(12) United States Patent
`Brenner
`
`(10) Patent N0.:
`(45) Date of Patent:
`
`US 8,197,807 B2
`Jun. 12, 2012
`
`US008197807B2
`
`Inventor:
`
`(75)
`(73)
`
`(*)
`
`Notice:
`
`(54) NICOTINAMIDE RIBOSIDE KINASE
`COMPOSITIONS AND METHODS FOR
`USING THE SAME
`Charles M. Brenner, Lyme, NH (U S)
`Assignee: Trustees of Dartmouth College,
`Hanover, NH (U S)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 213 days.
`11/912,400
`Apr. 20, 2006
`PCT/US2006/015495
`
`(21)
`(22)
`(86)
`
`Appl. No .:
`PCT Filed:
`
`PCT No.:
`§ 371 (0X1),
`(2), (4) Date:
`
`Nov. 20, 2007
`
`(87) PCTPub.No.: WO2006/116322
`
`PCT Pub. Date: Nov. 2, 2006
`
`(65)
`
`(51)
`
`(52)
`(58)
`
`(56)
`
`Prior Publication Data
`
`Aug. 28, 2008
`
`US 2008/0206221 A1
`Int. Cl.
`(2006.01)
`A61K 38/45
`(2006.01)
`C07H 17/00
`(2006.01)
`A61P 35/00
`U.S. Cl. ............ .. 424/945; 514/45; 514/25; 435/15
`Field of Classi?cation Search ...................... .. None
`See application ?le for complete search history.
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`7,491,743 B2 *
`
`2/2009 Cuny et al. .................. .. 514/397
`
`WO
`
`FOREIGN PATENT DOCUMENTS
`0132888
`5/2001
`
`OTHER PUBLICATIONS
`
`Saunders et al 1 TiaZofurin is phosphorylated by three enzymes from
`Chinese Hamster Ovary Cells. Cancer Research 50, 5269-5274, Sep.
`1, 1990*
`Saunders et al 2 Phosphorylation of 3-Deazaguanosine by
`nicotinamide riboside kinase in Chinese Hamster Ovary Cells.*
`Tanimori et al, Bioorganic. Med. Chem Lett. 12:1135-1137, 2002.*
`Stubber?eld et al NAD+ depletion and cytotoxicity in isolated
`hepatocytes. Biochemical Pharmacology 1988; 37(20): 3967-74
`Abstract included.*
`Stubber?eld et al (NAD+ depletion and cytotoxicity in isolated
`hepatocytes (Biochemical Pharmacology 1988; 37(20): 3967-74 see
`abstract.*
`Berger NA et al. (Poly (ADP-ribose in the cellular response to DNA
`damage), Radiation research Jan. 1985; 101(1): 4-15 see abstract.*
`Berger et al., “Modulation of Nicotinarnide Adenine Dinucleotide
`and Poly(Adenosine Dipho sphoribo se) Metabolism by the Synthetic
`“C” Nucieoside Analogs, Tiazofurin and SelenaZofurin”, J. Clin.
`Invest. 1985 75:702-705.
`Han et al., “Cellular Uptake Mechanism of Amino Acid Ester
`Prodrugs in Caco-2/hPEPT1 Cells Overexpressing a Human Peptide
`Transporter”, Pharmaceutical Research 1998 15(9):1382-1386.
`Genbank Accession No. AK001663 Jan. 9, 2008.
`Genbank Accession No. YNL129WiNov. 7, 2005.
`NCBI Accession No. NPi060351 [gi:8923530] with Revision His
`tory Jul. 4, 2000-Jun. 3, 2007.
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`NCBI Accession No. NPi733778 [gi:24762248] With Revision His
`toryiNov. 2, 2002-Jul. 5, 2007.
`NCBI Accession No. NMi017881 [gi:8923529] With Revision His
`toryiJul. 4, 2000-Nov. 17, 2006.
`NCBI Accession No. AK000566 [gi:7020748] with Revision His
`toryiFeb. 22, 2000-Sep. 12, 2006.
`NCBI Accession No. BC001366 [gi: 33876100] With Revision His
`toryiDec. 12, 2000-Jul. 15, 2006.
`NCBI Accession No. BC036804 [gi:22477870] with Revision His
`toryiAug. 26, 2002-Mar. 25, 2004.
`NCBI Accession No. BC026243 [gi:20072207] with Revision His
`toryiApr. 8, 2002-Mar. 25, 2004.
`NCBI Accession No. NMi170678 [gi:24762247] With Revision
`History Nov. 7, 2002-Nov. 17, 2006 NMi170678.2 which replaces
`NMi170678 is provided.
`NCBI Accession No. CAG61927 [gi:49528270] With Revision His
`toryiJun. 30, 2004-Nov. 14, 2006.
`NCBI Accession No. Z71405 [gi:1302065] With Revision Historyi
`May 6, 1996v-Aug. 11, 1997.
`NCBI Accession No. AX877238 [gi:40031974]iDec. 17, 2003.
`NCBI Genbank Accession No. AX877238 [gi:40031974]Dec. 17,
`2003.
`Bieganowski et al., “Discoveries of Nicotinamide Riboside as a
`Nutrient and Conserved NRK Genes Establish a Preiss-Handler
`Independent Route to NAD+ in Fungi and Hunams”, Cell 2004
`117:495-502.
`Farquhar et al., “Synthesis and antitumor evaluatiobn of
`bis[(pivaloyloxy)methyl]2‘-deoxy-t-?uoroudine 5‘-monophosphate
`(FdUMP):a stategy to introduce nucleotides into cells”, J Med Chem
`1994 37(23):3902-3909.
`Sasiak et al., “Puri?cation and Properties of a Human Nicotinarnide
`Ribonucleoside Kinas”, Archives of Biochemistry and Biophysics
`1996 333(2):414-418.
`Saunders et al., “Phosphorylation of 3-Deazaguanosine by
`Nicotinamide Riboside Kinse in Chinese Hamster Ovary Cells”,
`Cancer Research 1989 49:6593-6599.
`Bieganowski et al., “Eukaryotic NAD+ Synthetase Qnsl Contains an
`Essential,
`Obligate
`Intramolecular
`Thiol
`Glutamine
`Amidotransferase Domain Related to Nitrilase”, J. Biol. Chem. 2003
`278(35):33049-33055.
`Boon et al., “An anatomy of normal and malignant gene expression”,
`Proc. Natl. Acad. Sci. 2002 99(17):11287-11292.
`Burkle, Alexander, “Physiology and pathophysiology of poly(ADP
`ribosyl)ation”, BioEssays 2001 23:795-806.
`Fleischmann et al., “Whole-Genome Random Sequencing and
`Assembly of Haemophilus in?uenzae Rd”, Science 1995 269:496
`5 12.
`Gingrich et al., “Codehydrogenase I and other Pyridinium Com
`pounds as V-Factor for Hemophi/us in?uenzae and H.
`parain?uenzae”, J. Bacteriol. 1944 47:535-550.
`Godek et al., “In Vitro Evaluation of Nicotinamide Riboside Analogs
`against Haemophi/us in?uenzae”, Antimicrobal Agents and Chemo
`therapy 1990 34(8):1473-1479.
`(Continued)
`Primary Examiner * KagneW H Gebreyesus
`(74) Attorney, Agent, or Firm * Licata & Tyrrell PC.
`(57)
`ABSTRACT
`The present invention relates to isolated nicotinamide ribo
`side kinase (Nrk) nucleic acid sequences, vectors and cul
`tured cells containing the same, and Nrk polypeptides
`encoded thereby. Methods for identifying individuals or
`tumors susceptible to nicotinamide riboside-related prodrug
`treatment and methods for treating cancer by administering
`an Nrk nucleic acid sequence or polypeptide in combination
`With a nicotinamide riboside-related prodrug are also pro
`vided. The present invention further provides screening meth
`ods for isolating a nicotinamide riboside-related prodrug and
`identifying a natural source of nicotinamide riboside.
`
`3 Claims, 1 Drawing Sheet
`
`Elysium Health Exhibit 1001
`Page 1 of 30
`
`

`

`US 8,197,807 B2
`Page 2
`
`OTHER PUBLICATIONS
`HoldsWorth et al., “A fraction derived from brewer’s yeast inhibits
`cholesterol synthesis by rat liver preparations in vitro”, British Jour
`nal ofNutrition 1991 65:285-299.
`Leder et al., “Synthesis of Nicotinamide Mononucleotide by Human
`Erythrocytes in Vitro”, J. Biol. Chem. 1951 189:889-899.
`Li et al., “A Novel Muscle-speci?c Betal Integrin Binding Protein
`(MIBP) that Modulates Myogenic Differentiation”, J. Cell Biol. 1999
`147: 1391-1397.
`Li et al., “The muscle integrin binding protein (MIBP) interacts With
`alpha7Beta1 integrin and regulates cell adhesion and laminin matrix
`deposition”, Developmental Biology 2003 261 1209-219.
`
`Saunders et al., TiaZofurin Is Pho sphorylated by Three Enzymes from
`Chinese Hamster Ovary Cells, Cancer Research 1990 50: 5269-5274.
`Shifrine et al., “A Growth Factor for Haemophilus Species secreted
`by a Pseudomonad”, Nature 1960 1871623.
`Ziegler, Mathais, “New functions of a long-known moleculei
`Emerging roles of NAD in cellular signaling”, Eur. J. Biochem. 2000
`267:1550-1564.
`Sasiak et al., “Puri?cation and Properties of a Human Nicotinamide
`Ribonucleoside Kinase”, Archives of Biochemistry and Biophysics
`1996 333(2):414-418.
`
`* cited by examiner
`
`Elysium Health Exhibit 1001
`Page 2 of 30
`
`

`

`U.S. Patent
`
`Jun.12,2012
`
`US 8,197,807 B2
`
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`Elysium Health Exhibit 1001
`
`Page 3 0f 30
`
`Elysium Health Exhibit 1001
`Page 3 of 30
`
`

`

`US 8,197,807 B2
`
`1
`NICOTINAMIDE RIBOSIDE KINASE
`COMPOSITIONS AND METHODS FOR
`USING THE SAME
`
`INTRODUCTION
`
`This invention Was made in the course of research spon
`sored by the National Cancer Institute (Grant No. CA77738).
`The US. government may have certain rights in this inven
`tion.
`This application claims bene?t of priority to PCT/US2006/
`015495, ?led Apr. 20, 2006, Which claims bene?t from US.
`patent application Ser. No. 11/113,701, ?led Apr. 25, 2005,
`noW abandoned Which is a continuation-in-part of PCT appli
`cation No. PCT/US2005/004337, ?led Feb. 9, 2005, Which
`claims bene?t under 35 U.S.C. §119 to US. Provisional
`Patent Application Ser. No. 60/543,347, ?led on Feb. 10,
`2004, Whose contents are incorporated herein by reference in
`their entireties.
`
`BACKGROUND OF THE INVENTION
`
`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 (Krehl, 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 phospho
`ribosylated to nicotinic acid mononucleotide (NaMN), Which
`is then adenylylated to form nicotinic acid adenine dinucle
`otide (NaAD), Which in turn is amidated to form NAD+
`(Preiss and Handler (1958) .1. Biol. Chem. 233:488-492; Pre
`iss and Handler (1958b) J. Biol. Chem. 233:493-50).
`
`20
`
`25
`
`30
`
`2
`NAD+ Was initially characterized as a co-enZyme for oxi
`doreductases. 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. cerevi
`siae and its homologs deacetylate lysine residues With con
`sumption 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 repres
`sion 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 4181344
`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(AD
`Pribose) 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 231795-806).
`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 4231181
`1 85) depicting convergence of the ?ux to NAD+ from de novo
`synthesis, nicotinic acid import, and nicotinamide salvage at
`NaMN (Scheme 1).
`
`NaMN
`
`Schemel
`
`O
`
`NaAD+
`
`O
`
`NAD+
`
`DE NOVO
`Bnal-6
`
`\ 0'
`
`\ 0'
`
`\ NHZ
`
`N+
`
`Prbo
`
`Nmal, 2
`KN
`ATP PPi
`
`N*
`|
`ADPrbo
`
`Qnsl
`m N*
`ATP ATP
`|
`Gln
`PPi
`ADPrbo
`Gln
`
`PPi
`
`PrboPP
`
`SALVAGE
`N t1
`p
`
`Nicotinic acid
`
`O
`
`-
`0
`
`/
`5+
`
`LysAc
`
`L +
`V5
`ADPrboAc
`
`NHZ
`
`nicotinamide
`
`O
`
`P 1
`no
`
`m
`NH4+ H20
`
`5.
`
`IMPORT
`
`Plasma membrane
`
`Tnal
`
`Elysium Health Exhibit 1001
`Page 4 of 30
`
`

`

`US 8,197,807 B2
`
`3
`SUMMARY OF THE INVENTION
`
`It has now been shown that nicotinamide riboside, which
`was known to be an NAD+ precursor in bacteria such as
`Haemophilus in?uenza (Gingrich and Schlenk (1944) .1. Bac
`leriol. 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 speci?c
`functions in NAD+ metabolism are provided herein. The
`speci?city of these enZymes indicates that they are the long
`sought tiaZofurin kinases that perform the ?rst step in con
`verting cancer drugs such as tiaZofurin and benZamide ribo
`side and their analogs into toxic NAD+ analogs. Further,
`yeast mutants of de?ned genotype were used to identify
`sources of nicotinamide riboside and it is shown that milk is
`a source of nicotinamide riboside.
`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 N011, 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 ribo
`side kinase polypeptide; or (c) a nucleotide sequence encod
`ing 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-trans
`lated nucleotide sequences.
`The present invention is also an expression vector contain
`ing an isolated nucleic acid encoding a eukaryotic nicotina
`mide riboside kinase polypeptide. In one embodiment, the
`expression vector is part of a composition containing a phar
`maceutically acceptable carrier. In another embodiment, the
`composition further contains a prodrug wherein the prodrug
`is a nicotinamide riboside-related analog that is phosphory
`lated by the expressed nicotinamide riboside kinase thereby
`performing the ?rst step in activating said prodrug.
`The present invention is also an isolated eukaryotic nico
`tinamide 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
`N014, SEQ ID NO:5 or SEQ ID NO:6 or a functional frag
`ment thereof.
`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.
`Still further, the present invention is a composition con
`taining 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 ana
`log that is phosphorylated by the nicotinamide riboside
`kinase thereby performing the ?rst step in activating said
`prodrug.
`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 nicotina
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`mide riboside kinase polypeptide or expression vector con
`taining an isolated nucleic acid sequence encoding an eukary
`otic nicotinamide riboside kinase polypeptide wherein the
`nicotinamide riboside kinase polypeptide phosphorylates the
`prodrug thereby performing the ?rst step in activating the
`prodrug so that the signs or symptoms of said cancer are
`decreased or eliminated.
`The present invention is further a method for identifying a
`natural or synthetic source for nicotinamide riboside. The
`method involves contacting a ?rst 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+ syn
`thetase and nicotinamide riboside kinase with the isolated
`extract; and detecting growth of the ?rst cell compared to the
`growth of the second cell, wherein the presence of growth in
`the ?rst 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’s milk.
`Further, the present invention is a dietary supplement com
`position containing nicotinamide riboside identi?ed in accor
`dance with the methods of the present invention and a carrier.
`Moreover, the present invention is a method for preventing
`or treating a disease or condition associated with the nicoti
`namide 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 symp
`toms of the disease or condition are prevented or reduced. In
`one embodiment, the nicotinamide riboside is neuroprotec
`tive. In another embodiment the nicotinamide riboside is
`anti-fungal. In a further embodiment, the nicotinamide ribo
`side is administered in combination with tryptophan, nico
`tinic acid or nicotinamide.
`The present invention is also an in vitro method for iden
`tifying a nicotinamide riboside-related prodrug. The method
`involves contacting a nicotinamide riboside kinase polypep
`tide with a nicotinamide riboside-related test agent and deter
`mining whether said test agent is phosphorylated by said
`nicotinamide riboside kinase polypeptide wherein phospho
`rylation of said test agent is indicative of said test agent being
`a nicotinamide riboside-related prodrug. A nicotinamide
`riboside-related prodrug identi?ed by this method is also
`encompassed within the present invention.
`The present invention is further a cell-based method for
`identifying a nicotinamide riboside-related prodrug. This
`method involves contacting a ?rst 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 ?rst and second test cells,
`wherein sensitivity of the ?rst cell and not the second cell is
`indicative of a nicotinamide riboside-related prodrug. A nico
`tinamide riboside-related prodrug identi?ed by this method is
`also encompassed within the context of the present invention.
`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 expres
`sion 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.
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`5
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows the amino acid sequence alignment and con
`sensus sequence (SEQ ID NO134) of human Nrk1 (SEQ ID
`NO15), human Nrk2 (SEQ ID NO16), S. cerevisiae Nrk1
`(SEQ ID NO14), S. pombe nrk1 (SEQ ID NO17), as compared
`to portions of S. cerevisiae uridine/cytidine kinase Urk1 (SEQ
`ID NO18) and E. coli pantothenate kinase (SEQ ID NO:9).
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`6
`Given that mammalian pharmacology provided no useful
`clue to the identity of a putative fungal Nrk, it Was considered
`Whether the gene might have been conserved With the Nrk of
`Haemophilus in?uenza. The Nrk domain of H. in?uenza is
`encoded by amino acids 225 to 421 of the NadR gene product
`(the amino terminus of Which is NMN adenylyltransferase).
`Though this domain is structurally similar to yeast thymidy
`late kinase (Singh, et al. (2002) J. Biol. Chem. 277133291
`33299), sensitive sequence searches revealed that bacterial
`Nrk has no ortholog in yeast. Genomic searches With the Nrk
`domain of H. in?uenza NadR have identi?ed a groWing list of
`bacterial genomes predicted to utilize nicotinamide riboside
`as an NAD+ precursor (Kurnasov, et al. (2002) J Bacteriol.
`18416906-6917). Thus, had fungi possessed NadR Nrk-ho
`mologous domains, comparative genomics Would have
`already predicted that yeast can salvage nicotinamide ribo
`side.
`To identify the Nrk of S. cerevisiae, an HPLC assay for the
`enzymatic activity Was established and used in combination
`With a biochemical genomics approach to screen for the gene
`encoding this activity (Martzen, et al. (1999) Science 2861
`1153-1155). Sixty-four pools of 90-96 S. cerevisiae open
`reading frames fused to glutathione S-transferase (GST),
`expressed in S. cerevisiae, Were puri?ed as GST fusions and
`screened for the ability to convert nicotinamide riboside plus
`ATP to NMN plus ADP. Whereas mo st pools contained activi
`ties that consumed some of the input ATP, only pool 37
`consumed nicotinamide riboside and produced NMN. In pool
`37, approximately half of the 1 mM ATP Was converted to
`ADP and the 500 uM nicotinamide riboside peak Was almost
`entirely converted to NMN. Examination of the 94 open
`reading frames that Were used to generate pool 37 revealed
`that YNL129W (SEQ ID NO11) encodes a predicted 240
`amino acid polypeptide With a 187 amino acid segment con
`taining 23% identity With the 501 amino acid yeast uridine/
`cytidine kinase Urk1 and remote similarity With a segment of
`E. coli pantothenate kinase panK (Yun, et al. (2000) J. Biol.
`Chem. 275128093-28099) (FIG. 1).After cloningYNL129W
`into a bacterial expression vector it Was ascertained Whether
`this homolog of metabolite kinases Was the eukaryotic Nrk.
`The speci?c activity of puri?ed YNL129W Was ~100-times
`that of pool 37, consistent With the idea that all the Nrk
`activity of pool 37 Was encoded by this open reading frame.
`To test genetically Whether this gene product pho sphorylates
`nicotinamide riboside in vivo, a deletion of YNL129W Was
`created in the qns1 background. It Was found that nicotina
`mide riboside rescue of the qns1 deletion strain Was entirely
`dependent on this gene product. Having shoWnbiochemically
`and genetically that YNL129W encodes an authentic Nrk
`activity, the gene Was designated NRKl.
`A PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res.
`2513389-3402) comparison Was conducted on the predicted
`S. cerevisiae Nrk1 polypeptide and an orthologous human
`protein Nrk1 (NPi060351; SEQ ID NO15; FIG. 1) Was
`found. The human NPi060351 protein encoded at locus
`9q21.31 is a polypeptide of 199 amino acids and is annotated
`as an uncharacterized protein of the uridine kinase family. In
`addition, a second human gene product Nrk2 (N Pi733778;
`SEQ ID NO16; FIG. 1) Was found that is 57% identical to
`human Nrk1. Nrk2 is a 230 amino acid splice form of What
`Was described as a 186 amino acid muscle integrin beta 1
`binding protein (ITGB1BP3) encoded at 19p13.3 (Li, et al.
`(1999).]. CellBiol. 14711391-1398; Li, et al. (2003)Dev. Biol.
`2611209-219). Amino acid conservation betWeen S. cerevi
`siae, S. pombe and human Nrk homologs and similarity With
`fragments of S. cerevisiae Urk1 and E. coli panK is shoWn in
`FIG. 1. Fungal and human Nrk enzymes are members of a
`
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`25
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`A Saccharomyces cerevisiae QNS1 gene encoding
`glutamine-dependent NAD+synthetase has been character
`ized and mutation of either the glutaminase active site or the
`NAD+ synthetase active site resulted in inviable cells (Bie
`ganoWski, et al. (2003) J. Biol. Chem. 278133049-33055).
`Possession of strains containing the qns1 deletion and a plas
`mid-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 meta
`bolic pathWays to NAD+ in S. cerevisiae. The pathWays
`depicted in scheme 1 suggest that: nicotinamide is deami
`dated 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-con
`taining precursors through the pathWay; and QNS1 is com
`mon 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 qnsl
`mutants even at 1 or 10 mM, nicotinamide riboside functions
`as a vitamin form of NAD+ at 10 uM.
`Anticancer agents such as tiazofurin (Cooney, et al. (1983)
`Adv. Enzyme Regul. 211271-303) and benzamide riboside
`(Krohn, et al. (1992) J. Med. Chem. 351511-517) have been
`shoWn to be metabolized intracellularly to NAD+ analogs,
`taizofurin adenine dinucleotide and benzamide adenine
`dinucleotide, Which inhibit IMP dehydrogenase the rate-lim
`iting enzyme for de novo purine nucleotide biosynthesis.
`Though an NMN/NaMN adenylyltransferase is thought to
`be the enzyme that converts the mononucleotide intermedi
`ates to NAD+ analogs and the structural basis for this is
`knoWn (Zhou et al. (2002) supra), several different enzymes
`including adenosine kinase, 5' nucleotidase (Fridland, et al.
`(1986) CancerRes. 461532-537; Saunders, et al. (1990) Can
`cer Res. 5015269-5274) and a speci?c nicotinamide riboside
`kinase (Saunders, et al. (1990) supra) have been proposed to
`be responsible for tiazofurin phosphorylation in vivo. A puta
`tive nicotinamide riboside kinase (Nrk) activity Was puri?ed,
`hoWever no amino acid sequence information Was obtained
`and, as a consequence, no genetic test Was performed to
`assess its function (Sasiak and Saunders (1996) Arch. Bio
`chem. Biophys. 3331414-418).
`Using a qns1 deletion strain that Was additionally deleted
`for yeast homologs of candidate genes encoding nucleoside
`kinases proposed to phosphorylate tiazofurin, i.e., adenosine
`kinase ado1 (Lecoq, et al. (2001) Yeast 181335-342), uridine/
`cytidine kinase urk1 (Kern (1990) Nucleic Acids Res.
`1815279; Kurtz, et al. (1999) Curr. Genet. 361130-136), and
`ribokinase rbk1 (Thierry, et al. (1990) Yeast 61521-534), it
`Was determined Whether the nucleoside kinases are uniquely
`or collectively responsible for utilization of nicotinamide
`riboside. It Was found that despite these deletions, the strain
`retained the ability to utilize nicotinamide riboside in an
`anabolic pathWay independent of NAD+ synthetase.
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`7
`metabolite kinase superfamily that includes pantothenate
`kinase but is unrelated to bacterial nicotinamide riboside
`kinase. Robust complementation of the failure of qns1 nrk1 to
`grow on nicotinamide riboside-supplemented media Was pro
`vided by human NRKl and human NRK2 cDNA even When
`expressed from the GAL1 promoter on glucose.
`As shoWn in Table 1, puri?cation of yeast Nrkl and human
`Nrkl and Nrk2 revealed high speci?city for phosphorylation
`of nicotinamide riboside and tiaZofurin.
`
`TABLE 1
`
`Nicotinarnide
`'b
`'d
`n 081 e
`
`f '
`T'
`1520 um
`
`C 'd'
`U 'd'
`n m yn me
`
`Human Nrkl
`Human Nrkz
`Yeast Nrkl
`
`275 I17
`2320 I 20
`535 1 60
`
`538 I 27
`2150 I 210
`1129 1134
`
`19-3 11-7
`2220 I 170
`15.2 1 3.4
`
`35-5 I 6-4
`222 I 8
`82.9 14.4
`
`8
`Whey preparation of coWls milk. Unlike the original screen
`for vitamins in protein-depleted extracts of liver for reversal
`of black-tongue in starving dogs (Elvehjem, et al. (1938) J.
`Biol. Chem. 123:137-149), this assay is pathWay-speci?c in
`identifying NAD+ precursors. Because of the qns1 deletion,
`nicotinic acid and nicotinamide do not score positively in this
`assay. As the factor from milk requires nicotinamide riboside
`kinase for groWth, the nutrient is clearly nicotinamide ribo
`side and not NMN or NAD+.
`A revised metabolic scheme for NAD+, incorporating
`Nrkl homologs and the nicotinamide riboside salvage path
`.
`.
`.
`.
`Way is shown in Scheme 2 wherein double arroWs dep1ct
`
`15 metabolic steps common to yeast and humans (With yeast
`ene names) and sin le arroWs de ict ste s uni ue to humans
`g
`_
`_
`_ g
`_P
`P
`q
`(PBEF, n1cot1nam1de phosphonbosyltransferase) and yeast
`(Pnc 1, nicotinamidase).
`
`Nmal, 2
`Qnsl
`Nm ,
`Bnal-6
`:> NaMN :} NaAD+ I} NAD+ <: NMN
`
`Schemel
`
`Nptl
`
`Na
`
`H.s.
`SirZ PBEF
`
`S.c. Pncl
`<— N
`
`Nrkl
`
`Nr
`
`Speci?c activity is expressed in nmole mg'l min“1 for phos
`phorylation of nucleoside substrates.
`In the cases of yeast and human Nrkl enzymes, the
`enZymes preferred tiaZofurin to the natural substrate nicoti
`namide riboside by a factor of tWo and both enZymes retained
`less than 7% of their maximal speci?c activity on uridine and
`cytidine. In the case of human Nrk2, the 230 amino acid form
`Was essentially equally active on nicotinamide riboside, tia
`Zofurin and uridine With less than 10% of corresponding
`activity on cytidine. Conversely, the 186 amino acid integrin
`beta 1 binding protein form Was devoid of enZymatic activity
`in this in vitro assay and Was not functional as an Nrk in vivo.
`HoWever, both the 186 and 230 amino acid isoforms function
`in vivo in a yeast nicotinamide riboside utiliZation assay.
`Thus, though Nrk2 may contribute additionally to formation
`of uridylate, these data demonstrate that fungi and mammals
`possess speci?c nicotinamide riboside kinases that function
`to synthesiZe NAD+ through NMN in addition to the Well
`knoWn pathWays through NaMN. Identi?cation of Nrk enZy
`matic activities thus accounts for the dual speci?city of fungal
`and mammalian NaMN/NMN adenylyltransferases.
`On the basis of SAGE data, NRKl is a rare message in
`many tissues examined While NRK2 is highly expressed in
`heart and skeletal muscle and has loWer level expression in
`retinal epithelium and placenta (Boon, et al. (2002) Proc.
`Natl. Acad. Sci. USA 99: 1 1287-1 1292). From cancer cell line
`to cancer cell line the expression levels are quite variable
`(Boon, et al. (2002) supra). Thus, in individuals Whose tumors
`are NRKl, NRK2-loW, tiaZofurin conversion to NAD+ may
`occur more extensively in the patients hearts and muscles
`than in tumors. In tumors that are NRKl and/ or NRK2 -high,
`a substantial amount of tiaZofurin may be converted to tiaZo
`furin adenine dinucleotide in tumors.
`A yeast qns1 mutant Was used to screen for natural sources
`of nicotinamide riboside Wherein it Was identi?ed in an acid
`
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`60
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`65
`
`A difference betWeen humans and yeasts concerns the
`organisms’ uses of nicotinamide and nicotinic acid, the tWo
`niacins that Were co-identi?ed a