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WO2007136744A1 - Structure cristalline d'un substrat complexe de nampt/pbef/visfatine - Google Patents

Structure cristalline d'un substrat complexe de nampt/pbef/visfatine Download PDF

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WO2007136744A1
WO2007136744A1 PCT/US2007/011898 US2007011898W WO2007136744A1 WO 2007136744 A1 WO2007136744 A1 WO 2007136744A1 US 2007011898 W US2007011898 W US 2007011898W WO 2007136744 A1 WO2007136744 A1 WO 2007136744A1
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nampt
agent
crystal
nmn
nad
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Tao Wang
Cynthia Wolberger
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Johns Hopkins University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides

Definitions

  • the invention relates generally to the crystal structure of nicotinamide phosphoribosyltransferase (Nampt), and more particularly to the identification of inhibitors of Nampt for treatment of Nampt-associated disorders.
  • Nampt nicotinamide phosphoribosyltransferase
  • Nicotinamide adenine dinucleotide (NAD) and its derivative compounds are essential coenzymes in cellular redox reactions in all living organisms. NAD also participates in a number of important signaling pathways in mammalian cells, including poly(ADP-ribosyl)ation in DNA repair, mono-ADP-ribosylation in the immune response and in G protein-coupled signaling, and synthesis of cyclic ADP-ribose and nicotinate adenine dinucleotide phosphate (NAADP) in intracellular calcium signaling. Recently, it has also been demonstrated that NAD and its derivatives play an important role in transcriptional regulation.
  • NAD biosynthesis is markedly different from that of yeast and invertebrates. It has long been known that mammals predominantly use nicotinamide rather than nicotinic acid as a precursor for NAD biosynthesis. Despite significant numbers of studies about NAD, biosynthesis in the 1950's and 1960's, the mammalian NAD biosynthesis enzymes have remained poorly characterized. The first mammalian enzyme to be fully characterized was the human nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat), an enzyme required to convert nicotinamide mononucleotide (NMN) and NaMN to NAD in the nucleus.
  • Nmnat human nicotinamide/nicotinic acid mononucleotide adenylyltransferase
  • nicotinamide phosphoribosyltransferase Nampt
  • nicotinamide mononucleotide adenylyltransferase Nmnat
  • Nampt has very ancient origins as an NAD biosynthetic enzyme. The entire pyrdine nucleotide salvage cycle containing Nampt, Nmnat, and Sir2 homologues exists in the vibriophage. Despite its ancient origins, Nampt has a peculiar phylogenetic distribution. No other organisms between bacteria and vertebrates have obvious homologs of Nampt, except for one sponge species, and the homology of Nampt proteins between bacteria and vertebrates is unusually high.
  • PBEF is indeed mammalian Nampt.
  • Nampt/PBEF has also been re-identified as a "new visceral fat-derived hormone" named visfatin.
  • visfatin was found to exert insulin-mimetic effects in cultured cells and to lower plasma glucose levels in mice by binding to and activating the insulin receptor.
  • the physiological relevance of visfatin remains controversial because its plasma concentration is 40 to 100-fold lower than that of insulin.
  • the present invention relates to nicotinamide phosphoribosyltransferase (Nampt) and the identification of the crystal structure of a complex thereof. Accordingly, the invention provides a crystal of Nampt, for example, having the X-ray diffraction characteristics and the three dimensional atomic coordinates exemplified as set forth in Table 1. Also provided is a crystal of Nampt that is co-crystallized or soaked with another molecule, such as a nicotinamide-based molecule.
  • An exemplary nicotinamide-based molecule includes, but is not limited to, nicotinamide mononucleotide (NMN).
  • the invention also provides a crystal of a complex comprising Nampt and NMN that effectively diffracts X-rays for the determination of the atomic coordinates of the complex to a resolution of about 1.94 A.
  • the invention also provides a method of analyzing a Nampt-NMN complex.
  • the method employs X-ray crystal lographic diffraction data from the Nampt-NMN complex and a three-dimensional structure of Nampt-NMN, to generate a Fourier electron density map of the complex, the three-dimensional structure being defined by atomic coordinate data according to Table 1.
  • the invention further provides a computer system intended to generate structures and/or perform rational drug design for Nampt and Nampt-substrate complexes.
  • the system includes either (a) atomic coordinate data according to Table 1 , the data defining the three-dimensional structure of Nampt, or (b) structure factor data for Nampt, wherein the structure factor data is derivable from the atomic coordinate data of Table 1.
  • the invention further provides a method of growing a crystal of a complex comprising Nampt and NMN.
  • the method includes contacting Nampt and NMN under conditions in which a Nampt-NMN complex is formed and thereafter growing a crystal by hanging dropping of the Nampt-NMN complex using a reservoir solution at a temperature from about 18°C.
  • the method further includes replacing the reservoir solution with a cryoprotectant and flash-freezing the crystal in liquid nitrogen.
  • the reservoir solution includes about 0.1 M HEPES pH 7.8, about 0.1 M Na acetate, and about 14.5% PEG4000 in a 1: 1 ratio.
  • the cryoprotectant may be 20% glycerol.
  • the invention further provides a method of identifying an agent that interacts with Nampt by the crystal structure of Nampt-NMN and one or more molecular modeling techniques to select or design a potential agent.
  • the agent is then contacted with Nampt to determine if the agent binds to the active site or any inactive site of Nampt.
  • Agents useful in the methods of the invention include, but are not limited to, small molecules, peptides, inositol-based molecules, and nicotinamide-based molecules.
  • Molecular modeling techniques useful in the methods of the invention include, but are not limited to graphic molecular modeling and computational chemistry.
  • the method further includes screening for Nampt activity. The screens include both competitive and noncompetitive type assays against known agents.
  • the invention further provides a method of identifying an agent compound which modulates Nampt activity.
  • the method includes providing a candidate agent compound and forming a complex of Nampt and NMN and the candidate agent compound.
  • the method may include forming a complex of Nampt and the candidate agent compound. Analysis of the complex by X-ray crystallography or NMR spectroscopy will determine the ability of the candidate agent compound to interact with Nampt. In one embodiment, the interaction is at the active site. In another embodiment, the modulation is inhibition. In another embodiment, it will be appreciated by those skilled in the art that NMN may be substituted for another nicotinamide compound.
  • the invention also provides a method of identifying an agent that inhibits Nampt by using the crystal structure of Nampt-NMN and one or more molecular modeling techniques to select or design a potential agent.
  • the agent is then contacted with a cell or cell extract containing Nampt and NMN. Any detected increase in NAD+ level in the cell as compared to the NAD+ level in the cell prior to contacting with the agent is indicative of an agent that inhibits Nampt.
  • the invention also provides a method of treating an Nampt-associated disorder by administering to a subject having an Nampt-associated disorder, an agent identified by any of the methods of the invention.
  • the invention further provides a method of diagnosing an Nampt-associated disorder in a subject.
  • the method includes contacting a test sample from the subject and a corresponding normal sample with an agent identified by any of the methods of the present invention. Thereafter, the Nampt, NAD+, and/or NMN level in the test sample is compared to the Nampt, NAD+, and/or NMN level in the normal sample to detect a greater increase in the respective level in the test sample as compared to the respective level in the normal sample. Any greater increase in Nampt, NAD+, and/or NMN level in the test sample is indicative of an Nampt-associated disorder in the subject.
  • the invention further provides a method for monitoring a therapeutic regimen for treating a subject having an Nampt-associated disorder, comprising determining a change in Nampt, NAD+, and/or NMN levels during the treatment of the invention.
  • the invention further provides an agent identified by any of the methods of the invention.
  • Nampt-associated disorders include cancer, multiple sclerosis, neurodegeneration or Huntington's disease.
  • the current invention relates to the crystal structure of Nampt/PBEF/visfatin, including the biochemical function of both the intra- and extracellular forms of this protein.
  • the structures of the mouse Nampt apoenzyme and the product complex of Nampt with NMN demonstrate that Nampt is a dimeric type II phosphoribosyltransferase.
  • the enzyme contains dual active sites that lie at the enzyme's extensive dimer interface. Thus, mutation at the dimer interface is shown to destabilize the dimer and reduce its enzymatic activity, suggesting that dimerization is required for Nampt enzymatic activity.
  • Nampt In contrast with nicotinic acid phosphoribosyltransferases and quinolinic acid phosphoribosyltransferase, Nampt has an aspartic acid in its active pocket that forms hydrogen bonds to the amide group of nicotinamide. This may govern the specificity for nicotinamide over nicotinic acid or quinolinic acid. In addition to the known activity of Nampt in converting nicotinamide and PRPP into NMN and PPi, Nampt was found to also hydrolyzes ATP and autophosphorylates itself.
  • Figures IA- 1C are pictorial diagrams showing major NAD biosynthetic pathways in yeast and mammals and the enzymatic reaction of nicotinamide phosphoribosyltransferase (Nampt/PBEF/visfatin).
  • NAD nicotinamidase
  • Nptl nicotinic acid phosphoribosyltransferase
  • Nmal and Nma2 nicotinic acid mononucleotide adenylyltransferase 1 and 2
  • Qnsl NAD synthetase
  • NIC nicotinamide
  • NA nicotinic acid
  • NaMN nicotinic acid mononucleotide.
  • B The NAD biosynthetic pathway from nicotinamide in mammals. The pathways from tryptophan, nicotinic acid, and nicotinamide riboside are not shown in this scheme.
  • Nampt nicotinamide phosphoribosyltransferase
  • Nmnat nicotinamide mononucleotide adenylyltransferase
  • NMN nicotinamide mononucleotide.
  • C The reaction catalyzed by Nampt.
  • PPi inorganic pyrophosphate.
  • FIGS 2A-2D are pictorial diagrams showing structure comparison of mouse Nampt with NMN (A), Thermoplasma acidophilum NaPRTase (TaNaPRTase) with NaMN (PDB entry IYTK)(B), Mycobacterium tuberculosis QaPRTase with NaMN (IQPN)(C) and yeast Nptl (D).
  • Nptl forms a closed monomer, while the remaining enzymes are dimers.
  • FIGS 3A and 3B are pictorial diagrams showing active sites of Nampt complexed with NMN.
  • A One monomer is colored violet and the other green. D219, which forms hydrogen bonds with the nicotinamide (NAM) amide and is found only in Nampt, is designated by italics. The remaining side chains shown are active site residues in Nampt that are also conserved in TaNaPRTase and in Nptl . S280 is replaced with threonine in the other enzymes, while Y281 is either tyrosine or phenylalanine; all other residues are identical in Nampt, TaNaPRTase and Nptl.
  • B Schematic two-dimensional Ligplot diagram of the interactions between NMN and two monomers denoted by A and B. Dashed lines and associated numbers represent hydrogen bonds and their distances, and red semicircles exhibit van der Waals interactions.
  • FIGS 4A and 4B are pictorial and graphical diagrams showing that dimerization is important for Nampt activity.
  • A The Structure of Nampt dimerization interface. This figure shows the center of the interface where Ser 199, Ser200, and Thr203 from one monomer form tight hydrogen bonds with the corresponding residues from the other monomer.
  • B Gel-filtration analysis on the sizes of His-tagged Nampt proteins including wild type (upper), S199D (middle) and S200D (lower). The proteins were applied onto SP200 10/300 GL analytical sizing column using buffer of 2OmM Tris (pH 8.0), 10OmM NaCl, 5mM DTT, and 10% glycerol. The sizes of the proteins were calculated based on a standard curve obtained under the same conditions. Wild type His-tagged Nampt is a dimer; S199D is a mixture of monomer and dimer; S200D is a monomer.
  • FIGS 5A-5C are pictorial diagrams showing that high levels of intra- and extracellular Nampt are produced by differentiated brown adipocytes (A), but not by differentiated white adipocytes (B).
  • Intracellular and extracellular Nampt iNampt and eNampt were analyzed in cell extracts and culture supernatants, respectively, during differentiation of (A) HIB-IB brown preadipocytes and (B) 3T3-L1 white preadipocytes.
  • Middle panels 20 ⁇ l of each culture supernatant collected under regular culture conditions was analyzed for eNampt at the indicated days.
  • Lower panels Differentiated HIB-IB and 3T3-L1 cells were cultured in media without serum but supplemented with differentiation inducers from day 6 to day 8, and the culture supernatant was collected at day 8 and analyzed for eNampt.
  • 10 ⁇ g of the cell extract from Nampt-overexpressing NIH3T3 fibroblasts (Namptl) was loaded as a reference for iNampt.
  • C eNampt in mouse plasma has a slightly larger molecular weight than iNampt. Plasma samples were collected from three C57BL/6 mice.
  • FIGS. 6A-6E are pictorial and graphical diagrams showing that intra- and extracellular Nampt produced and secreted by differentiated HIB-IB brown adipocytes have high Nampt enzymatic activity.
  • A The expression of Nampt-FLAG was confirmed with both anti-Nampt and anti-FLAG antibodies in extracts from undifferentiated HIB-IB cells compared to a vector-only control.
  • the Nampt-FLAG protein is secreted by differentiated Nampt-FLAG HIB-IB cells. The protein was immunoprecipitated with an anti-FLAG antibody from 8 ml of each culture supernatant and blotted with an anti-Nampt antibody.
  • C eNampt-FLAG co-immunoprecipitates with untagged eNampt from culture media. The eNampt-FLAG protein was immunoprecipitated with an anti-FLAG antibody from 8 ml of each culture supernatant and blotted with either the same antibody or an anti- Nampt antibody. The culture supernatants of the vector-transfected cells were used as a control.
  • Figures 7A and 7B are pictorial and graphical diagrams showing autophosphorylation of His-tagged recombinant Nampt.
  • A In a 10 ⁇ l reaction, 10 ⁇ g of His-tagged Nampt was incubated with 1 mM cold ATP and 0.3 ⁇ M (lane C) or 1.3 ⁇ M (lane D) ( ⁇ - 32 ⁇ ) ATP (10 ⁇ Ci/ ⁇ L, 3000 Ci/mmol) at 25°C for 1 hr. Lanes A and B show controls without ( ⁇ - 32 p) ATP and the enzyme, respectively. Incorporation of ( ⁇ - 32 p) radiolabel was detected by autoradiography after SDS-PAGE analysis of the reaction mixtures.
  • Figure 7B shows ATP hydrolysis catalyzed by Nampt.
  • 5 ⁇ l 2Ox buffer was mixed with 20 ⁇ l MESG, 1 ⁇ l DTT (200 mM), 1 ⁇ l purine nucleotide phosphorylase (PNP), 3 ⁇ l Nampt (13 mg/ml), 3 ⁇ l ATP (10 mM), and 67 ⁇ l water.
  • the reaction mixture was scanned at OD360nm for the detection of inorganic phosphate. Inorganic phosphate is converted to 2-amino-6-mercapto-7 methyl purine that has a maximum absorbance at 360 nm.
  • the upper figure shows the scan for a control reaction without Nampt.
  • the middle figure shows the scan for a control reaction without ATP.
  • the bottom figure shows the positive ATP hydrolysis reaction.
  • FIGS 8A-8C are pictorial and graphical diagrams showing tissue distribution of Nampt and its fasting-induced increase in brown adipose tissue.
  • A Distribution of Nampt in mouse tissues. 22.5 ⁇ g of each tissue extract from a C57BL/6 mouse was analyzed by Western blotting with Nampt- and actin-specific antibodies. WAT, white adipose tissue; BAT, brown adipose tissue.
  • B Fasting increases the amount of Nampt in BAT. 45 ⁇ g of each BAT extract from fed and fasted C57BL/6 mice was analyzed by Western blotting.
  • C Quantitation of the Western blot results shown in B. *, PO.05.
  • Figure 9 is a pictorial diagram showing that intracellular Nampt-FLAG (iNampt- FLAG) co-immunoprecipitates with untagged iNampt from cell extracts of differentiated HIB-IB brown adipocytes.
  • the iNampt-FLAG protein was immunoprecipitated with an anti-FLAG antibody and blotted with either the same antibody or an anti-Nampt antibody.
  • the cell extracts of the vector-transfected cells were used as a control.
  • Figure 1OA is a graphical diagram showing the results of ATP hydrolysis catalyzed by Nampt.
  • ATP hydrolysis assay was performed using EnzCheck Phosphate Assay Kit (Molecular PROBES). According to the assay, in the presence of inorganic phosphate, substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is converted enzymatically by purine nucleotide phosphorylase (PNP) to 2-amino-6-mercapto- 7 methyl purine, which has a maximum absorbance at 360nm.
  • ESG 2-amino-6-mercapto-7-methylpurine ribonucleoside
  • PNP purine nucleotide phosphorylase
  • 25 ⁇ l 2Ox buffer (1.0 M Tris-HCl, 20 mM MgCl 2 , pH 7.5) was mixed with 365 ⁇ l water, 100 ⁇ l MESG (1 mM), 1 ⁇ l DTT (IM) 5 5 ⁇ l PNP, 10 ⁇ l Nampt (13 mg/ml), and 10 ⁇ l ATP (10 mM).
  • the reaction mixture was scanned at OD 360nm for the detection of inorganic phosphate.
  • the blue line shows the scan for the control reaction without ATP.
  • the brown line shows the positive ATP hydrolysis reaction.
  • Figure 1OB is a pictorial diagram showing the results of autophosphorylation of His-tagged Nampt.
  • 10 ⁇ g of His- tagged Nampt (Lane C), Nampt H247E (Lane D) or H247A (Lane E) respectively was incubated with 0.2 mM ( ⁇ - 32 P) ATP (2 ⁇ Ci/ ⁇ l, 3 Ci/mmol, Amersham) at O 0 C for 10 min.
  • the present invention is based on the identification of the crystal structure of nicotinamide phosphoribosyltransferase (Nampt) and Nampt complexes, which are useful in the identification of inhibitors of Nampt. Inhibition of Nampt reduces or halts synthesis of nicotinamide mononucleotide (NMN) and nicotinamide adenine dinucleotide (NAD4-).
  • Nampt nicotinamide phosphoribosyltransferase
  • Nampt nicotinamide phosphoribosyltransferase
  • Nicotinamide adenine dinucleotide is an essential coenzyme that is also consumed by Sir2 enzymes. Sir2-mediated deacetylation and ADP ribosylation both produce nicotinamide from NAD+ in the first step of the reaction. Nicotinamide is then used as a precursor for NAD+ synthesis through the NAD+ salvage pathway. Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the mammalian NAD+ salvage pathway, which is the conversion of nicotinamide and phosphoribosylpyriphosphate (pRPP) into nicotinamide mononucleotide (NMN).
  • NAD+ nicotinamide and phosphoribosylpyriphosphate
  • Nampt The human nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nmnat) converts NM into NAD+ in the nucleus.
  • the inventors previously showed that Nampt is the rate-limiting component in the NAD+ biosynthesis pathway and regulates the function of the mammalian Sir2 ortholog, Sirtl, in mammalian cells.
  • Human Nampt was originally isolated as a presumptive cytokine named pre-B cell colony-enhancing factor (PBEF), and other studies showed that PBEF is indeed mammalian Nampt.
  • PBEF pre-B cell colony-enhancing factor
  • Nampt/PBEF was identified as a "new visceral fat-derived hormone" named visfatin which was found to exert insulin-mimetic effects in cultured cells and lower plasma glucose levels in mice by binding to and activating the insulin receptor.
  • visfatin a new visceral fat-derived hormone
  • NAD biosynthesis pathways have been characterized mainly in the prokaryotes, Escherichia coli and Salmonella typhimurium, and more recently in yeast.
  • NAD is synthesized by the de novo pathway via quinolinic acid and by the salvage pathway via nicotinic acid.
  • yeast the de novo pathway begins with tryptophan, which is converted to nicotinic acid mononucleotide (NaMN) through six enzymatic steps and one non-enzymatic reaction.
  • NaMN nicotinic acid mononucleotide
  • the de novo pathway converges with the salvage pathway at the step of NaMN synthesis from quinolinic acid by quinolinic acid phosphoribosyltransferase (QaPRTase), which is encoded by the QPTI gene.
  • the salvage pathway begins with the breakdown of NAD into nicotinamide and ADP- ribose, which is catalyzed in yeast mainly by the Sir2 proteins ( Figure IA). Nicotinamide is then deamidated to nicotinic acid by nicotinamidase, which is encoded by the PNCl gene (Anderson, R.M., et al., Nicotinamide and PNCI govern lifespan extension by calorie restriction in Saccharomyces cerevisiae.
  • Nicotinic acid phosphoribosyltransferase (Npt), encoded by the NPTl gene, converts nicotinic acid to NaMN, which is eventually converted back to NAD through the sequential reactions of nicotinamide/nicotinic acid mononucleotide adenylyltransferase (encoded by the NMAl and/or NMA2 genes) and NAD synthetase (encoded by the QNSl gene) ( Figure IA).
  • Npt Nicotinic acid phosphoribosyltransferase
  • Nampt which allows for a more direct pathway to NAD biosynthesis from nicotinamide (Figure IB)
  • Figure IB clearly distinguishes NAD biosynthesis in vertebrates from that in invertebrates.
  • the gene encoding human Nampt was originally isolated as a presumptive cytokine named pre-B cell colony-enhancing factor (PBEF), although the PBEF function has never been reconfirmed.
  • PBEF pre-B cell colony-enhancing factor
  • Nampt which synthesizes NMN from nicotinamide and phosphoribosylpyrophosphate ( Figure 1 C)
  • Figure 1 C phosphoribosylpyrophosphate
  • the present invention provides the crystal structure of mouse Nampt, which is 95% identical to human Nampt. Also provided is the crystal structure of Nampt in complex with its substrate NMN.
  • disorder refers to any condition associated with altered levels of Nampt in a subject.
  • disorder refers to any condition associated with altered levels of NAD+ in a subject.
  • Nampt-associated disease or “Nampt-associated disorder” is used herein to refer specifically to a condition in which the level of Nampt is increased or decreased above the level of Nampt in a corresponding normal cell.
  • Nampt-associated disorders include, but are not limited to, cancer, multiple sclerosis, neurodegeneration and Huntington's disease.
  • Inhibition of Nampt can therefore be used to target all processes mediated by human SirTl, SirT2, SirT4, SirT5, SirT6, and SirT7 enzymes, all of which require NAD+ for activity, and are involved in the mammalian NAD+ biosynthetic pathway.
  • Nampt/PBEF/visfatin is a dimeric type II phosphoribosyltransferase enzyme that exhibits several important features.
  • two active sites lie at the dimer interface ( Figure 2A), suggesting that dimerization of Nampt is required for its enzymatic activity.
  • This notion was confirmed by the mutagenesis of two adjacent serine residues (S199 and S200) that, along with T203, mediate a central network of intermolecular hydrogen bonds between two monomers ( Figure 4 and Table 2).
  • Nampt Co-immunoprecipitation of tagged and untagged Nampt proteins from both cell extracts and culture supernatants also support that dimerization occurs intra- and extracellularly under more physiological conditions (Figure 6 and Figure 9).
  • the active site of Nampt contains features that are highly conserved among both dimeric and monomeric enzymes of this class, such as bacterial and yeast NaPRTases. conserveed key residues of these enzymes ( Figure 3) contribute to the formation of an acidic pocket structure that holds the reaction product.
  • Nampt becomes autophosphorylated in its enzymatic reaction ( Figures 7 A and 7B), suggesting that the mammalian Nampt enzyme likely shares a highly similar enzymatic mechanism with the dimeric bacterial NaPRTase and the monomelic yeast Nptl enzymes, whose reactions also require autophosphorylation (Gross, J., et al., Energy coupling in Salmonella typhimurium nicotinic acid phosphoribosyltransferase: identification of His-219 as site of phosphorylation.
  • Nampt While the mammalian Nampt enzyme shares structural and mechanistic features similar to other members in this enzyme family, Nampt also exhibits a unique biological feature, namely, the existence of intra- and extracellular forms in mammals (iNampt and eNampt). Both forms are highly induced during the differentiation of brown preadipocytes (Figure 5), consistent with the observed high expression of Nampt in mouse brown adipose tissue ( Figures 8A-8C).
  • the histidine residue is located in an acidic pocket amidst a cluster of two conserved aspartic acid residues ( Figure 3A). Phosphorylation of the histidine would give rise to additional negative charge in this region, which could be used to promote cleavage of pyrophosphate from PRPP by stabilizing formation of a positively charged oxacarbenium intermediate that could then react with nicotinamide, forming NMN. Either a purely dissociative mechanism or one in which the intermediate had partial oxacarbenium character could be promoted in this way, by analogy with uracil DNA glycosylase (Werner, R.M.
  • the present invention provides the unique extracellular form of Nampt, which had been identified as a presumptive cytokine named PBEF (Samal, B., et al., Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. MoI. Cell. Biol. 14, 1431-1437 (1994)) or a visceral fat- derived insulin-mimetic hormone named visfatin (Fukuhara, A., et al., Visfatin: a protein secreted by visceral fat that mimics the effects of insulin. Science 307, 426-430 (2005)), and functions as an enzymatically active dimer that has significantly higher activity than the intracellular form (Figure 6E).
  • PBEF presumptive cytokine
  • visfatin a visceral fat- derived insulin-mimetic hormone
  • iNampt is the rate-limiting enzyme in the NAD biosynthetic pathway from nicotinamide in mammalian cells (Revollo, J.R., et al., The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J. Biol. Chem. 279, 50754-50763 (2004)), one interesting idea is that the main physiological function of eNampt may be to catalyze NAD biosynthesis in extracellular compartments as well.
  • the present invention provides the crystal structure of Nampt/BEF/visfatin in the presence and absence of the product, NMN.
  • the structure of the 491 -amino acid mouse enzyme was determined at a resolution of 1.94 A.
  • wild type Nampt contains only one methionine in addition to the initiator methionine
  • a quadruple mutant containing four additional methionine residues and possessing near-wild type enzymatic activity was used to solve the apoenzyme structure by MAD phasing.
  • the structure of wild type Nampt bound to NMN was determined by molecular replacement at a resolution of 2.1 A from crystals grown in the presence of NMN. Both Nampt crystals contain a dimer in the asymmetric unit and the protein is also dimeric in solution.
  • Nampt belongs to the dimeric class of type II phosphoribosyltransferases ( Figure 2). While Nampt bears no significant sequence identity to other phosphoribosyltransferase enzymes, a search of the structure database with the DALI server identified Nampt as a structural homologue of the dimeric nicotinic acid phosphoribosyltransferase (NaPRTase) from Thermoplasma acidophilum (Z-score 27.4), which shares 11% sequence identity with Nampt.
  • Nampt forms a dimer with an extensive intermolecular interface ( Figure 2A) 5 burying a total surface area of 8,077 A 2 (as calculated by summing the surface area on each monomer that is buried by complex formation).
  • Nampt also bears some structural similarity to the yeast NaPRTase, called Nptl, a closed monomer that shares 11% sequence identity with Nampt and that had previously been shown to contain structural similarity with QaPRTase.
  • Figure 2 shows a side-by-side comparison of the structures. In contrast with TaNaPRTase and QaPRTase crystals, which contain a trimer of dimers, there is no evidence that mouse Nampt forms higher-order oligomers.
  • the present invention provides a method of identifying an agent that inhibits Nampt activity by using the crystal structure provided herein and molecular modeling techniques to select or design a potential agent.
  • Candidate inhibitors identified according to the methods of the invention are characterized by their suitability for binding to a particular binding site or sites.
  • the binding cavity can therefore be regarded as a type of binding site framework or negative template with which the candidate inhibitors correlate in the manner described herein.
  • a potential modulator of Nampt activity can be examined through the use of computer modeling using a docking program such as SYBYL., GRAM, DOCK, or AUTODOCK (Tripos Inc., Walters et al., Drug Discovery Today, Vol.3, No.4, (1998), 160-178, and Dunbrack et al., Folding and Design, 2, (1997), 27-42) to identify candidate inhibitors of Nampt.
  • This procedure can include computer fitting of candidate inhibitors to Nampt to ascertain how well the shape and the chemical structure of the candidate inhibitor will bind to the enzyme. Binding may be either at the active site or elsewhere on the enzyme.
  • the invention provides a system, particularly a computer system, intended to generate structures and/or perform rational drug design for Nampt and Nampt-substrate complexes, the system containing either (a) atomic coordinate data according to Table 1 , the data defining the three-dimensional structure of Nampt, or (b) structure factor data for Nampt, wherein the structure factor data is derivable from the atomic coordinate data of Table 1.
  • the present invention provides computer readable media with either (a) atomic coordinate data according to Table 1, the data defining the three-dimensional structure of Nampt, or (b) structure factor data for Nampt, wherein the structure factor data is derivable from the atomic coordinate data of Table 1, or (c) structure factor data for Nampt, wherein the structure factor data is derived from the X-ray diffraction of Nampt crystals.
  • test agent and test compound refer to any compound that is being examined for the ability to inhibit Nampt activity.
  • a test agent and an agent that modulates Nampt activity identified by a method of the invention
  • the agent may have insulin-like properties.
  • Peptides also can be useful as test agents.
  • peptide is used broadly herein to refer to a molecule containing two or more amino acids or amino acid analogs (or modified forms thereof) linked by peptide bonds or a single amino acid or amino acid analog (or modified forms thereof).
  • peptide test agents can contain one or more D-amino acids and/or L-amino acids; and/or one or more amino acid analogs, for example, an amino acid that has been derivatized or otherwise modified at its reactive side chain.
  • antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. Antibodies are characterized, in part, in that they specifically bind to an antigen, particularly to one or more epitopes of an antigen.
  • a screening assay of the invention can further include a step of determining an amount by which the agent increases or decreases Nampt, NAD+, and/or NMN levels in a subject.
  • a method of the invention can further include determining an amount by which the agent increases NAD+ levels above a basal level in a subject, which should ideally approach the level of a corresponding normal sample.
  • Such an agent can be identified by measuring the amount of NAD+ in a single sample both before adding the test agent and after adding the test agent, or can be identified for example, using two samples, wherein one sample serves as a control (no test agent added) and the other sample includes the test agent.
  • a method of the invention provides a means to obtain agents or panels of agents that variously modulate Nampt activity.
  • a "corresponding normal sample” is any sample taken from a subject of similar species that is considered healthy or otherwise not suffering from an Nampt-associated disorder. Such corresponding normal cells can, but need not be, from an individual that is age-matched and/or of the same sex as the individual providing the cells being examined.
  • a normal/standard level of NAD+ denotes the level of NAD+ present in a sample from the normal sample.
  • a normal level of NAD+ can be established by taking samples of body fluids or cell extracts from a statistically significant number of normal healthy subjects, preferably human, and determining the level of NAD+ to obtain an average level. Levels of NAD+ in a subject, control, and disease samples can be compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • subject refers to any individual or patient to which the subject methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.
  • rodents including mice, rats, hamsters and guinea pigs
  • cats dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc.
  • primates including monkeys, chimpanzees, orangutans and gorillas
  • the terms “sample” and “biological sample” refer to any sample suitable for the methods provided by the present invention.
  • the biological sample of the present invention is a tissue sample, e.g., a biopsy specimen such as samples from a needle biopsy.
  • the biological sample of the present invention is a sample of bodily fluid, e.g., serum, plasma, cerebral spinal fluid, sputum, urine, and ejaculate.
  • the term "computer system” is meant to refer to the hardware means, software means and data storage means used to analyze atomic coordinate data.
  • the minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualize structure data.
  • the data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are computer workstations running Linux available, from Dell, Hewlett Packard, IBM, etc, or microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows NT or IBM OS/2 operating systems.
  • computer readable media refers to any media that can be read and accessed directly by a computer e.g., so that the media is suitable for use in the above- mentioned computer system.
  • the media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • terapéuticaally effective amount or “effective amount” means the amount of a compound or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • compositions when used in reference to a carrier, means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the terms “reduce” and “inhibit” are used together because it is recognized that, in some cases, a decrease, for example, in Nampt activity can be reduced below the level of detection of a particular assay. As such, it may not always be clear whether the activity is “reduced” below a level of detection of an assay, or is completely “inhibited”. Nevertheless, it will be clearly determinable, following a treatment according to the present methods, that the level of Nampt is at least reduced from the level before treatment.
  • the invention provides a method for treating an Nampt- associated disorder in a subject.
  • the method includes administering to an individual or a cell, an inhibitor of Nampt activity, thereby changing the NAD+ and/or NMN level in the subject.
  • the methods of the invention are also useful for providing a means for practicing personalized medicine, wherein treatment is tailored to a subject based on the particular characteristics of the Nampt-associated disorder in the subject. Once disease is established and a treatment protocol is initiated, the methods of the invention may be repeated on a regular basis to evaluate whether the level of Nampt, NAD+ and/or NMN in the subject begins to approximate that which is observed in a normal subject.
  • the invention is also directed to methods for monitoring a therapeutic regimen for treating a subject having a Nampt-associated disorder.
  • a comparison of the level of Nampt, NAD+ and/or NMN prior to and during therapy indicates the efficacy of the therapy. Therefore, one skilled in the art will be able to recognize and adjust the therapeutic approach as needed.
  • All methods may further include the step of bringing the agents into association with a pharmaceutically acceptable carrier, which constitutes one or more accessory ingredients.
  • a pharmaceutically acceptable carrier useful for formulating an agent for administration to a subject are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.
  • a pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of the conjugate.
  • physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the physico-chemical characteristics of the therapeutic agent and on the route of administration of the composition, which can be, for example, orally or parenterally such as intravenously, and by injection, intubation, or other such method known in the art.
  • the pharmaceutical composition also can contain a second (or more) compound(s) such as a diagnostic reagent, nutritional substance, toxin, or therapeutic agent, for example, a cancer chemotherapeutic agent and/or vitamin(s).
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the subject's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • compositions containing the inhibitors of the invention will depend, in part, on the chemical structure of the molecule.
  • Polypeptides and polynucleotides are not particularly useful when administered orally because they can be degraded in the digestive tract.
  • methods for chemically modifying polynucleotides and polypeptides, for example, to render them less susceptible to degradation by endogenous nucleases or proteases, respectively, or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., Trends Anal. Chem. 14:83-92, 1995; Ecker and Crook, BioTechnology, 13:351-360, 1995).
  • Exemplary routes of administration include, but are not limited to, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraperitoneal Iy, intrarectally, intracisternally or, if appropriate, by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
  • the pharmaceutical composition can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment, or active, for example, using a nasal spray or inhalant, in which case one component of the composition is an appropriate propellant.
  • the total amount of an agent identified by the methods of the invention to be administered in practicing a treatment method of the invention can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which multiple doses are administered over a prolonged period of time.
  • One skilled in the art would know that the amount of the Nampt inhibitor to treat an Nampt-associated disorder in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose as necessary.
  • the formulation of the pharmaceutical composition and the routes and frequency of administration are determined, initially, using Phase I and Phase II clinical trials.
  • Nampt 491 -amino acid mouse nicotinamide phosphoribosyltransferase (Nampt) protein was determined in the presence and absence of the product, NMN. Since wild type Nampt contains only one methionine in addition to the initiator methionine, a quadruple mutant containing four additional methionine residues and near-wild type enzymatic activity was used to solve the structure by MAD phasing. The structure was determined at 1.94 A resolution from crystals grown in the presence of nicotinamide.
  • Expression plasmids A plasmid directing expression of the full-length Nampt protein with a cleavable N-terminal HiS 6 tag was constructed by sub cloning Nampt cDNA into a pET28a(+) vector through EcoRI site and Ndel sites. The plasmid expresses Nampt with three extra amino acid residues at N-terminus of Nampt following cleavage with thrombin to remove the Hise tag. Plasmids expressing protein with one or more mutations were generated using the Quick-Change mutagenesis kit (Stratagene).
  • the quadruple- substituted LM 1467 protein used for MAD phasing contained substitutions of Leul3, Leul77, Leu318, and Leu432 with methionine.
  • pCXN2-Nampt and pCXN2-Nampt-FLAG were used for Nampt expression in mammalian cells.
  • the C-terminally FLAG-tagged Nampt cDNA fragment was created by amplifying the previously cloned mouse Nampt cDNA with the following forward and reverse primers containing EcoRI sites: Forward, TTAGAATTCAGCCCATTTTTCTCCTTGCT (SEQ ID NO: 1); Reverse, AATGAATTCTACTTATCGTCGTCATCCTTGTAATCTCCTCCATGAGGTGCCACGT CCTGCTCGATGTT (SEQ ID NO: 2). All cDNA fragments were confirmed by sequencing.
  • Plasmid pET28a(+)-Nampt was transformed into BL21(DE3)pLysS cells, which were grown overnight at 37°C in 10 ml LB with 50 ⁇ g/ml kanamicin and 37 ⁇ g/ml chloramphenicol. The overnight cell culture was used to inoculate (1: 100) LB containing 50 ⁇ g/ml kanamicin and 37 ⁇ g/ml chloramphenicol. The culture was grown at 37°C until it reached an OD 6 0 0 of 0.8-0.9 and was then induced by addition of IPTG to a final concentration of 0.8 mM.
  • the temperature was lowered to 20 0 C and the cells were harvested following growth overnight.
  • the cell pellet was resuspended 20 mM Tris pH 8.0, 5 mM MgCl 2 , 500 mM NaCl, 5 mM DTT, 10% glycerol, and EDTA-free complete protease inhibitor cocktail (Roche) and lysed with a Microfluidizer. Cell debris was pelleted by centrifugation at 12,000 rpr ⁇ and the supernatant was applied to a 5 ml Histrap column, which retained the Hise-tagged Nampt.
  • the column was washed with 20 mM Tris pH 8.0, 500 mM NaCl, 5 mM DTT, and 10% glycerol and the Nampt eluted with a 0-500 mM imidazole gradient.
  • the eluted His-tagged Nampt was incubated overnight with 1 unit/ml thrombin in order to remove the His 6 -tag.
  • the protein was dialyzed against 20 mM Tris pH 8.0, 100 mM NaCl, 5 mM DTT and 10% glycerol and applied to a MonoQ HR 10/10 anion exchange column. The flow through of the MonoQ column was applied to a 5 ml Histrap column and Nampt was eluted with an imidazole gradient.
  • Nampt Fractions of Nampt with greater than 99% purity were pooled and dialyzed against 20 mM Tris pH 8.0, 100 Mm NaCl, 5 mM OTT and 10% glycerol. Nampt was concentrated to 10 mg/ml and stored at -80 0 C until use. Mutant proteins were purified by the same protocol as the wild type.
  • Se-Met protein expression and purification Overnight cultures were grown as described above, after which the cells were pelleted by centrifugation, resuspended in 2 ml of M9 minimal media, and added to 1 L M9 minimal media with kanamycin and chloramphenicol. The culture was grown at 37°C until it reached an OD 6O0 of 0.7, after which 100 mg each of lysine, phenylalanine, and threonine, 50 mg each of isoleucine, leucine, and valine; and 60 mg of selenomethionine were added to the cell culture. After 15 minutes, protein expression was induced by addition of 0.8 mM IPTG and the cells grown at 2O 0 C overnight. Purification of selenomethionine-substituted protein proceeded as for the native protein.
  • Crystals of the Se-Met Nampt in complex with NMN were grown at 18°C by the hanging drop method by mixing 5 mg/ml Se-Met Nampt with 10 mM added NMN and reservoir solution containing 0.1 M HEPES pH 7.8, 0.1 M Na acetate, and 14.5% PEG4000 in a 1: 1 ratio in a total initial drop volume of 2 ⁇ l . Crystals were transferred into a cryoprotectant solution containing reservoir solution plus 20% glycerol before flash- freezing by plunging a loop containing a crystal into liquid nitrogen. Diffraction data were collected at beamline GM/CA-CAT at the Advanced Photon Source and processed with HKL.
  • the structure of the apoenzyme was determined by the method of multi wavelength anomalous dispersion from crystals of selenomethionine-substituted LM 1467 protein grown in the presence of nicotinamide. Location of heavy atoms and phase calculation and refinement were carried out using the programs SOLVE and RESOLVE, which were used to produce an electron density map. An initial round of automated model building with ARP/wARP produced an initial model with about 90% of the amino acids correctly placed in the map. The structure was further refined with CNS and subjected to several rounds of manual model building and phase calculation using O and the CCP4 suite.
  • the final model consists of the non-crystallographic dimer containing residues 8-42, 53-483 of chain A and of chain B, two sulfate ions, and 315 water molecules. Since nicotinamide could not be located unambiguously in the map, this structure has been referred to as that of the apoenzyme.
  • the structure of the complex containing wild type protein bound to NMN was solved at 2.1 A by isomorphous replacement using phases calculated from the structure of the quadruply-substituted LM 1467 protein.
  • the final model of the NMN complex contains residues 8-42 and 53-482 of chain A, residues 9-42 and 53-483 of chain B, 2 NMN molecules and 163 water molecules.
  • the nicotinamide phosphoribosyltransferase activity of Nampt, Se-Met Nampt, and Se-Met LM 1467 was measured as previously described (Revollo, et al., 2004). For kinetics studies on His-tagged Nampt wild type and mutants (at least 95% pure after one-step Histrap purification).
  • Each reaction consists of 100 ⁇ l 20 mM Tris, pH 8.0, 50 mM NaCl, 2 mM DTT, and 12.5 mM MgCl 2 , 2.5 mM ATP, 0.03% BSA, 1.5% ethanol, 13 ⁇ g/ml His-tagged Nmnat, 100 ⁇ g/ml alcohol dehydrogenase, 500 ⁇ M PRPP, and varying concentrations of nicotinamide. Depending on different catalytic abilities, different amounts of His-Nampt were used for the wild type and the mutants.
  • Catalytic constants K m and Ic 08I were determined for His ⁇ -tagged mutant and wild type proteins by analyzing plots of initial rate measurements at 25°C under varying nicotinamide concentrations with the program, KaleidaGraph 3.6 (Synergy Software), which calculates constants K m and k cat automatically.
  • Size-exclusion chromatography Size exclusion chromatography was carried out on a SP200 global 10/300 GL column calibrated with standard protein markers. Native Nampt, His-tagged Nampt, His-tagged Nampt S199D, and His-tagged Nampt S200D were each applied to SP200 Global 10/300 GL run in 20 mM Tris, pH 8.0, 100 mM NaCl, 5 mM OTT, and 10% glycerol.
  • HIB-IB, 3T3-L1, NIH3T3, Hepal-6, and HEK293 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics.
  • HIB-IB preadipocytes were differentiated by adding 5 ⁇ g/ml insulin, 0.5 mM isobutylmethylxan thine, 1 ⁇ M dexamethasone, and 1 nM triiodothyronine (Sigma) to confluent cultures for 2 days and then re-feeding them with media containing 5 ⁇ g/mL insulin and 1 nM triodothyronine every other day for 6 more days.
  • 3T3-L1 preadipocytes were differentiated similarly, except that triodothyronine was absent in the media through the whole differentiation process.
  • Nampt- and Nampt-FLAG-overexpressing cells were established by transfecting cells with the pCXN2-Nampt and pCXN-Nampt-FLAG plasmids, respectively, and selecting the transfectants with 500 ⁇ g/mL G418 for 2 weeks.
  • HIB-IB culture supematants were collected after incubating differentiated HIB-IB cells overnight with DMEM without fetal bovine serum but supplemented with insulin and triodothyronine, filtered through a 0.22- ⁇ m PES membrane, concentrated with Amicon Ultra- 15 columns (Millipore, MA), and mixed with anti-FLAG beads for 3-4 hours at 4 0 C. Immunoprecipitates were washed twice with the IP buffer and twice with PBS.
  • the structure of the 491-amino acid mouse nicotinamide phosphoribosyltransferase (Nampt) protein was determined in the presence and absence of the product, NMN. Since wild type Nampt contains only one methionine in additional to the initiator methionine, a quadruple mutant containing four additional methionine residues and near-wild type enzymatic activity was used to solve the structure by MAD phasing (see Methods). The structure was determined at 1.94 A resolution from crystals grown in the presence of nicotinamide. However, since it was not possible to locate unambiguous electron density corresponding to nicotinamide, this structure is referred to as that of the apoenzyme.
  • the structure of wild type Nampt bound to NMN was determined by isomorphous replacement at a resolution of 2.1 A from crystals grown in the presence of NMN.
  • the crystals of the apoenzyme and the NMN complex are isomorphous and contain a dimer in the asymmetric unit.
  • Table 1 summarizes data collection and refinement statistics. Coordinates have been deposited with accession codes 2H3B (Nampt apoenzyme) and 2H3D (Nampt + NMN).
  • Table 1 Data collection, phasing and refinement statistics
  • Nampt belongs to the dimeric class of type II phosphoribosyltransferases ( Figures 2A-2D). While Nampt bears no significant sequence identity with other phosphoribosyltransferase enzymes ( ⁇ 12% identity), a search of the strcture database with the DALI server identified Nampt as a structural homologue of the dimeric nicotinic acid phosphoribosyltransferase (NaPRTase) from Thermoplasma acidophilum (PDB: IYTK, Z-score 27.4) (Rychlewski, L., et al., Comparison of sequence profiles.
  • PDB Thermoplasma acidophilum
  • Nampt forms a dimmer with an extensive intermolecular interface ( Figure 2A), burying a total surface area of 8,077 A 2 (as calculated by summing the surface area on each monomer that is buried by complex formation). Nampt also bears some structural similarity to the yeast NaPRTase, called Nptl, a closed monomer that shares 11% sequence identity with Nampt and that had previously been shown to contain structural similarity with QaPRTase (Chappie, et al., 2005).
  • Figure 2 shows a side-by-side comparison of the structures.
  • NaPRTase crystals Shin, D.H., et al., Crystal structure of a nicotinate phosphoribosyltransferase from Thermoplasma acidophilum. J Biol Chem 280, 18326-35 (2005), which contain a trimer of dimers, there is no evidence that mouse Nampt forms higher-order oligomers.
  • the active sites of Nampt lie at the dimer interface, where the two NMN product molecules bind (Figure 2A). Each active site in the Nampt dimer is therefore composed of residues contributed by each monomer ( Figure 3).
  • the nicotinamide moiety lies deeper in the pocket, while the phosphate moiety is located at the entrance of the pocket ( Figure 3A).
  • a sulfate ion binds in place of the phosphate.
  • the binding of the product, NMN, to Nampt has many features in common with the binding of NaMN to T. acidophilus NaPRTase (Shin, et al., 2005).
  • the additional negative charge arising from histidine phosphorylation could be used to promote cleavage of pyrophosphate from PRPP by stabilizing formation of a positively charged oxacarbenium intermediate, which could then react with nicotinamide to form NMN.
  • a positively charged oxacarbenium intermediate which could then react with nicotinamide to form NMN.
  • Either a purely dissociative mechanism or one in which the intermediate had partial oxacarbenium character could be promoted in this way, by analogy with uracil DNA glycosylase.
  • the analogous reaction could be used to form NaMN by reaction with nicotinic acid in the cast of bacterial and yeast NaPRTases.
  • Nampt contains a central network of intermolecular hydrogen bonds near the dimer twofold that are mediated by S 199, S200 and T203 in one monomer with the analogous residues in the opposing monomer ( Figure 4A). Point mutations S199D and S200D were generated and assayed for dimerization and enzymatic activity.
  • Nampt A unique feature of Nampt is the hydrogen bonding between D219 and the amide of nicotinamide (Figure 3B).
  • This acidic residue would be incompatible with hydrogen bonding with nicotinic or quinolinic acid, and is indeed not conserved in QaPRTase (Eads, et al., 1997) and NaPRTase (Shin, et al., 2005). It was reasoned that this acidic side chain could play a role in specifying nicotinamide as a substrate by excluding alternative, negatively charged substrates. Therefore this residue was mutated to alanine and the effect on enzymatic activity was examined (Table 2).
  • the mutant D219A enzyme has an increased K M (roughly two orders of magnitude higher than wild type) and a similar k ⁇ at- These observations are consistent with a role for this residue in substrate recognition, thereby suggesting that the nicotinamide substrate may also bind in a position similar to that of the nicotinamide moiety in the NMN product.
  • Nampt has autophosphorylation activity.
  • Yeast Nptl is known to catalyze ATP hydrolysis and becomes autophosphorylated at His232 (Gross, J. W., et al., Kinetic mechanism of nicotinic acid phosphoribosyltransferase: implications for energy coupling. Biochemistry 37, 4189-4199 (1998)).
  • Autophosphorylation of the yeast enzyme raises the nicotinic acid phosphoribosyltransferase activity 10-30 fold (Rajavel, M:, et al., . Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase.
  • Nampt nicotinamide mononucleotide pyrophosphorylase from rat erythrocytes. J Biol Chem 247, 8016-22 (1972)).
  • His247 is in a position that is structurally analogous to His232 in yeast Nptl, suggesting that this histidine residue may playa similar role in the mammalian enzyme.
  • This is the histidine residue noted above that lies at the center of a conserved cluster of active site residues ( Figure 3A). Accordingly, it was determined whether Nampt could also catalyze ATP hydrolysis by monitoring the formation of inorganic phosphate in the mixture of Nampt and ATP and found that Nampt indeed induces ATP hydrolysis.
  • Nampt also becomes autophosphorylated.
  • the autophosphorylated form appears ( Figures 7A and 7B). Therefore, the autophosphorylation activity appears to be conserved in this class of phosphoribosyltransferase enzymes.
  • Differentiated brown adipocytes produce and secrete high levels of Nampt.
  • Nampt exhibits both intra- and extracellular forms in mammals.
  • the former synthesizes NMN (Revollo, et al., 2004), an intermediate in NAD biosynthesis, in mammalian cells, while the latter has been presumed to be a cytokine, PBEF (Samal, et al., 1994; and Jia, et al. Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis. J. Clin. Invest. 113, 1318-1327 (2004)), or an insulin- mimetic hormone, visfatin (Fukuhara, et al., 2005).
  • an appropriate cell culture model was first established.
  • Nampt expression and secretion levels were compared between HIB-IB and 3T3-L1, mouse preadipocyte cell lines that can differentiate into brown and white adipocytes, respectively.
  • Intracellular Nampt iNampt
  • eNampt extracellular Nampt
  • differentiated HIB-IB cells provide a useful model for examining the biochemical function of both intra- and extracellular Nampt/PBEF/visfatin.
  • Differentiated brown adipocytes produce and secrete an activated form of Nampt.
  • differentiated HIB-IB brown adipocytes as a model system, the enzymatic activities of intra- and extracellular Nampt were analyzed.
  • a HIB-IB cell line that expresses C-terminally FLAG-tagged Nampt (Nampt-FLAG, Figure 6A) was initially established.
  • Nampt-enzymatic activity of the bacterially produced recombinant Nampt and the intracellular Nampt-FLAG (iNampt-FLAG) immunoprecipitated from HIB-IB and NIH- 3T3 cellular extracts were also- measured with their Ic 031 values calculated.
  • the eNampt-FLAG secreted from differentiated HIB-IB cells showed a significantly higher kc at value (0.380/s) than recombinant Nampt (0.035/s, ⁇ 11-fold), iNampt-FLAG from NIH3T3 cells (0.082/s, ⁇ 5-fold), and iNampt-FLAG from differentiated HIB-IB cells (0. 182/s, ⁇ 2- fold) ( Figure 6E). It should be noted that iNampt-FLAG from differentiated HIB-IB cells also showed higher enzymatic activity than iNampt-FLAG from NIH3T3 cells that do not secrete Nampt (Figure 6E).

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Abstract

Selon la présente invention, la nicotinamide phosphoribosyltransférase (Nampt) synthétise un mononucléotide de nicotinamide (NMN) à partir de nicotinamide par une voie biosynthétique de NAD+ de mammifère et est nécessaire pour une activité de SirTl in vivo. On a également présumé que la Nampt était une cytokine (PBEF) ou une hormone (visfatine). La structure cristalline de Nampt en présence et absence de NMN montre que la Nampt est une phosphoribosyltransférase dimère de type II et donne un enseignement pour le mécanisme enzymatique. La structure enseigne également la base de la spécificité du substrat et suggère des routes pour le développement d'inhibiteurs de Nampt spécifiques pour le traitement de troubles liés à la Nampt.
PCT/US2007/011898 2006-05-19 2007-05-18 Structure cristalline d'un substrat complexe de nampt/pbef/visfatine Ceased WO2007136744A1 (fr)

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WO2011135332A3 (fr) * 2010-04-27 2011-12-22 Babraham Institute Modulateur de la nmn
WO2012024478A3 (fr) * 2010-08-19 2012-05-10 Opko Curna Llc Traitement de maladies associées à la nicotinamide phosphoribosyltransférase (nampt) par inhibition de produit de transcription antisens naturel vers nampt
CN108697722A (zh) * 2015-10-02 2018-10-23 麦德龙国际生物科技有限责任公司 β-烟酰胺单核苷酸的晶体形式
US10189872B2 (en) 2015-03-09 2019-01-29 W. R. Grace & Co.-Conn Crystalline form of nicotinamide riboside
US10233207B2 (en) 2014-07-24 2019-03-19 W. R. Grace & Co.—Conn. Crystalline form of nicotinamide riboside
US11414407B2 (en) 2017-12-22 2022-08-16 Elysium Health, Inc. Crystalline forms of nicotinamide riboside chloride
JP2023001064A (ja) * 2021-06-18 2023-01-04 邦泰生物工程（深▲セン▼）有限公司 ニコチンアミドモノヌクレオチド共結晶を調製する方法
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WO2011135332A3 (fr) * 2010-04-27 2011-12-22 Babraham Institute Modulateur de la nmn
WO2012024478A3 (fr) * 2010-08-19 2012-05-10 Opko Curna Llc Traitement de maladies associées à la nicotinamide phosphoribosyltransférase (nampt) par inhibition de produit de transcription antisens naturel vers nampt
US10233207B2 (en) 2014-07-24 2019-03-19 W. R. Grace & Co.—Conn. Crystalline form of nicotinamide riboside
US10323058B2 (en) 2014-07-24 2019-06-18 W. R. Grace & Co.-Conn. Crystalline form of nicotinamide riboside
US10189872B2 (en) 2015-03-09 2019-01-29 W. R. Grace & Co.-Conn Crystalline form of nicotinamide riboside
CN108697722A (zh) * 2015-10-02 2018-10-23 麦德龙国际生物科技有限责任公司 β-烟酰胺单核苷酸的晶体形式
US11414407B2 (en) 2017-12-22 2022-08-16 Elysium Health, Inc. Crystalline forms of nicotinamide riboside chloride
US12043616B2 (en) 2017-12-22 2024-07-23 Elysium Health, Inc. Crystalline forms of nicotinamide riboside chloride
JP2023001064A (ja) * 2021-06-18 2023-01-04 邦泰生物工程（深▲セン▼）有限公司 ニコチンアミドモノヌクレオチド共結晶を調製する方法
JP7394410B2 (ja) 2021-06-18 2023-12-08 邦泰生物工程（深▲セン▼）有限公司 ニコチンアミドモノヌクレオチド共結晶を調製する方法
WO2023191204A1 (fr) * 2022-03-28 2023-10-05 주식회사 랩투메디씨알오 Composition peptidique ayant une activité angiogénique, et composition pharmaceutique l'utilisant et composition cosmétique l'utilisant

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