A Continuous Fluorogenic Sirtuin 2 Deacylase Assay

• Journal List
• Philos Trans R Soc Lond B Biol Sci
• v.373(1748); 2018 Jun 5
• PMC5915714

Philos Trans R Soc Lond B Biol Sci. 2018 Jun 5; 373(1748): 20170070.

Identification of a novel small molecule that inhibits deacetylase but not defatty-acylase reaction catalysed by SIRT2

Norio Kudo

¹Seed Compounds Exploratory Unit for Drug Discovery Platform, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Akihiro Ito

²Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

³School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Mayumi Arata

¹Seed Compounds Exploratory Unit for Drug Discovery Platform, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Akiko Nakata

¹Seed Compounds Exploratory Unit for Drug Discovery Platform, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Minoru Yoshida

¹Seed Compounds Exploratory Unit for Drug Discovery Platform, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

²Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

⁴Chemical Genetics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

⁵Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Tokyo 113-8657, Japan

Abstract

SIRT2 is a member of the human sirtuin family of proteins and possesses NAD⁺-dependent lysine deacetylase/deacylase activity. SIRT2 has been implicated in carcinogenesis in various cancers including leukaemia and is considered an attractive target for cancer therapy. Here, we identified NPD11033, a selective small-molecule SIRT2 inhibitor, by a high-throughput screen using the RIKEN NPDepo chemical library. NPD11033 was largely inactive against other sirtuins and zinc-dependent deacetylases. Crystallographic analysis revealed a unique mode of action, in which NPD11033 creates a hydrophobic cavity behind the substrate-binding pocket after a conformational change of the Zn-binding small domain of SIRT2. Furthermore, it forms a hydrogen bond to the active site histidine residue. In addition, NPD11033 inhibited cell growth of human pancreatic cancer PANC-1 cells with a concomitant increase in the acetylation of eukaryotic translation initiation factor 5A, a physiological substrate of SIRT2. Importantly, NPD11033 failed to inhibit defatty-acylase activity of SIRT2, despite its potent inhibitory effect on its deacetylase activity. Thus, NPD11033 will serve as a useful tool for both developing novel anti-cancer agents and elucidating the role of SIRT2 in various cellular biological processes.

This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Keywords: SIRT2, NAD⁺-dependent lysine deacetylase, deacylase, crystal structure, anti-cancer drug development, high-throughput screening

1. Introduction

Nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases constitute an enzyme family called sirtuin, which is conserved from bacteria to humans. These enzymes control a wide variety of cellular processes such as ageing, metabolism and gene silencing. The human genome encodes seven sirtuin members (SIRT1–7), which exhibit different subcellular localizations and substrate specificities [1]. SIRT2 is a predominantly cytoplasmic protein [2], which acts as a deacetylase for a variety of protein substrates, including α-tubulin [3], histones [2], transcriptional factors such as Foxo3a [4], Foxo1 [5] and p65 [6], metabolic enzymes such as lactate dehydrogenase A [7], phosphoglycerate mutase [8] and pyruvate kinase [9], a checkpoint kinase, BubR1 [10] and the actin-binding protein cortactin [11,12]. Through the deacetylation of these substrates, SIRT2 plays physiological roles in diverse biological processes such as the cell-cycle, differentiation, energy metabolism, ageing and cell migration. In addition to its deacetylase activity, emerging evidence suggests that SIRT2 acts as a deacylase that removes various acyl groups on lysine residues, including crotonylation and long-chain fatty acyl modifications such as myristoylation, and 4-oxononanoylation [13–16], although the physiological roles of SIRT2 as a deacylase remain to be elucidated.

A number of SIRT2 inhibitors, including AGK2 [17], salermide [18] and the macrocyclic peptide S2iL5 [19], have been reported. However, isozyme selectivity and/or cellular potency of most of these compounds appear to be poor. Recently, Jung and co-workers discovered SirReal2, a potent and selective SIRT2 inhibitor [20]. Target enzyme selectivity of SirReal2 is likely based on its unique mechanism of SIRT2 inhibition, by which SirReal2 binds to the hydrophobic cavity named 'selectivity pocket' created by conformational changes in the zinc-binding small domain [20]. In this study, we identified NPD11033, a potent, selective, and cellular effective inhibitor of SIRT2, with a structural framework different from existing SIRT2 inhibitors. X-ray crystallographic analyses revealed a unique mechanism of selective SIRT2 inhibition similar to that of SirReal2. The cofactor-binding loop adopts a new conformation different from other SIRT2–inhibitor complexes, owing to the hydrogen-bonding network between NPD11033 and SIRT2. Interestingly, NPD11033 was inactive at inhibiting the demyristoylation reaction catalysed by SIRT2, despite its potent inhibitory effect on deacetylation by SIRT2. Thus, NPD11033 may serve as a novel chemical tool for elucidating functions of SIRT2, a dual-specificity deacylase in cells.

2. Material and methods

(a) Material and cell culture

NPD11033 and RK-0310020 were purchased from Pharmeks Ltd (Moscow, Russia) through a local distributor. SirReal2 and nicotinamide (NA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Trichostatin A (TSA) was prepared as described previously [21]. Mouse monoclonal eIF5A antibody was purchased from BD Bioscience (San Jose, CA, USA). Rabbit monoclonal SIRT2 (EPR1667) antibody was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibody against acetylated eIF5A (Ac-eIF5A) was raised as described previously [22]. Mouse monoclonal α-tubulin antibody (B-5-1-2) was obtained from Sigma-Aldrich. PANC-1 cells were cultured in DMEM medium (Wako Pure Chemical Industries, Ltd, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum and antibiotics at 37°C, 5% CO₂ in a humidified incubator.

(b) siRNA transfection

AllStars Negative Control siRNA (Qiagen, Hilden, Germany) was used as a non-targeting control small interfering RNA (siRNA) oligo. Silencer Select siRNAs (Thermo Fisher Scientific Inc., Waltham, MA, USA) were used for SIRT2-knockdown (s22707 and s22708). The following target sequences of two SIRT2 siRNA oligos were used: SIRT2 siRNA#1 (s22707), 5′-AGAAACAUCCGGAACCCUUTT-3′; SIRT2 siRNA#2 (s22708), 5′-GCUCAUCAACAAGGACAAATT-3′. Cells were transfected with siRNA oligos using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific Inc.).

(c) Expression, purification and crystallization of SIRT2

The gene encoding human SIRT2 (Uniprot {"type":"entrez-protein","attrs":{"text":"Q8IXJ6","term_id":"38258608","term_text":"Q8IXJ6"}}Q8IXJ6, residues 54–356) was cloned into pGEX-4T3 vector using BamHI and EcoRI sites. The GST-fused SIRT2 was overexpressed in Escherichia coli BL21(DE3) cells overnight at 30°C. Overexpression was induced with isopropyl-beta-d-thiogalactoside (0.3 mM) at an OD₆₀₀ of 0.8–1.0. Cells were harvested and resuspended in BugBuster protein extraction reagent (Merck Millipore), and purified using Glutathione Sepharose 4B resin (GE Healthcare). After thrombin digestion, SIRT2 was further purified using a Resource Q column (GE Healthcare, 20 mM Tris-HCl pH 8.0 and 0–1 M NaCl). The purified SIRT2 has two residues (Gly-Ser) at the N-terminus derived from the thrombin recognition sequence of pGEX-4T3. These two residues are identical to the Gly52 and Ser53 residues of SIRT2. The purified protein consists of residues 52–356 of SIRT2.

Purified SIRT2 (17 mg ml⁻¹) and NPD11033 (final concentration of 1 mM) were mixed, and crystallized. Crystals for X-ray crystallography were obtained using 25% (w/v) PEG3350 and 0.1 M BisTris buffer, pH 5.5, at 20°C.

(d) Data collection, structure determination and refinement

Crystals were frozen with liquid nitrogen, using 10–15% glycerol as a cryoprotectant. X-ray diffraction data were collected at 100 K in a nitrogen gas stream at the synchrotron beamlines, PF-AR NW12A and PF-17A at Photon Factory, KEK, Japan. Data were processed and scaled with the HKL2000 program [23]. The crystal structures were determined by the molecular replacement method with molrep [24], using the structure of SIRT2 in complex with H3K9-myr peptide (PDB entry 4Y6 L [25]). Refinement and model building were performed with refmac5 [26,27], arp/warp [28] and Coot [29]. The geometric quality of the model was assessed with MolProbity [30]. Data collection and refinement statistics are listed in table 1. Structural illustrations were generated using PyMol (Schrödinger).

Table 1.

Diffraction data and refinement statistics.

crystallization
co-crystallized with	NPD11033
data collection statistics
X-ray source	PF-AR NW12A
space group	P21
unit cell
a (Å)	34.9
b (Å)	115.7
c (Å)	71.1
α (°)	90.0
β (°)	89.9
γ (°)	90.0
wavelength (Å)	1.000
resolution (Å)	50.0–1.99
	(2.02–1.99)
unique reflections	38 720
completeness (%)a	99.9 (98.6)
R merge (%)a	3.9 (7.5)
I/σ(I)a	35.8 (17.1)
Wilson B (Å2)	22.3
refinement statistics
resolution range (Å)	20.0–2.0
no. of reflections	36 188
no. of non-hydrogen atoms	4825
R work (%)	21.0
R free (%)	24.2
RMS deviations
bond length (Å)	0.008
bond angle (°)	1.38
B-factors (Å2)
protein	28.4
inhibitor	27.8
waters	24.8
Ramachandran plot (%)
favoured region	97.4
allowed region	2.6
outlier region	0.0

(e) In vitro lysine deacetylase and deacylase assays

Enzymatic activities of zinc-dependent histone deacetylases (HDACs) HDAC1 and HDAC6 were measured by a fluorogenic assay as described previously [31]. Measurements of in vitro deacetylase activities of human sirtuins SIRT1, 2 and 3 were carried out by an electrophoretic mobility shift assay or a fluorogenic assay as described previously [32]. Measurements of in vitro deacylase activities of SIRT2 were performed by a fluorogenic assay as described previously with modifications [33]. Briefly, recombinant SIRT2 proteins were incubated with an aminomethylcoumarin (AMC)-conjugated myristoylated-lysine-peptide (50 µM Ac-RHKK(Myr)-MCA; MCA = methylcoumarinamide) and 0.1 mM NAD in 20 µl of assay buffer (50 mM Tris-HCl (pH 9.0), 4 mM MgCl₂, 0.2 mM DTT) at 37°C for 60 min. The reaction was stopped by the addition of 20 µl of trypsin (20 mg ml⁻¹) and incubated at 37°C for 15 min. The released AMC was measured using a fluorescence plate reader. The 50% inhibitory concentrations (IC₅₀) were determined as the means with s.d. calculated from at least three independent dose–response curves using the Origin software (OriginLab, Northampton, MA, USA).

(f) Immunoblotting

Whole cell lysates were prepared by directly adding 1× SDS-PAGE sample loading buffer and heating at 95°C for 10 min. Immunoblotting was carried out as described previously [12].

(g) Statistical analysis

To determine significance of differences between two groups, the Student's t-test was applied using Microsoft Excel. **p < 0.01.

(h) Cell viability assay

PANC-1 cells were grown in a 96-well white plate (5 × 10³ cells per well). Cell viability was measured with the ATPlite luminescence assay system (PerkinElmer, Waltham, MA, USA) using a Synergy H4 hybrid reader (BioTeck, Tokyo, Japan).

3. Results

(a) Identification of a selective SIRT2 inhibitor

In order to identify small molecules that inhibit the deacetylase activity of SIRT2, we screened the RIKEN natural products depository (NPDepo) chemical library, mostly consisting of natural products and their synthetic derivatives [34], using an in vitro fluorogenic assay [35] and identified NPD11033 as a novel SIRT2 inhibitor (figure 1 a). We performed an electrophoretic mobility shift assay using a fluorescently labelled acetyl-lysine-peptide substrate [36] for further validation of in vitro SIRT2 inhibition. NPD11033 inhibits SIRT2 in a concentration-dependent manner with an IC₅₀ value of 0.46 ± 0.056 µM (figure 1 b). On the other hand, NPD11033 had only a marginal effect on other human sirtuin members including SIRT1 and SIRT3 (figure 1 b). In addition, NPD11033 did not inhibit enzymatic activities of zinc-dependent HDACs, such as HDAC1 and HDAC6, despite strong inhibition by TSA, a pan-inhibitor for zinc-dependent HDACs (figure 1 c). Thus, NPD11033 is a selective inhibitor specific to SIRT2.

Selective inhibition of SIRT2 by NPD11033 in vitro. (a) Chemical structure of NPD11033. (b,c) Effect of NPD11033 on lysine deacetylase activities in vitro. The inhibitory activity of NPD11033 against SIRT1, 2 and 3 (b) or HDAC1 and 6 (c) was estimated using an electrophoretic mobility shift assay or a fluorogenic assay, respectively. Error bars represent the standard deviation of three independent assays.

(b) Structure of SIRT2–inhibitor complex

To gain detailed information about the mechanism of inhibition, we crystallized SIRT2 in complex with NPD11033, and determined its crystal structure by X-ray crystallography at 2.0 Å resolution (table 1, figure 2 a,b). We screened several SIRT2 constructs suitable for crystallization and finally obtained a construct of the SIRT2 catalytic domain from Gly52 to Ser356. The electron density maps are well defined for SIRT2 with one NPD11033 molecule. The crystal structure has a typical sirtuin structure composed of two domains: a zinc-binding small domain and the Rossmann-fold NAD⁺-binding large domain. Between the two domains, a large groove exists for NAD⁺ and substrate binding. NPD11033 is encompassed by hydrophobic residues in a cavity created upon its binding to SIRT2. The cavity is located deep within the acetyl-substrate-binding site, behind the active site residue His187 (figure 2 c,d).

(a) Overall structure of human SIRT2 in complex with NPD11033, in a ribbon representation. NPD11033 and zinc ion are drawn in space-filled spheres in which yellow, blue, red and grey represent C, N, O and Zn atoms, respectively. (b) Right-side view of panel (a). (c) Molecular surface of SIRT2 in complex with NPD11033 cut at the level of the cavity. The molecular surface and the cross-section of SIRT2 are drawn in brown and grey, respectively. The arrows indicate viewpoints of panel (e) and figure 3 c. (d) Close-up view of NPD11033 in the hydrophobic cavity. Red dotted line shows a cofactor-binding loop (residues 92–104) (e) Hydrogen-bonding network around NPD11033. (f) Molecular surface of SIRT2 in complex with H3K9-myr (PDB entry 4Y6 L). (g) Close-up view of SIRT2 in complex with SirReal2 and acetyl-lysine substrate (PDB entry 4RMH).

(c) Hydrogen-bonding network

The newly generated cavity consists of hydrophobic residues such as phenylalanines (Phe96, Phe119, Phe131, Phe190, Phe235) and isoleucines (Ile93, Ile169, Ile232), and almost no specific interaction was observed within the cavity (figure 2 d). At the entrance of this cavity, an oxygen atom at the oxopyridyl group of NPD11033 is present within a hydrogen-bonding distance of 2.8 Å to the N_ε of His187 (figure 2 e). The N_ε of His187 is also close to the carbonyl oxygen of Gln167, and N_η1 of Arg97, which is proximal to the amide nitrogen of Gln167. The main chain carbonyl oxygen of Gln267 forms two hydrogen bonds with the N_η2 of Arg97 and the amide nitrogen of Gln167. Arg97 extends to the Rossmann-fold NAD⁺-binding domain and is fixed by these interactions in SIRT2 complexed with NPD11033.

(d) Structure and activity relationship analysis

NPD11033 is a synthetic compound derived from the natural alkaloid cytisine, a known nicotine receptor ligand [37]. A substituted phenyl moiety with two 1,1-dimethylpropyl groups of NPD11033 was placed at the bottom of the hydrophobic cavity (figures2 c and ​ 3 c). We measured the inhibitory activities of the NPD11033 derivative, RK-0310020, of which 1,1-dimethylpropyl groups are changed to methyl-groups (figure 3 a). RK-0310020 lost the inhibitory activity (figure 3 b), suggesting that the bulky 1,1-dimethylpropyl groups are important for its action. Thus, these hydrophobic interactions are key to the inhibition of SIRT2 deacetylase activity.

Structure–activity relationship of NPD11033. The activity of the NPD11033 derivative RK-0310020 (a) to inhibit the deacetylase activity of SIRT2 was estimated using an electrophoretic mobility shift assay (b). Error bars represent the standard deviation of three independent assays. (c) Two tert-pentyl groups (red dotted lines) shows accommodation of NPD11033 in a hydrophobic cavity.

(e) Inability of NPD11033 to inhibit deacylation

Recently, SIRT2 has been shown to remove not only acetyl groups but also other acyl groups on lysine residues. The newly identified activity includes long-chain fatty acyl deacylation such as demyristoylation [14,15]. Therefore, we tested whether NPD11033 can inhibit the demyristoylation reaction catalysed by SIRT2 using an in vitro assay with fluorogenic myristoyl-lysine peptides as substrates [33]. In contrast to the strong inhibition of deacetylation (figure 1 b), NPD11033 was inactive at inhibiting the demyristoylation activity of SIRT2, even at very high concentration (1 mM) (figure 4 a). SirReal2, a known specific inhibitor of SIRT2 [20], also failed to block the demyristoylation reaction of SIRT2 as reported previously (figure 4 b,c) [33]. On the other hand, suramin, a symmetric polyanionic naphthylurea that had been reported to inhibit SIRT2 [38,39], could inhibit both the deacetylase and defatty-acylase activities of SIRT2 with comparable IC₅₀ values (figure 4 d,e). Because suramin interacts with both the NAD⁺- and substrate-binding sites in SIRT5 [39], the differential activities of these SIRT2 inhibitors may be ascribable to the difference in their modes of SIRT2 binding.

Effect of on NPD11033 on demyristoylation reaction of SIRT2. The activity of NPD11033 (a), SirReal2 (b,c) and suramin (d,e) to inhibit defatty-acylase activity (a,b,d) or deacetylase activity (c,e) of SIRT2 was estimated using a fluorogenic assay. Error bars represent the standard deviation of three independent assays.

(f) In vivo inhibition of SIRT2 by NPD11033

We next examined the effect of NPD11033 on in vivo SIRT2 inhibition by investigating the acetylation of eukaryotic translation initiation factor 5A (eIF5A), a known physiological substrate of SIRT2. We previously demonstrated that both HDAC6 and SIRT2 regulate the acetylation of eIF5A and that simultaneous inhibition of both enzymes is necessary, but inhibition of either one of them alone is insufficient for a marked increase in eIF5A acetylation in cells [22,40]. Therefore, we tested for the level of eIF5A acetylation in the presence of TSA, which inhibits HDAC6 but not SIRT2, in order to assess the in vivo inhibition of SIRT2 by NPD11033. SirReal2 was used as a positive control. Human pancreatic cancer PANC-1 cells were treated with NPD11033 at various concentrations together with TSA, and the acetylation level of eIF5A was evaluated by western blotting using an antibody that specifically recognizes the acetylated form of eIF5A [22]. As previously reported, treatment with TSA alone did not have an impact on eIF5A acetylation, but treatment with NPD11033 in the presence of TSA increased eIF5A acetylation in a concentration-dependent manner (figure 5 a). Importantly, RK-0310020, which lacks in vitro activity to inhibit SIRT2 (figure 3), did not alter the eIF5A acetylation level, confirming that the increase in eIF5A acetylation by NPD11033 is due to the inhibition of SIRT2 (figure 5 a). For further validation of in vivo inhibition of SIRT2 by NPD11033, we examined the expression level of the checkpoint kinase BubR1; its abundance is upregulated by SIRT2-mediated deacetylation [10]. Treatment with NPD11033 but not RK-0310020 reduced the level of BubR1 expression in a dose-dependent manner (figure 5 b), supporting the notion of inhibition of cellular SIRT2 by NPD11033.

Effect of NPD11033 on cellular activities of SIRT2. (a) Effect of NPD11033 on the acetylation of eIF5A in PANC-1 cells. PANC-1 cells were treated with different concentrations of NPD11033, 20 µM RK-0310020 or 20 µM SirReal2 as a positive control in the presence of 1 µM TSA for 8 h. The lysates were immunoblotted with the indicated antibodies. (b) Effect of NPD11033 on BubR1 expression level in HeLa cells. HeLa cells were treated with the indicated concentrations of NPD11033, 30 µM of RK-0310020 or 20 mM nicotinamide (NA) as a positive control for 24 h. The lysates were immunoblotted with the indicated antibodies. (c,d) Effect of knockdown of SIRT2 on cell viability in PANC-1 cells. PANC-1 cells were transfected with non-targeting control siRNA oligos (siCtrl) or siRNA oligos targeting SIRT2 (siSIRT2 #1 and #2). After 5 days transfection, SIRT2 expression levels were determined by western blotting (c). Cell viability was measured using the ATPlite luminescence assay 7 days after transfection (d). Error bars represent the standard deviation of four independent assays. **p = 0.01. (e) Effect of NPD11033 on cell viability in PANC-1 cells. PANC-1 cells were treated with various concentrations of SirReal2, NPD11033 or RK-0310020 for 72 h. Cell viability was measured by the ATPlite luminescence assay. Error bars represent the standard deviation of three independent assays.

Next, we examined the effect of NPD11033 on pancreatic cancer cell growth. We chose PANC-1, a human pancreatic cancer cell line, because knockdown of SIRT2 significantly reduced the cell viability of PANC-1 cells (figure 5 c,d). We found that NPD11033 as well as SirReal2 reduced the cell viability of human pancreatic cancer PANC-1 cells in a dose range similar to that required for the increase in eIF5A acetylation (figure 5 e). The IC₅₀ value of NPD11033 for cell growth of PANC-1 cells was 7.5 ± 1.3 µM. Importantly, RK-0310020, an inactive analogue (figure 3), failed to inhibit cell growth of PANC-1 cells up to 60 µM (figure 5 c). These results suggest that NPD11033 kills PANC-1 cells by inhibiting cellular SIRT2 activity.

4. Discussion

NPD11033 was identified as a novel SIRT2 inhibitor during the course of our screening programme, and its mode of inhibition was analysed by X-ray crystallography of the SIRT2–inhibitor complex. The inhibitor was accommodated in a hydrophobic cavity that was created upon its binding. Structure–activity relationship analysis revealed the importance of the hydrophobic interaction in inhibiting SIRT2 deacetylase activity. A similar mode of inhibition by the compound SirReal2 had already been reported by the Jung group in 2015 [20]. In contrast to NPD11033, however, SirReal2 had no direct hydrogen-bonding interaction with SIRT2. The heterocyclic ring moiety of NPD11033 protrudes from the hydrophobic cavity and interacts with His187, the active site residue, while the naphthyl moiety of SirReal2 does not (figure 2 e,g). Indeed, we tried to crystallize NPD11033 and acetyl-lysine substrate peptide, but no crystals were obtained, while a ternary complex of SIRT2, SirReal2 and acetyl-lysine substrate was reported [20]. More recently, a crystal structure of SIRT2 with a 2-anilinobenzamide compound, another class of SIRT2-specific inhibitors, was solved, which demonstrated that the inhibitor was also accommodated in the hydrophobic cavity without forming any direct hydrogen bond to SIRT2 [41]. Thus, interaction with His187 appears to be one of the unique molecular mechanisms by which NPD11033 inhibits SIRT2.

In comparison with crystal structures of SIRT2 in the apo-form [42] or in complex with inhibitors [19,20,43–45] that have so far been reported, NPD11033 binding induces a unique conformational change of a loop (residues 92–104), which is called the 'cofactor-binding loop' [19,46] (figure 2 d). With this conformational change, the side chain of Phe96 is positioned toward the bottom side of the cavity in the SIRT2–NPD11033 structure, which was not seen in other. SIRT2-inhibitor structures (figure 2 d,g). The side chain occupies the space for the nicotinamide (NA) moiety of NAD⁺ binding [20,47]. In other structures, the side chain of Phe96 is towards the opposite side, and there is a room for NA binding or water.

For example, in the SIRT2–SirReal2 structure, a water molecule is situated there, and forms a water-mediated hydrogen bond between SirReal2 and the main chain carbonyl group of Pro94 (figure 2 g). Note that Phe96 is called a 'gate-keeper residue' for its work in the uptake or release of NAD⁺ [42,48]. The subsequent residue, Arg97, also in the cofactor-binding loop, adopts a characteristic conformation. In the SIRT2–NPD11033 structure, Arg97 forms hydrogen bonds with Gln167 and Gln267 of the Rossmann-fold NAD⁺-binding domain (figure 2 e). Thus, the cofactor-binding loop is pulled towards the Rossmann-fold domain via these hydrogen bonds, and the loop adopts a compact conformation. Even though the second-generation compound derived from SirReal2 forms hydrogen bonds with Arg97 through the extended triazole moiety, the cofactor-binding loop adopts a similar conformation to the original SirReal2 compound [49]. Based on its unique enzyme-inhibitor structure, NPD11033 will provide new insights for further development of specific inhibitors.

SIRT2 is active in removing the long-chain fatty acyl groups from acyl-modified lysine residues such as myristoyl-lysine [14,15]. We previously reported crystal structures of SIRT2 in complex with myristoyl-lysine-peptide [25], in which the acyl chain of myristoyl-lysine exists in a similar hydrophobic cavity with a different shape (figure 2 c,f). Importantly, while NPD11033 effectively inhibited deacetylase activity (figure 1 b), it was inactive at inhibiting the defatty-acylase activity of SIRT2 (figure 4 a). Because it is likely that the acyl-substrate and inhibitor compete with each other for binding to the hydrophobic cavity, the mechanism of the NPD11033-resistant defatty-acylase activity of SIRT2 is still unknown. It seems possible that both the acyl-substrate and the inhibitor can exist in the same hydrophobic cavity with a further conformational change, thereby allowing the defatty-acylation reaction to occur (retention model). Alternatively, it is also possible that the inhibitor present in the cavity can be kicked-out by repulsion from the acyl chain of the substrate (kick-out model). Further study is needed to determine which model is feasible.

It has been reported that Sirt2-deficient mice develop hepatocellular carcinoma, suggesting that SIRT2 functions as a tumour suppressor [50]. On the other hand, recent growing evidence suggests that SIRT2 is involved in tumorigenesis. Indeed, pharmacological or genetic inhibition of SIRT2 exhibited anti-cancer activities against some types of cancers including leukaemia [51,52] and breast cancer [53,54]. Thus, it seems possible that SIRT2 inhibits and promotes tumorigenesis depending on cancer cell type. In pancreatic cancer, SIRT2 protein is upregulated in cancer tissues compared with adjacent tissues [7], suggesting that SIRT2 possesses some oncogenic functions in pancreatic cancer. Indeed, we demonstrated that both pharmacological and genetic inhibition of SIRT2 by NPD11033 and siRNA oligos, respectively, reduced cell growth of pancreatic cancer PANC-1 cells (figure 5). Thus, our findings suggest that SIRT2 is an attractive target for pancreatic cancer therapy and that NPD11033 will serve as a useful starting point for development of an anti-pancreatic cancer drug targeting SIRT2.

Acknowledgements

This work was performed under the approval of the Photon Factory Program Advisory Committee (proposal nos 2013G674 and 2015G615). We are grateful to the RIKEN NPDepo for supplying chemical libraries, the Biomolecular Characterization Unit, RIKEN CSRS for technical help with N-terminal sequencing, the Support Unit for Bio-Material Analysis, RIKEN BSI Research Resources Center for DNA sequencing analysis, Dr Fumiyuki Shirai for technical advice, Dr Asad Ali Shah, Dr Tariq Mohammad, Ms Satoko Maeda for technical assistance, and Mr Elliot Bradshaw for critical reading.

Data accessibility

Atomic coordinates and structural factors have been deposited in the Protein Data Bank (PDB entry 5Y0Z).

Authors' contributions

N.K. carried out structural studies and drafted the manuscript. A.I. carried out biochemical studies and drafted the manuscript. M.A. and A.N. carried out biochemical experiments. M.Y. coordinated the study and helped draft the manuscript. All authors gave final approval for publication.

Competing interests

We declare we have no competing interests.

Funding

This work was supported, in part, by the Japan Society for the Promotion of Science (JSPS) under Grants-in-Aid for Scientific Research (S) (26221204) and for Challenging Exploratory Research (16K14674), and the Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was inspired by the JSPS Asian Chemical Biology Initiative.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915714/

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