USOO8383O86B2
`
`(12) United States Patent
`Brenner
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,383,086 B2
`*Feb. 26, 2013
`
`(54) NICOTINAMIDE RIBOSIDE KINASE
`COMPOSITIONS AND METHODS FOR
`USING THE SAME
`
`(75) Inventor: Charles M. Brenner, Lyme, NH (US)
`(73) Assignee: Trustees of Dartmouth College,
`Hanover, NH (US)
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`This patent is Subject to a terminal dis
`claimer.
`
`(*) Notice:
`
`(21) Appl. No.: 13/445.289
`(22) Filed:
`Apr. 12, 2012
`
`(65)
`
`Prior Publication Data
`US 2012/O251463 A1
`Oct. 4, 2012
`
`Related U.S. Application Data
`(63) Continuation of application No. 1 1/912,400, filed as
`application No. PCT/US2006/015495 on Apr. 20,
`2006, now Pat. No. 8,197,807.
`
`(51) Int. Cl.
`(2006.01)
`A6 IK38/45
`(2006.01)
`A6 IK3I/7088
`(2006.01)
`C07H 17/00
`(2006.01)
`A6IP35/00
`(52) U.S. Cl. ........... 424/48; 424/94.5; 435/15; 435/194;
`514/25; 514/44 R
`(58) Field of Classification Search ........................ None
`See application file for complete search history.
`
`(56)
`
`WO
`
`References Cited
`
`FOREIGN PATENT DOCUMENTS
`O132888
`5, 2001
`
`OTHER PUBLICATIONS
`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 Humans.” Cell 2004
`117:495-5O2.
`Bieganowski et al., “Eukaryotic NAD+ Synthetase Qns 1 Contains an
`Essential,
`Obligate
`Intramolecular
`Thiol
`Glutamine
`Amidotransferase Domain Related to Nitrilase,” J. Biol. Chem. 2003
`278 (35):33049-33055.
`Berger et al., “Modulation of Nicotinamide Adenine Dinucleotide
`and Poly(Adenosine Diphosphoribose) Metabolism by the Synthetic
`“C” Nucleoside Analogs, Tiazofurin and Selenazofurin.” J. Clin.
`Invest. 1985 75:702-705.
`Berger et al., “Poly ADP-ribose in the cellular response to DNA
`damage.” Radiation Research Jan. 1985:101 (1):4-15 (see abstract).
`Boon et al., “An anatomy of normal and malignant gene expression.”
`Proc. Natl. Acad. Sci. 200299 (17): 11287-11292.
`Burkle, A. “Physiology and pathophysiology of poly(ADP-ribosyl)a-
`tion.” BioEssays 2001 23:795-806.
`Farquhar et al., “Synthesis and antitumor evaluation of
`bis(pivaloyloxy)methyl]2'-deoxy-t-5-fluorouridine
`
`5'-monophosphate (FdUMP): a strategy to introduce nucleotides into
`cells.”J Med Chem 1994 37(23):3902-3909.
`Fleishchmann et al., “Whole-Genome Random Sequencing and
`Assembly of Haemophilus Influenzae Rd.” Science 1995 269:496
`512.
`Gingrich et al., “Codehydrogenase I and Other Pyridinium Com
`pounds as V-Factor for Hemophilus Influenzae and H.
`Parainfluenzae, J. Bacteriol. 199447:535-550.
`Godek et al., “In Vitro Evaluation of Nicotinamide Riboside Analogs
`Against Haemophilus Influenzae.” Antimicrobal Agents and Chemo
`therapy 199034(8): 1473-1479.
`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.
`Holdsworth et al., “A fraction derived from brewer's yeast inhibits
`cholesterol synthesis by rat liver preparations in vitro.” Br. J. Nutr.
`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-specific Beta 1 Integrin Binding Protein
`(MIBP) that Modulates Myogenic Differentiation.” J. Cell Biol. 1999
`147: 1391-1397.
`Liet al., “The muscle integrin binding protein (MIBP) interacts with
`alpha7beta1 integrin and regulates cell adhesion and laminin matrix
`deposition.” Developmental Biology 2003 261:209-219.
`Sasiak et al., “Purification and Properties of a Human Nicotinamide
`Ribonucleoside Kinase.” Archives of Biochemistry and Biophysics
`1996 333(2):414-418.
`Saunders et al., “Phosphorylation of 3-Deazaguanosine by
`Nicotinamide Riboside Kinase in Chinese Hamster Ovary Cells.”
`Cancer Research 1989 49:6593-6599.
`Saunders et al., “Tiazofurin Is Phosphorylated by Three Enzymes
`from Chinese Hamster Ovary Cells', Cancer Research 1990
`50:5269-5274
`Shifrine et al., “Bacteriology—A Growth Factor for Haemophilus
`Species secreted by a Pseudomonad'. Nature 1960 187:623.
`Stubberfield et al., “NAD+ depletion and cytotoxicity in isolated
`hepatocytes”, Biochem. Pharm. 199837(20):3967-3974.
`Tanimori et al., “An Efficient Chemical Synthesis of Nicotinamide
`Riboside (NAR) and Analogues.” Bioorganic. Med. Chem. Lett.
`2002 12:1135-1137.
`Ziegler, M., “New functions of a long-known molecule—Emerging
`roles of NAD in cellular signaling.” Eur, J. Biochem. 2000 267: 1550
`1564.
`NCBI Accession No. NP 060351 gi:8923530 with Revision His
`tory Jul. 4, 2000-Jun. 3, 2007.
`NCBI Accession No. NP 733778 gi:24762248 with Revision His
`tory Nov. 2, 2002-Jul. 5, 2007.
`NCBI Accession No. NM 017881 gi:8923529 with Revision His
`tory Jul. 4, 2000-Nov. 17, 2006.
`NCBI Accession No. AKO00566 gi:7020748 with Revision His
`tory Feb. 22, 2000-Sep. 12, 2006.
`NCBI Accession No. BC001366 gi:33876100 with Revision His
`tory Dec. 12, 2000-Jul. 15, 2006.
`(Continued)
`Primary Examiner — Kagnew H Gebreyesus
`(74) Attorney, Agent, or Firm — Licata & Tyrrell P.C.
`(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
`prvided. The present invention further provides screening
`methods for isolating a nicotinamide riboside-related prodrug
`and identifying a natural source of nicotinamide riboside.
`5 Claims, 1 Drawing Sheet o
`
`THORNE - EXHIBIT 1024
`
`

`

`US 8,383,086 B2
`Page 2
`
`OTHER PUBLICATIONS
`NCBI Accession No. BC036804 gi:22477870 with Revision His
`tory—Aug. 26, 2002-Mar. 25, 2004.
`NCBI Accession No. BC026243 gi:20072207 with Revision His
`tory—Apr. 8, 2002-Mar. 25, 2004.
`NCBI Accession No. NM 170678 gi:24762247 with Revision
`History Nov. 7, 2002-Nov. 17, 2006 NM 170678.2 which replaces
`NM 170678 is provided.
`NCBI Accession No. CAG61927 gi:49528270 with Revision His
`tory–Jun. 30, 2004-Nov. 14, 2006.
`
`NCBI Accession No. Z71405 gi: 1302065 with Revision History
`May 6, 1996-Aug. 11, 1997.
`NCBI Accession No. AX877238 gi:40031974 Dec. 17, 2003.
`Genbank Accession No. AK001663 Jan. 9, 2008.
`Genbank Accession No. YNL 129W Nov. 7, 2005.
`Written Opinion in International Application No. PCT/US2006/
`0 15495 mailed Nov. 23, 2007.
`International Search Report in international Application No. PCT/
`US2006/015495 mailed Nov. 23, 2007.
`European Search Report from EP Application No. 06751269 dated
`Dec. 29, 2008.
`
`

`

`U.S. Patent
`
`Feb.26,2013
`
`US 8,383,086 B2
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`

`US 8,383,086 B2
`
`1.
`NCOTNAMIDE RIBOSIDE KNASE
`COMPOSITIONS AND METHODS FOR
`USING THE SAME
`
`2
`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,
`5 et al. (2000) Cold Spring Harb. Symp. Ouant. 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 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(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 23:795-806).
`The non-coenzymatic properties of NAD+ has renewed
`Nicotinic acid and nicotinamide, collectively niacins, are 20
`interest in NAD+ biosynthesis. Four recent publications have
`the vitamin forms of nicotinamide adenine dinucleotide
`Suggested what is considered to be all of the gene products
`(NAD+). Eukaryotes can synthesize NAD+ de novo via the
`and pathways to NAD+ in S. cerevisiae (Panozzo, et al.
`kynurenine pathway from tryptophan (Krehl, et al. (1945)
`(2002) FEBS Lett. 517: 97-102; Sandmeier, et al. (2002)
`Science 101:489-490; Schutz and Feigelson (1972) J. Biol.
`Chem. 247:5327-5332) and niacin supplementation prevents 25 Genetics 160:877-889; Bitterman, et al. (2002).J. Biol. Chem.
`the pellagra that can occur in populations with a tryptophan-
`277:45099-45107: Anderson, et al. (2003) Nature 423:181
`poor diet. It is well-established that nicotinic acid is phospho-
`185) depicting convergence of the flux to NAD+ from de novo
`ribosylated to nicotinic acid mononucleotide (NaMN), which
`synthesis, nicotinic acid import, and nicotinamide Salvage at
`is then adenylylated to form nicotinic acid adenine dinucle-
`NaMN (Scheme 1).
`
`INTRODUCTION
`
`This application is a continuation of U.S. patent applica-
`tion Ser. No. 1 1/912,400 filed Nov. 20, 2007 now U.S. Pat.
`No. 8,197,807, which is the National Stage of International
`Application No. PCT/US2006/015495 filed Apr. 20, 2006,
`which claims benefit of priority to U.S. patent application Ser.
`No. 11/113,701 filed Apr. 25, 2005, the teachings of which are
`incorporated herein by reference in their entireties.
`This invention was made with government support under
`grant number CA77738 awarded by the National Cancer
`Institute. The government has certain rights in the invention.
`BACKGROUND OF THE INVENTION
`
`O
`
`Scheme 1
`
`O
`
`O
`
`DENOVO
`Bna1-6
`
`-e-
`
`N
`
`Nma1.2
`
`N 9
`
`Qns1
`
`N NH2
`
`N
`
`Prbd.
`
`NaMN
`
`ATP
`
`PP
`
`N
`
`ADPribo
`NaAD+
`
`ATP
`Gln
`
`N
`
`ATP
`PP, ADPribo
`Glu
`NAD+
`
`PP
`
`PrboPP
`
`SALWAGE
`Npt1
`
`O
`
`N O-
`2
`N
`H
`
`Nicotinic acid
`
`IMPORT
`
`Tnal
`
`Plasma membrane
`
`LySAc
`Sir2
`Lys + ADPriboAc
`O
`
`Pnc1
`-N
`NH HO
`
`N NH2
`2
`N
`H
`
`nicotinamide
`
`otide (NaAD), which in turn is amidated to form NAD+ 9
`(Preiss and Handler (1958).J. Biol. Chem. 233:488-492; Pre
`It has now been shown that nicotinamide riboside, which
`iss and Handler (1958b).J. Biol. Chem. 233:493-50).
`was known to be an NAD+ precursor in bacteria such as
`NAD+ was initially characterized as a co-enzyme for oxi-
`Haemophilus influenza (Gingrich and Schlenk (1944).J. Bac
`doreductases. Though conversions between NAD+, NADH,
`NADP and NADPH would not be accompanied by a loss of 65 teriol. 47:535-550; Leder and Handler (1951).J. Biol. Chem.
`total co-enzyme, it was discovered that NAD+ is also turned
`189:889-899: Shifrine and Biberstein (1960) Nature 187:
`over in cells for unknown purposes (Maayan (1964) Nature
`623) that lack the enzymes of the de novo and Preiss-Handler
`
`SUMMARY OF THE INVENTION
`
`

`

`3
`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 con
`Verting cancer drugs such as tiaZoflurin and benzamide ribo
`side 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.
`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, SEQID NO:2 or SEQ ID NO:3; (b) a nucleotide
`sequence that hybridizes to a nucleotide sequence of SEQID
`NO:1, SEQID NO:2 or SEQID 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 first 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
`NO:4, SEQID 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 first 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
`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 first step in activating the
`prodrug so that the signs or symptoms of said cancer are
`decreased or eliminated.
`
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`50
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`65
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`US 8,383,086 B2
`
`10
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`4
`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+ Syn
`thetase 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’s milk.
`Further, the present invention is a dietary Supplement com
`position containing nicotinamide riboside identified 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 identified 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 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 nico
`tinamide riboside-related prodrug identified 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.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`FIG. 1 shows the amino acid sequence alignment and con
`sensus sequence (SEQID NO:34) of human Nrk1 (SEQ ID
`NO:5), human Nrk2 (SEQ ID NO:6), S. cerevisiae Nrk1
`(SEQID NO:4), S. pombe nrk1 (SEQID NO:7), as compared
`
`

`

`US 8,383,086 B2
`
`5
`to portions of S. cerevisiae uridine/cytidine kinase Urk1 (SEQ
`ID NO:8) and E. coli pantothenate kinase (SEQID NO:9).
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`10
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`
`35
`
`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. 278:33049-33055).
`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) area 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 qns 1 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 qns 1 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 uM.
`Anticancer agents such as tiazofurin (Cooney, et al. (1983)
`Adv. Enzyme Regul. 21:271-303) and benzamide riboside
`(Krohn, et al. (1992).J. Med. Chem. 35:511-517) 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/NaMNadenylyltransferase 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) Cancer Res. 46:532-537; Saunders, et al. (1990) Can
`40
`cer Res. 50:5269-5274) and a specific nicotinamide riboside
`kinase (Saunders, et al. (1990) supra) have been proposed to
`be responsible fortiazofurin phosphorylation in vivo. A puta
`tive nicotinamide riboside kinase (Nrk) activity was purified,
`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. 333:414-418).
`Using a qns 1 deletion strain that was additionally deleted
`for yeast homologs of candidate genes encoding nucleoside
`kinases proposed to phosphorylate tiazofurin, i.e., adenosine
`kinase adol (Lecoq et al. (2001) Yeast 18:335-342), uridine/
`cytidine kinase urk1 (Kern (1990) Nucleic Acids Res.
`18:5279; Kurtz, et al. (1999) Curr. Genet. 36:130-136), and
`ribokinase rbk1 (Thierry, et al. (1990) Yeast 6:521-534), 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.
`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 influenza. The Nrk domain of H. influenza 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
`
`50
`
`45
`
`55
`
`60
`
`65
`
`6
`late kinase (Singh, et al. (2002).J. Biol. Chem. 277:33291
`33299), sensitive sequence searches revealed that bacterial
`Nrk has no ortholog in yeast. Genomic searches with the Nrk
`domain of H. influenza NadR have identified a growing list of
`bacterial genomes predicted to utilize nicotinamide riboside
`as an NAD+ precursor (Kurnasov, et al. (2002) J. Bacteriol.
`184:6906-6917). 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 286:
`1153-1155). Sixty-four pools of 90-96 S. cerevisiae open
`reading frames fused to glutathione S-transferase (GST),
`expressed in S. cerevisiae, were purified as GST fusions and
`screened for the ability to convert nicotinamide riboside plus
`ATP to NMNplus ADP. Whereas most 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 NO:1) 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 Urkland remote similarity with a segment of
`E. coli pantothenate kinase panK (Yun, et al. (2000).J. Biol.
`Chem. 275:28093-28099) (FIG. 1). After cloningYNL129W
`into a bacterial expression vector it was ascertained whether
`this homolog of metabolite kinases was the eukaryotic Nrk.
`The specific activity of purified 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 phosphorylates
`nicotinamide riboside in vivo, a deletion of YNL129W was
`created in the qns 1 background. It was found that nicotina
`mide riboside rescue of the qns 1 deletion strain was entirely
`dependent on this gene product. Having shown biochemically
`and genetically that YNL129W encodes an authentic Nrk
`activity, the gene was designated NRK1.
`A PSI-BLAST (Altschul, et al. (1997) Nucleic Acids Res.
`25:3389-3402) comparison was conducted on the predicted
`S. cerevisiae Nrk1 polypeptide and an orthologous human
`protein Nrk1 (NP 060351; SEQ ID NO:5; FIG. 1) was
`found. The human NP 060351 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 (NP 733778:
`SEQ ID NO:6; 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).J. Cell Biol. 147: 1391-1398; Li, et al. (2003) Dev. Biol.
`261:209-219). Amino acid conservation between S. cerevi
`siae, S. pombe and human Nrk homologs and similarity with
`fragments of S. cerevisiae Urkl and E. coli panK is shown in
`FIG. 1. Fungal and human Nrk enzymes are members of a
`metabolite kinase Superfamily that includes pantothenate
`kinase but is unrelated to bacterial nicotinamide riboside
`kinase. Robust complementation of the failure of qns 1 nrk1 to
`grow on nicotinamide riboside-supplemented media was pro
`vided by human NRK1 and human NRK2 cDNA even when
`expressed from the GAL1 promoter on glucose.
`
`

`

`US 8,383,086 B2
`
`8
`
`Scheme 2
`
`7
`As shown in Table 1, purification of yeast Nrk1 and human
`Nrk1 and Nrk2 revealed high specificity for phosphorylation
`of nicotinamide riboside and tiazofurin.
`
`TABLE 1.
`
`5
`
`Nicotinamide
`riboside
`
`Tiazofurin
`
`Uridine
`
`Cytidine
`
`Human Nrk1
`Human Nrk2
`Yeast Nrk1
`
`275 - 17
`232O20
`535 - 60
`
`S38 27
`2150 210
`1129 - 134
`
`19.3 1.7 355 6.4
`2220 17O 2228
`15.23.4 82.944
`
`10
`
`Specific activity is expressed in nmole mg min for phosphorylation of nucleoside sub
`strates,
`
`Nma1.2
`Qns1
`Nma1.2
`Bna1-6
`–> NaMN –> NaAD" --> NAD" (– NMN
`Na a- N 5-1 is
`H.S.
`Npt1
`sin
`PBEF
`
`S.c. Pnc1
`
`Nr
`
`A difference between humans and yeasts concerns the
`organisms uses of nicotinamide and nicotinic acid, the two
`niacins that were co-identified as anti-black tongue factor
`(Elvehjem, et al. (1938) supra). Humans encode a homolog of
`the Haemophilus ducreyi nadV gene, termed pre-B-cell
`colony enhancing factor, that may convert nicotinamide to
`NMN (Rongvaux, et al. (2002) Eur: J. Immunol. 32:3225
`3234) and is highly induced during lymphocyte activation
`(Samal, et al. (1994) Mol. Cell. Biol. 14:1431-1437). In con
`trast, S. cerevisiae lacks a homolog of nad V and instead has a
`homolog of the E. coli pncA gene, termed PNC1, that con
`verts nicotinamide to nicotinic acid for entry into the Preiss
`Handler pathway (Ghislain, et al. (2002) Yeast 19:215-224:
`Sandmeier, et al. (2002) supra). Though the Preiss-Handler
`pathway is frequently considered a salvage pathway from
`nicotinamide, it technically refers to the steps from nicotinic
`acid to NAD+ (Preiss and Handler (1958) supra; Preiss and
`Handler (1958) supra). Reports that nicotinamidase had been
`purified from mammalian liver in the 1960s (Petrack, et al.
`(1965).J. Biol. Chem. 240:1725-1730) may have contributed
`to the sense that fungal and animal NAD+ biosynthesis is
`entirely conserved. However, animal genes for nicotinami
`dase have not been identified and there is no compelling
`evidence that nicotinamide and nicotinic acid are utilized as
`NAD+ precursors through the same route in mammals. The
`persistence of “niacin' as a mixture of nicotinamide and
`nicotinic acid may attest to the utility of utilizing multiple
`pathways to generate NAD+ and indicates that Supplementa
`tion with nicotinamide riboside as third importable NAD+
`precursor can be beneficial for certain conditions.
`First reported in 1955, high doses of nicotinic acid are
`effective at reducing cholesterol levels (Altschul, et al. (1955)
`Arch. Biochem. Biophys. 54:558-559). Since the initial
`report, many controlled clinical studies have shown that nico
`tinic acid preparations, alone and in combination with HMG
`CoA reductase inhibitors, are effective in controlling low
`density lipoprotein cholesterol, increasing high-density lipo
`protein cholesterol, and reducing triglyceride and lipoprotein
`a levels in humans (Pasternak, et al. (1996) Ann. Intern. Med.
`125:529-540). Though nicotinic acid treatment effects all of
`the key lipids in the desirable direction and has been shown to
`reduce mortality in target populations (Pasternak, et al.
`(1996) supra), its use is limited because of a side effect of heat
`and redness termed “flushing,” which is significantly effected
`by the nature of formulation (Capuzzi, et al. (2000) Curr.
`Atheroscler. Rep. 2:64-71). Thus, nicotinamide riboside
`Supplementation could be one route to improve lipid profiles
`in humans. Further, nicotinamide is protective in animal mod
`els of stroke (Klaidman, et al. (2003) Pharmacology 69:150
`157) and nicotinamide riboside could be an important supple
`ment for acute conditions such as stroke. Additionally,
`regulation of NAD+ biosynthetic enzymes could be useful in
`sensitizing tumors to compounds Suchastiazofurin, to protect
`normal tissues from the toxicity of compounds such as tiaZo
`furin adenine dinucleotide, and to stratify patients for the
`most judicious use of tiazofurin chemotherapy.
`
`In the cases of yeast and human Nrk1 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 specific activity on uridine and
`cytidine. In the case of human Nrk2, the 230 amino acid form
`was essentially equally active on nicotinamide ri