Interactions between sirtuins and fluorogenic small-molecule substrates offer insights into inhibitor design

Article abstract of DOI:10.1039/c7ra05824a

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent lysine deacylases regulating metabolism and stress responses and are involved in human pathologies such as neurodegeneration. In this study, four fluorogenic small-molecule substrates, i.e., acetyl-(AcBKA), crotonyl-(CrBKA), succinyl-(SuBKA), and myristoyl-(MyBKA)-containing substrates, were synthesized and tested against three representative sirtuin isoforms (i.e., SIRT2, SIRT5, and SIRT6). Enzyme kinetic results indicate that the fluorogenic small-molecule substrates have similar sirtuin-isoform preference as compared to peptide substrates. ITC analyses reveal that AcBKA or MyBKA binding to SIRT2 is mainly driven by entropy, whereas SuBKA binding to SIRT5 is driven by enthalpy. The SIRT5:SuBKA complex crystal structure reveals a new substrate-binding mode that is different from peptide substrate binding modes, but involves Tyr102, Arg105, and other catalytically important residues on Loop S; this indicates that SuBKA is desuccinylated by SIRT5 probably through the catalytic mechanism proposed for peptide substrates. The biophysical and structural results presented herein will provide thermodynamic insights and key pharmacophore features for the development of selective sirtuin isoform-specific inhibitors.

Full text of DOI:10.1039/c7ra05824a

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PAPER
Interactions between sirtuins and fluorogenic
small-molecule substrates offer insights into
inhibitor design†
Cite this: RSC Adv., 2017, 7, 36214
Hua-Li Wang,‡ Sha Liu,‡ Zhu-Jun Yu, Chengyong Wu, Linna Cheng, Yuxi Wang,
*
*
Kai Chen, Shu Zhou, Qiang Chen, Yamei Yu and Guo-Bo Li
Sirtuins are nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases regulating metabolism
and stress responses and are involved in human pathologies such as neurodegeneration. In this study, four
fluorogenic small-molecule substrates, i.e., acetyl-(AcBKA), crotonyl-(CrBKA), succinyl-(SuBKA), and
myristoyl-(MyBKA)-containing substrates, were synthesized and tested against three representative
sirtuin isoforms (i.e., SIRT2, SIRT5, and SIRT6). Enzyme kinetic results indicate that the fluorogenic small-
molecule substrates have similar sirtuin-isoform preference as compared to peptide substrates. ITC
analyses reveal that AcBKA or MyBKA binding to SIRT2 is mainly driven by entropy, whereas SuBKA
binding to SIRT5 is driven by enthalpy. The SIRT5:SuBKA complex crystal structure reveals a new
substrate-binding mode that is different from peptide substrate binding modes, but involves Tyr102,
Arg105, and other catalytically important residues on Loop S; this indicates that SuBKA is desuccinylated
by SIRT5 probably through the catalytic mechanism proposed for peptide substrates. The biophysical
and structural results presented herein will provide thermodynamic insights and key pharmacophore
features for the development of selective sirtuin isoform-specific inhibitors.
Received 24th May 2017
Accepted 5th July 2017
DOI: 10.1039/c7ra05824a
rsc.li/rsc-advances
and glutarylation,3,8 whereas SIRT6 prefers long-chain fatty acyl
groups such as myristoyl-lysine residues.9,10
Introduction
Sirtuins are nicotinamide adenine dinucleotide (NAD+)-depen-
dent lysine deacylases (Fig. 1).1 Humans have seven sirtuin
enzymes (SIRT1-7) that are divided into four classes: SIRT1-3
belong to class I, SIRT4 belongs to class II, SIRT5 belongs to
class III, and SIRT6-7 belong to class IV.2 All sirtuins have
a conserved catalytic core domain, but distinct N- or C-terminal
extensions.3 Originally, sirtuins were known as deacetylases
acting on histones, transcription factors, and metabolic
enzymes.1,2 However, recently, sirtuins have been found to be
able to catalyze the enzymatic removal of other acyl modica-
tions besides acetyl moieties (Table S1†). For example, SIRT1-3
can remove propionyl and butyryl groups;4 more interestingly,
they can also remove long-chain acyl groups and crotonyl
moieties.5–7 Especially, SIRT5 can highly efficiently hydrolyze
acidic acyl modications such as succinylation, malonylation,
Due to their multifaceted catalytic activities on various
substrate proteins (Table S1†), sirtuins are pivotal regulators in
various cellular processes, including transcription, genome
stability, and energy metabolism,1,2 and are involved in human
pathologies such as diabetes,11,12 cancer,13–16 and neuro-
degeneration.2,17 These features make sirtuins potentially
attractive targets for drug discovery. Thus, sirtuin modulators
are sought as chemical tools and potential therapeutics.14,18–20
Current activity assays for the identication of new sirtuin
modulators are based on quantitative analysis of substrates/
products via HPLC/CE,21,22 uorescence resonance energy
transfer (FRET)-based peptide substrates,23 or uorogenic
peptide substrates.18,24,25 The latter has been most widely used
and requires a trypsin-coupled assay in which the release of
a
uorophore, e.g. 7-amino-4-methylcoumarin (AMC), is
Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education,
West China School of Pharmacy, and State Key Laboratory of Biotherapy, Sichuan
University, Chengdu, 610041, China. E-mail: liguobo@scu.edu.cn; yamei_yu@scu.
edu.cn
† Electronic supplementary information (ESI) available: Crystallographic data
collection and renement statistics, additional gures and tables. See DOI:
10.1039/c7ra05824a
Fig. 1 Outlined mechanisms for sirtuin-catalysed deacylations.
‡ H.-L. Wang and S. Liu contributed equally to this work.
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(class I), SIRT5 (class III), and SIRT6 (class IV), with the aim of
comparing their sensitivity and selectivity with those of peptide
substrates. We then used isothermal titration calorimetry (ITC)
analyses to investigate the inner drives of substrate binding and
performed crystallographic analyses to investigate how SuBKA
binds to its preferred sirtuin isoform SIRT5.
Results and discussion
The template molecule BKA (5) and the substrates AcBKA (6a),
SuBKA (6b), CrBKA (6c), and MyBKA (6d) were synthesized via
the synthetic route shown in Scheme 1. Briey, all target
compounds were prepared from the commercially available N2-
(((9H-uoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-
lysine (1), which reacted with 7-amino-4-methyl-2H-chromen-2-
one to afford compound 2 in the presence of POCl3. Then, 2 was
successively subjected to de-protection reaction and condensa-
tion reaction with benzyl carbonochloridate to obtain the
intermediate 4 in good yield. Subsequently, 4 was treated with
triuoroacetic acid in DCM to obtain the key intermediate 5,
which reacted with acetyl chloride, succinic anhydride, crotonic
acid, and myristic acid, respectively, to produce the corre-
sponding target compounds 6a, b, c, and d in moderate yields.
We rst tested whether BKA could be efficiently hydrolysed
by trypsin. When treated with saturating trypsin to different
concentrations of BKA for 24 h, we observed a clear linear
relationship between BKA concentrations and measured uo-
rescence values (RFU) (Fig. S3†); this implied that BKA
Scheme 1 Reagents and conditions: (a) pyridine, POCl3, THF, 0 ꢀC–
RT, yield: 83%; (b) piperidine, MeCN, 4 ꢀC, 16 h; DCM, RT, 8 h, yield:
80%; (c) Na2CO3, H2O/1,4-dioxane, 0 ꢀC–RT, yield: 75%; (d) TFA, DCM,
0 ꢀC–RT, yield: 71%; (e) for 6a: acetyl chloride, pyridine, THF, RT, yield:
43%; for 6b: DIPEA, succinic anhydride, THF, 0 ꢀC–RT, yield: 38.5%; for
6c/6d: crotonic acid/myristic acid, EDCI, HOBT, NMM, DCM, yield:
48–54%.
continuously monitored (Fig. S1†). Compared with uorogenic
peptide substrates that are derived from sirtuin isoform-specic
substrate proteins, small-molecule substrates for sirtuins have
been rarely studied. In this study, we synthesized four types of
uorogenic small-molecule substrates, i.e. acetyl-(AcBKA), cro-
tonyl-(CrBKA), succinyl-(SuBKA), and myristoyl-(MyBKA)-
containing substrates (Scheme 1 and Fig. S2†), and tested
them against three representative sirtuin proteins, i.e. SIRT2
Fig. 2 Deacylase activities of SIRT2, SIRT5, and SIRT6 on small-molecule substrates. (a, b, and c) Fluorescence values obtained by treating SIRT2,
SIRT5, and SIRT6 with 100 mM and 10 mM substrates, respectively. (d–f) Michaelis–Menten kinetic curves of SIRT2 on AcBKA (300 mM–0.58 mM),
SIRT2 on MyBKA (75 mM–0.58 mM), and SIRT5 on SuBKA (500 mM–0.98 mM).
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Table 1 Comparison of enzyme kinetic data of small-molecule substrates with that of peptide substrates
Literature data for peptide substrates
[Enzyme]
(mM)
KM
(mM)
Vmax
kcat/KM
Peptide
substrate
kcat/KM
Entry
Enzyme
SIRT2
Substrate
(nM minꢁ1
)
kcat (sꢁ1
)
(Mꢁ1 sꢁ1
)
KM (mM)
(Mꢁ1 sꢁ1
)
Ref.
1
2
3
AcBKA
MyBKA
SuBKA
0.5
0.5
0.2
273.5
1.7
13.3
29.3
1.63
21.7
9.8 ꢃ 10ꢁ4
5.4 ꢃ 10ꢁ5
2.6 ꢃ 10ꢁ3
3.5 ꢃ 101
3.2 ꢃ 101
1.6 ꢃ 102
Ac-ETDKAc
Ac-ETDKMy
Ac-LGKSu
750
1.8
33
8.1 ꢃ 101
8.5 ꢃ 103
9.2 ꢃ 102
26
26
27
SIRT5
ꢀ
concentrations could be quantitatively monitored via uores- with sirtuins for 4 h at 37 C, and then trypsin was added (2 U
cence values (Fig. S3†). To examine hydrolysis efficiency, we mLꢁ1) to release the AMC uorophore. The results revealed that
obtained the initial rate velocities of trypsin (2 U mLꢁ1) hydro- SIRT2 could hydrolyse the acetyl and myristoyl moieties from
lyzing BKA (30 mM to 0.05 mM) by acquiring the uorescence AcBKA and MyBKA, respectively, at both 100 mM and 10 mM
values every 30 seconds. We observed that the reaction rate (Fig. 2a); obviously, SIRT2 had much stronger deacetylation
increases with the increasing concentrations of BKA (Fig. S4†); activity than demyristoylation activity. No or very weak catalytic
for 30 mM BKA, the rate of hydrolysis by trypsin is 0.038 ꢂ 0.001 activity on CrBKA and SuBKA was observed for SIRT2 (Fig. 2a).
mM sꢁ1 (Fig. S4†), indicating that trypsin can efficiently hydro- In contrast, SIRT5 showed robust desuccinylation activity for
lyze BKA.
SuBKA (Fig. 2b), but had no or very weak deacylation activities
We then examined the deacylation activities of SIRT2, SIRT5, for AcBKA, CrBKA, and MyBKA (Fig. 2b); these ndings reected
and SIRT6 on the substrates AcBKA, CrBKA, SuBKA, and MyBKA a highly selective desuccinylation activity of SIRT5. Note that for
(see the Experimental section). Preliminarily, these substrates SIRT6, we only observed weak catalytic activities for AcBKA and
were tested at the concentrations of 100 mM and 10 mM, reacted MyBKA (Fig. 2c); this nding indicated that all these small-
Fig. 3 ITC analyses for small-molecule substrates to the preferred sirtuin isoforms. The curves of molar ratio to thermal effects of (a) AcBKA
titrating to SIRT2, (b) MyBKA to SIRT2, (c) SuBKA to SIRT5, and (d) specific thermodynamic parameters, showing that the binding of AcBKA/MyBKA
to SIRT2 is mainly driven by entropy, whereas SuBKA binding to SIRT5 is driven by enthalpy.
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molecule substrates might not be suitable for high-throughput reecting the exposure of buried hydrophobic surfaces and
screening assays for SIRT6. hydrophobic interactions of AcBKA/MyBKA with SIRT2. The
Then, enzyme kinetic studies were carried out for SIRT2 and enthalpy and entropy changes of SuBKA binding to SIRT5 are
SIRT5 with their sensitive small-molecule substrates. The ꢁ15.48 kJ molꢁ1 and ꢁ13.33 kJ molꢁ1, respectively, and the
kinetic constants were determined by measuring reaction rate calculated binding free energy is ꢁ28.81 kJ molꢁ1 (Fig. 3d). The
velocities at varying substrate concentrations, and the data was results reveal that the binding of SuBKA to SIRT5 is driven by
tted to the Michaelis–Menten equation to obtain KM and Vmax enthalpy (|DH| > |ꢁTDS|), uncovering the main contributions of
values (Table 1); the kcat values were calculated from the rela-
tionship kcat ¼ Vmax/[Enzyme]. For SIRT2, we observed that the
KM values for AcBKA and MyBKA were ꢄ273.5 mM (unsaturation
curve, Fig. 2d) and 1.7 mM (Fig. 2e and Table 1), respectively,
which were similar to those for Ac-ETDKAc (KM ¼ 750 mM)26 and
Ac-ETDKMy (KM ¼ 1.8 mM)26 peptide substrates, respectively. The
catalytic efficiencies (kcat/KM) for AcBKA and MyBKA were 3.5 ꢃ
101 Mꢁ1 sꢁ1 and 3.2 ꢃ 101 Mꢁ1 sꢁ1 (Table 1), respectively. The
observed SIRT2 deacetylation activity for AcBKA is similar to
that for the peptide substrate Ac-ETDKAc (kcat/KM ¼ 8.1 ꢃ 101
Mꢁ1 sꢁ1),26 but the demyristoylation activity for MyBKA (kcat/KM
¼ 8.5 ꢃ 103 Mꢁ1 sꢁ1
)
is apparently lower than that for Ac-
26
ETDKMy (Table 1) probably due to low water solubility of
MyBKA. In contrast, SIRT5 was shown to have excellent catalytic
efficiency for its sensitive substrate SuBKA (KM ¼ 13.3 mM, kcat
/
KM ¼ 1.6 ꢃ 102 Mꢁ1 sꢁ1) (Fig. 2f and Table 1), which is similar to
that for the peptide substrate Ac-LGKSu (KM ¼ 33 mM, kcat/KM
¼
9.2 ꢃ 102 Mꢁ1 sꢁ1).27,28 In addition, we observed that sirtuin-
catalysed deacylations for AcBKA and MyBKA are highly asso-
ciated with the concentrations of NAD+ (Fig. S5†); this further
revealed the evidence for the NAD+-dependent catalysis nature
of sirtuins.17,29 The enzyme kinetic results indicate that small-
molecule substrates (along with precise acyl substituents)
have similar sirtuin-isoform sensitivity as compared to peptide
substrates.
We then used isothermal titration calorimetry (ITC) method
to determine the binding affinity and thermodynamic param-
eters (e.g., enthalpy/entropy contributions) of AcBKA and
MyBKA binding to SIRT2 as well as SuBKA binding to SIRT5 (for
details, please check the Experimental section). The curves of
molar ratio to thermal effects are shown in Fig. 3a–c. From the
tting data, we obtained the equilibrium dissociation constants
(Ka) of AcBKA and MyBKA binding to SIRT2; the Ka values of
AcBKA and MyBKA were 4.41 ꢃ 104 ꢂ 2.84 ꢃ 104 Mꢁ1 (Fig. 3a)
and 1.24 ꢃ 105 ꢂ 5.26 ꢃ 104 Mꢁ1 (Fig. 3b), respectively, and the
corresponding binding affinities (Kd ¼ 1/Ka) were 22.67 mM and
8.06 mM, respectively, consistent with the KM values of AcBKA
(273.5 mM, Table 1) and MyBKA (1.7 mM, Table 1), respectively.
By contrast, the equilibrium dissociation constant Ka of SuBKA
binding with SIRT5 is 1.11 ꢃ 105 ꢂ 2.83 ꢃ 104 Mꢁ1 (Fig. 3c), and Fig. 4 Crystallographic analyses reveals how SuBKA binds to SIRT5. (a)
The AMC moieties of SuBKA in two neighboring SIRT5 molecules were
observed to form p–p stacking interactions (PDB ID 5XHS). (b) View
from the SIRT5:SuBKA complex structure, in which SuBKA is posi-
tioned to make hydrogen-bonding interactions with SIRT5-specific
the binding affinity Kd is 9.01 mM, which is close to the KM value
of SIRT5 hydrolyzing SuBKA (13.3 mM, Table 1).
The ITC analyses reveal that the enthalpy (DH) changes of
AcBKA and MyBKA binding to SIRT2 are ꢁ2.96 ꢂ 1.08 kJ molꢁ1
residues Tyr102 and Arg105, and the residues Val221, Gly224, and
and ꢁ7.05 ꢂ 1.13 kJ molꢁ1 (Fig. 3d), respectively; the entropy Glu225 on Loop S. (c) Comparison of SIRT5:SuBKA and SIRT5:LGKsu
-
(ꢁTDS) changes are ꢁ23.55 kJ molꢁ1 and ꢁ22.03 kJ molꢁ1
(Fig. 3d); and the calculated binding free energy DG (DG ¼ DH ꢁ
TDS) is ꢁ26.51 ꢂ 1.08 kJ molꢁ1 and ꢁ29.08 ꢂ 1.13 kJ molꢁ1
(Fig. 3d). The results indicate that both AcBKA and MyBKA
AMC structures (PDB ID 5BWL) reveals that although AMC moieties of
SuBKA and LGKsuAMC bind in different manners, SuBKA and LGKsu
-
AMC have similar hydrogen-bonding/electrostatic interactions with
SIRT5-specific residues and other catalytically important residues on
Loop S. SuBKA is shown by green sticks, and LGKsuAMC is shown by
binding to SIRT2 are mainly driven by entropy (|ꢁTDS| > |DH|), orange sticks.
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hydrogen-bonding/electrostatic interactions and complemen-
On superimposing the SIRT5:SuBKA structure with the
tarity between SuBKA and SIRT5, consistent with that observed SIRT5:LGKsuAMC (a uorogenic peptide substrate; PDB ID
by subsequent crystallographic analyses (Fig. 4). In addition, 5BWL) structure, we observed that SuBKA bound to SIRT5 via
these results can offer thermodynamic insights into the design a new mode, which was different from that of LGKsuAMC
of sirtuin isoform-specic inhibitors.
(Fig. 4c). The lysine N-/C-terminus of SuBKA and LGKsuAMC
We then performed crystallographic and molecular docking bound in opposite directions (Fig. 4c); this suggested the
analyses to investigate how the small-molecule substrates bind possibility of incorporation of D-lysine as sirtuin substrates.
to their preferred sirtuin isoforms. We attempted co- Note that SuBKA and LGKsuAMC form hydrogen-bonding/
crystallization and soaking, but failed to obtain a SIRT2 struc- electrostatic interactions with the residues (Val221, Gly224,
ture complexed with AcBKA or MyBKA probably due to their and Glu225) on the Loop S and the SIRT5-specic residues
limited solubility under the used crystallizing conditions. The (Tyr102 and Arg105) (Fig. 4c). This may reect the most
crystal structure of SIRT5:SuBKA complex was obtained via co- important binding features of the SIRT5 substrates and also
crystallization (see Experimental section) and determined to provide possible common pharmacophore features for the
˚
2.19 A resolution (Table S2†). The SIRT5:SuBKA structure crys- design of new small-molecule inhibitors for SIRT5.
tallized with one molecule in the asymmetric unit (space group
Comparison of SIRT5:SuBKA (PDB ID 5XHS) and SIRT5:Succ-
P21221) (PDB ID 5XHS, Table S2†). Interestingly, the AMC IDH2:Carba-NAD (PDB ID 4G1C)30 structures reveals that like that
moieties of SuBKA in two neighboring SIRT5 molecules formed of the peptide substrate Succ-IDH2, the amide bond of SuBKA
p–p stacking interactions, which might contribute to crystal approaches Carba-NAD (Fig. 5a). Through superimposition of the
packing (Fig. 4a).
SIRT5:SuBKA structure with the SIRT5:bicyclic-intermediate
In the SIRT5:SuBKA structure, we observed that SuBKA was (PDB ID 4F56)31 structure, we observed that SuBKA appears
positioned to make hydrogen-bonding interactions with the likely to form a bicyclic intermediate with the cofactor NAD+
main chains of Gly224, Glu225, and Val221 on the Loop S and (Fig. 5b), which is highly similar with the peptide substrate
hydrophobic interactions with the side chains of Val221 and GTSKsuRAT. Together, the crystallographic analyses indicate that
Phe223 (Fig. 4b). The carboxylate group of SuBKA was observed SuBKA is desuccinylated by SIRT5 probably through the catalytic
to form hydrogen bonding and electrostatic interactions with mechanism proposed for peptide substrates.27,28
Tyr102 and Arg105 (Fig. 4b), both of which were unique residues
of SIRT5. This, to a large extent, explained the specicity of
SIRT5 for catalytically recognizing/hydrolyzing SuBKA.
Fig. 5 Comparisons of the SIRT5:SuBKA structure with those of (a) the
SIRT5:Succ-IDH2:Carba-NAD (PDB ID 4G1C)30 and (b) SIRT5:bicyclic- Fig. 6 Predicted binding modes of (a) AcBKA and (b) MyBKA with
intermediate (PDB ID 4F56)31 reveal that SuBKA appears likely to SIRT2 using molecular docking simulations (for details, please check
undergo the same catalytic mechanism as the peptide substrates.
the Experimental section).
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We used molecular docking to predict the binding modes of
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tert-Butyl (S)-(5-amino-6-((4-methyl-2-oxo-2H-chromen-7-yl)-
amino)-6-oxohexyl) carbamate (3). A suspension of N2-(((9H-
AcBKA and MyBKA with SIRT2. The results show that AcBKA
and MyBKA are likely positioned to interact with the catalyti-
cally important residues of SIRT2 such as Phe235, Val233,
Glu237, and Gln267 (Fig. 6a and b). Compared to AcBKA,
MyBKA likely formed stronger interactions with the residues
(e.g., Ile169, Phe119, Tyr139, and Phe96) at the SIRT2 hydro-
phobic site (Fig. 6b); this may explain why MyBKA binds to
SIRT2 more tightly (Fig. 3d). Moreover, the molecular docking
results revealed the similarity between the SIRT2-catalyzed
mechanisms for AcBKA/MyBKA and the peptide substrates
(e.g. H3K9myr, Fig. S6†).32
uoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxy
carbonyl)-L-
lysine (1, 997 mg, 1.0 equiv.) and 7-amino-4-methyl-2H-
chromen-2-one (AMC, 438 mg, 1.17 equiv.) in dry THF (40 mL)
was cooled to 0 ꢀC, and dry pyridine (1.83 mL, 10.6 equiv.) was
added dropwise. Then, POCl3 (0.716 mL, 3.7 equiv.) was added
slowly aer the mixture was stirred for 0.5 h. The reaction was
ꢀ
continued for 1.5 h at 0 C and for 1 h at room temperature.
Aer completion (monitored by TLC), the mixture was poured
into ice and saturated NaHCO3 solution (v/v, 1 : 1), and the
organic solvent was removed under reduced pressure and par-
titioned between water and ethyl acetate (3ꢃ). The organic layer
was dried over anhydrous MgSO4 and concentrated in vacuo.
The residue was puried by column chromatography (eluent
gradient DCM/MeOH ¼ 200/1) to obtain (9H-uoren-9-yl)methyl
Conclusions
In summary, enzyme kinetic analyses reveal that the four types
of small-molecule substrates presented in this study have
similar sirtuin-isoform sensitivity to peptide substrates. ITC
analyses reveal that the binding of AcBKA or MyBKA to SIRT2 is
mainly driven by entropy, whereas SuBKA binding to SIRT5 is
driven by enthalpy, which also provide thermodynamic insights
into inhibitor design. The SIRT5:SuBKA complex structure
reveals that SuBKA binds to SIRT5 via a mode different from
that of the peptide substrates, but it involves SIRT5-specic
residues Tyr102 and Arg105 and other catalytically important
residues on Loop S. The crystallographic analyses also indicate
that SuBKA undergoes the same desucciylation mechanism as
peptide substrates. Comparison analyses of the SIRT5:SuBKA
structure and other SIRT5 complex structures revealed the
common pharmacophore features of the SIRT5 substrates,
which offer insights into the design of selective SIRT5 inhibi-
tors. This study will aid further efforts not only for developing
new small-molecule substrates but also for designing new
selective sirtuin isoform-specic inhibitors as tools and
therapeutics.
tert-butyl
(6-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-6-
oxohexane-1,5-diyl) (S)-dicarbamate (2, 1.102 g) in 83% yield.
A mixture of the intermediate 2 (1.0 g) and catalytic amount
of piperidine (0.184 mL) in 33 mL MeCN was stirred for 16 h at
4
ꢀC. Aer the addition of dichloromethane (16 mL), the
mixture was allowed to warm to room temperature and stirred
for another 8 h. The resulting solution was concentrated to
dryness and the residue was puried by silica gel column
chromatography (eluent gradient DCM/MeOH/NH4OH ¼ 100/1/
0.25) to obtain the white compound 3 (526 mg) in 80% yield. 1H-
NMR (400 MHz, DMSO-d6) d 7.83 (s, 1H), 7.76 (d, J ¼ 8.4 Hz, 1H),
7.57 (d, J ¼ 8.4 Hz, 1H), 6.77 (br s, 1H), 6.29 (s, 1H), 3.85 (t, J ¼
6.0 Hz, 1H), 2.89 (s, 2H), 2.40 (s, 3H), 1.79–1.73 (m, 2H), 1.41–
1.29 (m, 13H) ppm. LCMS m/z: 404.2 [M + H]+.
Benzyl
tert-butyl(6-((4-methyl-2-oxo-2H-chromen-7-yl)-
amino)-6-oxohexane-1,5-diyl) (S)-dicarbamate (4). To a solution
of compound 3 (400 mg, 1.0 equiv.) in H2O (12 mL) and 1,4-
dioxane (24 mL) in the presence of Na2CO3 (312.5 mg, 3 equiv.) at
ꢀ
0 C, benzyl carbonochloridate (Cbz-Cl, 176 mL, 1.3 equiv.) was
added, and the mixture was allowed to warm to room tempera-
ture and stirred for 4 h. Upon completion of the reaction, as
determined by TLC, the resulting solution was concentrated and
extracted with ethyl acetate; the combined organic extracts were
dried (MgSO4) and concentrated under reduced pressure to yield
the crude product, which was puried by silica gel column
chromatography (eluent gradient DCM/MeOH ¼ 200/1) to obtain
Experimental
Chemical synthesis
All chemicals and solvents were commercially purchased and
used as supplied without further purication. Room tempera-
ture refers to 20–25 ꢀC. All reactions were monitored using thin
layer chromatography (TLC) on silica gel F-254 TLC plates.
Column chromatography was carried out using silica gel
1
the white compound 4 (438.3 mg) in 75% yield. H-NMR (400
MHz, DMSO-d6) d 10.49 (br s, 1H), 7.78 (s, 1H), 7.73 (d, J ¼ 8.8 Hz,
1H), 7.67 (d, J ¼ 7.6 Hz, 1H), 7.51 (d, J ¼ 8.8 Hz, 1H), 7.37–7.31 (m,
5H), 6.78 (br s, 1H), 6.27 (s, 1H), 5.03 (s, 2H), 4.18–4.09 (m, 1H),
2.93–2.85 (m, 2H), 2.40 (s, 3H), 1.72–1.58 (m, 2H), 1.42–1.29 (m,
13H) ppm. LCMS m/z: 538.2 [M + H]+.
1
(300ꢁ400 mesh). H-NMR and 13C-NMR spectra were obtained
using a Bruker AV-400 spectrometer or Bruker DRX 600 spec-
trometer. Chemical shis (d) were obtained in parts per million
(ppm) relative to tetramethylsilane as the internal standard, and
coupling constants (J) were obtained in hertz (Hz). Low-
resolution mass spectral (MS) data were acquired using an
Agilent 1100 series LC-MS instrument with UV detection at
254 nm in the electrospray ionization (ESI) mode. All the target
compounds were puried to >95% purity, as determined by
high-performance liquid chromatography (HPLC). HPLC anal-
ysis was performed via a Waters 2695 HPLC system equipped
with a Kromasil C18 column (4.6 mm ꢃ 250 mm, 5 mm).
Benzyl
(S)-(6-amino-1-((4-methyl-2-oxo-2H-chromen-7-yl)
suspension of
amino)-1-oxohexan-2-yl)carbamate (5).
A
compound 4 (700 mg, 1.0 equiv.) in anhydrous dichloro-
methane (28 mL) was treated with triuoroacetic acid (TFA, 2.4
mL, 25 equiv.) and stirred for 3 h at ambient temperature. Aer
completion of the reaction (monitored by TLC), the mixture was
concentrated and extracted with saturated NaHCO3 and ethyl
acetate (3ꢃ), and the combined organic extracts were dried
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(MgSO4) and concentrated under reduced pressure. The residue (pH 5–6), which was extracted several times with ethyl acetate.
was puried by silica gel column chromatography (eluent The combined organic extracts were dried (Na2SO4), concen-
gradient DCM/MeOH ¼ 20/1) to afford compound 5 as a white trated under reduced pressure, and puried by silica gel column
solid (407.9 mg) in 71% yield. 1H-NMR (400 MHz, DMSO-d6) chromatography (eluent gradient DCM/MeOH ¼ 20/1) to afford
d 10.63 (br s, 1H), 7.80 (s, 1H), 7.74 (d, J ¼ 8.8 Hz, 1H), 7.70 (d, J compound 6c as a light yellow solid (47.3 mg) in 39% yield.
¼ 7.6 Hz, 1H), 7.54 (d, J ¼ 8.8 Hz, 1H), 7.41–7.32 (m, 4H), 6.27 (s, HPLC purity: 97.8%. 1H-NMR (600 MHz, DMSO-d6) d 10.54 (br s,
1H), 5.04 (s, 2H), 4.21–4.13 (m, 1H), 2.76 (t, J ¼ 7.2 Hz, 2H), 2.40 1H), 7.83 (t, J ¼ 5.4 Hz, 1H), 7.78 (s, 1H), 7.73 (d, J ¼ 9.0 Hz, 1H),
(s, 3H), 1.78–1.60 (m, 2H), 1.60–1.50 (m, 2H), 1.49–1.32 (m, 7.69 (d, J ¼ 7.8 Hz, 1H), 7.51 (d, J ¼ 9.0 Hz, 1H), 7.39–7.35 (m,
2H) ppm. LCMS m/z: 438.2 [M + H]+.
3H), 7.34–7.29 (m, 1H), 6.27 (s, 1H), 5.03 (s, 2H), 4.17–4.11 (m,
Benzyl (S)-(6-acetamido-1-((4-methyl-2-oxo-2H-chromen-7-yl)- 1H), 3.00 (t, J ¼ 5.4 Hz, 2H), 2.40 (s, 3H), 2.38 (t, J ¼ 7.2 Hz, 2H),
amino)-1-oxohexan-2-yl)carbamate (6a). To a suspension of 2.26 (t, J ¼ 7.2 Hz, 2H), 1.71–1.59 (m, 2H), 1.46–1.35 (m, 2H),
compound 5 (100 mg, 1.0 equiv.) in anhydrous THF (6 mL), 1.34–1.25 (m, 2H) ppm. LCMS (ESI) m/z: 538.3 [M + H]+.
acetyl chloride (24.3 mL, 1.5 equiv.) was added in the presence of
Benzyl
(S)-(1-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-1-
ꢀ
pyridine (DIPEA, 36.9 mL, 2 equiv.) and stirred for 3 h at 0 C. oxo-6-tetradecanamidohexan-2-yl)carbamate (6d). Compound
Upon completion of the reaction, as determined by TLC, the 6d (62.7 mg, 54%) was prepared from myristic acid (19.7 mg, 1.0
reaction solution was concentrated to dryness, and the residue equiv.) in the same manner as described for 6b. HPLC purity:
was puried by silica gel column chromatography to obtain the 96.9%. 1H-NMR (600 MHz, DMSO-d6) d 10.48 (br s, 1H), 7.78
white nal compound 6a in 86% yield. HPLC purity: 98.6%. 1H- (br s, 1H), 7.74–7.71 (m, 2H), 7.64 (d, J ¼ 7.2 Hz, 1H), 7.50 (d, J ¼
NMR (400 MHz, DMSO-d6) d 10.48 (s, 1H), 7.81–7.78 (m, 2H), 8.4 Hz, 1H), 7.40–7.34 (m, 3H), 7.33–7.30 (m, 1H), 6.27 (s, 1H),
7.73 (d, J ¼ 8.8 Hz, 1H), 7.65 (d, J ¼ 7.6 Hz, 1H), 7.50 (dd, J ¼ 5.03 (s, 2H), 4.17–4.11 (m, 1H), 3.05–2.97 (m, 2H), 2.40 (s, 3H),
8.8 Hz, J ¼ 1.6, 1H), 7.38–7.35 (m, 5H), 6.27 (s, 1H), 5.04 (s, 2H), 1.99 (t, J ¼ 7.2 Hz, 3H), 1.70–1.61 (m, 2H), 1.47–1.39 (m, 4H),
4.17–4.11 (m, 1H), 3.03–2.96 (m, 2H), 2.40 (s, 3H), 1.76 (s, 3H), 1.38–1.14 (m, 22H), 0.84 (t, J ¼ 7.2 Hz, 3H) ppm. 13C-NMR (101
1.67–1.59 (m, 2H), 1.40–1.29 (m, 4H), ppm. LCMS (ESI) m/z: MHz, DMSO-d6) d 172.41, 160.48, 156.62, 154.14, 153.53, 142.73,
480.2 [M + H]+.
137.42, 128.81, 128.27, 128.19, 126.36, 115.73, 115.53, 112.78,
Benzyl
(S,E)-(6-(but-2-enamido)-1-((4-methyl-2-oxo-2H- 107.46, 106.19, 67.09, 65.97, 56.05, 38.56, 35.93, 31.76, 29.53,
chromen-7-yl)amino)-1-oxohexan-2-yl)carbamate
(6b).
A
29.50, 29.48, 29.41, 29.36, 29.32, 29.17, 25.77, 24.03, 23.84,
mixture of compound 5 (100 mg, 1.0 equiv.), (E)-but-2-enoic acid 23.45, 22.56, 18.44, 14.41 ppm. LCMS (ESI) m/z: 648.4 [M + H]+.
(19.7 mg, 1.0 equiv.), 1-hydroxybenzotriazole (HOBT, 37.1 mg,
1.2 equiv.), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide
hydrochloride (EDCI, 52.6 mg, 1.2 equiv.), and 4-methyl-
Protein cloning, expression, and purication
morpholine (NMM, 31.2 mL, 2.2 equiv.) in dichloromethane (5
Cloning. The human SIRT2 (residues 56–356), human SIRT5
mL) was stirred at ambient temperature for 6 h. Upon (residues 34–269), and full-length human SIRT6 (residues 1–
completion of the reaction, as determined by TLC, the mixture 355) were PCR amplied and cloned into PET28 vector; this
was concentrated and partitioned between water and DCM (4ꢃ). resulted in a construct with N-terminal His-tag and a TEV
The organic layer was dried over anhydrous MgSO4, ltered, and protease cleavage site.
concentrated in vacuo. The products were puried by column
Protein expression and purication. Human SIRT2 (residues
chromatography (eluent gradient DCM/MeOH ¼ 80/1) to afford 56–356), human SIRT5 (residues 34–269), and human SIRT6
compound 6b as a white solid (62.7 mg) in 54% yield. HPLC (residues 1–355) were expressed in E. coli Transetta(DE3) cells
purity: 96.6%. 1H-NMR (400 MHz, DMSO-d6) d 10.49 (br s, 1H), (Novagen) overnight at 16 C in an LB medium. Overexpression
ꢀ
7.86 (br s, 1H), 7.77 (s, 1H), 7.73 (d, J ¼ 8.8 Hz, 1H), 7.67 (d, J ¼ was induced with isopropyl-b-D-thiogalactoside (0.3 mM) at an
7.6 Hz, 1H), 7.50 (d, J ¼ 8.8 Hz, 1H), 7.40–7.29 (m, 4H), 6.61–6.51 OD600 of 0.6–0.8. The cells were harvested and resuspended in lysis
(m, 1H), 6.27 (s, 1H), 5.87 (d, J ¼ 15.2 Hz, 1H), 5.03 (s, 2H), 4.18– buffer A (20 mM Tris–HCl, 250 mM NaCl, and pH 8.0), and then
4.09 (m, 1H), 3.10–3.05 (m, 2H), 2.40 (s, 3H), 1.75 (d, J ¼ 6.4 Hz, lysed using an ultrahigh-pressure homogenizer (JNBIO). The
3H), 1.70–1.61 (m, 2H), 1.50–1.29 (m, 4H) ppm. 13C-NMR (101 cellular debris was removed by centrifugation of the lysate at
MHz, DMSO-d6) d 172.44, 165.24, 160.49, 156.64, 154.13, 153.56, 15 000 rpm for 30 min. The supernatant was applied onto a Ni-
142.72, 137.77, 137.43, 128.82, 128.29, 128.21, 126.46, 126.40, NTA column (Roche), and the resin was washed with 20
115.74, 115.54, 112.78, 106.19, 65.98, 56.08, 38.60, 31.77, 29.36, volumes of the buffer B (20 mM Tris–HCl, 250 mM NaCl, 10 mM
23.56, 18.45, 17.73 ppm. LCMS (ESI) m/z: 506.2 [M + H]+.
(S)-4-((5-(((Benzyloxy)carbonyl)amino)-6-((4-methyl-2-oxo-2H- proteins. Recombinant proteins were eluted with buffer C (20 mM
chromen-7-yl)amino)-6-oxohexyl)amino)-4-oxobutanoic acid Tris–HCl, 250 mM NaCl, 250 mM imidazole, and pH 8.0). The
imidazole, and pH 8.0) to remove nonspecically binding
(6c). To a suspension of compound 5 (100 mg, 1.0 equiv.) in proteins of human SIRT2 (residues 56–356), human SIRT5 (resi-
anhydrous THF (6 mL), succinic anhydride (34.4 mg, 1.5 equiv.) dues 34–269), and human SIRT6 (residues 1–355) were pooled,
was added in the presence of ethyldiisopropylamine (DIPEA, concentrated, and desalted using a HiTrap Desalting column (GE
75.6 mL, 2 equiv.) and stirred for 1 h at 0 ꢀC. Then, the reaction Healthcare) into reaction buffer for enzyme kinetic analyses
mixture was allowed to warm to room temperature and stirred (SIRT2: 25 mM Tris–HCl, 150 mM NaCl, and pH 8.0; SIRT5: 20 mM
for 4 h. Aer completion of the reaction (monitored by TLC), the Tris–HCl, 150 mM NaCl and 5% (w/v) glycerol, and pH 8.0; SIRT6:
mixture was concentrated and acidied with NH4Cl solution 20 mM Tris–HCl, 150 mM NaCl, 5% (w/v) glycerol and 0.5 mM
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TCEP, and pH 8.0). The target proteins were concentrated to 24 mg
Crystallographic analyses
mLꢁ1 using Amicon Ultra 10K (Millipore) and stored at ꢁ80 C.
ꢀ
The SIRT5 proteins (13 mg mLꢁ1 in the buffer of 20 mM Tris/HCl
pH 8.0, 150 mM NaCl) were co-crystallized with SuBKA (100 mM
stocks dissolved in DMSO) at 16 ꢀC. The protein and SuBKA (2%
nal DMSO concentration for crystallization) mixtures were
prepared in a 1 : 5 molar ratio and incubated for 2 h at 4 ꢀC. The 2
mL crystallization drops constituted of a 1 : 1 ratio of pro-
tein : reservoir solutions (reservoir: 23–25% PEG 3350, 0.1 M Bis-
Tris, pH 5.5–6.5, and 0.2 M NaCl). Crystals appeared in 2–3 days.
The crystals were transferred to a cryo-protectant drop composed
of the reservoir, supplemented with 19% glycerol prior to ash-
freezing using liquid nitrogen. Data were obtained at the
Shanghai Synchrotron Radiation Facility and processed using
HKL2000 (Otinowski and Minor, 1997).
The human SIRT5 (residues 34–269) for crystallization were
digested with TEV protease (1 : 50) in the presence of 0.1% b-ME
(b-mercaptoethanol) overnight (4 ꢀC). The results of the TEV
digestion were veried by 12% SDS-PAGE. His-Tags, TEV protease,
and uncleaved protein removed by an Ni-NTA column (HiTrap™,
chelating HP, GE Healthcare). The digests were concentrated
using an Amicon Ultra 10K (Millipore), loaded onto a gel-ltration
column (Superdex 75 10/300 GL, GE Healthcare), and eluted at 0.5
mL minꢁ1 with buffer D (20 mM Tris–HCl 150 mM NaCl, and pH
¨
8.0) using an AKTA explorer device (GE Healthcare). Fractions
containing SIRT5 were pooled and concentrated to 13 mg mLꢁ1
ꢀ
and stored at ꢁ80 C. All purication steps were monitored via
12% SDS-PAGE, and the concentration was determined via
a NanoDrop 2000 spectrophotometer (Thermo Scientic).
Isothermal titration calorimetry (ITC) analysis
The ITC binding assay was carried out via a MicroCal ITC200
calorimeter (GE Healthcare) at 25 ꢀC. AcBKA (500 mM) and
MyBKA (750 mM) were titrated with SIRT2 (50 mM), separately,
using a buffer of 20 mM HEPES, 100 mM NaCl, and pH 7.4;
SuBKA (600 mM) was titrated with SIRT5 (60 mM) using the
SIRT5 buffer (20 mM Tris, pH 8.0, 20 mM NaCl, and 5% glyc-
erol). Both the cell and syringe of the calorimeter contained
0.6% DMSO. The system was equilibrated until the cell
temperature reached 25 ꢀC, and an additional delay of 60 s was
applied. All titrations were conducted using a preliminary
injection of 0.2 mL of 600 mM SuBKA and then a series of 19
individual injections of 2 mL at time intervals of 150 s. The
titration cell was stirred continuously at 750 rpm. The obtained
curve tting to a single binding site model was performed by
ITC data analysis module of Origin 7.5 (OriginLab).
Enzyme kinetic assays
In enzyme kinetic assays, all enzymes, cofactor NAD+, and
substrates were dissolved in the assay buffer: 50 mM Tris,
137 mM NaCl, 2.7 mM KCl, and pH 8.0. The assays were per-
formed in a 96-well black microplate (Corning No. 3603) with
a reaction volume of 60 mL per well. Fluorescence intensity was
measured using a microplate reader (BioTek Cytation 3, lex
390 nm, and lem ¼ 460 nm).
¼
Trypsin hydrolysing BKA assays. The saturated trypsins
(Sigma-Aldrich No. T8003; ꢄ20 U mLꢁ1) were reacted with BKA
with different concentrations (30 mM–0.058 mM; 2-fold dilu-
tions) for 24 h in the assay buffer (a total of 60 mL solution) to
obtain the calibration curve (Fig. S2†). To obtain the initial rate
velocities, BKA (30 mM–0.058 mM; 2-fold dilutions) was mixed
with 2U mLꢁ1 trypsins; moreover, the uorescence intensity was
obtained every 30 seconds (Fig. S3†).
Molecular docking analyses
The AutoDock Vina program33 was used for molecular docking
simulations. AcBKA and MyBKA were prepared as pdbqt le
using AutoDockTools. The crystal structure of SIRT2 complexed
with myristoylated peptide (H3K9myr) (PDB ID: 4Y6L)32 was
selected as the protein model. Then, all the water molecules and
solvent molecules were removed, Gasteiger–Marsili charges
were added, and non-polar hydrogens were then merged onto
their respective heavy atoms, as previously described.34,35 A total
of 20 docking poses were generated for AcBKA and MyBKA, and
the other parameters for Vina were set as default. The docking
results were viewed using the PyMOL program.
Preliminary activity test for sirtuins with substrates. Reac-
tion mixtures of the enzymes (SIRT2: 0.5 mM; SIRT5: 0.2 mM; or
SIRT6: 1 mM) with substrates (AcBKA, SuBKA, CrBKA, or MyBKA:
10 mM or 100 mM) and NAD+ (200 mM) were incubated for 4 h at
37 ꢀC and 140 rpm. Then, a stop solution (60 mL) containing ꢄ2
U mLꢁ1 trypsin and 8 mM nicotinamide was added to terminate
the reactions, followed by further incubation for 20 min at 37 ꢀC
and 140 rpm. Then, the uorescence was obtained by a micro-
plate reader.
Enzymatic kinetics of SIRT2 with AcBKA and MyBKA. The
SIRT2 enzymes (0.5 mM) were reacted with AcBKA (300 mM–0.58
mM) or MyBKA (75 mM–0.58 mM) and NAD+ (200 mM) in the assay
ꢀ
buffer (a total of 60 mL solution) for 4 h at 37 C and 140 rpm.
Conflicts of interest
Then, trypsin (ꢄ2 U mLꢁ1, 60 mL) containing nicotinamide (8
mM) was added and incubated for 20 min at 37 ꢀC and 140 rpm.
The uorescence was then obtained by a microplate reader.
Enzymatic kinetics of SIRT5 with SuBKA. The SIRT5 enzymes
(0.2 mM) were reacted with SuBKA (500 mM–0.49 mM) and NAD+
There are no conicts of interest to declare.
Acknowledgements
(200 mM) in the assay buffer (a total of 60 mL solution) for 4 h at The authors thank the staff of BL19U1 beamline of the National
37 ꢀC and 140 rpm. Then, trypsin (ꢄ2 U mLꢁ1, 60 mL) containing Center for Protein Science Shanghai at Shanghai Synchrotron
nicotinamide (8 mM) was added and incubated for 20 min at Radiation Facility for assistance during data collection. This
37 ꢀC and 140 rpm. The uorescence was then obtained by work was supported by the National Natural Science Founda-
a microplate reader.
tion of China (Grant No.: 81502989 and 81501368), the
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Scientic Research Foundation of Sichuan University (Grant 17 M. S. Bonkowski and D. A. Sinclair, Nat. Rev. Mol. Cell Biol.,
No.: 20822041A4193), and the China Postdoctoral Science
Foundation Funded Project (Grant No.: 2015M570789).
2016, 17, 679–690.
18 M. Schiedel, T. Rumpf, B. Karaman, A. Lehotzky,
´
S. Gerhardt, J. Ovadi, W. Sippl, O. Einsle and M. Jung,
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K. Weiser, M. Carbonaro, Y. L. Chiang, T. Southard,
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36222 | RSC Adv., 2017, 7, 36214–36222
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