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`
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`
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`
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`APPLICATION NUMBER: 60/543,347
`FILING DATE: February 10, 2004
`RELATED PCT APPLICATION NUMBER: PCT/US05/0433 7
`
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`a; '
`
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`.u_iven Name (first and middle [if any])
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`
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`NICOTINAMIDE RIBOSIDE KINASE COMPOSITIONS AND METHODS FOR USING THE SAME
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`(2 copies);
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`

`\
`.
`we:
`
`I",
`
`DC-0253
`
`-1-
`
`PATENT
`
`5
`
`NICOTINAMIDE RIBOSIDE KINASE COMPOSITIONS AND METHODS FOR
`USING THE SAME
`
`Introduction
`
`10
`
`This
`
`invention was made
`
`in the
`
`course of
`
`research
`
`sponsored
`
`by
`
`the National Cancer
`
`Institute
`
`(Grant No.
`
`CA77738). The U.S.
`
`government may have certain rights in
`
`this invention.
`
`15
`
`Background of the Invention
`
`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 phosphoribosylated to nicotinic acid mononucleotide
`
`(NaMN), which is then adenylylated to form nicotinic acid
`
`adenine dinucleotide (NaAD), which jJ1
`
`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).
`
`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)
`
`Amture
`
`204:1169—1170).
`
`Sirtuin
`
`enzymes
`
`such
`
`as
`
`Sir2
`
`of
`
`S.
`
`cerevisiae and
`
`its homologs deacetylate
`
`lysine
`
`residues
`
`20
`
`25
`
`30
`
`35
`
`0005
`
`0005
`
`

`

`I!
`
`DC-0253
`
`-2-
`
`PATENT
`
`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).
`
`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).
`
`10
`
`15
`
`20
`
`25
`
`0006
`
`0006
`
`

`

`n
`
`.
`
`DC-0253
`
`-3-
`
`A
`
`PATENT
`
`O
`
`O‘
`
`O
`
`O‘
`Nmal,2
`
`'
`
`l
`
`NaAD+
`
`\\\.
`
`/’
`+
`
`ATP
`ADPrbO Gln,
`
`,
`
`NaMN
`
`DE NOVO
`Bnal’6 I
`
`\\\
`
`//
`+
`
`g
`PrboP
`
`Nicotinic acid
`
`T ATP
`Prbo
`PPi
`_
`
`PPi
`
`Q
`SALVAGE’
`Nptl
`
`\\\
`
`//
`§+
`
`0'
`Qnsl
`
`.
`
`NAD+
`
`\\\
`
`//
`+
`
`T
`ATP
`PPi ADPrbO
`Glu
`
`NH
`
`2
`
`LysAc
`Sir2
`'
`:g: g A
`r o c
`NH2
`
`.
`
`nicotinamide
`
`"
`
`
`
`10
`
`15
`
`20
`
`25
`
`IMPORT
`
`Tnal
`
`Plasma membrane
`
`Scheme 1
`
`Brief Description of the Drawings
`
`Figure
`
`1
`
`shows
`
`the
`
`nucleotide
`
`sequences
`
`encoding
`
`Saccharomyces cerevisiae Nrkl
`
`(SEQ ID NO:1,
`
`Figure
`
`1A),
`
`human Nrkl
`
`(SEQ ID NO:2, Figure 18), and human Nrk2 (SEQ ID
`
`NO:3, Figure 1C).
`
`the amino: acid sequence alignment of
`shows
`Figure 2
`human Nrkl
`(SEQ ID NO:5),
`human Nrk2
`(SEQ ID NO:6),
`S.
`
`30
`
`cerevisiae Nrkl
`
`(SEQ ID NO:4),
`
`s. pombe nrkl
`
`(SEQ ID NO:7),
`
`and portions of
`
`.S. cerevisiae uridine/cytidine kinase Urkl
`
`(SEQ ID NO:8)
`
`and E.
`
`coli pantothenate kinase
`
`(SEQ ID
`
`NO:9).
`
`0007
`
`0007
`
`

`

`(I
`
`DC-0253
`
`-4-
`
`PATENT
`
`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
`
`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, Nrkl,
`
`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
`
`converting
`
`cancer
`
`drugs
`
`such
`
`as
`
`10
`
`15
`
`the
`
`first
`
`step
`
`in
`
`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
`
`20
`
`nicotinamide riboside.
`
`Accordingly, one aspect of
`
`the present
`
`invention is an
`
`isolated nucleic acid encoding a eukaryotic nicotinamide
`
`riboside kinase polypeptide. Said isolated nucleic acid is:
`
`(a)
`
`a nucleotide sequence of SEQ ID NO:1,
`
`SEQ ID
`
`25
`
`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
`
`3O
`
`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
`
`0008
`
`0008
`
`

`

`H
`
`DC-0253
`
`-5-
`
`PATENT
`
`(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.
`
`Another
`
`aspect
`
`of
`
`the
`
`present
`
`invention
`
`is
`
`an
`
`expression
`
`vector
`
`containing
`
`an
`
`isolated nucleic
`
`acid
`
`encoding
`
`a
`
`eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide.
`
`In one
`
`embodiment,
`
`said expression vector
`
`is
`
`part
`
`of
`
`a
`
`composition
`
`containing
`
`a
`
`pharmaceutically
`
`acceptable carrier.
`
`In another embodiment,
`
`said composition
`
`further
`
`contains
`
`a
`
`prodrug wherein
`
`said 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.
`
`10
`
`15
`
`A
`
`further
`
`aspect
`
`of
`
`the present
`
`invention is
`
`an
`
`isolated
`
`eukaryotic
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide.
`
`In one embodiment,
`
`said isolated nicotinamide
`
`riboside
`
`kinase polypeptide
`
`is
`
`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.
`
`A still further aspect of
`
`the present
`
`invention is a
`
`cultured cell containing an isolated nucleic acid encoding
`
`a eukaryotic nicotinamide riboside kinase polypeptide or a
`
`polypeptide encoded thereby.
`
`Another
`
`aspect
`
`of
`
`the
`
`present
`
`invention
`
`is
`
`a
`
`composition containing an isolated eukaryotic nicotinamide
`
`riboside
`
`kinase
`
`polypeptide
`
`and
`
`a
`
`pharmaceutically
`
`acceptable carrier.
`
`In one
`
`embodiment,
`
`said 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.
`
`20
`
`25
`
`3O
`
`0009
`
`0009
`
`

`

`DC-0253
`
`-6-
`
`PATENT
`
`A further aspect of
`
`the present
`
`invention is‘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
`
`said.
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide
`
`phosphorylates
`
`said prodrug
`
`thereby performing the first step in activating the prodrug
`
`so that
`
`the signs Or
`
`symptoms of said cancer are decreased
`
`or eliminated.
`
`A still further aspect of
`
`the present
`
`invention is a
`
`method for
`
`identifying a natural or synthetic source for
`
`nicotinamide
`
`riboside.
`
`The method
`
`involves
`
`contacting a
`
`10
`
`15
`
`NAD+
`
`first cell
`
`lacking la
`
`functional glutamine-dependent
`
`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's
`
`20
`
`25
`
`milk.
`
`Another aspect of
`
`the present
`
`invention is a dietary
`
`supplement
`
`composition
`
`containing
`
`nicotinamide
`
`riboside
`
`identified in accordance with the methods of
`
`the present
`
`30
`
`invention and a carrier.
`
`A still further aspect of
`
`the present
`
`invention is a
`
`method for preventing’ or
`
`treating a disease or condition
`
`associated with the nicotinamide riboside kinase pathway of
`
`0010
`
`0010
`
`

`

`n
`
`DC-0253
`
`-7-
`
`PATENT
`
`NAD+ biosynthesis. The method involves administering to a
`
`patient having a disease or condition associated with the
`
`Inicotinamide
`
`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 condition
`
`is
`
`stroke
`
`or dyslipidemia.
`
`In
`
`another
`
`embodiment,
`
`the
`
`nicotinamide
`
`riboside
`
`is
`
`further
`
`administered
`
`combination
`
`with
`
`tryptophan,
`
`nicotinic
`
`acid
`
`in
`
`or
`
`10
`
`nicotinamide.
`
`A further aspect of
`
`the present
`
`invention is an in
`
`Vitro method
`
`for
`
`identifying
`
`a
`
`nicotinamide
`
`riboside—
`
`related
`
`prodrug.
`
`The
`
`method
`
`involves
`
`contacting
`
`nicotinamide
`
`riboside
`
`kinase
`
`polypeptide
`
`with
`
`a
`
`a
`
`15
`
`20
`
`25
`
`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 yet another aspect of the invention.
`
`A still further aspect of
`
`the present
`
`invention is 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
`
`30
`
`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 yet
`
`another aspect of the invention.
`
`0011
`
`0011
`
`

`

`u
`
`DC-0253
`
`-8-
`
`PATENT
`
`Another aspect of
`
`the present
`
`invention is 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.
`
`Detailed Description of the Invention
`
`A
`
`Saccharomyces
`
`cerevisiae
`
`QNSl
`
`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 13f strains containing the qnsl deletion
`
`and a plasmid—borne QNSl gene allowed a determination of
`
`whether
`
`the canonical de novo,
`
`import and salvage pathways
`
`for
`
`NAD+
`
`of
`
`Scheme
`
`1
`
`(Panozzo,
`
`et
`
`al.
`
`(2002)
`
`supra;
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`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
`
`qnsl
`
`mutants
`
`by
`
`shunting
`
`nicotinamide-containing
`
`precursors
`
`through the
`
`pathway;
`
`and QNSl
`
`is
`
`common
`
`to the
`
`three
`
`pathways,
`
`thus there may be no NAD+ precursor that
`
`rescues
`
`qnsl mutants. However,
`
`it has
`
`now been found that while
`
`0012
`
`0012
`
`

`

`n
`
`DC-0253
`
`-9-
`
`PATENT
`
`nicotinamide does not
`
`rescue qnsl mutants even at
`
`J. 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.
`
`212271-303)
`
`and
`
`benzamide
`
`riboside
`
`(Krohn, et al.
`
`(1992)
`
`J1 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—limiting
`
`enzyme
`
`for
`
`'de
`
`novo
`
`purine
`
`nucleotide
`
`biosynthesis.
`
`Though an NMN/NaMN adenylyltransferase is thought
`
`to
`
`be
`
`the
`
`enzyme
`
`that
`
`converts
`
`the
`
`mononucleotide
`
`intermediates to NAD+ analogs and the structural basis for
`
`this is known
`
`(Zhou et al.
`
`(2002) supra), several different
`
`enzymes
`
`including
`
`adenosine
`
`kinase,
`
`5’
`
`nucleotidase
`
`10
`
`15
`
`20
`
`25
`
`30
`
`(Fridland, et al.
`
`(1986) Cancer Res.
`
`462532—537; Saunders,
`
`et al.
`
`(1990) Cancer Res.
`
`50:5269—5274)
`
`and
`
`a
`
`specific
`
`nicotinamide
`
`riboside
`
`kinase
`
`(Saunders,
`
`et
`
`al.
`
`(1990)
`
`supra) have been proposed to be responsible for tiazofurin
`
`phosphorylation in Vivo.
`
`A putative 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. Biochem. Biophys. 333:414—418).
`
`Using
`
`a qnsl 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
`
`urkl
`
`(Kern
`
`(1990)
`
`Nucleic Acids Res.
`
`18:5279; Kurtz,
`
`et al.
`
`(1999) Curr.
`
`Genet.
`
`36:130—136),
`
`and ribokinase rbkl
`
`(Thierry,
`
`et al.
`
`(1990) Yeast
`
`6:521—534),
`
`it was determined whether
`
`the
`
`0013
`
`0013
`
`

`

`DC-0253
`
`-10-
`
`PATENT
`
`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
`
`thymidylate kinase
`
`(Singh, et al.
`
`(2002)
`
`J.
`
`Biol.
`
`Chem.
`
`277:33291-33299),
`
`sensitive
`
`sequence
`
`searches
`
`revealed that bacterial Nrk has
`
`no ortholog in
`
`10
`
`15
`
`yeast. Genomic searches with the Nrk domain of H.
`
`influenza
`
`NadR have
`
`identified a growing list of bacterial genomes
`
`an
`
`NAD+
`
`20
`
`25
`
`30
`
`predicted to utilize nicotinamide
`
`riboside
`
`as
`
`precursor
`
`(Kurnasov, et al.
`
`(2002)
`
`J. Bacteriol.
`
`18426906—
`
`6917).
`
`Thus,
`
`had
`
`fungi
`
`possessed
`
`NadR Nrk—homologous
`
`domains,
`
`comparative genomics would have already predicted
`
`that yeast can salvage nicotinamide riboside.
`
`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
`
`cerevisiae open
`
`reading frames
`
`fused to glutathione
`
`S.
`
`S—
`
`transferase
`
`(GST),
`
`expressed
`
`in
`
`S.
`
`cerevisiae,
`
`were
`
`purified as GST
`
`fusions
`
`and screened for
`
`the ability to
`
`convert nicotinamide
`
`riboside plus ATP
`
`to NMN plus ADP.
`
`Whereas most pools contained activities that consumed some
`
`of
`
`the
`
`input ATP,
`
`only
`
`pool
`
`37
`
`consumed nicotinamide
`
`0014
`
`0014
`
`

`

`DC-0253
`
`-11-
`
`PATENT
`
`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
`
`N021;
`
`Figure
`
`1A)
`
`encodes
`
`a predicted 240
`
`amino
`
`acid
`
`polypeptide with a
`
`187
`
`amino acid -segment containing 23%
`
`identity with the
`
`501
`
`amino acid yeast uridine/cytidine
`
`kinase Urkl and remote similarity with a segment of E. coli
`
`pantothenate kinase panK (Yun, et al.
`
`(2000)
`
`J. Biol. Chem.
`
`275:28093—28099)
`
`(Figure 2). After cloning YNL129W 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
`
`a deletion of
`
`10
`
`15
`
`20
`
`25
`
`30
`
`phosphorylates nicotinamide riboside in Vivo,
`
`YNL129W was created in the qnsl background.
`
`It was
`
`found
`
`that nicotinamide
`
`riboside
`
`rescue
`
`of
`
`the
`
`qnsl 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 NRKJ.
`
`A
`
`PSI—BLAST
`
`(Altschul,
`
`et al.
`
`(1997) Nucleic Acids
`
`Res.
`
`25:3389—3402)
`
`comparison
`
`was
`
`conducted
`
`on
`
`the
`
`predicted S.
`
`cerevisiae Nrkl polypeptide and.
`
`an apparent
`
`orthologous
`
`human protein Nrkl
`
`(NP_060351;
`
`SEQ ID NO:5;
`
`Figure 2) was
`
`found. The human NP_060351 protein encoded at
`
`locus 9q21.3l
`
`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; Figure 2) was
`
`found that
`
`is
`
`57% identical
`
`to human Nrkl. Nrk2
`
`is
`
`a
`
`230
`
`amino acid
`
`0015
`
`0015
`
`

`

`DC-0253
`
`-12-
`
`PATENT
`
`splice form of what twas described as
`
`a
`
`186
`
`amino acid
`
`muscle integrin beta 1 binding protein (ITGBlBP3)
`
`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. cerevisiae, S. pombe and human Nrk
`
`homologs
`
`and
`
`similarity with fragments of
`
`S.
`
`cerevisiae
`
`Urkl
`
`and E.
`
`coli panK is
`
`shown in Figure 2. Fungal
`
`and
`
`human Nrk
`
`enzymes
`
`are members
`
`of
`
`a metabolite
`
`kinase
`
`superfamily
`
`that
`
`includes
`
`pantothenate
`
`kinase
`
`but
`
`is
`
`10
`
`unrelated to bacterial nicotinamide riboside kinase. Robust
`
`complementation of
`
`the failure of qnsl nrkl
`
`to grow on
`
`nicotinamide
`
`riboside—supplemented media was
`
`provided by
`
`human NRKl even when expressed from the GAL1 promoter on
`
`glucose.
`
`As
`
`shown in Table 1, purification of yeast. Nrkl and
`
`human Nrkl
`
`and
`
`Nrk2
`
`revealed
`
`high
`
`specificity
`
`for
`
`phosphorylation of nicotinamide riboside and tiazofurin.
`
` TABLE 1
`
`Human Nrkl
`Human Nrk2
`Yeast Nrkl
`
`Nicotlnamide Tiazofurin
`rlbOSlde
`275il7
`538i 27
`2320:20
`2150:210
`535:60
`1129i134
`
`Uridine
`19.3i
`1.7
`2220
`i170
`15.2i
`3.4
`
`Cytidine
`35.516.4
`222
`:8
`82.9:4.4
`
`expressed in nmole mg"I min"I
`Specific activity is
`phosphorylation of nucleoside substrates.
`
`for
`
`In the
`cases of yeast
`and human Nrkl
`enzymes,
`the
`enzymes
`preferred tiazofurin to
`the natural
`substrate
`
`nicotinamide 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
`
`riboside,
`
`tiazofurin and uridine with
`
`less
`
`than 10% of corresponding activity on cytidine. Conversely,
`
`15
`
`20
`
`25
`
`30
`
`0016
`
`0016
`
`

`

`DC-0253
`
`-13-
`
`PATENT
`
`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
`
`specific nicotinamide
`
`riboside kinases
`
`that
`
`function to
`
`synthesize NAD+
`
`through NMN :hi addition 11)
`
`the well—known
`
`pathways
`
`through
`
`NaMN.
`
`Identification of Nrk
`
`enzymatic
`
`activities thus accounts for the dual specificity of fungal
`
`and mammalian NaMN/NMN adenylyltransferases.
`
`On the basis of SAGE data, NRKl
`
`is a rare message in
`
`many
`
`tissues
`
`examined while NRKQ is highly expressed in
`
`heart and skeletal muscle and has lower
`
`level expression in
`
`retinal epitheliwn and placenta (Boon, et al.
`
`(2002) Proc.
`
`Natl. Acad. Sci. USA 99:11287—11292). From cancer cell
`
`line
`
`the
`
`expression
`
`levels
`
`are
`
`quite
`
`10
`
`15
`
`to
`
`cancer
`
`cell
`
`line
`
`20
`
`25
`
`3O
`
`variable (Boon, et al.
`
`(2002)
`
`supra). Thus,
`
`in individuals
`
`whose
`
`tumors are NRKl, NRKQ—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 tiazofurin adenine dinucleotide in tumors.
`
`A yeast qnsl mutant was used to screen for natural
`
`sources of nicotinamide riboside wherein it was
`
`identified
`
`in an acid whey preparation of
`
`cow’s 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—specific
`
`in identifying NAD+ precursors.
`
`Because
`
`of
`
`the
`
`qnsl
`
`deletion,
`
`nicotinic
`
`acid
`
`and
`
`nicotinamide do not score positively in this assay. As
`
`the
`
`0017
`
`0017
`
`

`

`DC-0253
`
`-
`
`-14-
`
`PATENT
`
`factor
`
`from milk requires nicotinamide riboside kinase for
`
`growth,
`
`the nutrient
`
`is clearly nicotinamide riboside and
`
`not NMN or NAD+.
`
`A
`
`revised metabolic
`
`scheme
`
`for NAD+,
`
`incorporating
`
`Nrkl homologs and the nicotinamide riboside salvage pathway
`
`is shown in Scheme 2 wherein double arrows depict metabolic
`
`steps
`
`common
`
`to yeast
`
`and humans
`
`(with yeast gene names)
`
`and
`
`single arrows depict
`
`steps unique
`
`to humans
`
`(PBEF,
`
`nicotinamide
`
`phosphoribosyltransferase)
`
`and
`
`yeast
`
`(Pncl,
`
`10
`
`nicotinamidase).
`
`Nmal,2
`Qnsl
`Nma1,2
`Bnal—6
`_—_> NaMN Z) NaAD+ :> NAD+ (Z NMN
`H.s.
`
`Nptl
`
`Na
`
`S.c. Pncl
`N
`
`Sir2 fl PBEF
`
`Nrkl
`
`Nr
`
`i Scheme 2‘
`
`A difference between humans
`
`and yeasts
`
`concerns
`
`the
`
`organisms'
`
`uses of nicotinamide 'and nicotinic acid,
`
`the
`
`compounds
`
`that were
`
`co—identified
`
`as
`
`anti-black tongue
`
`a
`
`15
`
`20
`
`factor
`
`(Elvehjem,
`
`et al.
`
`(1938)
`
`supra). Humans
`
`encode
`
`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) Mbl. Cell Biol.
`
`14:1431—1437).
`
`In
`
`contrast, S. cerevisiae lacks a homolog of nadV and instead
`
`has
`
`a homolog of
`
`the E. coli pncA gene,
`
`termed PNCl,
`
`that
`
`converts nicotinamide to nicotinic acid for entry into the
`
`Preiss-Handler
`
`pathway
`
`(Ghislain,
`
`et
`
`al.
`
`(2002)
`
`Yeast
`
`192215—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
`
`25
`
`3O
`
`0018
`
`0018
`
`

`

`DC-0253
`
`-15-
`
`PATENT
`
`(1958)
`
`supra; Preiss
`
`and Handler
`
`(1958)
`
`supra). Reports
`
`that nicotinamidase had been purified from mammalian liver
`
`in the
`
`19603
`
`(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 nicotinamidase
`
`have
`
`not
`
`been
`
`identified
`
`and
`
`there
`
`is
`
`no
`
`compelling
`
`evidence
`
`nicotinamide
`
`and nicotinic
`
`acid
`
`are utilized as
`
`that
`
`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
`
`supplementation with
`
`nicotinamide
`
`riboside
`
`as
`
`third
`
`importable
`
`NAD+ precursor
`
`can be beneficial
`
`for certain
`
`10
`
`15
`
`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 nicotinic acid preparations, alone and in combination
`
`with
`
`HMG
`
`CoA
`
`reductase
`
`inhibitors,
`
`are
`
`effective
`
`in
`
`controlling low—density lipoprotein cholesterol,
`
`increasing
`
`high—density
`
`lipoprotein
`
`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
`
`term