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Nicotinamide riboside kinase compositions and methods for using the sameRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero RingNicotinamide riboside kinase compositions and methods for using the same description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070027095, Nicotinamide riboside kinase compositions and methods for using the same. Brief Patent Description - Full Patent Description - Patent Application Claims
INTRODUCTION [0001] This application is a division of U.S. Ser. No. 11/113,701 filed Apr. 25, 2005, which is a continuation-in-part and claims benefit of priority under 35 U.S.C. .sctn.371 to PCT application No. PCT/US2005/004337, filed Feb. 9, 2005, which claims benefit under 35, U.S.C. .sctn.119 to U.S. Provisional Patent Application Serial No. 60/543,347, filed on Feb. 10, 2004, whose contents are incorporated herein by reference in their entireties. BACKGROUND OF THE INVENTION [0003] Nicotinic acid and nicotinamide, collectively niacins, are the vitamin forms of nicotinamide adenine dinucleotide (NAD+). Eukaryotes can synthesize NAD+de novo via the kynurenine pathway from tryptophan (Keel, et al. (1945) Science 101:489-490; Schutz and Feigelson (1972) J. Biol. Chem. 247:5327-5332) and niacin supplementation prevents the pellagra that can occur in populations with a tryptophan-poor diet. It is well-established that nicotinic acid is phosphoribosylated to nicotinic acid mononucleotide (NaMN), which is then adenylylated to form nicotinic acid adenine dinucleotide (NaAD), which in turn is amidated to form NAD+ (Preiss and Handler (1958) J. Biol. Chem. 233:488-492; Preiss and Handler (1958b) J. Biol. Chem. 233:493-50). [0004] NAD+ was initially characterized as a co-enzyme for oxidoreductases. Though conversions between NAD+, NADH, NADP and NADPH would not be accompanied by a loss of total co-enzyme, it was discovered that NAD+ is also turned over in cells for unknown purposes (Maayan (1964) Nature 204:1169-1170). Sirtuin enzymes such as Sir2 of S. cerevisiae and its homologs deacetylate lysine residues with consumption of an equivalent of NAD+ and this activity is required for Sir2 function as a transcriptional silencer (Imai, et al. (2000) Cold Spring Harb. Symp. Quant. Biol. 65:297-302). NAD+-dependent deacetylation reactions are required not only for alterations in gene expression but also for repression of ribosomal DNA recombination and extension of lifespan in response to calorie restriction (Lin, et al. (2000) Science 289:2126-2128; Lin, et al. (2002) Nature 418:344-348). NAD+ is consumed by Sir2 to produce a mixture of 2' and 3' O-acetylated ADP-ribose plus nicotinamide and the deacetylated polypeptide (Sauve, et al. (2001) Biochemistry 40:15456-15463). Additional enzymes, including poly(ADPribose) polymerases and cADPribose synthases are also NAD+-dependent and produce nicotinamide and ADPribosyl products (Ziegler (2000) Eur. J. Biochem. 267:1550-1564; Burkle (2001) Bioessays 23:795-806). [0005] The non-coenzymatic properties of NAD+ has renewed interest in NAD+ biosynthesis. Four recent publications have suggested what is considered to be all of the gene products and pathways to NAD+ in S. cerevisiae (Panozzo, et al. (2002) FEBS Lett. 517:97-102; Sandmeier, et al. (2002) Genetics 160:877-889; Bitterman, et al. (2002) J. Biol. Chem. 277:45099-45107; Anderson, et al. (2003) Nature 423:181-185) depicting convergence of the flux to NAD+ from de novo synthesis, nicotinic acid import, and nicotinamide salvage at NaMN (Scheme 1). SUMMARY OF THE INVENTION [0006] It has now been shown that nicotinamide riboside, which was known to be an NAD+ precursor in bacteria such as Haemophilus influenza (Gingrich and Schlenk (1944) J. Bacteriol. 47:535-550; Leder and Handler (1951) J. Biol. Chem. 189:889-899; Shifrine and Biberstein (1960) Nature 187:623) that lack the enzymes of the de novo and Preiss-Handler pathways (Fleischmann, et al. (1995) Science 269:496-512), is an NAD+ precursor in a previously unknown but conserved eukaryotic NAD+ biosynthetic pathway. Yeast nicotinamide riboside kinase, Nrk1, and human Nrk enzymes with specific functions in NAD+ metabolism are provided herein. The specificity of these enzymes indicates that they are the long-sought tiazofurin kinases that perform the first step in converting cancer drugs such as tiazofurin and benzamide riboside and their analogs into toxic NAD+ analogs. Further, yeast mutants of defined genotype were used to identify sources of nicotinamide riboside and it is shown that milk is a source of nicotinamide riboside. [0007] Accordingly, the present invention is an isolated nucleic acid encoding a eukaryotic nicotinamide riboside kinase polypeptide. A eukaryotic nicotinamide riboside kinase nucleic acid encompasses (a) a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; (b) a nucleotide sequence that hybridizes to a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or its complementary nucleotide sequence under stringent conditions, wherein said nucleotide sequence encodes a functional nicotinamide riboside kinase polypeptide; or (c) a nucleotide sequence encoding an amino acid sequence encoded by the nucleotide sequences of (a) or (b), but which has a different nucleotide sequence than the nucleotide sequences of (a) or (b) due to the degeneracy of the genetic code or the presence of non-translated nucleotide sequences. [0008] The present invention is also an expression vector containing an isolated nucleic acid encoding a eukaryotic nicotinamide riboside kinase polypeptide. In one embodiment, the expression vector is part of a composition containing a pharmaceutically acceptable carrier. In another embodiment, the composition further contains a prodrug wherein the prodrug is a nicotinamide riboside-related analog that is phosphorylated by the expressed nicotinamide riboside kinase thereby performing the first step in activating said prodrug. [0009] The present invention is also an isolated eukaryotic nicotinamide riboside kinase polypeptide. In one embodiment, the isolated nicotinamide riboside kinase polypeptide has an amino acid sequence having at least about 70% amino acid sequence similarity to an amino acid sequence of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 or a functional fragment thereof. [0010] The present invention is further a cultured cell containing an isolated nucleic acid encoding a eukaryotic nicotinamide riboside kinase polypeptide or a polypeptide encoded thereby. [0011] Still further, the present invention is a composition containing an isolated eukaryotic nicotinamide riboside kinase polypeptide and a pharmaceutically acceptable carrier. In one embodiment, the composition further contains a prodrug wherein said prodrug is a nicotinamide riboside-related analog that is phosphorylated by the nicotinamide riboside kinase thereby performing the first step in activating said prodrug. [0012] The present invention is also a method for treating cancer by administering to a patient having or suspected of having cancer an effective amount of a nicotinamide riboside-related prodrug in combination with an isolated eukaryotic nicotinamide riboside kinase polypeptide or expression vector containing an isolated nucleic acid sequence encoding an eukaryotic nicotinamide riboside kinase polypeptide wherein the nicotinamide riboside kinase polypeptide phosphorylates the prodrug thereby performing the first step in activating the prodrug so that the signs or symptoms of said cancer are decreased or eliminated. [0013] The present invention is further a method for identifying a natural or synthetic source for nicotinamide riboside. The method involves contacting a first cell lacking a functional glutamine-dependent NAD+ synthetase with an isolated extract from a natural source or synthetic; contacting a second cell lacking functional glutamine-dependent NAD+ synthetase and nicotinamide riboside kinase with the isolated extract; and detecting growth of the first cell compared to the growth of the second cell, wherein the presence of growth in the first cell and absence of growth in the second cell is indicative of the presence of nicotinamide riboside in the isolated extract. In one embodiment, the natural source is cow' milk. [0014] Further, the present invention is a dietary supplement composition containing nicotinamide riboside identified in accordance with the methods of the present invention and a carrier. [0015] Moreover, the present invention is a method for preventing or treating a disease or condition associated with the nicotinamide riboside kinase pathway of NAD+ biosynthesis. The method involves administering to a patient having a disease or condition associated with the nicotinamide riboside kinase pathway of NAD+ biosynthesis an effective amount of a nicotinamide riboside composition so that the signs or symptoms of the disease or condition are prevented or reduced. In one embodiment, the nicotinamide riboside is neuroprotective. In another embodiment the nicotinamide riboside is anti-fungal. In a further embodiment, the nicotinamide riboside is administered in combination with tryptophan, nicotinic acid or nicotinamide. [0016] The present invention is also an in vitro method for identifying a nicotinamide riboside-related prodrug. The method involves contacting a nicotinamide riboside kinase polypeptide with a nicotinamide riboside-related test agent and determining whether said test agent is phosphorylated by said nicotinamide riboside kinase polypeptide wherein phosphorylation of said test agent is indicative of said test agent being a nicotinamide riboside-related prodrug. A nicotinamide riboside-related prodrug identified by this method is also encompassed within the present invention. [0017] The present invention is further a cell-based method for identifying a nicotinamide riboside-related prodrug. This method involves contacting a first test cell which expresses a recombinant Nrk polypeptide with a nicotinamide riboside-related test agent; contacting a second test cell which lacks a functional Nrk polypeptide with the same test agent; and determining the viability of the first and second test cells, wherein sensitivity of the first cell and not the second cell is indicative of a nicotinamide riboside-related prodrug. A nicotinamide riboside-related prodrug identified by this method is also encompassed within the context of the present invention. [0018] The present invention is also a method for identifying an individual or tumor which is susceptible to treatment with a nicotinamide riboside-related prodrug. This method involves detecting the presence of mutations in, or the level of expression of, a nicotinamide riboside kinase in an individual or tumor wherein the presence of a mutation or change in expression of nicotinamide riboside kinase in said individual or tumor compared to a control is indicative of said individual or tumor having an altered level of susceptibility to treatment with a nicotinamide riboside-related prodrug. BRIEF DESCRIPTION OF THE DRAWINGS [0019] FIG. 1 shows the amino acid sequence alignment of human Nrk1 (SEQ ID NO:5), human Nrk2 (SEQ ID NO:6), S. cerevisiae Nrk1 (SEQ ID NO:4), S. pombe nrk1 (SEQ ID NO:7), and portions of S. cerevisiae uridine/cytidine kinase Urk1 (SEQ ID NO:8) and E. coli pantothenate kinase (SEQ ID NO:9). DETAILED DESCRIPTION OF THE INVENTION [0020] A Saccharomyces cerevisiae QNS1 gene encoding glutamine-dependent NAD+ synthetase has been characterized and mutation of either the glutaminase active site or the NAD+ synthetase active site resulted in inviable cells (Bieganowski, et al. (2003) J. Biol. Chem. 278:33049-33055). Possession of strains containing the qns1 deletion and a plasmid-borne QNS1 gene allowed a determination of whether the canonical de novo, import and salvage pathways for NAD+ of Scheme 1 (Panozzo, et al. (2002) supra; Sandmeier, et al. (2002) supra; Bitterman, et al. (2002) supra; Anderson, et al. (2003) supra) are a complete representation of the metabolic pathways to NAD+ in S. cerevisiae. The pathways depicted in scheme 1 suggest that: nicotinamide is deamidated to nicotinic acid before the pyridine ring is salvaged to make more NAD+, thus supplementation with nicotinamide may not rescue qns1 mutants by shunting nicotinamide-containing precursors through the pathway; and QNS1 is common to the three pathways, thus there may be no NAD+ precursor that rescues qns1 mutants. However, it has now been found that while nicotinamide does not rescue qns1 mutants even at 1 or 10 mM, nicotinamide riboside functions as a vitamin form of NAD+ at 10 .mu.M. Continue reading about Nicotinamide riboside kinase compositions and methods for using the same... 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