (1).
  • the nucleotide and amino acid sequences of the human isoform 1 are set forth in GenBank Accession numbers NM_002520 and NP_002511, respectively.
  • Isoform 2 lacks an alternate in-frame exon, compared to variant 1, resulting in a shorter protein (isoform 2) that lacks an internal segment, compared to isoform 1.
  • the nucleotide and amino acid sequences of the human isoform 2 are set forth in GenBank Accession numbers NM_199185 and NP_954654, respectively.
  • Isoform 3 utilizes an alternate 3'-terminal exon, compared to variant 1, resulting in a shorter protein (isoform 3) with a distinct C-terminus.
  • the nucleotide and amino acid sequences of the human isoform 3 are set forth in GenBank Accession numbers NM__001037738 and NP_001032827, respectively.
  • Sirt ⁇ Another target protein of Sirt ⁇ is Alternative Reading Frame (ARF) protein, which is a tumor suppressor, as well as p53.
  • ARF Alternative Reading Frame
  • the ink4a/arf locus encodes two cell cycle regulatory proteins - the cyclin-dependent kinase inhibitor (pl6(ink4a)), and the p53 activator (ARF),
  • the nucleotide and amino acid sequences of human pl6(ink4a) are set forth in GenBank Accession Nos. AF115544 and AAD11437, respectively, and those of human p53 are set forth in GenBank Accession Nos. NM_000546 and NP_000537, respectively.
  • Yet another target is a sirtuin itself, e.g., Sirt ⁇ , since it has been shown that Sirt ⁇ may auto-ribosylate (Liszt et al. > infra).
  • Table 1 provides GenBank Accession numbers for Sirt4, Sirt ⁇ and Sirt 7 proteins and nucleic acids of various species. Human Sirt nucleic acids and proteins are referred to as "SIRT.”
  • a method for identifying an agent that modulates the activity of a sirtuin ribosyltransferase may be a cell based or a cell-free assay.
  • a cell free assay may comprise (i) combining a sirtuin ribosyltransferase or a functional homolog thereof with a sirtuin ribosyltransferase target, labeled NAD+ and a test agent; and (ii) detecting labeled target.
  • a difference in the amount of labeled target in a reaction mixture comprising the test agent relative to the amount of labeled target in a reaction mixture that does not comprise the test agent indicates that the test agent modulates the activity of a sirtuin ribosyltransferase.
  • a difference in amount may be a factor of at least about 50%, 2 fold, 3 fold, 5 fold, 10 fold, 30 fold, 50 fold or more. If the amount of label in the presence of the test agent is higher relative to the absence of the test agent, the test agent is an agent that stimulates the activity of a sirtuin ribosyltransferase.
  • the test agent is an agent that inhibits the activity of a sirtuin ribosyltransferase.
  • the reagents used in an assay for identifying modulators of sirtuin ribosyltransferases may be added simultaneously or sequentially in a reaction mixture. For example, the enzyme and NAD+ may be combined first, followed by the addition of the test agent and then the target peptide.
  • SIRT6 substrate or SIRT6 itself (to assess auto-ADP-ribosylation activity), may be covalently linked to a solid support matrix present in each well of a multiwell, e.g., 96-well, plate.
  • Various techniques may be applied to cross-link the SIRT6 substrate to the plate to render it immobile, including simple absorption of the protein to the plastic surface.
  • recombinant SER.T6 may be added to each well (in the presence or absence of potential chemical activators/inhibitors) in ribosylation buffer (Liszt et al. J. Biol. Chem., June 3, 2005; 280(22): 21313 - 21320), supplemented with a 1:10 dilution of FITC- NAD (6- Fluorescein- 17-nicotinamide-adenine-dinucleotide) (available from Trevigen: 4673-500-01).
  • FITC- NAD 6- Fluorescein- 17-nicotinamide-adenine-dinucleotide
  • SIRT6 will catalytically transfer an ADP-ribose-FITC fluorometric moiety onto the target molecule.
  • SIRT6 may be covalently bound on the sorbent matrix, and ribosylation buffer + NAD-FITC may be added in solution (in addition to any chemical agents being tested).
  • ribosylation buffer + NAD-FITC may be added in solution (in addition to any chemical agents being tested).
  • an incubation time sufficient for allowing the reaction to take place e.g., 1 hour, at 37 0 C
  • the liquid contents of each well may be aspirated off to remove any un- reacted NAD-FITC.
  • the 96-well plate may be washed 3x in ribosylation buffer.
  • the amount of covalently labeled substrate may be assessed using a fluorometric spectrophotometer (see, e.g., Trevigen product information for appropriate excitation/emission wavelengths).
  • the intensity of the signal will be proportional to the activity of SIRT6 under the defined conditions (activators/inhibitors).
  • a standard curve using different amounts of STRT6 protein can also be constructed.
  • the aforementioned assay may be used to screen for chemical activators and inhibitors of SER.T6 ADP-ribosylation activity. It can be used to examine both intrinsic auto- ADP ribosylation of SIRT6 itself, and external protein targets, depending on which variation of the assay is used.
  • a sirtuin ribosyltransferase may be any sirtuin having ribosyltransferase activity, e.g., Sirt4, 6 and 7.
  • a homolog of a sirtuin ribosyltransferase includes proteins (e.g., peptides and polypeptides) comprising, consisting essentially of, or consisting of an amino acid sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity with the amino acid sequence of a sirtuin ribosyltransferase.
  • a homolog may also be a protein that is encoded by a nucleic acid comprising, consisting essentially of, or consisting of a nucleotide sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity with a nucleotide sequence encoding a sirtuin ribosyltransferase or the coding sequence thereof.
  • a homolog may also be a protein that is encoded by a nucleic acid that hybridizes, e.g., under stringent hybridization conditions, to a nucleic acid encoding a sirtuin ribosyltransferase, or the coding sequence thereof.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
  • Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • FASTA FASTA
  • BLAST BLAST
  • ENTREZ is available through the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Md.
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • MPSRCH uses a Smith-Waterman algorithm to score sequences on a massively parallel computer. This approach improves ability to pick up distantly related matches, and is especially tolerant of small gaps and nucleotide sequence errors. Nucleic acid-encoded amino acid sequences can be used to search both protein and DNA databases.
  • homologs may be encoded by nucleic acids that hybridize under high stringency conditions of 0.2 to 1 x SSC at 65 0 C followed by a wash at 0.2 x SSC at 65 0 C to a nucleic acid encoding a sirtuin ribosyltransferase.
  • Nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature to nucleic acid encoding a sirtuin ribosyltransferase or a portion thereof can be used.
  • hybridization conditions include 3 x SSC at 40 or 50 0 C, followed by a wash in 1 or 2 x SSC at 20, 30, 40, 50, 60, or 65 0 C.
  • Hybridizations can be conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40% or 50%, which further increases the stringency of hybridization. Theory and practice of nucleic acid hybridization is described, e.g., in S.
  • conservative amino acid changes may be made, which do not normally or significantly alter its function.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine (in positions other than proteolytic enzyme recognition sites); phenylalanine, tyrosine.
  • Homologs of a sirtuin ribosyltransferase also include portions or fragments of a naturally occurring sirtuin ribosyltransferase or homolog thereof, such as portions comprising one or more conserved domains, e.g., the catalytic domain that sirtuin ribosyltransferases share with the other sirtuins (Frye et al. (1999) Biochem. Biophys. Res. Comm. 260:273 and Liszt et al, infra).
  • the catalytic domain of human Sirt4 corresponds to about amino acids 55-314 of SEQ ID NO: 2 (Frye et al. (1999) BBRC 260:273.
  • the catalytic domain of human Sirt6 (SIRT6) corresponds to about amino acids 45 to 271 of SEQ ID NO: 4 (see Liszt et al., infra).
  • a "functional homolog" of a sirtuin ribosyltransferase refers to a homolog of sirtuin ribosyltransferase having at least one biological activity of the protein, e.g., ribosyltransferase activity or deacetylase activity. Whether a homolog is a functional homolog can be determined according to methods known in the art. For example, a ribosyl transferase activity can be determined as further described herein.
  • An exemplary functional homolog of SIR.T6 comprises, consists essentially of, or consists of an amino acid sequence that is at least about 80%, 95%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth as SEQ ID NO: 4 or of a portion thereof, wherein the functional homolog has ribosyltransferase activity.
  • a functional homolog of Sirt6 is not the full length Sirt ⁇ protein and may lack from about 1-5, 1-10, 1-15, 1-20, 1-25, 1-50 or more amino acids at the N- and/or C- terminus of the protein.
  • a functional homolog of SIRT6 comprises, consists essentially of, or consists of about amino acids 45 to 271 of SEQ ID NO: 4, which functional homolog may in certain embodiments, not comprise about amino acids 1-10, 1- 20, 1-30, 1-40 or 1-44 and/or about amino acids 272-355, 275-355, 280-355, 300-355, 320- 355 or 330-355 of SEQ ID NO: 4.
  • a sirtuin ribosyltransferase target for using in an assay may be a full length target protein, e.g., a sirtuin, a nucleoplasmin, ARF, p53 or GDH. It may also be a functional homolog thereof, e.g., a homolog of a naturally occurring target or a fragment thereof that is sufficient for receiving a ribosyl group.
  • a "homolog" of a sirtuin ribosyltransferase target may be any protein that differs from a sirtuin ribosyltransferase target protein in the same manner as described above in the context of a sirtuin ribosyltransferase, e.g., a protein that is a fragment of a target protein and/or that has a certain percentage identity to it.
  • a target may comprise or consist of 1-5 amino acids, about 5-10, about 10-15, about 15-20, about 20-25, about 25-30, about 11-40 amino acids, more than 30 amino acids, about 1-50, about 1-100, about 1-150 or 1-200 amino acids, wherein the target comprises a lysine and/or an arginine.
  • a target may lack about 1-5, 1-10, 1-15, 1-20, 1-30, 1-50 or more amino acids from the N- and/or C-terminus of the naturally occurring target.
  • Labeled NAD + or "tagged NAD +” refers to an NAD + molecule that is labeled on one or more atoms of its ribosyl group, such that if the ribosyl portion of the NAD + molecule is transferred onto a target protein, the target protein becomes labeled.
  • An NAD + molecule may be labeled with any type of label, e.g., one that is directly detectable or one that is indirectly detectable, such as further described herein.
  • Exemplary labeled NAD + molecules include ⁇ -biotin- ⁇ -nicotinamideadenine-dinucleotide, 6-biotin-10- nicotinamideadenine-dinucleotide and S-biotin- ⁇ -nicotinamideadenine-dinucleotide.
  • Labeled NAD + molecules may be prepared and at least some are also commercially available. For example, biotinylated NAD + is available from Trevigen and R&D systems.
  • a label may be a detectable label, e.g., a molecule capable of detection, including, but not limited to, fluorophores, chemiluminescent moieties, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens) and the like.
  • the label may be isotopic or nonisotopic.
  • the label can be a part of a catalytic reaction system such as enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, or catalysts; part of a chromogen system such as fiuorophores, dyes, chemiluminescers, luminescers, or sensitizers; a dispersible particle that can be non-magnetic or magnetic, a solid support, a liposome, a ligand, a receptor, a hapten radioactive isotope, and so forth.
  • a catalytic reaction system such as enzymes, enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, or catalysts
  • part of a chromogen system such as fiuorophores, dyes, chemiluminescers, luminescers, or sensitizers
  • a dispersible particle that can be non-magnetic or magnetic, a solid support, a liposome, a ligand, a receptor, a hapten radio
  • labels include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH, ⁇ -galactosidase, ⁇ -galactosidase and horseradish peroxidase, europium cryptate and a 105 kDa phycobiliprotein.
  • the enzyme or coenzyme employed provides the desired amplification by producing a product, which absorbs light, e.g., a dye, or emits light upon irradiation, e.g., a fluorescer.
  • a product which absorbs light, e.g., a dye, or emits light upon irradiation, e.g., a fluorescer.
  • the catalytic reaction can lead to direct light emission, e.g., chemiluminescence.
  • a large number of enzymes and coenzymes for providing such products are indicated in U.S. Pat. No. 4,275,149, columns 19 to 23, and U.S. Pat. No.
  • saccharide oxidases e.g., glucose and galactose oxidase
  • heterocyclic oxidases such as uricase and xanthine oxidase
  • an enzyme which employs the hydrogen peroxide to oxidize a dye precursor that is, a peroxidase such as horse radish peroxidase, lactoperoxidase, or microperoxidase.
  • hydrolases When a single enzyme is used as a marker, such enzymes that may find use are hydrolases, transferases, lyases, isomerases, ligases or synthetases and oxidoreductases, preferably, hydrolases.
  • luciferases may be used such as firefly luciferase and bacterial luciferase.
  • Exemplary enzymes, based on the I.U.B. classification are: Class 1. oxidoreductases and Class 3. Hydrolases; particularly in Class 1, the enzymes of interest are dehydrogenases of Class 1.1, more particularly 1.1.1, 1.1.3, and 1.1.99 and peroxidases, in Class 1.11. Of the hydrolases, particularly Class 3.1, more particularly 3.1.3 and Class 3.2, more particularly 3.2.1.
  • Illustrative dehydrogenases include malate dehydrogenase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase.
  • glucose oxidase is exemplary.
  • peroxidases horse radish peroxidase is illustrative.
  • hydrolases alkaline phosphatase, beta-glucosidase and lysozyme are illustrative.
  • the label can also be fluorescent either directly or by virtue of fluorescent compounds or fluorescers bound to a particle or other molecule in conventional ways.
  • the fluorescent labels will be bound to, or functionalized to render them capable of binding (being conjugated) to, optionally through a linking group, NAD+.
  • the fluorescers of interest will generally emit light at a wavelength above about 350 nm, usually above about 400 run and preferably above about 450 ran. Desirably, the fluorescers have a high quantum efficiency, a large Stokes shift, and are chemically stable under the conditions of their conjugation and use.
  • the term luminescent marker or label is intended to include substances that emit light upon activation by electromagnetic radiation, electro chemical excitation, or chemical activation and includes fluorescent and phosphorescent substances, scintillators, and chemiluminescent substances.
  • Fluorescers of interest fall into a variety of categories having certain primary functionalities. These primary functionalities include 1- and 2-aminonaphthalene, p,p- diaminostilbenes, pyrenes, quaternary phenanthridine salts, 9-aminoacridines, p,p'- diaminostilbenes imines, anthracenes, oxacarboxyanine, merocyanine, 3-aminoequilenin, perylene, bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol, bis-3- aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene, 7-hydroxycoumarin, 4,5- benzimidazoles, phenoxazine, salicylate, stroph
  • a label or marker may be a chemiluminescent compound.
  • the chemiluminescent source involves a compound, which becomes electronically excited by a chemical reaction and may then emit light which serves as the detectable signal or donates energy to a fluorescent acceptor.
  • a diverse number of families of compounds have been found to provide chemiluminescence under a variety of conditions.
  • One family of compounds is 2,3- dihydro-l,4-phthalazinedione.
  • the most popular compound is luminol, which is the 5- amino analog of the above compound.
  • Other members of the family include the 5-amino- 6,7,8-trimethoxy- and the dimethylamine-[ca]benzo analog.
  • Chemi luminescent analogs include para-dimethylamino- and para- methoxy-substituents. Chemiluminescence may also be obtained with geridinium esters, dioxetanes, and oxalates, usually oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogen peroxide, under basic conditions.
  • luciferins may be used in conjunction with luciferase or lucigenins.
  • An assay may comprise using a reagent or an agent that is linked to a solid support or surface.
  • the target protein is linked covalently or not to a solid support.
  • the method may then comprise determining the presence and/or amount of label that is linked to the solid support.
  • the method may further comprise washing off any unbound labeled NAD+ prior to determining the presence or amount of label.
  • An exemplary method is set forth in Fig. 1.
  • a solid support may be the inner surface of a reaction container (e.g., a reaction tube or a well of a microtiter plate).
  • a solid phase is a porous or non-porous water insoluble material.
  • the solid phase can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber containing papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly( vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available
  • the surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like.
  • the surface will usually be polyfunctional or be capable of being polyfunctionalized or be capable of binding a reagent, e.g., a target, through specific or non-specific covalent or non-covalent interactions.
  • a reagent is attached by using a suitable coupling agent, and in others by using appropriate linker substances, such as biotin and (strept)avidin.
  • the immobilization of a reagent is carried out after the incubation with one or more reagents, thereby allowing the reaction between the reagents to proceed in the liquid phase.
  • a target may be added to a reaction comprising a sirtuin ribosyltransferase or a functional homolog thereof, a labeled NAD+ and a test agent.
  • a solid surface can be added that will capture the target.
  • the target may be linked to the surface when it is first added to the reaction.
  • the assay may further comprise rinsing steps, e.g., a step wherein the excess free labeled NAD+ is removed prior to the detection step.
  • rinsing steps e.g., a step wherein the excess free labeled NAD+ is removed prior to the detection step.
  • Methods for removing free labeled NAD+ from labeled target when the target is not linked to a solid surface include methods based on size separation, such as chromatographic methods, as well as dialysis.
  • Labeled target protein may be detected and/or measured by any manner that allows detection of the label.
  • detecting labeled target may comprise contacting the labeled target with a binding partner that recognizes the label.
  • the binding partner carries a marker allowing its detection.
  • the binding partner is an antibody, e.g., a mouse antibody (either polyclonal or monoclonal) that binds specifically to the label, e.g., a goat anti-mouse IgG containing an enzyme moiety.
  • a substrate for the label e.g., enzyme
  • the label's substrate solution is added for a time that should be sufficiently long to allow a substantial enzymatic conversion of the label's substrate into a measurable reaction product.
  • the intensity of the reaction product which is proportional to the immobilized amount of the target, maybe measured, e.g., by optical means, such as a photometer that measures the absorbance at a proper wavelength.
  • the method is a quantitative method to determine the concentration or amount of the target in the reaction.
  • the method is not restricted to quantitative measurements, however, and may be useful to qualitatively determine the presence of labeled target.
  • the label is an enzyme and the detection means comprises a substrate of the enzyme.
  • the method may be an immunoassay, e.g., an ELISA method.
  • a horseradish peroxidase may be used as the enzyme and 3,3'-diaminobenzidine as the substrate.
  • the methods extend to other enzyme-substrate combinations capable of producing a precipitate, such as an alkaline phosphatase as the enzyme and 5- bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium as substrate.
  • methods include any other combination of label and detection means that is capable of producing a precipitate on a solid phase that carries said label.
  • formation of a precipitate may be the result of nucleation, by nucleated growth of metal, or non-metal particles, or the result of chain or polymerization reactions, of, e.g. unsaturated hydrocarbons which can be polymerized by exposure to ultraviolet radiation, etc.
  • the label may comprise an unsaturated hydrocarbon and the detection means a further amount of the same or a different hydrocarbon together with means to initiate polymerization (such as UV radiation).
  • the label could comprise colloidal particles of a metal, e.g. gold, silver, etc., or a non-metal, e.g.
  • the detection means comprises a source of a further amount of the same or a different metal or non-metal, together with means (e.g. a suitable reducing agent, such as a borohydride compound, capable of liberating the metal from a chemical compound containing said metal) to promote growth of the colloidal particles used as the label.
  • a suitable reducing agent such as a borohydride compound, capable of liberating the metal from a chemical compound containing said metal
  • An agent may be may be any type of molecule, such as a small molecule or a macromolecule, e.g., a nucleic acid, oligonucleotide, protein, peptide, peptide nucleic acids, peptidomimetics, carbohydrates, lipids, combination thereof.
  • a "small molecule” may be a composition which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu. Small molecules may be, for example organic (carbon containing) or inorganic molecules.
  • small organic molecule refers to a small molecule that is often identified as being an organic or medicinal compound, and does not include molecules that are exclusively nucleic acids, peptides or polypeptides.
  • An agent may be a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.
  • Agents may be identified as having a particular activity by screening assays described herein below. The activity of such agents may render it suitable as a "therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • compositions comprising more than one agent, e.g., 2, 3, 5, 10, 15, 20, 30, 50, 100 or more agents.
  • agents e.g., 2, 3, 5, 10, 15, 20, 30, 50, 100 or more agents.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays described herein.
  • a method for identifying an agent that modulates the ribosyltransferase activity of a sirtuin is a cell based assay.
  • a method may comprise (i) contacting a cell expressing a sirtuin ribosyltransferase or a functional homolog thereof with labeled NAD+ and a test agent; and (ii) comparing the level of labeled proteins in a cell that was contacted with the test agent relative to a cell that was not contacted with the test agent.
  • a different level of labeled proteins in a cell that was contacted with a test agent relative to a cell that was not contacted with a test agent indicates that the test agent is an agent that modulates the activity of a sirtuin ribosyltransferase.
  • a difference in amount may be a factor of at least about 50%, 2 fold, 3 fold, 5 fold, 10 fold, 30 fold, 50 fold or more. If the amount of label in the presence of the test agent is higher relative to the absence of the test agent, the test agent is an agent that stimulates the activity of a sirtuin ribosyltransferase. If the amount of label in the presence of the test agent is lower relative to the absence of the test agent, the test agent is an agent that inhibits the activity of a sirtuin ribosyltransferase.
  • a cell may be any cell, e.g., a mammalian cell, such as a human cell, a murine cell, a rat cell, or a non-human simian cell.
  • the cell may be a normal cell, a transformed cell, a cell in culture (e.g., of a cell line), a cell from a primary cell culture, or a stem cell.
  • the cell comprises a heterologous nucleic acid (one that is not naturally part of the cell) encoding the sirtuin ribosyltransferase or functional homolog thereof.
  • the nucleic acid may be present in an integrated form in a chromosome of the cell or it may be present as an extrachromosomal nucleic acid, e.g., as an episome.
  • the nucleic acid may be introduced into the cell by stable or transient transfection.
  • the nucleic acid may be part of a plasmid or a vector, e.g., an expression vector.
  • the portion encoding the sirtuin ribosyltransferase or functional homolog thereof may be under the control of an endogenous or exogenous promoter and/or enhancer and/or other regulatory element.
  • an “endogenous” regulatory element refers to a regulatory element that is part of the cell
  • an “exogenous” or “heterologous” regulatory element refers to a regulatory element that is not part of the cell, but that is part of a nucleic acid that was introduced into the cell.
  • a regulatory element e.g., a promoter or enhancer
  • Inducible promoters include those that are inducible by heavy metals, hormones, e.g., steroid hormones, and TetR.
  • a sirtuin ribosyltransferase or functional homolog thereof may be linked to a detectable tag, allowing its detection and/or isolation.
  • Exemplary tags include any peptide for which an antibody is available; polyhistidine tags; and myc tags. Labels further described herein may also be used.
  • a sirtuin ribosyltransferase or functional homolog thereof may be expressed at a level that is essentially normal for a cell, i.e., the level at which a cell normally expresses a sirtuin ribosyltransferase.
  • sirtuin ribosyltransferase or functional homolog thereof may be over-expressed, e.g., expressed at least 50%, 2 fold, 3 fold, 5 fold, 10 fold or more relative to the normal level of expression of the sirtuin ribosyltransferase in a cell.
  • a cell based screening method may comprise contacting a cell expressing a sirtuin ribosyltransferase with a test agent and labeled NAD+ simultaneously or consecutively (sequentially). For example, a cell may be contacted with a test agent and then with labeled NAD+.
  • a screening method may also be conducted with a cell lysate or cell extract, instead of a cell.
  • a cell lysate may be prepared from whole cells, or may be fractions of such lysates.
  • a cell lysate or extract may comprise a sirtuin ribosyltransferase or functional homolog thereof.
  • a lysate may be prepared from a cell that comprises a heterologous nucleic acid encoding a sirtuin ribosyltransferase or a functional homolog thereof.
  • a cell lysate or extract does not comprise a sirtuin ribosyltransferase or functional homolog thereof, and it is added to the lysate or extract.
  • a screening assay is a fiuorimetric assay.
  • an assay comprises using a target peptide comprising a lysine or arginine residue located adjacent to a fluorescent group, e.g., dimethylcoumarin, and a trypsin cleavage site (see, e.g., Figure 17).
  • Lysine and arginine are target amino acids for ribosylation by sirtuin ribosyltransferases. Trypsin cleaves peptides on the C-terminal side of lysine and arginine amino acid residues.
  • the rate of hydrolysis is slower if an acidic residue is on either side of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site.
  • Ribosylation of either amino acid prevents cleavage by trypsin.
  • Target peptides may comprise stretches of amino acids around the site of ribosylation.
  • the arginine or lysine that is modified may be immediately on the N-terminal side of the fluorescent group that is attached to the C-terminus of the peptide. Cleavage will liberate the labeled group.
  • a fluorimetric assay may also be described as follows.
  • a method for screening for potential activators/inhibitors of SIRT6 involves a target peptide which contains a fluorescent group (i.e. Dimethylcoumarin) adjacent to a target ribosylation lysine or arginine residue which may function as a trypsin cleavage site (K, R).
  • K, R trypsin cleavage site
  • the fluorescent moiety will be placed on the C- terminus of the peptide (which may be derived from a SHRT6 substrate - i.e. Histones).
  • the output of the assay will be a fluorometric measurement - activity of SIRT6 will be inversely proportional to the amount of fluorescence (i.e. If SIRT6 is fully active, no cleavage will occur, and there will be no fluorescence).
  • SIRT6 will be incubated with the aforementioned peptide construct in the presence of ribosylation buffer (Liszt et al., 2006) for a time sufficient for ribosylation, e.g., about one hour.
  • reaction may be be stopped by heating at 90°C for about 10 minutes.
  • reaction will then be subject to a tryptic (or similar endroproteolytic) digest in accordance with the manufacturer's protocol, and quantified using one of the methods detailed herein.
  • exemplary target peptides for use in a fluorimetric assay may comprise about 1, 2,
  • a target peptide may also be from about 5 or from about 10 to about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids.
  • a lysine or arginine may be located at the last or second to last amino acid of the sequence.
  • the fluorescent group may be attached to the last amino acid of the peptide, immediately adjacent to the lysine or arginine.
  • the amino acid sequence of the target peptide is that of a naturally occurring target peptide, e.g., a histone or Sirt6 for a Sirt6 target peptide; the second to last amino acid is a lysine or an arginine and the fluorescent group is attached to the last amino acid.
  • the last amino acid of the peptide is an arginine or a lysine and the fluorescent group is linked to an amino acid that is linked immediately C-terminal to the arginine or lysine.
  • the following peptides may be used for an assay using Sirt6 (the amino acid in parenthesis is an optional last amino acid of the peptide for linkage of the fluorescent group, which amino acid may be replaced by another): PEIFDPPEELER(K), corresponding to amino acids 21 to 33 of SEQ ID NO: 4; HGVWTMEER(G), corresponding to amino acids 68 to 77 of SEQ ID NO: 4; LQPTKHDR(H), corresponding to amino acids 241-249 of SEQ ID NO: 4.
  • PEIFDPPEELER(K) corresponding to amino acids 21 to 33 of SEQ ID NO: 4
  • HGVWTMEER(G) corresponding to amino acids 68 to 77 of SEQ ID NO: 4
  • LQPTKHDR(H) corresponding to amino acids 241-249 of SEQ ID NO: 4.
  • this technique could be modified to use fluorescent polarization (FP) or mass spectrometry.
  • FP fluorescent polarization
  • the position of the attached group can be varied and does not need to be on the very end of the peptide.
  • an FP group may be incorporated on the C-terminus. Shortening of the peptide by cleavage would result in a change in polarization.
  • the Arg or lysine does not need to be adjacent, as long as the peptide becomes shorter when cleaved so it spins faster and alters polarization.
  • the lysine or arginine may be placed about mid-way (although it may not be exactly centered, so as to generate different lengths peptides) within a peptide, e.g., of about 30-40 amino acids.
  • the absence of ribosylation allows the peptide to be cleaved by trypsin, which results in faster spinning of the molecules because they are smaller, resulting in light depolarization or low fluorescence polarization.
  • ribosylation of the target peptide results in the absence of cleavage by the enzyme, resulting in slower spinning of the molecules because they are larger, and emission of high fluorescence polarization.
  • An exemplary method may comprise contacting a sirtuin ribosyltransferase or a functional homolog thereof with a target peptide comprising an arginine or lysine residue located N-terminallyto and adjacent to a fluorescent group; NAD+ and a test agent. These reagents are incubated for a time sufficient for the ribosyl group of NAD+ to be transferred onto a target peptide in the absence of the agent, e.g., about 1, 5, 10, 20, 30, 45, 60, 75, 90, or 120 minutes, with 60 minutes being preferred.
  • the method further comprises adding to the reaction mixture trypsin that cleaves at the C-terminus of the arginine or lysine residue in the absence of ribosylation at the arginine or lysine residue. Trypsin is preferably added after incubation of the other reagents together, such that the peptide is not cleaved before it is ribosylated, if at all.
  • the enzyme is incubated for a time sufficient for the enzyme to cleave essentially all of the target peptides that are not ribosylated, e.g., about 1, 5, 10, 20, 30, 45 or 60 minutes, with 10 minutes being preferred.
  • the method then comprises determining the presence and/or amount of fluorescence.
  • test agent is an agent that inhibits the sirtuin ribosyltransferase.
  • a lower amount of fluorescence in a reaction that was incubated with a test agent relative to a reaction that was not incubated with a test agent indicates that the test agent is an agent that stimulates the sirtuin ribosyltransferase.
  • the fluorescent group on the target peptide may be dimethylcoumarin, fluorescein, tetramethylrhodamine, Texas Red, as well as longer- wavelength dyes and others described herein.
  • the fluorescent group could also be one that works in a fluorescent polarization (FP) assay, such as BOD EPY fluorescent dyes (InVitrogen).
  • an enzyme other than trypsin is used, in which case, the peptide must include an additional enzymatic cleavage site to be cleaved by the enzyme.
  • An enzyme cleavage site may be a site that is recognized and cleaved by an enzyme, e.g., a restriction enzyme, e.g. a peptidase or protease that cleaves a peptide or protein at specific recognition sites.
  • Exemplary peptidases and proteases include: aminopeptidase Y (also known as aminopeptidase Co; cobalt-activated aminopeptidase and lysyl aminopeptidase), clostripain (also known as clostridiopeptidase B), chymotrypsin, enterokinase, LysC lysyl endopeptidase (also known as Achromobacter proteinase I) and trypsin.
  • aminopeptidase Y also known as aminopeptidase Co; cobalt-activated aminopeptidase and lysyl aminopeptidase
  • clostripain also known as clostridiopeptidase B
  • chymotrypsin also known as clostridiopeptidase B
  • enterokinase LysC lysyl endopeptidase
  • trypsin trypsin.
  • Another screening method comprises (i) contacting a cell expressing a sirtuin ribosyltransferase or a functional homolog thereof with labeled NAD+ and a test agent for an amount of time and under conditions appropriate for transfer of the ribosyl group of NAD+ onto a target protein in the absence of the test agent; and (ii) determining whether there is a difference between the number and/or amount of labeled proteins in a cell that was contacted with a test agent relative to a cell that was not contacted with a test agent. The presence of a difference indicates that the test agent is an agent that modulates a rib osyl transferase.
  • the assay may comprise examining all or most of the proteins in the cell, or a portion thereof, such as the proteins within a particular range of sizes.
  • the assay may also comprise analyzing specific proteins, e.g., those that are known targets of sirtuin ribosyltransferases. Labeled proteins may be detected via Western blot and/or spectrometry or as further described herein.
  • the cell may be any cell as further described herein, e.g., a cell comprising a heterologous nucleic acid encoding the sirtuin ribosyltransferase or functional homolog thereof.
  • the assays described herein may be accompanied by control reactions.
  • a control reaction may be a reaction using a cell or lysate of a cell that does not express a heterologous sirtuin ribosyltransferase.
  • Such a control may indicate whether any difference in labeled target is specific to a sirtuin ribosyltransferase, as opposed to any other ribosyltransferase that may be present in a cell or lysate.
  • the method may be followed by the fractioning or subfractioning of the composition.
  • the same or a different screening assay may then be conducted on the one or more fractions of the composition. These steps may be repeated (or reiterated) until a sole agent has been identified. For example, the steps of screening and fractioning maybe repeated 2, 3, 5, 10, 25, 50, 100 times or more.
  • Screening assays may be conducted in any type of container or vial.
  • screening assays may be conducted in microtiter plates, e.g., 96 well plates.
  • Methods for identifying targets of sirtuin ribosyltransferases may be cell-based or cell-free methods.
  • a method for identifying a target protein of a sirtuin ribosyltransferase may comprise:
  • test peptide may be any peptide or protein comprising an arginine or a lysine.
  • a test peptide may be about 10, 15, 20, 25, 30, 35, 40, 50 or more amino acids long.
  • the arginine or lysine is preferably located at least about 1, 2, 3, 4, 5, 6,
  • Test peptides may be portions of proteins, e.g., naturally occurring proteins, or they may be artificial peptides, e.g., from a library. Test peptides may also be full length proteins.
  • a method may then further comprise identifying which peptide is labeled, e.g., by isolating the peptide that is labeled, and determining its amino acid sequence.
  • each peptide may be tagged with a unique identifier, in which case, one would identify the identifier to determine the identity of a test peptide that was labeled.
  • a method comprises (i) contacting a cell expressing a sirtuin ribosyltransferase or a functional homolog thereof with labeled NAD+ for an amount of time and under conditions appropriate for transfer of the ribosyl group of NAD+ onto a target protein; and
  • the cell may be any cell as further described herein, e.g., a cell comprising a heterologous nucleic acid encoding the sirtuin ribosyltransferase or functional homolog thereof.
  • the method may be used for detecting changes in the ribosylation of either a specific target or the entire proteome.
  • ELISA type detection strategies may be used with either streptavidin-conjugated enzyme, e.g., horse radish peroxidase or anti-biotin antibodies.
  • streptavidin-conjugated enzyme e.g., horse radish peroxidase or anti-biotin antibodies.
  • the ribosylated targets may be irnmunoprecipitated by pull down using streptavidin conjugated beads followed by mass spectrometric analysis. Targets may be completely or partially sequenced.
  • Determining the identity of a protein that is labeled may comprise isolating one or more proteins that are labeled, e.g., using a reagent that interacts with the label, and subjecting the one or more proteins or portions thereof to a method of detection, e.g., mass spectroscopy.
  • a method for identifying a target protein of a sirtuin ribosyltransferase comprises contacting a cell comprising, e.g., over-expressing, a sirtuin ribosyltransferase or a functional homolog thereof with biotin-NAD+, for a time sufficient for target proteins to become biotinylated.
  • the method then comprises lysing the cells and adding to the cell lysate avidin coated surfaces, e.g., beads, under conditions in which avidin binds to biotin.
  • the avidin-coated surfaces are then separated from the reaction mixture to thereby isolate the proteins that are biotinylated.
  • the proteins that are linked to the avidin-coated surfaces can then be stripped from the surfaces and subjected to an analytical method, e.g., a Western blot in which the biotinylated proteins are detected with avidin, or a silver stained gel. Proteins from the silver stained gel may be extracted from the gel and subjected to a method of identification, e.g., mass spectroscopy (see Example 1).
  • an analytical method e.g., a Western blot in which the biotinylated proteins are detected with avidin, or a silver stained gel. Proteins from the silver stained gel may be extracted from the gel and subjected to a method of identification, e.g., mass spectroscopy (see Example 1).
  • a method of identifying a sirtuin ribosylation target protein may further be followed by an experiment that confirms that the identified protein is indeed a sirtuin ribosylation target.
  • a method may further comprise combining a sirtuin ribosyltransferase with labeled NAD+ and the suspected target protein or a functional homolog thereof; and determining whether the suspected target protein or the functional homolog thereof is labeled, wherein the presence of label on the suspected target protein confirms that the suspected target protein is a target protein of a sirtuin ribosyltransferase.
  • Target proteins of sirtuin ribosyltransferases may be used in screening assays for identifying modulators of sirtuin ribosyltransferases, e.g., as further described herein. Such modulators may then be used to treat or prevent diseases or conditions that are associated with particular target proteins.
  • a target protein or functional homolog thereof may be administered to a subject to interfere with or augment the function of the naturally-occurring protein in a cell, e.g., to treat or prevent a disease or condition associated with a sirtuin ribosyltransferase.
  • the assay could also be used as a biomarker of a phenotype/symptorn/pathway.
  • the assays can be conducted using a single sirtuin ribosyltransferase or functional homolog thereof, a single target, or a single compound in one assay, the assays may also be conducted in a high throughput screening mode, with, e.g., a plurality (e.g., about 1-5, 1-10, 1-20 or 1-50) of sirtuin ribosyltransferases or functional homologs thereof, targets, and/or compounds (See generally, High Throughput Screening: The Discovery of Bioactive Substances (Devlin, Ed.) Marcel Dekker, 1997; Sittampalam et al, Curr. Opin. Chem.
  • the assay can be conducted in a multi-well (e.g., 24-, 48-, 96-, or 384-well), chip or array format.
  • a multi-well e.g., 24-, 48-, 96-, or 384-well
  • kits e.g., kits for screening assays.
  • a kit may comprise one or more components described herein and any other reagent that may be necessary or helpful in conducting an assay.
  • a method may comprise administering to a subject in need thereof a therapeutically effective amount of an agent that inhibits a sirtuin ribosyltransferase, e.g., Sirt ⁇ or a sirtuin ribosyltransferase, e.g., Sirt ⁇ , dependent ribosylation pathway.
  • a method may decrease the level of protein or activity of a Sirt ⁇ protein.
  • a decrease of the level of protein or activity may be by a factor of at least about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 100 fold or more.
  • Treating" a condition or disease refers to curing as well as ameliorating at least one symptom of the condition or disease or preventing the condition or disease from worsening.
  • a subject in need of treatment may be a subject who has been diagnosed as having or likely to develop a hyper-proliferating disease.
  • a subject may be a vertebrate, such as a mammal, e.g., a human, a bovine, an ovine, a sheep, porcine, a canine, a feline, a mouse or a rat.
  • An agent that inhibits a sirtuin dependent ribosylation or ribosylation pathway may be any type of molecule, such as a small molecule or a macromolecule, e.g., a nucleic acid, oligonucleotide, s ⁇ RNA, antisense RNA, triplex RNA or other. 1.
  • exemplary small molecule inhibitors of sirtuin dependent ribosylation pathways The following molecules may be inhibitors of Sirt ⁇ and/or Sirt4.
  • the agent is sirtinol (2-[(2-hydroxy-naphthalen-l-ylmethylene)-amino]- N-(l-phenyl-ethy- l)-benzamide; Grozinger et al. (2001) J. Biol. Chem. 276:38837), splitomycin (Bedalov et al.
  • nicotinamide (NAM), suranim; NF023 (a G-protein antagonist); NF279 (a purinergic receptor antagonist); Trolox (6-hydroxy- 2,5,7,8,tetramethylchroman-2-carboxylic acid); (-)-epigallocatechin (hydroxy on sites 3,5,7,3',4', 5'); (-)-epigallocatechin (hydroxy on sites 3,5,7,3',4',5'); (-)-epigallocatechin gallate (Hydroxy sites 5,7,3',4',5' and gallate ester on 3); cyanidin choloride (3,5,7,3',4 * - pentahydroxyflavylium chloride); delphinidin chloride (3,5,7,3 ⁇ 4 ⁇ 5'-hexahydroxyflavylium chloride); myricetin (cannabiscetin; 3,5,7,3',4',5'-hexahydroxyfiavone); 3,
  • a Sirt ⁇ inhibitory compound may have the formula I:
  • R 3 is hydrogen, alkyl, aryl, or aralkyl;
  • R b is hydrogen, hydroxyl, alkoxyl, amine, alkyl, aryl, or aralkyl; Ri is aryl;
  • R 2 is hydrogen, alkyl, aryl, or aralkyl
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl, or sulfoxido;
  • R4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl, or sulfoxido; provided that when X is -C(O)-; Y is -N(H)-; Z is -CH(CH 3 )-; R 2 is hydrogen; R 3
  • Ri is not and the compound is achiral or, when chiral, is a single stereoisomer or a mixture of stereoisomers.
  • a Sirt6 inhibitory compound may be a compound of formula II:
  • R 3 is hydrogen, alkyl, aryl, or aralkyl;
  • R b is hydrogen, hydroxyl, alkoxyl, amine, alkyl, aryl, or aralkyl;
  • Ri is aryl;
  • R 2 is hydrogen, alkyl, aryl, or aralkyl;
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R 4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaral
  • R a is hydrogen, alkyl, aryl, or aralkyl;
  • Rb is hydrogen, hydroxyl, alkoxyl, amine, alkyl, aryl, or aralkyl;
  • R 1 is aryl;
  • R 2 is hydrogen, alkyl, aryl, or aralkyl
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R 4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido; and the compound is achiral or, when chiral, is a single stereoisomer or a mixture of stereoisomers.
  • Sirt ⁇ inhibitory compounds are represented by I, II, or III and the attendant definitions, wherein Z is -CH(CH3)-. In certain embodiments, Sirt ⁇ inhibitory compounds are represented by I, II, or III and the attendant definitions, wherein R 2 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by I, II, or III and the attendant definitions, wherein R3 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by I, II, or III and the attendant definitions, wherein R 4 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by I, II, or III and the attendant definitions, wherein R 2 is hydrogen; R 3 is hydrogen; and R 4 is hydrogen.
  • a Sirt ⁇ inhibitory compound may be a compound of formula TV:
  • R a is hydrogen, alkyl, aryl, or aralkyl
  • R b is hydrogen, hydroxyl, alkoxyl, amine, alkyl, aryl, or aralkyl
  • Ri is aryl
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R 4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • Rs is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • a Sirt6 inhibitory compound may be a compound of formula V:
  • R 3 is hydrogen, alkyl, aryl, or aralkyl
  • R b is hydrogen, hydroxyl, alkoxyl, amine, alkyl, aryl, or aralkyl
  • Ri is aryl
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • Rs is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralky
  • R O is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido; and the compound is achiral or, when chiral, is a single stereoisomer or a mixture of stereoisomers.
  • a Sirt ⁇ inhibitory compound may be a compound of formula VI:
  • Ra is hydrogen, alkyl, aryl, or aralkyl
  • Rb is hydrogen, hydroxyl, alkoxyl, amine, alkyl, aryl, or aralkyl
  • Ri is aryl
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R 4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R 5 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • Re is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido; and the compound is achiral or, when chiral, is a single stereoisomer or a mixture of stereoisomers.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein Z is -C(Ra) 2 --
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein Z is -CH(R a )-; and R a is alkyl. In certain embodiments, Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein Z is -CH(CHs)-.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 3 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 4 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 5 is hydroxyl. In certain embodiments, Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 6 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by IV, V 3 or VI and the attendant definitions, wherein R 5 is hydroxyl; and Rg is hydrogen. In certain embodiments, Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 5 is hydroxyl; R 6 is hydrogen; and R 4 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 5 is hydroxyl; R ⁇ is hydrogen; R 4 is hydrogen; and R 3 is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R5 is hydroxyl; Re is hydrogen; R4 is hydrogen; R3 is hydrogen; Z is -CH(R a )-; and R a is alkyl.
  • Sirt ⁇ inhibitory compounds are represented by IV, V, or VI and the attendant definitions, wherein R 5 is hydroxyl; R 6 is hydrogen; R 4 is hydrogen; R 3 is hydrogen; and Z is -CH(CH 3 )-.
  • a Sirt ⁇ inhibitory compound may be a compound of formula VII:
  • Y is -N(H)-, -CH 2 - or -C(O)-;
  • a Sirt ⁇ inhibitory compound may be a compound of formula VIII:
  • a Sirt ⁇ inhibitory compound may be a compound of formula IX:
  • Rs is hydrogen, hydroxyl or alkoxyl; and the compound is achiral or, when chiral, is a single stereoisomer or a mixture of stereoisomers.
  • Sirt ⁇ inhibitory compounds are represented by VII, VIII, or IX and the attendant definitions, wherein R 5 is hydroxyl.
  • Sirt ⁇ inhibitory compounds are represented by VII, VIII, or IX and the attendant definitions, wherein R 5 is hydroxyl; X is -S-; and Y is -CH 2 -. In certain embodiments, Sirt ⁇ inhibitory compounds are represented by VII, VIII, or
  • R 5 is hydroxyl
  • Y is -N(H)-.
  • Sirt ⁇ inhibitory compounds are represented by VII, VIII, or IX and the attendant definitions, wherein the compound is a single enantiomer or steroisomer.
  • a Sirt ⁇ inhibitory compound may be a compound of formula X:
  • Ri is aryl
  • R 2 is hydrogen, alkyl, aryl, or aralkyl
  • R 3 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido; R4 is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkyn
  • a Sirt ⁇ inhibitory compound may be a compound of formula XI:
  • Sirt ⁇ inhibitory compounds are represented by X or XI and the attendant definitions, wherein R 4 is -CN.
  • Sirt ⁇ inhibitors include those described in Solomon et al. (2006) MoI. Cell. Biol. 26:28 and referred to as EX-519, EX-527, EX-586, EX-589, EX-622 and EX-635, derivatives and analogs thereof.
  • EX-527 has the following structure:
  • a Sirt ⁇ inhibitory compound may be a compound of formula XII:
  • A is O, S or N(R 1 );
  • X is C(R) 2 ;
  • Y is N or C(R);
  • R" is hydrogen, alkyl, alkenyl. alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; p is 0, 1, 2 or 3; and the stereochemical configuration at any stereocenter is R, S, or a mixture of these configurations.
  • Sirt ⁇ inhibitory compounds are represented by XII and the attendant definitions, wherein A is N(R 1 ).
  • Sirt6 inhibitory compounds are represented by XEI and the attendant definitions, wherein Y is C(R).
  • Sirt ⁇ inhibitory compounds are represented by XII and the attendant definitions, wherein p is 0.
  • a Sirt ⁇ inhibitory compound may be a compound of formula XIII:
  • X is C(R) 2 ;
  • R is hydrogen, halogen, alkyl, alkenyl, alkynyU aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamide, sulfamoyl, sulfonyl or sulfoxido;
  • R" is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl;
  • p is 0, 1 , 2 or 3 ; and the stereochemical configuration at any stereocenter is R, S, or a mixture of these configurations.
  • Sirt ⁇ inhibitory compounds are represented by XTII and the attendant definitions, wherein R' is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by XIII and the attendant definitions, wherein p is 0.
  • a Sirt ⁇ inhibitory compound may be a compound of formula XIV:
  • Z is -OR" or -N(R') 2 ;
  • R is hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkyenyl, aralkynyl, heteroaralkyl, heteroaralkyenyl, heteroaralkynyl, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, acylamino, amido, alkylthio, carboxyl, carbamoyl, alkoxyl, sulfonate, sulfate, sulfonamido, sulfamoyl, sulfonyl or sulfoxido;
  • R" is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and the stereochemical configuration at any stereocenter is R, S, or a mixture of these configurations.
  • Sirt ⁇ inhibitory compounds are represented by XIV and the attendant definitions, wherein Z is -N(R) 2 .
  • Sirt ⁇ inhibitory compounds are represented by XIV and the attendant definitions, wherein R' is hydrogen.
  • Sirt ⁇ inhibitory compounds are represented by XIV and the attendant definitions, wherein Z is -N(H) 2 .
  • Sirt6 inhibitory compounds are represented by XIV and the attendant definitions, wherein R is halogen, cyano, nitro, sulfhydryl, hydroxyl, sulfonyl, amino, alkoxyl, or trifluoromethyl.
  • Sirt6 inhibitory compounds are represented by XIV and the attendant definitions, wherein R is -Cl, -Br, -I or -F.
  • Sirt6 inhibitory compounds are represented by XIV and the attendant definitions, wherein R is -Cl.
  • both the cis (Z) and trans (E) isomers are contemplated herein.
  • the compounds may exist in tautomeric forms, such as keto-enol tautomers,
  • Prodrugs of the Sirt6 inhibitory compounds of formulas I-XI are considered to be any covalently bonded carriers that release the active parent drug in vivo. Metabolites, e.g., degradation products, of these compounds are also included.
  • a compound is included within the generic structures of formula I-XI with the proviso that the compound is not a particular compound, such as a naturally occuring compound or that the compound is not present in a particular form, such as a naturally-occurring form.
  • a compound may have a binding affinity for a Sirt6 of about 10 "9 M, 10 "10 M, 10 " 11 M, 10 "12 M or less.
  • a compound may have an EC 50 for inhibiting an activity of a Sirt ⁇ , such as its NAD-ribosyltransferase activity, of less than about 1 nM, less than about 10 nM, less than about 100 nM, less than about 1 ⁇ M, less than about 10 ⁇ M, less than about 100 ⁇ M, or from about 1-10 nM, from about 10-100 nM, from about 0.1-1 ⁇ M, from about 1-10 ⁇ M or from about 10-100 ⁇ M.
  • a Sirt ⁇ such as its NAD-ribosyltransferase activity
  • a compound may inhibit an activity of a Sirt ⁇ by a factor of at least about 50%, 2, 5, 10, 20, 30, 50, or 100, as measured in an acellular assay or in a cell based assay.
  • a compound may cause at least a 10%, 30%, 50%, 80%, 2 fold, 5 fold, 10 fold, 50 fold or 100 fold greater inhibition of an activity of Sirt ⁇ relative to the same concentration of sirtinol or other compound described herein.
  • the qualitative or quantitative effect of a compound on the NAD-ribosyltransferase activity of Sirt ⁇ may be determined as described, e.g., in Liszt et al. (2005) J. Biol. Chem. 22:21313.
  • a Sirt ⁇ protein or a functional homolog thereof may be contacted with a compound in vitro, e.g., in a solution, in a cell or in a cell extract, hi one embodiment, a Sirt ⁇ protein of functional homolog thereof is contacted with a compound and a ribosyltransferase target peptide in a solution and the amount of ribosyl that was transferred onto the target peptide is determined.
  • a target peptide may be a peptide from a histone or core histones or a functional homolog thereof.
  • a target peptide may also be nucleoplasmin, also referred to as nucleolar phosphoprotein B23, numatrin, and as chromatin decondensation protein or a functional homolog thereof. Targets are further described herein. These substrate or target proteins and functional homolog thereof may also be used in the assays. Similar assays may also be used to identify other inhibitors of Sirt ⁇ .
  • Sirt ⁇ includes proteins comprising, consisting essentially of, or consisting of an amino acid sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity with the amino acid sequence of the protein of interest.
  • a homolog may also be a protein that is encoded by a nucleic acid comprising, consisting essentially of, or consisting of a nucleotide sequence that has at least about 70%, 80%, 90%, 95%, 98% or 99% identity with a nucleotide sequence encoding the protein of interest or the coding sequence thereof.
  • a homolog may also be a protein that is encoded by a nucleic acid that hybridizes, e.g., under stringent hybridization conditions, to a nucleic acid encoding a protein of interest, or the coding sequence thereof.
  • homologs may be encoded by nucleic acids that hybridize under high stringency conditions of 0.2 to 1 x SSC at 65 0 C followed by a wash at 0.2 x SSC at 65 0 C to a nucleic acid encoding a protein of interest.
  • Nucleic acids that hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature to nucleic acid encoding a protein of interest or a portion thereof can be used.
  • hybridization conditions include 3 x SSC at 40 or 50 0 C, followed by a wash in 1 or 2 x SSC at 20, 30, 40, 50, 60, or 65 0 C.
  • Hybridizations can be conducted in the presence of formaldehyde, e.g., 10%, 20%, 30% 40% or 50%, which further increases the stringency of hybridization. Theory and practice of nucleic acid hybridization is described, e.g., in S.
  • Homologs of proteins described herein, such as Sirt ⁇ or a substrate thereof may also be analogs, e.g., that differ from the naturally occurring protein by conservative amino acid sequence differences or by modifications that do not affect sequence, or by both. Analogs can differ from naturally occurring proteins by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. Any number of procedures may be used for the generation of mutant, derivative or variant forms of a protein of interest using recombinant DNA methodology well known in the art such as, for example, that described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York) and Ausubel et al. (1997, Current Protocols in Molecular Biology, Green & Wiley, New York).
  • Homologs of a protein of interest also includes portions thereof, such as portions comprising one or more conserved domains, e.g., the catalytic domain that Sirt6 shares with the other sirtuins (Frye et al. (1999) Biochem. Biophys. Res. Comm. 260:273 and Liszt et al, infra).
  • the catalytic domain of human Sirt6 corresponds to about amino acids 45 to 271 of SEQ ID NO: 2 (see Liszt et al., infra).
  • a “functional homolog" of a protein of interest refers to a homolog of the protein having at least one biological activity of the protein.
  • a functional homolog of Sirt6 may be a protein having an ribosyl transferase or deacetylase activity, or other biological activities. Whether a homolog is a functional homolog can be determined according to methods known in the art. For example, a ribosyl transferase activity can be determined as further described herein.
  • a compound may inhibit more efficiently Sirt6 relative to one or more other sirtuins.
  • a compound may inhibit more efficiently Sirt ⁇ than the other sirtuins from the same species, e.g., SIRTl, SIRT2 ⁇ 1, SIRT2i2, SIRT3ia, SIRT3ib, SIRT4,
  • SIRT5il, SIRTi2 and SIRT7 or non human homologs thereof may inhibit more efficiently a sirtuin from a particular species relative to the homologous sirtuin from another species.
  • a compound may inhibit more efficiently a sirtuin from a microorganism, such as a pathogen, relative to the same sirtuin from humans.
  • a sirtuin inhibiting compound may be more efficient in inhibiting one sirtuin relative to another by a factor of at least about 50%, 2 fold, 5 fold, 10, fold, 20 fold, 50 fold, or 100 fold.
  • a compound may traverse the cytoplasmic membrane of a cell.
  • a compound may have a cell-permeability of at least about 20%, 50%, 75%, 80%, 90% or 95%.
  • a compound having a cell-permeability of at least about 20% means that at least about 20% of these compounds will enter a cell within a certain time frame when a given amount of these compounds is contacted with the cell.
  • a compound may have a normal half-life under normal atmospheric conditions of at least about 30 days, 60 days, 120 days, 6 months, or 1 year.
  • One compound maybe more stable in solution than another compound, e.g., sirtinol, by a factor of at least about 50%, 2 fold, 5 fold, 10 fold, 30 fold, 50 fold, or 100 fold.
  • a cell is obtained from a subject following administration of an inhibitory compound to the subject, such as by obtaining a biopsy, and the activity of the sirtuin is determined in the biopsy.
  • the cell may be any cell of the subject, but in cases in which an inhibitory compound is administered locally, the cell is preferably a cell that is located in the vicinity of or at the site of administration.
  • stereoisomers is art-recognized and refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • enantiomers refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • Diastereomers refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
  • a “stereoselective process” is one which produces a particular stereoisomer of a reaction product in preference to other possible stereoisomers of that product.
  • An “enantioselective process” is one which favors production of one of the two possible enantiomers of a reaction product.
  • the term "regioisomers” is art-recognized and refers to compounds which have the same molecular formula but differ in the connectivity of the atoms. Accordingly, a “regioselective process” is one which favors the production of a particular regioisomer over others, e.g., the reaction produces a statistically significant increase in the yield of a certain regioisomer.
  • esters are art-recognized and refers to molecules with identical chemical constitution and containing more than one stereocenter, but which differ in configuration at only one of these stereocenters.
  • Treating refers to curing as well as ameliorating at least one symptom of the condition or disease or preventing the condition or disease from worsening.
  • structure-activity relationship or "(SAR)” is art-recognized and refers to the way in which altering the molecular structure of a drug or other compound alters its biological activity, e.g., its interaction with a receptor, enzyme, nucleic acid or other target and the like.
  • aliphatic is art-recognized and refers to a linear, branched, cyclic alkane, alkene, or alkyne. In certain embodiments, aliphatic groups in the present compounds are linear or branched and have from 1 to about 20 carbon atoms.
  • heteroatom is art-recognized and refers to an atom of any element other than carbon or hydrogen.
  • Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
  • alkyl is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., C1-C 30 for straight chain, C 3 -C 3 0 for branched chain), and alternatively, about 20 or fewer.
  • cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.
  • lower alkyl refers to an alkyl group, as defined above, but having from one to about ten carbons, alternatively from one to about six carbon atoms in its backbone structure.
  • lower alkenyl and “lower alkynyl” have similar chain lengths.
  • aralkyl is art-recognized and refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • alkenyl and alkynyl are art-recognized and refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • aryl is art-recognized and refers to 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine,. and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or “heteroaromatics.”
  • the aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, - CF 3 , -CN, or the like.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • ortho, meta and para are art-recognized and refer to 1 ,2-, 1 ,3- and 1 ,4- disubstituted benzenes, respectively.
  • the names 1 ,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.
  • heterocyclyl refers to 3- to about 10-membered ring structures, alternatively 3- to about 7-membered rings, whose ring structures include one to four heteroatoms.
  • Heterocycles may also be polycycles.
  • Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboiine, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine,
  • the heterocyclic ring may be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, - hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, - hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,
  • polycyclyl or “polycyclic group” are art-recognized and refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle may be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF 3 , -CN, or the like.
  • the term "carbocycle” is art-recognized and refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
  • nitro is art-recognized and refers to -NO 2 ;
  • halogen is art- recognized and refers to -F, -Cl, -Br or -I;
  • sulfhydryl is art-recognized and refers to -SH;
  • hydroxyl means -OH;
  • sulfonyl is art-recognized and refers to -SO2 " .
  • Halide designates the corresponding anion of the halogens, and
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:
  • R50, R51 and R52 each independently represent a hydrogen, an alkyl, an alkenyl, - (CH 2 )m-R61 , or R50 and R51 , taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure;
  • R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to S.
  • R50 and R51 (and optionally R52) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH 2 ) m -R61.
  • alkylamine includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R50 and R51 is an alkyl group.
  • acylamino is art-recognized and refers to a moiety that may be represented by the general formula:
  • R50 is as defined above
  • R54 represents a hydrogen, an alkyl, an alkenyl or - (CH 2 ) m -R61, where m and R61 are as defined above.
  • amino is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH 2 ) m -R61, wherein m and R61 are defined above.
  • Representative alkylthio groups include methylthio, ethyl thio, and the like.
  • carboxyl is art recognized and includes such moieties as may be represented by the general formulas:
  • X50 is a bond or represents an oxygen or a sulfur
  • R55 and R56 represents a hydrogen, an alkyl, an alkenyl, -(CH 2 ) m -R61or a pharmaceutically acceptable salt
  • R56 represents a hydrogen, an alkyl, an alkenyl or -(CH 2 ) m -R61, where m and R61 are defined above.
  • X50 is an oxygen and R55 or R56 is not hydrogen
  • the formula represents an "ester”.
  • X50 is an oxygen
  • R55 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R55 is a hydrogen, the formula represents a "carboxylic acid".
  • X50 is an oxygen, and R56 is hydrogen
  • the formula represents a "formate".
  • the oxygen atom of the above formula is replaced by sulfur
  • the formula represents a "thiolcarbonyl” group.
  • X50 is a sulfur and R55 or R56 is not hydrogen
  • the formula represents a "thiolester.”
  • X50 is a sulfur and R55 is hydrogen
  • the formula represents a "thiolcarboxylic acid.”
  • X50 is a sulfur and R56 is hydrogen
  • the formula represents a "thiolformate.”
  • X50 is a bond, and R55 is not hydrogen
  • the above formula represents a "ketone” group.
  • X50 is a bond, and R55 is hydrogen
  • the above formula represents an "aldehyde” group.
  • oxime and "oxime ether” are art-recognized and refer to moieties that may be represented by the general formula:
  • R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, or -(CH 2 ) m -R61.
  • the moiety is an "oxime” when R is H; and it is an “oxime ether” when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl., aralkyl, or -(CH 2 ) m -R61.
  • alkoxyl or "alkoxy” are art-recognized and refer to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O ⁇ (CH 2 ) m -R61, where m and R61 are described above.
  • R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
  • sulfate is art recognized and includes a moiety that may be represented by the general formula:
  • sulfamoyl is art-recognized and refers to a moiety that may be represented by the general formula:
  • sulfonyl is art-recognized and refers to a moiety that may be represented by the general formula:
  • R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.
  • sulfoxido is art-recognized and refers to a moiety that may be represented by the general formula:
  • phosphoryl is art-recognized and may in general be represented by the formula:
  • Q50 represents S or O
  • R59 represents hydrogen, a lower alkyl or an aryl.
  • the phosphoryl group of the phosphorylalkyl may be represented by the general formulas:
  • Q50 and R59 each independently, are defined above, and Q51 represents O, S or N.
  • Q50 is S
  • the phosphoryl moiety is a "phosphorothioate”.
  • R60 represents a lower alkyl or an aryl.
  • Analogous substitutions may be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
  • each expression e.g. alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • sel is art-recognized and refers to an alkyl group having a substituted seleno group attached thereto.
  • exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and - Se-(CH2)m-R61 , m and R61 being defined above.
  • triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, /?-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
  • triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, /7-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
  • Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, /7-toluenesulfonyl and methanesulfonyl, respectively.
  • a more comprhensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations.
  • compositions of the present invention may exist in particular geometric or stereoisomeric forms.
  • polymers of the present invention may also be optically active.
  • the present invention contemplates all such compounds, including cis- and trans-isomers, R- and 5-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction.
  • substituted is also contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • the permissible substituents may be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed (Greene, T. W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 3 rd ed.; Wiley: New York, 1999). Protected forms of the inventive compounds are included within the scope of this invention.
  • protecting group is art-recognized and refers to temporary substituents that protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed by Greene and Wuts in Protective Groups in Organic Synthesis (2 nd ed., Wiley: New York, 1991).
  • hydroxyl-protecting group is art-recognized and refers to those groups intended to protect a hydroxyl group against undesirable reactions during synthetic procedures and includes, for example, benzyl or other suitable esters or ethers groups known in the art.
  • carboxyl-protecting group refers to those groups intended to protect a carboxylic acid group, such as the C-terminus of an amino acid or peptide or an acidic or hydroxyl azepine ring substituent, against undesirable reactions during synthetic procedures and includes.
  • Examples for protecting groups for carboxyl groups involve, for example, benzyl ester, cyclohexyl ester, 4-nitrobenzyl ester, t-butyl ester, 4-pyridylmethyl ester, and the like.
  • amino-b locking group refers to a group which will prevent an amino group from participating in a reaction carried out on some other functional group, but which can be removed from the amine when desired.
  • amino groups are discussed by in Ch. 7 of Greene and Wuts, cited above, and by Barton, Protective Groups in Organic Chemistry ch. 2 (McOmie, ed., Plenum Press, New York, 1973).
  • acyl protecting groups such as, to illustrate, formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl, methoxysuccinyl, benzyl and substituted benzyl such as 3,4-dimethoxybenzyl, o-nitrobenzyl, and triphenylmethyl; those of the formula -COOR where R includes such groups as methyl, ethyl, propyl, isopropyl, 2,2,2-trichloroethyl, 1- methyl-1-phenylethyl, isobutyl, t-butyl, t-amyl, vinyl, allyl, phenyl, benzyl, p-nitrobenzyl, o-nitrobenzyl, and 2,4-dichlorobenzyl; acyl groups and substituted acyl such as formyl, acetyl, chloroacetyl, dichloroacetyl,
  • Preferred amino-blocking groups are benzyl (-CH 2 CeH 5 ), acyl [C(O)Rl] or SiRl 3 where Rl is C1-C4 alkyl, halomethyl, or 2-halo-substituted-(C2-C 4 alkoxy), aromatic urethane protecting groups as, for example, carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (FMOC).
  • aromatic urethane protecting groups as, for example, carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups such as t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (FMOC).
  • the term "electron-withdrawing group” is art-recognized, and refers to the tendency of a substituent to attract valence electrons from neighboring atoms, i.e., the substituent is electronegative with respect to neighboring atoms.
  • ⁇ Hammett sigma
  • This well known constant is described in many references, for instance, March, Advanced Organic Chemistry 251-59 (McGraw Hill Book Company: New York, 1977).
  • Exemplary electron- withdrawing groups include nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride, and the like.
  • Exemplary electron- donating groups include amino, methoxy, and the like.
  • the term "small molecule” is art-recognized and refers to a composition which has a molecular weight of less than about 2000 amu, or less than about 1000 amu, and even less than about 500 amu. Small molecules may be, for example, nucleic acids, peptides, polypeptides, peptide nucleic acids, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.
  • the term "small organic molecule” refers to a small molecule that is often identified as being an organic or medicinal compound, and does not include molecules that are exclusively nucleic acids, peptides or polypeptides.
  • chemical entity refers to chemical compounds, complexes of two or more chemical compounds, and fragments of such compounds or complexes.
  • chemical entities exhibiting a wide range of structural and functional diversity, such as compounds exhibiting different shapes (e.g., flat aromatic rings(s), puckered aliphatic rings(s), straight and branched chain aliphatics with single, double, or triple bonds) and diverse functional groups (e.g., carboxylic acids, esters, ethers, amines, aldehydes, ketones, and various heterocyclic rings).
  • a sirtuin ribosyltransferase inhibitor may also be a siRNA, anti-sense RNA, or a ribozyme that can decrease the expression or level of the sirtuin ribosyltransferase.
  • siRNA anti-sense RNA
  • ribozyme that can decrease the expression or level of the sirtuin ribosyltransferase.
  • RNAi otherwise known as double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA)
  • dsRNAi double-stranded RNA interference
  • siRNA small interfering RNA
  • dsRNA can be delivered to cells or to an organism to antagonize a sirtuin or other protein described herein.
  • a dsRNA that is complementary to a Sirt ⁇ nucleic acid can silence protein expression of Sirt6.
  • the dsRNA can include a region that is complementary to a coding region of a Sirt ⁇ nucleic acid, e.g., a 5' coding region, a region encoding a sirtuin core domain, a 3' coding region, or a non-coding region, e.g., a 5' or 3' untranslated region.
  • dsRNA can be produced, e.g., by transcribing a cassette (in vitro or in vivo) in both directions, for example, by including a T7 promoter on either side of the cassette.
  • the insert in the cassette is selected so that it includes a sequence complementary to the Sirt ⁇ nucleic acid.
  • the sequence need not be full length, for example, an exon, or between 19-50 nucleotides or 50-200 nucleotides.
  • the sequence can be from the 5' half of the transcript, e.g., within 1000, 600, 400, or 300 nucleotides of the ATG. See also, the HISCRIBETM. RNAi Transcription Kit (New England Biolabs, MA) and Fire, A. (1999) Trends Genet. 15, 358-363.
  • dsRNA can be digested into smaller fragments. See, e.g., U.S. patent application 2002-0086356 and 2003-0084471.
  • Sirt ⁇ levels are decreased by administration of or expression in a subject, e.g., in cells or a tissue of the subjet, of one or more Sirt ⁇ siRNAs.
  • siRNA short interfering RNAs
  • siRNA refers to any nucleic acid molecule capable of mediating RNAi or gene silencing.
  • the term siRNA encompasses various naturally generated or synthetic compounds, with RNAi function. Such compounds include, without limitation, duplex synthetic oligonucleotides, of about 21 to 23 base pairs with terminal overlaps of 2 or 3 base pairs; hairpin structures of one oligonucleotide chain with sense and complementary, hybridizing, segments of 21-23 base pairs joined by a loop of, e.g., 3-5 base pairs; and various genetic constructs leading to the expression of the preceding structures or functional equivalents.
  • siRNAs is equivalent to any term in the art defined as a molecule capable of mediating sequence-specific RNAi.
  • dsRNA double-stranded RNA
  • mRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post-transcriptional gene silencing RNA
  • a composition comprising an siRNA effective to inhibit S ⁇ rt ⁇ expression may include an RNA duplex comprising a sense sequence of Sirt ⁇ .
  • An RNA duplex may comprise a first strand comprising a sense sequence of Sirt ⁇ and a second strand comprising a complement of the sense sequence of Sirt ⁇ .
  • the sense and/or complement sequence of Sirt ⁇ comprise of from 10 to 25 nucleotides in length. More preferably, the sense and/or complement sequence of Sirt ⁇ comprise of from 19 to 25 nucleotides in length. Most preferably, the sense and/or complement sequence of Sirt ⁇ comprise of from 21 to 23 nucleotides in length.
  • the sense sequence of Sirt ⁇ may comprise a sequence of Sirt ⁇ containing a translational start site, or may comprise a portion of Sirt ⁇ sequence within about 1000, 600, 400 or 300 nucleotides of the ATG.
  • the complement sequence need not be perfectly complementary to the sense sequence. They may differ in one or more nucleotide substitutions, deletions or additions. Similarly, the sense and/or complement sequences of Sirt ⁇ may differ in one or more nucleotide substitutions, additions and deletions from the Sirt ⁇ sequence in a target cell, provided that the siRNA is sufficiently specific for targeting Sirt ⁇ expression.
  • Exemplary human Sirt ⁇ target sequences for siRNAs may comprise, consist essentially of or consist of one of the following sequences: AAGCGGCCTCAACAAGGGAAA (starting at nucleotide 12); AACAAGGGAAACTTTATTGTT (starting at nucleotide 22) ; AAGGGAAACTTTATTGTTCCC (starting at nucleotide 25); AAACTTTATTGTTCCCGTGGG (starting at nucleotide 30); AATTACGCGGCGGGGCTGTCG (starting at nucleotide 70); AAGTTCGACACCACCTTTGAG (starting at nucleotide 301); AAACTGGCAGAGCTCCACGGG (starting at nucleotide 442); and
  • AAGGCAAGGGGGCTGCGAGCC starting at nucleotide 568.
  • Any other target sequence may be identified according to methods known in the art.
  • software for identifying siRNA target sequences is available at www.ambion.com/techlib/misc/siRNA_finder.html and several other sites for designing siRNAs are set forth in www.maiweb.com.
  • An siRNA may comprise one or more chemical modifications and/or nucleotide analogues.
  • the modification and/or analogue may be any modification and/or analogue, respectively, that does not negatively affect the ability of the siRNA to inhibit IMP3 expression.
  • the inclusion of one or more chemical modifications and/or nucleotide analogues in an siRNA may be preferred to prevent or slow nuclease digestion, and in turn, create a more stable siRNA for practical use.
  • Chemical modifications and/or nucleotide analogues which stabilize RNA are known in the art.
  • Phosphorothioate derivatives which include the replacement of non-bridging phosphoroyl oxygen atoms with sulfur atoms, are one example of analogues showing increased resistance to nuclease digestion.
  • Sites of the siRNA which may be targeted for chemical modification include the loop region of a hairpin structure, the 5' and 3' ends of a hairpin structure (e.g. cap structures), the 3' overhang regions of a double-stranded linear siRNA, the 5' or 3' ends of the sense strand and/or antisense strand of a linear siRNA, and one or more nucleotides of the sense and/or anti sense strand.
  • siRNAs may be administered to a cell or tissue or subject.
  • a nucleic acid encoding an siRNA is introduced or administred to a cell or tissue or subject, e.g., using a vector, e.g., a viral vector.
  • a vector may comprise a constitutive or an inducible promoter.
  • siRNA delivery systems include viral and non-viral systems.
  • Suitable viral systems include adenoviral vectors, adeno-associated virus, lentivirus, poxvirus, retroviral vectors, vaccinia, herpes simplex virus, HFV, the minute virus of mice, hepatitis B virus and influenza virus.
  • non-viral delivery systems include, for example, uncomplexed DNA or RNA, DNA or RNA-liposome complexes, DNA or RNA-protein complexes and DNA or RNA-coated gold particles, bacterial vectors such as salmonella, and other technologies such as those involving VP22 transport protein, Co-X-gene, and replicon vectors.
  • RNAi technology Publications describing RNAi technology include: U.S. Pat. No. 6,686,463, U.S. Pat. No. 6,673,611, U.S. Pat. No. 6,623,962, U.S. Pat. No. 6,506,559, U.S. Pat. No. 6,573,099, and U.S. Pat. No. 6,531,644; U.S. publication Nos: 20030153519,
  • the level of Sirt6 or Sirt ⁇ expression is reduced or decreased by administration or the expression of antisense molecules in a subject or tissue or cell thereof.
  • Antisense molecules may be antisense DNA or RNA or those resulting in triple-helix formation
  • An antisense nucleic acid molecule which is complementary to a nucleic acid molecule encoding Sirt6 can be designed based on the known Sirt ⁇ nucleotide sequences.
  • An antisense nucleic acid molecule can comprise a nucleotide sequence which is complementary to a coding strand of a nucleic acid, e.g. complementary to an mRNA sequence, constructed according to the rules of Watson and Crick base pairing, and can hydrogen bond to the coding strand of the nucleic acid.
  • the antisense sequence complementary to a sequence of an mRNA can be complementary to a sequence in the coding region of the mRNA or can be complementary to a 5' or 3' untranslated region of the mRNA.
  • an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element.
  • An antisense nucleic acid may be complementary to a region preceding or spanning the initiation codon or in the 3' untranslated region of an mRNA.
  • ribozymes are used to inhibit expression of Sirt ⁇ .
  • agents that can be used to inhibit Sirt ⁇ expression or reduce Sirt ⁇ protein levels or activity include anti-Sirt6 antibodies, e.g., intrabodies, single chain antibodies, and aptamers. Aptamers can be produced using the methodology disclosed in a U.S. Pat. No. 5,270,163 and WO 91/19813.
  • An antibody (or other inhibitors or intrabody) can be administered intracellularly as described in, e.g., Marasco and Haseltine in PCT WO94/02610.
  • An antibody may comprise a nuclear localization sequence, e.g., an SV40 nuclear localization signal.
  • Sirt ⁇ inhibitors include dominant negative mutants of Sirt ⁇ , e.g., a SIRT6 protein or portion thereof, in which the histidine at position 133 or the serine residue at position 56 is changed to another amino acid.
  • a dominant negative mutant of human Sirt ⁇ is a human Sirt ⁇ or portion thereof having one or both of the following mutations: S56A and H133Y (see Liszt et al., infra).
  • Other Sirt ⁇ inhibitors include Sirt ⁇ target proteins or peptides thereof.
  • Sirt ⁇ target peptides are fragments of Sirt ⁇ target proteins that are ribosylated by Sirt ⁇ , a high level of which in a cell, would titrate out the activity of Sirt ⁇ , preventing ribosylation of the target proteins in the cell.
  • Peptides may be about 5-10; 10-15, 15-20, 20-25 amino acids long or longer.
  • Target peptides maybe identified according to methods known in the art.
  • Sirt ⁇ inhibitors may be identified by screening methods.
  • a screening method may involve Sirt ⁇ or a portion thereof or a functional homolog thereof.
  • Exemplary assays for determining the ability of a compound to inhibit Sirt ⁇ , such as its function as a ribosyltransferase, are further described herein as well as in, e.g., in Liszt et al. (2005) J. Biol. Chem. 280:21313.
  • an agent that inhibits Sirt ⁇ is an agent that inhibits Sirt6 activity by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to the Sirt ⁇ activity in the absence of the agent.
  • Target proteins of Sirt ⁇ ribosylation include histones, nucleoplasmin, and the two tumor suppressors, ARF and p53. Nucleoplasmins are involved in histone binding, chromatin remodeling, embryonic development, fertilization, oocyte differentiation, regulation of meiosis, regulation of translation, and they interact with the reverse transcriptase and Tat of human immunodeficiency virus 1 (HIV-I). Accordingly, modulators of Sirt ⁇ ribosylation activity may be used for affecting reproduction, aging, cell growth, and for treating or preventing HIV-I infections. Based on the fact that Sirt ⁇ ribosylates tumor suppressors, Sirt ⁇ modulators may be used for treating or preventing cancer.
  • RNA stuin a sirtuin
  • a method comprises administering to a subject in need thereof a therapeutically effective amount of an agent that inhibits Sirt ⁇ or a Sirt ⁇ dependent ribosylation pathway, such as an agent that decreases Sirt ⁇ activity or protein level.
  • Treating a subject refers to curing, or improving at least one symptom of the disease or preventing the disease or a symptom thereof to worsen.
  • treating cancer in a subject includes reducing or maintaining tumor load; reducing metastasis; or curing the subject.
  • the method may also be used prophylactically to prevent the occurrence of a disease, e.g., cancer.
  • an “effective amount” of an agent refers to an amount of an agent which, when applied as part of a desired dosage regimen brings about a decrease in the rate of cell proliferation and/or the state of the disease, so as to produce a result according to clinically acceptable standards for the disorder to be treated.
  • the agents described herein can be administered in a "growth inhibitory amount,” i.e., an amount of the compound that is pharmaceutically effective to inhibit or decrease proliferation of target cells.
  • the agents described herein can be used to normalize, e.g., inhibit or block the proliferation of cells, in particular, cells that are subject to abnormal growth.
  • “Abnormal growth of cells” means cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition), including abnormal growth resulting form expression of an oncogene.
  • the agents described herein may be used for reducing or eliminating excessive cell proliferation.
  • the phrase "excessive cell proliferation,” used interchangeably herein with “hyper-proliferation" of cells refers to cells, which divide more often than their normal or wild-type counterpart or which are not sensitive to normal mechanisms of growth control. For example, cells are excessively proliferating when they double in less than 24 hours if their normal counterparts double in 24 hours.
  • Excessive proliferation can be detected by simple counting of the cells, with or without specific dyes, or by detecting DNA replication or transcription, such as by measuring incorporation of a labeled molecule or atom into DNA or RNA.
  • unwanted cell proliferation can be reduced or eliminated as described herein.
  • Unwanted cell proliferation refers to cell proliferation that is undesirable. Unwanted cell proliferation can refer to cells that are proliferating normally and to cells which are proliferating abnormally, such as cancerous cells. For example, a wart is a tissue in which unwanted epithelial cell proliferation is occurring. Methods described herein may be used for inhibiting cell proliferation, i.e., for decreasing the rate of cell division, by arresting or slowing down the cell cycle.
  • the phrase refers to complete blockage of cell proliferation, i.e., cell cycle arrest, as well as to a lengthening of the cell cycle.
  • the period of a cell cycle can be increased by about 10%, about 20%, about 30, 40, 50, or 100%.
  • the duration of the cell cycle can also be augmented by a factor of two, three, 4, 5, 10 or more.
  • Methods described herein may also be used for normalizing cell proliferation, i.e., reducing the rate of cell proliferation of a cell that proliferates excessively relative to that of its normal or wild-type counterpart. Methods described herein may also be used for suppressing an oncogenic phenotype of a cell, i.e., reducing the transforming, tumorigenic or metastatic potential of the cell.
  • a "transformed cell” refers to a cell which was converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite , number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. Transformed cells include cancer cells, cells infected by a microorganism, e.g., viruses, such as retroviruses.
  • Methods described herein may also be used to decrease the rate of proliferation of immortalized cells, e.g., cells which have been altered via chemical and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture.
  • immortalized cells e.g., cells which have been altered via chemical and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture.
  • Exemplary diseases or disorders that may be treated by the methods described herein include “proliferative disorders” and “hyper-proliferative disorders,” i.e., any disease/disorder of a tissue marked by unwanted or aberrant proliferation of at least some cells in the tissue. Whether a proliferative disorder is a hyper-proliferative disorder depends on how excessive the cell growth is. For example cancer is a hyper-proliferative disorder. Other proliferative diseases include benign diseases or disorders, such as warts or other benign tumors.
  • a preferred therapeutic effect provided by the instant composition is the treatment of cancer and specifically the inhibition of cancerous tumor growth and/or the regression of cancerous tumors.
  • Exemplary cancers include carcinomas, e.g., basal cell carcinomas, squamous cell carcinomas, carcinosarcomas, adenocystic carcinomas, epidermoid carcinomas, nasopharyngeal carcinomas, renal cell carcinomas, papillomas, and epidermoidomas.
  • carcinomas e.g., basal cell carcinomas, squamous cell carcinomas, carcinosarcomas, adenocystic carcinomas, epidermoid carcinomas, nasopharyngeal carcinomas, renal cell carcinomas, papillomas, and epidermoidomas.
  • Exemplary cancers are those of the brain including glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas; kidney; colon; lung; liver; pancreas; endometrium; spleen; small intestine; stomach; skin; head and neck; esophagus; hormone-dependent cancers including breast, prostate, testicular, and ovarian cancers; lymphomas (lymph node); and leukemias including cancer of blood cells and bone marrow.
  • cancers that can be treated include acral lentiginous melanoma, actinic keratoses, adenocarcinoma, adenoid cycstic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinomas, capillary, carcinoids, carcinoma, carcinosarcoma, cavernous, cholangiocarcinoma, chondrosarcoma, choriod plexus papilloma/carcinoma, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal, epitheloid, Ewing's sarcoma, fibrolamellar, focal nodular hyperplasia
  • a Sirt ⁇ inhibitory agent may be administered directly into the tumor.
  • Cancer of blood cells e.g., leukemia can be treated by administering a Sirt ⁇ inhibitory agent into the blood stream or into the bone marrow.
  • reducing SIRT6 reduces the migration of cancerous cells and suppresses their invasiveness, which are markers of metastasis.
  • the methods described herein may also be used for treating or preventing metastasis of tumors, in addition to treating or preventing primary tumors. Methods may also be used for reducing or preventing migration of cancer cells and/or their invasiveness.
  • proliferative disorders that can be treated according to the invention include non malignant cell proliferative disorders, e.g., benign cancers, neurofibromatosis; glaucoma; psoriasis; rheumatoid arthritis; restenosis; inflammatory bowel disease; chemotherapy-induced alopecia and mucositis; keratoacanthoma and actinic keratosis; smooth muscle cell hyper-proliferation, e.g., in atherosclerosis and restenosis; inhibiting vascularization, e.g., in tumors; cell hyper-proliferations stimulated by, e.g., hepatitis C or delta and related viruses, and papilloma viruses (HPV); hyperplastic epidermal conditions, such as keratosis; autoimmune diseases; atopic dermatosis; dermatitis; lens epithelial cell proliferation, e.g., to prevent post-operative complications of extracapsular cataract extraction;
  • proliferative skin disorders e.g., any disease/disorder of the skin marked by unwanted or aberrant proliferation of cutaneous tissue, e.g., X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, epidermodysplasia, epidermolysis, and seborrheic dermatitis.
  • autoimmune diseases examples include active chronic hepatitis, addison's disease, anti-phospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydra autoimmune, celiac disease, Crohn's disease, cushing's syndrome, dermatomyositis, diabetes (type I), discoid lupus, erythematosis, goodpasture's syndrome, grave's disease, hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes, lambert-eaton syndrome, lupoid hepatitis, some cases of lymphopenia, mixed connective tissue disease, multiple sclerosis, pemphigoid, pemphigus vulgaris, pernicious anema, phacogenic uveitis, polyarteritis nodosa, polyglandular auto, syndromes, primary biliary
  • the compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice.
  • the compounds can be administered orally or parenterally, including intravenously, intramuscularly, intraperitoneally, subcutaneously, rectally and topically.
  • one or more compounds are injected directly into a tumor of a subject to be treated.
  • a subject in need of therapy may be a subject having been diagnosed with a disease, e.g., cancer.
  • a subject may also be a subject who has been determined as being likely to develop cancer, e.g., a subject having a gene indicating susceptibility of developing the disease, or a subject in whose family the disease is more frequent than normal.
  • a subject in need of Sirt ⁇ inhibitory therapy may also be a subject having cancer that is likely to metastasize.
  • cells can be obtained from a subject, e.g., a human or other mammal, treated ex vivo according to the methods of the invention to remove undesirable cells, e.g., cancer cells, and then administered to the same or a different subject.
  • cells or tissues may be obtained from a donor, treated ex vivo as described herein and administered to the same or different subject.
  • cells are incubated with an agent described herein in the presence of a drug, e.g., a chemotherapeutic drug, such as to increase the susceptibility of the cells to the effect of the drug.
  • a drug e.g., a chemotherapeutic drug
  • an effective amount of an agent that inhibits Sirt ⁇ may be an amount that reduces the level and/or rate of cell proliferation and/or reduces tumor mass by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, or at least about 90%, or more, compared to the level and/or rate of cell proliferation and/or tumor mass in the absence of treatment with an agent that inhibits Sirt ⁇ .
  • a cell may also be contacted with more than one agent, e.g., a compound.
  • a cell may be contacted with at least 2, 3, 5, or 10 different agents.
  • a cell may be contacted simulatenously or sequentially with different agents.
  • a Sirt6 inhibitory agent is administered as part of a combination therapy with another therapeutic agent.
  • the second therapeutic agent is another Sirt ⁇ inhibitory agent, such as nicotinamide or sirtinol, and/or an agent that kills cells.
  • Such combination therapies may be administered simultaneoulsy (e.g., more than one therapeutic agent administered at the same time) or sequentially (e.g., different therapeutic agents administered at different times during a treatment regimen).
  • Chemotherapeutic agents that may be coadministered with agents, e.g., compounds, described herein include: aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradi
  • chemotherapeutic agents may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan
  • Radiation therapy including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with an agent described herein to treat cancer.
  • Modulators of sirtuin ribosyltransferases may be used for treating or preventing a variety of diseases or disorders.
  • a target protein of Sirt4 is glutamate dehydrogenase (GDH)
  • agents that modulate ribosylation activity of Sirt4 may be used for treating or preventing disorders that are associated with GDH.
  • GDH has, in particular, been shown to play an important role in insulin secretion, as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonernia syndrome (GDH-HI) and sensitize beta-cells to leucine stimulation (Li et al. J Biol Chem. 2006 Jun 2;281 (22): 15064).
  • Sirt4 ribosylation modulators may be used to regulate insulin production and treat or prevent, e.g., diabetes such as type I diabetes. Since ribosylation of GDH inhibits or represses it, and that Sirt4 ribosylates GDH, Sirt4 inhibits or represses GDH. Therefore, Sirt4 and agents that stimulate Sirt4 or increase its protein level may be used for treating or preventing diseases that are associated with a hyperactive GDH 5 e.g., GDH-HI, and may be used for reducing insulin levels. On the other hand, inhibitors of Sirt4 may be used for treating or preventing diseases that are associated with a hypoactive GDH and may be used for increasing insulin levels. Other metabolic diseases may also be treated.
  • a hyperactive GDH 5 e.g., GDH-HI
  • inhibitors of Sirt4 may be used for treating or preventing diseases that are associated with a hypoactive GDH and may be used for increasing insulin levels. Other metabolic diseases may also be treated.
  • a method may comprise contacting a cell with an agent that modulates the protein level or activity of Sirt ⁇ in the cell.
  • Signalling pathways and genes that may be modulated includee IGFBP3, Adenylate cyclase 3, Braf, Rho GTPase activating protein 1 and the vitamin D receptor (see Fig. 14).
  • a method may comprise determining the level of expression of 1, 2, 3, 4, 5, or from about 1-5, 1-10, 1-20, 1-50 genes listed in Figures 12-14, and comparing their expression level to that in a control, e.g., corresponding wild-type cells, wherein a difference in expression of these one or more genes relative to that in the control indicates that the expression or activity of Sirt ⁇ is either higher or lower relative to the control.
  • a control e.g., corresponding wild-type cells
  • a difference in expression of these one or more genes relative to that in the control indicates that the expression or activity of Sirt ⁇ is either higher or lower relative to the control.
  • cells in which Sirt ⁇ expression or activity is down-regulated will have a profile of gene expression that is significantly similar to that described in the Examples. Determining the level of expression of the genes that are biomarkers can be done, e.g., by measuring their mRNA level, such as with microarray analysis, Western blot or PCT, e.g., RT-PCR.
  • kits All the essential materials and reagents required for administering the agents described herein may be assembled together in a kit.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the agents may be provided in combination with one or more other drugs, e.g., chemo- or radiotherapeutic agent.
  • drugs e.g., chemo- or radiotherapeutic agent.
  • These normally will be a separate formulation, but may be formulated into a single pharmaceutically acceptable composition.
  • the container means may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, or injected into an animal, or even applied to and mixed with the other components of the kit.
  • compositions of these kits also may be provided in dried or lyophilized forms.
  • reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
  • the kits of the invention may also include an instruction sheet defining administration of the agent and, e.g., explaining how the agent will decrease proliferation of cells.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention also may comprise, or be packaged with a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • Other instrumentation includes devices that permit the reading or monitoring of compound levels or reactions in vitro.
  • Cells over-expressing SIRT6 or SIRT7 or no sirtuin (control) were contacted with biotin-NAD+ for 60 minutes at 37°C.
  • the cells were first lysed in the following hypotonic lysis buffer is : Buffer A: 10 mM Hepes, pH 7.9, 1OmM KCl, 1.5mM MgC12, 0.5mM DTT.
  • Proteins visualized on the silver stained gel were then separated from the gel and subjected to spectroscopy as follows. Proteins were visualized on silver-stained polyacrylamide gels (5-20%) and individual bands that were greater in intensity than the
  • Cells from the TRAMP-C2 prostate cancer cell line were stably transformed with a lenti viral shRNA vector targeting the Sirt ⁇ gene.
  • This cell line is a mouse transgenic prostate cancer model driven by the large T antigen.
  • the lentiviral vector is described in Araki et al. (2004) Science 305:1010. This vector was generated from the FUGW backbone by replacing the ubiquitin promoter and GFP cDNA with the human U6 promoter and Pol I termination signal followed by the SV40 promoter-puromycin-N-acetyl transferase gene.
  • the nucleotide sequence from Sirt ⁇ and its complement that were inserted into the vector for expression of siRNA corresponded to nucleotides 1390-1408 of human Sirt ⁇ nucleotide sequence. These were under the control of the U6 promoter (S2). A fixed number of cells were grown in culture and subsequently stained with Crystal Violet dye.
  • TRAMP-C2 cells were transformed by viral infection (overnight incubation in the presence of polybrene), followed by selection with puromycin 48 hours after infection.
  • SIRT6 was stably knocked down to about 30-40% in TRAMP prostate cells (confirmed via Western blot and quantitative RT-PCR) (see Fig. 10), in order to further investigate the observations of Fig. 9.
  • Example 3 SIRT6 knockdown reduces chemotaxis and invasive potential
  • Fig. 11 Cell migration (chemotaxis) was measured using a commercial assay from Cell Biolabs. As show in Fig. 11 panel A, knockdown of SIRT6 significantly reduced migration through a polycarbonate membrane (about 50%). Invasive potential through a Boyden chamber was measured using a kit from Cell Biolabs. As shown in Fig. 11, panels B and C, knockdown of SIRT6 suppressed invasiveness by approximately 50%. Increased cellular migration/invasion are markers for metastatic cancer.
  • Example 4 Inhibition of Sirt ⁇ in a xenograft reduces its cancerous growth
  • TRAMP-C2 prostate cancer cells stably transformed with a lentiviral shRNA vector targeting the Sirt ⁇ gene were injected into subcutaneous tissue on the right flank of mice. One month after injection, the tumors were removed from the mice and their size was determined. As shown in Fig. 8, cells that contained a vector only generated a tumor that was much larger than the tumor that was generated after injection of cells in which Sirt 6 was downregulated. These results show that inhibiting Sirt ⁇ in a tumor reduces its growth potential.
  • Microarray was also used for comparing the expression of genes involved in cell motility and chemotaxis in prostate cancer (TRAMP) cells. The results, which are shown in
  • Figs. 13 and 14 show that genes involved in cell motility and chemotaxis are also modified when SIRT6 is knocked down. Several of these genes have been reported to play a role in cancer cell metastasis.
  • Example 6 Identification of signalling pathways that are deregulated when SIRT6 is knocked down
  • the role of the genes identified in the microarray experiments described above was further investigated via qRT-PCR.
  • the experssion of the genes set forth in Fig. 15 was measured via qRT-PCR in control TRAMP cells and SIRT6 knockdown TRAMP cells.
  • the results are shown in Fig. 15 and indicate that several signalling pathways are deregulated when SIRT6 is knocked down.
  • the Braf oncogene which regulates the MAPK cascade, is decreased by about 50% in SIRT6 knockdown TRAMP cells. It is our hypothesis that suppression of MAPK signalling by knockdown of SIRT6 results in a decreased invasive potential and decreased transformative potential of prostate cancer cells.
  • Example 7 SIRT6 is expressed at the protein level in both normal and cancerous prostate tissue
  • SIRT6 is expressed at the protein level in both normal and cancerous prostate tissue, thus making it a viable target for inactivation with small molecules.

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Abstract

La présente invention concerne des procédés permettant d'identifier des agents qui modulent l'activité des ADP-ribosyltransférases de type sirtuine, notamment la Sirt4 et la Sirt6. L'invention concerne également des procédés permettant de traiter une maladie hyperproliférative, consistant, par exemple, à administrer à un sujet nécessitant des soins un agent qui réduit l'activité ou le niveau protéique d'une ADP-ribosyltransférase, notamment la Sirt6.
PCT/US2007/017461 2006-08-07 2007-08-06 Procédés et compositions fondés sur l'adp-ribosyltransférase Ceased WO2008021048A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008138943A3 (fr) * 2007-05-14 2009-04-09 Univ Bruxelles Utilisation prophylactique et thérapeutique d'inhibiteurs de la sirtuine dans des pathologies à médiation par tnf-alpha
KR20140110559A (ko) * 2013-03-08 2014-09-17 한국화학연구원 Sirt7단백질의 탈아세틸화 활성을 정량분석하는 방법 및 상기 방법을 이용한 활성을 저해하는 억제제의 스크리닝 방법
KR102043303B1 (ko) * 2013-03-08 2019-11-12 한국화학연구원 Sirt7단백질의 탈아세틸화 활성을 정량분석하는 방법 및 상기 방법을 이용한 활성을 저해하는 억제제의 스크리닝 방법
US8933239B1 (en) 2013-07-16 2015-01-13 Dow Global Technologies Llc Bis(aryl)acetal compounds
US8962779B2 (en) 2013-07-16 2015-02-24 Dow Global Technologies Llc Method of forming polyaryl polymers
US9063420B2 (en) 2013-07-16 2015-06-23 Rohm And Haas Electronic Materials Llc Photoresist composition, coated substrate, and method of forming electronic device
US9410016B2 (en) 2013-07-16 2016-08-09 Dow Global Technologies Llc Aromatic polyacetals and articles comprising them
CN114958960A (zh) * 2022-01-07 2022-08-30 河南省人民医院 一种靶向组蛋白去乙酰化酶sirt6调节剂的药物筛选方法
CN114958960B (zh) * 2022-01-07 2025-07-25 河南省人民医院 一种靶向组蛋白去乙酰化酶sirt6调节剂的药物筛选方法

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