Identification of Selective Inhibitors of NAD+-dependent Deacetylases Using Phenotypic Screens in Yeast

Hirao, Maki; Posakony, Jeffrey J.; Nelson, Melisa; Hruby, Henning; Jung, Manfred; Simon, Julian A.; Bedalov, Antonio

doi:10.1074/jbc.m308966200

Cited by 68 publications

(49 citation statements)

References 48 publications

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“…Using a cell-based chemical screen, Schreiber and colleagues (28) have identified several putative Sir2 inhibitors, including sirtinol, containing substructures that are similar to the adenine and nicotinamide groups of NAD ϩ . Another small-molecule Sir2 inhibitor, splitomicin, and its derivatives have been identified through library screening (29,30).…”

Section: Resultsmentioning

confidence: 99%

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases

Zhao

Harshaw²,

Chai³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

175

202

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Sir2 enzymes are broadly conserved from bacteria to humans and have been implicated to play roles in gene silencing, DNA repair, genome stability, longevity, metabolism, and cell physiology. These enzymes bind NAD ؉ and acetyllysine within protein targets and generate lysine, 2-O-acetyl-ADP-ribose, and nicotinamide products. To provide structural insights into the chemistry catalyzed by Sir2 proteins we report the high-resolution ternary structure of yeast Hst2 (homologue of Sir two 2) with an acetyllysine histone H4 peptide and a nonhydrolyzable NAD ؉ analogue, carba-NAD ؉ , as well as an analogous ternary complex with a reaction intermediate analog formed immediately after nicotinamide hydrolysis, ADP-ribose. The ternary complex with carba-NAD ؉ reveals that the nicotinamide group makes stabilizing interactions within a binding pocket harboring conserved Sir2 residues. Moreover, an asparagine residue, N116, strictly conserved within Sir2 proteins and shown to be essential for nicotinamide exchange, is in position to stabilize the oxocarbenium intermediate that has been proposed to proceed the hydrolysis of nicotinamide. A comparison of this structure with the ADP-ribose ternary complex and a previously reported ternary complex with the 2-O-acetyl-ADP-ribose reaction product reveals that the ribose ring of the cofactor and the highly conserved ␤1-␣2 loop of the protein undergo significant structural rearrangements to facilitate the ordered NAD ؉ reactions of nicotinamide cleavage and ADP-ribose transfer to acetate. Together, these studies provide insights into the chemistry of NAD ؉ cleavage and acetylation by Sir2 proteins and have implications for the design of Sir2-specific regulatory molecules. T he Sir2 (silent information regulator 2) or sirtuin family of deacetylases requires NAD ϩ as a cofactor to hydrolyze the acetyl moiety of an acetyllysine within protein targets to regulate diverse biological functions, including gene silencing, genome stability, longevity, metabolism, and cell physiology (for reviews, see refs. 1 and 2). The mechanism for Sir2 activity has been extensively studied at both structural and enzymatic levels. Structural studies reveal that the Sir2 proteins contain a structurally conserved elongated core domain containing a large Rossmann fold at one end, a structurally more variable zincbinding motif at the opposite end, and a series of loops connecting these regions and forming a cleft in the central region of the core domain (3-6). The acetyllysine and NAD ϩ cosubstrates bind to opposite sides of the cleft and highlight the region of the core domain containing the highest degree of sequence conservation within the Sir2 proteins, implying a conserved catalytic mechanism (3, 7). Biochemical and structural studies reveal that the deacetylation of acetyllysine is coupled to the hydrolysis of NAD ϩ to nicotinamide and 2Ј-O-acetyl-ADP-ribose (8, 9).A detailed structural understanding of how the Sir2 proteins mediate nicotinamide cleavage and ADP-ribose transfer to acetate has been hampered...

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Section: Resultsmentioning

confidence: 99%

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases

Zhao

Harshaw²,

Chai³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

175

202

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“…Splitomicin-resistant mutants contain point mutations in a small helical domain of Sir2 adjacent to the peptide binding site and distal to the NAD binding site. A single amino acid difference in the small helical domain of yeast Sir2 and its homologue Hst1, was found to underlie the ability of splitomicin to differentiate these two highly related enzymes (31). Cambinol, like splitomicin, discriminates between Sir2 family members as it is selective for SIRT1 and SIRT2.…”

Section: Discussionmentioning

confidence: 99%

“…30), a potent inhibitor of NAD-dependent HDACs, through a yeast cell-based screen for inhibitors of telomeric silencing. Splitomicin and its derivatives inhibit Sir2 and Sir2 homologues in yeast in vivo (30)(31)(32)(33). However, the hydrolysis of splitomicin and related lactone compounds at neutral pH (i.e., half-life 30 minutes at pH 7.4; ref.…”

Section: Introductionmentioning

confidence: 99%

Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes

Heltweg¹,

Gatbonton²,

et al. 2006

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SIRT1 and other NAD-dependent deacetylases have been implicated in control of cellular responses to stress and in tumorigenesis through deacetylation of important regulatory proteins, including p53 and the BCL6 oncoprotein. Hereby, we describe the identification of a compound we named cambinol that inhibits NAD-dependent deacetylase activity of human SIRT1 and SIRT2. Consistent with the role of SIRT1 in promoting cell survival during stress, inhibition of SIRT1 activity with cambinol during genotoxic stress leads to hyperacetylation of key stress response proteins and promotes cell cycle arrest. Treatment of BCL6-expressing Burkitt lymphoma cells with cambinol as a single agent induced apoptosis, which was accompanied by hyperacetylation of BCL6 and p53. Because acetylation inactivates BCL6 and has the opposite effect on the function of p53 and other checkpoint pathways, the antitumor activity of cambinol in Burkitt lymphoma cells may be accomplished through a combined effect of BCL6 inactivation and checkpoint activation. Cambinol was well tolerated in mice and inhibited growth of Burkitt lymphoma xenografts. Inhibitors of NADdependent deacetylases may constitute novel anticancer agents. (Cancer Res 2006; 66(8): 4368-77)

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“…Next, the effect of splitomicin, a selective and cell-permeable inhibitor of SIRT1 (38), was determined. Like nicotinamide, activation of AMPK by resveratrol was markedly diminished by splitomicin in HepG2 cells (Fig.…”

Section: Pharmacological Inhibition Of Sirt1 Attenuates Polyphenol-inmentioning

confidence: 99%

SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

Hou

Maitland-Toolan

et al. 2008

Journal of Biological Chemistry

709

572

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Resveratrol may protect against metabolic disease through activating SIRT1 deacetylase. Because we have recently defined AMPK activation as a key mechanism for the beneficial effects of polyphenols on hepatic lipid accumulation, hyperlipidemia, and atherosclerosis in type 1 diabetic mice, we hypothesize that polyphenol-activated SIRT1 acts upstream of AMPK signaling and hepatocellular lipid metabolism. Here we show that polyphenols, including resveratrol and the synthetic polyphenol S17834, increase SIRT1 deacetylase activity, LKB1 phosphorylation at Ser 428 , and AMPK activity. Polyphenols substantially prevent the impairment in phosphorylation of AMPK and its downstream target, ACC (acetyl-CoA carboxylase), elevation in expression of FAS (fatty acid synthase), and lipid accumulation in human HepG2 hepatocytes exposed to high glucose. These effects of polyphenols are largely abolished by pharmacological and genetic inhibition of SIRT1, suggesting that the stimulation of AMPK and lipid-lowering effect of polyphenols depend on SIRT1 activity. Furthermore, adenoviral overexpression of SIRT1 stimulates the basal AMPK signaling in HepG2 cells and in the mouse liver. AMPK activation by SIRT1 also protects against FAS induction and lipid accumulation caused by high glucose. Moreover, LKB1, but not CaMKK␤, is required for activation of AMPK by polyphenols and SIRT1. These findings suggest that SIRT1 functions as a novel upstream regulator for LKB1/AMPK signaling and plays an essential role in the regulation of hepatocyte lipid metabolism. Targeting SIRT1/LKB1/ AMPK signaling by polyphenols may have potential therapeutic implications for dyslipidemia and accelerated atherosclerosis in diabetes and age-related diseases. AMPK (AMP-activated protein kinase)2 serves as a sensor of cellular energy status, being activated by increased AMP/ATP ratio or by the upstream kinases, LKB1 (the tumor suppressor kinase), CaMKK␤ (Ca 2ϩ /calmodulin-dependent protein kinase kinase ␤), and TAK1 (transforming growth factor-␤-activated kinase-1) (1-7). Our previous studies demonstrated that dysfunction of hepatic AMPK induced by hyperglycemia represents a key mechanism for hepatic lipid accumulation and hyperlipidemia associated with diabetes (8, 9). Also, metformin, an antidiabetic drug, lowers systemic and hepatic lipids via activating LKB1/AMPK signaling (2, 8, 10). Our recent studies with human hepatocytes and type 1 diabetic LDL receptor-deficient (LDLR Ϫ/Ϫ ) mice have shown that polyphenols strongly stimulate hepatic AMPK and reduce lipid accumulation, which in turn attenuates hyperlipidemia and atherosclerosis in diabetic mice (9). Therefore, AMPK activation by polyphenols or metformin may be at least partially responsible for their therapeutic benefits on hyperlipidemia in diabetes (2,8,9). Resveratrol also stimulates AMPK in neurons (11). However, rapid activation of AMPK by polyphenols has been shown to be independent of altered adenine nucleotide levels (9, 11). Also, resveratrol activates AMPK in intact cells via an indirect mech...

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Identification of Selective Inhibitors of NAD+-dependent Deacetylases Using Phenotypic Screens in Yeast

Cited by 68 publications

References 48 publications

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases

Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes

SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

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Identification of Selective Inhibitors of NAD+-dependent Deacetylases Using Phenotypic Screens in Yeast

Cited by 68 publications

References 48 publications

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD + -dependent Sir2 histone/protein deacetylases

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD + -dependent Sir2 histone/protein deacetylases

Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes

SIRT1 Regulates Hepatocyte Lipid Metabolism through Activating AMP-activated Protein Kinase

Contact Info

Product

Resources

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Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases

Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD ⁺ -dependent Sir2 histone/protein deacetylases