The chemical biology of sirtuins.

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The chemical biology of sirtuins Cite this: DOI: 10.1039/c4cs00373j

Bing Chen,† Wenwen Zang,† Juan Wang,† Yajun Huang,† Yanhua He,‡ Lingling Yan,‡ Jiajia Liu‡ and Weiping Zheng* The sirtuin family of enzymes are able to catalyze the Ne-acyl-lysine deacylation reaction on histone and non-histone protein substrates. Over the past years since the discovery of its founding member (i.e. the yeast silent information regulator 2 (sir2) protein) in 2000, the sirtuin-catalyzed deacylation reaction has been demonstrated to play an important regulatory role in multiple crucial cellular processes such as transcription, DNA damage repair, and metabolism. This reaction has also been regarded as a current therapeutic target for human diseases such as cancer, and metabolic and neurodegenerative diseases. The unique b-nicotinamide adenine dinucleotide (b-NAD+ or NAD+)-dependent nature of the sirtuin-catalyzed deacylation reaction has also engendered extensive mechanistic studies, resulting in a mechanistic view of the enzyme chemistry supported by several lines of experimental evidence. On the journey toward these knowledge advances, chemical biological means have constituted an important functional arsenal; technically, a variety of chemical probes and modulators (inhibitors and activators) have been developed and some of them have been employed toward an enhanced mechanistic and functional (pharmacological) understanding of the sirtuincatalyzed deacylation reaction. On the other hand, an enhanced mechanistic understanding has also facilitated

Received 4th November 2014 DOI: 10.1039/c4cs00373j

the development of a variety of chemical probes and modulators. This article will review the tremendous accomplishments achieved during the past few years in the field of sirtuin chemical biology. It is hoped that this would also help to set a stage for how outstanding mechanistic and functional questions for the sirtuin-

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catalyzed deacylation reaction could be addressed in the future from the chemical biology perspective.

School of Pharmacy, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, Jiangsu Province, P. R. China. E-mail: [email protected]; Fax: +86-511-8503-8451 ext. 806; Tel: +86-151-8912-9171 † These authors are the equal first authors. ‡ These authors are the equal second authors.

Weiping Zheng obtained his PhD in Medicinal Chemistry from University of Tennessee, USA, under the supervision of Prof. Duane D. Miller; and was a post-doctoral fellow in Bioorganic Chemistry with Prof. Philip A. Cole at Johns Hopkins University School of Medicine, USA. Before joining the faculty at the Pharmacy School of Jiangsu University, China, he was the James L. and Martha Weiping Zheng J. Foght Assistant Professor in Biochemistry at University of Akron, USA. Prof. Zheng’s research interests center on the Chemical Biology and Medicinal Chemistry of the biomedically important post-translational modifications, especially those important in epigenetic processes.

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1. Introduction One type of important post-translational modification is the lysine side chain Ne-acylation which can be reversed by deacylation of

Juan Wang obtained her BS in Biological Engineering from Hefei University, China. She is currently pursuing her graduate research in Prof. Zheng’s laboratory, specifically on the chemical biology and medicinal chemistry of the sirtuin family of protein Ne-acyl-lysine deacylase enzymes.

Juan Wang

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Fig. 1

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The post-translational lysine side chain Ne-acylation on proteins and its reversal by deacylation.

the resulting Ne-acyl-lysine (Fig. 1).1–5 While the typical acyl group is acetyl, other bulkier acyl groups have also been recently identified, such as succinyl, crotonyl, and myristoyl. This acylation/deacylation switch is functionally important in both prokaryotes and eukaryotes. The acylation is typically realized via an enzymatic reaction catalyzed by, for example, protein lysine (K) Ne-acetyltransferases (KATs); however, acetylation and succinylation in the mitochondrial matrix could also be achieved non-enzymatically, due to the relatively high pH (B8) and the acyl donor acyl-CoA concentrations (mM range) inside the mitochondrial matrix.2,6 It has also been recently shown that acetyl-phosphate could also act as an acetyl donor in bacteria for a non-enzymatic protein lysine Ne-acetylation.2,7 For deacylation, two families of the protein Ne-acyl-lysine (K) deacylases (KDACs) have been known, namely, the Zn2+containing metallohydrolase family and the b-nicotinamide adenine dinucleotide (b-NAD+ or NAD+)-dependent sirtuin family.4,5,8,9 The members of the former family constitute the phylogenetic classes I, II, and IV KDACs and are able to hydrolyze the Ne-acyl-lysine side chain amide functionality with the help of a catalytic Zn2+; whereas the sirtuin family members constitute the phylogenetic class III KDACs and achieve Ne-acyllysine side chain deacylation via transferring the acyl group onto ADP-ribose (ADPR) (Fig. 2). Of note, ADP-ribosylation could also be catalyzed by sirtuins, in which the ADPR group of NAD+ gets transferred onto nucleophilic amino acid side

chains on themselves or their protein substrates (Fig. 3); however, this ADP-ribosyltransferase activity may not be physiologically relevant.10 Nevertheless, it is worth noting that it could be still an important activity for the human parasitic sirtuins.11 Also, as shown in Fig. 3, both Ne-acetyl-lysine-dependent and -independent mechanisms could be possible for such enzymatic ADP-ribosyl transfer.12 Since the discovery in 2000 that yeast and mouse transcriptional silencing sir2 (silent information regulator 2) proteins exhibited a NAD+-dependent histone deacetylase activity;13 being able to specifically deacetylate the Ne-acetyl-lysine side chain at positions 9 and 14 of the acetylated histone H3 protein and position 16 of the acetylated histone H4 protein, other NAD+-dependent histone deacetylases have also been identified, collectively forming the sirtuin family. The sirtuin family enzymes are present in organisms from all three evolutionary domains of life, i.e. bacteria, archaea, and eukarya.14 Native sirtuin substrates include both histone and non-histone proteins.15 Moreover, a couple of sirtuins were found to be more proficient in catalyzing the removal of the acyl groups of Ne-acyl-lysine that are bulkier than acetyl, including the succinyl group removal by SIRT516,17 and the myristoyl group removal by SIRT6.18 Table 1 summarizes the major enzymatic activity, the native histone substrates, the select native non-histone substrates, and the intracellular locations for the seven mammalian sirtuins (i.e. SIRT1-7).15,18–21 Jiajia Liu, Yajun Huang, Bing Chen, Wenwen Zang, Lingling Yan, and Yanhua He (from left to right) are also currently pursuing their graduate research in Prof. Zheng’s laboratory on sirtuin chemical biology and medicinal chemistry. Jiajia Liu, Yajun Huang, and Lingling Yan obtained their BS from Jiangsu University, respectively, in Pharmaceutical Formulation (Jiajia Liu), Pharmacy (Yajun Huang), and Pharmaceutics (Lingling Yan). Bing Chen and Wenwen Zang obtained their BE (Bachelor of Engineering) from Jiangsu University in Pharmaceutical Engineering. Yanhua He obtained her BS in Pharmacy from Anhui University of Traditional Chinese Medicine, China.

Jiajia Liu, Yajun Huang, Bing Chen, Wenwen Zang, Lingling Yan and Yanhua He (from left to right)

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Fig. 2

The Ne-acyl-lysine deacylation catalyzed by sirtuin enzymes and the Zn2+-containing deacylases. 2 0 -O-AADPR, 2 0 -O-acyl-ADP-ribose.

Fig. 3

The Ne-acetyl-lysine-dependent and -independent mechanisms for the sirtuin-catalyzed ADP-ribosylation reaction. Nu, nucleophile.

The sirtuin family members share a chemically and structurally conserved catalytic core in general,22 despite the possible existence of subtle active site infrastructure differences which could account for the reported ADP-ribosyltransferase activity in addition to the deacylase activity for certain sirtuins.11,12 Of note, in contrast to the conserved catalytic core, the N- and C-termini of sirtuins are fairly variable in length, chemical composition, and susceptibility to post-translational modifications (typically phosphorylation). This could account for the divergence in function and the catalytic activity regulatory mechanism during evolution, due to the differential interactions between these intrinsically disordered termini with the sirtuin catalytic core or


other protein partners including the native sirtuin substrates.23 The conserved catalytic core and the variable N-/C-termini are schematically illustrated in Fig. 4 with the seven human sirtuins. The three-dimensional structures have been solved for the catalytic core from many sirtuins such as the bacterial sirtuin Sir2Tm, the yeast sirtuin Hst2, and the five human sirtuins (SIRT1-3, SIRT5, and SIRT6).22,25 Consistent with the notion that sirtuins have a structurally and chemically conserved catalytic core, all the currently known core structures share the same overall two-domain structural topology and the same repertoire of catalytically important active site amino acid residues. Fig. 5 shows the two-domain catalytic core structural topology

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Review Article Table 1

Mammalian sirtuins

SIRT2

SIRT3a

SIRT4

Major enzymatic Deacetyl activity

Deacetyl

Deacetyl, decrotonyl19

Deacetyl, ADP- Deacetyl, demalonyl, Deacetyl, Deacetyl ribosyl transfer desuccinyl, deglutaryl demyristoyl

Native histone substrate

H3, H4, H1

H3, H4

H3, H4

Unknown or Unknown or non-applicable non-applicable

Native nonhistone substratec

p53, BCL6, Ku70, a-Tubulin, AceCS2, HMGCS2, MCD,20 GD Tat, AceCS1, HMGCS1, PEPCK1, FoxO3a, GD, LCAD, IDH2, p300, Tip60, DNMT1 p53, NF-kB MnSOD

Intracellular location

Nucleus, cytosole

Sirtuin


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SIRT1

Nucleus, cytosol

Nucleus,f mitochondrion f

SIRT5b

CPS1

Mitochondrion Mitochondrion

SIRT6b

SIRT7

H3

H3

CtIP, TNFa18

p53,d GABPb121

Nucleus

Nucleus

a The decrotonylase activity of SIRT3 is weaker than its deacetylase activity. b The deacetylase activity of SIRT5 and SIRT6 is weaker than their deacylase activities (demalonylase/desuccinylse/deglutarylase and demyristoylase, respectively). c These are the selected examples of such substrates. Also see ref. 15 for a fuller coverage. Abbreviations: AceCS, acetyl-coenzyme A synthetase; HMGCS, 3-hydroxy-3-methylglutaryl-coenzyme A synthase; DNMT1, DNA methyltransferase 1; GD, glutamate dehydrogenase; LCAD, long-chain acyl-coenzyme A dehydrogenase; IDH2, isocitrate dehydrogenase 2; MnSOD, manganese superoxide dismutase; PEPCK1, phosphoenolpyruvate carboxykinase 1; MCD, malonyl-CoA decarboxylase; CPS1, carbamoyl phosphate synthetase 1; CtIP, C-terminal binding protein (CtBP) interacting protein; TNFa, tumor necrosis factor a; GABPb1, GA binding protein b1. d The p53 protein is a possible SIRT7 native substrate based on the in vitro experiments with lysine Ne-acetylated p53 peptides. e Despite being primarily a nuclear protein, SIRT1 can also be present in the cytosol due to the nucleocytoplasmic shuttling. f The nuclear-encoded full-length SIRT3 initially resides in the nucleus, however, due to the presence of the mitochondrial targeting signal, it is ultimately transported into the mitochondrial matrix followed by an enzymatic removal of its N-terminus (1101) catalyzed by matrix processing peptidase.

Fig. 4 The sequence illustration of the seven full-length human sirtuins. The grey bar in each sequence refers to the conserved catalytic core. Modified from Fig. 1 in ref. 24.

from a representative sirtuin (Sir2Tm).26 In this X-ray crystal structure (Protein Data Bank code: 2H4F), two substrates (an lysine Ne-acetylated peptide and NAD+) are both bound at the Sir2Tm active site with the formation of a Michaelis complex. The overall structure is composed of the large Rossmann-fold domain and the small domain which includes the a-helical subdomain and the Zn2+ binding subdomain. Substrate binding takes place at the interface of the large and small domains. Since its discovery, the sirtuin-catalyzed deacylation reaction, deacetylation reaction in particular, has been demonstrated to play an important regulatory role in multiple crucial cellular processes such as transcription, DNA damage repair, and metabolism. This reaction has also been regarded as a current therapeutic target for human diseases such as cancer, and metabolic and neurodegenerative diseases. The NAD+-dependent nature of the sirtuincatalyzed deacylation reaction has also engendered extensive mechanistic studies, resulting in a mechanistic view of the enzyme chemistry supported by several lines of experimental evidence.

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On the way of this knowledge acquisition, chemical biology has played an important role: a variety of chemical probes and modulators (inhibitors and activators) have been developed and employed toward an enhanced mechanistic and functional (pharmacological) understanding of the sirtuin-catalyzed deacylation reaction. In the following sections, the tremendous accomplishments achieved during the past few years in the field of sirtuin chemical biology will be presented and a perspective will be offered as to how outstanding mechanistic and functional questions for the sirtuin-catalyzed deacylation reaction could be addressed in the future from the chemical biology standpoint.

2. Sirtuin substrate recognition behavior As afore-mentioned, the sirtuin family members possess physicochemically variable and intrinsically disordered N- and C-termini.


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Fig. 5 The X-ray co-crystal structure of Sir2Tm with bound Ne-acetyllysine peptide substrate and NAD+. This two-domain catalytic core structure is composed of the depicted large Rossmann-fold domain and small domain containing the depicted a-helical subdomain and Zn2+ binding subdomain. The Ne-acetyl-lysine substrate and NAD+ are depicted in stick model and are shown to bind at the interface of the large and small domains. This structural view was adopted from ref. 26 (used with permission).

While a role of the variable termini in regulating sirtuin native substrate selection has not yet been experimentally corroborated even though it is possible, their role in regulating the sirtuin catalytic activity has received experimental support. Specifically, in two recent studies,27,28 the SIRT1 deacetylase activity was observed to be potentiated by its N- and C-termini in cis. A common finding from these studies is that the native C-terminal sequence of SIRT1 was able to physically interact with the catalytic core, thus promoting substrate binding and leading to activity potentiation. Even though this activity auto-regulatory mechanism seemed to be unique to SIRT1 among the seven mammalian sirtuins, non-conserved N- and C-terminal segments could still be employed by certain other sirtuins to achieve a sirtuin isoform-specific functional regulation. Also as afore-mentioned, the sirtuin family members share a chemically and structurally conserved catalytic core; however, the active site infrastructure details could still be different among different sirtuins. In terms of this active site infrastructure variation among sirtuins, perhaps the most compelling observation is that, while sirtuins prefer b-NAD+ as the universal co-substrate, they can exhibit a quite different preference for the acyl group in the Ne-acyl-lysine substrate. For example, as shown in Table 1, among the seven mammalian sirtuins, SIRT5 was found to prefer the Ne-malonyl-lysine, Ne-succinyl-lysine, and Ne-glutaryl-lysine substrates to the Ne-acetyl-lysine substrate, and SIRT6 was found to prefer the Ne-myristoyl-lysine substrate to the Ne-acetyl-lysine substrate. Similarly, the preferred substrates for the Plasmodium falciparum sirtuin PfSir2A were found to be the Ne-octanoyl-lysine and Ne-myristoyl-lysine substrates instead of the Ne-acetyl-lysine substrate.29 Moreover, several sirtuins were found to be able to catalyze depropionylation and debutyrylation on the Ne-propionyl-lysine and Ne-butyryl-lysine substrates, respectively, however, with different catalytic efficiencies relative


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to their catalytic deacetylation.30 Specifically, relative to its deacetylase activity, the yeast sirtuin Hst2 was found to exhibit a comparable depropionylase activity while a much weaker (B2%) debutyrylase activity. However, relative to their deacetylase activity, human SIRT1, SIRT2, and SIRT3 were all found to exhibit an B2–3-fold lower depropionylase activity, whereas they still exhibited an only B5-fold lower debutyrylase activity. The above sirtuin substrate specificity findings are summarized in Fig. 6, in which the only structural variation from = (CH2)4) Ne-acetyl-lysine (X = NH, Y = O, Z = CH2, R3 = H, is the different R3 groups preferred by different sirtuins. In terms of this acyl head specificity, it was also found that Ne-formyllysine (X = NH, Y = O, Z = H, = (CH2)4) was a very weak SIRT1 substrate.31 As an extension of this acyl head specificity to also vary the X, Y, and Z atoms(groups), a family of compounds known as the catalytic mechanism-based sirtuin inhibitors32 have proved to be quite useful. These compounds are sirtuin substrates, however, sirtuins can catalyze their transformation into sirtuin inhibitory species. Therefore, the active site-directed substrate specificity could also be assessed from the inhibitory actions of these compounds. Since 2006, various sirtuin catalytic mechanism-based inhibitory warheads have been successfully developed,32 which include Ne-thioacetyl-lysine (X = NH, Y = S, Z = CH2, R3 = H, = (CH2)4), Ne-thiocarbamoyl-lysine = (CH2)4), L-2-amino-7(X = NH, Y = S, Z = NH, R3 = H, carboxamidoheptanoic acid (X = CH2, Y = O, Z = NH, R3 = H, = (CH2)4), and ethyl Ne-malonyl-lysine (X = NH, Y = O, = (CH2)4). Very recently, Z = CH2, R3 = C(QO)OC2H5, e N -thiomethylcarbamoyl-lysine (X = NH, Y = S, Z = NH, R3 = CH3, = (CH2)4) was found in our laboratory to be a stronger sirtuin inhibitory warhead than Ne-thiocarbamoyl-lysine (W. Zang, Y. Hao, Z. Wang, and W. Zheng, manuscript in preparation). In addition, L-2-amino-7-ethylcarboxamidoheptanoic acid (X = CH2, Y = O, Z = NH, R3 = C2H5, = (CH2)4) was also very recently found in our laboratory to be a stronger sirtuin inhibitory warhead than L-2-amino-7-carboxamidoheptanoic acid (Y. He, L. Yan, W. Zang, and W. Zheng, manuscript in preparation). The chemical structures of the acyl parts of these warheads are depicted in Fig. 6. The sirtuin inhibitors containing these warheads can be taken up by sirtuins as substrates and are able to support the sirtuin-catalyzed nicotinamide cleavage from NAD+ with the formation of the true sirtuin inhibiting species. Therefore, it could be concluded that these further acyl groups can also be accommodated at the sirtuin active site. In addition to the above-described studies on acyl head specificity, efforts have also been invested to probe the impact of side chain length variation in Ne-acetyl-lysine on the sirtuincatalyzed nicotinamide cleavage and deacetylation. It should be noted that we found that the D-isomer of Ne-acetyl-lysine was not a sirtuin substrate,33 therefore, this chemical probing was performed with Ne-acetyl-lysine and its close structural analogs with subtly varied side chains as shown in Fig. 6. The depicted analogs have subtly varied extended distances between the side chain amide oxygen and the a-carbon than that in Ne-acetyl-lysine.

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Fig. 6 Substrate recognition specificity at the sirtuin active site. Note: (i) the acyl head groups shown are those that can be accommodated at the sirtuin active site, and many of them are also found as endogenous lysine Ne-modifications, including acetyl, propionyl, butyryl, octanoyl, myristoyl, crotonyl, malonyl, succinyl, and glutaryl. (ii) For side chain length probing, Ne-acetyl-lysine and its close structural analogs harboring the depicted side chains were each introduced into model peptides for study.33,34 The singly boxed Ne-acetyl-lysine analog was used in ref. 35. The doubly boxed Ne-acetyl-lysine analog was used in ref. 36. (iii) The ‘‘neighboring amino acid specificity’’ was suggested from the studies on Ne-acetyl-lysine substrates. Ne-thiotrifluoroacetyl-lysine was used in ref. 41.

The study in our laboratory33 with human SIRT1 and the peptides (into which Ne-acetyl-lysine or its analogs were incorporated) showed that, while the Ne-acetyl-lysine-containing peptide exhibited robust enzymatic nicotinamide cleavage and deacetylation, none of the analog peptides was able to support the enzymatic nicotinamide cleavage or deacetylation under the same experimental condition. Another study34 with yeast Hst2 and acetyl-poly-ornithine also found no enzymatic nicotinamide cleavage and deacetylation. Of note, ornithine is an analog of lysine with one less side chain methylene (CH2). Therefore, it seems that the sirtuin (e.g. SIRT1 or Hst2)-catalyzed nicotinamide cleavage and deacetylation (or deacylation in general) have a fairly stringent requirement for the side chain length between the a-carbon and the acetamido group, and that of Ne-acetyl-lysine (or Ne-acyl-lysine in general) is optimal. This notion is also consistent with two recent findings: (i) the rate of the SIRT2-catalyzed deacetylation of a peptide harboring the singly boxed analog depicted in Fig. 6 was about 1/3 that of the corresponding Ne-acetyl-lysine peptide.35 (ii) The kcat of the bacterial Sir2Tm-catalyzed deacetylation of a peptide harboring the doubly boxed analog depicted in Fig. 6 was about 1/8 that of the corresponding Ne-acetyl-lysine peptide,36 even though here the presence of an electron withdrawing NH adjacent to the C(QO)NH moiety of acetylhydrazide may also contribute to the observed rate decrease. For a discussion on the chemical mechanism of the sirtuin-catalyzed deacylation reaction, see Section 3.2. As depicted in Fig. 6, another level of the substrate specificity for the sirtuin-catalyzed deacylation reaction is whether and how

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the amino acid residues immediately surrounding Ne-acyl-lysine influence the substrate recognition at the sirtuin active site. The corresponding research on this topic has been focused on probing the amino acid residues relative to Ne-acetyl-lysine. The major observations from this research are briefly described below, and the corresponding suggestions are summarized in Fig. 6. (i) Ne-acetyl-lysine and its four immediately neighboring amino acid residues (two on each side) in a peptide substrate make predominant binding interactions with a sirtuin enzyme. (ii) Ne-acetyl-lysine and its four immediately neighboring amino acid residues (two on each side) form a b-strand in a sirtuinbound peptide substrate, and this b-strand forms an anti-parallel b-sheet with two b-strands from a sirtuin enzyme. (iii) The amino acid side chains immediately neighboring Ne-acetyl-lysine in a peptide substrate play an important role in selective substrate recognition at the sirtuin active site. The first two phenomena above were first observed during an X-ray structural analysis37 of the archaeal sirtuin Sir2-Af2 complexed with an Ne-acetyl-lysine peptide derived from the human p53 protein, and an in vitro deacetylase assay38 with multiple sirtuins (SIRT1, SIRT2, yeast Sir2, Trypanosoma brucei Sir2) and a panel of 10 Ne-acetyl-lysine histone H3 peptides with varied lengths. The third phenomenon above has been observed in multiple structural and biochemical studies. Specifically, (i) yeast sirtuins Sir2 and Hst2, and human SIRT2 were shown to exhibit varying substrate activities (kcat/Km) toward histone H3 and H4 peptides bearing a single Ne-acetyl-lysine at different


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sites;39 (ii) the amino acid side chain interactions at the 1 and +2 positions relative to Ne-acetyl-lysine in a target sequence were shown with the bacterial Sir2Tm as a model sirtuin to be important in contributing selective substrate recognition at the sirtuin active site;40 (iii) by high-throughput screening of a onebead, one-compound library whose members were all 5-amino acid peptides with Ne-acetyl-lysine at the central position, and the subsequent in-solution assays on hits and non-hits, the contextdependent substrate specificity was demonstrated for SIRT1.38 It was found in the study that the SIRT1 preference for the side chain at a given position relative to Ne-acetyl-lysine is contingent upon the side chain identities at neighboring positions on the peptide. It is worth noting that, while the above-described substrate recognition behavior at the sirtuin active site would help to identify the physiological substrates for the sirtuin-catalyzed deacylation reaction, a chemical biological approach to such identification would also be desirable. The following is a recent study in this regard. Specifically, in this study,41 combinatorial 9-amino acid peptide libraries were constructed on cellulose membranes, screened for high-affinity SIRT3 binding peptides, and then subjected to machine learning analysis to establish binding trends and to make a binding affinity prediction for all the lysine sites in the entire mitochondrial proteome. Strikingly, the predicted SIRT3 binding affinities for a set of 24 Ne-acetyl-lysine peptides were found during the in-solution kinetic validation stage to correlate with their kcat/Km values. One key feature of this strategy is the use of Ne-thiotrifluoroacetyl-lysine (structure depicted in Fig. 6) as the central residue of the peptides in the constructed libraries. This sirtuin tight-binding close structural analog of Ne-acetyl-lysine could be regarded as a chimera of Ne-thioacetyl-lysine and Ne-trifluoroacetyl-lysine. It presumably would also be able to very weakly support the sirtuin-catalyzed nicotinamide cleavage, like Ne-trifluoroacetyl-lysine (vide infra), therefore, it binds tightly to the sirtuin active site yet will be very inefficiently processed by a sirtuin. Importantly, by using the mitochondrial SIRT3 as a model sirtuin, this study not only established an un-biased screening strategy, but also uncovered multiple potential native substrates of SIRT3 in the mitochondrial proteome. Moreover, the results from this study also reinforced the notion that the side chains immediately neighboring Ne-acetyl-lysine in a peptide substrate are important in conferring selective substrate recognition at the sirtuin active site. In addition to substrate identification for the sirtuin-catalyzed deacylation reaction, catalytic activity-based sirtuin protein profiling is another important area where chemical biology has played a role. Specifically, in a recent study,42 a Ne-thioacetyl-lysine-based approach was found to be able to detect and isolate the enzymatically active sirtuin proteins. The key features of this approach are the formation of a stalled a-1 0 -S-alkylamidate intermediate at the sirtuin active site from an Ne-thioacetyl-lysine-containing substrate and a NAD+ analog carrying an affinity tag, and the subsequent pulling down (via the affinity tag on the NAD+ analog) of the sirtuin protein whose active site has the bound a-1 0 -Salkylamidate intermediate. In terms of the ability of a sirtuin to catalyze the formation of the stalled a-1 0 -S-alkylamidate intermediate from an Ne-thioacetyl-lysine-containing substrate and NAD+, see Section 3 for detailed discussion.


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In addition, identifying the sirtuin(s) responsible for the deacylation of a particular post-translational lysine Ne-acylation is also an important area where chemical biology has played a role. Specifically, in order to capture the transient interaction between Ne-acyl-lysine and a sirtuin that is able to catalyze its deacylation, photoaffinity labeling has been employed.19,43–45 Such a photoaffinity label has been constructed by installing an appropriately placed photoreactive group onto the native Ne-acyllysine peptide sequence. Benzophenone and diazirine have both been employed as the photoreactive group. In addition, the label also contains a terminal alkyne as a bio-orthogonal handle for the subsequent selective detection/isolation of the captured sirtuin(s). This photoaffinity-labeling-assisted sirtuin profiling strategy is schematically illustrated in Fig. 7A. It is conceivable that a labeling reagent would be recognized by its sirtuin as a substrate. Therefore, the success of this photoaffinity-labeling-assisted strategy would be consistent with an ordered sequential kinetic mechanism for the sirtuin-catalyzed deacylation reaction (Ne-acyl-lysine substrate binding prior to NAD+) (see above). This kinetic mechanism dictates that the specific binding of the Ne-acyl-lysine substrate at the sirtuin active site does not require a prior binding of NAD+. Fig. 7B depicts the photoaffinity labels that have been constructed and employed to selectively label the sirtuins that ought to be able to catalyze the corresponding Ne-acyl-lyine deacylation. The depicted labels based on Ne-succinyl-lysine,43 Ne-malonyllysine,44 and Ne-acetyl-lysine44 were found to be able to selectively label desuccinylase/demalonylase SIRT5 and deacetylase SIRT3, respectively. When the depicted Ne-myristoyl-lysine-based label was used,45 the authors of the study found that, in addition to labeling the then known demyristoylase SIRT6, this reagent was able to more proficiently label SIRT2, suggesting that SIRT2 was a robust in vitro demyristoylase. In conjunction with the use of alk-14 (HCRC–(CH2)13–COOH, a chemical reporter for the intracellular protein fatty-acylation),46 SIRT2 small interfering RNA (siRNA), and AGK2 (a SIRT2 inhibitor, see Section 4.2), SIRT2 was found in the study to be likely also an in vivo Ne-fatty-acyl-lysine deacylase. However, whether demyristoylation is also an in vivo deacylase activity of SIRT2 is yet to be determined. When the depicted Ne-crotonyl-lysine-based label was used in a study in combination with the stable isotope labeling of amino acids in cell culture (SILAC) technology, SIRT3 was identified as a native Ne-crotonyl-lysine decrotonylase.19 It should be noted that all the labeling reagents depicted in Fig. 7B were not only able to robustly label the purified sirtuins, but also able to selectively label them in the context of an entire proteome.

3. The catalytic mechanisms of the sirtuin-catalyzed Ne-acyl-lysine deacylation reaction As afore-mentioned, the evolutionarily conserved sirtuin family members share a chemically and structurally conserved catalytic core, with a conserved overall two-domain structural topology and a conserved ensemble of catalytically important active

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Fig. 7 (A) A schematic illustration of the photoaffinity-labeling-assisted sirtuin profiling strategy. (B) The photoaffinity labels that have been constructed and employed in the indicated literature to selectively label the sirtuins for the corresponding Ne-acyl-lyine.

site amino acid residues. One implication of this is the catalytic mechanism conservation for sirtuin family members from different organisms. The following sections will be covering the current knowledge of the kinetic and chemical mechanisms for the sirtuincatalyzed deacylation reaction, with an emphasis on the chemical biological mechanistic interrogation of the chemical mechanism. It should be noted that, while the up-to-date mechanistic studies were mostly performed for the sirtuin-catalyzed Ne-acetyl-lysine deacetylation, the insights obtained ought to be applicable to the whole spectrum of the sirtuin-catalyzed Ne-acyl-lysine deacylations, since it is believed that these deacylation reactions employ the same catalytic mechanisms as those for the deacetylation reaction.

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3.1.

Kinetic mechanism

The sirtuin-catalyzed deacylation reaction has three substrates (the Ne-acyl-lysine substrate, NAD+, and H2O) and three products (nicotinamide, 2 0 -O-acyl-ADP-ribose (2 0 -O-AADPR), and deacylated product) (Fig. 2). The kinetic mechanism has been elucidated for the Ne-acetyl-lysine deacetylation catalyzed by yeast Hst2 and human SIRT2, and it was found that this enzymatic deacetylation reaction obeys a sequential ternary complex kinetic mechanism (Fig. 8).39 Specifically, substrate binding follows the order of the Ne-acetyl-lysine substrate prior to NAD+, and from the resulting ternary substrate–sirtuin complex does the


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Fig. 8 The sequential kinetic mechanism for the sirtuin-catalyzed deacylation reaction. This mechanistic scheme was suggested from a kinetic study on the Ne-acetyl-lysine deacetylation reaction catalyzed by yeast Hst2 and human SIRT2.39 E, a sirtuin; NAM, nicotinamide; 2 0 -O-AADPR, 20 -O-acyl-ADP-ribose.

first chemical step (nicotinamide cleavage) take place; following the release of thus formed nicotinamide, the other two products (20 -O-AADPR and the deacetylated product) are formed and yet released randomly from the sirtuin active site. As part of the kinetic mechanistic elucidation of an enzymatic reaction, the rate-limiting step for the overall deacetylation catalysis was demonstrated to be the product release from the active site of yeast Hst2.47 However, for Plasmodium falciparum sirtuin PfSir2A, the rate-limiting step for the overall deacetylation catalysis was found to be the further chemical transformation of the a-1 0 -O-alkylamidate intermediate formed along the sirtuin catalytic coordinate (ref. 12 and Section 3.2). These observations offer another example for the afore-mentioned idea that active site infrastructure variation is present among sirtuin family members. 3.2.

Chemical mechanism

Fig. 9 depicts the current version of the chemical mechanism of the sirtuin-catalyzed Ne-acyl-lysine deacylation reaction. This multi-step mechanistic scheme is consistent with the abovedescribed kinetic mechanism in that the first chemical step

(i.e. the nicotinamide cleavage from NAD+) occurs only after the binding of the Ne-acyl-lysine substrate and NAD+ at the sirtuin active site with the formation of the corresponding ternary complex and the Michaelis complex. The following paragraphs will provide a description of how chemical biological means have been judiciously designed and exploited for an enhanced mechanistic interrogation of the chemical mechanism for the sirtuin-catalyzed deacylation reaction. As shown in Fig. 9, the first chemical step for the sirtuincatalyzed deacylation reaction is the depicted nucleophilic substitution reaction between the Ne-acyl-lysine side chain amide oxygen and the electrophilic C10 of NAD+. This reaction route could be hinted to be possible from the sirtuin structural studies, with one example being the afore-mentioned X-ray co-crystal structure of the bacterial Sir2Tm with the bound Ne-acetyllysine substrate and NAD+ (Fig. 5).26 In this ternary complex structure, the Ne-acetyl-lysine side chain amide oxygen was found to be positioned at the a-face of NAD+’s nicotinamide ribose within van der Waals distance (3.2 Å) from the C1 0 position of NAD+. This relative positioning of the two potential

Fig. 9 The current version of the chemical mechanism of the sirtuin-catalyzed Ne-acyl-lysine deacylation reaction. See Fig. 6 for currently known R groups whose corresponding acyl head groups can be accommodated at the sirtuin active site and get deacylated by sirtuin. ADP, adenosine diphosphate; 2 0 -O-AADPR, 2 0 -O-acyl-ADP-ribose; B: refers to general base. Note: the stereochemistry at the tetrahedral carbon of the dioxo ring in the bicyclic intermediate is inferred from the observed stereochemistry at the corresponding carbon in the a-1 0 -S-bicyclic intermediate shown in Fig. 13 (see below).


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reaction sites hinted that the depicted direct interaction of the bound Ne-acetyl-lysine substrate and NAD+ is possible with the formation of the a-1 0 -O-alkylamidate intermediate (Fig. 9). Considering that the amide oxygen is a weak nucleophile, the capability of the Ne-acyl-lysine side chain amide oxygen to directly interact with the electrophilic C1 0 of NAD+ seems to be an unusual instance. However, this mode of direct interaction has been more or less directly ascertained via employing two close structural analogs of Ne-acetyl-lysine, i.e. Ne-[18O]acetyl-lysine and Ne-thioacetyl-lysine (Fig. 10). Specifically, when a histone H3 peptide installed with Ne-[18O]acetyl-lysine was used as a substrate in a Hst2-catalyzed deacetylation assay, the 18O label was found to be transferred onto the C10 position of NAD+, affording 1 0 -18OH-2 0 -O-acetyl-ADPribose (10 -18OH-20 -O-AADPR, Fig. 10).47 This finding strongly argued for the direct covalent interaction between the Ne-acyl-lysine side chain amide oxygen and the electrophilic C1 0 of NAD+ as an obligatory step toward the sirtuin-catalyzed deacylation reaction. When the crystal of the bacterial Sir2Tm complexed with a Ne-thioacetyl-lysine p53 peptide was soaked with a NAD+ cryoprotective solution, the in crystallo catalysis of Sir2Tm was found to result in the formation of a covalent bond between the side chain thioacetyl sulfur of Ne-thioacetyl-lysine and the C10 of NAD+, with the formation of the a-1 0 -S-alkylamidate intermediate that was found to be trapped within the crystal.48 This Sir2Tm co-crystal structure (Protein Data Bank code: 3D81, Fig. 10) offers the first structure-based conclusive argument for the existence of a direct covalent interaction between the Ne-acyl-lysine side chain amide oxygen and the electrophilic C10 of NAD+, which would lead to the formation of the a-1 0 -O-alkylamidate intermediate along the sirtuin catalytic deacylation coordinate (Fig. 9). It should be noted that, while Fig. 10 only depicts the Sir2Tmcatalyzed formation of an a-1 0 -S-alkylamidate intermediate, such a

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trapped intermediate was also subsequently observed when the co-crystal of human SIRT3 and a Ne-thioacetyl-lysine-containing acetyl-CoA synthetase 2 peptide was soaked with NAD+ (Protein Data Bank code: 3GLT).49 Moreover, a stalled a-1 0 -S-alkylamidate intermediate has been known to be detectable using mass spectrometry; the pioneering work in this regard was the detection of that formed from the Hst2-catalyzed reaction between an Ne-thioacetyl-lysine histone H3 peptide and NAD+.50 The next mechanistic question is how a sirtuin enzyme can catalyze the direct covalent interaction between the Ne-acyl-lysine side chain amide oxygen and the electrophilic C1 0 of NAD+, since the amide oxygen is a weak nucleophile. It is known that a nucleophilic substitution reaction could proceed via SN1-like or SN2-like mechanisms. When applied to the sirtuin-catalyzed reaction, the SN1-like mechanism would entail the formation of an enzyme-stabilized strongly electrophilic fully dissociated oxacarbenium ion intermediate following the complete departure of nicotinamide, and its ensuing reaction with amide oxygen; whereas the SN2-like mechanism would entail a direct displacement of the nicotinamide of NAD+ as the leaving group with the side chain amide oxygen of the Ne-acyl-lysine substrate in the absence of a discrete oxacarbenium ion intermediate. For a SN1like mechanism, a nucleophilic participation from the amide oxygen would be minimal. However, for a SN2-like mechanism, two scenarios would be possible: one is an associative displacement of amide oxygen for nicotinamide with a strong nucleophilic participation in the transition state of the reaction; the other is a displacement reaction with a loosely dissociative transition state with a minimal nucleophilic participation, yet this transition state has a strong oxacarbenium ion character. Fig. 11A depicts the above three possible mechanisms for the first step nucleophilic substitution reaction (or the nicotinamide cleavage reaction) along the sirtuin-catalyzed deacylation coordinate.

Fig. 10 A schematic illustration of using peptides harboring Ne-[18O]acetyl-lysine or Ne-thioacetyl-lysine as chemical probes for the direct covalent interaction between the Ne-acyl-lysine side chain amide oxygen and C10 of NAD+ during the first step of sirtuin deacylation catalysis depicted in Fig. 9. Shown on the right hand side of the figure are the two key mechanistic species formed via sirtuin-catalyzed transformation on these probes, i.e. 1 0 -18OH-2 0 -Oacetyl-ADP-ribose and the a-10 -S-alkylamidate intermediate (in stick model) trapped at the Sir2Tm active site. The close-up view showing the trapped a-10 -Salkylamidate intermediate and its interactions with Sir2Tm active site amino acid residues was adopted from ref. 48 (used with permission).

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Fig. 11 (A) A schematic illustration of the three possible mechanisms for the first step nucleophilic substitution reaction along the sirtuin deacylation coordinate. ADP, adenosine diphosphate; NAM, nicotinamide. Note: routes (a), (b), and (c) respectively denote the stepwise SN1-like mechanism, the associative SN2-like mechanism, and the dissociative SN2-like mechanism. Due to a strong oxacarbenium ion character in the loosely dissociative transition state in route (c), the C1 0 position was depicted to have a formal +1 charge. (B) A schematic illustration of the peptides harboring Ne-acetyl-lysine or its three fluorinated analogs that have been employed to investigate if there is a nucleophilic participation for the sirtuin-catalyzed nicotinamide cleavage.

To determine whether the SN1-like or the SN2-like mechanism is operative, the fluorinated analogs of Ne-acetyl-lysine shown in Fig. 11B were employed in a nicotinamide cleavage assay with yeast Hst2.51 When histone H3 peptides installed with Ne-acetyl-lysine or its fluorinated analogs were assayed with yeast Hst2 for their ability to support nicotinamide cleavage, the nicotinamide formation rate was found to be inversely correlated with the number of the strongly electron withdrawing fluorine atom. Therefore, the rate of nicotinamide formation is positively correlated with the nucleophilicity of the side chain amide oxygen. In other words, the more fluorine atoms substituted on the acetyl a-carbon of Ne-acetyl-lysine, the weaker the nucleophilicity of the side chain amide oxygen will be, and the lower the rate of nicotinamide formation will be. From the Kd measurement in the study, the Hst2 active site was also found to be able to bind favorably to the fluorinated analogs of Ne-acetyllysine. Therefore, the observed decrease in the nicotinamide formation rates ought to be mainly due to the decreased nucleophilicity of the side chain amide oxygen. It could be thus concluded that there was a nucleophilic participation of the side


chain amide oxygen of Ne-acetyl-lysine in the transition state for the sirtuin-catalyzed first step nucleophilic substitution reaction (or the nicotinamide cleavage), and a SN2-like mechanism was suggested. To examine the transition state structure for the SN2-like mechanism suggested from the above study for the sirtuincatalyzed nicotinamide cleavage reaction, both computational (e.g. ab initio QM/MM molecular dynamics simulation) and experimental (kinetic isotope effect measurements) studies with model sirtuins (e.g. bacterial Sir2Tm and archeal Sir2Af2) have been performed.52–54 These studies suggested that this enzymatic nucleophilic substitution reaction follows a concerted yet dissociative SN2-like mechanism that involves a highly dissociative transition state with a strong oxacarbenium ion character and a mild nucleophilic participation from the side chain amide oxygen of Ne-acetyl-lysine. It is worth noting that, while the above described biochemical/ structural findings have supported the existence of the a-1 0 -Oalkylamidate intermediate and suggested how it could be generated along the sirtuin deacylation catalytic coordinate,

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the corresponding biochemical/structural evidence for the existence and the generation route of the bicyclic intermediate depicted in Fig. 9 has also been obtained. So far the most direct pieces of the experimental evidence for this came from the following two recent studies.55,56 First, our laboratory recently discovered that, when using a peptide installed with L-2-amino-7-carboxamidoheptanoic acid, a close structural analog of Ne-acetyl-lysine, in a sirtuin deacetylation assay, the corresponding bicyclic intermediate (Intermediate II in Fig. 12) was long-lived enough to be detected using mass spectrometry.55 Of note, this analog is an isostere of Ne-acetyllysine through swapping the NH and CH3 groups of the acetamide portion in Ne-acetyl-lysine. As described above in Section 2, the studies on probing the impact of side chain length variation in Ne-acetyl-lysine on the sirtuin-catalyzed nicotinamide cleavage and deacetylation suggested that the side chain length of Ne-acetyl-lysine represents the optimal length in supporting these two sirtuin-catalyzed reactions. Therefore, the observed sirtuin-catalyzed formation of Intermediate II from a peptide harboring L-2-amino-7-carboxamidoheptanoic acid (Fig. 12) would imply that the side chain length of this analog is close to that of Ne-acetyl-lysine. Our observation argues strongly for the existence and the formation route of the bicyclic intermediate as depicted in Fig. 9. Second, similar to the above-described solution of a Sir2Tm co-crystal structure with the trapped a-1 0 -S-alkylamidate intermediate (Fig. 10) formed from the Sir2Tm-catalyzed processing of a peptide installed with Ne-thioacetyl-lysine, a co-crystal structure of SIRT5 complexed with a trapped bicyclic intermediate (Protein Data Bank code: 4F56, Fig. 13) formed apparently from the SIRT5catalyzed processing of a peptide containing Ne-thiosuccinyllysine was also recently solved.56 Of note, Ne-thiosuccinyl-lysine is the Ne-succinyl-lysine analog with the side chain amide oxygen replaced with sulfur. This observation provides the first structurebased direct argument for the existence and the route of

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formation of the bicyclic intermediate along the sirtuin deacylation catalytic coordinate (Fig. 9). This study also argues strongly for chemical mechanism conservation for the sirtuin-catalyzed deacylation reactions. Moreover, the successful solution of this SIRT5 co-crystal structure with a trapped bicyclic intermediate would offer an opportunity to analyze its formation and downstream catalytic transformation with molecular details. From the above description, it becomes clear that a simple isosteric replacement of the Ne-acyl group in a sirtuin substrate with Ne-thioacyl or carboxamide has afforded fairly effective sirtuin mechanistic probes (i.e. Ne-thioacetyl-lysine, Ne-thiosuccinyllysine, or L-2-amino-7-carboxamidoheptanoic acid) whose use has furnished profound mechanistic insights. These isosteric replacements and others could find broader use in biochemical systems besides the sirtuin-catalyzed deacylation reaction. As implied in Fig. 9, the route of the further transformation of the bicyclic intermediate into the deacylated product and 2 0 -O-AADPR is still unknown. However, it is known that water is involved in the resolution of the bicyclic intermediate. One piece of convincing experimental evidence for this notion is the observed transfer of the 18O label from H218O onto the 2 0 -Oacetyl carbonyl oxygen of 2 0 -O-AADPR when a sirtuin deacetylation assay was performed in H218O.47,57 The study with the use of H218O further suggested that water was involved in a regioselective nucleophilic attack at the tetrahedral carbon of the dioxo ring in the bicyclic intermediate. In terms of how the bicyclic intermediate would be resolved in the presence of water to afford the deacylated product and 2 0 -O-AADPR, while further studies are needed to ascertain the responsible pathway or pathways, the three pathways depicted in Fig. 14 could be candidates responsible for the resolution of the bicyclic intermediate in the presence of water. One piece of informative experimental evidence for pathways A and B came from our study with SIRT1 and Sir2Tm and a peptide installed with L-2-amino-7-carboxamidoheptanoic acid (vide supra). In addition

Fig. 12 The proposed sirtuin processing of a peptide containing L-2-amino-7-carboxamidoheptanoic acid (shown in red). This peptide was shown to be converted to Intermediate II and (Intermediate III and/or End Product) which were detectable by mass spectrometry.55 Intermediates II and possibly III were stalled species along the sirtuin catalytic coordinate. Note: the stereochemistry at the tetrahedral carbon of the dioxo ring in Intermediate II is inferred from the observed stereochemistry at the corresponding carbon in the a-1 0 -S-bicyclic intermediate shown in Fig. 13 (see below), and that in Intermediate III is not explicitly indicated.

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Fig. 13 A schematic illustration of using a histone H3 peptide harboring Ne-thiosuccinyl-lysine as a chemical probe for the formation of the proposed bicyclic intermediate during the sirtuin deacylation catalysis depicted in Fig. 9. The a-1 0 -S-bicyclic intermediate (the boxed structure) formed via sirtuincatalyzed transformation on the probe is shown trapped at the SIRT5 active site (in stick model). The close-up view showing this trapped intermediate and its interactions with SIRT5 active site amino acid residues was adopted from ref. 56 (used with permission). Note: the observed stereochemistry at the tetrahedral carbon of the a-1 0 -S-associated five-membered ring in the a-1 0 -S-bicyclic intermediate is also indicated in the boxed structure.

Fig. 14 The three possible pathways for the collapse of the bicyclic intermediate depicted in Fig. 9 in the presence of water to afford the deacylated product and 2 0 -O-AADPR. Note: the stereochemistry at the tetrahedral carbon of the dioxo ring in the boxed structure is not explicitly indicated.

to a successful mass spectral detection of the bicyclic Intermediate II depicted in Fig. 12, Intermediate III could also be relatively long-lived and detectable by mass spectrometry.55 It is important to note that Intermediate III is a mimic of the boxed intermediate depicted in Fig. 14 on the path of the collapse of the bicyclic intermediate toward the deacylated product and 2 0 -O-AADPR. It would then be a tantalizing idea that a structural study with a peptide harboring L-2-amino-7-carboxamidoheptanoic acid would possibly be able to provide a crystal structure of a sirtuin with trapped longer-lived Intermediate III, thus furnishing more direct evidence for how the bicyclic


intermediate is collapsed to form the deacylated product and 2 0 -O-AADPR. The afore-mentioned previous success in using Ne-thioacyl-lysine-containing peptides to structurally elucidate the sirtuin deacylation reaction intermediate steps suggests that similar endeavors for trapping Intermediate III could also be successful. For pathways A and B, when considering the kinetic mechanism for the sirtuin-catalyzed deacylation reaction (vide supra), it seems to be difficult to appreciate a random release of the deacylated product and 2 0 -O-AADPR from the sirtuin active site unless the deacylated product is able to be held within the

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sirtuin active site until the release of 2 0 -O-AADPR. However, it would be possible that the kinetic mechanism(s) of SIRT1 and Sir2Tm is different from that of SIRT2 and Hst2, which could be possible, given the subtle active site infrastructure difference among different sirtuins. On the other hand, a pathway such as pathway C could be another possibility, and its operation seems to be consistent with the random release of the deacylated product and 2 0 -O-AADPR, since this pathway would lead to the simultaneous formation of the deacylated product and 2 0 -O-AADPR from the splitting of the depicted tetrahedral intermediate. It is clear that further studies are needed to tease out these possibilities and to ascertain the one(s) responsible for the collapse of the bicyclic intermediate to form the deacylated product and 2 0 -O-AADPR.

4. The (patho)physiological roles played by the sirtuin-catalyzed Ne-acyl-lysine deacylation reaction During the past years since the discovery of the Ne-acetyl-lysine deacetylation reaction catalyzed by the sirtuin family founding member (i.e. the yeast sir2 protein) in 2000, the sirtuin-catalyzed deacylation, deacetylation in particular, has been demonstrated to play an important regulatory role in multiple crucial cellular processes including transcription, DNA damage repair, and metabolism; and has been regarded as a current therapeutic target for human diseases including cancer, and metabolic and neurodegenerative diseases. The following section will briefly present the roles of the sirtuin-catalyzed deacylation reaction in three example (patho)physiological conditions, i.e. cancer, metabolism, and neurodegeneration. A key pre-requisite for a successful chemical biological functional interrogation of the sirtuin-catalyzed deacylation reaction is to develop potent, selective, and cell permeable chemical modulators for this enzymatic reaction. Therefore, a brief description of the different types of the chemical modulators that have been developed during the past few years for this enzymatic reaction will also be provided. It should be noted that some of the developed modulators have been used to explore/validate the therapeutic potentials of targeting the sirtuin-catalyzed deacylation reaction, the relevant efforts will therefore also be presented. 4.1. The sirtuin-catalyzed deacylation reaction in cancer, metabolism, and neurodegeneration It is clear from Table 1 that endogenous sirtuin substrates are present in major intracellular compartments (nucleus, mitochondrion, and cytosol), and therefore, it would be not difficult to appreciate that the sirtuin-catalyzed deacylation reaction is involved in regulating a variety of crucial fundamental cellular processes such as transcription and metabolism. Since the hallmark of cancer is an unchecked cell proliferation, the sirtuincatalyzed deacylation reaction could well be exploited by cancer cells to provide an underlying support for their survival. On the other hand, the sirtuin-catalyzed deacylation reaction could

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also be functioning to suppress the growth of cancer cells. Indeed, one debate in the field was whether the sirtuin-catalyzed deacylation reaction supports or suppresses cancer cell growth.24,58 It is now generally concurred that SIRT1, SIRT2, and SIRT3 could be tumour suppressors or promoters, contingent upon the cellular contexts and cell types. In normal cells, the deacylation reaction catalyzed by these three sirtuins would help to suppress tumour formation via genome maintenance, however, in cancer cells already established by certain oncogenic events, this enzymatic reaction and its functional consequences would actually help the cancer cells to grow. Based on our current understanding, SIRT4 and SIRT6 are both tumor suppressors; SIRT7 is a tumor promoter. The role of the SIRT5-catalyzed deacylation reaction in cancer is currently still unknown. Among the seven mammalian sirtuins, the SIRT1- and SIRT3catalyzed deacetylation reaction has been known to play an important role in regulating metabolic processes and maintaining cellular energy homeostasis.59–61 Even though SIRT1 can be present in the nucleus and cytosol, and the mature form of SIRT3 is a mitochondrial sirtuin, they are both able to catalyze the deacetylation of proteins that are important in regulating metabolic pathways. Since oxidative catabolic pathways in a eukaryotic cell occur primarily in its mitochondria, the other two mitochondrial sirtuins (i.e. SIRT4 and SIRT5) could be therefore also important in regulating cellular metabolism.20,61 Indeed, via catalyzing the deacetylation of malonyl-CoA decarboxylase, SIRT4 has been recently found to be able to decrease the enzymatic activity of this enzyme that can be present in both cytosol and mitochondria. Because malonyl-CoA decarboxylase catalyzes the conversion of malonyl-CoA back to acetyl-CoA, and malonyl-CoA and acetyl-CoA are the two starting metabolites for fatty acid biosynthesis, this activity decrease of malonyl-CoA decarboxylase would lead to an increased abundance of malonyl-CoA and an increased fatty acid biosynthesis and lipogenesis. Furthermore, because malonyl-CoA is an inhibitor of carnitine palmitoyl transferase I which facilitates the transport of the cytosolic fatty acyl-CoA into mitochondrial matrix for its b-oxidation, the accumulation of malonyl-CoA would inhibit fatty acid oxidation. It should be noted that SIRT4 is also known to be able to catalyze the ADP-ribosylation of glutamate dehydrogenase, an mitochondrial enzyme that catalyzes the conversion of glutamate to a-ketoglutarate. This enzymatic ADP-ribosylation reaction decreases the activity of glutamate dehydrogenase and consequently the amino acid-facilitated insulin secretion. SIRT5 was initially known to be a Ne-acetyl-lysine deacetylase able to catalyze the deacetylation of its native protein substrate carbamoyl phosphate synthetase 1, with the consequent activation of this urea cycle enzyme. It is now known that SIRT5 is able to more proficiently catalyze demalonylation, desuccinylation, and deglutarylation of carbamoyl phosphate synthetase 1.61,62 The SIRT5-catalyzed deglutarylation also activates carbamoyl phosphate synthetase 1. Another pathological condition in which the sirtuin-catalyzed deacylation reaction plays a regulatory role is neurodegeneration.63 This involvement is not as apparent as those in cancer and metabolism, however, the sirtuin-catalyzed deacylation can impact


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the cellular protein homoeostasis, in that it can inhibit the formation of toxic protein aggregates such as amyloid b, hyperphosphorylated tau, a-synuclein, and huntingtin. The formation of such protein aggregates is a hallmark of neurodegenerative diseases in which specific neural networks are impaired by these protein aggregates. In addition to suppressing the accumulation of toxic protein aggregates, the sirtuin-catalyzed deacylation reaction also exhibits other beneficial effects such as improvement of neural plasticity via promoting the transcription of those genes that are important for learning and memory. Therefore, activation of the sirtuin-catalyzed deacylation reaction would confer a protection against neurodegenerative diseases; and this activation could be realized by genetic or pharmacological means. 4.2. The chemical modulators for the sirtuin-catalyzed deacylation reaction During the past few years, inhibitors with different structural classes have been developed for the sirtuin-catalyzed deacylation reaction, as illustrated with the representative ones in Fig. 15.32,64–71 Major lead discovery strategies that have been considered for the development of such inhibitors include chemical library screening and the catalytic mechanism-based design. It should be noted that such approaches are quite often combined with traditional structure–activity–relationship (SAR) elaboration. One notable recent endeavor is the discovery of potent and selective SIRT2 inhibitors by employing a fragment-based approach.66 The best compound in the study (i.e. 64 in Fig. 15, compound numbering from the original paper) was found under the experimental conditions to exhibit IC50 values of 12 000, 48.3, and 44 200 nM against SIRT1, SIRT2, and SIRT3, respectively. Compound 64 resulted from a two-step SAR elaboration: (i) construction and screening of two libraries of ‘‘fragments’’; (ii) guided by the SAR information derived from the screening, binary compounds composed of the hit ‘‘fragments’’ (respectively from the two libraries) were then constructed and subjected to further SAR elaboration. Another notable recent study disclosed the discovery of a class of exceptionally potent (low nM) SIRT1/2/3 pan-inhibitors with the novel thieno[3,2-d]pyrimidine-6-carboxamide core functional scaffold.67 The best compound in the study (i.e. 31 in Fig. 15, compound numbering from the original paper) was found under the experimental conditions to exhibit IC50 values of 4.3, 1.1, and 7.2 nM against SIRT1, SIRT2, and SIRT3, respectively. Compound 31 resulted from a SAR elaboration on the hit compounds from a screening campaign on a DNA encoded small molecule library. Very recently, SirReal2 (Fig. 15) was identified from a chemical library screening campaign to be a potent and very selective SIRT2 inhibitor.68 With a HPLC-based sirtuin deacylation assay, SirReal2 was found in the study to exhibit a SIRT2 inhibitory IC50 value of 140 nM, which was 41000-fold more potent than inhibiting SIRT1, SIRT3, SIRT4, SIRT5, and SIRT6. Through structural analysis via X-ray crystallography and homology modeling, this potent and exceptionally selective SIRT2 inhibition of SirReal2 could be accounted for by its ability to assume a rigid conformation at the SIRT2 active site via an intramolecular


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hydrogen bond between amide N–H and one pyrimidine N, and thus to behave as a ‘‘molecular wedge’’ locking SIRT2 in a conformation preventing the normal catalysis. The formation of such a stabilized rigid conformation of SirReal2 seems to be unique to the SIRT2 active site, since other sirtuin active sites were predicted not to be able to support the formation of the intramolecular hydrogen bond between amide N–H and pyrimidine N. The ultimate discriminative sirtuin structural feature seems to be the ‘‘selectivity pocket’’ induced by the binding of the dimethylmercaptopyrimidine part of SirReal2 to a sirtuin active site, which is lined with significantly different amino acid residues among the active sites of the sirtuin family members. It is also worth noting that certain natural product-based sirtuin inhibitors have also been found, as illustrated with Amurensin G in Fig. 15.71 Among the lead discovery strategies, the catalytic mechanismbased design has proved to be quite successful since this approach has been found to be able to quickly and cost-effectively furnish potent inhibitory lead compounds for the deacylation reaction catalyzed by many sirtuin family members. In our opinion, it ought to be applicable to the deacylation reaction catalyzed by all the sirtuin family members since this approach is based on the conserved catalytic mechanism of the sirtuin-catalyzed deacylation reaction. The success with the above-described lead discovery strategies has also successfully circumvented the lack of a rich 3-dimensional structural information for human sirtuins. Indeed, while 3-dimensinal structures have been solved for the catalytic core from SIRT1-3, SIRT5, and SIRT6,22,25 only a limited number of ligand-enzyme co-crystal structures have been solved for human sirtuins, with perhaps the most significant one being the SIRT5 structure with the trapped bicyclic intermediate within its active site (vide supra). Of note, as afore-mentioned, a SIRT3 structure with a trapped a-10 -S-alkylamidate intermediate within its active site was also solved a few years ago.49 The successful trapping of these sirtuin-catalyzed intermediates ought to be ascribed to the use of the thioacyl-type of the catalytic mechanism-based inhibitory warheads shown in Fig. 15 (thiosuccinyl and thioacetyl, respectively). Together with what is described in Section 3.2, it is clear that the catalytic mechanism-based lead discovery approach entertains an additional advantage over the one based on chemical library screening in that the inhibitors developed with the former approach could also be effective mechanistic probes for the sirtuin-catalyzed deacylation reaction. The above-described lead discovery approaches have also found use in identifying activators for the sirtuin-catalyzed deacylation reaction as potential therapeutics for neurodegeneration and metabolic diseases such as type 2 diabetes and nonalcoholic fatty liver disease.32,60,63,70,72 Fig. 16 depicts representative compounds that have been claimed to be activators for the sirtuin-catalyzed deacetylation reaction.32,70,72 Of note, SIRT1 has been the primary sirtuin toward which past efforts have been directed in terms of identifying sirtuin activating compounds. It should also be pointed out that, while isonicotinamide has been shown to be a catalytic

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Fig. 15 The representative inhibitors for the sirtuin-catalyzed deacylation reaction. These inhibitors were discovered via the three indicated lead generation approaches, combined with further medicinal chemistry efforts. Note: the catalytic mechanism-based inhibitors of the Ne-thioacyl type (i.e. the depicted Ne-thioacetyl, Ne-thiosuccinyl, Ne-thiomyristoyl, and Ne-thiourea compounds), the carboxamide type, and the ethyl malonyl type all achieved inhibition against the sirtuin deacylation reaction via a sirutin-catalyzed formation of stalled catalytic intermediates, as illustrated in Fig. 10, 12, 13; for the ethyl malonylbased inhibitor, the a-carbon of the malonyl portion (instead of the amide oxygen) serves as the nucleophile during the sirtuin-catalyzed first step nucleophilic substitution reaction, affording the corresponding stalled intermediate. Of note, the catalytic mechanism-based sirtuin inhibitory warheads are highlighted in red, and several of them are also depicted in Fig. 6. Nicotinamide is another catalytic mechanism-based sirtuin inhibitor in that it is able to intercept the a-10 -Oalkylamidate intermediate along the sirtuin catalytic coordinate (depicted in Fig. 9) with the regeneration of NAD+ and the lysine Ne-acylated substrate; in this reaction, the pyridine ring N atom of nicotinamide nucleophilically attacks the C10 position of the a-10 -O-alkylamidate intermediate.

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Fig. 16 The representative compounds that have been claimed to be activators for the sirtuin-catalyzed deacetylation reaction. These compounds were discovered via the three indicated lead discovery approaches, combined with further medicinal chemistry efforts.

mechanism-based direct sirtuin activator,32,73 whether or not the other compounds shown in Fig. 16 also behave as direct sirtuin activators has been debated.58,70,72 Nevertheless, a recent study demonstrated that a few compounds including resveratrol, SRT1720, and SRT1460 (depicted in Fig. 16) were able to activate SIRT1 via directly binding to an N-terminal region on SIRT1; however, this allosteric activation was only observed with the use of certain SIRT1 native peptide substrates each harboring a bulky hydrophobic amino acid residue (Tyr, Trp, or Phe) at the position immediately C-terminal to the Ne-acetyl-lysine residue to be deacetylated.74 The finding from this study ought to be in line with the initial in vitro demonstration of a SIRT1 activation by the above-mentioned compounds with the use of certain artificial SIRT1 peptide substrates each harboring the bulky hydrophobic fluorophore (i.e. 7-amino-4-methylcoumarin) immediately C-terminal to the Ne-acetyl-lysine residue to be deacetylated. It should be noted that fluorophore 7-amino-4-methylcoumarin would mimic the above-mentioned bulky hydrophobic amino acid residues, and the initially observed direct SIRT1 activation by resveratrol and other compounds triggered a debate in the field because other researchers were subsequently unable to observe a SIRT1 activation with the same compounds when using the SIRT1 peptide substrates without a bulky hydrophobic moiety (amino acid side chain or other group) immediately C-terminal to the Ne-acetyl-lysine residue to be deacetylated.58,70,72 Isonicotinamide has been shown to be able to compete with nicotinamide for binding to sirtuin active site yet is unable to promote the regeneration of the two substrates because the pyridine ring N atom of the bound isonicotinamide would be positioned too far away to interact with the C1 0 position of the a-1 0 -O-alkylamidate intermediate.32,73 As described above, catalytic mechanism-based sirtuin inhibitors have also constituted a class of fairly effective chemical biological probes for an enhanced mechanistic understanding of the sirtuin-catalyzed deacylation reaction. Another level of the chemical biological investigation on sirtuins is to use their chemical modulators (inhibitors or activators) to facilitate our functional understanding of the sirtuin-catalyzed deacylation reaction. For this latter type of chemical biological investigation, a pre-requisite is to have cell permeable sirtuin modulators with sufficient potency and selectivity for a given sirtuin family member versus other sirtuin family members and the off-targets outside of the sirtuin family. It is worth noting that, even though fairly potent and/or selective sirtuin inhibitors have been developed, with three


notable examples being described above within this section, this type of the chemical biological investigation on sirtuins has not yet been seriously undertaken. Nevertheless, some of the existing sirtuin inhibitors have been employed to explore/validate the anti-cancer therapeutic potential of inhibiting the sirtuincatalyzed deacylation reaction. For example, an anti-cancer potential has been demonstrated on animal models with cambinol and tenovin-6, the two sirtuin inhibitors shown in Fig. 15.75,76 Since cambinol and tenovin-6 are dual SIRT1/SIRT2 inhibitors, it seems to be necessary to inhibit both SIRT1 and SIRT2 in order to realize an anti-cancer potential. A strong piece of evidence supporting this notion comes from the cellular studies with a few sirtuin inhibitors including salermide and EX-527 which are depicted in Fig. 15. Of note, while salermide is a SIRT1/SIRT2 dual inhibitor, EX-527 is the up-to-date still most potent and selective SIRT1 inhibitor versus SIRT2 and SIRT3. It was found that salermide, rather than EX-527, was effective in inhibiting cancer cell growth.77 As another example of the pharmacological use of sirtuin inhibitors in in vivo settings, an anti-Parkinsonism effect was demonstrated with the use of the relatively potent and selective SIRT2 inhibitor AGK2 shown in Fig. 15.78

5. Future perspectives Since the discovery of the NAD+-dependent histone Ne-acetyllysine deacetylase activity for the yeast sir2 protein in 2000, the founding member of the sirtuin family of deacylase enzymes, it has been about 15 years now, and this period of time has witnessed a steady increase in our understanding of these fantastic enzymes whose activities bridge many cellular processes especially gene expression and metabolism via the famous metabolite NAD+. Specifically, the sirtuin-catalyzed deacylatiom reaction has been demonstrated to play an important regulatory role in multiple crucial cellular processes such as transcription, DNA damage repair, and metabolism. In the meantime, mechanistic studies have also produced a clearer overall picture of the fascinating deacylation chemistry of sirtuins. Active site infrastructure variation and its impact on substrate recognition among different sirtuin family members have also been more and more appreciated. On the journey toward these knowledge advances, chemical biological means have constituted an important functional arsenal; technically, a variety of chemical probes and modulators

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(inhibitors and activators) have been developed and employed toward an enhanced mechanistic, substrate recognition behavior, and pharmacological understanding of the sirtuin-catalyzed deacylation reaction. With these having been described, however, our understanding of sirtuins is still limited overall in that: (i) the mechanistic picture for the sirtuin-catalyzed deacylation reaction is still incomplete, concerning how the bicyclic intermediate collapses in the presence of water to afford the deacylated product and 2 0 -O-AADPR (see Fig. 9); (ii) the regulatory role of the variable N-/C-termini of sirtuin proteins is still poorly understood; (iii) the therapeutic potential for the sirtuin-catalyzed deacylation reaction has been under-explored. In order to address these outstanding questions, an important future step would be to develop superior chemical probes with unique chemical and enzymatic properties, superior sirtuin direct modulators (activators and inhibitors). The availability of these reagents (both the cell impermeable and permeable types) would facilitate the mechanistic investigation and therapeutic potential exploration for the sirtuin-catalyzed deacylation reaction. The availability of these reagents (of the cell permeable type) would also facilitate the further functional dissection for the sirtuin-catalyzed deacylation reaction, given the unique advantage of chemical biology means being a reversible and conditional protein functional knockdown technology.79 We will also see if chemical biology means could be developed to assess the regulatory role of the variable N-/C-termini of sirtuin proteins. In addition, to devise and use novel chemical biology means to directly identify endogenous substrates of the sirtuin-catalyzed deacylation reaction ought to be also a rewarding future endeavor in the field.

Acknowledgements The work in the laboratory of the corresponding author (W. Zheng) on sirtuins at Jiangsu University has been supported by the National Natural Science Foundation of China (grant no. 21272094), the Jiangsu provincial specially-appointed professorship, the Jiangsu provincial ‘‘innovation and venture talents’’ award plan, and Jiangsu University. The work in his laboratory on sirtuins at University of Akron was supported by the U.S. National Institutes of Health (CA152972), the James L. and Martha J. Foght Endowment, and University of Akron.

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